小鼠GITR真核表达载体的构建及表达  

Construction and expression of eukaryotic expression vectors of mouse GITR

作　　者：沈培[1] 郑东[1] 刘艳[1] 田洁[1] 赵银霞[1] 刘青玲[1] 朱晨露[1] 马洁[1] 苏兆亮[1] 马斌[1] 许化溪[1] 王胜军[1] 

机构地区：[1]江苏大学基础医学与医学技术学院免疫学研究室,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2011年第4期329-332,共4页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(81072453;30972748;30871193;30910103087);江苏省自然科学基金资助项目(08KJB320002);江苏省医学科研基金资助项目(200952)

摘　　要：目的:构建小鼠GITR(mGITR)的真核表达载体,转染COS-7细胞表达小鼠GITR蛋白。方法:用TRIzol试剂抽提小鼠脾细胞总RNA,经RT-PCR扩增出GITR全长基因,构建真核重组载体pIRES2-EGFP-mGITR,经鉴定后采用脂质体介导DNA法转染COS-7细胞。结果:从小鼠脾细胞中成功扩增得到GITR全长基因。重组载体pIRES2-EGFP-mGITR转染COS-7细胞后,经荧光显微镜观察可见绿色荧光,提取蛋白后经蛋白质印迹鉴定有目的蛋白mGITR。结论:成功构建了小鼠GITR基因的真核表达载体pIRES2-EGFP-mGITR,转染COS-7细胞后mGITR蛋白获得成功表达。Objective： To construct eukaryotic expression vecter of mouse GITR gene fragment.The eukaryotic recombinant vectors then were transfected into COS-7 cells to express mGITR protein.Methods： The DNA fragment encoding mouse GITR was amplified by RT-PCR from the total RNA which was extracted by TRIzol agent from mouse spleen cells.We constructed the eukaryotic recombinant vectors of mGITR,which were pIRES2-EGFP-mGITR.Being correctly identified,the vectors were transfected into COS-7 cells by the lipofectin reagent.Results： We successfully extracted the total RNA containing mGITR gene from mouse spleen cells and amplified the mGITR gene fragment by RT-PCR.After the recombinant vectors pIRES2-EGFP-mGITR were transfected into COS-7 cells,the green fluorescent light was seen and the protein extracted that was identified as the target protein mGITR by Western-blot.Conclusion： The eukaryotic expression vectors pIRES2-EGFP-mGITR was successfully constructed.mGITR protein was expressed after the expression vectors pIRES2-EGFP-mGITR were transfected into COS-7 cells.

关 键 词：mGITR 真核表达 转染 COS-7细胞 

分 类 号：R393[医药卫生—基础医学]