本發(fā)明涉及基因工程和酶工程領(lǐng)域，具體涉及一種新型的葡聚糖蔗糖酶及其催化制備水不溶性葡聚糖的方法。
背景技術(shù)：
：葡聚糖（Glucan）是一種以葡萄糖為單體的聚合物，在醫(yī)藥、精細(xì)化工、石油開采、食品、化妝品等領(lǐng)域有著廣泛的應(yīng)用，其中根據(jù)糖苷鍵的類型可分為α,葡聚糖和β-葡聚糖。α-葡聚糖中研究使用較多的為右旋糖酐（dextran），右旋糖酐具有較高的分子量，主要由D-葡糖吡喃糖以α,1-6鍵連接，支鏈點(diǎn)有α,1-2、1-3、1-4糖苷鍵連接形成的。隨著微生物種類和生長條件的不同，其結(jié)構(gòu)也有差別。由于結(jié)構(gòu)的不同，葡聚糖可分為水不溶性葡聚糖和水溶性葡聚糖。水不溶性葡聚糖在代血漿、藥物載體、凝膠分離、色譜分離柱等領(lǐng)域都有重要的應(yīng)用。葡聚糖蔗糖酶（Glucansucrase，亦稱蔗糖-6-葡萄糖基轉(zhuǎn)移酶）（EC.2.4.15）是一種糖苷轉(zhuǎn)移酶，屬于糖苷水解酶第70家族。腸膜明串珠菌分泌的葡聚糖蔗糖酶屬于誘導(dǎo)型酶，誘導(dǎo)物和底物都為蔗糖，且在一定范圍內(nèi)，酶的產(chǎn)量與蔗糖濃度成正比。葡聚糖的生產(chǎn)方法有兩種：微生物直接發(fā)酵法和酶合成法。直接發(fā)酵生產(chǎn)葡聚糖時(shí)，發(fā)酵液成分復(fù)雜，導(dǎo)致產(chǎn)物分離困難、葡聚糖分子大小難以控制、細(xì)胞核蛋白類雜質(zhì)多等缺點(diǎn)，而利用純酶催化制備葡聚糖，可以克服這些不足，生產(chǎn)出高產(chǎn)品質(zhì)量的葡聚糖。技術(shù)實(shí)現(xiàn)要素：本發(fā)明的第一目的是提供一種新型的葡聚糖蔗糖酶。為實(shí)現(xiàn)上述目的，本發(fā)明采用如下技術(shù)方案：一種葡聚糖蔗糖酶DsrU，其氨基酸序列如SEQIDNO.2所示。本發(fā)明還提供了編碼該葡聚糖蔗糖酶DsrU的基因，其核苷酸序列如SEQIDNO.1所示。本發(fā)明所述葡聚糖蔗糖酶DsrU獲取自腸膜明串珠菌LeuconostocmesenteroidesM8，所述腸膜明串珠菌LeuconostocmesenteroidesM8為本實(shí)驗(yàn)室從泡菜發(fā)酵液中，通過自然篩選、鑒定得到的菌株。本發(fā)明對(duì)腸膜明串珠菌LeuconostocmesenteroidesM8進(jìn)行了全基因組測序，通過對(duì)其基因組序列分析和已報(bào)道的葡聚糖蔗糖酶的基因序列比對(duì)，得到一種未見報(bào)道的新型葡聚糖蔗糖酶基因。本發(fā)明的另一目的是提供含有上述DsrU編碼基因的表達(dá)載體及基因工程菌。所述表達(dá)載體的出發(fā)載體選用pET-28a，構(gòu)建含DsrU基因的表達(dá)質(zhì)粒pET28a-DsrU作為表達(dá)載體。選用宿主菌E.coliBL21(DE3)構(gòu)建含有DsrU基因的基因工程菌E.coliBL21(DE3)-pET28a-DsrU，高效表達(dá)該葡聚糖蔗糖酶基因。所述的基因工程菌E.coliBL21(DE3)-pET28a-DsrU的構(gòu)建方法為：將葡聚糖蔗糖酶DsrU基因?qū)氲捷d體pET28a中進(jìn)行外源表達(dá)。構(gòu)建得到pET28a-DsrU表達(dá)質(zhì)粒，然后將表達(dá)質(zhì)粒pET28a-DsrU轉(zhuǎn)化到大腸桿菌E.coliBL21(DE3)中，，通過選擇性培養(yǎng)基挑選得到重組菌E.coliBL21(DE3)-pET28a-DsrU。本發(fā)明的又一目的是提供上述葡聚糖蔗糖酶DsrU在制備水不溶性葡聚糖中的應(yīng)用。本發(fā)明提供了一種葡聚糖蔗糖酶DsrU制備水不溶性葡聚糖的具體方法，其技術(shù)路線包括如下步驟：（1）將葡聚糖蔗糖酶DsrU基因?qū)氲捷d體pET28a中進(jìn)行外源表達(dá)。構(gòu)建得到pET28a-DsrU表達(dá)質(zhì)粒，將該質(zhì)粒轉(zhuǎn)入大腸桿菌感受態(tài)細(xì)胞中，通過選擇性培養(yǎng)基挑選得到正確的基因工程菌。（2）將重組菌E.colipET28a-DsrU接種至種子培養(yǎng)基，進(jìn)行種子培養(yǎng)。（3）將培養(yǎng)好的種子培養(yǎng)液接種至發(fā)酵培養(yǎng)基中，30℃培養(yǎng)0~4h，加0.1~1.0mMIPTG，繼續(xù)在25~30℃下培養(yǎng)24h后停止發(fā)酵，離心菌液后棄上清，用磷酸緩沖液重懸沉淀下來的細(xì)胞，超聲破碎細(xì)胞，獲得葡聚糖蔗糖酶粗酶液。（4）向葡聚糖蔗糖酶的粗酶液中加入不同濃度的蔗糖，并調(diào)節(jié)pH至5~7，反應(yīng)一段時(shí)間后6~12h后，向反應(yīng)液中加入冷乙醇，離心得葡聚糖沉淀，40-60℃下干燥得葡聚糖。（5）將得到的葡聚糖加水復(fù)溶，溶解12h后，離心，取上清液，加入冷乙醇，離心，無沉淀，可判斷上清液中無葡聚糖，及所述的葡聚糖為水不溶性葡聚糖。所述步驟（1）中含葡聚糖蔗糖酶基因?qū)氲妮d體為pET28a，胞內(nèi)表達(dá)葡聚糖蔗糖酶DsrU有利于酶的回收和純化。所述步驟（1）中基因工程菌的構(gòu)建方法：將葡聚糖蔗糖酶基因DsrU連接到質(zhì)粒pET28a上，得到包含葡聚糖蔗糖酶基因的重組質(zhì)粒pET28a-DsrU，然后將重組質(zhì)粒pET28a-DsrU轉(zhuǎn)化到大腸桿菌E.coliBL21(DE3)中，得到重組菌E.coliBL21(DE3)-pET28a-DsrU。所述步驟（3）IPTG濃度為0.1mM~1.0mM。所述步驟（4）中pH的初始值為7.0。所述步驟（4）中需控制pH不低于5.0。所述步驟（4）中加入冷乙醇的量是上清液的2-3倍，離心條件9,000rpm，10min。所述步驟（5）中加入冷乙醇的量是上清液的2-3倍，離心條件9,000rpm，10min。本發(fā)明的積極進(jìn)步效果在于：本發(fā)明提供了一種來自于腸膜明串珠菌LeuconostocmesenteroidesM8的葡聚糖蔗糖酶DsrU，并提供了利用葡聚糖蔗糖酶DsrU合成水不溶性葡聚糖的方法。該酶在特定的催化條件下可高效催化制備水不溶性葡聚糖，同時(shí)葡聚糖得率高達(dá)90%以上。本發(fā)明提供的新型葡聚糖蔗糖酶應(yīng)用前景廣闊，和以其制備的水不溶性葡聚糖的可被引用于藥物載體、凝膠分離系統(tǒng)、分離色譜柱制備等領(lǐng)域。附圖說明圖1是E.coliBL21(DE3)-pET28a-DsrU重組菌株鑒定圖；其中，Line1為DNAmarker，Line2為重組菌菌落PCR產(chǎn)物。具體實(shí)施方式下面通過實(shí)施例的方式進(jìn)一步說明本發(fā)明，但并不因此將本發(fā)明限制在所述的實(shí)施例范圍之中。實(shí)施例1產(chǎn)葡聚糖蔗糖酶的菌株篩選與鑒定步驟1：取泡菜發(fā)酵液1mL于9mL的滅菌生理鹽水中，并梯度稀釋為10-4、10-5、10-6、10-7、10-8，取0.1mL的各梯度稀釋液接入選擇性平板中，于28℃培養(yǎng)2~3d，菌落長出后，挑取典型菌落，在含有萬古霉素（30μl/mL）的MRS平板上劃線，置于28℃培養(yǎng)箱中厭氧培養(yǎng)2~3d，觀察菌落生長狀況，若菌落生長，則進(jìn)一步選取單菌落于含有萬古霉素（30μl/mL）的MRS固體培養(yǎng)基上劃線培養(yǎng)，重復(fù)上述步驟2~3次后，通過鏡檢確認(rèn)為純培養(yǎng)物、并具有典型明串珠菌細(xì)胞形態(tài)的菌株轉(zhuǎn)入MRS固體培養(yǎng)基，低溫保存?zhèn)溆?。步驟2：將步驟1中分離得到各菌株分別劃線接種于葡聚糖生成固體培養(yǎng)基表面，置于28℃培養(yǎng)1~3d，隨時(shí)觀察葡聚糖生成情況，選取能夠在培養(yǎng)基表面形成粘稠狀多糖的菌株。最終挑取一株在能夠在葡聚糖生成培養(yǎng)基上產(chǎn)生較多粘稠狀的菌株，接入MRS液體培養(yǎng)基，30℃培養(yǎng)8-10h后，轉(zhuǎn)入含有15%的甘油的MRS中，低溫保存，備用，命名為腸膜明串珠菌LeuconostocmesenteroidesM8。實(shí)施例2產(chǎn)葡聚糖蔗糖酶重組大腸桿菌的構(gòu)建步驟1：將腸膜明串株菌LeuconostocmesenteroidesM8進(jìn)行試管培養(yǎng)活化，試管裝液量為5mL，接種量為1%，培養(yǎng)溫度為30℃，培養(yǎng)時(shí)間為10-12h，得到菌株生長旺盛的菌液，10000rpm離心獲取菌泥，利用革蘭氏陽性菌的提基因組試劑盒提取腸膜明串株菌LeuconostocmesenteroidesM8的基因組作為PCR模板。步驟2：產(chǎn)葡聚糖蔗糖酶DsrU基因的獲取及線性化克隆載體的制備上游引物：5’-CGCGGATCCGAATTCATGTTAGTAACAGCTGGTATTTTTTC-3’下游引物：5’-ACGGAGCTCGAATTCTTATATAGTATTTAAACGCTGTCTCTCTC-3’以LeuconostocmesenteroidesM8的基因組DNA為模板，以上述引入PCR擴(kuò)增DsrU基因片段，反應(yīng)條件為：94℃，5min；（94℃45s，50℃45s，72℃300s，35個(gè)循環(huán)），72℃，10min，擴(kuò)增出DsrU基因，將PCR反應(yīng)液進(jìn)行純化回收，備用。將pET28a質(zhì)粒用EcoRⅠ酶進(jìn)行單酶切，酶切產(chǎn)物進(jìn)行膠回收，作為線性化克隆載體，備用。步驟3：制備E.coliBL21(DE3)感受態(tài)細(xì)胞將取5μL的E.coliBL21菌體加入5mL的LB試管中，37℃，200rpm，培養(yǎng)12h，取1mL培養(yǎng)液加入裝有100mLLB培養(yǎng)基的搖瓶中，37℃，200rpm，待菌體OD600為0.5-0.6時(shí)，取出搖瓶，冰上放置10min，4℃，4100rpm離心10min，棄上清，用2mL含有15%甘油的0.05MCaCl2溶液混懸沉淀，分裝后于-80℃保存，備用。步驟4：利用一步克隆技術(shù)構(gòu)建目標(biāo)菌株將步驟2中回收的DsrU基因和線性化pET28a質(zhì)粒載體，通過一部克隆轉(zhuǎn)入E.coliBL21(DE3)中，具體方法如下：將裝有200μL制作好的E.coliBL21(DE3)感受態(tài)細(xì)胞冰盒上解凍5-10min；加入20μL冷卻的一步克隆反應(yīng)液：手指輕彈，混勻，冰上放置30min；42℃，水浴90s，冰浴2min；加入900μL新鮮LB培養(yǎng)基，復(fù)蘇細(xì)胞，37℃，200rpm，培養(yǎng)1h。取100μL涂布在抗性平板上，37℃培養(yǎng)箱中培養(yǎng)10-12h，挑取菌落，使用步驟2中的引物進(jìn)行PCR驗(yàn)證，能擴(kuò)增出目的基因片段的菌落即為pET28a-DsrU質(zhì)粒成功表達(dá)的菌株。如圖1所示，DsrUPCR產(chǎn)物大小約為4500bp與預(yù)期一致，說明構(gòu)建菌株正確。實(shí)施例3構(gòu)建菌株發(fā)酵制備葡聚糖蔗糖酶DsrU種子培養(yǎng)基（g/L）：酵母粉5，蛋白胨10，NaCl10，pH7.0，121℃滅菌15min。發(fā)酵培養(yǎng)基（g/L）：酵母粉24，蛋白胨12，KH2PO42.31，K2HPO416.43，葡萄糖10，121℃滅菌15min。生產(chǎn)方法：取平板上單菌落接種于裝液量為5mL的試管中，30℃、200rpm培養(yǎng)12h。制備一級(jí)種子液，將一級(jí)種子液接種于裝有100mL發(fā)酵培養(yǎng)液的500mL三角瓶中，200rpm，30℃培養(yǎng)0~4h后加入0.1~1.0mMIPTG誘導(dǎo)DsrU目的蛋白的表達(dá)，繼續(xù)在25~30℃下培養(yǎng)至24h，停止發(fā)酵后，將培養(yǎng)液于10000r/min離心10min，棄上清，用pH為6.2的磷酸緩沖液重懸沉淀，超聲破碎細(xì)胞獲得粗酶液。如表1所示，不同IPTG誘導(dǎo)條件下，DsrU最高酶活可達(dá)4.9U。表1不同IPTG濃度誘導(dǎo)重組菌株對(duì)DsrU酶活的影響酶活測定方法：葡聚糖蔗糖酶的活力表示為在pH5.4，30℃的條件下，1min中內(nèi)反應(yīng)生成1μmol果糖所需的酶量為一個(gè)酶活單位U。酶活反應(yīng)體系：30℃，20mM乙酸鈉緩沖液（pH5.4），0.05g/LCaCl2，1g/LNaNO3，100g/L蔗糖。具體方法為：（1）取7支比色管編號(hào)0-7，分別加入濃度為1mg/mL的果糖標(biāo)準(zhǔn)液0mL，0.2mL，0.4mL，0.6mL，0.8mL，1.0mL，1.2mL，補(bǔ)足蒸餾水至2.0mL，加入1.5mLDNS試劑，配成不同果糖濃度的反應(yīng)液，將比色管搖勻后在沸水浴中加熱5min，取出，冷卻至室溫，蒸餾水定容至20mL，顛倒混勻，用0號(hào)管調(diào)零，540nm下測定吸光值，以吸光值為橫坐標(biāo)，果糖含量（μmol）為縱坐標(biāo)制定標(biāo)準(zhǔn)曲線。（2）取500μL待測酶液與2.5mL反應(yīng)體系混合，混勻后放入30℃水浴中反應(yīng)20min，采用DNS測定反應(yīng)體系中0min和20min的還原糖（即為葡聚糖蔗糖酶催化反應(yīng)生成的果糖）含量。（3）取葡聚糖蔗糖酶反應(yīng)體系的樣品500μL，加蒸餾水至2.0mL，加入1.5mLDNS，沸水浴5min，取出冷卻至室溫，蒸餾水定容至20mL，顛倒混勻，測定540nm下測定吸光值，對(duì)照標(biāo)曲計(jì)算得到相應(yīng)還原糖的含量（μmol）。（4）葡聚糖蔗糖酶活力（U,μmol/min）=(20min反應(yīng)體系中還原糖總量-0min反應(yīng)體系中還原糖總量)/(20min*反應(yīng)體系中加入的酶液體積)。實(shí)施例4葡聚糖蔗糖酶催化蔗糖生產(chǎn)不溶性葡聚糖步驟一：向葡聚糖蔗糖酶的粗酶液中加入不同濃度的蔗糖，使反應(yīng)體系中蔗糖終濃度分別為50g/L，100g/L，150g/L，200g/L，30℃，反應(yīng)時(shí)間為6~12h，優(yōu)選8h；反應(yīng)體系pH為5.0~7.0，優(yōu)選6.0；反應(yīng)溫度為30℃~40℃，優(yōu)選30℃；酶添加量為1~10U，優(yōu)選10U，利用葡聚糖蔗糖酶催化蔗糖制備葡聚糖的產(chǎn)量如表2所示。表2利用葡聚糖蔗糖酶催化蔗糖在優(yōu)選條件下制備葡聚糖蔗糖濃度葡聚糖產(chǎn)量葡聚糖得率*50g/L23.6g/L94%100g/L48.2g/L96%150g/L72.7g/L97%200g/L93.5g/L95%*得率按照生產(chǎn)每g葡聚糖消耗的蔗糖中的葡萄糖量計(jì)算得出。該新型酶具有高葡聚糖催化得率的特性，在50~200g/L蔗糖條件下，葡聚糖得率均大于90%。步驟二：向步驟一的反應(yīng)液中加入冷乙醇，加入冷乙醇的體積為反應(yīng)液的2-3倍，冰上靜置30~60min，9000rpm離心10~20min得沉淀物，棄上清，50-55℃下干燥至恒重（W1）得葡聚糖。步驟三：將步驟二中干燥所得的葡聚糖，加水于40℃溶解12h，將溶解液9000rpm離心10min得沉淀物，50-55℃下干燥至恒重（W2），并收集上清，加入2-3倍體積的冷乙醇，冰上靜置30~60min，9000rpm離心10~20min，經(jīng)比較，W2和步驟三中的W1值基本一致，且離心后并無沉淀產(chǎn)生，由此可得，步驟二中所產(chǎn)生的葡聚糖為水不溶性葡聚糖。序列表<110>南京工業(yè)大學(xué)<120>一種葡聚糖蔗糖酶及其應(yīng)用<130>xb16110401<160>2<170>PatentInversion3.5<210>1<211>4518<212>DNA<213>ArtificialSequence<220><223>1<220><221>CDS<222>(1)..(4518)<400>1atgttagtaacagctggtattttttctgctgtgatatttggcgtttcc48MetLeuValThrAlaGlyIlePheSerAlaValIlePheGlyValSer151015atagctaacgtaagcgctgatagtattaacaatactagtattgcggtg96IleAlaAsnValSerAlaAspSerIleAsnAsnThrSerIleAlaVal202530gcgcaatcaaaaaatatagcagtggccacaacgacagctacaatggac144AlaGlnSerLysAsnIleAlaValAlaThrThrThrAlaThrMetAsp354045aaagtaactgatacgacagctacaacggacaaagtaactgatacgaca192LysValThrAspThrThrAlaThrThrAspLysValThrAspThrThr505560gctacgacagataaagtaactgatacgacagctacaacggacaaagta240AlaThrThrAspLysValThrAspThrThrAlaThrThrAspLysVal65707580actgatacgacagctacgacagataaagtaactgacacggcagctacg288ThrAspThrThrAlaThrThrAspLysValThrAspThrAlaAlaThr859095acggacaaagtggctgacacgacagctacaacggacaaagtaactgat336ThrAspLysValAlaAspThrThrAlaThrThrAspLysValThrAsp100105110acgacagctacaacgggcaaagtaactgacacgacagctacgacggac384ThrThrAlaThrThrGlyLysValThrAspThrThrAlaThrThrAsp115120125aaagtggctgacacgacagctacgacagataaagtggctgacacgaca432LysValAlaAspThrThrAlaThrThrAspLysValAlaAspThrThr130135140gctacaacggacaaagtaactgacacggcagctacaacggataaagca480AlaThrThrAspLysValThrAspThrAlaAlaThrThrAspLysAla145150155160actgacacggcagctacaacggacaaagtagctgatacaacagctacg528ThrAspThrAlaAlaThrThrAspLysValAlaAspThrThrAlaThr165170175acagataaagcagcgaacacaacagttacaccttcagaaaaatcaaaa576ThrAspLysAlaAlaAsnThrThrValThrProSerGluLysSerLys180185190agtattaagcaaatcgatggtaaaacatatttcattggtgatgatggt624SerIleLysGlnIleAspGlyLysThrTyrPheIleGlyAspAspGly195200205cagcccaagaaaaattttacagccattgtggacggtcaagtattatat672GlnProLysLysAsnPheThrAlaIleValAspGlyGlnValLeuTyr210215220ttcgacaaagataccggtactttgacatcaaatagtagtcaatatacc720PheAspLysAspThrGlyThrLeuThrSerAsnSerSerGlnTyrThr225230235240gatggtttggtcaatataggaaatgagcataatgcggcttattcattg768AspGlyLeuValAsnIleGlyAsnGluHisAsnAlaAlaTyrSerLeu245250255tcttcggacagttttacacaagttgatggctatctaacggctaacagt816SerSerAspSerPheThrGlnValAspGlyTyrLeuThrAlaAsnSer260265270tggtatcgacctaaagatatattgaaaaatggtacaacatggacagct864TrpTyrArgProLysAspIleLeuLysAsnGlyThrThrTrpThrAla275280285tcaacagcaaatgattttcgaccattgttgatgtcttggtggccggat912SerThrAlaAsnAspPheArgProLeuLeuMetSerTrpTrpProAsp290295300aaagatacccaagtttcatacttaaaatatatgcaatctgttggatta960LysAspThrGlnValSerTyrLeuLysTyrMetGlnSerValGlyLeu305310315320ttatcagatgacgttgtactatcaaacaaggatagtatgaatagtttg1008LeuSerAspAspValValLeuSerAsnLysAspSerMetAsnSerLeu325330335acagctatggcactgactgttcaaaaaaatattgaagagaaaataggc1056ThrAlaMetAlaLeuThrValGlnLysAsnIleGluGluLysIleGly340345350ctattaggtaccactgactggcttaagactgatatggaccaaatggtt1104LeuLeuGlyThrThrAspTrpLeuLysThrAspMetAspGlnMetVal355360365gactcacaatcaaattggaacattagtagtgagtctaaaggaacagat1152AspSerGlnSerAsnTrpAsnIleSerSerGluSerLysGlyThrAsp370375380catttgcaaggtggtgcgctcctatatgtgaatagtgatttgacacca1200HisLeuGlnGlyGlyAlaLeuLeuTyrValAsnSerAspLeuThrPro385390395400aatgctaattctgattatcgtttattaaatcgaacgccaacgaaccaa1248AsnAlaAsnSerAspTyrArgLeuLeuAsnArgThrProThrAsnGln405410415aaaggtcaaattacaacag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ggtgtagagttacaagatgcttatttgtcggatggt3474LeuProAsnGlyValGluLeuGlnAspAlaTyrLeuSerAspGly114511501155actaatgtatattattacagtagtacaggtcgtcaaattactaat3519ThrAsnValTyrTyrTyrSerSerThrGlyArgGlnIleThrAsn116011651170caatattatcgagattcagatagcaaatggcattacttcttctca3564GlnTyrTyrArgAspSerAspSerLysTrpHisTyrPhePheSer117511801185gatgggcatatggccgttggtttaacaacaattactgcagataac3609AspGlyHisMetAlaValGlyLeuThrThrIleThrAlaAspAsn119011951200ggcacggctaatcaacaatattttgacgttaatggtatacaactt3654GlyThrAlaAsnGlnGlnTyrPheAspValAsnGlyIleGlnLeu120512101215aagggtgttgctgttaaagacactgatggaaatgttcgttatttt3699LysGlyValAlaValLysAspThrAspGlyAsnValArgTyrPhe122012251230gatggtaacacaggaaacttggttgtcagtaactggggtaaaacg3744AspGlyAsnThrGlyAsnLeuValValSerAsnTrpGlyLysThr123512401245gctgatggttcatggttatacctaaatgataaagggatagcagta3789AlaAspGlySerTrpLeuTyrLeuAsnAspLysGlyIleAlaVal125012551260acgggacagcaaaatattaatggccaaaatgtctattttaacgaa3834ThrGlyGlnGlnAsnIleAsnGlyGlnAsnValTyrPheAsnGlu126512701275gatggtgttcaagtgaagggcgaagccattacggatactaatggc3879AspGlyValGlnValLysGlyGluAlaIleThrAspThrAsnGly128012851290aatgtacattactatgatcgtagtactggaaatatggtgactaat3924AsnValHisTyrTyrAspArgSerThrGlyAsnMetValThrAsn129513001305tcatgggcagagttaccggacagctcgtggatgtatctagatgtt3969SerTrpAlaGluLeuProAspSerSerTrpMetTyrLeuAspVal131013151320aatggcgttgctgctattggtgctcaaaaaattaatggtctggaa4014AsnGlyValAlaAlaIleGlyAlaGlnLysIleAsnGlyLeuGlu132513301335ttttatttcgataataacggtaaacaagttaagaacgacaaagtc4059PheTyrPheAspAsnAsnGlyLysGlnValLysAsnAspLysVal134013451350attaatgatgatggaacaataaactattacacaggtatgagcggt4104IleAsnAspAspGlyThrIleAsnTyrTyrThrGlyMetSerGly135513601365gaaaaactaaaaaatgattttggtgaattgccagacggatcatgg4149GluLysLeuLysAsnAspPheGlyGluLeuProAspGlySerTrp137013751380atgtacttggataatcaaggtaatgctgtaataggcgacaaaaaa4194MetTyrLeuAspAsnGlnGlyAsnAlaValIleGlyAspLysLys138513901395attaatggtcagaatctatacttcaagatagacggacaacaggtt4239IleAsnGlyGlnAsnLeuTyrPheLysIleAspGlyGlnGlnVal140014051410aagggtgaaacctatatagatggagttggcaagatgcgtttctat4284LysGlyGluThrTyrIleAspGlyValGlyLysMetArgPheTyr141514201425caagctgccagcggtgaaatggtgacgaatcaattcgaacaagtt4329GlnAlaAlaSerGlyGluMetValThrAsnGlnPheGluGlnVal143014351440gctgatggcaaatgggcttactttggtgctgatggtgtggctgtc4374AlaAspGlyLysTrpAlaTyrPheGlyAlaAspGlyValAlaVal144514501455actggagagcaatatattgatggtcaggatcttttctttgaccca4419ThrGlyGluGlnTyrIleAspGlyGlnAspLeuPhePheAspPro146014651470actggttatcaagtgaagggtgacaaacgcacaattgacagcgtt4464ThrGlyTyrGlnValLysGlyAspLysArgThrIleAspSerVal147514801485ctctatagctttgataaagacagtggagagagacagcgtttaaat4509LeuTyrSerPheAspLysAspSerGlyGluArgGlnArgLeuAsn149014951500actatataa4518ThrIle1505<210>2<211>1505<212>PRT<213>ArtificialSequence<220><223>SyntheticConstruct<400>2MetLeuValThrAlaGlyIlePheSerAlaValIlePheGlyValSer151015IleAlaAsnValSerAlaAspSerIleAsnAsnThrSerIleAlaVal202530AlaGlnSerLysAsnIleAlaValAlaThrThrThrAlaThrMetAsp354045LysValThrAspThrThrAlaThrThrAspLysValThrAspThrThr505560AlaThrThrAspLysValThrAspThrThrAlaThrThrAspLysVal65707580ThrAspThrThrAlaThrThrAspLysValThrAspThrAlaAlaThr859095ThrAspLysValAlaAspThrThrAlaThrThrAspLysValThrAsp100105110ThrThrAlaThrThrGlyLysValThrAspThrThrAlaThrThrAsp115120125LysValAlaAspThrThrAlaThrThrAspLysValAlaAspThrThr130135140AlaThrThrAspLysValThrAspThrAlaAlaThrThrAspLysAla145150155160ThrAspThrAlaAlaThrThrAspLysValAlaAspThrThrAlaThr165170175ThrAspLysAlaAlaAsnThrThrValThrProSerGluLysSerLys180185190SerIleLysGlnIleAspGlyLysThrTyrPheIleGlyAspAspGly195200205GlnProLysLysAsnPheThrAlaIleValAspGlyGlnValLeuTyr210215220PheAspLysAspThrGlyThrLeuThrSerAsnSerSerGlnTyrThr225230235240AspGlyLeuValAsnIleGlyAsnGluHisAsnAlaAlaTyrSerLeu245250255SerSerAspSerPheThrGlnValAspGlyTyrLeuThrAlaAsnSer260265270TrpTyrArgProLysAspIleLeuLysAsnGlyThrThrTrpThrAla275280285SerThrAlaAsnAspPheArgProLeuLeuMetSerTrpTrpProAsp290295300LysAspThrGlnValSerTyrLeuLysTyrMetGlnSerValGlyLeu305310315320LeuSerAspAspValValLeuSerAsnLysAspSerMetAsnSerLeu325330335ThrAlaMetAlaLeuThrValGlnLysAsnIleGluGluLysIleGly340345350LeuLeuGlyThrThrAspTrpLeuLysThrAspMetAspGlnMetVal355360365AspSerGlnSerAsnTrpAsnIleSerSerGluSerLysGlyThrAsp370375380HisLeuGlnGlyGlyAlaLeuLeuTyrValAsnSerAspLeuThrPro385390395400AsnAlaAsnSerAspTyrArgLeuLeuAsnArgThrProThrAsnGln405410415LysGlyGlnIleThrThrAspGlyAsnGlnGlyGlyTyrGluMetLeu420425430LeuAlaAsnAspValAspAsnSerAsnProIleValGlnAlaGluGln435440445LeuAsnTrpLeuTyrTyrMetMetAsnIleGlySerIleValGlnAsn450455460AspProThrAlaAsnPheAspGlyTyrArgValAspAlaValAspAsn465470475480ValAsnAlaAspLeuLeuGlnIleAlaGlyAspTyrPheLysAlaAla485490495TyrGlyThrAspLysSerAspAlaAsnAlaAsnAsnHisIleSerIle500505510LeuGluAspTrpAspAsnAsnAspProAlaTyrValLysSerGlnGly515520525AsnAsnGlnSerThrMetAspPheProMetHisLeuAlaLeuLysTyr530535540SerLeuAsnMetProSerSerAlaArgSerGlyLeuGluProAspIle545550555560ValThrSerLeuValAsnArgSerGluAspSerThrGluAsnGluAla565570575GlnProAsnTyrSerPheIleArgAlaHisAspSerGluValGlnThr580585590ValIleAlaGlnIleIleLysAspLysIleAsnProSerSerAspAsp595600605GlyLeuThrValSerThrAspGluIleAlaLysAlaPheGluIleTyr610615620AsnAlaAspGluLeuLysAlaAspLysGluTyrThrAlaTyrAsnIle625630635640ProSerSerTyrAlaLeuMetLeuThrAsnLysAspThrIleProArg645650655ValTyrTyrGlyAspLeuPheThrAspAspGlyGlnTyrMetSerAla660665670LysSerProTyrTyrAspAlaLeuThrSerLeuLeuGlnSerArgVal675680685LysTyrValSerGlyGlyGlnSerMetAsnMetAlaTyrLeuHisAsn690695700AsnGlnGlyLeuLeuThrSerValArgTyrGlyLysGlyAlaMetThr705710715720AlaThrAspThrGlyThrSerGluThrArgThrGlnGlyIleGlyLeu725730735IleValSerAsnLysThrAspLeuAsnLeuAsnAsnAspGluGlnIle740745750ValLeuAsnMetGlyAlaValHisLysAsnGlnAlaTyrArgAlaLeu755760765MetLeuSerThrLysAspGlyLeuLysIleTyrAsnSerAspAspAsp770775780AlaProLeuAlaTyrThrAspAspGlnGlyArgLeuThrPheLysSer785790795800AspMetValPheGlyValSerAspAlaGlnValSerGlyTyrLeuAla805810815AlaTrpValProValGlyAlaThrAspAspGlnAspAlaArgAsnGln820825830SerSerThrIleAlaSerThrAspGlyAsnThrTyrHisSerAsnAla835840845AlaLeuAspSerGlnValIleTyrGluGlyPheSerAsnPheGlnAla850855860MetProThrGlnThrAsnGluTyrThrAsnValLysIleAlaGlnAsn865870875880AlaGlnLeuPheLysAsnLeuGlyIleThrSerPheGluLeuAlaPro885890895GlnTyrArgSerSerThrAspAsnSerPheLeuAspAlaValValGln900905910AsnGlyTyrAlaPheThrAspArgTyrAspIleGlyTyrAsnThrPro915920925ThrLysTyrGlyThrValAspGlnLeuLeuAspAlaLeuArgAlaLeu930935940HisAlaGlnAspIleGlnAlaIleAsnAspTrpValProAspGlnIle945950955960TyrAsnLeuProSerGluGluIleValThrAlaSerArgThrAsnGly965970975SerGlyLysIleAsnGluThrSerValIleAsnAsnThrLeuTyrAsp980985990SerHisThrValGlyGlyGlyGluTyrGlnAlaPheTyrGlyGlyAla99510001005PheLeuAspLysLeuLysGlnAspPheProGluLeuPheGluThr101010151020LysGlnIleSerThrGlyGluAlaMetAsnProAspValLysIle102510301035ThrGluTrpSerAlaLysTyrPheAsnGlySerAsnIleGlnGly104010451050ArgGlyAlaTrpTyrValLeuLysAspTrpAlaThrAsnGlnTyr105510601065PheAsnValSerSerGlySerLysPheLeuProLysGlnLeuLeu107010751080GlyGluLysThrSerThrGlyPheThrAsnValAspAsnGlyLys108510901095ThrGluPheTyrSerThrSerGlyTyrGlnAlaLysAsnThrPhe110011051110IleGlnAspAsnAspAsnTrpTyrTyrPheAspAsnAspGlyTyr111511201125MetValValGlyGlyGlnGluIleAsnGlyLysLysTyrTyrPhe113011351140LeuProAsnGlyValGluLeuGlnAspAlaTyrLeuSerAspGly114511501155ThrAsnValTyrTyrTyrSerSerThrGlyArgGlnIleThrAsn116011651170GlnTyrTyrArgAspSerAspSerLysTrpHisTyrPhePheSer117511801185AspGlyHisMetAlaValGlyLeuThrThrIleThrAlaAspAsn119011951200GlyThrAlaAsnGlnGlnTyrPheAspValAsnGlyIleGlnLeu120512101215LysGlyValAlaValLysAspThrAspGlyAsnValArgTyrPhe122012251230AspGlyAsnThrGlyAsnLeuValValSerAsnTrpGlyLysThr123512401245AlaAspGlySerTrpLeuTyrLeuAsnAspLysGlyIleAlaVal125012551260ThrGlyGlnGlnAsnIleAsnGlyGlnAsnValTyrPheAsnGlu126512701275AspGlyValGlnValLysGlyGluAlaIleThrAspThrAsnGly128012851290AsnValHisTyrTyrAspArgSerThrGlyAsnMetValThrAsn129513001305SerTrpAlaGluLeuProAspSerSerTrpMetTyrLeuAspVal131013151320AsnGlyValAlaAlaIleGlyAlaGlnLysIleAsnGlyLeuGlu132513301335PheTyrPheAspAsnAsnGlyLysGlnValLysAsnAspLysVal134013451350IleAsnAspAspGlyThrIleAsnTyrTyrThrGlyMetSerGly135513601365GluLysLeuLysAsnAspPheGlyGluLeuProAspGlySerTrp137013751380MetTyrLeuAspAsnGlnGlyAsnAlaValIleGlyAspLysLys138513901395IleAsnGlyGlnAsnLeuTyrPheLysIleAspGlyGlnGlnVal140014051410LysGlyGluThrTyrIleAspGlyValGlyLysMetArgPheTyr141514201425GlnAlaAlaSerGlyGluMetValThrAsnGlnPheGluGlnVal143014351440AlaAspGlyLysTrpAlaTyrPheGlyAlaAspGlyValAlaVal144514501455ThrGlyGluGlnTyrIleAspGlyGlnAspLeuPhePheAspPro146014651470ThrGlyTyrGlnValLysGlyAspLysArgThrIleAspSerVal147514801485LeuTyrSerPheAspLysAspSerGlyGluArgGlnArgLeuAsn149014951500ThrIle1505當(dāng)前第1頁1 2 3