丙型肝炎病毒中和抗原表位与乙型肝炎病毒S抗原嵌合基因重组真核表达质粒的构建及其在293T细胞中的表达

目的构建丙型肝炎病毒(hepatitis C virus,HCV)中和抗原表位与乙型肝炎病毒(hepatitis B virus,HBV)S抗原嵌合基因真核表达质粒,并在293T细胞中进行表达。方法从含HBV全序列的质粒pHBV中扩增HBV S抗原基因,在HBV S疏水区127和128位氨基酸序列处引入AgeⅠ酶切位点,将HCV E1和E2区保守的线性中和抗原表位及HVR1的模拟表位基因分别插入该位点,获得嵌合HCV中和抗原表位的重组HBV S基因,将该基因克隆至真核表达载体pCI-neo中,构建重组真核表达质粒pCI-HBSE1～4。将4种重组真核表达质粒转染293T细胞,间接免疫荧光和Western blot检测嵌合基因的表达。结果 4种重组真核表达质粒经双酶切证实构建正确;4种重组质粒转染的293T细胞胞浆内可见较强的绿色荧光,Western blot显示,在相对分子质量约27 000处可见蛋白条带。结论成功构建了HCV中和抗原表位与HBV S抗原嵌合基因真核表达质粒,其在293T细胞中可有效表达,为进一步制备嵌合HCV中和抗原表位的HBV S抗原VLP,研究中和抗体对HCV假病毒颗粒(HCVpp)和JFH-1 HCV体外培养系统(HCVcc)感染的抑制作用奠定了实验基础。

Construction of eukaryotic expression vector for chimeric gene of hepatitis C virus neutralizing epitopes with hepatitis B virus S antigen and its expression in 293T cells

Objective To construct a eukaryotic expression vector for chimeric gene of hepatitis C virus(HCV) neutralizing epitopes with hepatitis B virus(HBV)S antigen and express in 293T cells.Methods HBV S antigen gene was amplified from plasmid pHBV containing full-length HBV gene,into which the restriction site of Age I was introduced between amino acids 127 and 128.Conservative HCV linear neutralizing epitope genes in E1 and E2 regions and mimotope of hypervariable region 1 were inserted to the restriction site of Age I,and the obtained chimeric gene of HCV neutralizing epitopes with HBV S antigen was cloned into eukaryotic expression vector pCI-neo.293T cells were transfected with the constructed recombinant plasmids pCI-HBSE1 ～ 4 and determined for expression of chimeric gene by indirect IFA and Western blot.Results Restriction analysis proved that recombinant plasmids pCI-HBSE1 ～ 4 were constructed correctly.Strong green fluorescence was observed in the cytoplasm of 293T cells transfected with the recombinant plasmids.Western blot showed a protein band with a relative molecular mass of about 27 000.Conclusion A eukaryotic expression vector for chimeric gene of HCV neutralizing epitopes with HBV S antigen was successfully constructed and effectively expressed in 293T cells,which laid an experimental foundation of further preparation of chimeric HBV S antigen VLPs with HCV neutralizing epitope and study on inhibitory effect of neutralizing antibody on infections with HCV pseudovirus particles(HCVpp)and JFH-1 HCV in vitro culture system(HCVcc).