应用TmAdh3重组蛋白建立绵羊脑多头蚴病间接ELISA诊断方法的研究  

Development of indirect ELISA for the diagnosis of cerebral coenurosis in sheep based on recombinant protein TmAdh3

作　　者：刘垠鞠 李文卉[1] 岳龙 张念章[1] 金启旺 秦洪涛 贾万忠[1] 姚强[3] 付宝权[1,4] LIU Yin-ju;LI Wen-hui;YUE Long;ZHANG Nian-zhang;JIN Qi-wang;QIN Hong-tao;JIA Wan-zhong;YAO Qiang;FU Bao-quan(State Key Laboratory for Animal Disease Control and Prevention/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Wuhan Animal Disease Prevention and Control Center,Wuhan 430016,China;China Animal Disease Prevention and Control Center,Beijing 100125,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]中国农业科学院,兰州兽医研究所,动物疫病防控全国重点实验室,甘肃兰州730046 [2]武汉市动物疫病预防控制中心,湖北武汉430016 [3]中国动物疫病预防控制中心,北京100125 [4]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2023年第9期1082-1088,共7页Chinese Veterinary Science

基　　金：国家重点研发计划项目(政府间国际科技创新合作)(2021YFE0191600);国家自然科学基金项目(31702228)。

摘　　要：为评价多头带绦虫TmAdh3重组蛋白作为脑多头蚴病诊断抗原的潜力,根据多头带绦虫TmAdh3基因组序列,人工优化合成TmAdh3基因ORF序列并连接至表达载体pGEX-4T-1,转化至大肠杆菌BL21中诱导表达,利用Western-blot分析纯化的重组蛋白的反应原性,优化检测条件,建立脑多头蚴病间接ELISA诊断方法并进行评价。结果显示,成功表达了分子质量大小为39.0 ku的重组蛋白rTmAdh3。间接ELISA检测的最适反应条件为:5.00μg/m L重组蛋白rTmAdh3在4℃包被过夜;1∶100稀释血清在37℃孵育30min;1∶10 000稀释的HRP标记的酶标二抗在37℃孵育45 min,底物避光显色10 min。应用建立的方法可检测到人工感染脑多头蚴后第3~8周的绵羊血清;对40份自然感染脑多头蚴的绵羊(剖检确认脑部有包囊)血清的阳性检出率为22.5%(9/40)。与细粒棘球蚴感染绵羊血清和细粒棘球蚴Eg95免疫绵羊血清交叉反应率为5%(4/80)。结果表明,本研究建立的以TmAdh3重组蛋白作为检测抗原的间接ELISA诊断方法可用于绵羊脑多头蚴感染早期的筛选。To evaluate the diagnostic potential of recombinant protein TmAdh3 for cerebral coenurosis in sheep,the TmAdh3 gene open reading frame(ORF)was synthesized and ligated into pGEX-4T-1 expression vector.The expected molecular mass of 39.0 ku of the recombinant protein TmAdh3 was successfully expressed in prokaryotic system.Subsequently,the reactogenicity of the purified protein was analyzed by Western-blot.The optimal indirect ELISA reaction conditions were:5.00μg/mL rTmAdh3 coated antigen at 4℃for one night,1∶100 diluted serum was incubated at 37℃for 30 min,1∶10000 diluted HRP secondary antibody was incubated at 37℃for 45 min,the substrate was developed protection from light for 10 min.The rTmAdh3-based ELISA can recognize the positive sera from the infected sheep post 3 to 8 weeks withTaenia multiceps.The positive percentage of the naturally infected sheep was 22.5%(9/40)(cysts were confirmed by necropsying in the brain),and the cross-reactivity percen tage of the serum from sheep which were experimentally infected with Echinococcus granulosus and immunized with Eg95 recombinant antigen was 5%(4/80).In this study,the recombinant antigen TmAdh3 built indirect ELISA could be used for early diagnosis of coenurus infection in sheep.

关 键 词：多头带绦虫 脑多头蚴病 TmAdh3 间接ELISA 

分 类 号：S852.734[农业科学—基础兽医学]