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目的 探讨表达乙型肝炎病毒 (HBV)双靶区反义RNA的逆转录病毒载体对HBV复制和表达的影响。方法 用分子克隆法构建表达HBVX、P双靶区正、反义RNA的重组载体质粒 ,转染PA317细胞 ,用NIH3T3细胞扩增法检测假病毒颗粒滴度 ,以假病毒颗粒感染 2 2 15细胞 ,用酶标法检测其上清HBsAg和HBeAg,并以荧光聚合酶链反应定量方法检测 2 2 15细胞内DNA和RNA含量。结果 HBVX、P双靶区反义RNA抗HBV的作用较单靶区反义RNA更明显 ,且对HBVS、C、P三个开放读码区的DNA和RNA抑制作用大 ,正义RNA无明显抑制作用。结论 细胞内表达HBV反义RNA具有明显抗HBV复制和表达的作用 ,双靶区反义RNA作用更明显。
Objective To investigate the effect of retroviral vector expressing dual target antisense RNA of Hepatitis B virus on HBV replication and expression. Methods Recombinant vector expressing positive and negative RNA of HBVX and P target region was constructed by molecular cloning and transfected into PA317 cells. The NIH3T3 cells were used to detect the titer of pseudovirions and the infected cells were infected with pseudovirus The HBsAg and HBeAg of the supernatant were detected by enzyme-linked immunosorbent assay. The contents of DNA and RNA in 2 215 cells were detected by fluorescence polymerase chain reaction. Results The anti-HBV effect of HBVX and P double-target antisense RNA was more obvious than that of single-target antisense RNA. The inhibitory effect on DNA and RNA of three open reading frames of HBVS, C and P was not significant effect. Conclusion Intracellular expression of HBV antisense RNA has obvious effect on anti-HBV replication and expression, and the effect of antisense RNA in double-target region is more obvious.