小鼠TIM-3原核表达载体的建立及多克隆抗体的制备与鉴定  

Prokaryotic expression of mouse TIM-3 protein and preparation of its polyclonal antibody

作　　者：马德强[1] 郝友华[1] 刘敏[1] 徐春利[1] 郭艳[1] 陈悦[2] 陆孟吉 杨东亮[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院临床免疫研究室,武汉430030 [2]郧阳医学院附属十堰市太和医院,十堰442000 [3]德国ESSEN大学病毒学研究所,埃森市45122

出　　处：《中国免疫学杂志》2011年第2期158-162,共5页Chinese Journal of Immunology

基　　金：国家科技重大专项(2008ZX10002-0117)

摘　　要：目的:克隆小鼠TIM-3基因,构建原核表达载体,制备相应的多克隆抗体并初步鉴定。方法:以小鼠脾细胞总RNA为模板,用RT-PCR方法,扩增得到TIM-3基因编码区,构建pRSET-B-TIM-3原核表达载体;经IPTG诱导表达并纯化重组蛋白;然后常规免疫家兔,制备多克隆抗体;用ELISA方法检测抗体的效价,用Western blot、免疫组化、流式细胞术检测抗体的特异性。结果:成功构建的原核表达载体pRSET-B-TIM-3在大肠杆菌中诱导后可以高效表达TIM-3蛋白;免疫获得的多克隆抗体经过ELISA检测,抗体效价为1∶320 000,经Western blot、免疫组化和流式细胞术等鉴定,抗体的特异性较好。结论:成功克隆出小鼠的TIM-3基因,构建了原核表达载体,制备的兔抗小鼠TIM-3多克隆抗体具有较高的效价和良好的特异性。Objective：To construct a prokaryotic expression vector of mouse Tim-3 molecule and prepare the polyclonal antibody against mouse TIM-3.Methods：The extracellular TIM-3 gene was cloned from the spleen cells of BALB/c mice by RT-PCR and inserted into the vector pRSET-B in order to construct prokaryotic expression vector pRSET-B-TIM-3.Mouse TIM-3 protein was expressed by IPTG induction and purified by Ni-NTA column.Polyclonal antibody against TIM-3 protein was generated by immunizing rabbit with the routine method.The specificity and sensitivity of this antibody was confirmed by ELISA,Western blot,immunohistochemistry assay and FACS.Results：The fragment of 510 bp extracellular DNA sequence was obtained from the spleen cells of BALB/c mice by RT-PCR,and the prokaryotic expression vector pRSET-B-TIM-3 was constructed successfully.Induced by IPTG,the recombinant TIM-3 protein could be expressed effectively.After immunization of rabbit with the recombinant TIM-3 protein,the polyclonal antibody against mouse TIM-3 protein with a high affinity and specificity was obtained.Conclusion：The TIM-3 gene is cloned and the prokaryotic expression vector pRSET-BTIM-3 is constructed successfully.The polyclonal antibody with high affinity and specificity is generated successfully.It can be used to study the mouse TIM-3 effectively.

关 键 词：TIM-3 蛋白 多克隆抗体 

分 类 号：R392.33[医药卫生—免疫学]