非洲猪瘟病毒p72重组蛋白单克隆抗体的制备及双抗体夹心ELISA方法的建立  

Preparation of monoclonal antibody against recombinant protein of African swine fever virus p72 and establishment of double antibody sandwich ELISA method

作　　者：谢林梅 唐子木 钱新杰 陈君[1] 邓浩东 邹前萍 朱世锋 魏毓卿 魏小冬 花慧颖 丁能水[1,2] 邬向东 李细林[3] 丁珍 胡睿铭[1] XIE Linmei;TANG Zimu;QIAN Xinjie;CHEN Jun;DENG Haodong;ZOU Qianping;ZHU Shifeng;WEI Yuqing;WEI Xiaodong;HUA Huiying;DING Nengshui;WU Xiangdong;LI Xilin;DING Zhen;HU Ruiming(College of Animal Science&Technology,Jiangxi Agriculturual University,Nangchang 330045,China;Key Laboratory of Swine Nutrition and Feed Science,Zhangzhou 363000,China;Fuzhou Dongxiang District Agricultural Science and Technology Research Center,Fuzhou 344000,China)

机构地区：[1]江西农业大学动物科学技术学院,南昌330045 [2]福建省生猪营养与饲料重点实验室,福建漳州363000 [3]抚州市东乡区农业科学技术研究中心,江西抚州344000

出　　处：《黑龙江畜牧兽医》2023年第13期5-11,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：江西省教育厅科技计划项目(GJJ190192);江西省科技计划项目(20202BAB205004);福建傲农生物科技集团股份有限公司项目“江西生猪产业体系”(JXARS-01)。

摘　　要：为了建立一种快速检测非洲猪瘟病毒(African swine fever virus,ASFV)的方法,试验利用GenBank中ASFV的p72基因(登录号为MK128995.1)序列构建原核重组表达质粒pGEX-6P-1-p72和真核重组表达质粒pCAGGS-myc-p72,通过大肠杆菌原核表达系统获得p72重组蛋白,将其纯化后免疫小鼠,获得阳性杂交瘤细胞,通过Western-blot和间接免疫荧光鉴定筛选单克隆细胞株,并用单克隆抗体亚类鉴定试剂盒鉴定各单克隆抗体的亚型。通过对p72兔源多克隆抗体包被浓度及纯化的3株单克隆抗体稀释度进行优化,初步建立检测ASFV的双抗体夹心ELISA方法,并用该方法测试纯化的3株单克隆抗体分别与p72兔源多克隆抗体两两组合后所能检测的p72重组蛋白的灵敏度。结果表明:p72重组蛋白大小为99 ku;以纯化的原核表达的p72重组蛋白作为抗原免疫3只小鼠,抗体效价均大于1∶10 000;经细胞融合、克隆和筛选共获得5株阳性杂交瘤细胞,将其分泌的单克隆抗体分别命名为2C4C8、2C4B8、5C11F11、5C11D12、7D10C9,5株单克隆抗体与真核表达的p72重组蛋白均可发生特异性反应;单克隆抗体2C4B8和2C4C8的亚型为IgG1,5C11D12和5C11F11的亚型为IgG2b,7D10C9的亚型为IgG2a,轻链亚类均为κ;单克隆抗体2C4C8、5C11D12、7D10C9所能检测的p72重组蛋白最低浓度分别为0.25,0.25,0.05μg/mL。说明以纯化的3株单克隆抗体建立的双抗体夹心ELISA方法可用于检测p72重组蛋白抗原,且该检测方法有较好的灵敏度。To establish a rapid etiological detection method for African swine fever virus(ASFV),the prokaryotic recombinant expression plasmid pGEX-6P-1-p72 and the eukaryotic recombinant expression plasmid pcags-Myc-P72 were constructed by using the sequence of p72 gene(accession number MK128995.1) of ASFV in GenBank.The recombinant p72 protein was obtained by E.coli prokaryotic expression system,purified and mice were immunized to obtain positive hybridoma cells.Monoclonal cell lines were screened by Western-blot and indirect immunofluorescence,and subtypes of monoclonal antibodies were identified by monoclonal antibody subclass identification kit.The coated concentration of p72 rabbit polyclonal antibody and the dilution of three purified monoclonal antibodies were optimized.A double-antibody sandwich ELISA method was established to detect ASFV,and the sensitivity of p72 recombinant protein could be detected by combining three purified monoclonal antibodies with p72 rabbit polyclonal antibody.The results showed that the size of p72 recombinant protein was 99 ku.Three mice were immunized with purified p72 recombinant protein as antigen,and the antibody titers were all greater than 1∶10 000.Five positive hybridoma cells were obtained by cell fusion,cloning and screening,which were named 2C4C8,2C4B8,5C11F11,5C11D12,7D10C9,respectively.The five monoclonal antibodies could react specifically with the recombinant p72 protein expressed in eukaryotes.The subtype of monoclonal antibody 2C4B8 and 2C4C8 was IgG1;the subtype of 5C11D12 and 5C11F11 was IgG2b;the subtype of 7D10C9 was IgG2a and the light chain subtype was κ.The lowest concentrations of p72 recombinant protein detected by monoclonal antibodies 2C4C8,5C11D12 and 7D10C9 were 0.25,0.25 and 0.05 μg/mL,respectively.These results indicated that the double antibody sandwich ELISA based on three purified monoclonal antibodies could be used for the detection of p72 recombinant protein antigen,and the detection method had good sensitivity.

关 键 词：非洲猪瘟病毒 p72蛋白 单克隆抗体 双抗体夹心ELISA 灵敏度 

分 类 号：S852.659.1[农业科学—基础兽医学] S852.42[农业科学—兽医学]