人心肌肌钙蛋白Ⅰ基因的原核表达及抗体制备  

Expression of Human Cardiac Troponin Ⅰ Gene in E.coli and Preparation of Its Specific Polyclonal Antibody

作　　者：刘红梅[1] 刘汉才[1] 屈伸[2] 

机构地区：[1]咸宁学院医学院生物化学与分子生物学教研室,湖北咸宁437100 [2]华中科技大学同济医学院基础医学院生物化学与分子生物学系,武汉430030

出　　处：《现代检验医学杂志》2008年第5期15-17,共3页Journal of Modern Laboratory Medicine

摘　　要：目的进行人心肌肌钙蛋白I(cardiac troponin Ⅰ,cTnⅠ)基因原核表达载体的构建,获得高纯度的心肌肌钙蛋白I并制备相应的多克隆抗体。方法利用RT-PCR方法从人心肌细胞的总RNA中克隆出编码人心肌肌钙蛋白的cDNA片段,将其插入原核表达载体形成重组体并导入宿主菌BL21(DE3)中,经IPTG诱导,表达出带6个组氨酸标签的融合蛋白,Ni-NTA树脂纯化,纯化后的融合蛋白免疫小鼠,制备抗血清。通过ELISA,Western blot鉴定血清效价和特异性。结果成功构建了cTnⅠ的原核表达载体,并在大肠埃希菌中获得了高效表达。Western blot检测证实获得了抗cTnI抗体。结论成功表达了cTnⅠ蛋白并制备了其特异性抗体。Objective To express human cardiac troponin I in E coil and to prepare antibody against it. Methods cDNA fragment was amplified by RT-PCR from human cardiac total RNA and inserted into pET-28c （＋） to construct an prokaryotic expressing vector. The recombinant was transformed into BL21 （DE3） bacteria. The histidine-tagged fusion protein expressed by IPTG-induced was purified by Ni-NTA resin. The cTnI protein was used to immunize mice. The specific antibody against cTnI was identified by ELISA and western blot. Results The constructor carrying cTnI cDNA was expressed in E. coli and its specific polyclonal antibody was prepared. Conclusion The cTnI protein was expressed in E. coli and its specific polyclonal antibody was obtained.

关 键 词：人心肌肌钙蛋白I 表达 多克隆抗体 

分 类 号：Q786[生物学—分子生物学] R392-33[医药卫生—免疫学]