乙型肝炎病毒外膜蛋白真核表达质粒的构建和表达

Construction of the Plasmids Expressing Pcmv-Tag2B-HBs Gene and Their Expression

目的:构建乙型肝炎病毒(HBV)S基因(包括Pre-S1,Pre-S2和S)的真核表达质粒.方法:将PCR克隆获得的Large surface antigen of HBV,Middle surface antigen of HBV和Small surface antigen of HBV基因片段通过T-A克隆插入真核表达载体Pcmv-Tag2B,构建重组质粒Tag2B-LS,Tag2B-MS和Tag2B-SS,经测序后转染293FT细胞,收取蛋白并鉴定.结果:分别双酶切构建的重组质粒,获得相应大小的DNA片段,且测序证实为有完整读码框的S基因,重组质粒分别转染293FT细胞并表达,经Western Blot鉴定证实.结论:成功构建真核表达质粒Tag2B-LS,Tag2B-MS和Tag2B-SS,并能正确表达,为进一步研究乙型肝炎病毒外膜蛋白的功能打下基础.

To construct the eukaryotic expression plasmids of the S gene of HBV virus, we cloned the gene fragments large surface antigen of HBV（LS） ,middle surface antigen of HBV（MS） and small surface antigen of HBV（SS）through PCR. The eukaryotic expression plasmids named Tag2B-LS, Tag2B-MS and Tag2B-SS were constructed with the respective gene fragments and Pcmv-Tag2B vectors. DNA sequencing identified the recombinant plasmids constructed including the complete reading frame of the S gene. Then the plasmids were transfected into 293FT cells using lipofecting 2000. Western blot confirmed that the gene fragments were expressed effectively. All the results showed that the eukaryotic expression plasmids of the S gene of HBV virus were constructed and expressed successfully and this could lay a foundation for the further research on the function of the HBVlsurface proteins.