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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。
Objective To prepare human cardiac troponin I (cTnI) peptide and identify its quality. Methods Based on the human cTnI amino acid sequence and computer software analysis, the hydrophilicity, antigenicity and homology of cTnI were predicted, and the amino acid peptide sequence of peptide 98-110 of cTnI was synthesized by solid phase peptide synthesis (13 peptide, RQLHARVDKVDEE ). Anti-cTnI-KLH antibody was prepared by conjugating with carrier protein KLH as an immunogen, and its immunogenicity was identified by indirect ELISA. Results The cTnI synthetic peptide (13 peptide) synthesized by peptide synthesis was purified by HPLC and the purity of cTnI-KLH was 95%. Immunological experiments confirmed that the antibody titer reached 1: 100 000. Conclusion The peptide linker prepared in this study is highly immunogenic and has good cross-reactivity with human cTnI. The development of cTnI synthetic peptide lays a foundation for the localization of clinical cTnI detection reagent.