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本研究旨在构建家兔出血症病毒VP60基因特异性噬菌体展示肽库,并选用已知表位序列的单克隆抗体对VP60基因特异性肽库进行鉴定,以确定所构建肽库的特异性和丰富性。利用DNA酶Ⅰ随机消化法构建VP60基因噬菌体随机肽库。采用生物淘选和噬菌体原位杂交技术,选用1B8、1D4、5H3和A3C 4株纯化的单克隆抗体对该肽库进行抗原表位的鉴定。结果显示,所构建多肽库的滴度约为2.6×1012 PFU/m L,能够满足鉴定VP60蛋白的抗原表位的要求,同时鉴定到4条与VP60蛋白高度同源的序列,分别为N(326)PISQV(331)、D(338)MSFV(342)、K(562)STLVFNL(569)和G(25)TTTDGMDPGVVAA(38),与已知单抗所对应的表位序列高度同源。本研究成功构建了家兔出血症病毒VP60基因特异性随机肽库,选用已知表位序列的单克隆抗体对构建的肽库进行鉴定,鉴定到的表位与目前已经报道的单抗针对的表位序列高度同源,表明所构建肽库的特异性和丰富性良好,为进一步筛选和丰富VP60抗原表位奠定基础,还可用于VP60与受体结合域的鉴定,对阐述RHDV的感染、致病和免疫保护机制具有重要意义。
The purpose of this study was to construct the VP60 gene-specific phage display peptide library of rabbit hemorrhagic disease virus and to identify VP60 gene-specific peptide library by using monoclonal antibodies with known epitope sequences to determine the specificity and Richness Construction of phage random peptide library of VP60 gene by DNase Ⅰ random digestion. Biotinylation and phage in situ hybridization were used to identify epitopes of the peptide library using monoclonal antibodies against 1B8, 1D4, 5H3 and A3C. The results showed that the titer of the constructed peptide library was about 2.6 × 1012 PFU / m L, which could meet the requirement of identifying epitopes of VP60 protein. Four highly homologous sequences of VP60 protein were identified, which were N ( 326) PISQV (331), D (338) MSFV (342), K (562) STLVFNL (569) and G (25) TTTDGMDPGVVAA (38) were highly homologous to the epitope sequences corresponding to known mAbs. In this study, we successfully constructed a random peptide library of VP60 gene of rabbit hemorrhagic disease virus. The constructed peptide library was identified by the monoclonal antibody with known epitope sequence. The identified epitopes are specific to the currently reported mAbs The sequences of the epitopes are highly homologous, indicating that the constructed peptide library has good specificity and richness, which lays the foundation for further screening and enriching VP60 epitopes, and can also be used for identifying the binding domain of VP60 and the receptor, and expresses the infection of RHDV, Pathogenesis and immune protection mechanisms are of great importance.