Epstein-Barr病毒DNA多聚酶基因扩增和表达载体构建

Amplification and Construction of Expression Vector of the Epstein-Barr Virus DNA Polymerase Gene

【目的】扩增Epstein Barr病毒 (EBV)DNA多聚酶基因 ,构建高效表达载体。【方法】根据EBV全基因序列 ,在EBVDNA多聚酶基因的 3′、5′端设计一对附加EcoRⅠ和HindⅢ酶切位点的特异性引物 ,以B95 8细胞DNA为模板 ,采用改良PCR方法扩增出EBVDNA多聚酶基因 (3 0 44bp)。用EcoRⅠ和HindⅢ酶切该基因片断并克隆至表达载体 pMAL p2 ,采用蓝白斑试验筛选阳性菌落。EcoRⅠ和HindⅢ酶切分析、鉴定重组体 pEBP。【结果】通过电泳鉴定 ,PCR产物为 3 0 44bp ,与EBVDNA多聚酶基因大小一致。经蓝白斑试验及限制性DNA内切酶分析 ,成功地筛选到EBVDNA多聚酶基因重组表达质粒 pEBP。【结论】本实验成功地扩增出完整EBVDNA多聚酶基因 ,并构建了高效表达重组体 ,为进一步在体外表达、制备EBVDNA多聚酶蛋白奠定了基础。

Objective To construct an efficient expression vector of the Epstein-Barr virus DNA polymerase gene. Methods Using B 95-8 cell DNA as template, the EBV DNA ploymerase gene was amplified by modified PCR with a pair of specific primers which contain the restrictive sites of Eco RⅠ and Hin dⅢ. The amplified fragment of EBV DNA polymerase gene was digested with Eco RⅠ and Hin dⅢ, and then cloned into plasmid pMAL-p2. E.Coli TB1 was used as host cells for transformation.The positive recombinant clones were screened by blue-white test.The recombinant plasmids were identified by restriction endonuclease enzyme analysis. Results The agarose gel electrophoresis showed that the amplified fragments were the same size of 3 044 bp as the entire EBV DNA polymerase gene.The recombinant plasmid pEBP containing EBV polymerase gene was successfully selected. Conclusion The modified double enzyme PCR method conducts to amplify a big fragment DNA.The establishment of the recombinant efficient expression vector carrying EBV DNA polymerase gene has provided a base for producing EBV DNA polymerase protein in vitro.