人CTLA-4Ig融合基因减毒鼠伤寒沙门菌真核表达载体的构建及表达  

Construction of eukaryotic expression vector of attenuated Salmonella typhimurium containing hCTLA4Ig gene and expression in COS-7 cells

作　　者：周春丽[1] 郝进[1] 唐书谦[1] 钟白玉[1] 吴军[2] 贺伟峰[2] 郝飞[1] 

机构地区：[1]第三军医大学西南医院皮肤科,重庆市皮肤性病研究所,重庆400038 [2]第三军医大学西南医院全军烧伤研究所创伤烧伤与复合伤国家重点实验室,重庆400038

出　　处：《第三军医大学学报》2006年第3期243-246,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目(30200258)~~

摘　　要：目的构建能稳定表达人CTLA4 Ig融合基因的减毒鼠伤寒沙门菌真核表达载体,探讨其在哺乳动物细胞中的表达并对表达产物进行鉴定。方法用touchdown PCR法扩增CTLA-4 Ig融合基因,将PCR产物连接真核表达载体pcDNA3.1(+),构建pcDNA3.1(+)-CTLA-4 Ig,用电穿孔仪转入减毒鼠伤寒沙门菌SL7207。将表达质粒转染COS-7细胞,SDS-PAGE、W estern b lot检测转染细胞裂解上清中目的蛋白的表达。结果酶切鉴定和基因序列测定显示重组质粒构建成功。在pcDNA3.1(+)-CTLA-4 Ig质粒转染后48 h细胞裂解上清中,检测到CTLA-4 Ig融合蛋白的表达,该蛋白能与抗人CTLA-4单抗特异结合。结论成功构建了能稳定表达人CTLA4 Ig融合基因的减毒鼠伤寒沙门菌真核表达载体,并在哺乳动物细胞中成功表达有生物学活性的重组人CTLA-4 Ig蛋白。Objective To construct eukaryotic expression vector of attenuated Salmonella typhimurium containing hCTLA-4Ig eDNA and identify its expression in COS-7 cells for the further study of function in SLE models. Methods Touchdown PCR was used to amplify hCTLA-4Ig eDNA. The PCR product was ligated into the multiple clone site of eukaryotic expression vector pcDNA3.1 （ ＋ ） by gene recombination technique. Then the recombinated plasmid pcDNA3.1 （ ＋ ） -CTLA-4Ig was transfected into COS-7 cells using DOTAP. The expression of interest protein in the supernatant of the cell disruption was detected with SDS-PAGE and Western blotting. Results Restriction analysis and DNA sequence analysis showed that the CTLA-4Ig eDNA had been successfully inserted into pcDNA3.1 （ ＋ ） eukaryotic expression vector. The interest protein could be detected in the supematant of cell disruption 48h after the transfection of pcDNA3.1 （ ＋ ） -CTLA-4Ig. This protein specifically bound with human CTLA-4 monoclonal antibody. Conclusion The eukaryotic expression vector containing hCTLA-4Ig gene was successfully constructed and bioactive interest protein could be successfully expressed in mammalian cells.

关 键 词：CTLA-4IG 减毒鼠伤寒沙门菌 真核表达载体 基因表达 

分 类 号：R378.23[医药卫生—病原生物学] R392.33[医药卫生—基础医学]