【摘要】 目的　克隆人乳头瘤病毒 6b型E7(humanpapillomavirus 6bE7)基因 ,构建原核表达载体 ,并进行蛋白表达和纯化。方法　将HPV 6bE7基因经PCR扩增和亚克隆 ,与GST原核表达载体pGEX 4T 2重组 ,转化E .coliP2 3 92 菌表达蛋白 ,经GlutathioneSepharose 4B亲和层析纯化GST HPV 6bE7融合蛋白。结果　限制性酶切和DNA测序表明 ,HPV 6bE7DNA正确克隆于 pGEX 4T 2多克隆位点。经IPTG诱导表达的GST HPV 6bE7融合蛋白主要存在于可溶性上清。经亲和层析 ,每升诱导菌回收 8.4mg融合蛋白 ,10 %SDS PAGE分析融合蛋白表观分子量约 3 7kDa。结论　成功构建了HPV 6bE7基因原核表达载体 ,并大量表达和纯化了GST HPV 6bE7融合蛋白更多还原

【Abstract】 ObjectiveTo clone HPV 6b E7 gene into express ion vector pGEX-4T-2 and express GST-HPV 6b E7 fusion protein in E.coli.MethodsHPV 6b E7 gene amplified by PCR was cloned and li gated with the expression vector pGEX-4T-2 to form pGEX-4T-2/HPV 6b E7 recom binants that transformed E.coli P 2392 .A high level GST-HPV 6b E7 fusi on protein was induced and purified by Glutathione Sepharose 4B affinity (GST:Gl utathione S Transferase).ResultsRestriction enzyme analy sis and DNA sequencing showed HPV 6b E7 DNA was inserted into the expression vec tor with correct sequence.The expressed GST-HPV 6b E7 fusion protein induced by IPTG mainly exists in the supernatant with a yield of 8.4 mg per liter of induc ed cultures after Glutathione Sepharose 4B affinity.Apparent molecular weight of GST-HPV 6b E7 fusion protein estimated by 10% SDS-PAGE analysis was approxima tely 37 kDa.ConclusionWe succeeded in constructing the e xpression recombinant of HPV 6b E7 gene and acquiring GST-HPV 6b E7 fusion prot ein in large amount.The results may lead to further study of the peptide based v accine to cure condyloma acuminata.更多还原