A型塞内卡病毒VP1蛋白单克隆抗体制备及胶体金试纸条的试制  

Preparation of Monoclonal Antibody Based on VP1 Protein Against Senecavirus A(SVA)and Trial Production of Colloidal Gold Test Strips for SVA Detection

作　　者：张涛[1,2] 王会宝 孙燕燕 翟国元 崔燕 ZHANG Tao;WANG Hui-Bao;SUN Yan-Yan;ZHAI Guo-Yuan;CUI Yan(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;China Agricultural Vet.Bio.Science and Technology CO.,Ltd.,Lanzhou 730046,China;Lanzhou Animal Research Biotechnology Co.,Ltd.,Lanzhou 730046,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中农威特生物科技股份有限公司,兰州730046 [3]兰州兽研生物科技有限公司,兰州730046

出　　处：《农业生物技术学报》2022年第11期2255-2266,共12页Journal of Agricultural Biotechnology

基　　金：甘肃省青年科学基金(20JR10RA475)。

摘　　要：A型塞内卡病毒(Senecavirus A,SVA)是近年新发现的一种病原体,严重危害着我国养猪业的健康发展。为制备SVA病毒结构蛋白1(viral structural protein 1,VP1)单克隆抗体(monoclonol aitibody,McAbs)及建立一种快速、高效、简便的SVA检测方法,本研究利用RT-PCR方法扩增SVA VP1基因,将其克隆到表达载体pET-28a(+)中,诱导表达并纯化得到重组VP1蛋白。以重组VP1蛋白免疫BALB/c小鼠(Mus musculus),筛选获得2株稳定分泌抗SVA VP1蛋白单克隆抗体的杂交瘤细胞株(2E10,3E4)。通过2E10和3E4免疫小鼠获得腹水型McAbs并纯化,利用间接免疫荧光实验(indirect immunofluorescence assay,IFA)和Western blot鉴定McAbs。以2E10 McAbs作为捕捉抗体,山羊抗鼠IgG与3E4 McAbs分别作为质控线(C线)与检测线(T线)试制SVA胶体金试纸条。结果显示,重组SVA VP1蛋白主要以包涵体形式表达,分子质量约为43 kD。抗体亚型鉴定结果显示,2E10重链亚型为IgG2b,3E4重链亚型为IgG2a,轻链均为κ链。IFA和Western blot结果显示,制备的McAbs与SVA特异性结合。试纸条检测结果显示,试纸条能特异性地检测SVA,而与口蹄疫病毒(Foot and mouth disease virus,FMDV)、猪繁殖障碍与呼吸症病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪瘟病毒(Classical swine fever virus,CSFV)和猪圆环病毒2型病毒(Porcine cirovirus type 2,PCV2)无交叉反应;最低检测病毒含量为5×10TCID/mL;该方法与qPCR方法检测临床样品的符合率达96.55%。以上结果表明,本研究制备的基于VP1单克隆抗体的胶体金试纸条为SVA的即时检测提供了新方法。Senecavirus A(SVA),an emerging pathogen in recent years,is posing a severe danger to the healthy development of pig(Sus domesticus)industry in China.Viral structural protein 1(VP1),the main structural protein of SVA,can induce the body to produce specific antibodies.In order to prepare monoclonal antibodies(McAbs)against SVA VP1 and establish a rapid,efficient and simple method for the detection of SVA,the SVA VP1 gene was amplified by RT-PCR and cloned into the expression vector pET-28a(+).After induction,expression and purification,the recombinant SVA VP1 protein was obtained.BALB/c mice(Mus musculus)were immunized with recombinant SVA VP1 protein,2 hybridoma cell lines(2E10 and 3E4)stably secreting monoclonal antibodies against SVA VP1 protein were obtained.Ascites type McAbs were obtained and purified by immunizing mice with cell lines 2E10 and 3E4.McAbs were identified by indirect immunofluorescence assay(IFA)and Western blot.The prepared 2E10 McAbs were used as capture antibody,Goat anti-mouse IgG and 3E4 McAbs were used as quality control band(C band)and detection band(T band)to produce SVA colloidal gold test strip.The results showed that the recombinant SVA VP1 protein was mainly expressed in the form of inclusion bodies,with a molecular weight of about 43 kD.The results of antibody subtype identification showed that the heavy chain subtypes of 2E10 and 3E4 were IgG2b and IgG2a,respectively,and the light chains were bothκchain.The results of IFA and Western blot showed that the obtained McAbs were specific bound to SVA.The test of strip showed that colloidal gold test strips could specifically detect SVA,and had no cross reaction with Foot and mouth disease virus(FMDV),Porcine reproductive and respiratory syndrome virus(PRRSV),Classical swine fever virus(CSFV)and Porcine circovirus type 2(PCV2);The minimum detected viral content was 5×10TCID/mL;The positive coincidence rate between this method and qPCR was 96.55%.The above results indicate that the prepared colloidal gold test paper based on VP1 monocl

关 键 词：A型塞内卡病毒(SVA) 病毒结构蛋白1(VP1) 单克隆抗体 胶体金试纸条 

分 类 号：S855.3[农业科学—临床兽医学]