人AITRL胞外区基因克隆表达及重组蛋白生物活性分析  

Expression and bioactivity analysis of an soluble extracellular fragment of human AITRL

作　　者：毛朝明[1] 王胜军[1] 仝佳[1] 马洁[1] 杨敏[1] 许小朋[1] 邱谷风[1] 唐莉[1] 邵启祥[1] 李龙[1] 许化溪[1] 

机构地区：[1]江苏大学医学技术学院免疫学研究室江苏大学附属医院核医学科,镇江212013

出　　处：《中国免疫学杂志》2007年第12期1059-1063,共5页Chinese Journal of Immunology

基　　金：江苏省自然科学基金(BK2004405);江苏大学高级人才基金(05JDG042);国家自然科学基金(30300169)资助

摘　　要：目的:获得重组人活化诱导的肿瘤坏死因子超家族配体胞外区蛋白(sAITRL),分析AITRL蛋白的生物学功能。方法:从全长质粒AITRL-pMD18-T中扩增sAITRL cDNA,亚克隆至表达载体pQE30;将重组质粒转化至E.coliM15,IPTG诱导蛋白表达;表达蛋白用Ni2+-IMAC柱纯化、复性并Western blot鉴定,流式细胞仪检测sAITRL重组蛋白的结合活性,增殖试验分析sAITRL的生物学活性。结果:成功构建了sAITRL-pQE30原核表达载体;表达相对分子质量约15kD的重组蛋白;sAITRL能与活化淋巴细胞上的天然受体AITR结合,低浓度的sAITRL能促进淋巴细胞的增殖,而在高浓度下则抑制了淋巴细胞的增殖。结论:成功表达了具有生物学活性的sAITRL重组蛋白,AITR-AITRL相互作用在调节淋巴细胞增殖过程中起重要作用。Objective:To obtain recombinant protein of an soluble extracellular fragment of activation-inducible tumor necrosis factor ligand ( sAITRL), and to analyze its bioactivity.Methods:sAITRL gene was amplified by PCR from the full-length plasmid AITRL-pMD18-T and subcloned into the pQE30 vector. The recombinant plasmid was transformed into E.coli M15, and the expression of recombinant protein was induced by IPTG. After purified and refolded with Ni2+-IMAC column, identification of the recombinant protein by Western blot was performed. The binding ability of sAITRL protein to natural AITR was evaluated by FCM, and the bioactivity was estimated by proliferation assay in vitro.Results:The prokaryotic expression vector for sAITRL-pQE30 was constructed successfully. sAITRL protein with molecular mass about 15 kD was expressed in E.coli M15 after IPTG induction. This recombinant sAITRL,which was able to bind to natural AITR on surface of activated lymphocytes,significantly stimulated proliferation of lymphocytes at low doses, but had an opposite effect at higher concentrations.Conclusion:Recombinant sAITRL with bioactivity is expressed successfully. AITR-AITRL interaction appears to be crucial in regulation the ongoing immune responses.

关 键 词：活化诱导的肿瘤坏死因子超家族配体 基因克隆 原核表达 重组蛋白 

分 类 号：R392.12[医药卫生—免疫学]