HBV信号肽区热点变异真核表达载体的构建及对HBeAg表达的影响

采用分子生物学方法，设计引入KpnⅠ和HindⅢ酶切位点的引物扩增HBV的Pre－C／C片段（1814－2452），经酶切后连于EBO真核表达载体，利用定点突变技术分别对连接载体进行了T1862，A1896，A189，A1896＋A1899点的定点诱变。经错配PCR－RFLP和测序, 克隆。将突变前后的质粒以脂质体转染法转染HepG2细胞，检测不同点突变后HBeAg表达的改变，突变前后质粒转染HepG2细胞经稳定表达，结果未突变的EBO－PreC/C重组质粒HBeAg表达为强阳性，A1899突变的重组质粒HBeAg表达比未突变的重组质粒稍弱，而转染EBO空载体和T1862，A1896，A1986＋A1899重组质粒突变体的细胞培养上清HBeAg均为阴性，上述体的构建将为体外研究前C信号肽区热点变异对HBeAg在细胞中表达及对肝炎病毒基因组复制的影响奠定基础。

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTORS WITH HOT SPOT MUTATIONS IN HBV SIGNAL PEPTIDE REGION

Molecular biological methods were used to construct the eukaryotic expression vectors of precore and core gene named EBO PreC/C. Then T1862, A1896, A1899 and A1896+A1899 variants were constructed by site mutagenesis in vitro. By PCR RFLP and sequencing ,T1862,A1896,A1899 and A1896+A1899 variants were obtained. Wild type and variants were transfected to HepG2 cells, and HBeAg was tested to observe the difference of HBeAg expression between wild type and variants. After stable expression in HepG2 cells, HBeAg was detected to be positive in cells transfected wild type and A1899 variants, and negative in cells transfected with T1862, A1896,A1896+A1899 variants. The construction of these variants will play an important pole in studying the relation of PreC/C mutations and HBV expression and replication of HBV genome.