乙型肝炎病毒截短C基因及前S1基因在大肠杆菌中融合表达及纯化.docVIP

乙型肝炎病毒截短C基因及前S1基因在大肠杆菌中融合表达及纯化 　 作者：胡兴斌，尹文，韦三华，雷迎峰，吕欣，孙梦宁，杨敬，徐志凯 【关键词】 ,乙型肝炎病毒 Expression and purification of truncated C gene combined with preS1 gene from HBV in E.coli 　【Abstract】 AIM: To construct the prokaryotic recombinant plasmid to express HBV truncated C gene with preS1 gene and to acquire and purify the fused protein for antigenic analysis. METHODS: The target gene was amplified by PCR and digested by restrictive enzyme before cloned into pET28a. It was then transformed into E.coli DH5α and the positive recombinant plasmid named pET28aCtpreS1 was sequenced. The target protein was distinctly expressed after transformed into E.coli BL21 and induced with IPTG. The protein was scanned and purified on Ni2+NTA column and Western blot was performed after solubility analysis. RESULTS: The recombinant plasmid pET28aCtpreS1 was successfully constructed and could efficiently express the target gene. Protein production mainly was in inclusion body but could be purified through Ni2+NTA column. The purified protein maintained the antigen activity. CONCLUSION: The truncated HBcAg combined with preS1 antigen can efficiently be expressed and purified, which lays a foundation for further research of novel vaccine against HBV. 　　【Keywords】 hepatitis B virus; fused protein; prokaryotic expression; vaccine 　　【摘要】 目的: 构建乙型肝炎病毒(HBV)截短C基因和前S1基因重组原核表达质粒，获得融合蛋白的表达并进行抗原性分析. 方法: PCR扩增获得截短C基因片段和前S1基因，双酶切后克隆至原核表达质粒pET28a，转化E.coli DH5α，酶切鉴定得阳性重组质粒pET28a并测序；然后转化E.coli BL21，IPTG诱导融合蛋白表达,薄层扫描分析表达蛋白组成；可溶性分析后用Ni2+NTA凝胶亲和层析柱纯化、透析并浓缩融合蛋白，Western Blot分析特异性和抗原性. 结果: 成功构建了HBV 截短C基因和前S1基因融合的原核表达质粒pET28aCtpreS1，目的基因可高效表达，表达产物主要以包涵体形式存在，Ni2+NTA 纯化可获得目的蛋白，纯化蛋白具有良好的抗原性和特异性. 结论: HBV截短HBcAg和preS1抗原融合蛋白可高效表达并得到纯化，为研究新型乙肝疫苗奠定了基础. 　　【关键词】 乙型肝炎病毒；融合蛋白；原核表达；疫苗 　　0引言 　　乙型肝炎病毒(hepatitis B virus, HBV)导致的病毒性肝炎目前尚无十分有效的治疗措施，疫苗接种是控制和预防乙型肝炎的重要手段，但部分人群对现有的疫苗不应答或低应答而导致接种失败，因此需要研发新型HBV疫苗. 我们利用基因重组技术构建含截短C基因和preS1基因联合的原核表达载体，在大肠杆菌中表达融合蛋白并进行纯化，初步分析其免疫性和特异性，为比较不同来源的HBV候选疫苗分子和深入研究HBV新型疫苗奠定基础. 　　1材料和方法 　　1.1材料 　　E.coli D