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

发布时间：2018-03-31 10:03

本文选题：猪德尔塔冠状病毒　切入点：核衣壳蛋白　出处：《微生物学通报》2017年12期

【摘要】：【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PDCoV CHN-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p ET30a-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。
[Abstract]:[objective] Porcine Delta coronavirus (PDCoV) is a newly discovered porcine intestinal coronavirus in recent years. It first broke out in the United States in 2014, and was subsequently reported in many countries in Asia. The recombinant PDCoV nucleocapsid protein expressed by Escherichia coli was used as the coating antigen to establish an indirect ELISA method for the detection of PDCoV antibody. [methods] using genomic RNA of PDCoV CHN-HN-2014 strain as template, the full-length cDNA of PDCoV nucleocapsid protein N) gene was amplified by RT-PCR and inserted into prokaryotic expression vector pET-30a. The prokaryotic expression plasmid pET30a-N was constructed and transformed into E. coli Rosetta-DE3N. The expression was induced by Isopropyl-beta-beta-D-thiogactopyranoside IPTGG. The purified recombinant N protein was used as the encapsulated antigen to establish a method for the detection of PDCoV N-ELISA antibody and to evaluate its specificity, sensitivity and stability. [results] SDS-PAGE electrophoresis showed that the expressed recombinant N protein mainly existed in soluble form. The recombinant protein expressed by Western blotting had the reaction activity. The N-ELISA established by the purified recombinant protein had good specificity. Sensitivity and stability. 148 sera from immunized pigs and 102 clinical sera were detected simultaneously with neutralization test. The positive coincidence rate of the two methods was 88.99 and the negative coincidence rate was 92.90, and the total coincidence rate was 91.20.The positive rate of the two methods was 66.67 by using the established ELISA method. [conclusion] the established swine Delta coronavirus N-ELISA anti-physical examination. The coincidence rate between the test method and the neutralization test is high. It can be used for PDCoV serum antibody detection and epidemiological investigation.
【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;
【基金】：国家科技支撑计划项目(No.2015BAD12B02) 国家重点研发计划项目(No.2016YFD0500103)~~
【分类号】：S852.651

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