论文部分内容阅读
目的:构建hTERT启动子和保留HBV同源序列的缺失点变hTERT启动子(pGL3del445)的真核表达载体,为进一步研究HBV与hTERT启动子的关系奠定实验基础。方法:通过双酶切及PCR方法从pCRTRTP载体上将全长及含有HBV同源序列的hTERT启动子的基因序列克隆到pPLbasic多克隆位点上,构建真核表达载体pGL3TRTP与pGL3Bdel445,将该两质粒与pRLtk共转染不同细胞系,评估其端粒酶启动子活性。结果:经测序证明成功构建了全长及含有HBV同源序列的hTERT启动子的真核表达载体pGL3TRTP和pGL3Bdel445,共转染实验中,证实在永生细胞系中重组子高效表达,在非永生正常细胞中重组子不表达。结论:构建的两种重组表达载体具有端粒酶启动子活性,在不同端粒酶表型的细胞系中其表达具有选择性。
OBJECTIVE: To construct an eukaryotic expression vector of hTERT promoter and hTERT promoter (pGL3del445), which has the same sequence of hTERT, and lay the experimental foundation for further study on the relationship between HBV and hTERT promoter. Methods: The full length and the hTERT promoter containing HBV homologous sequence were cloned into the pPLbasic multi-cloning site by double enzyme digestion and PCR from the pCRTRTP vector, and the eukaryotic expression vectors pGL3TRTP and pGL3Bdel445 were constructed. Plasmids were cotransfected into different cell lines with pRLtk to assess their telomerase promoter activity. Results: The eukaryotic expression vectors pGL3TRTP and pGL3Bdel445 with full length and hTERT promoter containing HBV homologous sequences were successfully constructed. The co-transfection experiments confirmed that the recombinant eukaryotic expression vector was highly expressed in immortalized cell lines, Recombinant cells do not express. CONCLUSION: The two constructed recombinant vectors have the activity of telomerase promoter and their expression is selective in cell lines with different telomerase phenotypes.