真核表达载体pEGFP-N1-hSyntenin的构建及其在人脑胶质瘤细胞CHG-5中的表达  

Construction of eukaryotic expression vector pEGFP-N1-hSyntenin and its expression in CHG-5 cells

作　　者：钟东[1] 唐文渊[1] 冉建华[2] 杨刚[1] 晏怡[1] 

机构地区：[1]重庆医科大学附属第一医院神经外科,重庆400016 [2]重庆医科大学基础医学院解剖学教研室,重庆400016

出　　处：《第三军医大学学报》2010年第19期2080-2084,共5页Journal of Third Military Medical University

摘　　要：目的构建pEGFP-N1-hSyntenin基因真核表达载体,并观察其在人脑胶质瘤细胞CHG-5中的表达。方法采用RT-PCR技术从人脑肿瘤组织中扩增hSyntenin,并将PCR产物双酶切后定向克隆入pEGFP-N1的多克隆位点,构建pEGFP-N1-hSyntenin重组质粒。经过菌落PCR、双酶切及测序验证后,采用脂质体转染技术将pEGFP-N1-hSyntenin融合基因转染CHG-5细胞表达,G418筛选建立稳定表达hSyntenin的CHG-hS细胞系。采用荧光检测和免疫印迹技术分析hSyntenin在稳定转染的CHG-5细胞系中的表达。结果菌落PCR鉴定出1个阳性克隆,阳性克隆经双酶切鉴定得到分别与载体和目的片段大小一致的4.7kb和897bp的两条片段,测序证实目的基因hSyntenin正确连接到pEGFP-N1的多克隆位点;pEGFP-N1-hSyntenin重组体稳定转染CHG-5细胞后,细胞荧光检测结果显示hSyntenin在CHG-5细胞质发出绿色荧光,免疫印迹检测可见32×103处的阳性条带。结论成功构建pEGFP-N1-hSyntenin重组质粒,并在人脑胶质瘤细胞CHG-5中获得稳定表达。Objective To construct the enhanced green fluorescent protein（EGFP）-labeled eukaryotic expression recombinant vector of human syntenin（hSyntenin） gene,and observe its expression in CHG-5 cells.Methods hSyntenin was amplified from human glioma by RT-PCR.The PCR products were cloned into the eukaryotic expression vector pEGFP-N1 after Xho Ⅰ and BamH Ⅰ digestion.Recombinant plasmid pEGFP-N1-hSyntenin was identified by PCR,double enzyme digestion and DNA sequencing,and transfected into CHG-5 cells with Lipofectamine 2000.After screened with G418,a stably transfected CHG-5 cell line was established.Expression of hSyntenin in CHG-5 cells was identified by immunocytochemistry and immunoblot analysis,respectively.Results A positive clone was identified by PCR.The fragments were 4.7 kb and 897 bp in length,which were consistent with the carrier and purpose gene respectively by double enzyme digestion.The eukaryotic expression plasmid of pEGFP-N1-hSyntenin was confirmed by DNA sequencing and its stable transfectant into CHG-5 cell line was established.Fluorescence image revealed that the hSyntenin with green fluorescence was expressed in cytoplasm of CHG-5 cells.Immunoblot following SDS-PAGE showed the positive band at 32 × 103.Conclusion Recombinant eukaryotic expression vector of pEGFP-N1-hSyntenin,constructed in this study,is stab1y expressed in CHG-5 cell line.

关 键 词：pEGFP-N1-hSyntenin 稳定转染 CHG-5 

分 类 号：R394.33[医药卫生—医学遗传学] R394.2[医药卫生—基础医学]