乙肝病毒X基因真核表达载体的构建及人肝细胞株HL-7702转染

Construction of hepatitis B virus X gene expression vector in eucaryotic cells and its transfection in HL-7702 cells

目的:构建表达乙肝病毒(HBV)X基因的人肝细胞株. 方法:用PCR法扩增HBVX基因序列,将其添A后连至PUCmT载体上,用EcolⅠ和Hind Ⅲ双酶切PUCmT-X和PcDNA3载体,连接酶切片段PcDNA3及X片段以构建重组质粒PcDNA3-X.用脂质体转染法将PcDNA3-X及空质粒PcDNA3导入肝细胞HL-7702中,G418选择培养,RT- PCR鉴定其稳定表达. 结果:已构建的PcDNA3-X经序列测定含有完整的HBV X 基因片段,转入HL-7702细胞后经RT-PCR证实该细胞有稳定表达X蛋白. 结论:成功构建了表达HBV X基因的肝细胞株,为进一步探讨HBV X基因在肝炎与肝癌发生中的作用提供了理想的实验模型.

AIM: To establish a human hepatocyte cell line which can express hepatitis B virus (HBV) X gene. METHODS: HBV X gene was obtained through PCR technology. After A-tailing added, X gene was connected into vector PUCmT. Vector PUCmT-X and PcDNA3 were digested with EcoRI and HindⅢ. The fragments of X and PcDNA3 were connected to establish reconstituted plasmid PcDNA3-X. Then PcDNA3-X and PcDNA3 were transfected into HL-7702 cells by lipid-mediated transfection. After selected with G418, HL-7702/HBx cells were analysed by the reverse transcription-PCR to confirm the steady expression of X gene in HL-7702. RESULTS: Reconstituted plasmid PcDNA3-X included the anticipated fragment of HBV X gene was proved by auto-sequencing assay. RT-PCR analysis showed that reconstituted plasmid PcDNA3-X could express the X protein efficiently in HL-7702 cells. CONCLUSION: Hepatocyte can express HBV X gene, which is an ideal model to study the effect of HBV X gene on the development of hepatitis and hepatocelular carcinoma.