B、C基因型乙型肝炎病毒X蛋白在果蝇细胞中的表达与纯化  

Expression and purification of genotype B and C hepatitis B virus X proteins in Drosophila melanogaster cells

作　　者：郑瑾[1] 林万松[1] 林建银[1] 林旭[1] 

机构地区：[1]福建医科大学分子医学研究中心,福建省高校感染与肿瘤重点实验室,福建福州350004

出　　处：《中国病原生物学杂志》2009年第3期173-176,共4页Journal of Pathogen Biology

基　　金：全国优秀博士学位论文作者专项资金资助项目(No.200359);教育部新世纪优秀人才支持计划基金资助项目(No.NCET-05-0574);福建省高等学校科技创新团队培育计划基金资助项目(No.FMU-RT001)

摘　　要：目的获得高纯度B、C基因型乙型肝炎病毒X蛋白(HBx蛋白)。方法PCR扩增获得B、C基因型乙型肝炎病毒X基因,以AgeⅠ及BglⅡ位点将其克隆入果蝇表达载体pMT/BiP/V5/HisC。重组载体与筛选质粒pCoBlast以脂质体Cellfectine共转染果蝇S2细胞,25μg/ml Blasticidin筛选多克隆抗性细胞株。扩增细胞以CuSO4诱导HBx蛋白表达,Western blot鉴定表达产物并确定最佳蛋白表达时间和诱导浓度。200 ml大规模培养细胞并诱导表达,一步法及两步法分别纯化B、C基因型HBx蛋白。结果成功构建重组表达载体pMT/BiP-HBxb(含B基因型HBx基因)和pMT/BiP-HBxc(含C基因型HBx基因)。在果蝇S2细胞中,B、C基因型HBx蛋白与6×His融合表达(分别为HBxb-His及HBxc-His),在CuSO4诱导后72 h以及诱导浓度为500μmol/L^1 mmol/L时蛋白表达量最高。在200 ml培养液中,一步法纯化所得HBxb-His及HBxc-His蛋白总量分别为1.52和1.37 mg,纯度为85.23%和84.59%;二步法所得的HBxb-His及HBxc-His蛋白总量分别为5.38和6.42 mg,纯度分别为95.36%和94.62%。结论在果蝇表达系统中表达并获得高纯度B、C基因型乙型肝炎病毒X蛋白,为进一步探讨结构和功能的关系奠定了基础。Objective To obtain genotype B and C hepatitis B virus X proteins（HBx） with a high level of purity.Methods X genes of genotype B or C hepatitis B virus（HBV） were amplified by PCR and cloned into Drosophila expression vectors pMT/BiP/V5/HisC in AgeⅠ and BglⅡ sites.The recombinant plasmids were co-transfected by Cellfectine into S2 cells with the selection vector pCoBlast.Drug-resistant multiclonal cell lines were selected by blasticidin with a concentration of 25 μg/ml.Cells were cultured on a large scale and the target proteins were expressed by the induction of CuSO4.Western blot analysis was used to determine the optimal CuSO4 concentration and inducing interval for protein expression.After expression on a large scale of 200 ml of culture media,genotype B and C HBx with high levels of purity were obtained by two different methods of purification.Results The recombinant plasmids pMT/BiP-HBxb and pMT/BiP-HBxc,which harbor the genotype B and C HBx respectively,were successfully constructed.Genotype B or C HBx was expressed with a C terminal 6 × His tag（HBxb-His and HBxc-His,respectively） and peaked 72 h after induction when the inducing concentration of CuSO4 was between 500 μmol/L and 1 mmol/L.One-step purification from 200 ml culture media provided a total amount of 1.52 mg（HBxb-His） and 1.37 mg（HBxc-His） with a purity of 85.23% and 84.59%,respectively,while two-step purification provided 5.38 mg HBxb-His and 6.42 mg HBxc-His with a purity of 95.36% and 94.62% respectively.Conclusion Genotype B and C HBx are successfully expressed with high levels of purity in a Drosophila expression system,and this finding should prove helpful in the further study of the structural and functional differences between genotype B and C HBx.

关 键 词：乙型肝炎病毒 基因型 表达 纯化 果蝇 

分 类 号：R373.21[医药卫生—病原生物学]