人心肌肌钙蛋白I的重组表达及其单克隆抗体的制备  

Prokaryotic expression of human cTnI and preparation of anti-cTnI monoclonal antibody

作　　者：杨能[1] 徐霞[1] 

机构地区：[1]广州医学院检验系,510182

出　　处：《中华检验医学杂志》2005年第11期1207-1210,共4页Chinese Journal of Laboratory Medicine

基　　金：广东省医学科学技术研究基金资助项目(NO.A2000265)

摘　　要：目的构建人心肌肌钙蛋白I(cTnI)的表达载体,表达cTnI重组蛋白,并制备其单克隆抗体(mAb)。方法以化学方法合成cTnI基因并插入融合表达载体pBV220的多克隆位点,构建重组表达质粒pBV220/cTnI。以重组质粒转化大肠杆菌DH5α,筛选阳性重组子,经热激诱导目的蛋白的表达,表达产物的免疫学活性用Westernblot进行鉴定。以基因重组的cTnI蛋白为抗原,常规方法免疫BALB/c小鼠,取其脾细胞与NS1细胞融合,获得稳定分泌cTnImAb的杂交瘤细胞株,ELISA检测mAb的相对亲和力;Westernblot检测mAb的特异性。结果酶切鉴定和DNA测序显示cTnI重组表达载体中含有人cTnI全长编码序列。将该重组载体转化入大肠杆菌DH5α中表达所得蛋白经WesternBlot验证为目的蛋白。筛选出2株能稳定分泌特异性抗cTnI的mAb杂交瘤细胞株,免疫球蛋白亚类均为IgG类;Westernblot结果显示,两株单抗识别相对分子量为24000的cTnI单一条带;中和抑制试验表明培养上清中的抗体能明显被cTnI中和;cTnI单克隆抗体的亲和常数为Kaff=1.62×109(mol/L)-1。结论成功地构建了cTnI表达载体、表达出cTnI重组蛋白,制备出抗cTnImAb,为进一步用于cTnI的体外免疫学检测奠定了基础。Objective To prepare monoclonal antibody （mAb） against recombinant human cardiac troponin I （cTnI）. Methods The full-length gene encoding human cardiac troponin I （cTnI） was synthesized chemically and inserted into expression plasmid pBV220 to construct recombinant plasmid p pBV220/cTnI. The recombinant plasmid was transformed into E. coli DH5α which then expressed cTnI. The immunological activity of the expressed cTnI was analyzed by Western blot. Recombinant human cTnI protein was used as antigen to immunize BALB/c mice. Monoclonal anti-bodies against cTnI were prepared by normal hybridoma technology. The relative affinity of mAbs was determined by ELISA. Specificity of mAbs was analyzed by Western blot. Results Human cTnI gene was synthesized and confirmed by DNA sequencing, Positive recombinant clones were identified by restriction enzyme digestion analysis and DNA sequencing. Western blot analysis showed that the cTnI protein could be recognized by an anti- cTnI antibody. Two hybridmas producing antibodies against cTnI were obtained. IgG isotypes of two mAbs were IgG2a and IgG2b. Western blot showed that the antibodies were specific for cTnI. Neutrahsation test showed that these mAbs could be evidently neutralized by cTnI. Conclusion The recombinant expression plasmid of cTnI was constructed successfully and expressed in E. coli. The method of EL ISA established to test serum cTnI is to clinically useful. The cTnI mAb which using cTnI as antigen prepared in this paper can be used for cTnI immunoassay in vitro.

关 键 词：肌钙蛋白I 抗体 单克隆 

分 类 号：R446.6[医药卫生—诊断学]