牛病毒性腹泻-黏膜病病毒噬菌体单链抗体库的构建和筛选  

Construction and screening of ScFv against bovine viral diarrhea virus

作　　者：孔梓安 宋想胜 程如楠 甄思慧 吴竹青 吴清民[2] 王真[1] KONG Zian;SONG Xiangsheng;CHENG Runan;ZHEN Sihui;WU Zhuqing;WU Qingmin;WANG Zhen(College of Animal Science and Technolog/Beijing Key Laboratory of Veterinary Medicine(Chinese Veterinary Medicine),Beijing University of Agriculture,Beijing 102206,China;College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)

机构地区：[1]北京农学院动物科学技术学院/兽医学(中兽医)北京市重点实验室,北京102206 [2]中国农业大学动物医学院,北京100193

出　　处：《中国农业大学学报》2022年第9期64-71,共8页Journal of China Agricultural University

基　　金：北京市教委青年拔尖人才项目(CIT&TCD201904052);北京农学院青年基金项目(QNKJ202107);大北农青年基金项目(16ZK007)。

摘　　要：为筛选牛病毒性腹泻-黏膜病病毒(Bovine viral diarrhea virus,BVDV)gP48蛋白的单链抗体,本研究利用噬菌体展示技术构建了gP48蛋白单链抗体克隆文库,通过微孔筛选法对抗体库进行3轮富集淘筛,利用ELISA技术测定单链抗体的亲和力,对亲和力较高的克隆进行基因测序分析和结合活性测定,旨在获得gP48蛋白的单链抗体。结果表明:1)成功构建了库容量为4.3×10^(7)的噬菌体单链抗体库,其重组率为83.3%,经三轮筛选对噬菌体抗体库进行富集,富集倍数为1.43×10^(4);2)优化了间接ELISA方法,筛选到3株与BVDV gP48蛋白具有高亲和力的单链抗体以及其基因序列,通过IgBLAST分析显示,3株单链抗体均为鼠源IgG。综上,从gP48蛋白的单链抗体库中筛选获得3个具有高亲和力的单链抗体,可用于后续BVDV检测方法的建立。To screen the single chain antibody of gP48protein of bovine viral diarrhea mucosal disease virus(Bovine virtual diarrhea virus,BVDV),a single chain antibody clone library of gP48protein was constructed by phage display technology.The antibody library was enriched and screened for three rounds by microporous screening method.In order to obtain gP48protein single chain antibody to protein.The affinity of single chain antibody was determined by ELISA technology,and the clones with high affinity were sequenced and analyzed for binding activity.The results show that:1)The library capacity was 4.3×10^(7),and the recombination rate of phage scFv library was 83.3%.After three rounds of screening,the phage antibody library was enriched,and the enrichment multiple was 1.43×10^(4);2)The indirect ELISA method was optimized,and three scFvs with high affinity for BVDV gp48 protein and their gene sequences were screened.IgBLAST analysis showed that the three scFvs were mouse IgG.In conclusion,a total of three scFvs with high affinity were identified from the scFv Library of gP48protein,which can be used for the establishment of subsequent BVDV detection methods.

关 键 词：牛病毒性腹泻病毒 gP48蛋白 噬菌体抗体库 单链抗体 亲和筛选 

分 类 号：S855.3[农业科学—临床兽医学]