柯萨奇病毒A组16型抗原表位预测

Prediction of epitope of Coxsackievirus A16

目的应用模拟表位策略研究柯萨奇病毒A组16型(Coxsackievirus A16,CA16)抗原表位。方法分别以具有中和活性的抗CA16单克隆抗体2C6、4F9、1D8为靶分子,在噬菌体12肽库中淘选可与之特异性结合的阳性克隆,对淘选得到的阳性克隆进行序列测定和比对,并采用Phage ELISA法进行特异性鉴定。同时利用生物信息学方法对CA16衣壳蛋白理化性质进行分析,并与阳性噬菌体多肽序列和结构进行比对,预测CA16中和抗原表位。结果经噬菌体12肽库筛选,单克隆抗体2C6、4F9、1D8淘选阳性噬菌体富集率分别为2.39×103、1.32×104和3.762×10,获得可与单克隆抗体2C6、4F9、1D8结合的阳性克隆分别为10、11、6种,其中各组共有基序分别为K+X+WW和N/Q+S/T+Y/F/W、K+X+WW+D/E、T+X+P+W。Phage ELISA特异性鉴定阳性克隆肽CA1和CB1、FB3和FC3、DA1和DA2、DA5分别强特异性结合单克隆抗体2C6、4F9、1D8。经计算机预测分析,确定了主要位于CA16 VP1800-810区域,并涉及VP4、VP3上的多个中和表位区域。结论应用模拟表位策略成功筛选出了CA16中和抗原表位,为CA16蛋白抗原结构、新型表位疫苗及诊断试剂的进一步研究奠定了基础。

Objective To identify the antigenic epitope of Coxsackievirus A16(CA16)by mimotope strategy.Methods Serving CA16 monoclonal antibodies(mAbs)2C6,4F9 and 1D8 as target proteins,the positive phage clones screened from phage display 12 peptide library were sequenced,aligned,and tested for specificity by Phage ELISA.Meanwhile,the physiochemical properties of CA16 capsid protein was analyzed by bioinformatics tool,while the sequence and structure were aligned to those of positive phage clones to predict the neutralizing antigen epitope of CA16.Results The enrichment rates of mAbs 2C6,4F9 and 1D8 from phage display 12 peptide library were 2.39×103,1.32×104 and 3.76×10 respectively.There were 10,11 and 6 positive phages which could be recognized by mAb 2C6,4F9 and 1D8 respectively,of which the consensus motifs were K+X+WW,N/Q+S/T+Y/F/W,K+X+WW+D/E and T+X+P+W.Phage ELISA showed strong specific binding of positive cloning peptides CA1 and CB1 to mAb 2C6,while FB3 and FC3 to 4F9,and DA1,DA2 and DA5 to 1D8.The prediction by computer confirmed several neutralizing epitopes which were mainly located in VP1800-810 region and also located in VP4 and VP3.Conclusion The neutralizing antigen epitopes of CA16 were successfully screened by mimotope strategy,which laid a foundation of further study on the antigen structure of CA16 protein,novel epitope-based vaccine and diagnostic kit.