人乳头瘤病毒16型E6蛋白真核表达载体pGADT7-E6的构建与鉴定

Construction and Identification of Eukaryotic Expression Vector pGADT7-E6 of HPV16 E6 Protein

目的构建HPV16 E6基因的真核表达载体,为在酵母菌中表达以及进一步探讨HPV16E6基因与宫颈癌发生的关系奠定前期实验基础。方法用PCR扩增含EcoRⅠ和BamHⅠ酶切位点的E6基因序列,双酶切pGADT7载体与E6基因PCR产物,纯化回收后,用连接酶将二者连接并转化大肠杆菌JM109,筛选阳性克隆,并对其进行双酶切和测序鉴定,构建HPV16 E6基因的真核表达载体pGADT7-E6。结果双酶切pGADT7-E6后,琼脂糖凝胶电泳显示2个片段,分别在约500 bp与8.0 kb处。DNA测序结果经与GenBank登录的序列做BLAST分析,显示重组质粒含有477 bp的目的基因片段,读码框正确,无碱基错配和移码突变。结论成功构建了HPV16 E6基因真核表达载体pGADT7-E6。

Objective To construct the eukaryotic expression vector of E6 gene of HPV16 for expressing in the yeast and exploring the contribution of E6 gene to the uterine cervix cancer. Methods E6 gene with EcoR I and BamH I endoenzyrne sites was amplified by PCR, and cloned into pGADT7 vector. The positive clones were analyzed by endonuclease restriction digestion and sequencing. Results A BLAST search confirmed that the cloned fragment contained the complete open reading frame of E6 gene of HPV16. Conclusion Eukaryotic expression vector of E6 gene of HPV16 was constructed successfully.