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      治療和預(yù)防細(xì)菌感染的化合物和方法

      文檔序號(hào):1155479閱讀:527來源:國(guó)知局
      專利名稱:治療和預(yù)防細(xì)菌感染的化合物和方法
      有關(guān)聯(lián)邦政府資助研究的聲明本發(fā)明由國(guó)家衛(wèi)生協(xié)會(huì)津貼R37-AI22021和2T32-AI07410資助。政府享有由本發(fā)明所獲得的某些權(quán)利。
      背景技術(shù)
      總的來說,本發(fā)明涉及治療細(xì)菌感染,如炭疽感染的化合物和方法。
      當(dāng)作為生物戰(zhàn)或生物恐怖主義的媒介時(shí),炭疽(炭疽桿菌(Bacillus anthracis))病源媒介具有潛在的危險(xiǎn),這是由于霧化的炭疽桿菌(B.anthracis)孢子對(duì)哺乳動(dòng)物,如人類,來說是致命的。由這種生物所產(chǎn)生的主要?jiǎng)《拘砸蜃邮蔷?D-谷氨酸莢膜和炭疽毒素(ATx)。所述的莢膜和毒素均有利于細(xì)菌的群集和免疫逃避。僅單獨(dú)的Atx即可導(dǎo)致宿主死亡。接種抗毒素的疫苗可防止宿主被感染。
      炭疽毒素是一種細(xì)菌毒素,稱為A-B毒素。A-B毒素包括兩個(gè)組成成分,A組分是毒素的酶活性部分,其催化在靶細(xì)胞內(nèi)對(duì)胞漿的毒性作用。B組分結(jié)合于細(xì)胞受體,使A組分易于穿過細(xì)胞膜進(jìn)入胞漿。
      來自破傷風(fēng),肉毒梭菌,白喉和炭疽的A-B毒素的B組分全部在細(xì)胞膜上形成通道。假定這些通道可以作為A組分膜轉(zhuǎn)運(yùn)的導(dǎo)管。所述的炭疽毒素A和B組分作為不同的多肽,由細(xì)菌細(xì)胞中分泌出來。其它的A-B毒素作為單鏈多肽產(chǎn)生,或作為單獨(dú)的鏈產(chǎn)生再在釋放到細(xì)菌外之前組裝到寡聚體毒素中。有兩種可替換的炭疽毒素A亞基,分別稱為水腫因子(EF)和致死因子(LF)。EF或LF與保護(hù)性抗原(PA)B亞基的非共價(jià)復(fù)合物分別稱為水腫毒素和致死毒素。PA使EF和LF易于穿過細(xì)胞膜。
      PA作為83 kDa的單體多肽分泌出來。單體PA與哺乳動(dòng)物細(xì)胞表面受體結(jié)合,通過蛋白水解開裂。C-末端63 kDa片段(PA63)仍然與細(xì)胞相結(jié)合,N-末端的20 kDa(PA20)與PA63解離。這一蛋白水解切割與接下來的PA20解離賦予了PA63兩個(gè)新的特性(1)寡聚化成為稱為前孔的、SDS可解離的環(huán)狀七聚體結(jié)構(gòu)的能力(2)與EF和LF結(jié)合的能力。包含PA63-EF,PA63-LF,或PA63-EF和PA63-LF的結(jié)合的寡聚物經(jīng)胞吞并運(yùn)輸?shù)剿嵝詤^(qū)室,在那里PA63前孔插入到細(xì)胞膜中形成孔。在孔形成過程中或形成后,EF和LF穿過胞內(nèi)膜進(jìn)入細(xì)胞質(zhì)。EF是鈣調(diào)蛋白-依賴的腺苷酸環(huán)化酶,其可保護(hù)細(xì)菌不受吞噬細(xì)胞的破壞。LF是一種金屬蛋白酶,其可以殺死巨噬細(xì)胞或在低濃度下誘導(dǎo)巨噬細(xì)胞過量的產(chǎn)生細(xì)胞因子,其可能導(dǎo)致宿主死亡。
      這一中毒途徑的關(guān)鍵步驟是由PA形成孔。低pH引發(fā)PA63前孔向孔的轉(zhuǎn)化。這一轉(zhuǎn)化伴隨可使所述的寡聚體由SDS可解離狀態(tài)向抗SDS狀態(tài)的轉(zhuǎn)化并形成跨膜的14-鏈β桶。這些步驟對(duì)EF和LF穿越胞內(nèi)膜進(jìn)而進(jìn)行毒性作用是必需的。
      發(fā)明公開第一方面本發(fā)明提供由兩部分組成的形成孔的A-B毒素的B組分。所述的B組分帶有突變,導(dǎo)致其孔形成能力受到抑制。在一個(gè)優(yōu)選的實(shí)施方案中,這一突變導(dǎo)致在體內(nèi)該蛋白的孔形成能力受到抑制。在另一個(gè)優(yōu)選的實(shí)施例中,所述突變的B組分在體外和/或體內(nèi)缺乏孔形成能力。在又一優(yōu)選的實(shí)施方案中,所述的B組分是炭疽保護(hù)抗原(PA)。在再一優(yōu)選的實(shí)施方案中所述的PA突變體具有與天然存在的PA蛋白(如SEQ ID No.21;圖13所示)存在至少80%,90%,95%或98%同一性的氨基酸序列,并且其具有下述的替換之一K397A,K397D,K397C,K397Q,D425A,D425N,D425E,D425K,F(xiàn)427A,K397+D425K雙突變體,K395D+K397D+D425K+D426K四重突變體,K397D+D425K+F427A三重突變體,F(xiàn)427A+ΔD2L2雙突變體,K397D+F427A+ΔD2L2三重突變體,K397D+D425K+F427A+ΔD2L2四重突變體,F(xiàn)427D,或F427K。在另一優(yōu)選的實(shí)施方案中,所述的PA突變體與SEQID Nos.1-11和13-18(表1)中的任意序列具有序列同一性。其它優(yōu)選的PA突變體包括第397位是除賴氨酸外的任意氨基酸殘基的PA突變體(SEQ ID No.19),第425位是除天冬氨酸外的任意氨基酸殘基的PA突變體(SEQ ID No.20),第427位是除苯丙氨酸外的任意氨基酸殘基的PA突變體(SEQ ID No.23),在結(jié)構(gòu)域2(259-487位殘基)存在突變的PA突變體。再有其它優(yōu)選的突變體B組分包括一個(gè)或多個(gè)列于表6的替換或相應(yīng)于PA結(jié)構(gòu)域2存在突變的艱難梭菌(Clostridium difficile),產(chǎn)氣莢膜梭菌(C.perfringens),螺狀梭菌(C.spiroforme),肉毒梭菌(C.botulinum),蠟狀芽孢桿菌(Bacillus cereus)或蘇云金芽孢桿菌(B.Thuringiensis)毒素。上述突變體B組分的片段也包含在本發(fā)明的范圍內(nèi),其與天然存在的相應(yīng)毒素的B組分相比具有降低的孔形成能力。具有本發(fā)明的突變體B組分或其片段與另一多肽或蛋白共價(jià)結(jié)合的融合蛋白也包含在本發(fā)明的范圍內(nèi)。特別地從本發(fā)明的第一方面排除出去的是PA的302-325位氨基酸(D2L2環(huán))缺失。
      在本發(fā)明的第二方面中涉及含有第一方面中突變體B組分或ΔD2L2 PA(第302-325位氨基酸缺失的PA突變體)或其片段,以及在制藥學(xué)上可接受的載體的疫苗組合物。在一個(gè)優(yōu)選的實(shí)施方案中,所述的疫苗可以能通過化學(xué)或物理的方法滅活。
      第三方面,本發(fā)明涉及防止或治療哺乳動(dòng)物,如人細(xì)菌感染的方法。這一方法包括對(duì)所述哺乳動(dòng)物施用本發(fā)明第二方面中所述的疫苗。在一個(gè)優(yōu)選的實(shí)施方案中,所述的疫苗與制藥學(xué)上合適的載體或佐劑一起施用。所述的疫苗可通過口服,肌肉內(nèi),靜脈內(nèi),皮下,或通過吸入的方式施用,或者通過其它足以提供預(yù)防或治療細(xì)菌感染的途徑施用。在另一優(yōu)選的實(shí)施方案中,通過施用包含突變的炭疽保護(hù)抗原的疫苗以預(yù)防或治療炭疽感染。
      第四方面,本發(fā)明涉及由兩組分組成的孔形成A-B毒素的突變體B組分。所述的突變體B組分存在導(dǎo)致孔形成能力受到抑制。所述的突變體B組分也體內(nèi)和/體外抑制天然存在的相應(yīng)毒素的B組分的孔形成能力。在一個(gè)優(yōu)選的實(shí)施方案中,這一突變導(dǎo)致體內(nèi)該蛋白的孔形成能力受到抑制。在另一優(yōu)選的實(shí)施方案中,所述的突變體B組分在體內(nèi)和/或體外缺乏孔形成能力。在又一優(yōu)選的實(shí)施方案中所述的B組分是炭疽保護(hù)性抗原(PA)。所述的突變體B組分可以與相應(yīng)毒素的A組分結(jié)合。例如,某種PA突變體可以與致死因子或水腫因子A組分相結(jié)合。所述的突變體B組分可與天然存在的B組分競(jìng)爭(zhēng)結(jié)合于哺乳動(dòng)物細(xì)胞表面的受體。所述的突變體B組分也可以與天然存在的相應(yīng)毒素的B組分相結(jié)合。這種突變體B組分可以與天然存在的B組分寡聚化形成復(fù)合物以降低孔形成能力。在一個(gè)優(yōu)選的實(shí)施方案中,所述的復(fù)合物缺乏形成孔并進(jìn)一步使A組分(例如EF或LF)穿過細(xì)胞膜運(yùn)輸?shù)郊?xì)胞質(zhì)的能力。
      在第四方面的一個(gè)優(yōu)選的實(shí)施方案中,所述的突變體PA具有與天然存在的PA蛋白(例如SEQ ID No.21;圖13)存在至少80%,90%,95%,或98%同一性的氨基酸序列,并存在下述的替換之一K397D+D425K雙突變體,ΔD2L2(其中PA的第302-325位殘基缺失),K395D+K397D+D425K+D426K四重突變體,D425K,F(xiàn)427A,K397D+D425K+F427A三重突變體,F(xiàn)427A+ΔD2L2雙突變體,K397D+F427A+ΔD2L2三重突變體,K397D+D425K+F427A+ΔD2L2四重突變體,F(xiàn)427D,或F427K。在另一優(yōu)選的實(shí)施方案中,所述的PA突變體與SEQ ID Nos.8-18(表1)中的任意一項(xiàng)具有序列同一性。在又一優(yōu)選的實(shí)施方案中,天然存在的PA中第395,397,425,426位氨基酸,或其結(jié)合發(fā)生突變。在另一實(shí)施方案中,存在于PA結(jié)構(gòu)域2中的殘基發(fā)生突變。在又一優(yōu)選的實(shí)施方案中,所述的PA的D2L2環(huán)中或另一由兩組分構(gòu)成的孔形成A-B毒素的B組分的相應(yīng)區(qū)域具有至少2,至少10或至少20個(gè)氨基酸殘基缺失。所述的突變體具有全部或部分D2L2環(huán)缺失以及該環(huán)的N-或C-末端的氨基酸缺失。再有其它優(yōu)選的突變體B組分包括一個(gè)或多個(gè)列于表6的替換或相應(yīng)于PA結(jié)構(gòu)域2存在突變的艱難梭菌(Clostridium difficile),產(chǎn)氣莢膜梭菌(C.perfringens),螺狀梭菌(C.spiroforme),肉毒梭菌(C.botulinum),蠟狀芽孢桿菌(Bacillus cereus)或蘇云金芽孢桿菌(B.Thuringiensis)毒素。上述突變體B組分的片段也包含在本發(fā)明的范圍內(nèi),其與天然存在的相應(yīng)毒素的B組分相比具有降低的孔形成能力。由本發(fā)明的突變體B組分或其片段與另一多肽或蛋白共價(jià)結(jié)合的融合蛋白也包含在本發(fā)明的范圍內(nèi)。
      第五方面本發(fā)明涉及預(yù)防和治療哺乳動(dòng)物,如人的細(xì)菌感染的方法。該方法包括對(duì)哺乳動(dòng)物施用能抑制天然存在的B組分的孔形成能力的上述第四方面的突變體B組分或其片段。在一個(gè)實(shí)施方案中,上述第四方面的PA突變體或其片段施用于已暴露于炭疽桿菌(B.anthracis)孢子的哺乳動(dòng)物以預(yù)防或治療炭疽感染。在另一實(shí)施方案中,所述的蛋白作為預(yù)防性施用。在一個(gè)優(yōu)選的實(shí)施方案中,所述的突變體B組分與藥學(xué)上適合的載體一起施用。所述的疫苗可通過口服,肌肉內(nèi),靜脈內(nèi),皮下,或通過吸入的方式施用,或者通過其它足以提供預(yù)防或治療炭疽菌感染的劑量的途徑施用。在一個(gè)實(shí)施方案中,所述的方法還包括對(duì)哺乳動(dòng)物施用抗-B組分抗體,如可與天然存在的B組分結(jié)合,但不與顯性負(fù)B組分結(jié)合的抗體。在一個(gè)特定的實(shí)施方案中,所述的抗體可與天然存在的PA結(jié)合,但不與顯性負(fù)PA突變體結(jié)合。
      第六方面,本發(fā)明涉及編碼上述第一或四方面的突變體B組分(如PA突變體)的核酸。
      第七方面,本發(fā)明涉及具有上述第六方面的核酸的載體。
      第八方面,本發(fā)明涉及可與天然存在的PA或列于表1的PA突變體蛋白特異結(jié)合的純化抗體。在一個(gè)實(shí)施方案中,所述的抗體與PA蛋白的D2L2環(huán)、K397,D425,D426,或F427結(jié)合。所述的抗體可以是單克隆抗體也可以是多克隆抗體。在相關(guān)的方面,本發(fā)明涉及純化的抗體,其可以比結(jié)合于相應(yīng)毒素的本發(fā)明的B組分更強(qiáng)的親和力,特異性地與天然存在的由兩個(gè)組分組成的孔形成A-B毒素的B組分結(jié)合。另一相關(guān)的方面中,本發(fā)明涉及純化的抗體,其可以比結(jié)合于天然存在的由兩個(gè)組分組成的孔形成A-B毒素的B組分更強(qiáng)的親和力,特異性地與相應(yīng)毒素的本發(fā)明的B組分結(jié)合。
      在第一和第四方面的一個(gè)實(shí)施方案中,特異地排除那些具有唯一的替換,定位于跨膜孔疏水表面的半胱氨酸殘基突變的那些PA分子。在這些方面的另一實(shí)施方案中,那些定位于跨膜孔疏水表面的氨基酸殘基突變?yōu)槠湮ㄒ惶鎿Q的PA分子被特異排除在外。在一個(gè)實(shí)施方案中,特異地排除在所述第一和第四方面之外的是那些在天然存在的PA分子中Glu302,His304,Asn306,Glu308,His310,Ser312,Phe313,Phe314,Asp315,Gly317,Ser319,Ser321,Gly323,或Ser325處的突變作為唯一替換的PA分子。在這些方面的一個(gè)實(shí)施方案中,特異地排除了以在D2L2環(huán)的氨基酸序列中存在的突變作為其唯一替換的PA分子。在另一實(shí)施方案中,在形成跨膜孔的氨基酸中的突變或缺失作為其唯一替換的PA分子被排除在外。在又一實(shí)施方案中,特異地排除在外的是ΔD2L2PA。在又一實(shí)施方案中,特異地被排除在這些方面之一之外的是具有C-末端63 kDA胰蛋白酶片段(PA63)且以形成跨膜孔的氨基酸中的突變作為其唯一替換的PA分子。在其它不同的實(shí)施方案中,被特異地排除在這些方面之一之外的是具有相應(yīng)于其它被排除在外的PA突變體的突變的,其它由兩組分組成的孔形成A-B毒素。在這些方面的不同實(shí)施方案中,所述的突變體B組分,以比天然存在的B組分與來自相同A-B毒素的其它天然存在的分子相結(jié)合更高平衡常數(shù)(即更高的親和力)與來自天然存在的相同A-B毒素的B組分結(jié)合。在優(yōu)選的實(shí)施方案中,所述的突變PA蛋白以,比天然存在的PA與其它天然存在的PA分子的結(jié)合高出至少2,5,10,或20倍的親和力與天然存在的PA分子結(jié)合。
      應(yīng)當(dāng)理解,除炭疽毒素外的其它孔形成毒素也可在本發(fā)明的化合物和方法中使用。例如孔形成毒素,如其它具有抑制所述毒素孔形成能力的突變(點(diǎn)突變或缺失突變),或抑制天然存在毒素的孔形成能力突變的A-B毒素均包含在本發(fā)明的范圍內(nèi)。所述的帶有這些突變的孔形成毒素可用于本發(fā)明的疫苗組合物和方法中以預(yù)防或治療所述毒素的病源媒介的感染。
      在不對(duì)本發(fā)明作任何限制的前提下,也可以使用其它由兩組分構(gòu)成的A-B毒素;異源寡聚體毒素(AB5毒素),如霍亂毒素;或其它單一的多肽A-B毒素,如破傷風(fēng),肉毒梭菌,或白喉毒素。其它可以使用的毒素包括來自金黃色葡萄球菌(Staphylococcus aureus)的α-溶血素,來自嗜水氣單胞菌(Aeromonas hydrophila)的氣單胞菌溶素,來自敗毒梭菌(Clostridium septicum)的α-毒素,和來自銅綠假單胞菌(Pseudomonas aeruginosa)的細(xì)胞毒素。本發(fā)明還涉及其它孔形成毒素,如膽固醇依賴性的溶細(xì)胞素,六聚體毒素,或七聚體毒素。六聚體和七聚體毒素的例子包括與葡萄球菌相關(guān)的α-毒素。在一個(gè)實(shí)施方案中,來自幽門螺旋桿菌(Helicobacter pylori)的VacA毒素的缺失突變被特異地排除在外。
      “突變”指在核酸序列中的替換,由此由核酸序列編碼的氨基酸序列與天然存在的序列相比有至少一個(gè)氨基酸的替換。所述的突變可以是,但不限于插入,缺失,移碼突變或缺失突變。
      “孔形成毒素”是指形成跨膜含水孔的毒素。
      “孔形成A-B毒素”指具有兩個(gè)功能組分的孔形成毒素;一個(gè)組分(B)在所述宿主細(xì)胞屏障膜中形成孔,另一個(gè)(A)組分穿過所述的膜屏障并酶促修飾宿主細(xì)胞中的特異細(xì)胞內(nèi)底物。
      “由兩組分組成的孔形成A-B毒素的”是指孔形成A-B毒素,其中所述孔形成毒素的A和B組分分別存在于獨(dú)立的蛋白中,并在宿主細(xì)胞的中毒過程中相互作用。由兩個(gè)組分組成的毒素的例子是炭疽毒素。
      ″B組分″是指孔形成A-B毒素的組件,其與特異的宿主細(xì)胞表面受體結(jié)合,與所述毒素的A組分相互作用,并輔助A組分向所述細(xì)胞的內(nèi)在化。許多B組分,如PA也可形成跨膜的孔。
      ″保護(hù)性抗原(PA)″是指具有此處所述的炭疽PA多肽的生物活性的60%,70%,80%,或90%的至少之一多肽。所述的多肽可由Vodkin等(Cell 34693-697,1983)中報(bào)道的PA基因編碼。所述的多肽可以具有與Miller等(Biochemistry 38(32)10432-10441,1999)(SEQ IDNo.21)描述的野生型PA,或其它任何來自炭疽桿菌(Bacillusanthracis)的天然存在的PA多肽相同的特性。所述的PA多肽可以在異源宿主如大腸桿菌或枯草芽孢桿菌中克隆和表達(dá)。應(yīng)當(dāng)理解具有此處所述的炭疽PA特性的同源物或類似物也可用在本發(fā)明的方法中。
      ″PA63″指由PA多肽經(jīng)蛋白水解切除20 kDa N-末端節(jié)段所得的羧基末端部分。PA63形成七聚體的前孔并與兩個(gè)可替換的A組分,水腫因子(EF)和致命因子(LF)結(jié)合。所述的整個(gè)復(fù)合物運(yùn)輸?shù)絻?nèi)體,在那里PA63插入到細(xì)胞膜形成跨膜孔并將EF和LF轉(zhuǎn)移到宿主細(xì)胞的細(xì)胞質(zhì)中。
      “跨膜孔”是指跨越細(xì)胞膜的含水通道。例如所述的跨膜孔可以是β-筒通道,其是通過替換PA63的親水和疏水殘基,以使疏水殘基形成所述筒的外膜表面,親水殘基朝向跨越宿主細(xì)胞細(xì)胞膜的孔的含水腔。
      “跨膜孔的親水表面”是指朝向跨越宿主細(xì)胞細(xì)胞膜的孔的含水腔的PA的氨基酸。
      “形成跨膜孔的氨基酸”是指定位于跨膜孔β-筒通道的PA的氨基酸。
      ″D2L2環(huán)″是指連接此處所述的PA多肽的2β2和2β3鏈和PA63多肽的兩親環(huán)。
      ″抑制孔形成能力″是指降低膜中孔形成的量或降低運(yùn)輸?shù)剿拗骷?xì)胞細(xì)胞質(zhì)中的A組分(如EF或LF)的速率和數(shù)量。這種孔形成或毒素轉(zhuǎn)移的降低與此處所述的在細(xì)胞表面轉(zhuǎn)移的活性LFnDTA毒性、銣釋放分析下降正相關(guān),也可通過上述試驗(yàn)預(yù)測(cè)出。這一下降的活性可與下述情況聯(lián)系起來在細(xì)胞表面轉(zhuǎn)移試驗(yàn)中,放射性標(biāo)記的配體數(shù)量下降;在LFnDTA毒性試驗(yàn)中,由于配體轉(zhuǎn)移到細(xì)胞內(nèi)使蛋白合成的抑制的下降;或在銣釋放分析試驗(yàn)中,由細(xì)胞釋放的放射性標(biāo)記的離子的下降。另外這一下降的活性可與由于毒性配基轉(zhuǎn)移到細(xì)胞內(nèi)使得毒性下降聯(lián)系起來。在一個(gè)優(yōu)選的實(shí)施方案中,孔形成或A組分轉(zhuǎn)移的下降至少是相對(duì)于天然存在的相應(yīng)毒素的B組分的20%,更優(yōu)選至少40%,最優(yōu)選至少80%。在另一優(yōu)選的實(shí)施方案中,孔形成或PA突變體EF或LF轉(zhuǎn)移的下降是相對(duì)于天然存在的PA63的20%,更優(yōu)選至少40%,最優(yōu)選至少80%。
      ″缺乏孔形成能力″是指不能在膜中形成顯著數(shù)量的孔,或不能將顯著數(shù)量的EF或LF轉(zhuǎn)移到宿主細(xì)胞的細(xì)胞質(zhì)中。這種缺乏顯著的孔形成或毒素轉(zhuǎn)移能力與此處所述的在細(xì)胞表面運(yùn)輸,LFnDTA毒性,或銣釋放試驗(yàn)中顯著活性的缺乏正相關(guān),也可通過上述的實(shí)驗(yàn)預(yù)測(cè)。在一個(gè)優(yōu)選的實(shí)施方案中所形成的孔的數(shù)量和毒素轉(zhuǎn)移的數(shù)量比在沒有PA的對(duì)照試驗(yàn)中的少5倍。更優(yōu)選所述的數(shù)量比在沒有PA的對(duì)照試驗(yàn)中的少2倍。
      “片段”是指具有與PA突變體中的相應(yīng)區(qū)域同一的連續(xù)氨基酸區(qū)域的多肽。所述的片段與天然存在的PA相比或具有降低的孔形成或毒素轉(zhuǎn)移的能力。所述的片段也可以抑制天然存在的PA的孔形成能力。這種孔形成或毒素轉(zhuǎn)移的降低與此處所述的在細(xì)胞表面轉(zhuǎn)移的活性LFnDTA毒性、銣釋放試驗(yàn)中的下降正相關(guān),也可通過上述試驗(yàn)預(yù)測(cè)出。這一下降的活性可與下述情況聯(lián)系起來在細(xì)胞表面轉(zhuǎn)移試驗(yàn)中,放射性標(biāo)記的配體數(shù)量下降;在LFnDTA毒性試驗(yàn)中,由于配體轉(zhuǎn)移到細(xì)胞內(nèi)使蛋白合成的抑制的下降;或在銣釋放分析試驗(yàn)中,由細(xì)胞釋放的放射性標(biāo)記的離子的下降。在一個(gè)優(yōu)選的實(shí)施方案中,孔形成或EF或LF轉(zhuǎn)移的下降至少是相對(duì)于天然存在的PA63的20%。更優(yōu)選至少40%,最優(yōu)選至少80%。對(duì)天然存在的PA的孔形成能力的抑制與上述與單獨(dú)的天然存在的PA相比,用等摩爾的天然存在的PA和PA片段的試驗(yàn)中的活性下降正相關(guān),也可通過上述的試驗(yàn)預(yù)測(cè)出。在一個(gè)優(yōu)選的實(shí)施方案中,與僅用天然存在的PA相比,所述的下降是至少20,40,60,80,或99%。所期望的是,所述的片段是免疫原性的且能夠誘導(dǎo)針對(duì)天然存在的PA的保護(hù)性抗體產(chǎn)生。在另一優(yōu)選的實(shí)施方案中,如實(shí)施例9所述對(duì)哺乳動(dòng)物施用所述的片段,在至少1個(gè)月,優(yōu)選3個(gè)月,最優(yōu)選6個(gè)月的時(shí)間內(nèi)預(yù)防或減少炭疽感染。可能的片段的例子包括PA突變體的C-末端63kD胰蛋白酶片段或具有形成跨膜孔的氨基酸缺失的PA突變體。
      “經(jīng)純化的抗體”是指重量至少占60%的去除了下述物質(zhì)的抗體,所述的物質(zhì)是指上述抗體在天然狀態(tài)下與之結(jié)合的蛋白和天然存在的有機(jī)分子。預(yù)期上述制備物中的抗體至少占重量的75%,更優(yōu)選90%,最優(yōu)選99%。經(jīng)純化的抗體可以,例如利用重組蛋白或保守組分肽以標(biāo)準(zhǔn)的方法通過親和層析獲得。
      “特異結(jié)合”是指一種抗體識(shí)別并結(jié)合于天然狀態(tài)下包括蛋白的樣品,例如生物樣品中的野生型PA或PA突變體,但基本上不識(shí)別、結(jié)合非PA分子。優(yōu)選的抗體特異結(jié)合于列于表1#1-18中的任何PA突變體。其它優(yōu)選的抗體以與列于表1的一種或多種突變體結(jié)合高出至少2,5,10,或20倍的親和力與野生型的PA結(jié)合。
      序列同一性典型地使用序列分析軟件(例如Sequence AnalysisSoftware Package of the Genetics Computer Group,Universityof Wisconsin Biotechnology Center,1710 University Avenue,Madison,WI 53705),以其所特有的缺省參數(shù)進(jìn)行測(cè)定。這種軟件通過賦予不同的取代,缺失,和其它修飾以不同程度的同源性來比較序列相似性。保守取代典型地包括下述組中的取代甘氨酸,丙氨酸,纈氨酸,異亮氨酸,亮氨酸;天冬氨酸,谷氨酸,天冬酰胺,谷氨酰胺;絲氨酸,蘇氨酸;賴氨酸,精氨酸;和苯丙氨酸,酪氨酸。
      本發(fā)明的其它特征和有益之處將通過下述的詳細(xì)描述予以說明。


      圖1是ATx毒素中毒途徑的圖示說明。Atx中的PA組分與哺乳動(dòng)物細(xì)胞表面的受體結(jié)合,并如上所述向胞質(zhì)溶膠中運(yùn)送所述毒素的酶促A組分,水腫因子(EF)和致命因子(LF)。
      圖2A是SDS-PAGE凝膠圖,表明缺口PA突變體蛋白的形成和由野生型,K397Q,和ΔD2L2 PA形成抗-SDS的寡聚體。圖2B是天然的凝膠表明由野生型,K397A,和ΔD2L2 PA形成前孔。
      圖3是表明與野生型,K397A,或D425A PA一起培養(yǎng)后,與無PA的對(duì)照相比,86RB負(fù)載細(xì)胞釋放86Rb的量的柱形圖。
      圖4A是表明與野生型,K397A,或D425APA一起培養(yǎng)的細(xì)胞所結(jié)合的相似水平的35S-LFn(LF的PA結(jié)合結(jié)構(gòu)域N-末端1-255氨基酸)。圖4B與野生型PA相比由K397A或D425A PA介導(dǎo)的35S-LFn向細(xì)胞內(nèi)轉(zhuǎn)移的減少。
      圖5是表明在LFnDTA毒性試驗(yàn)中,通過與野生型,K397A,或D425APA共同培養(yǎng)后,不溶性的TCA組分(蛋白組分)中3H-Leu的百分比。包含與白喉毒素A鏈融合的LFn的LFnDTA轉(zhuǎn)運(yùn)到細(xì)胞中使得EF-2核糖基化,導(dǎo)致蛋白合成受到抑制,在蛋白組分中3H-Leu的量下降。
      圖6A是表明35S-LFn對(duì)與野生型,ΔD2L2,雙突變體K397D+D425K,或野生型和ΔD2L2或K397D+D425K PA的混合物一起培養(yǎng)的細(xì)胞的相似結(jié)合性的柱形圖。圖6B是表明由ΔD2L2或K397D+D425K PA,野生型PA-介導(dǎo)的35S LFn轉(zhuǎn)運(yùn)的下降的柱形圖。
      圖7表明與單獨(dú)的野生型PA相比,與ΔD2L2或K397D+D425K PA和野生型的PA一起培養(yǎng)后,在TCA不溶性組分中較高百分比的3H-Leu。這一結(jié)果與LFnDTA毒性試驗(yàn)中由野生型PA-介導(dǎo)的蛋白合成的抑制作用的降低相應(yīng)。
      圖8A表明LFnDTA毒性試驗(yàn)中由野生型PA-介導(dǎo)的蛋白合成的抑制作用的降低。突變PA蛋白濃度的升高減少了由野生型PA-介導(dǎo)的3H-Leu攝入到TCA不溶性組分中的抑制作用。圖8B表明與列于圖8A中的突變體需要量相比,需要更大量的相對(duì)于野生型PA的PA-SSR以降低野生型PA-介導(dǎo)的3H-Leu攝入的抑制。
      圖9表明在LFnDTA毒性試驗(yàn)中,野生型PA-介導(dǎo)的蛋白合成的抑制的降低是由于存在顯性負(fù)PA突變體濃度的升高。顯示在該圖中的是顯性負(fù)突變體K397D+D425K(□),ΔD2L2(■),F(xiàn)427A(○),D425K(△),和K397D(◇)和對(duì)照突變體SSSR(◆)的效果。
      圖10是表明在LFnDTA毒性試驗(yàn)中,野生型PA-介導(dǎo)的蛋白合成的抑制的降低是由于出現(xiàn)下述的一種顯性負(fù)PA突變體濃度升高K397D+D425K(■),F(xiàn)427A+ΔD2L2(□),K397D+D425K+F427A(○),和K397D+F427A+ΔD2L2(▲)。
      圖11是表明野生型PA與突變體PA(K397D+D425K,ΔD2L2,F(xiàn)427A,或D425K)混合形成異源七聚體,然后用胰蛋白酶裂解PA分子而使蛋白合成受到抑制的柱形圖。以1∶1混合的相應(yīng)突變體與野生型同源七聚體混合物對(duì)蛋白合成的抑制也進(jìn)行了測(cè)定。
      圖12是表明顯性負(fù)突變體K397D+D425K,ΔD2L2,F(xiàn)427A,和D425K對(duì)低-pH引發(fā)的35S LFN穿越質(zhì)膜的作用的柱形圖。所示的結(jié)果是三次實(shí)驗(yàn)的平均±SEM。
      圖13是用于此處所述試驗(yàn)的野生型PA蛋白的氨基酸序列(SEQ IDNo.21)。此處所述的PA突變體蛋白均基于該野生型序列。
      圖14是編碼用于此處所述試驗(yàn)的野生型PA蛋白的多核苷酸序列(SEQ ID No.22)。
      圖15是PA與其它由兩個(gè)組分組成且具有ADP核糖基轉(zhuǎn)移酶活性的A-B毒素的氨基酸序列對(duì)比。此處列出所述的來自梭狀芽孢桿菌艱難梭菌(Clostridium difficile)(″cdADPRT″),產(chǎn)氣莢膜梭菌(C.perfringens)(″cpiota″),螺狀梭菌(C.spiroforme)(″csiota′),和肉毒梭菌(C.botulinum)(″cbc2″)的毒素的氨基酸序列。產(chǎn)氣莢膜梭菌(C.perfringens)和螺狀梭菌(C.spiroforme)毒素通常是指ι(iota)毒素,而肉毒梭菌毒素是指C2。另外,上述對(duì)比包括由蠟狀芽孢桿菌(Bacillus cereus)(″VIP1″)產(chǎn)生的毒素,該毒素通常被認(rèn)為是VIP是植物殺蟲蛋白。
      圖16是PA的氨基酸序列與來自艱難梭菌(Clostridiumdifficile)(″cdADPRT″),產(chǎn)氣莢膜梭菌(C.perfringens)(″cpiota″),螺狀梭菌(C.spiroforme)(″csiota′),和肉毒梭菌(C.botulinum)(″cbc2″)的毒素的氨基酸序列的對(duì)比。這一對(duì)比顯示了所述毒素的全序列。
      發(fā)明詳述我們發(fā)現(xiàn)了一種阻止A-B毒素感染產(chǎn)生細(xì)菌的方法。由此,本發(fā)明提供用作對(duì)特定細(xì)菌感染,包括炭疽和壞疽感染解毒劑的組合物。由于該組合物是安全且具有免疫原性的,它也可以用作疫苗。
      對(duì)炭疽pA多突變體進(jìn)行構(gòu)建,表達(dá),純化,并通過試驗(yàn)確定其是否具有比野生型PA減弱的活性。具體地說是試驗(yàn)這些突變體結(jié)合PA配體和受體的能力;形成前孔,抗SDS-寡聚體,和孔的能力;和運(yùn)送配體穿越細(xì)胞膜的能力?;赑A的x-射線結(jié)構(gòu)預(yù)測(cè)將突變的殘基設(shè)計(jì)到PA前孔的腔中。具有減弱的或檢測(cè)不到的在膜上形成孔的能力的PA突變體或其片段可用作疫苗以誘導(dǎo)預(yù)防炭疽感染的保護(hù)性抗體。另外,這些突變體比野生型PA更有效地治療炭疽感染,這是由于它們具有減弱的轉(zhuǎn)移由受感染的哺乳動(dòng)物中的炭疽桿菌(Bacillusanthracis)的分泌的EF和LF的能力。
      研究這些點(diǎn)突變體,和先前報(bào)道的缺少結(jié)構(gòu)域2(ΔD2L2)的跨膜環(huán)2中第302-325位殘基的缺失突變體(Miller等,Biochemistry3810432-10441,1999)的特性,以進(jìn)一步確定是否他們可以通過減少野生型PA的孔形成而作為顯性負(fù)抑制劑。這一抑制可能是由于突變體與配體或受體結(jié)合,以使得很少的分子與野生型PA結(jié)合。所述的突變體也可以與野生型PA形成寡聚體,其具有減弱的或檢測(cè)不到的形成孔及轉(zhuǎn)運(yùn)配體的能力。如上所述,顯性負(fù)PA突變體及其片段可用作疫苗以引發(fā)預(yù)防或治療炭疽感染的保護(hù)性抗體。另外,具有顯性負(fù)活性的突變體或其片段可以通過抑制受感染的哺乳動(dòng)物中的炭疽桿菌(Bacillus anthracis)分泌的PA的活性作為治療炭疽感染的治療劑。因?yàn)轱@性負(fù)突變體可以誘導(dǎo)保護(hù)性抗體產(chǎn)生并抑制感染細(xì)菌產(chǎn)生的PA的活性,其可作為治療受炭疽感染、或有受炭疽感染危險(xiǎn)的個(gè)體的組合疫苗/治療劑。除了需要盡快消除毒素的作用,對(duì)暴露于霧化的炭疽桿菌(B.anthracis)孢子的個(gè)體進(jìn)行預(yù)防接種也同樣重要。這種預(yù)防接種對(duì)抵抗由于能夠在體內(nèi)長(zhǎng)期停留(至少一個(gè)月)的孢子萌發(fā)而引起的延遲收縮至關(guān)重要。
      在這一研究中,鑒定了幾種缺乏孔形成能力和轉(zhuǎn)運(yùn)配體能力的PA突變體,它們是潛在的預(yù)防和治療炭疽感染的疫苗(表1)。突變體#1-12能夠由蛋白水解激活形成易于SDS解離的PA63前孔狀態(tài),并結(jié)合于細(xì)胞受體EF,和LF。一些突變體阻止由前孔到抗SDS狀態(tài)的轉(zhuǎn)換(表1)。這些突變體(K397A,K397C,K397D,D425A,D425N,D425KD425E,D425K,K397D+D425K,和K395D+K397D+D425K+D426K)在孔形成和膜運(yùn)輸方面也存在缺陷。其它種類的突變體(ΔD2L2 PA,K397Q,和F427A)形成抗SDS寡聚體,但不經(jīng)過膜插入和孔形成。這些結(jié)果是出乎預(yù)料的。
      表1.保護(hù)性抗原突變體的活性

      這些PA突變體是如實(shí)施例1所述構(gòu)建的。
      突變體(ΔD2L2,K397D+D425K雙突變體,K395D+K397D+D425K+D426K四重突變體,D425K,F(xiàn)427A,K397D+D425K+F427A三重突變體,F(xiàn)427A+ΔD2L2雙突變體,K397D+F427A+ΔD2L2三重突變體,K397D+D425K+F427A+ΔD2L2四重突變體,F(xiàn)427D,和F427K)中的幾種抑制野生型PA-介導(dǎo)的配體跨膜轉(zhuǎn)運(yùn)。ΔD2L2和K397D+D425K PA突變體可與野生型PA形成寡聚體,其不能轉(zhuǎn)運(yùn)配體。這些結(jié)果是出乎預(yù)料的。在七聚體前孔內(nèi)存在這些突變體中的單一分子即足以阻止孔轉(zhuǎn)化。這種阻止由野生型PA的孔形成的能力,與和野生型PA競(jìng)爭(zhēng)結(jié)合細(xì)胞受體,并從循環(huán)中去除EF和LF的能力相偶聯(lián)使得這些突變體在治療和預(yù)防炭疽感染方面特別引人關(guān)注。
      PA中的其它殘基突變也可抑制孔的形成和產(chǎn)生顯性負(fù)活性。例如,可與帶電的側(cè)鏈Lys397或Asp425產(chǎn)生靜電相互作用的殘基也是PA孔形成中所需的,這些殘基中的一個(gè)或其組合的突變也可抑制孔的形成并產(chǎn)生顯性負(fù)活性。另外,小部分302-325 D2L2環(huán)缺失或該環(huán)的側(cè)翼氨基酸缺失,以及全部或部分302-325區(qū)域缺失能夠產(chǎn)生這種結(jié)果。
      獲得具有檢測(cè)不到的形成孔或轉(zhuǎn)運(yùn)配體能力的PA突變體,及可作為野生型PA顯性負(fù)抑制劑的突變體的能力表明在其它毒素中也可以獲得類似的突變體,如來自金黃色葡萄球菌(Staphylococcus aureus)的α-溶血素,來自嗜水氣單胞菌(Aeromonas hydrophila)的氣單胞菌溶素,來自敗毒梭菌(Clostridium septicum)的α-毒素,來自銅綠假單胞菌(Pseudomonas aeruginosa)的細(xì)胞毒素,異源寡聚體毒素(AB5毒素)或破傷風(fēng),肉毒梭菌或白喉毒素的B組分。另外,這些結(jié)果強(qiáng)調(diào)顯示了鑒定許多其它寡聚毒性因子,范圍從毒素到粘附素的顯性負(fù)形式的可能性。
      在炭疽毒素和其它裝配過程在與胞外環(huán)境的相互作用中出現(xiàn)的寡聚體系統(tǒng)中,外源添加的突變體亞單位可以大體上并入最終的結(jié)構(gòu)中,提高了這些亞單位用于治療的可能性。全身炭疽,盡管罕見作為天然疾病,但作為生物戰(zhàn)和恐怖主義的試劑則具有威脅性,顯性負(fù)PA看來是其中最有效的治療劑。假定施用的顯性負(fù)PA與炭疽桿菌(B.anthracis)在體內(nèi)產(chǎn)生的野生型PA任意混合,所述的蛋白將在細(xì)胞上共裝配以形成非活性的終端復(fù)合物,由此阻止LF和EF的作用。除對(duì)明顯的癥狀進(jìn)行預(yù)防外,顯性負(fù)突變體還可以保護(hù)專職的吞噬細(xì)胞不受損害,由此幫助宿主根除所述感染。向人體注射野生型PA后沒有觀察到明顯的副作用,由此該蛋白的突變體滅活形式將不會(huì)造成危害。
      顯性負(fù)PA還可以用作抗炭疽的新疫苗的基礎(chǔ)。如其名稱意味著的那樣,PA誘導(dǎo)針對(duì)炭疽的保護(hù)性抗體,事實(shí)上美國(guó)目前該疫苗的主要免疫原是得到許可的。在Fisher大鼠中,此處所述的ΔD2L2,K397D+D425K,和F427A突變體顯示極少或沒有有關(guān)相對(duì)于野生型PA的致免疫性降低。我們還發(fā)現(xiàn)突變體出乎預(yù)料的為顯性負(fù),因此以突變體比野生型PA 0.251的比率施用于大鼠模型不會(huì)引起可檢測(cè)到癥狀的炭疽感染。考慮用純化的野生型PA作為目前得到許可的疫苗的替代,如果PA的顯性負(fù)形式證明可有效地進(jìn)行治療,其也可以完成這一任務(wù),排除了發(fā)展兩種幾乎相同的藥劑的需要。
      下述的實(shí)施例用來說明本發(fā)明。它們不以任何形式對(duì)本發(fā)明進(jìn)行限制。除非另外提及,K397A和D425A PA突變體的數(shù)據(jù)作為獲得列于表1的PA突變體號(hào)1-12的數(shù)據(jù)的代表。
      實(shí)施例1一般方法細(xì)胞培養(yǎng),培養(yǎng)基和化學(xué)藥品中國(guó)倉(cāng)鼠卵巢-K1(CHO-K1)由美國(guó)典型培養(yǎng)物保藏中心獲得。所述的細(xì)胞在添加了10%牛血清,500單位/mL青霉素G,2mM L-谷氨酰胺和500單位/mL鏈霉素硫酸鹽的HAM′s F-12中生長(zhǎng),并于CO2含量5%的潮濕空氣中維持。在進(jìn)行試驗(yàn)前的16-18小時(shí)將細(xì)胞接種在24-或96-孔微滴定板(Costar,Cambridge,MA)。除非特別說明,所有用于細(xì)胞培養(yǎng)的培養(yǎng)基均由Gibco BRL獲得。除非特別說明所有的化學(xué)藥品均由Sigma化學(xué)試劑公司獲得。
      PA蛋白的構(gòu)建和純化所述的不包含PA的第302-325位氨基酸的ΔD2L2 PA突變體,如上所述進(jìn)行表達(dá)和純化(Miller等,Biochemistry 3810432-10441,1999)。表1中#1-11點(diǎn)突變是用QuickChange定點(diǎn)誘變方法,依照制造商所述的方法進(jìn)行的(Stratagene,La Jolla,CA)。Miller等(見上)編碼野生型PA的質(zhì)粒用作模板。將點(diǎn)突變體克隆到pET22-b(+)(Novagen)表達(dá)載體中,轉(zhuǎn)化到BL21(DE3)(Novagen)中表達(dá)。所述的點(diǎn)突變體依上述進(jìn)行表達(dá)、純化(Miller,1999)。簡(jiǎn)要地說,培養(yǎng)物在LB培養(yǎng)基中于37℃下培養(yǎng)至A600為1.0。添加β-D-異丙基硫代半乳吡喃糖苷(β-D-isopropylthiogalactopyranoside)至1mM以誘導(dǎo)重組蛋白表達(dá)。誘導(dǎo)后所述細(xì)胞在30℃下繼續(xù)培養(yǎng)3小時(shí),然后以8000xg離心10分鐘收集細(xì)胞。
      所述的蛋白通過滲透休克從胞質(zhì)中釋放出來。將所述細(xì)胞懸浮于20mM Tris-HCl,pH8.0,30%葡萄糖和1mM EDTA在室溫下溫育10分鐘并伴隨連續(xù)的攪拌。再次通過離心收集細(xì)胞,懸浮于含20mM芐脒的5mM MgSO4中,在4℃下溫育10分鐘并伴隨連續(xù)的攪拌。再次通過8000xg離心使細(xì)胞成顆粒狀,輕輕倒出提取出的perplasmic。加入Tris-HCL pH8.0至終濃度為20mM,將全部樣品上到Q-sepharoseHP柱中。用緩沖液A(20mM Tris,pH8.0)從柱中洗去未結(jié)合的蛋白。結(jié)合的蛋白用0%-25%線性梯度的緩沖液B(20mM Tris,pH8.0,1M NaCl)洗脫。濃縮含有PA的組分,用含有緩沖液A的pd-10柱(Amersham-Pharmacia)交換上述緩沖液。含有PA的洗脫物加到Mono-Q柱用0-25%梯度的緩沖液B洗脫。通過SDS-PAGE分析含有PA的組分并將其貯存于-80℃。用Bio-Rad蛋白分析試劑盒根據(jù)制造商所述的方法確定蛋白的濃度。所有的液相色譜均使用AKTA-凈化液相色譜系統(tǒng)(Amersham-Pharmacia)。
      其它的PA突變體(表1中#13-18)也類似地進(jìn)行表達(dá)和純化。
      PA的蛋白水解激活胰蛋白酶用于蛋白水解裂解PA83成為缺口PA(nPA)。將PA稀釋至濃度為0.5mg/ml用于前孔-形成試驗(yàn)或0.2mg/ml用于其它試驗(yàn)。添加胰蛋白酶至終胰蛋白酶濃度比PA的比率為1∶1,000(w∶w),所述的混合物于室溫下溫育20分鐘,然后用超過大豆胰蛋白酶抑制劑10摩爾的劑量抑制胰蛋白酶的作用。
      細(xì)胞表面轉(zhuǎn)運(yùn)試驗(yàn)如上所述用細(xì)胞表面轉(zhuǎn)運(yùn)試驗(yàn)測(cè)定PA-介導(dǎo)的放射性標(biāo)記的LFn(LF的PA結(jié)合結(jié)構(gòu)域N-末端1-255氨基酸)的轉(zhuǎn)運(yùn)(Wesche等,Biochemistry3715737,1998)。簡(jiǎn)要地說,nPA(2×10-8M)先結(jié)合于CHO細(xì)胞,然后與結(jié)合于細(xì)胞表面的PA63結(jié)合的35SLFn結(jié)合。去除過量的LFn,洗細(xì)胞,并在37℃下使細(xì)胞經(jīng)受pH5.0脈沖。所述的低pH模擬內(nèi)體的酸化作用并導(dǎo)致PA-介導(dǎo)的LFn轉(zhuǎn)運(yùn)轉(zhuǎn)過質(zhì)膜進(jìn)入細(xì)胞。將所述的樣品用鏈霉蛋白酶處理,所述蛋白酶可以通過蛋白水解降解胞外的35S-LFn,但不能降解已轉(zhuǎn)運(yùn)到細(xì)胞內(nèi)的35S-LFn。清洗所述細(xì)胞并進(jìn)行細(xì)胞溶解。為確定與細(xì)胞結(jié)合的35S-LFn的總量,一些細(xì)胞不用鏈霉蛋白酶處理。細(xì)胞溶解后,用閃爍計(jì)數(shù)器確定在上清液中的35S-LFn。如下所述計(jì)算轉(zhuǎn)運(yùn)的百分比(經(jīng)保護(hù)不受鏈霉蛋白酶影響的DPM)/(與細(xì)胞結(jié)合的DPM)×100=%已轉(zhuǎn)運(yùn)的。
      利用在經(jīng)胰蛋白酶處理前等摩爾結(jié)合的突變體和野生型PA,并且在加入細(xì)胞前將其稀釋到2×10-8M PA(每種蛋白1×10-8M)也進(jìn)行了上述試驗(yàn),以確定是否突變體PA蛋白抑制野生型的PA對(duì)LFn的轉(zhuǎn)運(yùn)。當(dāng)1×10-8M濃度的PA用作對(duì)照時(shí),與上述用2×10-8M野生型PA進(jìn)行的試驗(yàn)相比,轉(zhuǎn)運(yùn)效率僅稍稍受PA中的落差的影響,這表明任何轉(zhuǎn)運(yùn)和結(jié)合的降低都不是野生型PA濃度落差的結(jié)果。
      蛋白合成的抑制蛋白合成的LFnDTA抑制是用作測(cè)定PA-介導(dǎo)將配體轉(zhuǎn)運(yùn)到細(xì)胞內(nèi)的另一種方法(Milne等,Mol.Microbiol.1566,1995)。為對(duì)列于表1中的PA突變體#1-12進(jìn)行分析,在添加PA蛋白前16小時(shí),將CHO-K1細(xì)胞置于2.5×104細(xì)胞/孔的96孔板中。在存在1×10-8M LFnDTA的情況下,將PA83(1×10-12M到1×10-7M)與細(xì)胞一起培養(yǎng)4小時(shí)。去除所述的培養(yǎng)基更換為添加了1 mCi/ml3H-Leu的無亮氨酸的HAM′s F-12培養(yǎng)基。培養(yǎng)1小時(shí)后用冰冷的PBS接著用冰冷的三氯醋酸(10%)沉淀蛋白。摻入TCA不溶物質(zhì)中的3H-leu的數(shù)量用閃爍計(jì)數(shù)器確定,并作為新合成的蛋白的量的尺度。
      在這一實(shí)驗(yàn)中還測(cè)試了突變體PA蛋白的以證明其是否減輕野生型PA-介導(dǎo)的對(duì)3H-leu攝入的抑制。將10×10-9M野生型PA與1×10-8MLFnDTA添加到CHO細(xì)胞中。加入遞增量的突變體之一。將所述的細(xì)胞與毒素一起培養(yǎng)4小時(shí),如上所述加工樣品。
      類似地對(duì)列于圖9中的PA突變體進(jìn)行試驗(yàn)。在LFN-DTA(100pM)及不同量的單獨(dú)的PA突變體(K397D+D425K,ΔD2L2,F(xiàn)427A,D425K,K397D,或SSSR)存在下,CHO-K1細(xì)胞(2.5×104細(xì)胞/孔)與野生型PA(100pM)在96-孔板中于37℃下培養(yǎng)18小時(shí)。去除所述的培養(yǎng)基更換為添加了1μCi/ml3H-Leu的無亮氨酸HAM F-12培養(yǎng)基。37℃培養(yǎng)1小時(shí)后用冰冷的PBS及冰冷的三氯醋酸(TCA)(10%)洗細(xì)胞。測(cè)定摻入到TCA-沉淀物中的3H-leu的量,其表明在不存在PA的情況下?lián)饺氲陌俜直?。在所選擇的野生型PA和LFnDTA濃度下,在不存在突變體PA(虛線)的情況下,蛋白合成受到約90%的抑制。報(bào)道了三次試驗(yàn)±SEM的平均值。在開始培養(yǎng)4小時(shí)而非18的情況下所觀察到的結(jié)果是類似的。列于圖10的K397D+D425K+F427A,F(xiàn)427A+ΔD2L2,和K397D+F427A+ΔD2L2PA突變體也進(jìn)行了類似的試驗(yàn)。
      將野生型異源七聚體和突變體PA對(duì)PA-介導(dǎo)的蛋白合成的抑制與相應(yīng)的同源七聚體混合物的抑制作用進(jìn)行的比較。野生型PA63同源七聚體和K397D+D425K,ΔD2L2,F(xiàn)427A,K397D,及D425K突變體如上所述進(jìn)行制備。推定的異源七聚體通過,在經(jīng)胰蛋白酶處理和柱層析之前,將每種PA與野生型PA以1∶1的比例混合而制備的(圖11)。野生型PA(1nM),異源七聚體(H)(終濃度2nM),或相應(yīng)的突變體的同源七聚體和野生型的七聚體的等摩爾混合物(M)(每種1nM)與CHO-K1細(xì)胞一起,在LFnDTA(100pM)存在下培養(yǎng)18小時(shí),如上述測(cè)定其對(duì)蛋白合成的抑制,參見圖9。七聚體的濃度按單體PA63亞單元表示。蛋白合成用占不添加PA的對(duì)照百分比來表示。報(bào)道了三次實(shí)驗(yàn)SEM的平均值。培養(yǎng)4小時(shí)后所觀察到的結(jié)果類似。
      前孔和抗-SDS寡聚體的形成前孔和抗-SDS寡聚體的形成是通過將等摩爾量的LFn與nPA在室溫下一起培養(yǎng)30分鐘。為確定是否已形成了前孔,將所述樣品用4-12%自然梯度凝膠(FMC),以50mM CHES,pH 9.0,2mg/ml CHAPS作為電泳緩沖液進(jìn)行電泳。為確定是否低pH誘導(dǎo)抗-SDS的七聚體形成,向溶液中添加100mM醋酸鈉,pH4.5至所述溶液的pH達(dá)5.0然后將所述樣品于室溫下溫育30分鐘。然后將樣品溶于SDS-PAGE樣品緩沖液中在4-12% SDS-PAGE梯度凝膠中電泳。樣品中的蛋白可經(jīng)考馬斯亮藍(lán)染色觀察。
      銣的釋放CHO-K1細(xì)胞以2×105細(xì)胞/孔的密度接種于培養(yǎng)板在37℃下培養(yǎng)24小時(shí)。吸出上述培養(yǎng)基換為含有1μCi/ml86RbCl的培養(yǎng)基培養(yǎng)16小時(shí)。將所述細(xì)胞在冰上冷卻20分鐘,去除培養(yǎng)基。洗細(xì)胞,然后加入于HEPES緩沖培養(yǎng)基中的nPA(2×10-8M)。將所述的細(xì)胞與nPA在冰上處理2小時(shí),然后加入pH5.0的緩沖液。30分鐘后,收集上清液中的樣品,用閃爍計(jì)數(shù)器記數(shù)以確定所釋放的86Rb的量。
      這一標(biāo)準(zhǔn)試驗(yàn)還用于確定其它孔形成毒素有關(guān)86Rb的釋放量的效果。因此,本發(fā)明的其它突變體毒素也可以用這一試驗(yàn)進(jìn)行測(cè)定其是否具有減弱的孔形成能力。
      實(shí)施例2大多數(shù)突變體形成抗-SDS寡聚體失敗所有列于表1的PA突變體#1-12和野生型PA蛋白均依照上述經(jīng)胰蛋白酶水解缺刻,形成的缺口PA(nPA)蛋白在SDS-PAGE分析中作為低分子量樣品遷移(圖2A)。由列于表1的突變體#1-12形成可被SDS解離的前孔可以通過與單體nPA相比,與LFn復(fù)合的PA63七聚體在天然凝膠中的遷移率的降低來檢測(cè)(圖2B)。由K397A和D425A PA突變體形成的前孔進(jìn)一步得到下述結(jié)果的支持,由MonoQ柱洗脫上述前孔比洗脫單體PA所用的鹽濃度高。還對(duì)nPA突變體形成抗-SDS寡聚體進(jìn)行了分析。用LFn處理野生型PA作為陽性對(duì)照。低pH脈沖將野生型PA轉(zhuǎn)化成抗-SDS寡聚體,當(dāng)進(jìn)行SDS-PAGE分析時(shí),其作為高分子量復(fù)合物遷移。ΔD2L2(缺少第302-325位殘基的PA)和K397Q(圖2B)。當(dāng)用低pH處理野生型,K397Q,F(xiàn)427A,和ΔD2L2 PA形成的抗-SDS寡聚體(圖2A和表1)。
      實(shí)施例3突變體在膜上形成孔的失敗大多數(shù)的PA突變體形成抗-SDS寡聚體失敗表明在細(xì)胞膜上形成孔受到抑制。如實(shí)施例1中所述,將nPA蛋白與載有放射活性的鉀類似物86Rb的細(xì)胞相結(jié)合,經(jīng)低pH脈沖處理,再檢測(cè)釋放到培養(yǎng)基周圍的86Rb來檢測(cè)孔的形成。野生型的nPA誘導(dǎo)86Rb釋放是由于nPA插入到膜上形成離子能通過的孔。相反,列于表1中的任何突變體#1-12均不誘導(dǎo)86Rb的釋放(圖3和表1)。因此,使大多數(shù)PA突變體失去形成抗-SDS寡聚體(實(shí)施例2)的能力與這些突變體失去在膜上形成孔的能力相關(guān)。
      實(shí)施例4PA突變體跨膜轉(zhuǎn)運(yùn)LFn失敗孔的形成對(duì)依賴PA的配體(即,LF,EF或LFn)轉(zhuǎn)運(yùn)而言是必須的步驟。用細(xì)胞表面轉(zhuǎn)運(yùn)試驗(yàn)直接測(cè)定PA配體向細(xì)胞質(zhì)中的轉(zhuǎn)運(yùn)(實(shí)施例1)。任何列于表1中的PA突變體#1-12均沒有顯著下降的結(jié)合LFn的能力(圖4A);但在這一試驗(yàn)中所有被分析的突變體轉(zhuǎn)運(yùn)LFn的能力均明顯下降(圖4B和12)。SSSR對(duì)照突變體在此條件下幾乎不引起抑制。這些數(shù)據(jù)表明突變體保持了結(jié)構(gòu)完整性及與細(xì)胞受體和LFn結(jié)合的能力,但不能形成孔或跨膜轉(zhuǎn)運(yùn)配體。
      實(shí)施例5PA突變體跨膜轉(zhuǎn)運(yùn)LFnDTA失敗用于測(cè)定PA配體跨膜轉(zhuǎn)運(yùn)的另一種方法是LFnDTA毒性試驗(yàn)(實(shí)施例1)。在這一試驗(yàn)中,用PA和含有與白喉毒素DTA A-鏈(LFnDTA)融合的LFn的配體處理CHO細(xì)胞。經(jīng)轉(zhuǎn)運(yùn)的白喉毒素A-鏈ADP核糖基化細(xì)胞質(zhì)蛋白EF-2,使蛋白合成受到抑制并誘導(dǎo)細(xì)胞死亡。如實(shí)施例4中所測(cè)定的,這一試驗(yàn)是由與細(xì)胞表面相對(duì)的內(nèi)體區(qū)室轉(zhuǎn)運(yùn)配體的估算尺度。與LFnDTA和野生型或突變體PA一起培養(yǎng)后,洗細(xì)胞,然后在添加了3H-亮氨酸但無亮氨酸的培養(yǎng)基中培養(yǎng)。如果蛋白合成沒有受到抑制,3H-亮氨酸將摻入到新合成的蛋白中。如果蛋白合成受到LFnDTA抑制,就幾乎沒有3H摻入。所有的受試突變體在這一試驗(yàn)中均未明顯抑制蛋白合成(圖5)。這一結(jié)果進(jìn)一步支持了下述的假設(shè),即由于PA突變體明顯缺乏孔形成,則導(dǎo)致這些突變體PA配體膜轉(zhuǎn)運(yùn)能力下降。
      實(shí)施例6PA突變體抑制野生型PA孔形成由于所有列于表1的PA突變體#1-12都在孔形成方面存在缺陷,則測(cè)試這些突變體以確定它們是否可與野生型的PA形成失活的異源-寡聚體,從而抑制PA-介導(dǎo)的配體跨膜轉(zhuǎn)運(yùn)。ΔD2L2,K397D+D425K,和K395D+K397D+D425K+D426K PA以這種方式抑制野生型PA。當(dāng)與等摩爾的野生型PA混合時(shí),在細(xì)胞表面轉(zhuǎn)運(yùn)試驗(yàn)中,這三種突變體中的每一種均顯著地抑制35S-LFn向細(xì)胞內(nèi)的轉(zhuǎn)運(yùn)(圖6)。35S-LFn與細(xì)胞的結(jié)合并未受到抑制(圖6)。
      在LFnDTA試驗(yàn)中用野生型PA滴定突變體進(jìn)一步描述了野生型PA抑制作用的特性。將其中的一種突變體以遞增的量添加到帶有野生型PA和LFnDTA的細(xì)胞培養(yǎng)物中(圖8A)。所述的具有由164RKKR167到164SSSR167突變的弗林蛋白酶識(shí)別位點(diǎn)的突變體PA-SSSR包含于其中作為對(duì)照。所述的突變體不能被弗林蛋白酶或其它弗林蛋白酶-樣蛋白酶形成缺刻,因此不能形成孔。該突變體僅通過與受體的競(jìng)爭(zhēng)抑制PA。ΔD2L2和K397D+D425K在很大程度上抑制PA介導(dǎo)的轉(zhuǎn)運(yùn)。重要的是因?yàn)榕cPA-SSSR所需要的蛋白相比,這些突變體只需要極少的蛋白即可觀察到50%的抑制,所以這些突變體并不是通過競(jìng)爭(zhēng)受體而單獨(dú)地抑制(圖7B)。單一突變體包括K397D+D425K中的單一突變體組分與雙突變體抑制程度不同,但比PA-SSSR抑制作用強(qiáng)。綜合考慮這些數(shù)據(jù)表明ΔD2L2,K397D+D425K,和K395D+K397D+D425K+D426K PA是野生型PA的顯性負(fù)抑制劑。
      所述的F427A,D425K,K397D+D425K+F427A,F(xiàn)427A+ΔD2L2,K397D+F427A+ΔD2L2 PA突變體顯性負(fù)抑制劑也進(jìn)行了測(cè)定。在這一試驗(yàn)中,如上所述,遞增數(shù)量的PA突變體形式與衡量的野生型PA相混合。這一組中最有效的成員K397D+D425K+F427A三重突變體在毒素1∶1比率的突變體野生型PA的情況下幾乎完全阻止了毒素的活性。D2L2,K397D+D425K,F(xiàn)427A,F(xiàn)427A+ΔD2L2,和K397D+F427A+ΔD2L2 PA突變體也有抑制劑活性。K397D+D425K+F427A+ΔD2L2,F(xiàn)427D,和F427K PA突變體在LFnDTA毒性試驗(yàn)中也表現(xiàn)出顯性負(fù)活性。相反,另一種轉(zhuǎn)運(yùn)缺失突變體K397D在1∶1的比率下事實(shí)上沒有表現(xiàn)抑制,由此說明并非所有的這種類型的突變體均是強(qiáng)抑制劑(圖9)。所述的S SSR對(duì)照突變體即使在超過野生型PA10倍的情況下也不引起可檢測(cè)到的毒素抑制作用,暗示與受體的競(jìng)爭(zhēng)作用不對(duì)其它突變體的抑制劑活性起主要作用。
      用經(jīng)純化的同源-和異源七聚體驗(yàn)證了顯性負(fù)突變體的抑制作用依賴其PA63組分與野生型PA63形成雜種復(fù)合體的能力的假說。溶液中的PA可通過適度的胰蛋白酶作用在弗林蛋白酶位點(diǎn)切割,所得的片段在離子交換柱上通過胰蛋白酶-缺刻分子層析分離(Miller等,Biochemistry38,10432,1999)。經(jīng)此種方法分離的純化的PA63是七聚體,這表明低聚反應(yīng)平衡在很大程度上有利于此種形式,且可能與前孔在結(jié)構(gòu)上相似或同一。純化的同源七聚體是由野生型PA和K397D+D425K,ΔD2L2,F(xiàn)427A,D425K,和K397D轉(zhuǎn)運(yùn)缺陷PA突變體中的一種制備的。推定的異源七聚體是通過下述方式制備的將每種突變體PA與野生型PA以1∶1的比例混合,接下來使所述混合物胰蛋白酶化并在離子交換柱上將所述產(chǎn)物進(jìn)行層析。
      通過每一種異源七聚體和1∶1混合的相應(yīng)突變體和野生型同源七聚體測(cè)定了LFnDTA依賴的蛋白合成的抑制。含K397D+D425K,ΔD2L2,F(xiàn)427A和D425K突變體的異源七聚體不介導(dǎo)LFnDTA的作用,而相應(yīng)的同源七聚體混合物具有高度的活性(圖11)。與此相反,通過混合K397D和野生型PA形成的確定異源七聚體與由同源-K397D PA和同源-野生型PA混合形成的混合物一樣具有活性。這些結(jié)果與圖9中所示試驗(yàn)中這些突變體的性質(zhì)一致,并支持下述概念,即顯性負(fù)突變體的PA63通過與之共寡聚化滅活野生型蛋白。在異源七聚體制劑中K397D缺乏抑制劑活性反映出其與野生型蛋白共-寡聚化的能力的缺陷或在七聚體中抑制其活性的能力的缺陷。突變體同源七聚體不抑制野生型的活性的發(fā)現(xiàn)表明,在所述試驗(yàn)條件下幾乎不存在受體的競(jìng)爭(zhēng)并且在七聚體之間幾乎沒有或沒有亞單位的交換。
      如上所述,在這些LFnDTA毒性試驗(yàn)中K397D+D425K雙突變體幾乎完全封阻的活性表明所述突變體的單個(gè)分子滅活七聚體且寡聚體化是隨機(jī)的。ΔD2L2,D425K,和F427A突變體表現(xiàn)為稍弱的抑制性,暗示每七聚體這些突變體中不只一個(gè)分子對(duì)于滅活和/或它們與野生型PA的共寡聚體化不是純粹隨機(jī)的。其它的因素,例如向正生長(zhǎng)的七聚體復(fù)合物中添加B組分(例如,最先或最后添加的B組分)的順序也影響滅活作用。本說明書中所述的任何建議的有關(guān)抑制的機(jī)制均不應(yīng)視為對(duì)本發(fā)明的限制。
      實(shí)施例8含突變體和野生型PA的抗-SDS寡聚體的形成為檢測(cè)ΔD2L2和K397D+D425K突變體與野生型PA的相互作用,將等摩爾比例的突變體及野生型PA相混合,用胰蛋白酶斷裂形成缺刻,并通過SDS-PAGE分析抗-SDS寡聚體的形成。每當(dāng)一種突變體與野生型PA即形成一種新的抗-SDS PA。與單獨(dú)的野生型PA在凝膠中產(chǎn)生分散的高分子量成片條帶不同,突變體和野生型PA的混合物形成明顯的高分子量條帶。這一明顯條帶與所觀察到的單獨(dú)的突變體所形成的條帶也有區(qū)別單獨(dú)的K397D+D425K不形成抗-SDS寡聚體,單獨(dú)的ΔD2L2 PA所形成的寡聚體在凝膠中比同時(shí)存在野生型PA時(shí)形成的條帶遷移得更遠(yuǎn)。盡管確切的組合物以及這一條帶的性質(zhì)尚未確定了,但這一條帶進(jìn)一步暗示在抗-SDS寡聚體中突變體與野生型PA的相互作用導(dǎo)致寡聚體在凝膠中遷移率的改變。
      實(shí)施例9體內(nèi)毒素抑制在體外顯性負(fù)突變體表現(xiàn)出的性質(zhì)暗示它們應(yīng)當(dāng)在體內(nèi)抑制毒素作用。為驗(yàn)證這一假說在用于炭疽毒素的傳統(tǒng)體內(nèi)模型Fisher 344 rat(Ivins等,Appl.Environ.Microbiol.552098,1989)中檢測(cè)了這三種突變體(K397D+D425K,ΔD2L2,和F427A)的活性。靜脈注射了8μg LF和40μg PA混合物(約為最低致死劑量的10倍)的雄鼠(250-300g)在約90分鐘表現(xiàn)垂死狀態(tài)(表3)。用任意的顯性負(fù)突變體替換野生型PA時(shí),在所述動(dòng)物被處死前兩周的時(shí)間內(nèi)未表現(xiàn)出中毒癥狀。當(dāng)注射前將顯性負(fù)PA添加到野生型PA/LF混合物中時(shí),無論以相對(duì)于野生型PA1∶1的比例(40μg顯性負(fù)PA)還是0.25∶1的比例(10μg顯性負(fù)PA)注射的動(dòng)物均無癥狀地存活下來。所述的SSSR突變體對(duì)所述毒素的活性幾乎不起作用。這些結(jié)果與體外試驗(yàn)的結(jié)果一致,表明顯性負(fù)突變體甚至在相對(duì)于野生型PA的亞化學(xué)劑量(0.25∶1)下也可以在體內(nèi)切除炭疽毒素的作用。
      表3在體內(nèi)PA突變體對(duì)野生型PA的抑制蛋白總量(μg)WT ΔD2L2 K397D+D425KF427ASSR TTM40 -- - - 90±11分鐘- 40 - - -存活- -40- -存活- -- 40 -存活40 40 - - -存活40 -40- -存活40 -- 40 -存活
      40- --40 100±3分鐘4010---存活40 10 --存活40- -10 -存活比較了K397D+D425K雙突變體(″二聯(lián)體″)和K397D+D425K+F427A三重突變體(″三聯(lián)體″)在體內(nèi)抑制野生型PA活性的能力(表4)。如上所述用40μg野生型PA,10μg LF以及PBS或顯性負(fù)PA突變體的混合物注射大鼠進(jìn)行這一試驗(yàn)。
      表4在體內(nèi)PA突變體對(duì)野生型PA的抑制動(dòng)物突變體PA的量 TTMPBS2 -~100分鐘兩倍 2 40μg 存活三倍 2 40μg 存活兩倍 4 4μg存活三倍 4 4μg存活還用K397D+D425K,ΔD2L2,或F427A PA測(cè)定了對(duì)大鼠接種疫苗所產(chǎn)生的抗-PA和中和抗體滴度。為進(jìn)行這一鑒定,以6只動(dòng)物為一組的多組動(dòng)物分別在第0,3,6周接受了三次進(jìn)入后四分之一內(nèi)的肌內(nèi)注射疫苗接種,所注射的是于200μl Ribi Tri-Mix佐劑(Sigma)中的50μg蛋白。在第一次注射的兩天前和每次注射后的14天從每只動(dòng)物抽血收集血清。最后一次注射后16天用致死劑量LF(30μg PA+6μg LF)如表5所述進(jìn)行第IV次注射。用抗PA標(biāo)準(zhǔn)ELISA試驗(yàn)確定血清中的平均抗-PA抗體滴度。報(bào)告的是使血清反應(yīng)性終止的滴度的幾何平均數(shù)的倒數(shù)。在LFnDTA試驗(yàn)中,以1×10-10M PA和1×10-10M LFnDTA滴定中和抗體。在開始試驗(yàn)前,將抗體稀釋物與PA一起于37℃下培養(yǎng)1小時(shí)。如上所述用LFnDTA毒性試驗(yàn)測(cè)定蛋白合成抑制。所述的中和滴度代表抑制PA活性50%所需稀釋度的幾何平均數(shù)的倒數(shù)。如表5中所述,在Fisher大鼠中K397D+D425K,ΔD2L2,和F427A PA突變體相對(duì)于野生型PA幾乎沒有或沒有表現(xiàn)出免疫原性減弱。無論所用的免疫原如何,進(jìn)行三次注射后的中和和抗-PA抗體滴度相似,且經(jīng)疫苗接種的動(dòng)物在最后一次注射后的16天抵抗住所施用的致死劑量的野生型PA加LF,存活下來。表5由PA突變體對(duì)大鼠進(jìn)行疫苗接種所產(chǎn)生的抗-PA和中和抗體滴度動(dòng)物 抗-PA滴度中和滴度 TTMPBS6 <10 <10 74.2±1.5WT 5 43,3002,490 存活ΔD2L2 6 47,5003,350 存活K397D+ 6 65,5002,260 存活D425KF427A6 132,000 6,090 存活實(shí)施例10針對(duì)PA的抗體針對(duì)PA的抗體可以用于治療和/或診斷??梢詷?biāo)準(zhǔn)的方法通過PA蛋白或其片段免疫攻擊含B細(xì)胞的生物系統(tǒng),例如某種動(dòng)物如小鼠或兔,以刺激B細(xì)胞產(chǎn)生抗-PA抗體,接下來從所述生物系統(tǒng)中分離產(chǎn)生的抗體。為產(chǎn)生單克隆抗體,從具有對(duì)PA蛋白最高的ELISA確定的免疫應(yīng)答的動(dòng)物中收獲脾,將所述的B細(xì)胞與NS-1骨髓瘤細(xì)胞融合以產(chǎn)生雜交瘤。用標(biāo)準(zhǔn)的ELISA試驗(yàn)或Western印跡篩選分泌可與PA結(jié)合的抗體的雜交瘤。保留產(chǎn)生高抗體滴度并特異識(shí)別PA蛋白的單克隆細(xì)胞系。
      也可以篩選出所述的細(xì)胞系用于鑒別產(chǎn)生對(duì)突變體PA蛋白比對(duì)天然存在的PA具有更高親和力的抗體的細(xì)胞系。這些抗體可以通過對(duì)動(dòng)物施用包含如K397,D425,D426,或F427殘基的天然存在的PA片段而產(chǎn)生。篩選所得的抗體以確定那些可與天然存在的PA結(jié)合但不與K397,D425,D426,或F427中的一個(gè)或多個(gè)殘基突變或缺失的突變體PA蛋白結(jié)合。例如,可將所得的抗體可通過含有固定了突變體PA蛋白的柱,則可以篩選出不與突變體PA蛋白結(jié)合的抗體。也可以生產(chǎn)出可與D2L2環(huán)上的殘基反應(yīng)的;這些抗體可以如上所述通過向動(dòng)物施用含D2L2環(huán)的PA片段產(chǎn)生。可與天然存在的PA中的D2L2環(huán)上的殘基反應(yīng)的抗體也可以通過篩選不與D2L2環(huán)上一個(gè)或多個(gè)殘基缺失的突變體PA蛋白結(jié)合的抗體而產(chǎn)生。另外,也可以產(chǎn)生與對(duì)突變體PA比對(duì)天然存在的PA分子具有更強(qiáng)親和力的抗體,這樣的抗體可以如上所述通過向動(dòng)物施用突變體PA并篩選對(duì)突變體PA形式具有更強(qiáng)親和力的抗體。這些抗體可與不存在于天然PA中而存在與突變體PA中的殘基結(jié)合。
      抗-PA抗體可以用于檢測(cè)生物樣品,如血清中的PA蛋白,所述的檢測(cè)是將所述的樣品與所述抗體接觸,然后測(cè)定免疫復(fù)合物作為測(cè)定樣品中的PA蛋白的尺度。因此,這些抗體可以用作確定受試者是否已暴露于炭疽毒素的試劑盒。
      針對(duì)PA的抗體也可以用作治療或預(yù)防炭疽感染的治療劑。如果可與野生型PA結(jié)合但不與顯性負(fù)PA突變體結(jié)合的抗-PA抗體施用于受試者作為抗炭疽感染被動(dòng)免疫,顯性負(fù)PA突變體也可以施用于同一受試者作為抑制野生型PA活性的治療劑。由于所施用的抗-PA抗體不與治療性的顯性負(fù)PA突變體反應(yīng),抗-PA抗體不應(yīng)減弱顯性負(fù)PA突變體抑制野生型PA的能力。另外,不與治療劑反應(yīng)的抗-PA抗體可用于確定存在于從用顯性負(fù)PA突變體治療的受試者獲得的樣品中的野生型PA的量。
      也可以產(chǎn)生針對(duì)本發(fā)明的其它突變體B組分的類似抗體。
      實(shí)施例11PA蛋白和片段的施用施用本發(fā)明的片段或蛋白并不局限于特定的施用方式,劑量,或劑量頻率;目前的方式包括所有的施用方式,即包括口,肌肉內(nèi),靜脈內(nèi),皮下,經(jīng)吸入,或其它足以提供充分預(yù)防或治療炭疽感染的劑量。一種或多種突變體PA蛋白或片段可以在單劑量或多劑量中施用于哺乳動(dòng)物。當(dāng)多劑施用時(shí),每劑之間可以間隔,例如一周到一個(gè)月。應(yīng)當(dāng)理解,對(duì)于任何特定的受試者應(yīng)當(dāng)根據(jù)個(gè)體的需要施用以及或監(jiān)督所述組合物施用的人員的職業(yè)判斷,在整個(gè)過程中調(diào)整劑量方案。
      可以根據(jù)E.W.Martin在Remingtion′s PharmaceuticalSciences的描述制備包含一種或多種本發(fā)明的PA蛋白或片段的藥物組合物。可以使用藥物穩(wěn)定化合物,遞送載體,運(yùn)輸載體或佐劑。例如,也可以用人血清白蛋白或其它人或動(dòng)物蛋白。磷脂載體或脂質(zhì)體懸液是可能的藥用載體或遞送載體??捎糜诒景l(fā)明中的佐劑包括鋁化合物,如氫氧化鋁,磷酸鋁和羥基磷酸鋁。這些組合物可用本領(lǐng)域技術(shù)人員已知的方法制備。
      本發(fā)明的其它B組分或片段也可以類似地施用。
      實(shí)施例12其它孔形成突變體PA的晶體結(jié)構(gòu)確定了四個(gè)PA結(jié)構(gòu)域(Petosa等,Nature385(6619)833-838,1997)。結(jié)構(gòu)域2(第259-487位殘基)包含大的可變形的環(huán),其在由前孔到孔的轉(zhuǎn)換過程中經(jīng)歷主要的構(gòu)象改變。在這一區(qū)域突變,缺失或插入一個(gè)或多個(gè)氨基酸在體內(nèi)可導(dǎo)致該蛋白的孔形成能力受到抑制和/或?qū)е翽A突變體抑制天然存在的PA的孔形成能力。例如,與其它一個(gè)或多個(gè)孔形成毒素(如來自艱難梭菌(Clostridium difficile),產(chǎn)氣莢膜梭菌(C.perfringens),螺狀梭菌(C.spiroforme),肉毒梭菌(C.botulinum),蠟狀芽孢桿菌(Bacillus cereus),或蘇云金芽孢桿菌(B.Thuringiensis)的毒素;圖15和16)中的相應(yīng)的殘基相同的PA結(jié)構(gòu)域2中的殘基也可能發(fā)生突變。這些殘基可能在PA中突變或缺失以形成顯性負(fù)PA突變體。
      下述PA結(jié)構(gòu)域2中的殘基在列于圖15和16中由兩個(gè)組分組成的A-B毒素中是不變的A259,P260,V262,V264,M266,E267,S272E275 T298,N353,N361,N363 R365,Y366,N368,G370,T371,Y375,V377,P389,T380,T381,V384,T393,I394,P407,Y411,P412,A420,D425,F(xiàn)427,I432,N435,Q438,L450,T452,Q454,G457,G474,W477,和I484。這些殘基可以突變成其它任何氨基酸。例如,這些殘基可以變?yōu)閹в行〉膫?cè)鏈的氨基酸,如甘氨酸和丙氨酸,所述的氨基酸也可以變?yōu)閹в写蟮幕蛑ф渹?cè)鏈的氨基酸,如色氨酸,亮氨酸和甲硫氨酸。其它可用于替換天然存在的殘基的殘基的實(shí)施例列于表1。
      除炭疽毒素外,本發(fā)明還涉及其它孔形成毒素。這些毒素也可以突變以產(chǎn)生減弱的或可忽略的寡聚化,形成跨膜通道或轉(zhuǎn)運(yùn)配體能力的毒素。另外也可以產(chǎn)生其它孔形成突變體的顯性負(fù)突變體。相應(yīng)于此處所述的PA突變體的突變體也可以由其它與PA同源的毒素制備(如來自艱難梭菌(Clostriidum difficile),產(chǎn)氣莢膜梭菌(C.perfringens),螺狀梭菌(C.spiroforme),肉毒梭菌(C.botulinum),蠟狀芽孢桿菌(Bacillus cereus),或蘇云金芽孢桿菌(B.Thuringiensis)的毒素)(圖15和16和表6)。相應(yīng)于PA結(jié)構(gòu)域2中的殘基的其它毒素中的殘基也可以如上所述進(jìn)行突變。另外,在其它孔形成毒素中至少相應(yīng)于PA的D2L2環(huán)區(qū)域的1,3,5,8,10,15,20,或24個(gè)氨基酸可以缺失。在相應(yīng)于此處所述的突變PA殘基的殘基處可進(jìn)行一個(gè)或多個(gè)點(diǎn)突變。
      可將任何一種孔形成毒素的突變體施用于哺乳動(dòng)物以治療和預(yù)防產(chǎn)生相應(yīng)毒素的病原體(如,細(xì)菌)感染。
      表6.相應(yīng)于此處所述的炭疽PA中突變的其它孔形成毒素中的突變。在其它孔形成毒素中相應(yīng)于PA中突變的殘基的殘基也可以突變?yōu)槿魏纹渌被帷?br>

      另外,可以用標(biāo)準(zhǔn)的分子生物學(xué)方法對(duì)編碼孔形成突變體(如依賴膽固醇的溶細(xì)胞素或與葡萄球菌α-毒素相關(guān)的六聚體或七聚體的毒素)的核酸進(jìn)行隨機(jī)誘變。編碼的突變體毒素可以用標(biāo)準(zhǔn)的方法表達(dá)或選擇性純化。此處所述的銣釋放試驗(yàn)可用于鑒定具有減弱的形成跨膜通道能力的突變體毒素。另外動(dòng)物模型可以用于鑒定顯性負(fù)毒素突變體,當(dāng)所述的突變體和野生型毒素均施用于動(dòng)物時(shí),所述的突變體減弱相應(yīng)的野生型毒素的毒性。
      其它實(shí)施例在本說明書中所提及的所有出版物和專利申請(qǐng)均以相同的程度引入本申請(qǐng),此處,如同每篇單獨(dú)的出版物或?qū)@暾?qǐng)均特別地、單獨(dú)地列出以并入本文。
      雖然本發(fā)明通過特定的實(shí)施例進(jìn)行描述,應(yīng)當(dāng)理解本發(fā)明可以被進(jìn)一步修改,而本申請(qǐng)?jiān)噲D涵蓋一般根據(jù)本發(fā)明的原理所作的任何變更、應(yīng)用或修改,并包括下述的本發(fā)明的背離,所謂背離是指在本發(fā)明所從屬于的領(lǐng)域中所公知或常用的,適用于在上文中所述的本發(fā)明基本特征的,并遵循以下所附的權(quán)利要求書中所述的范圍的范圍內(nèi)。
      其它的實(shí)施方案包含在權(quán)利要求書中。
      序列表&lt;110&gt;哈佛大學(xué)校長(zhǎng)及研究員協(xié)會(huì)&lt;120&gt;治療和預(yù)防細(xì)菌感染的化合物和方法&lt;130&gt;00742/060CN2&lt;150&gt;PCT/US01/14372&lt;151&gt;2001-05-04&lt;150&gt;US 60/201,800&lt;151&gt;2000-05-04&lt;160&gt;35&lt;170&gt;FastSEQ,Windows版本4.0&lt;210&gt;1&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;1Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg
      100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu
      405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;2&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;2Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu
      210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala G1u Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys
      515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;3&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;3Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro
      20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His
      325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;4&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;4Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His ValThr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu
      130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu
      435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;5&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;5Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val
      245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val ValLys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;6&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;6Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val ValThr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile
      50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn
      355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly
      660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720lle Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;7&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;7Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro
      165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;8&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;8Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr
      275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg
      580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;9&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;9Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser15 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val
      85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu
      690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;10&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;10Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser
      195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp
      500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;11&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;11Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr
      610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;12&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;12Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys
      115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile
      420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx
      725 730 735&lt;210&gt;13&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;13Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly
      530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;14&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;14Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser
      35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu
      340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile
      645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;15&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;15Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn ValSer Pro Glu Ala Arg His Pro Leu Val245250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe
      450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;16&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;16Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser
      260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val Hi290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr
      565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val ValLys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;17&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;17Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val
      370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr
      675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;18&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;18Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr
      180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys
      485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;19&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;19Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His
      290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp
      595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;20&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt; 20Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg
      100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu
      405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;21&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;21Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu
      210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr ValAla Ile Asp His325 330335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys
      515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;22&lt;211&gt;2208&lt;212&gt;DNA&lt;213&gt;炭疽桿菌&lt;400&gt;22gaagttaaac aggagaaccg gttattaaat gaatcagaat caagttccca ggggttacta 60ggatactatt ttagtgattt gaattttcaa gcacccatgg tggttacctc ttctactaca 120ggggatttat ctattcctag ttctgagtta gaaaatattc catcggaaaa ccaatatttt 180caatctgcta tttggtcagg atttatcaaa gttaagaaga gtgatgaata tacatttgct 240acttccgctg ataatcatgt aacaatgtgg gtagatgacc aagaagtgat taataaagct 300tctaattcta acaaaatcag attagaaaaa ggaagattat atcaaataaa aattcaatat 360caacgagaaa atcctactga aaaaggattg gatttcaagt tgtactggac cgattctcaa 420aataaaaaag aagtgatttc tagtgataac ttacaattgc cagaattaaa acaaaaatct 480tcgaactcaa gaaaaaagcg aagtacaagt gctggaccta cggttccaga ccgtgacaat 540gatggaatcc ctgattcatt agaggtagaa ggatatacgg ttgatgtcaa aaataaaaga 600acttttcttt caccatggat ttctaatatt catgaaaaga aaggattaac caaatataaa 660tcatctcctg aaaaatggag cacggcttct gatccgtaca gtgatttcga aaaggttaca 720ggacggattg ataagaatgt atcaccagag gcaagacacc cccttgtggc agcttatccg 780attgtacatg tagatatgga gaatattatt ctctcaaaaa atgaggatca atccacacag 840aatactgata gtgaaacgag aacaataagt aaaaatactt ctacaagtag gacacatact 900agtgaagtac atggaaatgc agaagtgcat gcgtcgttct ttgatattgg tgggagtgta 960tctgcaggat ttagtaattc gaattcaagt acggtcgcaa ttgatcattc actatctcta 1020gcaggggaaa gaacttgggc tgaaacaatg ggtttaaata ccgctgatac agcaagatta 1080aatgccaata ttagatatgt aaatactggg acggctccaa tctacaacgt gttaccaacg 1140acttcgttag tgttaggaaa aaatcaaaca ctcgcgacaa ttaaagctaa ggaaaaccaa 1200ttaagtcaaa tacttgcacc taataattat tatccttcta aaaacttggc gccaatcgca 1260ttaaatgcac aagacgattt cagttctact ccaattacaa tgaattacaa tcaatttctt 1320gagttagaaa aaacgaaaca attaagatta gatacggatc aagtatatgg gaatatagca 1380acatacaatt ttgaaaatgg aagagtgagg gtggatacag gctcgaactg gagtgaagtg 1440ttaccgcaaa ttcaagaaac aactgcacgt atcattttta atggaaaaga tttaaatctg 1500gtagaaaggc ggatagcggc ggttaatcct agtgatccat tagaaacgac taaaccggat 1560atgacattaa aagaagccct taaaatagca tttggattta acgaaccgaa tggaaactta 1620caatatcaag ggaaagacat aaccgaattt gattttaatt tcgatcaaca aacatctcaa 1680aatatcaaga atcagttagc ggaattaaac gcaactaaca tatatactgt attagataaa 1740atcaaattaa atgcaaaaat gaatatttta ataagagata aacgttttca ttatgataga 1800aataacatag cagttggggc ggatgagtca gtagttaagg aggctcatag agaagtaatt 1860aattcgtcaa cagagggatt attgttaaat attgataagg atataagaaa aatattatca 1920ggttatattg tagaaattga agatactgaa gggcttaaag aagttataaa tgacagatat 1980gatatgttga atatttctag tttacggcaa gatggaaaaa catttataga ttttaaaaaa 2040tataatgata aattaccgtt atatataagt aatcccaatt ataaggtaaa tgtatatgct 2100gttactaaag aaaacactat tattaatcct agtgagaatg gggatactag taccaacggg 2160atcaagaaaa ttttaatctt ttctaaaaaa ggctatgaga taggataa 2208&lt;210&gt;23&lt;211&gt;736&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;23Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His ValThr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr
      275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg
      580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly Glx725 730 735&lt;210&gt;24&lt;211&gt;599&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;24Thr Asp Ser Gln Asn Lys Lys Glu Val Ile Ser Ser Asp Asn Leu Gln1 5 10 15Leu Pro Glu Leu Lys Gln Lys Ser Ser Asn Ser Arg Lys Lys Arg Ser20 25 30Thr Ser Ala Gly Pro Thr Val Pro Asp Arg Asp Asn Asp Gly Ile Pro35 40 45Asp Ser Leu Glu Val Glu Gly Tyr Thr Val Asp Val Lys Asn Lys Arg50 55 60Thr Phe Leu Ser Pro Trp Ile Ser Asn Ile His Glu Lys Lys Gly Leu65 70 75 80Thr Lys Tyr Lys Ser Ser Pro Glu Lys Trp Ser Thr Ala Ser Asp Pro
      85 90 95Tyr Ser Asp Phe Glu Lys Val Thr Gly Arg Ile Asp Lys Asn Val Ser100 105 110Pro Glu Ala Arg His Pro Leu Val Ala Ala Tyr Pro Ile Val His Val115 120 125Asp Met Glu Asn Ile Ile Leu Ser Lys Asn Glu Asp Gln Ser Thr Gln130 135 140Asn Thr Asp Ser Gln Thr Arg Thr Ile Ser Lys Asn Thr Ser Thr Ser145 150 155 160Arg Thr His Thr Ser Glu Val His Gly Asn Ala Glu Val His Ala Ser165 170 175Phe Phe Asp Ile Gly Gly Ser Val Ser Ala Gly Phe Ser Asn Ser Asn180 185 190Ser Ser Thr Val Ala Ile Asp His Ser Leu Ser Leu Ala Gly Glu Arg195 200 205Thr Trp Ala Glu Thr Met Gly Leu Asn Thr Ala Asp Thr Ala Arg Leu210 215 220Asn Ala Asn Ile Arg Tyr Val Asn Thr Gly Thr Ala Pro Ile Tyr Asn225 230 235 240Val Leu Pro Thr Thr Ser Leu Val Leu Gly Lys Asn Gln Thr Leu Ala245 250 255Thr Ile Lys Ala Lys Glu Asn Gln Leu Ser Gln Ile Leu Ala Pro Asn260 265 270Asn Tyr Tyr Pro Ser Lys Asn Leu Ala Pro Ile Ala Leu Asn Ala Gln275 280 285Asp Asp Phe Ser Ser Thr Pro Ile Thr Met Asn Tyr Asn Gln Phe Leu290 295 300Glu Leu Glu Lys Thr Lys Gln Leu Arg Leu Asp Thr Asp Gln Val Tyr305 310 315 320Gly Asn Ile Ala Thr Tyr Asn Phe Glu Asn Gly Arg Val Arg Val Asp325 330 335Thr Gly Ser Asn Trp Ser Glu Val Leu Pro Gln Ile Gln Glu Thr Thr340 345 350Ala Arg Ile Ile Phe Asn Gly Lys Asp Leu Asn Leu Val Glu Arg Arg355 360 365Ile Ala Ala Val Asn Pro Ser Asp Pro Leu Glu Thr Thr Lys Pro Asp370 375 380Met Thr Leu Lys Glu Ala Leu Lys Ile Ala Phe Gly Phe Asn Glu Pro385 390 395 400Asn Gly Asn Leu Gln Tyr Gln Gly Lys Asp Ile Thr Glu Phe Asp Phe405 410 415Asn Phe Asp Gln Gln Thr Ser Gln Asn Ile Lys Asn Gln Leu Ala Glu420 425 430Leu Asn Val Thr Asn Ile Tyr Thr Val Leu Asp Lys Ile Lys Leu Asn435 440 445Ala Lys Met Asn Ile Leu Ile Arg Asp Lys Arg Phe His Tyr Asp Arg450 455 460Asn Asn Ile Ala Val Gly Ala Asp Glu Ser Val Val Lys Glu Ala His465 470 475 480Arg Glu Val Ile Asn Ser Ser Thr Glu Gly Leu Leu Leu Asn Ile Asp485 490 495Lys Asp Ile Arg Lys Ile Leu Ser Gly Tyr Ile Val Glu Ile Glu Asp500 505 510Thr Glu Gly Leu Lys Glu Val Ile Asn Asp Arg Tyr Asp Met Leu Asn515 520 525Ile Ser Ser Leu Arg Gln Asp Gly Lys Thr Phe Ile Asp Phe Lys Lys530 535 540Tyr Asn Asp Lys Leu Pro Leu Tyr Ile Ser Asn Pro Asn Tyr Lys Val545 550 555 560Asn Val Tyr Ala Val Thr Lys Glu Asn Thr Ile Ile Asn Pro Ser Glu565 570 575Asn Gly Asp Thr Ser Thr Asn Gly Ile Lys Lys Ile Leu Ile Phe Ser580 585 590Lys Lys Gly Tyr Glu Ile Gly595&lt;210&gt;25&lt;211&gt;705&lt;212&gt;PRT&lt;213&gt;艱難梭菌&lt;400&gt;25Glu Leu Asp Gly Met Lys Lys Ile Ile Pro Glu Glu Asn Leu Phe Leu1 5 10 15Arg Asp Tyr Ser Asn Ile Glu Lys Asp Asp Pro Phe Ile Pro Asn Asn
      20 25 30Asn Phe Phe Asp Pro Lys Leu Met Ser Asp Trp Glu Asp Glu Asp Leu35 40 45Asp Thr Asp Asn Asp Asn Ile Pro Asp Ser Tyr Glu Arg Asn Gly Tyr50 55 60Thr Ile Lys Asp Leu Ile Ala ValLys Trp Glu Asp Ser Phe Ala Glu65 70 75 80Gln Gly Tyr Lys Lys Tyr Val Ser Asn Tyr Leu Glu Ser Asn Thr Ala85 90 95Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ser Gly Ser Phe Asp Lys100 105 110Ala Ile Lys Thr Glu Ala Arg Asp Pro Leu Val Ala Ala Tyr Pro Ile115 120 125Val Gly Val Gly Met Glu Lys Leu Ile Ile Ser Thr Asn Glu His Ala130 135 140Ser Thr Asp Gln Gly Lys Thr Val Ser Arg Ala Thr Thr Asn Ser Lys145 150155 160Thr Glu Ser Asn Thr Ala Gly Val Ser Val Asn Val Gly Tyr Gln Asn165 170 175Gly Phe Thr Ala Asn Val Thr Thr Asn Tyr Ser His Thr Thr Asp Asn180 185 190Ser Thr Ala Val Gln Asp Ser Asn Gly Glu Ser Trp Asn Thr Gly Leu195 200 205Ser Ile Asn Lys Gly Glu Ser Ala Tyr Ile Asn Ala Asn Val Arg Tyr210 215 220Tyr Asn Thr Gly Thr Ala Pro Met Tyr Lys Val Thr Pro Thr Thr Asn225 230 235 240Leu Val Leu Asp Gly Asp Thr Leu Ser Thr Ile Lys Ala Gln Glu Asn245 250 255Gln Ile Gly Asn Asn Leu Ser Pro Gly Asp Thr Tyr Pro Lys Lys Gly260 265 270Leu Ser Pro Leu Ala Leu Asn Thr Met Asp Gln Phe Ser Ser Arg Leu275 280 285Ile Pro Ile Asn Tyr Asp Gln Leu Lys Lys Leu Asp Ala Gly Lys Gln290 295 300Ile Lys Leu Glu Thr Thr Gln Val Ser Gly Asn Phe Gly Thr Lys Asn305 310 315 320Ser Ser Gly Gln Ile Val Thr Glu Gly Asn Ser Trp Ser Asp Tyr Ile
      325 330 335Ser Gln Ile Asp Ser Ile Ser AIa Ser Ile Ile Leu Asp Thr Glu Asn340 345 350Glu Ser Tyr Glu Arg Arg Val Thr Ala Lys Asn Leu Gln Asp Pro Glu355 360 365Asp Lys Thr Pro Glu Leu Thr Ile Gly Glu Ala Ile Glu Lys Ala Phe370 375 380Gly Ala Thr Lys Lys Asp Gly Leu Leu Tyr Phe Asn Asp Ile Pro Ile385 390 395 400Asp Glu Ser Cys Val Glu Leu Ile Phe Asp Asp Asn Thr Ala Asn Lys405 410 415Ile Lys Asp Ser Leu Lys Thr Leu Ser Asp Lys Lys Ile Tyr Asn Val420 425 430Lys Leu Glu Arg Gly Met Asn Ile Leu Ile Lys Thr Pro Thr Tyr Phe435 440 445Thr Asn Phe Asp Asp Tyr Asn Asn Tyr Pro Ser Thr Trp Ser Asn Val450 455 460Asn Thr Thr Asn Gln Asp Gly Leu Gln Gly Ser Ala Asn Lys Leu Asn465 470 475 480Gly Glu Thr Lys Ile Lys Ile Pro Met Ser Glu Leu Lys Pro Tyr Lys485 490 495Arg Tyr Val Phe Ser Gly Tyr Ser Lys Asp Pro Leu Thr Ser Asn Ser500 505 510Ile Ile Val Lys Ile Lys Ala Lys Glu Glu Lys Thr Asp Tyr Leu Val515 520 525Pro Glu Gln Gly Tyr Thr Lys Phe Ser Tyr Glu Phe Glu Thr Thr Glu530 535 540Lys Asp Ser Ser Asn Ile Glu Ile Thr Leu Ile Gly Ser Gly Thr Thr545 550 555 560Tyr Leu Asp Asn Leu Ser Ile Thr Glu Leu Asn Ser Thr Pro Glu Ile565 570 575Leu Asp Glu Pro Glu Val Lys Ile Pro Thr Asp Gln Glu Ile Met Asp580 585 590Ala His Lys Ile Tyr Phe Ala Asp Leu Asn Phe Asn Pro Ser Thr Gly595 600 605Asn Thr Tyr Ile Asn Gly Met Tyr Phe Ala Pro Thr Gln Thr Asn Lys610 615 620Glu Ala Leu Asp Tyr Ile Gln Lys Tyr Arg Val Glu Ala Thr Leu Gln625 630 635 640Tyr Ser Gly Phe Lys Asp Ile Gly Thr Lys Asp Lys Glu Met Arg Asn645 650 655Tyr Leu Gly Asp Pro Asn Gln Pro Lys Thr Asn Tyr Val Asn Leu Arg660 665 670Ser Tyr Phe Thr Gly Gly Glu Asn Ile Met Thr Tyr Lys Lys Leu Arg675 680 685Ile Tyr Ala Ile Thr Pro Asp Asp Arg Glu Leu Leu Val Leu Ser Val690 695 700Asp705&lt;210&gt;26&lt;211&gt;706&lt;212&gt;PRT&lt;213&gt;產(chǎn)氣莢膜梭菌&lt;400&gt;26Glu Leu Asn Gly Asn Lys Thr Val Ile Pro Glu Glu Asn Leu Phe Phe1 5 10 15Arg Asp Tyr Ser Lys Ile Asp Glu Asn Asp Pro Phe Ile Pro Asn Asn20 25 30Asn Phe Phe Asp ValArg Phe Phe Ser Ala Ala Trp Glu Asp Glu Asp35 40 45Leu Asp Thr Asp Asn Asp Asn Ile Pro Asp Ala Tyr Glu Lys Asn Gly50 55 60Tyr Thr Ile Lys Asp Ser Ile Ala Val Lys Trp Asn Asp Ser Phe Ala65 70 75 80Glu Gln Gly Tyr Lys Lys Tyr Val Ser Ser Tyr Leu Glu Ser Asn Thr85 90 95Ala Gly Asp Pro Tyr Thr Asp Tyr Gln Lys Ala Ser Gly Ser Ile Asp100 105 110Lys Ala Ile Lys Leu Glu Ala Arg Asp Pro Leu Val Ala Ala Tyr Pro115 120 125Val Val Gly Val Gly Met Glu Asn Leu Ile Ile Ser Thr Asn Glu His130 135 140Ala Ser Ser Asp Gln Gly Lys Thr Val Ser Arg Ala Thr Thr Asn Ser145 150 155160Lys Thr Asp Ala Asn Thr Val Gly Val Ser Ile Ser Ala Gly Tyr Gln165 170 175Asn Gly Phe Thr Gly Asn Ile Thr Thr Ser Tyr Sar His Thr Thr Asp180 185 190Asn Ser Thr Ala Val Gln Asp Ser Asn Gly Glu Ser Trp Asn Thr Gly195 200 205Leu Ser Ile Asn Lys Gly Glu Ser Ala Tyr Ile Asn Ala Asn Val Arg210 215 220Tyr Tyr Asn Thr Gly Thr Ala Pro Met Tyr Lys Val Thr Pro Thr Thr225 230 235 240Asn Leu Val Leu Asp Gly Glu Thr Leu Ala Thr Ile Lys Ala Gln Asp245 250 255Asn Gln Ile Gly Asn Asn Leu Ser Pro Asn Glu Thr Tyr Pro Lys Lys260 265 270Gly Leu Ser Pro Leu Ala Leu Asn Thr Met Asp Gln Phe Asn Ala Arg275 280 285Leu Ile Pro Ile Asn Tyr Asp Gln Leu Lys Lys Leu Asp Ser Gly Lys290 295 300Gln Ile Lys Leu Glu Thr Thr Gln Val Ser Gly Asn Tyr Gly Thr Lys305 310 315 320Asn Ser Gln Gly Gln Ile Ile Thr Glu Gly Asn Ser Trp Ser Asn Tyr325 330 335Ile Ser Gln Ile Asp Ser Val Ser Ala Ser Ile Ile Leu Asp Thr Gly340 345 350Ser Gln Thr Phe Glu Arg Arg Val Ala Ala Lys Glu Gln Gly Asn Pro355 360 365Glu Asp Lys Thr Pro Glu Ile Thr Ile Gly Glu Ala Ile Lys Lys Ala370 375 380Phe Ser Ala Thr Lys Asn Gly Glu Leu Leu Tyr Phe Asn Gly Ile Pro385 390 395 400Ile Asp Glu Ser Cys Val Glu Leu Ile Phe Asp Asp Asn Thr Ser Glu405 410 415Ile Ile Lys Glu Gln Leu Lys Tyr Leu Asp Asp Lys Lys Ile Tyr Asn420 425 430ValLys Leu Glu Arg Gly Met Asn Ile Leu Ile Lys Val Pro Ser Tyr435 440 445Phe Thr Asn Phe Asp Glu Tyr Asn Asn Phe Pro Ala Ser Trp Ser Asn
      450 455 460Ile Asp Thr Lys Asn Gln Asp Gly Leu Gln Ser Val Ala Asn Lys Leu465 470 475 480Ser Gly Glu Thr Lys Ile Ile Ile Pro Met Ser Lys Leu Lys Pro Tyr485 490 495Lys Arg Tyr Val Phe Ser Gly Tyr Ser Lys Asp Pro Ser Thr Ser Asn500 505 510Ser Ile Thr Val Asn Ile Lys Ser Lys Glu Gln Lys Thr Asp Tyr Leu515 520 525Val Pro Glu Lys Asp Tyr Thr Lys Phe Ser Tyr Glu Phe Glu Thr Thr530535 540Gly Lys Asp Ser Ser Asp Ile Glu Ile Thr Leu Thr Ser Ser Gly Val545 550 555 560Ile Phe Leu Asp Asn Leu Ser Ile Thr Glu Leu Asn Ser Thr Pro Glu565 570 575Ile Leu Lys Glu Pro Glu Ile Lys Val Pro Ser Asp Gln Glu Ile Leu580 585 590Asp Ala His Asn Lys Tyr Tyr Ala Asp Ile Lys Leu Asp Thr Asn Thr595 600 605Gly Asn Thr Tyr Ile Asp Gly Ile Tyr Phe Glu Pro Thr Gln Thr Asn610 615 620Lys Glu Ala Leu Asp Tyr Ile Gln Lys Tyr Arg Val Glu Ala Thr Leu625 630 635 640Gln Tyr Ser Gly Phe Lys Asp Ile Gly Thr Lys Asp Lys Glu Ile Arg645 650 655Asn Tyr Leu Gly Asp Gln Asn Gln Pro Lys Thr Asn Tyr Ile Asn Phe660 665 670Arg Ser Tyr Phe Thr Ser Gly Glu Asn Val Met Thr Tyr Lys Lys Leu675 680 685Arg Ile Tyr Ala ValThr Pro Asp Asn Arg Glu Leu Leu Val Leu Ser690695 700Val Asn705&lt;210&gt;27&lt;211&gt;712&lt;212&gt;PRT&lt;213&gt;螺狀梭菌&lt;400&gt;27Glu Leu Asn Gly Asp Lys Thr Leu Ile Pro Glu Lys Asn Leu Phe Leu1 5 10 15Arg Asp Tyr Ser Lys Ile Asp Glu Asn Asp Pro Phe Ile Pro Lys Asp20 25 30Asn Phe Phe Asp Leu Lys Leu Lys Ser Arg Ser Ala Arg Leu Ala Ser35 40 45Gly Trp Gly Asp Glu Asp Leu Asp Thr Asp Asn Asp Asn Ile Pro Asp50 55 60Ala Tyr Glu Lys Asn Gly Tyr Thr Ile Lys Asp Ser Ile Ala Val Lys65 70 75 80Trp Glu Asp Ser Phe Ala Gln Gln Gly Tyr Lys Lys Tyr Leu Ser Ser85 90 95Tyr Leu Glu Ser Asn Thr Ala Gly Asp Pro Tyr Thr Asp Tyr Gln Lys100 105 110Ala Ser Gly Ser Phe Asp Lys Ala Ile Lys Ala Glu Ala Arg Asp Pro115 120 125Leu Val Ala Ala Tyr Pro Val Val Gly Val Gly Met Glu Lys Leu Ile130 135 140Ile Ser Thr Asn Glu His Ala Ser Thr Asp Gln Gly Lys Thr Val Ser145 150 155 160Arg Asn Thr Thr Asn Ser Lys Thr Asp Ala Asn Thr Ala Gly Val Ala165 170 175Ile Asn Ile Ala Tyr Gln Asn Gly Phe Thr Gly Ser Ile Thr Thr Asn180 185 190Tyr Ser His Thr Thr Glu Asn Ser Thr Ala Val Gln Asn Ser Asn Gly195 200 205Glu Ser Trp Asn Thr Ser Leu Ser Ile Asn Lys Gly Glu Ser Ala Tyr210 215 220Ile Asn Ala Asn Val Arg Tyr Tyr Asn Thr Gly Thr Ala Pro Met Tyr225 230 235 240Lys Val Thr Pro Thr Thr Asn Leu Val Leu Asp Gly Asp Thr Leu Thr245 250 255Thr Ile Lys Ala Gln Asp Asn Gln Ile Gly Asn Asn Leu Ser Pro Asn260 265 270Glu Thr Tyr Pro Lys Lys Gly Leu Ser Pro Leu Ala Leu Asn Thr Met
      275 280 285Asp Gln Phe Ser Ser Arg Leu Ile Pro Ile Asn Tyr Asp Gln Leu Lys290 295 300Lys Leu Asp Ala Gly Lys Gln Ile Lys Leu Glu Thr Thr Gln Val Ser305 310 315 320Gly Asn Tyr Gly Ile Lys Asn Ser Gln Gly Gln Ile Ile Thr Glu Gly325 330 335Asn Ser Trp Ser Asp Tyr Ile Ser Gln Ile Asp Ser Leu Ser Ala Ser340 345 350Ile Ile Leu Asp Thr Gly Ser Asp Val Phe Glu Arg Arg Val Thr Ala355 360 365Lys Asp Ser Ser Asn Pro Glu Asp Lys Thr Pro Val Leu Thr Ile Gly370 375 380Glu Ala Ile Glu Lys Ala Phe Gly Ala Thr Lys Asn Gly Glu Ile Leu385 390 395 400Tyr Phe Asn Gly Met Pro Ile Asp Glu Ser Cys Val Glu Leu Ile Phe405 410 415Asp Gly Asn Thr Ala Asn Leu Ile Lys Glu Arg Leu Asn Ala Leu Asn420 425 430Asp Lys Lys Ile Tyr Asn Val Gln Leu Glu Arg Gly Met Lys Ile Leu435 440 445Ile Lys Thr Ser Thr Tyr Phe Asn Asn Phe Asp Gly Tyr Asn Asn Phe450 455 460Pro Ser Ser Trp Ser Asn Val Asp Ser Asn Asn Gln Asp Gly Leu Gln465 470 475 480Asn Ala Ala Asn Lys Leu Ser Gly Glu Thr Lys Ile Val Ile Pro Met485 490 495Ser Lys Leu Asn Pro Tyr Lys Arg Tyr Val Phe Ser Gly Tyr Leu Lys500 505 510Asn Ser Ser Thr Ser Asn Pro Ile Thr Val Asn Ile Lys Ala Lys Glu515 520 525Gln Lys Thr Tyr Asn Leu Val Ser Glu Asn Asp Tyr Lys Lys Phe Ser530 535 540Tyr Glu Phe Glu Thr Ile Gly Arg Asp Ala Ser Asn Ile Glu Ile Thr545 550 555 560Leu Thr Ser Ser Gly Thr Ile Phe Leu Asp Asn Leu Ser Ile Thr Glu565 570 575Leu Asn Ser Thr Pro Glu Ile Leu Lys Glu Pro Asp Ile Lys Val Pro
      580 585 590Ser Asp Gln Glu Ile Ile Asp Ala His Lys Lys Tyr Tyr Ala Asp Leu595 600 605Ser Phe Asn Gln Ser Thr Ala Asn Tyr Tyr Leu Asp Gly Leu Tyr Phe610 615 620Glu Pro Thr Gln Thr Asn Lys Glu Val Leu Asp Tyr Ile Gln Lys Tyr625 630 635 640Lys Val Glu Ala Thr Leu Glu Tyr Ser Gly Phe Lys Asp Ile Gly Thr645 650 655Lys Asp Lys Glu Leu Arg Asn Tyr Thr Gly Asp Ser Asn Gln Pro Lys660 665 670Thr Asn Tyr Val Asn Phe Arg Ser Tyr Phe Thr Ser Gly Glu Asn Val675 680 685Met Pro Tyr Lys Lys Leu Arg Ile Tyr Ala Ile Thr Pro Glu Asn Lys690 695 700Glu Leu Leu Val Leu Ser Ile Asn705 710&lt;210&gt;28&lt;211&gt;582&lt;212&gt;PRT&lt;213&gt;肉毒梭菌&lt;400&gt;28Glu Thr Ser Asp Ile Ile Lys Glu Ile Ile Pro Ser Glu Val Leu Leu1 5 10 15Lys Pro Asn Tyr Ser Asn Thr Asn Glu Lys Ser Lys Phe Ile Pro Asn20 25 30Asn Thr Leu Phe Ser Asn Ala Lys Leu Lys Ala Asn Ala Asn Arg Asp35 40 45Thr Asp Arg Asp Gly Ile Pro Asp Glu Trp Glu Ile Asn Gly Tyr Thr50 55 60Val Met Asn Gln Lys Ala ValAla Trp Asp Asp Lys Phe Ala Ala Asn65 70 75 80Gly Tyr Lys Lys Tyr Val Ser Asn Pro Phe Lys Pro Cys Thr Ala Asn85 90 95Asp Pro Tyr Thr Asp Phe Glu Lys Val Ser Gly Gln Ile Asp Pro Ser
      100 105 110Val Ser Met Val Ala Arg Asp Pro Met Ile Ser Ala Tyr Pro Ile Val115 120 125Gly Val Gln Met Glu Arg Leu Val Val Ser Lys Ser Glu Thr Ile Thr130 135 140Gly Asp Ser Thr Lys Ser Met Ser Lys Ser Thr Ser His Ser Ser Thr145 150 155 160Asn Ile Asn Thr Val Gly Ala Glu Val Ser Gly Ser Leu Gln Leu Ala165 170 175Gly Gly Ile Phe Pro Val Phe Ser Met Ser Ala Ser Ala Asn Tyr Ser180 185 190His Thr Trp Gln Asn Thr Ser Thr Val Asp Asp Thr Thr Gly Glu Ser195 200 205Phe Ser Gln Gly Leu Ser Ile Asn Thr Gly Glu Ser Ala Tyr Ile Asn210 215 220Pro Asn Ile Arg Tyr Tyr Asn Thr Gly Thr Ala Pro Val Tyr Asn Val225 230 235 240Thr Pro Thr Thr Thr Ile Val Ile Asp Lys Gln Ser Val Ala Thr Ile245 250 255Lys Gly Gln Glu Ser Leu Ile Gly Asp Tyr Leu Asn Pro Gly Gly Thr260 265 270Tyr Pro Ile Ile Gly Glu Pro Pro Met Ala Leu Asn Thr Met Asp Gln275 280 285Phe Ser Ser Arg Leu Ile Pro Ile Asn Tyr Asn Gln Leu Lys Ser Ile290 295 300Asp Asn Gly Gly Thr Val Met Leu Ser Thr Ser Gln Phe Thr Gly Asn305 310 315 320Phe Ala Lys Tyr Asn Ser Asn Gly Asn Leu Val Thr Asp Gly Asn Asn325 330 335Trp Gly Pro Tyr Leu Gly Thr Ile Lys Ser Thr Thr Ala Ser Leu Thr340 345 350Leu Ser Phe Ser Gly Gln Thr Thr Gln Val Ala Val Val Ala Pro Asn355 360 365Phe Ser Asp Pro Glu Asp Lys Thr Pro Lys Leu Thr Leu Glu Gln Ala370 375 380Leu Val Lys Ala Phe Ala Leu Glu Lys Lys Asn Gly Lys Phe Tyr Phe385 390 395 400His Gly Leu Glu Ile Ser Lys Asn Glu Lys Ile Gln Val Phe Leu Asp
      405 410 415Ser Asn Thr Asn Asn Asp Phe Glu Asn Gln Leu Lys Asn Thr Ala Asp420 425 430Lys Asp Ile Met His Cys Ile Ile Lys Arg Asn Met Asn Ile Leu Val435 440 445Lys Val Ile Thr Phe Lys Glu Asn Ile Ser Ser Ile Asn Ile Ile Asn450 455 460Asp Thr Asn Phe Gly Val Gln Ser Met Thr Gly Leu Ser Asn Arg Ser465 470 475 480Lys Gly Gln Asp Gly Ile Tyr Arg Ala Ala Thr Thr Ala Phe Ser Phe485 490 495Lys Ser Lys Glu Leu Lys Tyr Pro Glu Gly Arg Tyr Arg Met Arg Phe500 505 510Val Ile Gln Ser Tyr Glu Pro Phe Thr Cys Asn Phe Lys Leu Phe Asn515 520 525Asn Leu Ile Tyr Ser Ser Ser Phe Asp Lys Gly Tyr Tyr Asp Glu Phe530 535 540Phe Tyr Phe Tyr Tyr Asn Gly Ser Lys Ser Phe Phe Asn Ile Ser Cys545 550 555 560Asp Ile Ile Asn Ser Ile Asn Arg Leu Ser Gly Val Phe Leu Ile Glu565 570 575Leu Asp Lys Leu Ile Ile580&lt;210&gt;29&lt;211&gt;708&lt;212&gt;PRT&lt;213&gt;蠟狀芽孢桿菌&lt;400&gt;29Ile Asp Ser Gln Asn Gln Pro Gln Gln Val Gln Gln Asp Glu Leu Arg1 5 10 15Asn Pro Glu Phe Asn Lys Lys Glu Ser Gln Glu Phe Leu Ala Lys Pro20 25 30Ser Lys Ile Asn Leu Phe Thr Gln Gln Met Lys Arg Glu Ile Asp Glu35 40 45Asp Thr Asp Thr Asp Gly Asp Ser Ile Pro Asp Leu Trp Glu Glu Asn
      50 55 60Gly Tyr Thr Ile Gln Asn Arg Ile Ala Val Lys Trp Asp Asp Ser Leu65 70 75 80Ala Ser Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu Ser His85 90 95Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu100 105 110Asp Leu Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe115 120 125Pro Ser Val Asn Val Ser Met Glu Lys Val Ile Leu Ser Pro Asn Glu130 135 140Asn Leu Ser Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp Ser Tyr145 150 155 160Thr Asn Thr Glu Gly Ala Ser Val Glu Ala Gly Ile Gly Pro Lys Gly165 170 175Ile Ser Phe Gly Val Ser Val Asn Tyr Gln His Ser Glu Thr Val Ala180 185 190Gln Glu Trp Gly Thr Ser Thr Gly Asn Thr Ser Gln Phe Asn Thr Ala195 200 205Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly Thr210 215 220Gly Ala Ile Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Asn225 230 235 240Asp Thr Ile Ala Thr Ile Thr Ala Lys Ser Asn Ser Thr Ala Leu Asn245 250 255Ile Ser Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gln Asn Gly Ile Ala260 265 270Ile Thr Ser Met Asp Asp Phe Asn Ser His Pro Ile Thr Leu Asn Lys275 280 285Lys Gln Val Asp Asn Leu Leu Asn Asn Lys Pro Met Met Leu Glu Thr290 295 300Asn Gln Thr Asp Gly Val Tyr Lys Ile Lys Asp Thr His Gly Asn Ile305 310 315 320Val Thr Gly Gly Glu Trp Asn Gly Val Ile Gln Gln Ile Lys Ala Lys325 330 335Thr Ala Ser Ile Ile Val Asp Asp Gly Glu Arg Val Ala Glu Lys Arg340 345 350Val Ala Ala Lys Asp Tyr Glu Asn Pro Glu Asp Lys Thr Pro Ser Leu
      355 360 365Thr Leu Lys Asp Ala Leu Lys Leu Ser Tyr Pro Asp Glu Ile Lys Glu370 375 380Ile Glu Gly Leu Leu Tyr Tyr Lys Asn Lys Pro Ile Tyr Glu Ser Ser385 390 395 400Val Met Thr Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Thr Lys Gln405 410 415Leu Asn Asp Thr Thr Gly Lys Phe Lys Asp Val Ser His Leu Tyr Asp420 425 430Val Lys Leu Thr Pro Lys Met Asn Val Thr Ile Lys Leu Ser Ile Leu435 440 445Tyr Asp Asn Ala Glu Ser Asn Asp Asn Ser Ile Gly Lys Trp Thr Asn450 455 460Thr Asn Ile Val Ser Gly Gly Asn Asn Gly Lys Lys Gln Tyr Ser Ser465 470 475 480Asn Asn Pro Asp Ala Asn Leu Thr Leu Asn Thr Asp Ala Gln Glu Lys485 490 495Leu Asn Lys Asn Arg Asp Tyr Tyr Ile Ser Leu Tyr Met Lys Ser Glu500 505 510Lys Asn Thr Gln Cys Glu Ile Thr Ile Asp Gly Glu Ile Tyr Pro Ile515 520 525Thr Thr Lys Thr Val Asn Val Asn Lys Asp Asn Tyr Lys Arg Leu Asp530 535 540Ile Ile Ala His Asn Ile Lys Ser Asn Pro Ile Ser Ser Leu His Ile545 550 555 560Lys Thr Asn Asp Glu Ile Thr Leu Phe Trp Asp Asp Ile Ser Ile Thr565 570 575Asp Val Ala Ser Ile Lys Pro Glu Asn Leu Thr Asp Ser Glu Ile Lys580 585 590Gln Ile Tyr Ser Arg Tyr Gly Ile Lys Leu Glu Asp Gly Ile Leu Ile595 600 605Asp Lys Lys Gly Gly Ile His Tyr Gly Glu Phe Ile Asn Glu Ala Ser610 615 620Phe Asn Ile Glu Pro Leu Gln Asn Tyr Val Thr Lys Tyr Glu Val Thr625 630 635 640Tyr Ser Ser Glu Leu Gly Pro Asn Val Ser Asp Thr Leu Glu Ser Asp645 650 655Lys Ile Tyr Lys Asp Gly Thr Ile Lys Phe Asp Phe Thr Lys Tyr Ser
      660 665 670Lys Asn Glu Gln Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe675 680 685Lys Ile Asn Ala Ile Thr Tyr Asp Gly Lys Glu Met Asn Val Phe His690 695 700Arg Tyr Asn Lys705&lt;210&gt;30&lt;211&gt;735&lt;212&gt;PRT&lt;213&gt;炭疽桿菌&lt;400&gt;30Glu Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser1 5 10 15Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro20 25 30Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser35 40 45Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile50 55 60Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala65 70 75 80Thr Ser Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val85 90 95Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg100 105 110Leu Tyr Gln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys115 120 125Gly Leu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser145 150 155 160Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr
      180 185 190Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser195 200 205Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr225 230 235 240Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile Leu Ser260 265 270Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Gln Thr Arg Thr275 280 285Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser Glu Val His290 295 300Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile Gly Gly Ser Val305 310 315 320Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His325 330 335Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu Thr Met Gly Leu340 345 350Asn Thr Ala Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn355 360 365Thr Gly Thr Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val370 375 380Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln385 390 395 400Leu Ser Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile420 425 430Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu435 440 445Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val465 470 475 480Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys
      485 490 495Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp500 505 510Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys515 520 525Ile Ala Phe Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly530 535 540Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln545 550 555 560Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Val Thr Asn Ile Tyr Thr565 570 575Val Leu Asp Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg580 585 590Asp Lys Arg Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp595 600 605Glu Ser Val Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr610 615 620Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser625 630 635 640Gly Tyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly660 665 670Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr675 680 685Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly705 710 715 720Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly725 730 735&lt;210&gt;31&lt;211&gt;876&lt;212&gt;PRT&lt;213&gt;艱難梭菌&lt;400&gt;31Met Lys Ile Gln Met Arg Asn Lys Lys Val Leu Ser Phe Leu Thr Leu1 5 10 15Thr Ala Ile Val Ser Gln Ala Leu Val Tyr Pro Val Tyr Ala Gln Thr20 25 30Ser Thr Ser Asn His Ser Asn Lys Lys Lys Glu Ile Val Asn Glu Asp35 40 45Ile Leu Pro Asn Asn Gly Leu Met Gly Tyr Tyr Phe Ser Asp Glu His50 55 60Phe Lys Asp Leu Lys Leu Met Ala Pro Ile Lys Asp Gly Asn Leu Lys65 70 75 80Phe Glu Glu Lys Lys Val Asp Lys Leu Leu Asp Lys Asp Lys Ser Asp85 90 95Val Lys Ser Ile Arg Trp Thr Gly Arg Ile Ile Pro Ser Lys Asp Gly100 105 110Glu Tyr Thr Leu Ser Thr Asp Arg Asp Asp Val Leu Met Gln Val Asn115 120 125Thr Glu Ser Thr Ile Ser Asn Thr Leu Lys Val Asn Met Lys Lys Gly130 135 140Lys Glu Tyr Lys Val Arg Ile Glu Leu Gln Asp Lys Asn Leu Gly Ser145 150 155 160Ile Asp Asn Leu Ser Ser Pro Asn Leu Tyr Trp Glu Leu Asp Gly Met165 170 175Lys Lys Ile Ile Pro Glu Glu Asn Leu Phe Leu Arg Asp Tyr Ser Asn180 185 190Ile Glu Lys Asp Asp Pro Phe Ile Pro Asn Asn Asn Phe Phe Asp Pro195 200 205Lys Leu Met Ser Asp Trp Glu Asp Glu Asp Leu Asp Thr Asp Asn Asp210 215 220Asn lle Pro Asp Ser Tyr Glu Arg Asn Gly Tyr Thr Ile Lys Asp Leu225 230 235 240Ile Ala Val Lys Trp Glu Asp Ser Phe Ala Glu Gln Gly Tyr Lys Lys245 250 255Tyr Val Ser Asn Tyr Leu Glu Ser Asn Thr Ala Gly Asp Pro Tyr Thr260 265 270Asp Tyr Glu Lys Ala Ser Gly Ser Phe Asp Lys Ala Ile Lys Thr Glu275 280 285Ala Arg Asp Pro Leu Val Ala Ala Tyr Pro Ile Val Gly Val Gly Met
      290 295 300Glu Lys Leu Ile Ile Ser Thr Asn Glu His Ala Ser Thr Asp Gln Gly305 310 315 320Lys Thr Val Ser Arg Ala Thr Thr Asn Ser Lys Thr Glu Ser Asn Thr325 330 335Ala Gly Val Ser Val Asn Val Gly Tyr Gln Asn Gly Phe Thr Ala Asn340 345 350ValThr Thr Asn Tyr Ser His Thr Thr Asp Asn Ser Thr Ala Val Gln355 360 365Asp Ser Asn Gly Glu Ser Trp Asn Thr Gly Leu Ser Ile Asn Lys Gly370 375 380Glu Ser Ala Tyr Ile Asn Ala Asn Val Arg Tyr Tyr Asn Thr Gly Thr385 390 395 400Ala Pro Met Tyr Lys Val Thr Pro Thr Thr Asn Leu Val Leu Asp Gly405 410 415Asp Thr Leu Ser Thr Ile Lys Ala Gln Glu Asn Gln Ile Gly Asn Asn420 425 430Leu Ser Pro Gly Asp Thr Tyr Pro Lys Lys Gly Leu Ser Pro Leu Ala435 440 445Leu Asn Thr Met Asp Gln Phe Ser Ser Arg Leu Ile Pro Ile Asn Tyr450 455 460Asp Gln Leu Lys Lys Leu Asp Ala Gly Lys Gln Ile Lys Leu Glu Thr465470 475 480Thr Gln Val Ser Gly Asn Phe Gly Thr Lys Asn Ser Ser Gly Gln Ile485 490 495Val Thr Glu Gly Asn Ser Trp Ser Asp Tyr Ile Ser Gln Ile Asp Ser500 505 510Ile Ser Ala Ser Ile Ile Leu Asp Thr Glu Asn Glu Ser Tyr Glu Arg515 520 525Arg Val Thr Ala Lys Asn Leu Gln Asp Pro Glu Asp Lys Thr Pro Glu530 535 540Leu Thr Ile Gly Glu Ala Ile Glu Lys Ala Phe Gly Ala Thr Lys Lys545 550 555 560Asp Gly Leu Leu Tyr Phe Asn Asp Ile Pro Ile Asp Glu Ser Cys Val565 570 575Glu Leu Ile Phe Asp Asp Asn Thr Ala Asn Lys Ile Lys Asp Ser Leu580 585 590Lys Thr Leu Ser Asp Lys Lys Ile Tyr Asn Val Lys Leu Glu Arg Gly
      595 600 605Met Asn Ile Leu Ile Lys Thr Pro Thr Tyr Phe Thr Asn Phe Asp Asp610 615 620Tyr Asn Asn Tyr Pro Ser Thr Trp Ser Asn Val Asn Thr Thr Asn Gln625 630 635 640Asp Gly Leu Gln Gly Ser Ala Asn Lys Leu Asn Gly Glu Thr Lys Ile645 650 655Lys Ile Pro Met Ser Glu Leu Lys Pro Tyr Lys Arg Tyr Val Phe Ser660 665 670Gly Tyr Ser Lys Asp Pro Leu Thr Ser Asn Ser Ile Ile Val Lys Ile675 680 685Lys Ala Lys Glu Glu Lys Thr Asp Tyr Leu Val Pro Glu Gln Gly Tyr690 695 700Thr Lys Phe Ser Tyr Glu Phe Glu Thr Thr Glu Lys Asp Ser Ser Asn705 710 715 720Ile Glu Ile Thr Leu Ile Gly Ser Gly Thr Thr Tyr Leu Asp Asn Leu725 730 735Ser Ile Thr Glu Leu Asn Ser Thr Pro Glu Ile Leu Asp Glu Pro Glu740 745 750Val Lys Ile Pro Thr Asp Gln Glu Ile Met Asp Ala His Lys Ile Tyr755 760 765Phe Ala Asp Leu Asn Phe Asn Pro Ser Thr Gly Asn Thr Tyr Ile Asn770 775 780Gly Met Tyr Phe Ala Pro Thr Gln Thr Asn Lys Glu Ala Leu Asp Tyr785 790 795 800Ile Gln Lys Tyr Arg Val Glu Ala Thr Leu Gln Tyr Ser Gly Phe Lys805 810 815Asp Ile Gly Thr Lys Asp Lys Glu Met Arg Asn Tyr Leu Gly Asp Pro820 825 830Asn Gln Pro Lys Thr Asn Tyr Val Asn Leu Arg Ser Tyr Phe Thr Gly835 840 845Gly Glu Asn Ile Met Thr Tyr Lys Lys Leu Arg Ile Tyr Ala Ile Thr850 855 860Pro Asp Asp Arg Glu Leu Leu Val Leu Ser Val Asp865 870 875&lt;210&gt;32&lt;211&gt;875&lt;212&gt;PRT&lt;213&gt;產(chǎn)氣莢膜梭菌&lt;400&gt;32Met Asn Ile Gln Ile Lys Asn Val Phe Ser Phe Leu Thr Leu Thr Ala1 5 10 15Met Ile Ser Gln Thr Leu Ser Tyr Asn Val Tyr Ala Gln Thr Thr Thr20 25 30Gln Asn Asp Thr Asn Gln Lys Glu Glu Ile Thr Asn Glu Asn Thr Leu35 40 45Ser Ser Asn Gly Leu Met Gly Tyr Tyr Phe Ala Asp Glu His Phe Lys50 55 60Asp Leu Glu Leu Met Ala Pro Ile Lys Asn Gly Asp Leu Lys Phe Glu65 70 75 80Glu Lys Lys Val Asp Lys Leu Leu Thr Glu Asp Asn Ser Ser Ile Lys85 90 95Ser Ile Arg Trp Thr Gly Arg Ile Ile Pro Ser Glu Asp Gly Glu Tyr100 105 110Ile Leu Ser Thr Asp Arg Asn Asp Val Leu Met Gln Ile Asn Ala Lys115 120 125Gly Asp Ile Ala Lys Thr Leu Lys Val Asn Met Lys Lys Gly Gln Ala130 135 140Tyr Asn Ile Arg Ile Glu Ile Gln Asp Lys Asn Leu Gly Ser Ile Asp145 150 155 160Asn Leu Ser Val Pro Lys Leu Tyr Trp Glu Leu Asn Gly Asn Lys Thr165 170 175Val Ile Pro Glu Glu Asn Leu Phe Phe Arg Asp Tyr Ser Lys Ile Asp180 185 190Glu Asn Asp Pro Phe Ile Pro Asn Asn Asn Phe Phe Asp Val Arg Phe195 200 205Phe Ser Ala Ala Trp Glu Asp Glu Asp Leu Asp Thr Asp Asn Asp Asn210 215 220Ile Pro Asp Ala Tyr Glu Lys Asn Gly Tyr Thr Ile Lys Asp Ser Ile225 230 235 240Ala Val Lys Trp Asn Asp Ser Phe Ala Glu Gln Gly Tyr Lys Lys Tyr245 250 255Val Ser Ser Tyr Leu Glu Ser Asn Thr Ala Gly Asp Pro Tyr Thr Asp
      260 265 270Tyr Gln Lys Ala Ser Gly Ser Ile Asp Lys Ala Ile Lys Leu Glu Ala275 280 285Arg Asp Pro Leu Val Ala Ala Tyr Pro Val Val Gly Val Gly Met Glu290 295 300Asn Leu Ile Ile Ser Thr Asn Glu His Ala Ser Ser Asp Gln Gly Lys305 310 315 320Thr Val Ser Arg Ala Thr Thr Asn Ser Lys Thr Asp Ala Asn Thr Val325 330 335Gly Val Ser Ile Ser Ala Gly Tyr Gln Asn Gly Phe Thr Gly Asn Ile340 345 350Thr Thr Ser Tyr Ser His Thr Thr Asp Asn Ser Thr Ala Val Gln Asp355 360 365Ser Asn Gly Glu Ser Trp Asn Thr Gly Leu Ser Ile Asn Lys Gly Glu370 375 380Ser Ala Tyr Ile Asn Ala Asn Val Arg Tyr Tyr Asn Thr Gly Thr Ala385 390 395 400Pro Met Tyr Lys Val Thr Pro Thr Thr Asn Leu Val Leu Asp Gly Glu405 410 415Thr Leu Ala Thr Ile Lys Ala Gln Asp Asn Gln Ile Gly Asn Asn Leu420 425 430Ser Pro Asn Glu Thr Tyr Pro Lys Lys Gly Leu Ser Pro Leu Ala Leu435 440 445Asn Thr Met Asp Gln Phe Asn Ala Arg Leu Ile Pro Ile Asn Tyr Asp450 455 460Gln Leu Lys Lys Leu Asp Ser Gly Lys Gln Ile Lys Leu Glu Thr Thr465 470 475 480Gln Val Ser Gly Asn Tyr Gly Thr Lys Asn Ser Gln Gly Gln Ile Ile485 490 495Thr Glu Gly Asn Ser Trp Ser Asn Tyr Ile Ser Gln Ile Asp Ser Val500 505 510Ser Ala Ser Ile Ile Leu Asp Thr Gly Ser Gln Thr Phe Glu Arg Arg515 520 525Val Ala Ala Lys Glu Gln Gly Asn Pro Glu Asp Lys Thr Pro Glu Ile530 535 540Thr Ile Gly Glu Ala Ile Lys Lys Ala Phe Ser Ala Thr Lys Asn Gly545 550 555 560Glu Leu Leu Tyr Phe Asn Gly Ile Pro Ile Asp Glu Ser Cys Val Glu
      565 570 575Leu Ile Phe Asp Asp Asn Thr Ser Glu Ile Ile Lys Glu Gln Leu Lys580 585 590Tyr Leu Asp Asp Lys Lys Ile Tyr Asn Val Lys Leu Glu Arg Gly Met595 600 605Asn Ile Leu Ile Lys Val Pro Ser Tyr Phe Thr Asn Phe Asp Glu Tyr610 615 620Asn Asn Phe Pro Ala Ser Trp Ser Asn Ile Asp Thr Lys Asn Gln Asp625 630 635 640Gly Leu Gln Ser Val Ala Asn Lys Leu Ser Gly Glu Thr Lys Ile Ile645 650 655Ile Pro Met Ser Lys Leu Lys Pro Tyr Lys Arg Tyr Val Phe Ser Gly660 665 670Tyr Ser Lys Asp Pro Ser Thr Ser Asn Ser Ile Thr Val Asn Ile Lys675 680 685Ser Lys Glu Gln Lys Thr Asp Tyr Leu Val Pro Glu Lys Asp Tyr Thr690 695 700Lys Phe Ser Tyr Glu Phe Glu Thr Thr Gly Lys Asp Ser Ser Asp Ile705 710 715 720Glu Ile Thr Leu Thr Ser Ser Gly Val Ile Phe Leu Asp Asn Leu Ser725 730 735Ile Thr Glu Leu Asn Ser Thr Pro Glu Ile Leu Lys Glu Pro Glu Ile740 745 750Lys Val Pro Ser Asp Gln Glu Ile Leu Asp Ala His Asn Lys Tyr Tyr755 760 765Ala Asp Ile Lys Leu Asp Thr Asn Thr Gly Asn Thr Tyr Ile Asp Gly770 775 780Ile Tyr Phe Glu Pro Thr Gln Thr Asn Lys Glu Ala Leu Asp Tyr Ile785 790 795 800Gln Lys Tyr Arg Val Glu Ala Thr Leu Gln Tyr Ser Gly Phe Lys Asp805 810 815Ile Gly Thr Lys Asp Lys Glu Ile Arg Asn Tyr Leu Gly Asp Gln Asn820 825 830Gln Pro Lys Thr Asn Tyr Ile Asn Phe Arg Ser Tyr Phe Thr Ser Gly835 840 845Glu Asn Val Met Thr Tyr Lys Lys Leu Arg Ile Tyr Ala Val Thr Pro850 855 860Asp Asn Arg Glu Leu Leu Val Leu Ser Val Asn865870 875&lt;210&gt;33&lt;211&gt;879&lt;212&gt;PRT&lt;213&gt;螺狀梭菌&lt;400&gt;33Met Lys Asn Lys Lys Ile Leu Gly Leu Leu Thr Cys Thr Val Leu Val1 5 10 15Gly Gln Met Met Thr Tyr Pro Val Tyr Ala Lys Thr Ile Thr Gln Asn20 25 30Tyr Asp Asn Gln Glu Val Glu Thr Thr Asn Glu Lys Thr Val Ser Ser35 40 45Asn Gly Leu Met Gly Tyr Tyr Phe Ala Asp Glu His Phe Lys Asp Leu50 55 60Glu Leu Met Ala Pro Val Lys Asn Gly Glu Leu Lys Phe Glu Lys Asn65 70 75 80Lys Val Glu Lys Leu Leu Thr Glu Glu Lys Thr Asn Ile Lys Ser Ile85 90 95Arg Trp Thr Gly Arg Ile Ile Pro Ser Lys Asp Gly Glu Tyr Thr Leu100 105 110Ser Thr Asp Lys Asp Asn Val Leu Met Gln Ile Asn Ala Glu Gly Glu115 120 125Ile Ala Asn Thr Leu Lys Val Asn Met Ile Lys Gly Gln Glu Tyr Ser130 135 140Ile Arg Ile Glu Ile Gln Asp Lys Asp Ile Gly Tyr Val Asp Asp Leu145 150 155 160Ser Ser Pro Lys Leu Tyr Trp Glu Leu Asn Gly Asp Lys Thr Leu Ile165 170 175Pro Glu Lys Asn Leu Phe Leu Arg Asp Tyr Ser Lys Ile Asp Glu Asn180 185 190Asp Pro Phe Ile Pro Lys Asp Asn Phe Phe Asp Leu Lys Leu Lys Ser195 200 205Arg Ser Ala Arg Leu Ala Ser Gly Trp Gly Asp Glu Asp Leu Asp Thr210 215 220Asp Asn Asp Asn Ile Pro Asp Ala Tyr Glu Lys Asn Gly Tyr Thr Ile225 230 235 240Lys Asp Ser Ile Ala Val Lys Trp Glu Asp Ser Phe Ala Gln Gln Gly245 250 255Tyr Lys Lys Tyr Leu Ser Ser Tyr Leu Glu Ser Asn Thr Ala Gly Asp260 265 270Pro Tyr Thr Asp Tyr Gln Lys Ala Ser Gly Ser Phe Asp Lys Ala Ile275 280 285Lys Ala Glu Ala Arg Asp Pro Leu Val Ala Ala Tyr Pro Val Val Gly290 295 300Val Gly Met Glu Lys Leu Ile Ile Ser Thr Asn Glu His Ala Ser Thr305 310 315 320Asp Gln Gly Lys Thr Val Ser Arg Asn Thr Thr Asn Ser Lys Thr Asp325 330 335Ala Asn Thr Ala Gly Val Ala Ile Asn Ile Ala Tyr Gln Asn Gly Phe340 345 350Thr Gly Ser Ile Thr Thr Asn Tyr Ser His Thr Thr Glu Asn Ser Thr355 360 365Ala Val Gln Asn Ser Asn Gly Glu Ser Trp Asn Thr Ser Leu Ser Ile370 375 380Asn Lys Gly Glu Ser Ala Tyr Ile Asn Ala Asn Val Arg Tyr Tyr Asn385 390 395 400Thr Gly Thr Ala Pro Met Tyr Lys Val Thr Pro Thr Thr Asn Leu Val405 410 415Leu Asp Gly Asp Thr Leu Thr Thr Ile Lys Ala Gln Asp Asn Gln Ile420 425 430Gly Asn Asn Leu Ser Pro Asn Glu Thr Tyr Pro Lys Lys Gly Leu Ser435 440 445Pro Leu Ala Leu Asn Thr Met Asp Gln Phe Ser Ser Arg Leu Ile Pro450 455 460Ile Asn Tyr Asp Gln Leu Lys Lys Leu Asp Ala Gly Lys Gln Ile Lys465 470 475 480Leu Glu Thr Thr Gln Val Ser Gly Asn Tyr Gly Ile Lys Asn Ser Gln485 490 495Gly Gln Ile Ile Thr Glu Gly Asn Ser Trp Ser Asp Tyr Ile Ser Gln500 505 510Ile Asp Ser Leu Ser Ala Ser Ile Ile Leu Asp Thr Gly Ser Asp Val515 520 525Phe Glu Arg Arg Val Thr Ala Lys Asp Ser Ser Asn Pro Glu Asp Lys
      530 535 540Thr Pro Val Leu Thr Ile Gly Glu Ala Ile Glu Lys Ala Phe Gly Ala545 550 555 560Thr Lys Asn Gly Glu Ile Leu Tyr Phe Asn Gly Met Pro Ile Asp Glu565 570 575Ser Cys Val Glu Leu Ile Phe Asp Gly Asn Thr Ala Asn Leu Ile Lys580 585 590Glu Arg Leu Asn Ala Leu Asn Asp Lys Lys Ile Tyr Asn Val Gln Leu595 600 605Glu Arg Gly Met Lys Ile Leu Ile Lys Thr Ser Thr Tyr Phe Asn Asn610 615 620Phe Asp Gly Tyr Asn Asn Phe Pro Ser Ser Trp Ser Asn Val Asp Ser625 630 635 640Asn Asn Gln Asp Gly Leu Gln Asn Ala Ala Asn Lys Leu Ser Gly Glu645 650 655Thr Lys Ile Val Ile Pro Met Ser Lys Leu Asn Pro Tyr Lys Arg Tyr660 665 670Val Phe Ser Gly Tyr Leu Lys Asn Ser Ser Thr Ser Asn Pro Ile Thr675 680 685Val Asn Ile Lys Ala Lys Glu Gln Lys Thr Tyr Asn Leu Val Ser Glu690 695 700Asn Asp Tyr Lys Lys Phe Ser Tyr Glu Phe Glu Thr Ile Gly Arg Asp705 710 715 720Ala Ser Asn Ile Glu Ile Thr Leu Thr Ser Ser Gly Thr Ile Phe Leu725 730 735Asp Asn Leu Ser Ile Thr Glu Leu Asn Ser Thr Pro Glu Ile Leu Lys740 745 750Glu Pro Asp Ile Lys Val Pro Ser Asp Gln Glu Ile Ile Asp Ala His755 760 765Lys Lys Tyr Tyr Ala Asp Leu Ser Phe Asn Gln Ser Thr Ala Asn Tyr770 775 780Tyr Leu Asp Gly Leu Tyr Phe Glu Pro Thr Gln Thr Asn Lys Glu Val785 790 795 800Leu Asp Tyr Ile Gln Lys Tyr Lys Val Glu Ala Thr Leu Glu Tyr Ser805 810 815Gly Phe Lys Asp Ile Gly Thr Lys Asp Lys Glu Leu Arg Asn Tyr Thr820 825 830Gly Asp Ser Asn Gln Pro Lys Thr Asn Tyr Val Asn Phe Arg Ser Tyr
      835 840 845Phe Thr Ser Gly Glu Asn Val Met Pro Tyr Lys Lys Leu Arg Ile Tyr850 855 860Ala Ile Thr Pro Glu Asn Lys Glu Leu Leu Val Leu Ser Ile Asn865 870 875&lt;210&gt;34&lt;211&gt;721&lt;212&gt;PRT&lt;213&gt;肉毒梭菌&lt;400&gt;34Met Leu Val Ser Lys Phe Glu Asn Ser Val Lys Asn Ser Asn Lys Asn1 5 10 15Tyr Phe Thr Ile Asn Gly Leu Met Gly Tyr Tyr Phe Glu Asn Asp Phe20 25 30Phe Asn Leu Asn Ile Ile Ser Pro Thr Leu Asp Gly Asn Leu Thr Phe35 40 45Ser Lys Glu Asp Ile Asn Ser Ile Leu Gly Asn Lys Ile Ile Lys Ser50 55 60Ala Arg Trp Ile Gly Leu Ile Lys Pro Ser Ile Thr Gly Glu Tyr Ile65 70 75 80Leu Ser Thr Asn Ser Pro Asn Cys Arg Val Glu Leu Asn Gly Glu Ile85 90 95Phe Asn Leu Ser Leu Asn Thr Ser Asn Thr Val Asn Leu Ile Gln Gly100 105 110Asn Val Tyr Asp Ile Arg Ile Glu Gln Leu Met Ser Glu Asn Gln Leu115 120 125Leu Lys Asn Tyr Glu Gly Ile Lys Leu Tyr Trp Glu Thr Ser Asp Ile130 135 140Ile Lys Glu Ile Ile Pro Ser Glu Val Leu Leu Lys Pro Asn Tyr Ser145 150 155 160Asn Thr Asn Glu Lys Ser Lys Phe Ile Pro Asn Asn Thr Leu Phe Ser165 170 175Asn Ala Lys Leu Lys Ala Asn Ala Asn Arg Asp Thr Asp Arg Asp Gly180 185 190Ile Pro Asp Glu Trp Glu Ile Asn Gly Tyr Thr Val Met Asn Gln Lys
      195 200 205Ala Val Ala Trp Asp Asp Lys Phe Ala Ala Asn Gly Tyr Lys Lys Tyr210 215 220Val Ser Asn Pro Phe Lys Pro Cys Thr Ala Asn Asp Pro Tyr Thr Asp225 230 235 240Phe Glu Lys Val Ser Gly Gln Ile Asp Pro Ser Val Ser Met Val Ala245 250 255Arg Asp Pro Met Ile Ser Ala Tyr Pro Ile Val Gly Val Gln Met Glu260 265 270Arg Leu Val Val Ser Lys Ser Glu Thr Ile Thr Gly Asp Ser Thr Lys275 280 285Ser Met Ser Lys Ser Thr Ser His Ser Ser Thr Asn Ile Asn Thr Val290 295 300Gly Ala Glu Val Ser Gly Ser Leu Gln Leu Ala Gly Gly Ile Phe Pro305 310 315 320Val Phe Ser Met Ser Ala Ser Ala Asn Tyr Ser His Thr Trp Gln Asn325 330 335Thr Ser Thr Val Asp Asp Thr Thr Gly Glu Ser Phe Ser Gln Gly Leu340 345 350Ser Ile Asn Thr Gly Glu Ser Ala Tyr Ile Asn Pro Asn Ile Arg Tyr355 360 365Tyr Asn Thr Gly Thr Ala Pro Val Tyr Asn Val Thr Pro Thr Thr Thr370 375 380Ile Val Ile Asp Lys Gln Ser Val Ala Thr Ile Lys Gly Gln Glu Ser385 390 395 400Leu Ile Gly Asp Tyr Leu Asn Pro Gly Gly Thr Tyr Pro Ile Ile Gly405 410 415Glu Pro Pro Met Ala Leu Asn Thr Met Asp Gln Phe Ser Ser Arg Leu420 425 430Ile Pro Ile Asn Tyr Asn Gln Leu Lys Ser Ile Asp Asn Gly Gly Thr435 440 445Val Met Leu Ser Thr Ser Gln Phe Thr Gly Asn Phe Ala Lys Tyr Asn450 455 460Ser Asn Gly Asn Leu Val Thr Asp Gly Asn Asn Trp Gly Pro Tyr Leu465 470 475 480Gly Thr Ile Lys Ser Thr Thr Ala Ser Leu Thr Leu Ser Phe Ser Gly485 490 495Gln Thr Thr Gln Val Ala Val Val Ala Pro Asn Phe Ser Asp Pro Glu
      500 505 510Asp Lys Thr Pro Lys Leu Thr Leu Glu Gln Ala Leu Val Lys Ala Phe515 520 525Ala Leu Glu Lys Lys Asn Gly Lys Phe Tyr Phe His Gly Leu Glu Ile530 535 540Ser Lys Asn Glu Lys Ile Gln Val Phe Leu Asp Ser Asn Thr Asn Asn545 550 555 560Asp Phe Glu Asn Gln Leu Lys Asn Thr Ala Asp Lys Asp Ile Met His565 570 575Cys Ile Ile Lys Arg Asn Met Asn Ile Leu Val Lys Val Ile Thr Phe580 585 590Lys Glu Asn Ile Ser Ser Ils Asn Ile Ile Asn Asp Thr Asn Phe Gly595 600 605Val Gln Ser Met Thr Gly Leu Ser Asn Arg Ser Lys Gly Gln Asp Gly610 615 620Ile Tyr Arg Ala Ala Thr Thr Ala Phe Ser Phe Lys Ser Lys Glu Leu625 630 635 640Lys Tyr Pro Glu Gly Arg Tyr Arg Met Arg Phe Val Ile Gln Ser Tyr645650 655Glu Pro Phe Thr Cys Asn Phe Lys Leu Phe Asn Asn Leu Ile Tyr Ser660 665 670Ser Ser Phe Asp Lys Gly Tyr Tyr Asp Glu Phe Phe Tyr Phe Tyr Tyr675 680 685Asn Gly Ser Lys Ser Phe Phe Asn Ile Ser Cys Asp Ile Ile Asn Ser690 695 700Ile Asn Arg Leu Ser Gly Val Phe Leu Ile Glu Leu Asp Lys Leu Ile705 710 715 720Ile&lt;210&gt;35&lt;211&gt;1338&lt;212&gt;PRT&lt;213&gt;蠟狀芽孢桿菌&lt;400&gt;35Met Lys Arg Met Glu Gly Lys Leu Phe Met Val Ser Lys Lys Leu Gln1 5 10 15Val Val Thr Lys Thr Val Leu Leu Ser Thr Val Phe Ser Ile Ser Leu20 25 30Leu Asn Asn Glu Val Ile Lys Ala Glu Gln Leu Asn Ile Asn Ser Gln35 40 45Ser Lys Tyr Thr Asn Leu Gln Asn Leu Lys Ile Thr Asp Lys Val Glu50 55 60Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Glu Lys65 70 75 80Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Lys Met Asn Asn85 90 95Phe Leu Asp Asn Lys Asn Asp Ile Lys Thr Asn Tyr Lys Glu Ile Thr100 105 110Phe Ser Ile Ala Gly Ser Phe Glu Asp Glu Ile Lys Asp Leu Lys Glu115120 125Ile Asp Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Ser Ile Ile Thr130 135 140Tyr Lys Asn Val Glu Pro Thr Thr Ile Gly Phe Asn Lys Ser Leu Thr145 150 155 160Glu Gly Asn Thr Ile Asn Ser Asp Ala Met Ala Gln Phe Lys Glu Gln165 170 175Phe Leu Asp Arg Asp Ile Lys Phe Asp Ser Tyr Leu Asp Thr His Leu180 185 190Thr Ala Gln Gln Val Ser Ser Lys Glu Arg Val Ile Leu Lys Val Thr195 200 205Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val Ile210 215 220Leu Asn Asn Ser Glu Tyr Lys Met Leu Ile Asp Asn Gly Tyr Met Val225 230 235 240His Val Asp Lys Val Ser Lys Val Val Lys Lys Gly Val Glu Cys Leu245 250 255Gln Ile Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp Ile260 265 270Asn Ala Glu Ala His Ser Trp Gly Met Lys Asn Tyr Glu Glu Trp Ala275 280 285Lys Asp Leu Thr Asp Ser Gln Arg Glu Ala Leu Asp Gly Tyr Ala Arg290 295 300Gln Asp Tyr Lys Glu Ile Asn Asn Tyr Leu Arg Asn Gln Gly Gly Ser305 310 315 320Gly Asn Glu Lys Leu Asp Ala Gln Ile Lys Asn Ile Ser Asp Ala Leu325 330 335Gly Lys Lys Pro Ile Pro Glu Asn Ile Thr Val Tyr Arg Trp Cys Gly340 345 350Met Pro Glu Phe Gly Tyr Gln Ile Ser Asp Pro Leu Pro Ser Leu Lys355 360 365Asp Phe Glu Glu Gln Phe Leu Asn Thr Ile Lys Glu Asp Lys Gly Tyr370 375 380Met Ser Thr Ser Leu Ser Ser Glu Arg Leu Ala Ala Phe Gly Ser Arg385 390 395 400Lys Ile Ile Leu Arg Leu Gln Val Pro Lys Gly Ser Thr Gly Ala Tyr405 410 415Leu Ser Ala Ile Gly Gly Phe Ala Ser Glu Lys Glu Ile Leu Leu Asp420 425 430Lys Asp Ser Lys Tyr His Ile Asp Lys Val Thr Glu Val Ile Ile Lys435 440 445Gly Val Lys Arg Tyr Val Val Asp Ala Thr Leu Leu Thr Asn Ser Arg450 455 460Gly Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro465 470 475 480Ser Asp Ile Gly Ser Thr Met Lys Thr Asn Gln Ile Ser Thr Thr Gln485 490 495Lys Asn Gln Gln Lys Glu Met Asp Arg Lys Gly Leu Leu Gly Tyr Tyr500 505 510Phe Lys Gly Lys Asp Phe Ser Asn Leu Thr Met Phe Ala Pro Thr Arg515 520 525Asp Ser Thr Leu Ile Tyr Asp Gln Gln Thr Ala Asn Lys Leu Leu Asp530 535 540Lys Lys Gln Gln Glu Tyr Gln Ser Ile Arg Trp Ile Gly Leu Ile Gln545 550 555 560Ser Lys Glu Thr Gly Asp Phe Thr Phe Asn Leu Ser Glu Asp Glu Gln565 570 575Ala Ile Ile Glu Ile Asn Gly Lys Ile Ile Ser Asn Lys Gly Lys Glu580 585 590Lys Gln Val Val His Leu Glu Lys Gly Lys Leu Val Pro Ile Lys Ile595 600 605Glu Tyr Gln Ser Asp Thr Lys Phe Asn Ile Asp Ser Lys Thr Phe Lys
      610 615 620Glu Leu Lys Leu Phe Lys Ile Asp Ser Gln Asn Gln Pro Gln Gln Val625 630 635 640Gln Gln Asp Glu Leu Arg Asn Pro Glu Phe Asn Lys Lys Glu Ser Gln645 650 655Glu Phe Leu Ala Lys Pro Ser Lys Ile Asn Leu Phe Thr Gln Gln Met660 665 670Lys Arg Glu Ile Asp Glu Asp Thr Asp Thr Asp Gly Asp Ser Ile Pro675 680 685Asp Leu Trp Glu Glu Asn Gly Tyr Thr Ile Gln Asn Arg Ile Ala Val690 695 700Lys Trp Asp Asp Ser Leu Ala Ser Lys Gly Tyr Thr Lys Phe Val Ser705 710 715 720Asn Pro Leu Glu Ser His Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu725 730 735Lys Ala Ala Arg Asp Leu Asp Leu Ser Asn Ala Lys Glu Thr Phe Asn740 745 750Pro Leu Val Ala Ala Phe Pro Ser Val Asn Val Ser Met Glu Lys Val755 760 765Ile Leu Ser Pro Asn Glu Asn Leu Ser Asn Ser Val Glu Ser His Ser770 775 780Ser Thr Asn Trp Ser Tyr Thr Asn Thr Glu Gly Ala Ser Val Glu Ala785 790 795 800Gly Ile Gly Pro Lys Gly Ile Ser Phe Gly Val Ser Val Asn Tyr Gln805 810 815His Ser Glu Thr Val Ala Gln Glu Trp Gly Thr Ser Thr Gly Asn Thr820 825 830Ser Gln Phe Asn Thr Ala Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg835 840 845Tyr Asn Asn Val Gly Thr Gly Ala Ile Tyr Asp Val Lys Pro Thr Thr850 855 860Ser Phe Val Leu Asn Asn Asp Thr Ile Ala Thr Ile Thr Ala Lys Ser865 870 875 880Asn Ser Thr Ala Leu Asn Ile Ser Pro Gly Glu Ser Tyr Pro Lys Lys885 890 895Gly Gln Asn Gly Ile Ala Ile Thr Ser Met Asp Asp Phe Asn Ser His900 905 910Pro Ile Thr Leu Asn Lys Lys Gln Val Asp Asn Leu Leu Asn Asn Lys
      915 920 925Pro Met Met Leu Glu Thr Asn Gln Thr Asp Gly Val Tyr Lys Ile Lys930 935 940Asp Thr His Gly Asn Ile Val Thr Gly Gly Glu Trp Asn Gly Val Ile945 950 955 960Gln Gln Ile Lys Ala Lys Thr Ala Ser Ile Ile Val Asp Asp Gly Glu965 970 975Arg Val Ala Glu Lys Arg Val Ala Ala Lys Asp Tyr Glu Asn Pro Glu980 985 990Asp Lys Thr Pro Ser Leu Thr Leu Lys Asp Ala Leu Lys Leu Ser Tyr995 10001005Pro Asp Glu Ile Lys Glu Ile Glu Gly Leu Leu Tyr Tyr Lys Asn Lys101010151020Pro Ile Tyr Glu Ser Ser Val Met Thr Tyr Leu Asp Glu Asn Thr Ala1025103010351040Lys Glu Val Thr Lys Gln Leu Asn Asp Thr Thr Gly Lys Phe Lys Asp104510501055Val Ser His Leu Tyr Asp Val Lys Leu Thr Pro Lys Met Asn Val Thr106010651070Ile Lys Leu Ser Ile Leu Tyr Asp Asn Ala Glu Ser Asn Asp Asn Ser107510801085Ile Gly Lys Trp Thr Asn Thr Asn Ile Val Ser Gly Gly Asn Asn Gly109010951100Lys Lys Gln Tyr Ser Ser Asn Asn Pro Asp Ala Asn Leu Thr Leu Asn1105111011151120Thr Asp Ala Gln Glu Lys Leu Asn Lys Asn Arg Asp Tyr Tyr Ile Ser112511301135Leu Tyr Met Lys Ser Glu Lys Asn Thr Gln Cys Glu Ile Thr Ile Asp114011451150Gly Glu Ile Tyr Pro Ile Thr Thr Lys Thr Val Asn Val Asn Lys Asp115511601165Asn Tyr Lys Arg Leu Asp Ile Ile Ala His Asn Ile Lys Ser Asn Pro117011751180Ile Ser Ser Leu His Ile Lys Thr Asn Asp Glu Ile Thr Leu Phe Trp1185119011951200Asp Asp Ile Ser Ile Thr Asp Val Ala Ser Ile Lys Pro Glu Asn Leu120512101215Thr Asp Ser Glu Ile Lys Gln Ile Tyr Ser Arg Tyr Gly Ile Lys Leu
      122012251230Glu Asp Gly Ile Leu Ile Asp Lys Lys Gly Gly Ile His Tyr Gly Glu123512401245Phe Ile Asn Glu Ala Ser Phe Asn Ile Glu Pro Leu Gln Asn Tyr Val1250 1255 1260Thr Lys Tyr Glu Val Thr Tyr Ser Ser Glu Leu Gly Pro Asn Val Ser1265127012751280Asp Thr Leu Glu Ser Asp Lys Ile Tyr Lys Asp Gly Thr Ile Lys Phe128512901295Asp Phe Thr Lys Tyr Ser Lys Asn Glu Gln Gly Leu Phe Tyr Asp Ser130013051310Gly Leu Asn Trp Asp Phe Lys Ile Asn Ala Ile Thr Tyr Asp Gly Lys131513201325Glu Met Asn Val Phe His Arg Tyr Asn Lys1330133權(quán)利要求
      1.由兩組分組成的孔形成A-B毒素的B組分,其中所述的B組分包括抑制其孔形成能力的突變,其中所述的突變不是炭疽保護(hù)性抗原(SEQID NO.12)第302-325位氨基酸的缺失。
      2.如權(quán)利要求1所述的B組分,其中所述的B組分是炭疽保護(hù)性抗原。
      3 如權(quán)利要求1所述的B組分,其中所述的B組分缺乏孔-形成能力。
      4.如權(quán)利要求1所述的B組分,其中所述的B組分具有與SEQ ID No.21有至少80%同一性的氨基酸序列,并具有選自下述的替換a) K397A;b) K397D;c) K397C;d) K397Q ;e) D425A;f) D425N;g) D425E;h) D425K;i) F427A;j) K397+D425K雙突變;k) K395D+K397D+D425K+D426K四重突變;1) K397D+D425K+F427A三重突變;m) F427A+ΔD2L2雙突變;n) K397D+F427A+ΔD2L2三重突變;o) K397D+D425K+F427A+ΔD2L2四重突變;p) F427D;和q) F427K。
      5.一種疫苗組合物,其包含在藥學(xué)上可接受的載體上的由兩組分組成的孔形成A-B毒素的B組分或其片段,其中所述的B組分包含抑制其孔形成能力的突變。
      6.如權(quán)利要求5所述的疫苗組合物,其中所述的B組分是炭疽保護(hù)性抗原。
      7.如權(quán)利要求5所述的疫苗組合物,其中所述的B組分是經(jīng)化學(xué)或物理方式滅活的。
      8.一種預(yù)防哺乳動(dòng)物中細(xì)菌感染的方法,所述的方法包括對(duì)所述哺乳動(dòng)物施用含有包含在藥學(xué)上可接受的載體上的,由兩組分組成的孔形成A-B毒素的B組分或其片段的疫苗,其中,所述的B組分包含抑制其孔形成能力的突變。
      9.一種治療哺乳動(dòng)物中細(xì)菌感染的方法,所述的方法包括對(duì)所述哺乳動(dòng)物施用含有包含在藥學(xué)上可接受的載體上的,由兩組分組成的孔形成A-B毒素的B組分或其片段的疫苗,其中,所述的B組分包含抑制其孔形成能力的突變。
      10.如權(quán)利要求8或9所述的方法,其中所述的疫苗與佐劑一起施用。
      11.一種由兩組分組成的孔形成A-B毒素的突變體B組分,其中,所述的突變體B組分包含抑制其孔形成能力的突變體,其中所述的突變體B組分抑制所述毒素天然存在的B組分的孔形成能力。
      12.如權(quán)利要求11所述的突變體B組分,其中所述的突變體B組分是炭疽保護(hù)性抗原。
      13.如權(quán)利要求12所述的突變體B組分,其具有與致命因子或水腫因子結(jié)合的能力。
      14.如權(quán)利要求11所述的突變體B組分,其具有與所述天然存在的B組分競(jìng)爭(zhēng)結(jié)合哺乳動(dòng)物細(xì)胞表面受體的能力。
      15.如權(quán)利要求11所述的突變體B組分,其具有與所述天然存在的B組分相結(jié)合的能力。
      16.如權(quán)利要求11所述的突變體B組分,其具有與天然存在的B組分寡聚化以形成具有減弱的孔形成能力的復(fù)合物的能力。
      17.如權(quán)利要求16所述的突變體B組分,其中,所述的復(fù)合物缺少孔形成能力。
      18.如權(quán)利要求11所述的突變體B組分,其中所述的B組分具有與SEQ ID No.21有至少80%同一性的氨基酸序列,并具有選自下述的突變a) K397D+D425K雙突變;b) ΔD2L2;c) K395D+K397D+D425K+D426K四重突變;d) D425K;e) F427A;f) K397D+D425K+F427A三重突變;g) F427A+ΔD2L2雙突變;h) K397D+F427A+ΔD2L2三重突變;i) K397D+D425K+F427A+ΔD2L2四重突變;h) F427D;和i) F427K。
      19.如權(quán)利要求11所述的突變體B組分,其包含D2L2環(huán)的至少5個(gè)氨基酸的缺失。
      20.一種在哺乳動(dòng)物中預(yù)防細(xì)菌感染的方法,所述的方法包括對(duì)所述哺乳動(dòng)物施用由兩組分組成的孔形成A-B毒素的突變體B組分或其片段,其中所述的突變體B組分包含抑制其孔-形成能力的突變,其中所述的突變體B組分抑制所述毒素天然存在的B組分的孔形成能力。
      21.一種在哺乳動(dòng)物中治療細(xì)菌感染的方法,所述的方法包括對(duì)所述哺乳動(dòng)物施用由兩組分組成的孔形成A-B毒素的突變體B組分或其片段,其中所述的突變體B組分包含抑制其孔-形成能力的突變,其中所述的突變體B組分抑制所述毒素天然存在的B組分的孔形成能力。
      22.如權(quán)利要求8,9,20,或21所述的方法,其中所述的B組分或突變體B組分是炭疽保護(hù)性抗原,所述的細(xì)菌感染是炭疽感染。
      23.如權(quán)利要求8,9,20,或21所述的方法,其進(jìn)一步包括對(duì)所述哺乳動(dòng)物施用與天然存在的B組分結(jié)合的抗體。
      24.如權(quán)利要求8,9,20,或21所述的方法,其中所述的哺乳動(dòng)物是人。
      25.如權(quán)利要求24所述的方法,其中所述的哺乳動(dòng)物已暴露于炭疽桿菌(B.anthracis)孢子。
      26.一種純化的抗體,其以比與所述毒素的突變體B組分結(jié)合更高的親和力,與由兩組分組成的孔形成A-B毒素的B組分特異性地結(jié)合,其中所述的突變體B組分包含抑制其孔形成能力的突變。
      27.如權(quán)利要求26所述的抗體,其中,所述的突變體B組分抑制所述毒素天然存在的B組分的孔形成能力。
      全文摘要
      本發(fā)明提供孔-形成毒素的突變體形式。這些突變體毒素可用作預(yù)防細(xì)菌感染的疫苗。另外顯性負(fù)突變體可作為治療劑施用,以治療細(xì)菌感染。
      文檔編號(hào)A61P31/04GK1440419SQ01812400
      公開日2003年9月3日 申請(qǐng)日期2001年5月4日 優(yōu)先權(quán)日2000年5月4日
      發(fā)明者R·J·科利爾, B·R·塞爾曼 申請(qǐng)人:哈佛大學(xué)校長(zhǎng)及研究員協(xié)會(huì)
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