摘要
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 。
Objective To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of antisense RNA. Methods Two parts of HBV genome were reversedly recombined back into overlength HBV genome, which can produce HBV particle, to express antisense RNA complementary to S or S promoter region respectively. HepG 2.2.15 cell lines were transfected with these constructs and the empty vector pMEP4, then 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 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)%、(66.54±4.45)%(P<0.01) and (55.18±3.27)%(P<0.01) in group 2.2.15-pMEP4,2.2.15-SAS and 2.2.15-PAS respectively. The mean inhibitory rates of HBeAg were (4.46±4.25)%,(26.36±1.69)%(P<0.01) and (65.54±3.22)%(P<0.01) respectively. The inhibitory rates of HBc related HBV DNA were 17.0%、59.9% and 72.8% respectively. Recombinant HBV virion was detectable in the culture medium of group 2.2.15-SAS and 2.2.15-PAS. Conclusions Antisense RNA of S or S promoter region can efficiently suppress wt-HBV replication and the expressions of HBV antigens. With the help of wild-type HBV in 2.2.15 cell lines, the constructs can form and secret HBV like particles, and it may be a potential candidate for gene therapy against HBV infection.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2004年第2期90-93,共4页
Chinese Journal of Infectious Diseases
基金
国家自然科学基金资助项目 ( 3 0 170 85 4)
