反义乙肝病毒S基因真核载体构建及体外抗乙肝病毒作用

CONSTRUCTION OF ANTISENSE HBV S GENE MAMMALIAN VECTOR AND ITS ANTI-HBV EFFECT

目的:探讨反义 RNA 抗乙肝病毒(HBV)作用。方法:构建了正、反义 HBV S 基因重组 EB 病毒载体 pMEP4Ss、pMEP4Sas,DNA-磷酸钙共沉淀法将重组载体 DNA 转染2.2.15细胞,潮霉素筛选1个月得到抗性细胞克隆。ELISA、斑点杂交法分别检测抗性细胞培养上清 HBsAg、HBeAg、HBV DNA 水平。结果:转染后1、2月,反义载体对 HBsAg、HBeAg 的抑制率分别达75%、51.6%;70%、46%。反义载体组 HBV DNA 水平低于对照组。正义载体组 HBeAg、HBVDNA 水平与对照组无差别,HBsAg 高于对照组。在实验范围内,载体对2.2.15细胞无毒性作用。结论:反义 HBV S 基因重组载体转移能有效抑制 HBV 复制及抗原合成。

Objective:To explore the effect of antisense RNA on tlBV.Method:Sense and antisense HBV S gene recombinant EB virus vector pMFP4Ss and pMEP4Sas were constructed.Using DNA-calcium phosphat coprecipitation method,recombinant plasmids were transfered into HepG_2 2.2.15 cells,then posi- tive clones were selected by month one-hygromycine culture.ELISA and DNA dot blot were used to examine HBsAg,HBeAg and HBV DNA level in cuctured medium.Results:The inhibitions of antisense vector on HBsAg,HBeAg at one and two mouth after transfection were 75%,51.5% and 70%,46%. respec- tively.Meanwhile HBV DNA level in antisense group was lower than that in control.There were no significant differences of HBeAg and HBV DNA level befween sense group and control,but HBsAg in sense group was higher than that in controI.the recombinant plasminds within experimented concentrantions exhibited nontoxicitey on HepG_2 2.2.15 cells.Conclusion:The study demonstrated that recombinant