小鼠分泌型TIGIT慢病毒载体的构建及其功能研究  

Construction and functional analysis of a mouse secretory TIGIT lentiviral vector

作　　者：陈茜[1] 高义[2] 黄敏[3] 阿孜古丽.艾海提 陈忠华 刘喆隆[1] 余学锋[1] 何文涛[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院内分泌科,武汉430030 [2]华中科技大学同济医学院附属同济医院器官移植研究所,武汉430030 [3]华中科技大学同济医学院附属同济医院检验科,武汉430030

出　　处：《现代免疫学》2014年第1期36-41,共6页Current Immunology

基　　金：国家自然科学基金(81102260);中央高校基本科研业务费资助(HUST2012QN190)

摘　　要：本文旨在构建表达小鼠TIGIT胞外段与人IgG-Fc段融合基因TIG-Fc的慢病毒载体,验证TIG-Fc蛋白的功能,为进一步研究其免疫调节作用奠定基础。具体方法是将TIG-Fc融合基因克隆到pLOX载体质粒上,鉴定正确连接的克隆TIG-Fc-pLOX,并与pCMV△R8.2和pMD.G三质粒共转染人胚肾(HEK)293T细胞,收获上清中携带T1G-Fc基因的慢病毒Lenti-TIG-Fc。感染了Lenti-TIG-Fc的293T细胞与内毒素刺激成熟的小鼠树突状细胞(DC)共孵育后,ELISA检测上清中IL-10水平。结果显示成功构建的慢病毒Lenti-TIG-Fc感染293T细胞后,可使其稳定分泌TIG-Fc融合蛋白。融合蛋白可诱导成熟DC分泌IL-10明显增加。Our aims are to construct a lentiviral vector Lenti-TIG-Fc which encodes the extracellular domain of mouse TIGIT and the human IgG-Fc fusion gene, and to verify the functions of the fusion protein TIG-Fc and lay a foundation for the further study in immune regulation. The extracellular domain of mouse TIGIT and human IgG-Fc fusion gene was cloned into plasmid pLOX. Correctly connected cloning TIG-Fc-pLOX was identified and then co-transfeeted into HEN 293T cells together with pCMVAR8.2 and pMD. G. The supernatant containing lentivirus Lenti-TIG-Fc was harvested. 293T cells infected by the Lenti-TIG-Fc were co-cultured with LPS-induced mature dendritic cells （DCs） from BALB/c mice. IL 10 level secreted by DCs was detected by ELISA. The results showed that TIG-Fc fusion gene was successfully cloned and inserted to lentivirus plasmids. 293T cells infected hy the Lenti-TIG-Fc consistently secret TIG-Fc fusion protein and could significantly promote secretion of IL-10 by mature DCs,

关 键 词：TIGIT 树突状细胞 IL-10 慢病毒 

分 类 号：R516.7[医药卫生—内科学]