摘要
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 ratroviral vector expressing the dual target antisense RNA of hepatitis B virus (HBV) on the replication and expression of HBV.Methods Recombinant retroviral vector plasmids expressing dual target antisense RNA complementary to HBV X (1400-1430) and P(2375-2405) were constructed and transduced into 2.2.15 cells. The experimental cells were divided into PLXSN+X, PLXSN+P, PLXSN+X/P, and PLXSN+Xpos/P pos groups. The HBV expression was tested by ELISA method. HBV DNA and RNA were tested by FQ PCR. Results The inhibition rates of 2.2.15 cells transduced with recombinant vector plasmids (PLXSN+X group, PLXSN+P group, especially the PLXSN+X/P group) on expression of HBV DNA and RNA were higher than those of blank control group, PLXSN group, and PLXSN+Xpos/Ppos group. The expression of the three ORFs, S, C, and P were significantly inhibited. Conclusion The expression of HBV antisense RNA in 2.2.15 cells can inhibit the replciation and expression of HBV. The inhibitory effect of dual antisense RNA group is higher than that of single antisense RNA group. Dual antisense RNA has the potentiality in anti HBV gene therapy.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2001年第10期605-608,共4页
National Medical Journal of China
基金
国家自然科学基金资助项目 ( 3970 0 12 5 )
