Epstein-Barr病毒胸苷激酶(TK)基因表达质粒的构建

Construction of an expression system of Epstein-Barr Virus thymidine kinase(TK)

目的 :构建高效表达 EBVTK的重组克隆体系。方法 :以 p UC8X为模板 ,5’- CTGAATTCATG-GCTGGATTTCC- 3’及 5’CAACTGCAGCCTAGTCCCGATT- 3’为引物 ,用 PCR技术扩增出 EBV tk基因的 DNA片段。Eco R I/ Pst I双酶切 PCR产物和载体 p BV2 2 0 ,T4连接酶使目的基因 tk定向克隆至选定质粒 p BV2 2 0中。结果 :重组质粒 p BV2 2 0 - tk经 Eco R I/ Pst I双酶切后得两条电泳带分别位于 1.8kb、3.6 kb处 ;以 p BV2 2 0 - tk为模板 ,两引物同上进行 PCR扩增 ,产物电泳带于 1.8kb处。结论 :目的基因 tk已定向克隆至质粒 p BV2 2 0中。

Objective:To construct a recombinant plasmid which can express large amounts of the Epstein Barr virus coded thymidine kinase.Methods:The plasmid PUC8X was used as a template for PCR with the 5’primer GTGAATTCATGGCTGGATTTCC and the 3’primer CAACTGCAGCCTAGTCCCGATT to generate a fragment with a 5’EcoR I site and a 3’Pst I site.The DNA was cut to produce an EcoR I Pst I fragment that was ligated into the similarly cut vector pBV220 with T4 ligase.Results:The recombmant DNA pBV220 tk was constructed,which was cut by EcoR I and Pst I and analyzed by agarose gel electrophoresis.Two bands at 1.8 kb and 3.6 kb were obtained.The pBV220 tk was used as a template for PCR with two primers indicated above,the PCR generated fragment was at 1.8 kb analyzed by agarose gel electrophoresis.Conclusion:The aimed gene tk has been inserted into the pBV220 successfully.