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两种端粒酶逆转录酶启动子真核表达载体的构建
http://www.100md.com 《中国普通现代外科进展》 2005年第4期
肝炎病毒,,端粒酶逆转录酶·启动子·基因表达·基因融合·肝炎病毒,乙型,1材料与方法,2结果,3讨、论,参考文献
     【摘要】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(pGL3del445)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pCRTRTP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pPLbasic多克隆位点上,构建真核表达载体pGL3TRTP与pGL3Bdel445,将该两质粒与pRLtk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pGL3TRTP和pGL3Bdel445,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。

    【关键词】端粒酶逆转录酶·启动子·基因表达·基因融合·肝炎病毒,乙型

    Construction of two different recombinant plasmids containing human telomerase reverse transcriptase gene promoter

    LIU Hua,MA Chunhong,LIU Suxia,WANG Xiaoyan,GAO Li fen,HAN Lihui,ZHU Faliang,ZHANG Yan,YANG Yongmei,WU Weifang

    1Immunology Institute,2Institute of Biochemistry and Molecnlar Biology,School of

    Medicine,Shandong University (Jinan 250012,China)

    【ABSTRACT】Objective:To construct two eukaryotic expression vector containing human telomerase reverse transcriptase (hTERT) gene promoter.Methods:FULLlength and deleted hTERT promoter sequence which contain HBV homology sequence were respectively cloned into pGL3basic multiple cloning sites to construct recombinant plasmids pGL3TRTP and pGL3Bdel445.To detect the transcriptional activity of hTERT promoter in the plasmids,transient transfection was performed in different cells.pRLtk was used to mormalize the transfection efficiency.Results:The sequencing data of expression vectors was correspondence with the design.The results of transient showed that the two recombinant plasmids could highly expressed in immortal cells Lo2 and carcinoma cells BEL7402,but not in mortal cells.Conclusion:Two different recombinant plasmids containing hTERP promoter has been successfully constrcucted which could selectively express in hTERT positive cells. ......

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