本研究目的是构建HPV16E6真核表达载体,为细胞转染研究E6蛋白和p53Arg或p53Pro蛋白的相互作用以及作用后对细胞功能的影响奠定基础。应用PCR技术从质粒pSP64-HPV16E6上扩增出HPV16E6片断,根据Kozak序列对真核蛋白表达的影响,设计2条引物扩增出2个HPV16E6目的片断均插入真核表达载体pcDNA3.1/myc-His(-)A中,构建pcDNA3.1/myc-His(-)A-HPV16E6重组质粒。研究结果显示:经酶切和测序鉴定克隆的基因片段为HPV16E6 cDNA;建立了pcDNA3.1/myc-His(-)A-HPV16E6重组质粒。这表明,已成功构建HPV16E6真核表达载体pcDNA3.1/myc-His(-)A-HPV16E6。

To construct eukaryotic expression vector pcDNA3.1/myc-His(-) A-HPV16 E6 for investigation on the function and mechanism of HPV16 E6 in cell transfection.Methods: The encoding fragment of HPV16 E6(500 bp) containing the digestion site of BamHⅠ and HindⅢ was amplified from the plasmid pSP64-HPV16 E6 by means of PCR and then inserted into the eukaryotic expression vector pcDNA3.1/myc-His(-) A.Recombinant pcDNA3.1/myc-His(-) A-HPV16 E6 vectors were then transformed into E.coli DH5α of competence.The positive clones were selected and recombinant pcDNA3.1/myc-His(-) A-HPV16 E6 plasmids were extracted.The plasmids were digested with BamHⅠ and HindⅢ and identified by agarose gel electrophoresis.And DNA sequence code was checked with automatic sequenator.Results: A 500 bp specific fragment were obtained and DNA sequencing showed that the fragment was consistent with those of the published.After the recombinant expression plasmid was confirmed by enzymes digestion and sequence analysis,it showed that the eukaryotic expression vector of HPV16 E6 was constructed successfully.Conclusion:The protein of HPV16 E6 will be obtained for further study.Successful construction of recombinant pcDNA3.1/myc-His(-) A-HPV 16 E6 plasmids lays solid foundation experiments and clinical trials.