家兔出血症病毒VP60基因特异性噬菌体展示肽库的构建及鉴定  

Construction and identification of specific phage display libraries of rabbit hemorrhagic disease virus VP60 gene

作　　者：左园园[1,2] 宋艳华[2] 姜平[1] 王芳[1,2] 胡波[2] 范志宇[2] 魏后军[2] 薛家宾[2] 仇汝龙[2] 徐为中 

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室国家兽用生物制品工程技术研究中心,江苏南京210014

出　　处：《中国兽医科学》2016年第12期1511-1517,共7页Chinese Veterinary Science

基　　金：现代农业产业技术体系建设兔体系病毒病预防与控制岗位项目(CARS-44-C-1);国家自然科学基金项目(31502073);江苏省自然科学基金资助项目(BK20140740)

摘　　要：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 focused on constructing specific phage display peptide libraries of rabbit hemorrhagic disease virus（RHDV）VP60 gene,and then identified the richness and specificity of the libraries by using monoclonal antibody（MAbs）with known epitopes. Gene-targeted phage random peptide library of VP60 protein was constructed by DNase I random digestion method. Monoclonal antibody（MAbs）1B8,1D4,5H3,and A3 C were used to screen the epitopes in VP60 libraries by biopanning and using phage in situ hybridization.The titer of peptide libraries were about 2.6×10^（12） PFU/m L,and the peptide libraries could meet the requirements of identification epitopes of VP60 protein. Four sequences which were highly homologous with N（326）PISQV（331）,D（338）MSFV（342）,K（562）STLVFNL（569）and G（25）TTTDGMDPGVVAA（38）were selected by the MAbs,moreover the sequences were in accordance with the known antigen epitope. The specific phage display peptide librarie of rabbit hemorrhagic disease virus（RHDV）VP60 gene were constructed in this study. Identifying the libraries by using monoclonal antibody（MAbs）with known epitopes,and the epitope identified in this study were consistent with the previous papers. Results indicated that the richness and specificity of the libraries were well. It laid a foundation for further study of screening and enriching epitopes of VP60 protein,furthermore the libraries can be used to identify the receptor domain bind to VP60. And it has important significance in expounding the infection,pathogenesis and mechanism of immune protection of RHDV.

关 键 词：家兔出血症病毒 VP60基因 特异性噬菌体展示肽库 

分 类 号：S852.659.6[农业科学—基础兽医学]