抗水禽呼肠孤病毒σC蛋白抗体间接ELISA方法的建立及鹅群血清学调查  

Development of an Indirect ELISA Method for Antibodies Against Waterfowl ReovirusσC Protein and Serological Investigation of Goose Population

作　　者：陈稼龙 何莉 郑炜鸿 朱婉君 张济培 陈济铛 CHEN Jialong;HE Li;ZHENG Weihong;ZHU Wanjun;ZHANG Jipei;CHEN Jidang(School of Animal Science and Technology,Foshan University,Foshan Guangdong 528225;Foshan Tianmu Biotechnology Co Lt,Foshan Guangdong 528225)

机构地区：[1]佛山大学动物科技学院,广东佛山528225 [2]佛山天牧生物科技有限公司,广东佛山528225

出　　处：《广东畜牧兽医科技》2024年第6期8-15,共8页Guangdong Journal of Animal and Veterinary Science

基　　金：广东省普通高校科研项目重点领域专项(2020ZDZX1018);佛山天牧生物科技有限公司技术开发项目(KH22275);佛山万牧洲生物科技有限公司技术开发项目(KH23279)。

摘　　要：为提供水禽呼肠孤病毒病早期诊断的精准快速检测方法,该试验通过BL21(DE3)原核表达系统稳定表达水禽呼肠孤病毒σC蛋白,并将纯化的重组σC蛋白包被ELISA酶标板,经进一步反应条件优化。结果显示,该试验建立的ELISA检测方法最优反应条件是:抗原包被含量为1μg/孔,待检血清最佳稀释度为1∶50,孵育时间为45 min,酶标二抗最佳稀释度为1∶500,孵育时间为30 min,最佳底物显色时间为20 min,OD_(450)值≥0.3判定为阳性。该方法具有良好的特异性、敏感性和重复性,对水禽呼肠孤病毒基因Ⅰ型和基因Ⅱ型血清具有一定的交叉反应。对105份临床健康鹅群血清样品进行检测,阳性率高达77.33%。该研究提供了一种敏感高效的快速检测水禽呼肠孤病毒中和抗体的方法,为鹅群临床血清大规模检测提供方法,为研发商品化的ELISA检测试剂盒提供基础。To provide a serological detection method for the early diagnosis of reovirus disease in waterfowl,this study stably expressed theσC protein of waterfowl reovirus by BL21(DE3)prokaryotic expression system.The purified recombinantσC protein was coated with ELISA plate,and the reaction conditions were further optimized.The results showed that the optimal reaction conditions for the ELISA detection method established in this assay were as follows:antigen coating content of 1μg/well,the best dilution of serum to be tested was 1∶50,incubation time was 45 min,the best dilution of HRP-conjugated secondary antibody was 1∶500,incubation time was 30 min,the best substrate chromogenic time was 20 min.OD_(450)value≥0.3 was considered positive.This method has good specificity,sensitivity and repeatability,and has certain cross-reactivity against reovirus genotype I and genotypeⅡserotypes in waterfowl.The positive rate of 105 serum samples from healthy geese was 77.33%.This study provides a sensitive and efficient method for the rapid detection of neutralizing antibodies against reovirus in waterfowl,which provides a method for the large-scale detection of clinical serum in geese,and provides a basis for the development of commercial ELISA kits.

关 键 词：水禽呼肠孤病毒 σC蛋白 间接ELISA方法 血清学调查 

分 类 号：S852.657[农业科学—基础兽医学]