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| 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 | |
| 引用本文： | 马德强,郝友华,刘敏,徐春利,郭艳,陈悦,陆孟吉,杨东亮.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[J].中国免疫学杂志,2011,27(2):158-162. |
| 作者姓名： | 马德强 郝友华 刘敏 徐春利 郭艳 陈悦 陆孟吉 杨东亮 |
| 作者单位： | 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 |
| 基金项目： | 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。 |
| 关 键 词： | TIM-3 蛋白 多克隆抗体 |
Prokaryotic expression of mouse TIM-3 protein and preparation of its polyclonal antibody |
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| MA De-Qiang,HAO You-Hua,LIU Min,XU Chun-Li,GUO Yan,CHEN Yue,LU Meng-Ji,YANG Dong-Liang.Prokaryotic expression of mouse TIM-3 protein and preparation of its polyclonal antibody[J].Chinese Journal of Immunology,2011,27(2):158-162. | |
| Authors: | MA De-Qiang HAO You-Hua LIU Min XU Chun-Li GUO Yan CHEN Yue LU Meng-Ji YANG Dong-Liang |
| Institution: | MA De-Qiang,HAO You-Hua,LIU Min,XU Chun-Li,GUO Yan,CHEN Yue,LU Meng-Ji,YANG Dong-Liang.Division of Clinical Immunology,Tongji Hospital of Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China |
| Abstract: | 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 met... |
| Keywords: | TIM-3 Protein Olyclonal antibody |
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