乙肝病毒抗原基因真核表达载体的构建及表达

Construction and expression of eukaryotic expression vectors of HBV antigen genes

目的:构建乙肝病毒(Hepatitis B virus,HBV)相关抗原基因真核表达载体,检测各抗原基因在体外真核细胞中的表达情况。方法:以含有1.3倍的HBV全基因组序列的质粒PcDNA3.1-HBV为模板,PCR扩增HBV的各抗原基因片段,通过连接至中间克隆载体Peasy-T1-Simple,酶切及测序鉴定正确后,再将目的片段插入真核表达载体pcDNA3.1(+)中,构建包含HBV抗原基因的重组表达质粒pcDNA3.1(+)-HBs,pcDNA3.1(+)-HBe,pcDNA3.1(+)-HBc。阳性克隆用限制性内切酶酶切鉴定正确后用脂质体2000分别瞬时转染HepG2细胞,同时转染LO2细胞作为对照,Western blot和免疫荧光技术分别检测各抗原基因产物在细胞内的表达。结果:成功扩增出HBV各抗原基因片段,通过酶切测序鉴定证明成功构建了包含HBV抗原基因的重组表达质粒pcDNA3.1(+)-HBs,pcDNA3.1(+)-HBe,pcDNA3.1(+)-HBc,Western blot和免疫荧光检测到目的蛋白在转染细胞中的正确表达。结论:本实验成功构建包含HBV抗原基因的真核表达载体,并能在体外真核细胞中表达相关抗原蛋白,为进一步研究各抗原的生物学功能奠定了实验基础。

Objective：To analyze the expression of HBV antigen genes in eukaryotic cells in vitro,eukaryotic expression plasmids containing HBV antigen genes were constructed.Methods：The fragments of HBV antigen genes were amplified by PCR from the plasmid PcDNA3.1-HBV which contains 1.3 fold HBV whole genome sequence.Then the PCR fragments were cloned into Peasy-T1-Simple vector following the routine procedures.After identification by enzyme digestion and DNA sequence analysis,the interested gene fragments were inserted into the eukaryotic expression vector PcDNA3.1（＋）.The resultant recombinant plasmids were confirmed by restriction enzyme digestion and the correct ones were transiently transfected into HepG2 cells,and LO2 cells as a control.Western blot and Immunofluores-cence were used to analyze the expression of HBV gene products in the transfected cells.Results：The fragments of HBV antigen genes were correctly amplified.Restriction enzyme digestion and DNA sequence analysis confirmed that the recombinant plasmids containing HBV antigen genes were successfully constructed.Western blot and Immunofluorescence confirmed that the interested genes could be correctly expressed in transfected cells.Conclusion：The construction of eukaryotic expression plasmids containing HBV antigen genes and the antigen gene expression in eukaryotic cells in vitro provide a solid experimental foundation for further research on the biological function of HBV antigens.