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目的 探讨HBV作为基因治疗载体的可能性并检验其联合表达反义RNA和显性阴性突变体抗HBV的作用。方法 在表达完整HBV颗粒的质粒上,经基因修饰后联合表达S区反义RNA和核心-P蛋白的融合蛋白,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HBV DNA,PCR检测上清液中重组HBV颗粒。HBV全基因经删除包装信号ε区后,插入到G418抗性PCI-neo载体,转染HepG2细胞系,用G418筛选形成细胞克隆,检测表达HBsAg及HBcAg较多者作为HBV包装细胞系,进一步转染表达复制缺损型HBV的质粒,经两种抗生素同时筛选,PCR方法观察上清液中的病毒。结果 2.2.15-pMEP4组、2.2.15-CP组、2.2.15-SAS组和2.2.15-CPAS组,对HBsAg平均抑制率分别为2.74％±3.83％、40.08％±2.05％（t=35.5,P<0.01）、66.54％±4.45％（t=42.3,P<0.01）和73.68％±5.07％（t=51.9,P<0.01）;对HBeAg平均抑制率分别为4.46％±4.25％、52.86％±1.32％（t=36.2,P<0.01）、26.36％±1.69％（t=22.3,P<0.01）和 59.28％±2.10％（t=39.0,P<0.01）;对 HBV复制的抑制率分别为0、82.0％、59.9％和96.6％。在各治疗组培养上清液中均能检测出重组HBV颗粒。证明包装细胞系具有HBSAg和HBcAg表达,pMEP-CP
Objective To investigate the possibility of HBV as a gene therapy vector and to examine its anti-HBV effect by co-expressing antisense RNA and dominant negative mutants. Methods The fusion protein expressing antisense RNA and core-P protein of S region was genetically modified and integrated into 2.2.15 cells with HBV replication to form cell clones. The cell culture was detected by ELISA The HBsAg and HBeAg in the supernatant were detected by dot blot hybridization, and the recombinant HBV particles in the supernatant were detected by PCR. The whole gene of HBV was deleted after the packaging signal epsilon region and inserted into the G418-resistant PCI-neo vector, transfected into HepG2 cell line, and screened by G418 to form cell clones. HBsAg and HBcAg were detected as HBV packaging cell lines, The plasmid expressing replication-deficient HBV was stained by two antibiotics simultaneously, and the virus in the supernatant was observed by PCR. Results The average inhibitory rates of HBsAg in 2.2.15-pMEP4, 2.2.15-CP, 2.2.15-SAS and 2.2.15-CPAS groups were 2.74% ± 3.83% and 40.08% ± 2.05% (t = 35.5, P <0.01), 66.54% ± 4.45% (t = 42.3, P <0.01) and 73.68% ± 5.07% (t = 51.9, P <0.01). The average inhibitory rates of HBeAg were 4.46% ± 4.25% 52.86% ± 1.32% (t = 36.2, P <0.01), 26.36% ± 1.69% (t = 22.3, P <0.01) and 59.28% ± 2.10% Rates were 0,82.0%, 59.9% and 96.6% respectively. Recombinant HBV particles could be detected in the culture supernatant of each treatment group. The packaging cell lines proved to have HBSAg and HBcAg expression, pMEP-CP