PEDV S蛋白B细胞抗原表位的筛选和鉴定  

Screening and Identification of B Cell Antigenic Epitopes of Porcine Epidemic Diarrhea Virus Spike Glycoprotein by Phage Display

作　　者：孙东波[1] 朗洪武[2] 时洪艳[1] 陈建飞[1] 崔晓辰 王承宝[1] 佟有恩[1] 冯力[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所,兽医生物技术国家重点实验室,猪传染病研究室,哈尔滨150001 [2]中国兽医药品监察所,北京100081

出　　处：《生物化学与生物物理进展》2007年第9期971-977,共7页Progress In Biochemistry and Biophysics

基　　金：兽医生物技术国家重点实验室开放基金项目~~

摘　　要：以猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)S基因的免疫优势区S1(1～2367bp)为靶基因,利用基于gⅢ表达外源多肽的fd丝状噬菌体展示系统,构建S1基因噬菌体展示肽库,以纯化病毒制备的兔抗PEDV多克隆血清为靶蛋白,对S1基因噬菌体展示肽库进行3轮生物淘选,结果获得3个高亲和力的序列,分别命名为S1P1(248～280位氨基酸)、S1P2(442～499位氨基酸)和S1P3(697～742位氨基酸).ELISA和蛋白质印迹结果显示,S1P1、S1P2和S1P3短肽都能被兔抗PEDV多克隆血清识别,其中S1P3反应性最强.为了进一步揭示S1P1、S1P2和S1P3短肽的抗原性,制备了3个短肽GST融合蛋白和它们串连后GST融合蛋白的单因子血清,间接免疫荧光试验(IFA)结果证实,抗S1P2-GST、S1P3-GST和S1P123-GST融合蛋白的单因子血清能够识别Vero细胞培养物中天然的PEDV.S1 gene targeted libraries containing the major immunodominant region （1 -2 367 bp） of PEDV spike glycoprotein were constructed by phage-display vectors based on filamentous phage strain fd-tet, in which the exogenous polypeptides were expressed in the N-terminal of gene Ⅲ coat protein. The S 1 libraries were panned three times using the purified rabbit sera against PEDV. Three peptides, displayed on recombinant phages, showed strong binding affinity with the PEDV antisera, and were designated as S1P1 （248 -280aa）, S1P2 （442-499aa） and S1P3 （697-742aa） respectively. In ELISA and Western blot, the three peptides were all recognized by the PEDV antisera, but S1P3 showed strong binding activity. To further determine antigenicity of the three peptides, the antisera of S 1P 1-GST, S 1P2-GST, S 1P3-GST and their ligations GST fusion protein were prepared. In ELISA, S1P1-GST, S1P2-GST, S1P3-GST and S1P123-GST fusion proteins were able to induce the Sl-specific antisera. The result of indirect immunofluorescence assay （IFA） demonstrated that the antisera induced by S1P2-GST, S1P3-GST and S1P123-GST fusion proteins possessed binding ability to the native S protein of PEDV cultured in Vero cells.

关 键 词：猪流行性腹泻病毒 S蛋白 抗原表位 噬菌体展示 

分 类 号：S852.65[农业科学—基础兽医学] Q78[农业科学—兽医学]