心肌肌钙蛋白I(cTnI)合成肽制备、鉴定及抗体研究

The preparation and identification of human cardiac troponin I(cTnI) synthetic peptide and research of anti-cTnI antibody

目的制备人心肌肌钙蛋白I(cardiac troponin I,cTnI)合成肽并鉴定其质量。方法根据人cTnI氨基酸序列,结合计算机软件分析,预测cTnI分子的亲水性、抗原性和同源性,采用固相多肽合成法合成了cTnI分子98～110位氨基酸肽段序列(13肽,RQLHARVDKVDEE)。经与载体蛋白KLH偶联作为免疫原制备抗cTnI-KLH抗体,用间接ELISA方法鉴定其免疫原性。结果采用多肽合成法合成的cTnI合成多肽(13肽),经HPLC纯化后,以其制备的cTnI-KLH纯度达到95%。免疫学实验证实,制备的抗体效价达到1∶100 000。结论本研究制备的肽段连接物具有较强的免疫原性,并与生物提取人cTnI具有较好的交叉反应性。cTnI合成肽的研制为临床cTnI检测试剂的国产化奠定了基础。

At present,diagnostic agent for human cardiac troponin I（cTnI） detection is lack in China,which is necessary for diagnosis of myocardial ischemia.The present study aimed to prepare human cardiac troponin I（cTnI） synthetic peptide and identified its quality.Based on human cTnI amino-acid sequence and combined with computer software analysis,we predicted the hydrophilicity,antigenicity and autoploidy of cTnI,thus synthesized the 98-110 amino-acids peptide sequence（a 13-peptide,RQLHARVDKVDEE） of human cTnI molecule by using solid-phase peptide synthesis approach.Then the peptide was linked to a carrier protein KLH（keyhole limpet hemocyanin） as immunogen to prepare the antibody against cTnI-KLH,which was then identified by indirect ELISA method for antigenicity.Results showed that cTnI（a 13-peptide） was prepared successfully,and the purity of cTnI-KLH,which was prepared subsequently,was up to 95%.Immune test indicated that the titer of the antibody prepared was up to 1∶100 000.All result manifested that the cTnI synthetic peptide possessed stronger immunogenicity,and the antibody was of cross-reaction with cTnI from human cardiac muscle tissue.In conclusion,research and preparation of cTnI provides a basis for approach to prepare cTnI detection reagents in China.