A型塞内卡病毒VP2蛋白多克隆抗体制备与竞争ELISA方法的建立  

Preparation of Polyclonal Antibodies against SVA VP2 Protein andEstablishment of Competitive ELISA Method

作　　者：何启杰 农作荣 王豪 牛晨霞 莫勇芳 欧阳康[1,2,3,4] 陈樱 黄伟坚[1,2,3,4] 黄稳妃 韦祖樟[1,2,3,4] HE Qi-jie;NONG Zuo-rong;WANG Hao;NIU Chen-xia;MO Yong-fang;OUYang Kang;CHEN Ying;HUANG Wei-jian;HUANG Wen-fei;WEI Zu-zhang(College of Animal Science and Technology,Guangxi University,Nanning,Guangxi,530005,China;Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics,Nanning,Guangxi,530005,China;Guangxi Key Laboratory of Animal Breeding,Disease Control and Prevention,Nanning,Guangxi,530004,China;Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Diseases,Nanning,Guangxi,530005,China;Department of Animal Science and Technology,Guangxi Agricultural Vocational University,Nanning,Guangxi,530007,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530005 [2]广西壮族自治区兽用生物制品工程研究中心,广西南宁530005 [3]广西畜禽繁育与疾病防控重点实验室,广西南宁530004 [4]广西高校动物疫病预防与控制重点实验室,广西南宁530005 [5]广西农业职业技术大学动物科学技术系,广西南宁530007

出　　处：《动物医学进展》2025年第2期23-29,共7页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31972666);来宾市科学研究与技术开发计划项目(来科攻220823,来科产220826)。

摘　　要：依据A型塞内卡病毒(SVA)结构蛋白VP2基因序列构建原核表达质粒pET-30a-VP2,经转化和诱导,表达出VP2重组蛋白,纯化后免疫新西兰大白兔,采集血清获得VP2的多克隆抗体。用间接ELISA方法测定血清中VP2抗体效价,并用Western blot与IFA验证其特异性。随后建立竞争ELISA方法,并对实验室收集的2016-2019年广西11个地区64个规模猪场592份猪血清进行SVA抗体检测。结果显示,诱导表达出的重组VP2蛋白主要以可溶性形式表达,血清中VP2抗体效价大于1∶64000,且制备的VP2抗体能特异结合SVA感染细胞中的VP2蛋白。竞争ELISA检测结果显示广西地区猪场SVA血清阳性率高达76.6%,血清样品阳性率高达55.9%,研究结果为SVA的检测和研究奠定了基础。In this study,a prokaryotic expression plasmid pET-30a-VP2 was constructed based on the SVA structural protein VP2 gene sequence.After transformation and induction,the VP2 recombinant protein was expressed,and then purified and used to immunize New Zealand white rabbits.The serum was collected to obtain the VP2 polyclonal antibodies.The serum VP2 antibody titer was measured by indirect ELISA,and its specificity was verified by Western blot and IFA.Subsequently,a competitive ELISA method was established,and 592 pig serum samples collected from 64 pig farms in 11 regions of Guangxi from 2016 to 2019 were tested for SVA antibody.The results showed that the induced VP2 recombinant protein was mainly expressed in soluble form,the serum VP2 antibody titer was greater than 1∶64000,and the prepared VP2 antibody could specifically bind to the VP2 protein in SVA-infected cells.The competitive ELISA detection results showed that the SVA serum positive rate in Guangxi pig farms was as high as 76.6%,and the serum sample positive rate was 55.9%.The results laid a foundation for the detection and research of SVA.

关 键 词：A型塞内卡 VP2蛋白 原核表达 多克隆抗体 竞争ELISA 

分 类 号：S852.65[农业科学—基础兽医学]