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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 investigate antisense RNA anti-hepatitis B virus (HBV) effect. Methods: Positive and antisense HBV S gene recombinant EB virus vectors pMEP4Ss, pMEP4Sas, DNA-calcium phosphate co-precipitation method of recombinant vector DNA transfected 2.2.15 cells, hygromycin screening for 1 month to obtain resistant cell clones. The HBsAg, HBeAg and HBV DNA levels of the resistant cell culture supernatant were detected by ELISA and dot blot hybridization respectively. RESULTS: The antisense vector had 75%, 51.6%, 70% and 46% inhibition of HBsAg and HBeAg respectively at 1 and 2 months after transfection. The level of HBV DNA in the antisense vector group was lower than that in the control group. The positive carrier group HBeAg, HBVDNA levels and the control group no difference, HBsAg higher than the control group. In the experimental context, the vector has no toxic effect on 2.2.15 cells. Conclusion: The transfer of antisense HBV S gene recombinant vector can effectively inhibit HBV replication and antigen synthesis.