HBsAg逆转录病毒表达质粒的构建及其在真核细胞中的表达

Construction and Expression of a Recombinant Retrovirus Vector Inserted HBsAg Gene

目的 构建乙型肝炎表面抗原 (HBsAg)逆转录病毒表达质粒并探讨其在真核细胞中的表达 .方法 用限制性内切酶从重组质粒pBKS -S中切出HBsAg基因 ,并亚克隆到逆转录病毒表达质粒pLXSN ,构建成重组质粒pLXSN-S ;经限制性内切酶消化和DNA序列测定鉴定无误后 ,用阳离子脂质体转染法将重组质粒pLXSN -S分别转染HepG2、K5 6 2和EB病毒转化B淋巴母细胞 (EBVC) ;用ELISA法检测HBsAg在细胞培养上清中的表达 .结果 经酶切和DNA序列测定鉴定证实重组质粒构建正确 ;质粒pLXSN -S转染细胞培养上清均可检测到HBsAg .结论 逆转录病毒表达质粒pLXSN

Objective To construct a recombinant retrovirus expression plasmid inserted HBsAg gene and study its expression in eukaryotic cells. Methods The full length gene of HBsAg was obtained from the recombinant plasmid pBKS-S by digesting with EcoRⅠ and BamHⅠ. Then the gene was subcloned into retrovirus expression plasmid pLXSN. The accuracy of recombinant plasmid pLXSN-S was confirmed by restriction enzyme digestion and DNA sequencing;Finally, pLXSN-S was transfected into HepG2、K562 and EBV-immortalized B-cells(EBVC) by means of cationic liposome, respectively. Cells were selected with G418 and HBsAg in culture supernatants were assayed with enzyme-linked immunosorbent assay(ELISA)kit. Results Restriction enzyme digestion and DNA sequencing confirmed that the recombinant retrovirus plasmid inserted HBsAg gene(pLXSN-S)had been constructed correctly and HBsAg were readily detectable in culture supernatants of pLXSN-S transfected cells. Conclusions A retrovirus expression plasmid pLXSN-S has been constructed successfully. Transfection of eukaryotic cells with plasmid pLXSN-S lead to stable expression of HBsAg.