摘要
为快速检测猪传染性胃肠炎病毒(TGEV)抗体,本研究以原核表达系统串联表达的含有TGEV S蛋白抗原表位C的重组蛋白r-(C_1C_2)6为包被抗原建立TGEV血清抗体的Dot-ELISA检测方法。经反应条件优化结果显示,抗原最适包被量为62.5 ng/点;血清最佳工作浓度和工作时间分别为1∶80和45 min;羊抗猪IgG-HRP最佳工作浓度和工作时间分别为1∶800和45 min;最适封闭条件分别为5%脱脂乳37℃,45 min;血清和酶标抗体最适反应温度均为37℃;最佳显色时间7.5 min。结果显示该方法与猪轮状病毒和猪流行性腹泻病毒阳性血清无交叉反应,表明其具有良好的特异性。该方法对TGEV阳性血清最低检测限为1∶1 280。采用建立的Dot-ELISA对27份临床猪血清样品进行检测,结果显示Dot-ELISA与TGEV/PRCV抗体检测试剂盒检测结果的符合率为92.6%。本研究建立的Dot-ELISA检测方法简便、快速、灵敏、特异,适合用于TGE快速诊断。
In order to establish a rapid method for detection of antibodies to transmissible gastroenteritis virus （TGEV）, a Dot-ELISA was established with the coating antigen of a recombinant protein r-（C1C2）6, which was a multi-copy antigenic epitope C of TGEV spike （S） protein expressed in E.coli. The reaction condition for Dot-ELISA was optimized including： coating with the r-（C1C2）6 at the concentration of 62.5ng/spot, diluting the serum in 1：80 and incubating for 45min, detecting with goat anti-pig HRP-labeled antibody IgG in 1：800 dilution and incubating for 45 min, blocking with 5% skimmed milk incubating 45min under 37℃. Cross test showed the Dot-ELISA was specific for TGEV antibody detection. Sensitive test showed the Dot-ELISA was capable to detect the positive sera diluted up to 1：1,280. Tested on 27 clinical serum samples, the coincidence rate for detection between the Dot-ELISA and SvanovaTM TGEV/PRCV-Ab ELISA Kits were 92.6%. In conclusion, the Dot-ELISA detection method is comparatively convenient, rapid, sensitive and specific, which is suitable for rapid diagnosis of porcine transmissible gastroenteritis （TGE）.
作者
于天飞
董慧莹
张喜文
谢鹏宇
孙婉姝
王有祺
YU Tian-fei;DONG Hui-ying;ZHANG Xi-wen;XIE Peng-yu;SUN Wan-shu;WANG You-qi(College of Life Science and Agriculture and Forestry,Qiqihar University,Qiqihar 161006,China;Key Laboratory of Resistance Gene Engineering and Protection of Biodiversity in Cold Areas,Heilongjiang Province,Qiqihar 161006,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2018年第10期956-959,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
黑龙江省自然科学基金(QC2014C034)
黑龙江省普通本科高等学校青年创新人才培养计划(UNPYSCT-2015093)
齐齐哈尔大学研究生创新科研项目(YJSCX2017-030X)
