基于猪丁型冠状病毒重组S1蛋白间接ELISA抗体检测方法的建立与应用

doi:10.11843/j.issn.0366-6964.2019.08.013

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (8): 1642-1648.doi: 10.11843/j.issn.0366-6964.2019.08.013

基于猪丁型冠状病毒重组S1蛋白间接ELISA抗体检测方法的建立与应用

侯林杉¹, 贾敬亮², 顾文源³, 刘宝京¹, 师乾凯¹, 袁广富¹, 陈少杰¹, 范京惠^1*, 左玉柱^1*

1. 河北农业大学动物医学院, 保定 071001;
2. 衡水市畜牧技术推广站, 衡水 053000;
3. 河北省动物疫病预防控制中心, 石家庄 050035

收稿日期:2019-02-26 出版日期:2019-08-23 发布日期:2019-08-23
通讯作者: 左玉柱,主要从事动物传染病学的教学、科研和社会服务工作,E-mail:zuoyuzhu@163.com;范京惠,主要从事兽医微生物与免疫学的教学、科研工作,E-mail:jinghui76@163.com
作者简介:侯林杉(1993-),男,河南周口人,硕士生,主要从事微生物学与分子免疫学的研究,E-mail:644719595@qq.com
基金资助:
河北省科技创新资金项目（17C1303111001）；河北省农业产业技术体系生猪创新团队（HBCT2018110207）；河北省研究生创新能力培养资助项目（CXZZSS2018058）

Establishment and Application of an Indirect ELISA Based on Recombinant S1 Protein for the Detection of Antibodies against Porcine Deltacoronavirus

HOU Linshan¹, JIA Jingliang², GU Wenyuan³, LIU Baojing¹, SHI Qiankai¹, YUAN Guangfu¹, CHEN Shaojie¹, FAN Jinghui^1*, ZUO Yuzhu^1*

1. College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, China;
2. Hengshui Animal Husbandry Technology Promotion Station, Hengshui 053000, China;
3. Hebei Provincial Center for Animal Disease Control and Prevention, Shijiazhuang 050035, China

Received:2019-02-26 Online:2019-08-23 Published:2019-08-23

摘要/Abstract

摘要： 为了解猪丁型冠状病毒（PDCoV）的流行情况，为PDCoV血清抗体检测提供工具，本研究应用原核表达系统表达部分PDCoV纤突蛋白（S）作为检测抗原，建立了基于PDCoV S1蛋白的间接ELISA抗体检测方法，并用该方法检测了2017年1月至12月自河北地区部分猪场收集的待检血清样品570份。结果显示：本研究建立的PDCoV间接ELISA方法能够特异地检测血清中的PDCoV抗体；与猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪圆环病毒2型、猪繁殖与呼吸综合征病毒、猪瘟病毒及伪狂犬病病毒的抗血清无交叉反应；批内重复变异系数1.9%～5.4%、批间重复变异系数2.3%～5.1%。570份待检血样中105份呈阳性，阳性率为18.4%（105/570），高于之前本实验室检测2016年1月至2016年10月河北地区PDCoV IgG抗体阳性率（11%），提示河北地区PDCoV的感染率呈上升趋势。本研究建立的ELISA方法具有敏感性高、特异性强、重复性好的优点，可用于临床PDCoV血清抗体的检测。

Abstract: This study was conducted to understand porcine deltacoronavirus (PDCoV) epidemic situation, and provide a tool for PDCoV serum antibody detection and the epidemiological investigation. Prokaryotic expression of recombinant PDCoV partial spike (S) protein was used as detection antigen, and an indirect ELISA antibody detection method based on PDCoV S1 protein was established. The method was used to detect 570 serum samples collected from some pig farms in Hebei from January to December 2017. The results showed that the established ELISA method of PDCoV IgG antibody in this study could specifically detect PDCoV antibody; no cross-reaction with antisera against porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever (CSF) and pseudorabies virus (PRV) was found. The coefficients of variation (CV) of repeated in-batch test was 1.9%-5.4%, CV of batch-to-batch test was 2.3%-5.1%. In 570 blood samples, 105 were positive, and the positive rate was 18.4% (105/570), which was higher than the positive rate of PDCoV IgG antibody (11%) in Hebei from January 2016 to October 2016. It is suggested that the infection rate of PDCoV in Hebei is on the rise. The ELISA method established in this study had the advantages of high sensitivity, strong specificity and good reproducibility, and can be used for the detection of clinical PDCoV serum antibodies.

中图分类号:

S852.659.6

侯林杉, 贾敬亮, 顾文源, 刘宝京, 师乾凯, 袁广富, 陈少杰, 范京惠, 左玉柱. 基于猪丁型冠状病毒重组S1蛋白间接ELISA抗体检测方法的建立与应用[J]. 畜牧兽医学报, 2019, 50(8): 1642-1648.

HOU Linshan, JIA Jingliang, GU Wenyuan, LIU Baojing, SHI Qiankai, YUAN Guangfu, CHEN Shaojie, FAN Jinghui, ZUO Yuzhu. Establishment and Application of an Indirect ELISA Based on Recombinant S1 Protein for the Detection of Antibodies against Porcine Deltacoronavirus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(8): 1642-1648.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2019.08.013

https://www.xmsyxb.com/CN/Y2019/V50/I8/1642

参考文献

[1] WOO P C Y, LAU S K P, LAM C S F, et al. Discovery of seven novel Mammalian and avian coronaviruses in the genus Deltacoronavirus supports bat coronaviruses as the gene source of Alphacoronavirus,Betacoronavirus and avian coronaviruses as the gene source of Gammacoronavirus and Deltacoronavirus[J]. J Virol, 2012, 86(7):3995-4008.
[2] WANG L Y, BYRUM B, ZHANG Y. Detection and genetic characterization of deltacoronavirus in pigs, Ohio, USA, 2014[J]. Emerg Infect Dis, 2014, 20(7):1227-1230.
[3] 方谱县, 方六荣,董楠,等. 猪δ冠状病毒的研究进展[J]. 病毒学报, 2016, 32(2):243-248.
FANG P X, FANG L R, DONG N, et al. Research advances in the porcine deltacoronavirus[J]. Chinese Journal of Virology, 2016, 32(2):243-248. (in Chinese)
[4] JUNG K, HU H, EYERLY B, et al. Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs[J]. Emerg Infect Dis, 2015, 21(4):650-654.
[5] ZHANG M J, LIU D J, LIU X L, et al. Genomic characterization and pathogenicity of porcinedeltacoronavirus strain CHN-HG-2017 from China[J]. Arch Virol, 2019, 164(2):413-425.
[6] HU H, JUNG K, VLASOVA A N, et al. Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States[J]. J Clin Microbiol, 2015, 53(5):1537-1548.
[7] FANG P X, FANG L R, HONG Y Y, et al. Discovery of a novel accessory protein NS7a encoded by porcine deltacoronavirus[J]. J Gen Virol, 2017, 98(2):173-178.
[8] MA Y M, ZHANG Y, LIANG X Y, et al. Two-way antigenic cross-reactivity between porcine epidemic diarrhea virus and porcine deltacoronavirus[J]. Vet Microbiol, 2016, 186:90-96.
[9] SHANG J, ZHENG Y, YANG Y, et al.Cryo-electron microscopy structure of porcine deltacoronavirus spike protein in the prefusion state[J]. J Virol, 2018, 92(4):e01556-17.
[10] THACHIL A, GERBER P F, XIAO C T, et al. Development and application of an ELISA for the detection of porcine deltacoronavirus IgG antibodies[J]. PLoS One, 2015, 10(4):e0124363.
[11] LI Y F, ZHENG F Y, FAN B C, et al. Development of an indirect ELISA based on a truncated S protein of the porcine epidemic diarrhea virus[J]. Can J Microbiol, 2015, 61(11):811-817.
[12] LIN H X, ZHOU H, GAO L, et al. Development and application of an indirect ELISA for the detection of antibodies to porcine epidemic diarrhea virus based on a recombinant spike protein[J]. BMC Vet Res, 2018, 14:243.
[13] 刘宝京. 猪丁型冠状病毒HB-BD株分离鉴定及生物学特性研究[D]. 保定:河北农业大学, 2018.
LIU B J. Study on isolation, identification and biological characteristics of porcine deltacoronavirus HB-BD strain[D]. Baoding:Agricultural University of Hebei, 2018. (in Chinese)
[14] XU Z C, ZHONG H L, ZHOU Q F, et al. A highly pathogenic strain of porcine deltacoronavirus caused watery diarrhea in newborn piglets[J]. Virol Sin, 2018, 33(2):131-141.
[15] 张帆帆, 宋德平,周信荣,等. 新现猪Delta冠状病毒RT-PCR检测方法的建立及其应用[J]. 中国农业科学, 2016, 49(7):1408-1416.
ZHANG F F, SONG D P, ZHOU X R, et al. Establishment and application of a RT-PCR assay for detection of newly emerged porcine deltacoronavirus[J]. Scientia Agricultura Sinica, 2016, 49(7):1408-1416. (in Chinese)
[16] 陈建飞, 王潇博,焦贺勋,等. 国内首株猪德尔塔冠状病毒(Porcine deltacoronavirus)的分离鉴定[J]. 中国预防兽医学报, 2016, 38(3):171-174.
CHEN J F, WANG X B, JIAO H X, et al. Isolation and identification of the first Porcine deltacoronavirus strain in China[J]. Chinese Journal of Preventive Veterinary Medicine, 2016, 38(3):171-174. (in Chinese)
[17] 逄凤娇, 俞正玉,何孔旺,等. 猪Deltacoronavirus RT-PCR检测方法的建立及其应用[J]. 中国预防兽医学报, 2015, 37(9):683-686.
PANG F J, YU Z Y, HE K W, et al. Development and application of a RT-PCR assay for detection of porcine deltacoronavirus[J]. Chinese Journal of Preventive Veterinary Medicine, 2015, 37(9):683-686. (in Chinese)
[18] HOMWONG N, JARVIS M C, LAM H C, et al. Characterization and evolution of porcine deltacoronavirus in the United States[J]. Prev Vet Med, 2016, 123:168-174.
[19] 罗尚星, 范京惠,刘宝京,等. 猪丁型冠状病毒与猪流行性腹泻病毒双重实时荧光定量RT-PCR方法的建立和初步应用[J]. 畜牧兽医学报, 2018, 49(4):852-858.
LUO S X, FAN J H, LIU B J, et al. Establishment and application of the real-time reverse transcription quantitative PCR assay for porcine epidemic diarrhea virus and porcine deltacoronavirus[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(4):852-858. (in Chinese)
[20] 杨浩,方六荣,董楠,等. 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[J]. 微生物学通报, 2017, 44(12):2830-2838.
YANG H, FANG L R, DONG N, et al. Development and evaluation of an indirect ELISA based on recombinant nucleocapsid protein for detecting antibodies against porcine deltacoronavirus[J]. Microbiology China, 2017, 44(12):2830-2838. (in Chinese)
[21] 逄凤娇, 俞正玉,何孔旺,等. 猪丁型冠状病毒重组N蛋白间接ELISA抗体检测方法的建立[J]. 中国预防兽医学报, 2017, 39(6):461-465.
PANG F J, YU Z Y, HE K W, et al. Development of an indirect ELISA for detection antibody to porcine deltacoronavirus using recombinant N protein[J]. Chinese Journal of Preventive Veterinary Medicine, 2017, 39(6):461-465. (in Chinese)
[22] 张帆帆, 宋德平,郭楠楠,等. 以原核表达的猪δ冠状病毒N蛋白为包被抗原的间接ELISA方法的建立[J]. 中国预防兽医学报, 2016, 38(10):795-799.
ZHANG F F, SONG D P, GUO N N, et al. Establishment of a recombinant nucleoprotein-based ELISA for detection of antibodies against newly emerged porcine deltacoronavirus[J]. Chinese Journal of Preventive Veterinary Medicine, 2016, 38(10):795-799. (in Chinese)
[23] LUO S X, FAN J H, OPRIESSNIG T, et al. Development and application of a recombinant M protein-based indirect ELISA for the detection of porcine deltacoronavirus IgG antibodies[J]. J Virol Methods, 2017, 249:76-78.
[24] GIMENEZ-LIROLA L G, ZHANG J Q, CARRILLO-AVILA J A, et al. Reactivity of porcine epidemic diarrhea virus structural proteins to antibodies against porcine enteric coronaviruses:diagnostic implications[J]. J Clin Microbiol, 2017, 55(5):1426-1436.
[25] 李成,张雨迪,黄小波,等. 猪丁型冠状病毒S1基因的克隆、原核表达及多克隆抗体制备[J]. 畜牧兽医学报, 2018, 49(6):1256-1264.
LI C, ZHANG Y D, HUANG X B, et al. Cloning and prokaryotic expression of porcine deltacoronavirus S1 gene and preparation of polyclonal antibody[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(6):1256-1264. (in Chinese)
[26] 王经纬, 雷喜梅,覃盼,等. 猪丁型冠状病毒荧光定量RT-PCR与S1蛋白间接ELISA检测方法的建立及应用[J]. 生物工程学报, 2017, 33(8):1265-1275.
WANG J W, LEI X M, QIN P, et al. Development and application of real-time RT-PCR and S1 protein-based indirect ELISA for porcine deltacoronavirus[J]. Chinese Journal of Biotechnology, 2017, 33(8):1265-1275. (in Chinese)

基于猪丁型冠状病毒重组S1蛋白间接ELISA抗体检测方法的建立与应用

Establishment and Application of an Indirect ELISA Based on Recombinant S1 Protein for the Detection of Antibodies against Porcine Deltacoronavirus

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	宋雯妍, 张瀚文, 吴澳迪, 张丽燕, 刘照, 叶桐桐, 陈创夫, 盛金良. 猪繁殖与呼吸综合征病毒GP5蛋白纳米抗体的筛选及其对病毒复制的抑制效应[J]. 畜牧兽医学报, 2024, 55(1): 258-270.
[2]	王莹, 李佳康, 曾悦, 史开拓, 韦丽婷, 彭佳佳, 李秋燕, 曹龙龙, 周登元. 猫杯状病毒单克隆抗体的制备[J]. 畜牧兽医学报, 2024, 55(1): 401-405.
[3]	李惠惠, 董可儿, 刘馨博, 张春晓, 马超, 陈利苹, 钟旗, 姚刚, 马雪连. 牛诺瓦病毒和牛轮状病毒双重RAA-LFD快速检测方法的建立及应用[J]. 畜牧兽医学报, 2024, 55(1): 406-412.
[4]	周建浩, 王东方, 刘影, 王淑娟, 马震原, 谢彩华, 赵雪丽, 杨海波, 冯桂丹, 康台生, 胡煜锋, 李博文, 闫若潜. 猪流行性腹泻病毒微滴式数字PCR定量检测方法的建立及初步应用[J]. 畜牧兽医学报, 2024, 55(1): 413-418.
[5]	张家祺, 贾浠宁, 周群, 宋鑫, 朱晨曦, 李格格, 张斌. 基于E2蛋白BVDV-1病毒样颗粒的构建及对小鼠的免疫效力评估[J]. 畜牧兽医学报, 2023, 54(12): 5143-5153.
[6]	周立坤, 董鑫媛, 李家辉, 崔志莹, 赵士杰, 徐婕, 陈静, 张宜娜, 夏平安. MID2对猪繁殖与呼吸综合征病毒复制的调控作用[J]. 畜牧兽医学报, 2023, 54(11): 4735-4744.
[7]	郑如雯, 黄涛, 吴道义, 禹光美, 李芳芳, 隋鑫, 闵婷玉. 牛病毒性腹泻病毒LAMP-LFD检测方法的建立及初步应用[J]. 畜牧兽医学报, 2023, 54(11): 4745-4753.
[8]	贾艺泉, 于梦婷, 刘卫容, 方守国, 章松柏. 黄芩苷通过促进Ⅰ型干扰素表达抑制鸡传染性支气管炎病毒复制[J]. 畜牧兽医学报, 2023, 54(11): 4839-4850.
[9]	陈璐, 常新宇, 沈嘉忱, 沈瑞廷, 赵振华, 侯晓林. 鸡传染性支气管炎病毒感染HD11细胞的转录组分析[J]. 畜牧兽医学报, 2023, 54(11): 4860-4865.
[10]	贺叶青, 左百姣, 李庆豪, 孙娟, 金鑫, 夏璐, 魏战勇. 猪传染性胃肠炎病毒感染仔猪免疫器官的组织病理学观察[J]. 畜牧兽医学报, 2023, 54(11): 4872-4879.
[11]	陈文哲, 张向乐, 顾峰幸, 赵振翔, 李康丽, 薛钊宁, 郑海学, 张小丽, 朱紫祥. 猪GRK2蛋白抗口蹄疫病毒作用分析[J]. 畜牧兽医学报, 2023, 54(10): 4350-4361.
[12]	邹宏, 夏应菊, 李玲, 徐璐, 赵俊杰, 王团结, 张乾义, 宋振辉. 猪瘟病毒和牛病毒性腹泻病毒双重荧光定量PCR检测方法的建立和初步应用[J]. 畜牧兽医学报, 2023, 54(10): 4422-4427.
[13]	袁丽, 孙杨杨, 张路捷, 张杰, 孙海凤, 白娟, 姜平. 猪繁殖与呼吸综合征病毒GP3蛋白单克隆抗体制备及抗原表位鉴定[J]. 畜牧兽医学报, 2023, 54(8): 3424-3434.
[14]	曹丽艳, 孔祥雨, 李想通, 索学鹏, 段月月, 袁聪, 施磊, 张宇, 马国祥, 郑海学, 王琦. 猪急性腹泻综合征冠状病毒核衣壳蛋白单克隆抗体的制备及其抗体序列的分析[J]. 畜牧兽医学报, 2023, 54(6): 2487-2497.
[15]	张森豪, 王雪莹, 蔡李萌, 解伟纯, 匡虹迪, 王晓娜, 李佳璇, 崔文, 姜艳平, 周晗, 单智夫, 王丽, 乔薪瑗, 李一经, 唐丽杰. 猪流行性腹泻病毒重组酶聚合酶扩增快速诊断方法的建立和应用[J]. 畜牧兽医学报, 2023, 54(6): 2498-2508.