高特异性心肌肌钙蛋白Ⅰ适配体的ePCR技术筛选及其表征

Screening and characterization of high specificity cardiac troponinⅠaptamers by ePCR technique

目的筛选出与心肌肌钙蛋白I(cTnI)具有高亲和力和高特异性的核酸适配体,为诊断急性心肌梗死(AMI)提供实验基础。方法采用基于乳液PCR(ePCR)的SELEX技术筛选出能与cTnI特异性结合的3个适配体,并对ePCR条件进行优化,对适配体进行亲和力(Kd值)测定并与市售cTnI抗体进行比较,选出Kd值最小的适配体,进行特异性鉴定。结果SELEX过程中通过优化得到ePCR最佳条件,筛选得到的3个适配体均对cTnI具有较强的亲和力,其Kd值范围为2.04~16.85 nmol/L,其中适配体Apt-1的亲和力最强[Kd为(2.04±0.11)nmol/L]且优于市售抗体,特异性高,敏感性强,不与其他相关蛋白有非特异性结合。结论筛选出的适配体Apt-1能与cTnI产生高亲和力的特异性结合,可进一步用于临床样本中cTnI的检测。

Aim Nucleic acid aptamers with high affinity and specificity for cardiac troponin I(cTnI)were screened to provide experimental basis for the diagnosis of acute myocardial infarction(AMI).Methods Three aptamers specifically binding to cTnI were screened by SELEX technology and the ePCR conditions were optimized.The affinity(Kd value)of the aptamer was detected and compared with the existing antibody.The aptamer with the lowest Kd value was selected for specificity identification.Results The conditions of ePCR were optimized during SELEX.Three aptamersall had strong affinity for cTnI,with Kd value in the range of 2.04-16.85 nmol/L.Apt-1 had the strongest affinity(Kd=(2.04±0.11)nmol/L)and was superior to the existing antibody,with high specificity and strong sensitivity,and no non-specific binding with other related proteins.Conclusion Apt-1 can specifically bind to cTnI with high affinity,which can be further used for the detection of cTnI in clinical samples.