致马属动物流产沙门氏菌通用型间接ELISA抗体检测方法的建立与应用  

Development and Application of a Universal iELISA Antibody Assay for Abortion-Causing Salmonella in Equidae

作　　者：郭奎 张泽楠 李帅杰 初晓雨 王垚鑫 郭巍[1] 胡哲[1] 王晓钧[1] GUO Kui;ZHANG ZeNan;LI ShuaiJie;CHU XiaoYu;WANG YaoXin;GUO Wei;HU Zhe;WANG XiaoJun(Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences/State Key Laboratory for Animal Disease Control and Prevention,Harbin 150069)

机构地区：[1]中国农业科学院哈尔滨兽医研究所/动物疫病防控全国重点实验室,哈尔滨150069

出　　处：《中国农业科学》2023年第12期2421-2430,共10页Scientia Agricultura Sinica

基　　金：十四五国家重点研发计划重点专项(2021YFD1800500);“中国-哈萨克斯坦跨境动物疫病防控联合研究及一带一路科技合作平台建设”项目(2020YFE0203400)。

摘　　要：【目的】通过筛选引起马流产的4种沙门氏菌(Salmonella)共同的优势抗原,建立一种敏感高、特异强的通用型间接ELISA(iELISA)抗体检测方法,实现对马群中沙门氏菌抗体的快速高通量检测。【方法】首先利用马流产沙门氏菌阳性血清、阴性血清分别与马流产沙门氏菌全菌抗原进行免疫共沉淀(pull down)试验,筛选出马流产沙门氏菌优势抗原;根据质谱分析结果找到优势抗原基因,将优势抗原基因与其他3种致马流产沙门氏菌病的鼠伤寒沙门氏菌、都柏林沙门氏菌、肠炎沙门氏菌进行序列比对验证其保守性;其次根据抗原性分析对优势抗原编码基因进行分段表达,设计3对引物,利用聚合酶链式反应(polymerase chain reaction,PCR)扩增沙门氏菌优势抗原基因并将其分别克隆至原核表达载体pET28a;测序分析后将构建正确的重组质粒分别转化入大肠杆菌(Escherichia coli E.coli)Rosetta(DE3)中并加入0.6 mmol·L^(-1)异丙基-β-D-硫代半乳糖甘(Isopropy-β-D-thiogalactoside,IPTG)进行诱导表达,观察蛋白的表达形式;蛋白纯化后通过Western blot验证蛋白的反应原性和广谱性;然后利用纯化后蛋白作为包被抗原,通过对包被抗原量、血清和第二抗体浓度等条件进行优化建立马流产沙门氏菌病间接ELISA抗体诊断方法,并对该方法的特异性、敏感性进行评估。最后将该方法应用于试验感染马血清及临床样本的检测,并将检测结果同凝集试验结果比较。【结果】筛选出的马流产沙门氏菌优势抗原是ompA(Outer Membrane Protein,OMP),通过序列比对,该蛋白氨基酸序列与同属鼠伤寒沙门氏菌、都柏林沙门氏菌、肠炎沙门氏菌同源性99.4%—100%,具有很好的保守性;PCR扩增后成功获得3条与预期符合的目的基因;成功构建了pET28a-ompA1、pET28a-ompA2、pET28a-ompA33个重组质粒。SDS-PAGE结果表明,在24℃、0.6 mmol·L^(-1) IPTG诱导5 h下,含有pET28a-ompA1、pET【Objective】The aim for this study was to identify the predominant antigen of Salmonella and to develop a sensitive,specific and universal iELISA assay method for the rapid and accurate detection of Salmonella antibodies in equidae.【Method】For the purpose of screening out dominant antigens for Salmonella Abortusequi,the immunoprecipitation(pull down)tests were performed using Salmonella Abortusequi positive/negative sera with whole bacterium antigens of Salmonella Abortusequi.Then,amino acid sequence alignment of the dominant antigen were compared with Salmonella Typhimurium,Salmonella Dublin,and Salmonella Enteritidis to verify itsconservative.Three pairs of specific primers were designed and synthesized according to the nucleotide sequence of the full ompA gene published in GenBank.Three ompA genes with different lengths were amplified by PCR,and then cloned into pET28a vector and transformed Escherichia coli Rosetta(DE3)competent cell.The expressed products were analyzed by SDS-PAGE electrophoresis and Western-blot test after induced by IPTG.The reactivity of the purified protein was verified using S.Abortusequi,Salmonella Typhimurium,Salmonella Dublin(S.Dublin),and S.Enteritidis serums,and one negative serum by Western blot.An indirect ELISA method for the diagnosis of equine abortion salmonellosis was developed by optimizing the amount of coating antigen,serum and secondary antibody concentrations using the purified ompA3 protein as the coating antigen,and evaluating the specificity and sensitivity of the iELISA,and finally applying the iELISA to detection of clinical samples.【Result】In this study,the ompA dominant antigen of S.Abortusequi was screened.S.Abortusequi ompA was conservative and showed 99.4%-100%identical with Salmonella Typhimurium,Salmonella Dublin and S.Enteritidis strains at the amino acid level.Three target genes were successfully obtained by PCR amplification.Three recombinant plasmids,including pET28a-ompA1,pET28a-ompA2,and pET28a-ompA3 were successfully constructed.The express

关 键 词：马流产沙门氏菌病 OMPA 通用型 IELISA 诊断 

分 类 号：S852.61[农业科学—基础兽医学]