專利名稱：一種新型的日本血吸蟲優(yōu)勢(shì)抗原分子的制作方法
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聽聽聽聽聽浜斻€佺函鍖杛SjP38鍗曞厠闅嗘姉浣撶殑鍒跺1銆佸姩鐗╁厤鐤 鍒濇鍏嶇柅聽聽Ag聽10锝 00渭g鍔犵姘忓畬鍏ㄤ綈鍓傜毊涓嬪鐐規(guī)敞灝 聽聽聽聽聽聽聽聽聽聽聽聽聽(鎶楀師鍜屼綈鍓傜瓑浣撶Н娣峰悎)聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽鈫 鍛ㄥ悗絎簩嬈″厤鐤犅犲墏閲忓悓涓婏紝鍔犵姘忎笉瀹屽叏浣愬墏鐨笅鎴杋p聽聽聽聽聽聽聽聽聽聽聽聽聽(ip鍓傞噺涓嶅疁瓚呰繃0.5ml)聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽鈫 鍛ㄥ悗絎笁嬈″厤鐤犅犲墏閲忓悓涓婏紝涓嶅姞浣愬墏錛宨p聽聽聽聽聽聽聽(5锝 澶╁悗閲囪嫻嬪叾鏁堜環(huán)錛屾嫻嬪厤鐤晥鏋 聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽鈫 鍛ㄥ悗鍔犲己鍏嶇柅錛屽墏閲 0锝 00渭g涓哄疁錛宨p鎴杋v聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽聽鈫 澶╁悗鍙栬劸铻嶅悎(姝ゆ椂B娣嬪反姣嶇粏鑳?yōu)姣斾緥杈冨ぇ锛岃瀺鍚堢殑鎴愬姛鐜囪緝楂?聽聽聽聽聽聽聽聽聽緇撴灉錛歳SjP38鍏嶇柅灝忛紶琛€娓呮晥浠風(fēng)殑嫻嬪畾
緇撴灉錛
2)Westernblot媯€(gè)嫻嬬壒寮傛€ф姉浣揂6鐢 2錛呰兌緇廠DS-PAGE鐢墊吵120V鐩磋惤錛岀敤鍗婂共杞嵃浠 6V15min杞嚦PDFV鑶滀笂錛 7鈩冪儰騫插悗鍔犲叆鍗曟姉A6緇嗚優(yōu)涓婃竻錛屼簬37鈩冧綔鐢 h錛孴PBS媧 嬈★紝姣忔5min錛涘姞鍏RP鏍囪鐨勭緤鎶楅紶IgG(1鈭 500紼€閲 浜 7鈩冧綔鐢 h錛孴PBS媧 嬈★紝姣忔5min錛汥AB鏄捐壊5-10min瑙傚療鏈夋棤鐗瑰紓鎬ф潯甯︺€ 緇撴灉錛氬崟鎶桝6鍙壒寮傛€ц瘑鍒玶SjP38鍜屽ぉ鐒舵姉鍘烻EA涓殑38kDa鎶楀師錛岃鍥 8銆 銆 聽聽聽聽聽聽聽聽涓冦€乺SjP38鍗曞厠闅嗘姉浣揂6緇嗚優(yōu)鐨勫喕瀛樹笌澶ч噺鎵╁灝嗙粡媯€(gè)嫻嬪悗鐨勫崟鎶桝6緇嗚優(yōu)鍐誨瓨(鍐誨瓨娑詫細(xì)50錛呭皬鐗涜娓呫€ 0錛呬笉瀹屽叏鍩瑰吇娑層€ 0錛匘MSO)鍚屾椂鍦ㄥ皬榧犱綋鍐呰繘琛屽ぇ閲忔墿澧炪€ 鑵規(guī)按鐨勫埗澶囷細(xì)甯歌鏄厛鑵硅厰娉ㄥ皠0.5ml娑蹭綋鐭寵厞浜嶣ALB/c灝忛紶錛 锝 鍛 鍚庤吂鑵旀敞灝 脳106涓潅浜ょ槫緇嗚優(yōu)錛屾帴縐嶇粏鑳 锝 0澶╁悗鍙駭鐢熻吂姘達(dá)紝瀵嗗垏瑙傚療鍔ㄧ墿鐨勫仴搴風(fēng)姸鍐典笌鑵規(guī)按寰佽薄錛屽緟鑵規(guī)按灝藉彲鑳藉錛岃€屽皬榧犻浜庢浜′箣鍓嶏紝澶勬灝忛紶錛 鐢ㄦ淮綆″皢鑵規(guī)按鍚稿叆璇曠涓紝涓€鑸竴鍙皬榧犲彲鑾 锝 0ml鑵規(guī)按銆備篃鍙敤娉ㄥ皠鍣ㄦ娊鍙 鑵規(guī)按錛屽彲鍙嶅鏀墮泦鏁版銆傝吂姘翠腑鍗曞厠闅嗘姉浣撳惈閲忓彲杈 -20mg/ml銆
權(quán)利要求
1銆佹棩鏈鍚歌櫕铏嵉涓昏铔嬬櫧P38(SjP38)鐨刢DNA錛屽簭鍒楀叏闀 206bp錛 1-1065bp涓轟竴瀹屾暣鐨勫紑鏀捐鐮佹錛岀紪鐮 55aa錛岀悊璁洪嫻嬪垎瀛愰噺涓 9233Da錛岃泲鐧 璐ㄧ瓑鐢電偣pI錛 .06錛涘畠鐨勬牳鑻烽吀搴忓垪濡備笅錛 1聽聽聽聽聽聽GTTATGTCGG聽GTACACATCA聽AAACCATGCA聽GTTAGTATTC聽CTGTGAACCG聽TGAGCAATGT61聽聽聽聽聽TCATTTCCGA聽GGCATAGACA聽CGAACTGCTA聽CAGAATGTGG聽GGAAAGCCAG聽TAAAAGCGGT121聽聽聽聽CAGTTGGGTT聽CAGTAGTGCC聽ATACACGGAA聽GACTGGCCCA聽GTTCAGTGAG聽TAACTGGATA181聽聽聽聽GAGTCTTCGT聽TGAAAAGCTG聽GGACAAAGAA聽ATGGAACGTT聽TAAGGCGAGG聽AATGTTTGCT241聽聽聽聽CTGTTGCCAA聽TGGATCGCTT聽CATGGACGGA聽ATCCCACATG聽ATCCGTTGGG聽AATGATGCAT301聽聽聽聽GAAATGGATC聽GTCACATTGA聽GGAACTTCAA聽AATGGGATGG聽GTTTAAGTGC聽TGTGGCGCCG361聽聽聽聽CTTGGATCAA聽CGAGTGACTA聽CTTGAAGGAT聽GCCTACGAAG聽TCGGCGATGA聽TGGCAAGGTG421聽聽聽聽CATTTCAAGG聽TGAGATTTGA聽CGCACAGGGT聽TTTGCTCCAG聽AAGACATCAA聽TGTGACGTCG481聽聽聽聽AGTGACAACC聽GTGTGACAGT聽GCACGCGAAG聽AAGGAGACGA聽CAACAGGTGG聽TAAGAAGTGT541聽聽聽聽AGTCATGAGT聽TCTGTCGTAT聽GATCCAGTTG聽CCGAAGAGTA聽TTGAGAATAA聽CCAGTTGAAA601聽聽聽聽TGCCGTCTGA聽CTGATGATGG聽TGTTTTGATG聽TTGGAGGCTC聽CAGTGAAAGT聽TGGTGAGAAT661聽聽聽聽AAGTCGCTGA聽CAATGAACGA聽GTCTGGTCAG聽GTGGGTATTC聽AACCGAAATC聽AGCGAGTCAG721聽聽聽聽ATACAAGCAG聽TTCCTGCATC聽GCAAGCTCTC聽ACTGTGAAAG聽GTTGTCAAGG聽TCTGACGGTG781聽聽聽聽TTGGATGATG聽GTGCAGGTGG聽CAAGAAACTG聽CACGTTGAGG聽TTCAATTGGA聽CCCAGTGTAC841聽聽聽聽CGACCAGAAG聽ATTTGTGTGT聽GAATATTGAT聽TCGAATCGTG聽TTGTGGTTAG聽CGGACGTCAT901聽聽聽聽TACAAGCAAA聽AGACTGATGC聽AAGAAGAAAG聽TCGAGTTCAT聽TCGCAGAATT聽CAGTCAGTCG961聽聽聽聽TATTCGATTC聽CTGAGACTGT聽GGATCCGTTA聽ACTGTGTCTG聽CTCAGGTAGT聽TGACAATATG1021聽聽聽TTGGTTGTAG聽AGGCACCGAT聽GATGAAGCAA聽CACGAAATCG聽CTCATTAGAC聽GAAAATATGA1081聽聽聽TTGACTGAAA聽TCACAATAAT聽TATGGTAATG聽ATGATATTCA聽CTTGTGTTAT聽TTAACAAATA1141聽聽聽AATACATTTG聽CTTACTCAAA聽AAAAAAAAAA聽AAAAAAAAAA聽AAAACATGTC聽GGCCGCCT
2銆佹棩鏈鍚歌櫕铏嵉涓昏铔嬬櫧P38(SjP38)鐨勬皚鍩洪吀搴忓垪濡備笅錛 1聽聽聽聽聽VMSGTHQNHA聽VSIPVNREQC聽SFPRHRHELL聽QNVGKASKSG聽QLGSVVpYTE聽DWPSSVSNWI61聽聽聽聽ESSLKSWDKE聽MERLRRGMFA聽LLPMDRFMDG聽IPHDPLGMMH聽EMDRHIEELQ聽NGMGLSAVAP121聽聽聽LGSTSDYLKD聽AYEVGDDGKV聽HFKVRFDAQG聽FAPEDINVTS聽SDNRVTVHAK聽KETTTGGKKC181聽聽聽SHEFCRMIQL聽PKSIENNQLK聽CRLTDDGVLM聽LEAPVKVGEN聽KSLTMNESGQ聽VGIQPKSASQ241聽聽聽IQAVPASQAL聽TVKGCQGLTV聽LDDGAGGKKL聽HVEVQLDPVY聽RPEDLCVNID聽SNRVVVSGRH301聽聽聽YKQKTDARRK聽SSSFAEFSQS聽YSIPETVDPL聽TVSAQVVDNM聽LVVEAPMMKQ聽HEIAHTKILT361聽聽聽EITIIMVMMIFTCVIQINTF聽AYSKKKKKKK聽KNMSAA
3銆佸寘鎷潈鍒╄姹 鐨刢DNA鐨勮〃杈捐澆浣撱€
4銆佸寘鎷潈鍒╄姹 鐨刢DNA鐨勮〃杈捐澆浣損ET-32a(+)-SjP38銆
5銆佸埗澶囬噸緇勬棩鏈鍚歌櫕铏嵉涓昏铔嬬櫧P38(SjP38)鐨勬柟娉曪紝鍖呮嫭鐢ㄦ潈鍒╄姹 鎵€榪扮殑琛ㄨ揪杞戒綋杞寲瀵勪富緇嗚優(yōu)錛屽煿鍏昏漿鍖栦綋錛岃幏寰楅噸緇勭殑SjP38銆
6銆佹牴鎹潈鍒╄姹 鎵€榪扮殑鏂規(guī)硶錛屽叾涓瘎涓葷粏鑳?yōu)鏄ぇ鑲犳潌鑿屻€
7銆侀噸緇勬棩鏈鍚歌櫕铏嵉涓昏铔嬬櫧P38錛屽寘鎷敤鏉冨埄瑕佹眰3鎵€榪扮殑琛ㄨ揪杞戒綋杞寲瀵勪富緇嗚優(yōu)錛屽煿鍏昏漿鍖栦綋錛屼粠鍩瑰吇鐗╄幏寰楅噸緇勭殑SjP38鐨勬楠ょ殑鏂規(guī)硶鍒跺銆
8銆佸埗澶囨姉鏃ユ湰琛€鍚歌櫕铏嵉涓昏铔嬬櫧P38鍗曞厠闅嗘姉浣撶殑鏂規(guī)硶錛屽寘鎷敤綰寲鐨 閲嶇粍SjP38铔嬬櫧鍏嶇柅BABL/C灝忛紶錛岃幏寰楁姉SjP38鍗曞厠闅嗘姉浣撱€
9銆佹姉鏃ユ湰琛€鍚歌櫕铏嵉涓昏铔嬬櫧P38鍗曞厠闅嗘姉浣擄紝鍖呮嫭鐢ㄧ函鍖栫殑閲嶇粍SjP38铔嬬櫧鍏嶇柅BABL/C灝忛紶鐨勬楠ょ殑鏂規(guī)硶鍒跺銆
10銆佹姉鏃ユ湰琛€鍚歌櫕铏嵉涓昏铔嬬櫧P38鍗曞厠闅嗘姉浣擄紝鍙瘑鍒噸緇凷jP38铔嬬櫧鍜 澶╃劧铏嵉鍙憾鎬ц泲鐧 SEA)涓殑38kDa铔嬬櫧銆
全文摘要
本發(fā)明克隆表達(dá)了具有早期診斷價(jià)值的日本血吸蟲蟲卵主要蛋白P38(SjP38)基因，構(gòu)建了能高效可溶性表達(dá)P38基因的工程菌，建立了簡便快捷的重組蛋白純化方法，獲得了具有良好抗原活性的重組蛋白，制備了重組抗原特異的單克隆抗體，為血吸蟲病的診治研究提供了一種新型優(yōu)勢(shì)抗原分子，為實(shí)現(xiàn)新型的標(biāo)準(zhǔn)化、產(chǎn)業(yè)化生產(chǎn)的血吸蟲病診斷試劑奠定了工作基礎(chǔ)。
文檔編號(hào)C07K16/18GK1618965SQ20031011218
公開日2005年5月25日 申請(qǐng)日期2003年11月18日 優(yōu)先權(quán)日2003年11月18日
發(fā)明者陳曉光, 周曉紅, 吳錦雅 申請(qǐng)人:陳曉光, 周曉紅