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首页> 中文期刊> 《微生物学通报》 >基于猪德尔塔冠状病毒重组核衣壳蛋白的ELISA抗体检测方法的建立与评价

基于猪德尔塔冠状病毒重组核衣壳蛋白的ELISA抗体检测方法的建立与评价

         
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摘要

[Objective] Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus.PDCoV outbreak was first reported in the United States swine in 2014,and has subsequently been reported in many Asian countries.Nowadays,PDCoV has been becoming a great threat to swine industry worldwide.Using the recombinant nucleocapsid (N) protein expressed in E.coli Rosetta(DE3) as the coated antigen to establish an indirect ELISA for the detection of PDCoV antibody,providing a useful tool for antibody detection and epidemiology surveillance of PDCoV.[Methods] The full-length cDNA of PDCoV N gene was amplified by RT-PCR based on the PDCoV strain CHN-HN-2014.The RT-PCR production was inserted into prokaryotic expressing vector pET-30a to generate a recombinant plasmid pET30a-N.Then pET30a-N was transformed into E.coli Rosetta(DE3) and induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG).The expressed N protein was purified and used as coated antigen to develop an indirect ELISA for detecting antibodies against PDCoV.After the specificity,sensibility,and repeatability of the developed ELISA method were evaluated,this method was used to test clinical serum samples.[Results] SDS-PAGE and Western blotting analysis showed that the N protein was efficiently expressed in supernatant and was specific reactions with antiserum against PDCoV.The established N-ELISA was highly specific,sensitive and repeatable.A total of 148 serum samples collected from pigs with a known immunization history and 102 serum samples collected from pigs with unknown PDCoV status were detected using both N-ELISA and neutralization test.The results showed that the positive agreement rate was 88.99％,the negative agreement rate was 92.90％,and overall coincidence rate was 91.20％.The positive rate of 267 clinical serum samples examined by N-ELISA was 66.67％.[Conclusion] The established ELISA method can be used as a tool for antibody detection and epidemiology surveillance of PDCoV.%[目的]猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV)是近年来新发现的一种猪肠道冠状病毒,2014年首次暴发于美国,随后亚洲多个国家也相继报道,对养猪业构成了巨大威胁.以大肠杆菌表达纯化的PDCoV重组核衣壳(N)蛋白为包被抗原,建立检测PDCoV抗体的间接ELISA方法,为PDCoV的血清抗体检测和流行病学调查提供工具.[方法]以PDCoVCHN-HN-2014株的基因组RNA为模板,通过RT-PCR扩增PDCoV核衣壳蛋白(N)基因的全长cDNA,将其插入原核表达载体pET-30a中,构建原核表达质粒pET30a-N,转化大肠杆菌Rosetta(DE3),经异丙基-β-D-硫代半乳糖苷(Isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导表达,以纯化的重组N蛋白为包被抗原,建立PDCoV N-ELISA抗体检测方法,评估其特异性、敏感性、稳定性,并用于临床血清的检测.[结果]SDS-PAGE电泳检测证实表达的重组N蛋白主要以可溶性形式存在,Western blotting证实表达的重组蛋白具有反应活性.用纯化的重组蛋白建立的N-ELISA具有良好的特异性、敏感性、稳定性.与中和试验同时检测148份免疫猪血清和102份临床血清,两种方法的阳性符合率为88.99％,阴性符合率为92.90％,总符合率为91.20％.用建立的ELISA方法检测267份临床血清,PDCoV抗体阳性血清的比率为66.67％.[结论]建立的猪德尔塔冠状病毒N-ELISA抗体检测方法与中和试验的符合率高,可用于PDCoV血清抗体检测和流行病学调查.

著录项

  • 来源
    《微生物学通报》 |2017年第12期|2830-2838|共9页
  • 作者

    杨浩; 方六荣; 董楠; 刘静; 钱瑾; 刘寒; 王荡; 肖少波;

  • 作者单位

    华中农业大学动物医学院农业微生物学国家重点实验室 湖北武汉430070;

    华中农业大学动物医学院农业微生物学国家重点实验室 湖北武汉430070;

    华中农业大学动物医学院农业微生物学国家重点实验室 湖北武汉430070;

    华中农业大学动物医学院农业微生物学国家重点实验室 湖北武汉430070;

    华中农业大学动物医学院农业微生物学国家重点实验室 湖北武汉430070;

    华中农业大学动物医学院农业微生物学国家重点实验室 湖北武汉430070;

    华中农业大学动物医学院农业微生物学国家重点实验室 湖北武汉430070;

    华中农业大学动物医学院农业微生物学国家重点实验室 湖北武汉430070;

  • 原文格式 PDF
  • 正文语种 chi
  • 中图分类
  • 关键词

    猪德尔塔冠状病毒; 核衣壳蛋白; 间接ELISA; 中和试验;

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