重组真核质粒pEGFP-C2-hG3BP-Domain(1～5)的构建及表达  

Construction and Expression of pEGFP-C2-hG3BP-Domain (1-5) Recombinant Eukaryotic Plasmids

作　　者：朱梦瑜[1] 高星杰[2] 钱宝鑫[1] 葛林[3] 赵虹[1] 赵秀娟[1] 杨洁[2,3] 

机构地区：[1]天津医科大学免疫教研室,300070 [2]天津医科大学基础医学研究中心 [3]天津医科大学生化教研室

出　　处：《天津医药》2011年第11期990-992,1088,共4页Tianjin Medical Journal

基　　金：国家科技部"863"项目(项目编号:2007AA02Z115);国家自然科学基金资助项目(项目编号:30970582);天津市应用基础研究计划项目(项目编号:07JCZDJC07300)

摘　　要：目的:将人类G3BP(Ras-GTPase activating protein SH3 domain binding protein)蛋白Domain(1～5)基因片段分别定向连入pEGFP-C2质粒,使G3BP蛋白各功能片段与绿色荧光蛋白在HeLa细胞内融合表达。方法:以重组质粒pEGFP-C1-G3BP为模板,PCR法扩增出目的基因,利用EcoRⅠ和BamHⅠ双酶切法将目的片段连接到pEGFP-C2载体上,再将构建成功的pEGFP-C2-hG3BP-Domain(1～5)重组质粒转染入HeLa细胞内,以荧光显微镜及Western印迹法检测绿色荧光蛋白与目的蛋白的融合表达情况。结果:以单/双酶切及基因测序法鉴定构建的重组质粒均无误,荧光显微镜及Western印迹结果均检测到绿色融合蛋白的表达。结论:重组pEGFP-C2-hG3BP-Domain(1～5)质粒成功构建并表达。Objective：To construct eukaryotic green fluorescent protein （GFP） expressing recombinant plasmids,pEGFP-C2-hG3BP-Domain （1-5）,which contain domain （1-5） fragments of human G3BP.Methods：The genes of G3BP fragments were amplified by PCR from the recombinant pEGFP-C1-G3BP plasmid and inserted into pEGFP-C2 fluorescent expressing vector with EcoRI and BamHI sites.These recombinant pEGFP-C2-hG3BP-Domain（1-5） plasmids were transfected into HeLa cells and the expression of green fluorescent fusion proteins was examined by fluorescence microscope and Western blotting assay.Results：The domain （1-5） fragments of G3BP were sequenced correctly and detected in the products of the restriction single/double enzyme digestion.The green fluorescent fusion proteins were also detected in the transfected HeLa cells by fluorescence microscope and Western blotting assay.Conclusion：These recombinant eukaryotic plasmids of pEGFP-C2-G3BP-Domain （1-5） were constructed successfully and expressed effectively.

关 键 词：绿色荧光蛋白质类 质粒 基因表达 印迹法 蛋白质 重组融合蛋白质类 限制性内切酶图谱法 

分 类 号：R382.5[医药卫生—医学寄生虫学]