乙型肝炎病毒前S1蛋白反式激活蛋白2结合蛋白1基因的克隆及其反式调节基因的筛选

摘要

目的 克隆HBV前S1蛋白反式激活蛋白2结合蛋白1(PS1TP2BP1)基因,并筛选与其相互作用的反式激活基因.方法 应用聚合酶链反应(PCR)扩增PS1TP2BP1基因,鉴定正确后再将其亚克隆到真核表达载体pcDNATM3.1/myc-His A上;以真核表达质粒pcDNATM3.1/mycHis A-PS1TP2BP1转染HepG2细胞,构建cDNA消减文库;进行测序及同源性分析.结果 从HepG2细胞来源的cDNA中扩增出PS1TP2BP1基因,并成功进行TA克隆,酶切、测序均正确后,成功构建真核表达重组体.消减文库扩增后得到35个阳性克隆,经菌落PCR分析,得到15个含200～1000 bp插入片段的菌落.对所得片段测序,并进行间源性分析,显示15种已知基因编码蛋白可能是PS1TP2BP1反式激活靶基因.结论 成功克隆PS1TP2BP1,并构建了PS1TP2BP1反式激活基因差异表达的cDNA消减文库,为今后进一步分析、研究病毒蛋白的致病机制奠定了基础.%Objective To identify genes regulated by HBV preS1-transactivated protein 2 binding protein 1(PS1TP2BP1).Methods PS1TP2BP1 gene was amplified by polymerase chain reaction (PCR)technique and cloned into the eukaryotic expression vector pcDNATM3.1/my-c-His A.The mRNAs isolated from HepG2 cells transfected recombinant eukaryotic expression vector pcDNATM3.1/mycHisA-PS1TP2BP1 and pcDNATM3.1/myc-HisA empty vector were used to construct subtractive library.The differentially expressed genes were idenfied and analyzed.Results 35 differentially expressed clones were obtained.Colony PCR identified 15 clones with 200-1000 bp inserts.Sequence analysis identified 1 5 differentially expressed genes.Conclusion This study provides data for further characterize the function of PS1TP2BP1.