复制缺损型HBV表达显性阴性突变体抗HBV作用的研究

Study on anti-HBV effects of genetically engineered replication-defective hepatitis B virus expressing dominant negative mutants of core protein

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 delivery vector,and to test the anti-HBV effects by intracellular expression of dominant negative mutants of core protein. Methods Two kinds of full length mutant HBV genome,which express Core-partial P and Core-S fusion protein,were 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 and 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. Results The mean inhibitory rates of HBsAg were 2.74±3.83%,40.08±2.05% ( P <0.01) and 52.94±1.93% ( P <0.01) in group 2.2.15-pMEP4,2.2.15-CP and 2.2.15-CS,respectively. The mean inhibitory rates of HBeAg were 4.46±4.25%,52.86±1.32% ( P <0.01) and 41.60±1.65% ( P <0.01),respectively. The inhibitory rates of HBc related HBV DNA were 15.3%,82.0% and 67.2%, respectively. Recombinant HBV virion was detectable in the culture medium of only group 2.2.15-CP. Conclusion Dominant negative mutants of core protein can efficiently suppress wt-HBV replication and the expressions of HBV antigens. With the help of wild-type HBV,the recombinant 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.