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目的 探索利用乙型肝炎病毒 (HBV)作为基因治疗载体的可能性及检验其表达反义RNA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S或S启动子区的反义RNA ,整合于具有HBV复制的 2 .2 .15细胞 ,形成细胞克隆 ,酶联免疫吸附 (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 ,聚合酶链反应(PCR)法检测上清液中重组HBV颗粒。结果 2 .2 .15 pMEP4组、2 .2 .15 SAS组和 2 .2 .15 PAS组对HBsAg平均抑制率分别为 (2 .74± 3.83) %、(6 6 .5 4± 4 .4 5 ) % (P <0 .0 1)和 (5 5 .18± 3.2 7) % (P <0 .0 1) ;对HBeAg平均抑制率分别为 (4 .4 6± 4 .2 5 ) %、(2 6 .36± 1.6 9) % (P <0 .0 1)和 (6 5 .5 4± 3.2 2 ) % (P <0 .0 1) ;对HBV复制的抑制率分别为 17.0 %、5 9.9%和 72 .8%。 2 .2 .15 SAS组及 2 .2 .15 PAS组培养上清液中能检测出突变型HBV颗粒。结论 经过对HBV基因组的两处改造 ,分别在细胞内表达S区及S启动子区反义RNA具有干扰HBV复制及抑制HBV抗原表达的作用 ;在 2 .2 .15细胞中野生型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 Hepatitis B virus (HBV) as a gene therapy vector and to test the anti-HBV effect of expressing antisense RNA. Methods The antisense RNA of S or S promoter region was expressed by gene modification on the plasmid expressing the complete HBV particles and integrated into 2.2.15 cells with HBV replication to form cell clone and enzyme-linked immunosorbent assay (ELISA) ) Method was used to detect the HBsAg and HBeAg in the cell culture supernatant. HBVDNA was detected by dot blot hybridization in the nucleus of HBV nucleus and the recombinant HBV particles were detected by polymerase chain reaction (PCR) in the supernatant. Results The average inhibitory rates of HBsAg in pMEP4 group, 2.2.2 SAS group and 2.2.2 PAS group were (2.74 ± 3.83)%, (6.64 ± 4.44) 5)% (P <0.01) and (5 5 .18 ± 3.2 7)% (P <0.01), respectively. The average inhibitory rates of HBeAg were (4.46 ± 4.25)% , (26.36 ± 1.6 9)% (P <0.01) and (6.55 ± 3.2 2)% (P <0.01), respectively. The inhibition rates on HBV replication were 17.0% , 59.9% and 72.8% respectively. 2 .2 .15 SAS group and 2 .2 .15 PAS group can detect mutant HBV particles in the supernatant. Conclusions After two modifications of the HBV genome, antisense RNAs expressing S and S promoters in the cells respectively interfere with HBV replication and inhibit the expression of HBV antigens; in 2.2.15 cells, wild-type HBV , Still able to package and secrete intact HBV-like particles