非洲猪瘟病毒MGF360-9L蛋白多克隆抗体的制备及其对病毒复制的影响  

Preparation of polyclonal antibodies against MGF360-9L protein of African swine fever virus and its effect on virus replication

作　　者：杨博 申超超 张婷 郝雨 杨金柯 崔卉梅 赵登率 袁兴国 陈学辉 鲍恩东[1] 张克山[2] 郑海学[2] 刘湘涛[2] YANG Bo;SHEN Chao-chao;ZHANG Ting;HAO Yu;YANG Jin-ke;CUI Hui-mei;ZHAO Deng-shuai;YUAN Xing-guo;CHEN Xue-hui;BAO En-dong;ZHANG Ke-shan;ZHENG Hai-xue;LIU Xiang-tao(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;State Key Laboratory of Veterinary Etiological Biology/National Foot-and-Mouth Disease Reference Laboratory/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室国家口蹄疫参考实验室,甘肃兰州730046

出　　处：《中国兽医科学》2022年第3期324-332,共9页Chinese Veterinary Science

基　　金：甘肃省科技重大专项(20ZD7NA006);国家自然科学基金联合项目(31941002);国家重点研发计划项目(2018YFC0840400)。

摘　　要：为制备非洲猪瘟病毒(ASFV)MGF360-9L蛋白的多克隆抗体并评价其对病毒复制的影响,使用DNAStar 2.1软件预测MGF360-9L的抗原表位,选择并合成了符合抗原要求的MGF360-9L C端多肽CKNLSIAHKHYINDGFND,制备MGF360-9L家兔源多克隆抗体。随后采用实时荧光定量PCR方法、血细胞吸附试验和Western-blot方法等评价MGF360-9L的多克隆抗体对病毒复制的影响。结果显示,不同稀释倍数的MGF360-9L多克隆抗体在不同感染时间下均可显著降低病毒的拷贝数、效价以及D1133L和B646L(p72)两种病毒蛋白的表达,并且抗体稀释度越低,降低病毒拷贝数、效价以及病毒蛋白表达的作用越强。此外,不同稀释倍数的MGF360-9L多克隆抗体均可抑制感染重组病毒ASFV-GFP的猪肺泡巨噬细胞中绿色荧光蛋白的产生。结果表明,与阴性家兔血清相比,不同稀释倍数的MGF360-9L抗体均可显著抑制ASFV的复制。本研究为ASFV靶向药物和疫苗的研发提供了参考依据。To prepare polyclonal antibodies against African swine fever virus(ASFV)MGF360-9 L protein and evaluate its effect on virus replication,the antigenic epitope of MGF360-9 L was predicted by DNAStar 2.1 software,and the MGF360-9 L C-terminal polypeptide C-KNLSIAHKHYINDGFND was selected and synthesized to prepare MGF360-9 L rabbit polyclonal antibody.Then the effect of polyclonal antibody against MGF360-9 L on virus replication was evaluated by real-time quantitative PCR,50%hemadsorpting doses assay and Western-blot method.The results showed that MGF360-9 L polyclonal antibody of different dilution times could significantly reduce the virus copy,titer and the expression of D1133 L and B646 L(p72)protein in different infection time,and the lower the antibody dilution,the stronger the effect of reducing virus copy,titer and expression of virus protein.In addition,MGF360-9 L polyclonal antibody of different dilution times could inhibit the production of green fluorescence protein in porcine alveolar macrophages infected with recombinant virus ASFV-GFP.The results suggest that MGF360-9 L polyclonal antibody of different dilution times could significantly inhibit the replication of ASFV compared with negative rabbit serum.This study provides a reference basis for the research and development of targeted drugs and vaccines for ASFV.

关 键 词：非洲猪瘟病毒 MGF360-9L蛋白 多克隆抗体 抗体功能 猪肺泡巨噬细胞 

分 类 号：S852.659.1[农业科学—基础兽医学]