论文部分内容阅读
目的 探索HBV作为基因治疗载体的可能性及检验HBV点突变表达显性阴性突变体抗HBV的作用。方法 在表达完整HBV颗粒的质粒上 ,经基因修饰后分别表达核心 P蛋白及核心 表面抗原的融合蛋白 ,整合于具有HBV复制的 2 2 15细胞 ,形成细胞克隆 ,ELISA法检测细胞培养上清液中HBsAg和HBeAg ,斑点杂交法检测细胞内HBV核壳中HBVDNA ,PCR检测上清液中重组HBV颗粒。结果 2 2 15 pMEP4组、2 2 15 CP组、2 2 15 CS组 ,对HBsAg平均抑制率分别为 2 74 %±3 83%、4 0 0 8%± 2 0 5 % (P <0 0 1)和 5 2 94 %± 1 93% (P <0 0 1) ;对HBeAg平均抑制率分别为4 4 6 %± 4 2 5 %、5 2 86 %± 1 32 % (P <0 0 1)和 4 1 6 0 %± 1 6 5 % (P <0 0 1) ;对HBV复制的抑制率分别为 15 3%、82 0 %和 6 7 2 %。仅在 2 2 15 CP组培养上清液中能检测出突变型HBV颗粒。结论 在细胞内表达显性阴性突变体具有干扰HBV复制及抑制HBV抗原表达的作用 ;经修饰后的HBV基因组在野生型HBV辅助下 ,仍能包装并分泌完整的HBV样颗粒。
Objective To explore the possibility of using HBV as a gene therapy vector and to test the effect of HBV point mutation in expressing dominant negative mutant against HBV. Methods The fusion protein of core P protein and core surface antigen was expressed on the plasmid expressing the complete HBV DNA after gene modification and integrated into 2 215 cells with HBV replication to form cell clone. The cell culture supernatants HBsAg and HBeAg in HBsAg were detected by dot blot hybridization. HBV DNA in supernatant was detected by PCR. Results The mean inhibitory rates of HBsAg in 2 2 15 pMEP4, 2 2 15 CP and 2 2 15 CS groups were 2 74% ± 3 83% and 4 0 0 8% ± 2 0 5%, respectively (P 0 01 ) And 5 2 94% ± 1 93% (P 0 01) respectively. The average inhibitory rates of HBeAg were 446% ± 425% and 5286% ± 132%, respectively (P <0.01) And 4 1 6 0% ± 1 65% respectively (P 0 01). The inhibitory rates of HBV replication were 15 3%, 82 0% and 67 2%, respectively. Only mutant HBV particles were detected in 2 2 15 CP culture supernatants. Conclusion The expression of dominant negative mutant in the cell may interfere with HBV replication and inhibit the expression of HBV antigens. The modified HBV genome can still package and secrete the complete HBV-like particles with the help of wild-type HBV.