特异性结合于HBV X基因启动子的人工锌指蛋白真核表达载体的构建及表达  

Construction of eukaryotic expression vector of artificial zinc finger protein gene of specific binding to X gene promoter of HBV and its expression

作　　者：李鑫[1] 吴忠均[1] 陈聪[1] 

机构地区：[1]重庆医科大学附属第一医院肝胆外科,重庆400016

出　　处：《重庆医科大学学报》2011年第3期270-273,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金项目资助(编号:30772055)

摘　　要：目的:构建人工锌指蛋白(Putative zinc finger protein,ZFP)基因的真核表达载体,验证其在真核细胞内的表达情况,为进一步构建人工转录因子(Artificial transcription domain factor,ATF)所需的DNA结合结构域寻找合适的ZFP。方法:合成ZFP的全基因核酸序列并载入pEGFP-N1真核表达载体中,构建重组质粒pEGFP-N1/ZFP及pEGFP-N1/ZFP-Flag,通过菌液PCR、酶切和测序来鉴定重组质粒的正确性。脂质体Lipofectamine 2000将重组质粒转染于COS-7细胞中,使用RT-PCR、荧光显微镜、Western blot方法鉴定ZFP在该细胞中的表达。结果:成功构建了pEGFP-N1/ZFP及pEGFP-N1/ZFP-Flag重组质粒,并且在COS-7细胞中进行了ZFP的表达。结论:通过生物信息学工具获取的ZFP氨基酸序列,经过密码子优化得到的基因序列能够在真核细胞内成功表达。Objective： To construct eukaryotic expression vector of zinc finger protein（ZFP） gene,and to detect the eukaryotic expression vector of zinc ZFP gene expressed in eukaryotic cells and search the available ZFP as DNA binding domain of artificial transcription factor（ATF）.Methods：Full length ZFP gene was synthesized and cloned into pEGFP-N1 plasmids to construct the recombinant plasmids pEGFP-N1/ZFP and pEGFP-N1/ZFP-flag that were identified by colony PCR and sequencing.Recombinant plasmids were transformed into COS-7 cells by liposome Lipofectamine 2000.The expressed EGFP was observed under fluorescent microscope and the ZFP expression was detected by RT-PCR and Western blot.Results：Recombinant plasmids pEGFP-N1/ZFP and pEGFP-N1/ZFP-flag were constructed correctly which could express ZFP well in the transformed COS-7 cells.Conclusion：It is feasible that engineered artificial zinc finger proteins obtained by bioinformatics technique and codon optimization can be successfully expressed in eukaryotic cells.

关 键 词：人工锌指蛋白 真核表达载体 COS-7细胞 

分 类 号：Q782[生物学—分子生物学]