噬菌体随机肽库筛选HPV45亚型L1蛋白抗原表位

Screening of Epitopes of HPV45 L1 Protein from Random Peptide Library Displayed on Phage

高危型人乳头瘤病毒(human papilloma virus,HPV)可诱发女性宫颈癌,HPV45是HPV主要高危亚型之一,L1蛋白是主要的保护性蛋白。以大肠杆菌表达、纯化获得的重组蛋白SUMO-45 L1作为免疫原,通过杂交瘤技术制备抗HPV45 L1蛋白的单克隆抗体,并以单克隆抗体3C11为靶分子,使用噬菌体展示技术对随机十二肽库进行亲和淘选,三轮筛选后挑选12个与单抗3C11反应较强的克隆进行测序并分析。结果表明,噬菌体所展示的氨基酸序列与HPV45 L1蛋白序列“381VPNTYD386”相同,将其进行肽的合成,并通过斑点-酶联免疫吸附测定(dot enzyme-linked immunosorbent assay,Dot-ELISA)和间接竞争ELISA鉴定,最终鉴定出单克隆抗体3C11识别的L1蛋白线性表位“381VPNTYD386”。该表位的鉴定,为进一步分析HPV45 L1蛋白的结构和功能关系奠定了基础。

High risk human papillomavirus(HPV)could induce cervical cancer in women.HPV45 was one of the main high-risk subtypes of HPV,and L1 protein of HPV was the main protective protein.Expressed and purified was from E.coli.And then the monoclonal antibody 3C11 against HPV45 L1 protein was prepared by classical in hybridoma fusion technique using SUMO-45 L1 as immunogen.In order to identify the antigenic epitope of HPV45 L1 protein,monoclonal antibody 3C11 was used as coating material,the phage display random 12 peptide library was panned by indirect ELISA for three rounds,and 12 positive clones were screened and sequenced.The sequence analysis showed that the dominant sequence of mimotope was 381VPNTYD386.And the phage displaying this sequence could effectively inhibit the binding of monoclonal antibody 3C11 to HPV31 L1 protein by indirect competitive ELISA.The peptide was synthesized and identified by dot ELISA and indirect competitive ELISA.Finally,the linear epitope 381VPNTYD386 of L1 protein recognized by monoclonal antibody 3C11 was identified,which could provide useful information for the further study of the structure and function of HPV45 L1 protein.