逆转录病毒载体介导HBV前C/C反义RNA的体外抗HBV作用

Effect of Antisense HBV PreC/C RNA Delivered by Pseudo-Retrovirus Against HBV Replication and Antigen Synthesis

目的 了解反义乙型肝炎病毒 (HBV)基因转移、表达的抗HBV作用。方法 BamHⅠ酶切HBV质粒获取 1 5 0 4bp前C/C基因片段 ,将其正、反向插入逆转录病毒载体pDOR构建了正、反义前C/C重组体。将重组体转染逆转录病毒包装细胞PA31 7,G4 1 8筛选获取抗性细胞及假逆转录病毒。RT PCR检测了反义HBV基因表达的反义RNA。将重组假逆转录病毒感染 2 2 1 5细胞 ,ELISA及斑点杂交法分别检测被感染细胞培养上清中HBsAg、HBeAg及HBVDNA。结果 反义前C/C重组逆转录病毒载体在细胞内能转录HBV反义RNA。反义前C/C重组假病毒感染的 2 2 1 5细胞HBsAg、HBeAg的合成分别降低了 5 5 5 %、78% ;HBVDNA水平也明显低于空白对照组。该抑制具有一定特异性。增加假病毒感染次数在一定范围内能提高抑制作用。正义假逆转录病毒感染对 2 2 1 5细胞HBVDNA水平及抗原合成无影响。结论 逆转录病毒载体介导反义HBV基因转移表达的反义RNA能抑制HBV复制及抗原合成 。

Objective To explore the effect of antisense RNA on hepatitis B virus (HBV) replication and antigen synthesis.Methods Plasmid pHBV containing a copy of HBV genome was digested with BamHⅠto yield a 1504bp HBV PreC/C gene fragment. The PreC/C gene fragment was cloned into the BamHⅠsite of the retroviral vector pDOR to construct antisense and sense recombinant vector. The recombinant vector DNA and pDOR DNA were introduced into PA317 packaging cell by lipofectin method and the trnasfected PA317 cells and pseudovirus were obtained by selecting with G418. The antisense RNA transcribed from antisense PreC/C was detected by RT PCR. The pseudovirus was used to infect the 2 2 15 cell line. In infected 2 2 15 cells, the levels of HBsAg, HBeAg and HBV DNA were determined by ELISA and dot hybridization, respectively. Results The retroviral vector pDOR could efficiently deliver antisense HBV gene into eukaryotic cells and transcribe the antisense RNA. The antisense HBV PreC/C vector transfected 2 2 15 cells reduced synthesis of HBsAg and HBeAg by 55 5% and 78% respectively, while sense PreC/C transfected cells showed no reduction on HBsAg and HbeAg production. HBV DNA was significantly decreased in the supernatants of 2 2 15 cells infected with antisense pseudovirus than that infected with sense pseudovirus group and control group. The antisense pseudovirus demonstrated no toxic effect on the infected 2 2 15 cells. Conclusion Antisense HBV PreC/C recombinant pseudo retrovirus could be used to deliver and express the antisense RNA against HBV replication.This may be useful an agent for antiviral therapy of HBV infection.