基因重组HBV联合表达反义RNA和显性阴性突变体抗HBV作用及HBV包装细胞系的构建

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-CPAS质粒转染G418抗性包装细胞系,在细胞培养上清液中检出重组HBV,未检出野生型HBV.结论在同一载体上联合表达S区反义RNA及核心蛋白与部分P蛋白的融合蛋白,具有较单一机制更强的抗HBV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.包装细胞系能为复制缺损型HBV提供包装,但效率较低.

Anti-HBV effects of genetically engineered replication-defective HBV with combined expression of antisense RNAand dominant negative mutants of core protein and construction of first-generation packaging cell line for HBV vector

OBJECTIVE: To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular combined expression of antisense RNA and dominant negative mutants of core protein. METHODS: Full length of mutant HBV genome, which expresses core-partial P fusion protein and/or antisense RNA, was transfected into HepG2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method. Intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Free of packaging signal, HBV genome, which express the HBV structural proteins including core, pol and preS/S proteins, was inserted into pCI-neo vector. HepG2 cell lines were employed to transfect with the construct. G418 selection was done at the concentration of 400mug/ml in the culture medium. The G418-resistant clones with the best expression of HBsAg and HBcAg were theoretically considered as packaging cell lines and propagated under the same conditions. It was transfected with plasmid pMEP-CPAS and then selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. RESULTS: The mean inhibitory rates of HBsAg were 2.74% 3.83%, 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) in group 2.2.15-pMEP4, 2.2.15-CP, 2.2.15-SAS, and 2.2.15-CPAS, respectively. The mean 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% (t=39.0, P<0.01), respectively. The inhibitory rates of HBc related HBV DNA were 0, 82.0%, 59.9%, and 96.6%, respectively. Recombinant HB virion was detectable in the culture medium of all the three treatment groups. G418-resistant HBV packaging cell line, which harbored an HBV mutant whose packaging signal had been deleted, was generated. Expression of HBsAg and HBcAg was detectable. Transfected with plasmid pMEP-CPAS, it was found to secrete recombinant HB virion and no wild-type HBV was detectable in the culture medium. CONCLUSIONS: It has stronger anti-HBV effects by combined expression of antisense RNA and dominant negative mutants than by individual expression of them. With the help of wild-type HBV, the modified HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified. The packaging cell line can provide packaging for replication-defective HBV, but with low efficiency.