截短序列和全序列HBcAg基因以及HBV C-S融合基因的克隆与表达  

Cloning and expression of truncated HBcAg gene,whole-length HBcAg gene and HBc-HBsAg fusion gene in vitro

作　　者：朱翔[1] 路文明[1] 丁宁玲 叶建中[1] 王锋[1] 沙莉[1] 李扬[1] 高胜兰 

机构地区：[1]苏州大学附属传染病医院(苏州巿第五人民医院),江苏省苏州市210007

出　　处：《实用肝脏病杂志》2015年第1期20-23,共4页Journal of Practical Hepatology

基　　金：苏州市科学技术局科技支撑计划(社会发展)项目(编号:YJS0943)

摘　　要：目的构建截短序列和全序列HBcAg基因和HBc-HBs Ag融合基因原核表达质粒,研究目的蛋白在大肠杆菌中的表达及其免疫原性。方法利用HBV全基因(adr亚型)质粒pUCm T-HBV分别扩增HBs Ag截短基因、HBcAg截短基因和HBcAg全基因,构建成重组质粒p SK-HBs、p SK-HBc和p KS-HBV C,经DNA序列测定鉴定后,分别将HBcAg截短基因、HBcAg全基因及HBc-HBs Ag融合基因亚克隆至表达质粒PET-30a,在大肠杆菌BL21(DE3)中进行表达HBcAg截短基因、HBcAg全基因和HBc-HBs Ag融合基因产物,采用PAGE-SDS和免疫印迹法对表达产物进行鉴定。结果成功构建了含HBcAg截短基因、HBcAg全基因和HBc-HBs Ag融合基因的原核表达质粒;成功构建的质粒在大肠杆菌BL21(DE3)中能大量表达HBcAg蛋白和HBc-HBs Ag融合蛋白,免疫印迹分析结果显示表达产物具有免疫原性。结论成功构建的原核表达载体在大肠杆菌BL21(DE3)中能顺利表达HBcAg蛋白和HBc-HBs Ag融合蛋白,表达产物具有免疫原性,为慢性乙型肝炎特异性免疫治疗研究提供了实验基础。Objective To construct the prokaryotic recombinant plasmids carring truncated HBcAg gene, whole-length HBeAg gene and HBc-HBsAg fusion gene,and to observe the expression of target proteins in E.coli and their immunogenicity in vitro. Methods Truncated HBcAg gene,wbole-length HBcAg gene and HBc-HBsAg fusion gene were obtained from plasmid pUCmT-HBV containing whole-length HBV gene （subtype adr）and con- struct recombinant plasmids of pSK-HBs,pSK-HBc and pKS-HBV C. Truncated HBcAg gene,whole-length HBcAg gene and HBc-HBsAg fusion gene which were obtained by fusing truncated HBsAg and truncated HBcAg gene,were subcloned into a expression vector pET-30a respectively after confirmed by DNA sequencing. The gene products were expressed in E.coli BL21 （DE3） and identified by SDS-PAGE and Western blot. Results The prokaryotic expression plasmids expressing truncated HBcAg gene,whole-length HBcAg gene and HBc-HBsAg fu- sion gene were successfully constructed. High levels of HBcAg protein and HBc-HBsAg fusion protein were ob- served in E.coli BL21 （DE3） and their immunogenicity were confirmed by Western blot. Conclusion HBcAg protein and HBc-HBsAg fusion protein are successfully obtained by selected expressing vector in E.coli BL2I （DE3） and the gene products have immunogenicity. This study has provided an experimental basis for specific immunotherapy for chronic hepatitis B.

关 键 词：乙型肝炎病毒 HBSAG HBCAG 融合蛋白 原核表达质粒 

分 类 号：Q78[生物学—分子生物学] R512.62[医药卫生—内科学]