HBV基因T1862突变真核表达载体的构建及在Cos7细胞中的表达

Construction of eukarotic expression vector of HBV gene with nt 1862 mutation and its expression in Cos7 cells

目的研究ＨＢＶＴ１８６２变异的生物学意义。方法 采用分子生物学方法，构建 ＨＢＶ前Ｃ／Ｃ基因ＥＢ病毒真核表达 载体，利用体外定点突变技术诱导前Ｃ／Ｃ基因Ｔ１８６２变异，经ＰＣＲ－ＲＦＬＰ初筛并经测序终鉴定出阳性克隆后，以脂质体 介导方法将突变前后的重组质粒转染Ｃos７细胞，以 ＥＬＩＳＡ检测 ＨＢeＡg的表达量。结果 未突变的重组质粒可稳定表 达ＨＢｅＡｇ，突变后的重组质粒未能检测到ＨＢｅＡｇ表达。结论ＨＢＶ前Ｃ／Ｃ基因１８６２点突变的真核表达载体的构建， 为体外研究该点突变引起ＨＢＶ的一系列生物学改变奠定基础。

Objective To study the biological significance of HBV gene mutation at nucleotide (nt) 1862. Methods The EB virus eukarotic expression vector for HBV pre-C/C gene was constructed using molecular biological method, HBV pre-C/C gene mutation at nt 1862 was induced by way of site-specific mutation technique, and identified by PCR-RFLP and sequencing analysis. The resulted recombinant plasmid containing the HBV variant was subsequently transfected into Cos7 cell line mediated by lipofectin, to observe the expression of HBeAg. The cells transfected with the recombinant plasmid containing wild HBV Pre-C/C gene fragment served as control. Results HBeAg expression was detected in the cells transfected with wild recombinant plasmid but not in those with HBV variant transfection. Conclusion The success in the construction of eukarotic expression vector for HBV pre-C/C gene mutation at nt 1862 may pave the way for further studying a series of biological changes of HBV resulted from the mutation addressed in this study.