乙型肝炎病毒X基因-丙型肝炎病毒C基因融合表达蛋白对肝癌细胞端粒酶活性的影响

Effect of hepatitis B virus X gene and hepatitis C virus C gene coexpression protein on telomerase activity of HepG2 cells

目的建立HBVX-HCVC融合蛋白细胞表达模型,并探讨其对细胞端粒酶活性的影响。方法双酶切质粒pXT1-X,得到完整的HBVX基因片段后,将其插入到质粒PBK-CMV和PBK-HCVC的相应酶切位点,得到重组质粒PBK-X和PBK-X-C;再将质粒PBK-CMV、PBK-X、PBK-HCVC和PBK-X-C分别导入肝癌细胞株HepG2中,G418筛选,RT-PCR、蛋白印迹鉴定HBVX和HCVC蛋白表达。PCR-ELISA法检测端粒酶活性。结果质粒PBK-CMV、PBK-X、PBK-HCVC和PBK-X-C在HepG2细胞中有稳定表达。表达融合蛋白的细胞的端粒酶活性较转染空载体的细胞及单独表达HBVX、HCVC蛋白的细胞明显升高。结论HBVX-HCVC融合蛋白能显著上调端粒酶活性,提示HBV、HCV可能具有协同致癌作用。

Objective To establish an experimental model of HCV C-HBV X coexpression protein and explore its effect on tolemerase activity. Methods The HBV X gene was recovered by enzyme digestion, cloned into PBK-CMV and PBK-HCV C, and the recombinant plasmids PBK-X and PBK-X-C were obstained. The plasmids PBK-CMV, PBK-X, PBK-HCV C and PBK-X-C were transfected into HepG2 cells with liposome. After selected with G418, positive colonies were obtained. The reverse transcription PCR and Western blotting were used to detect HBV X and HCV core protein expression and PCR-ELISA for tolemerase activity. Results The recombinant plasmid PBK-X-C could express HBV X and HCV core protein efficiently. The telomerase activity of the cells coexpressed HCV C-HBV X protein was higher than that of cells expessed HBV X, HCV C and vector only. Conclusion HBV X-HCV C coexpression protein can increase the telomerase activity of HepG2 cells, which suggests that HBV and HCV can cooperate with carcinogenesis.