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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.
Objective: To construct eukaryotic expression plasmid of hepatitis B virus (HBV) S gene, including Pre-S1, Pre-S2 and S.Methods: The large surface antigen of HBV, Middle surface antigen of HBV and Small surface antigen of HBV gene fragment was inserted into the eukaryotic expression vector Pcmv-Tag2B by TA cloning to construct the recombinant plasmids Tag2B-LS, Tag2B-MS and Tag2B-SS, which were transfected into 293FT cells after sequencing and the proteins were collected and identified.Results: The recombinant plasmids were digested with restriction endonucleases to get the corresponding DNA fragments.The sequence was verified to be S gene with complete reading frame.The recombinant plasmids were transfected into 293FT cells respectively and confirmed by Western Blot.Conclusion: Tag2B-LS, Tag2B-MS and Tag2B-SS, which can be correctly expressed, laying a foundation for further study on the function of hepatitis B virus outer membrane protein.