重组组蛋白去乙酰化酶2-pEGFP-C2质粒的构建及表达  

Construction and expression of a pEGFP-C2-HDAC2 recombinant plamid

作　　者：章慧[1,2] 黄成[1,2] 卞尔保[1,2] 赵斌[1,2] 吴宝明[1,2] 刘昌伟[1,2] 陈小霞[1,2] 李俊[1,2] 

机构地区：[1]安徽医科大学药学院,安徽合肥230032 [2]安徽医科大学肝病研究所,安徽合肥230032

出　　处：《中国药理学通报》2014年第6期812-816,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81273526;81072686)

摘　　要：目的将组蛋白去乙酰化酶2(HDAC2)基因克隆入pEGFP-C2载体,探讨构建的质粒转染进非洲绿猴肾成纤维细胞COS-7株相关蛋白和mRNA的表达及蛋白分布部位的情况。方法从人肝癌细胞(HepG2)中获得组蛋白去乙酰化酶2(HDAC2)的cDNA,采用Xho I和BamH I双酶切,用T4连接酶将该cDNA连接pEGFP-C2质粒,将构建成功的pEGFP-C2-HDAC2质粒经PCR、限制性内切酶酶切和测序鉴定后转染非洲绿猴肾成纤维细胞(COS-7),荧光显微镜下观察绿色荧光蛋白表达,RT-PCR和Western blot鉴定HDAC2的表达。结果双酶切鉴定可见HDAC2质粒片段,转染重组质粒后可观察到绿色荧光蛋白的表达,PCR结果可检测到HDAC2 mRNA表达,同时Western blot可检测HDAC2蛋白和GFP蛋白的表达。结论成功构建了重组pEGFP-C2-HDAC2表达质粒,HDAC2蛋白可与绿色荧光蛋白在COS-7细胞中融合表达。Aims HDAC2 gene was cloned into pEGFP-C2 vector to explore the efficiency of the plasmid trans-fection in renal fibroblasts COS-7 cells to identify the expression of both mRNA and protein levels and to ob-serve the distribution of the protein. Methods The HDAC2 cDNA was amlified by PCR and cut with the double enzyme Xho I and BamH I, then inserted into the eukaryotic expression vector pEGFP-C2 with T4 en-zyme. The recombinant vector was verified by PCR, restriction enzymes cut and sequencing identification. Then it was transfected into COS-7 cells and the ex-pression of pEGFP-C2-HDAC2 was monitored by fluorescence microscope and PCR. Results Fragments of HDAC2 could be seen after dealt with double diges-tion, and GFP could also be detected in the transfected COS-7 cells. HDAC2 gene expression could be detec-ted by PCR and Western blot. The fusion expression of pEGFP-C2-HDAC2 could be detected by Western blot. Conclusion Eukaryotic expression vector of HDAC2 has been successfully constructed, the fusion expres-sion of HDAC2 and GFP protein can be detected in COS-7 cells.

关 键 词：组蛋白去乙酰化酶2 COS-7 融合蛋白 PEGFP-C2 HEPG2 绿色荧光蛋白 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R341[医药卫生—基础医学]