人工锌指蛋白真核表达载体的构建及表达  

Construction of eukaryotic expressing vector of artificial zinc finger protein gene and its expression

作　　者：魏勇[1] 应大君[1] 朱楚洪[1] 侯春丽[1] 崔晓萍[1] 

机构地区：[1]第三军医大学基础医学部解剖学教研室,重庆市生物力学与组织工程重点实验室,重庆400038

出　　处：《第三军医大学学报》2008年第2期134-137,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30472027)~~

摘　　要：目的构建人工锌指蛋白ZFP基因的真核表达载体,检测其在真核细胞的表达情况,从而探讨人工锌指蛋白作为人工转录因子DNA结合域的可能性。方法全基因合成人工锌指蛋白(putative zinc finger protein,ZFP)的核酸序列并克隆入pEGFP-N1及pIRES2-EGFP真核表达载体中,构建重组质粒pEGFP-N1/ZFP及pIRES2-EGFP/ZFP-Flag,通过酶切、菌落PCR及测序来鉴定重组载体的正确性。脂质体DOTAP体外转染重组质粒于COS-7细胞中,用荧光显微镜、RT-PCR、Western blot方法鉴定ZFP在该细胞中的表达。结果正确构建了pEGFP-N1/ZFP、pIRES2-EGFP/ZFP-flag重组表达质粒,并且在COS-7细胞中成功进行了人工锌指蛋白ZFP的表达。结论采用生物信息学手段获得的假定的锌指蛋白基因序列可以在真核细胞内正常表达。Objective To construct the recombinant plasmid pEGFP-N1/ZFP and pIRES2-EGFP/ZFP- flag and explore the possibility of engineering zinc finger protein （ZFP） as DNA binding domain of artificial transcription factor （ATF）. Methods Full length ZFP gene was synthesized and cloned into pEGFP-N1 and pIRES2-EGFP plasmids to construct the recombinant plasmids pEGFP-N1/ZFP and pIRES2-EGFP/ZFP-flag that were identified by colony PCR and sequencing. Recombinant plasmids were transformed into COS-7 cells by liposome DOTAP. The expressed EGFP was observed under fluorescent microscope and the ZFP expression was detected by RT-PCR and Western blot. Results Recombinant plasmids pEGFP-NI/ZFP and pIRES2-EG- FP/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 by bioinformatics technique can be expressed in eukaryotic cells.

关 键 词：人工锌指蛋白 真核表达载体 EGFP COS-7细胞 

分 类 号：R341[医药卫生—基础医学] R394-33