人LIGHT胞外区基因的克隆及融合蛋白的表达  

Cloning of extracellular domain fragment of human LIGHT gene and expression of fusion protein

作　　者：姚智燕[1] 赵瑞景[1] 朱铁年[1] 尹晓琳[1] 冯惠东[1] 王秀荣[1] 马翠卿[1] 

机构地区：[1]河北医科大学免疫学教研室,石家庄050017

出　　处：《中国免疫学杂志》2006年第1期37-40,共4页Chinese Journal of Immunology

基　　金：河北省科技厅基金资助(03276196D-45;LLL012761110;LL01547013D)

摘　　要：目的:克隆人LIGHT胞外区基因(hsLIGHT),构建其原核表达质粒,并诱导其在大肠杆菌中表达融合蛋白。方法:采用RT-PCR方法从HL60细胞中扩增hsLIGHT,将其克隆入原核表达质粒pGEX-4T-2,构建其重组表达质粒pGEX-4T- 2/hsLIGHT,以不同浓度IPTG诱导表达,于不同时间经SDS-PAGE和Western blot分析、鉴定。结果:经RT-PCR扩增获得的 hsLIGHT序列与Genebank报道的LIGHT基因胞外区序列完全一致;SDS-PAGE和Western blot分析证实重组质粒可表达出相对分子量为47 000的蛋白,与GST-hsLIGHT融合蛋白分子量一致。结论:成功完成了人LIGHT胞外区基因的克隆及其原核表达质粒的构建,在大肠杆菌E．coli BL21中经IPTG诱导表达了融合蛋白GST-hsLIGHT,为进一步探讨LIGHT的抗肿瘤生物学活性、探索肿瘤免疫治疗新方法奠定了基础。Objective： To clone human LIGHT extracellular domain （hsLIGHT） gene, construct its recombinant expression plasmid and induce the expression of fusion protein using a prokaryotic expression system. Methods：The hsLIGHT gene fragment was amplified by RT-PCR from HL60 cells and subcloned into the prokaryotic expression plasmid pGEX-4T-2 to form pGEX-4T-2/hs-LIGHT. After recombinant plasmid was induced by different concentrations of IPTG, the expressed proteins were analyzed by SDS-PAGE and confirmed by Western blot. Results：The sequence of hsLIGHT gene amplified by RT-PCR was the same as the sequence of LIGHT extracelluar domain in gene map of Genbank;SDS-PAGE and Western blot analysis showed that a protein was expressed, the molecular weight of this protein was 47 000,which was the same as the fusion protein GST-hsLIGHT. Conclusion ：The hsLIGHT was cloned and its recombinant expression plasmid was constructed successfully. The fusion protein GST-hsLIGHT was successfully expressed in the prokaryotic expression system E. coli BL21 induced by IPTG. This research laid a foundation for further studying on its antitumor effects and exploring new ways of immunotherapy of tumor.

关 键 词：人LIGHT基因 基因克隆 融合蛋白 原核表达 

分 类 号：R392.11[医药卫生—免疫学]