人类Munc18-1重组质粒的构建及蛋白表达和定位  

Construction of hMunc18-1 recombinant plasmid and the expression and localization of its recombinant protein

作　　者：吴怡[1] 王婧[2] 王岚[1] 杨卫静 

机构地区：[1]沈阳医学院病原生物学教研室,110034 [2]沈阳医学院附属中心医院神经内一科,110024 [3]沈阳医学院基础医学院临床医学系,110034

出　　处：《中国神经免疫学和神经病学杂志》2015年第6期430-433,共4页Chinese Journal of Neuroimmunology and Neurology

基　　金：辽宁省教育厅科学技术研究项目(L2013403);沈阳医学院青年基金项目(20132028)

摘　　要：目的构建人类Munc18-1(hMunc18-1)真核表达质粒pEGFP-hMunc18-1并检测其融合蛋白GFPhMunc18-1在细胞内表达及定位。方法以人胎脑cDNA文库中的cDNA为模板,聚合酶链反应PCR扩增hMunc18-1全长编码基因,亚克隆至绿色荧光蛋白真核表达载体pEGFP-C1中。将构建的重组质粒pEGFPhMunc18-1送到Invitrogen公司进行基因测序,根据NCBI数据库中的hMunc18-1基因序列判断所得重组质粒的基因序列是否正确。将测序正确的质粒pEGFP-hMunc18-1转染到非洲绿猴肾细胞COS-7细胞中,提取细胞蛋白以Western blot法检测融合蛋白GFP-hMunc18-1表达,利用共聚焦激光扫描显微镜观察其在非洲绿猴肾细胞COS-7内的定位。结果 hMunc18-1全长基因序列被成功克隆到绿色荧光蛋白真核表达载体pEGFP-C1中,经酶切后鉴定其片段大小为1785bp。Western blot检测到融合蛋白GFP-hMunc18-1相对分子质量约为94 000,主要分布在细胞质,尤其是细胞膜附近。结论成功构建了hMunc18-1全长基因真核表达质粒pEGFPhMunc18-1,其融合蛋白GFP-hMunc18-1主要定位于COS-7细胞质内,尤其是细胞膜附近。Objective To construct the recombinant plasmid pEGFP-hMunc18-1of hMunc18-1gene and identify the expression and localization of its fusion protein GFP-hMunc18-1in the cell.Methods The full-length hMunc18-1coding sequence was amplified by polymerase chain reaction（PCR）from cDNA of the human fetal brain cDNA library and was subcloned into the pEGFP-C1 vector.Then the recombinant plasmid was sequenced by the Invitrogen company,the sequencing result was determined according to hMunc18-1gene sequence in NCBI.The plasmid pEGFP-hMunc18-1with correct sequence was transfected into COS-7（African green monkey kidney）cell lines.The expression of the recombinant plasmid GFP-hMunc18-1in cells was proved by western blot method.The localization of fusion protein GFP-hMunc18-1in COS-7was observed by using laser scanning confocal microscopy.Results The full-length hMunc18-1coding sequence was successfully constructed into the expressing vector pEGFP-C1.The length of the fragment was 1785 bp which was identified by restriction enzymes digestion.The expression of GFP-hMunc18-1fusion protein was detected by western blot method,with a molecular weight of 94 000.The GFP-hMunc18-1protein was mainly localized in the cytoplasm,especially,adjacent to cell membrane.Conclusions The recombinant plasmid pEGFP-hMunc18-1 was successfully cloned into the eukaryotic expressing vector.The GFP-hMunc18-1fusion protein localized mainly in the cytoplasm of COS-7cell,especially,adjacent to cell membrane.

关 键 词：Munc18-1 WESTERN BLOT 绿色荧光蛋白 质粒构建 

分 类 号：Q28[生物学—细胞生物学]