【摘要】： 目的:克隆egG1Y162抗原基因,構建PET-41a/egG1Y162原核表達質(zhì)粒,并誘導表達egG1Y162重組蛋白及抗原性檢測。方法:根據(jù)emY162基因序列設計引物,分別從細粒棘球絳蟲原頭蚴和成蟲兩個發(fā)育階段,提取基因組DNA和總RNA,mRNA反轉(zhuǎn)錄為cDNA,利用PCR的方法以基因組DNA和cDNA為模板擴增egG1Y162基因;構建PUCm-T/egG1Y162重組質(zhì)粒,經(jīng)PCR、酶切及測序鑒定后,測序確定其正確性。利用定向克隆技術將egG1Y162抗原基因片段克隆至原核表達質(zhì)粒PET-41a上,通過酶切分析和PCR鑒定篩選出陽性克隆,測序確定序列。IPTG初步誘導和表達egG1Y162-GST重組蛋白,通過SDS-PAGE電泳和Western blot試驗分析鑒定。結果:從細粒棘球絳蟲的兩個不同發(fā)育階段均克隆出egG1Y162基因,從總DNA克隆得到片段長度1 680bp,從cDNA克隆得到片段長度459bp。相似性比較表明,egG1Y162基因序列與emY162相似性為91%,而egG1Y162基因cDNA與emY162相似性為95%。進一步分析顯示,egG1Y162基因序列由3個外顯子和2個內(nèi)含子組成,外顯子區(qū)域分別為1～70,1 064～1 380和1 577～1 648。位于疏水端1～16位氨基酸構成egG1Y162信號肽序列,35～115位氨基酸形成一個大的纖黏連蛋白剪接體FN3,133～152位氨基酸構成羧基端跨膜區(qū)域。測序結果顯示egG1Y162抗原基因長度為360 bp,編碼120個氨基酸。構建的PET-41a/egG1Y162原核表達質(zhì)粒,經(jīng)IPTG誘導后,SDS-PAGE檢測表明egG1Y162-GST重組蛋白得到成功表達,在相對分子量為44 KDa處有表達條帶。Western blot分析顯示陽性印跡條帶,分子量為44 KDa,egG1Y162重組蛋白能與細粒棘球蚴感染40天后犬的血清發(fā)生反應;與包蟲病患者血清也有陽性反應。結論:成功克隆egG1Y162抗原基因,序列對比分析顯示egG1Y162的cDNA與emY162的cDNA具有很高的相似性,基因的差異性主要存在于內(nèi)含子區(qū)域,egG1Y162抗原基因是一種新的基因。成功誘導表達出egG1Y162重組蛋白,并且egG1Y162重組蛋白具有很好的抗原性。
[Abstract]:Objective: To clone egG1Y162 antigen gene, construct PET-41a/egG1Y162 prokaryotic expression plasmid, induce the expression of egG1Y162 recombinant protein and detect its antigenicity. Methods: According to the sequence of emY162 gene, primers were designed to extract genomic DNA and total RNA from protozoan and adult stages of Echinococcus granulosus, and mRNA was retranscribed into cDNA. Methods The egG1Y162 gene was amplified with genomic DNA and cDNA as templates, and the PUCm-T/egG1Y162 recombinant plasmid was constructed and identified by PCR, restriction enzyme digestion and sequencing. The recombinant protein of egG1Y162-GST was induced and expressed by IPTG, and identified by SDS-PAGE electrophoresis and Western blot. Results: The egG1Y162 gene was cloned from two different developmental stages of Echinococcus granulosus, and the length of the fragment was 1 680 BP from the total DNA clone. The length of the fragment was 459 BP from the cDNA clone. The sequence similarity of egG1Y162 gene to emY162 was 91%, while that of egG1Y162 gene was 95%. The sequence analysis showed that the length of egG1Y162 antigen gene was 360 bp, encoding 120 amino acids. The constructed prokaryotic expression plasmid of ET-41a/egG1Y162 was induced by IPTG. SDS-PAGE analysis showed that the recombinant protein of egG1Y162-GST was obtained. Western blot analysis showed that the recombinant protein could react with the serum of dogs 40 days after infection with Echinococcus granulosus, and positive reaction with the serum of patients with hydatidosis. The analysis showed that the cDNA of egG1Y162 was highly similar to that of emY162. The gene difference mainly existed in the intron region. The egG1Y162 antigen gene was a new gene. The recombinant protein of egG1Y162 was successfully induced and expressed, and the recombinant protein of egG1Y162 had good antigenicity.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392

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