不同类型的HBsAg真核表达质粒在EBV永生化B淋巴母细胞中的表达

目的 探讨3种不同类型的HBsAg真核表达质粒在EBV永生化B淋巴母细胞中的表达。方法 用3种不同类型的质粒载体分别构建乙型肝炎表面抗原（HBsAg)真核表达质粒（pCI-S,pMEP4-S,pLXSN-S)；然后用阳离子脂质体介导的转染法分别转染正常人EBV永生化的B淋巴母细胞，经G418或HygromycinB筛选出抗性细胞克隆，用RT-PCR检测抗性细胞总RNA中目的基因在转录水平的表达；用ELISA检测抗性细胞培养上清和细胞裂解液中HB-sAg的含量。结果 3种不同类型HBsAg重组表达质粒转染的EBV永生化B淋巴母细胞，在转录水平上，可检出HB-sAgmRNA的表达；在蛋白水平上，细胞增养上清和细胞裂解液均可检出HBsAg；而以EB病毒表达质粒pMEP4-S表达最高，真核表达质粒pCI-S和逆转录病毒表达质粒pLXSN-S表达HBsAg的量无明显差异。结论 3种不同类型的HBsAg真核表达质粒均可在EBV永生化B淋巴母细胞中稳定表达，EB病毒表达质粒pMEP4-S表达的HBsAg明显高于真核表达质粒pCI-S和逆转录病毒表达质粒pLXSN-S。

Expression of HBsAg with Three Kinds of Eukaryotic Expression Plasmids Inserted HBsAg Gene in EBV-Immortalized B-cell Line

Objective To investigate the expression of HBsAg with three kinds of eukaryotic expression plasmids inserted HBsAg gene in Epstein-Barr virus-immortalized B-cell line. Methods Three kinds of plasmids inserted HBsAg gene were constructed (pCI-S?pMEP4-S? pLXSN-S) and their accuracy was confirmed by restriction enzyme digestion. Then these recombinant plasmids were transfected into EBV-immortalized B-cells by means of cationic liposome,respectively.Cells were selected with G418 or hygromycin B,and mRNA of HBsAg gene in these selected cells were detected by RT-PCR and HBsAg in culture supernatants and cell lysates were assayed with enzyme-linked immunosorbent assay(ELISA)kit. Results mRNA of HBsAg in selected cells transfected three kinds of eukaryotic expression plasmids inserted HBsAg gene all can been detected by RT-PCR, and these transfected cells produce HBsAg which is readily detectable in culture supernatants and cell lysates.But level of HBsAg expressived by pMEP4-S in EBV-immortalized B-cells is higher than those expressived by pCI-S or pLXSN-S. Conclusions Transfection of EBV-immortalized B-cell line with three kinds of plasmids inserted HBsAg gene all lead to stable expression of HBsAg. Level of HBsAg expressived by pMEP4-S in EBV-immortalized B-cells is higher than those expressived by pCI-S or pLXSN-S .