人LIGHT胞外段基因的克隆及在大肠杆菌中的表达  

Cloning and expression of the extracellular region of human LIGHT gene in E.coli

作　　者：郝文丽[1] 江文正[1] 闻洁君[1] 樊燕[1] 钱旻[1] 

机构地区：[1]华东师范大学生命科学学院,上海200062

出　　处：《细胞与分子免疫学杂志》2009年第12期1076-1078,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：上海市青年科技启明星计划资助项目(06QA14017)

摘　　要：目的:构建人LIGHT胞外段基因的原核表达载体,并在大肠杆菌中诱导表达。方法:从人外周血单核细胞来源的未成熟树突状细胞中提取总RNA,通过RT-PCR得到LIGHT胞外段基因,并将其克隆至pET32a(+)原核表达载体中,经双酶切鉴定及序列测定后的重组质粒转化入E.coli BL21,IPTG诱导表达目的蛋白,并用SDS-PAGE和Western blot进行检测。结果:RT-PCR扩增出了大小为543bp的LIGHT胞外段基因,经测序证明序列正确。SDS-PAGE和Western blot分析证实重组质粒可表达出Mr约为41000的蛋白。结论:成功地克隆了人LIGHT胞外段基因并在大肠杆菌中进行了表达为进一步研究LIGHT的功能打下了基础。AIM： To construct a recombinant prokaryotic expression vector containing the extracellular region of human LIGHT gene and perform the express in E. coil METHODS： Total RNA was extracted from human immature bone marrow-derived dendritic cells. The extracellular region of human LIGHT gene was amplified by RT-PCR and cloned into pET32a（ ＋ ） vector, the recombinant plasmid was identified by restriction endonuclease digestion analysis and DNA sequencing. After the recombinant plasmid was transformed into E. coil BL？.I and induced with IPTG, the expressed protein was analyzed by SDS-PAGE and Western blot. RESULTS： A .543 bp of the extracellular region of hu- man LIGHT gene was obtained and the sequence was confirmed correct by DNA sequencing. SDS-PAGE and Western blot analysis showed that a protein with molecular weight of 41 000 was expressed in E. coil BL21. CONCLUSION： The extracellular region of LIGHT gene is cloned successfully and expressed in E. coil BL21 and the elementary expression conditions were obtained, which lays a basis on the further functional research of LIGHT.

关 键 词：LIGHT 基因克隆 原核表达 大肠杆菌 

分 类 号：R392.11[医药卫生—免疫学]