猪传染性胃肠炎病毒N蛋白原核表达和间接ELISA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2016.09.012

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (9): 2296-2301.doi: 10.16431/j.cnki.1671-7236.2016.09.012

猪传染性胃肠炎病毒N蛋白原核表达和间接ELISA检测方法的建立

郭宏伟, 郑鸣, 乔宏兴, 马辉, 赵绪永, 王永芬

河南牧业经济学院生物工程学院, 郑州 450046

收稿日期:2016-02-29 出版日期:2016-09-20 发布日期:2016-09-20
通讯作者: 王永芬 E-mail:yongfwang@163.com
作者简介:郭宏伟(1977-),男,山西忻州人,讲师,研究方向:动物疫病防控,E-mail:ghw221@126.com
基金资助:
河南省科技攻关项目（142102310034、13A230382）；校科技创新团队项目（HUAHE2015001）

Prokaryotic Expression and Development of an Indirect ELISA Detection Method of Porcine Transmissible Gastroenteritis Virus N Protein

GUO Hong-wei, ZHENG Ming, QIAO Hong-xing, MA Hui, ZHAO Xu-yong, WANG Yong-fen

College of Biology Engineering, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China

Received:2016-02-29 Online:2016-09-20 Published:2016-09-20

摘要/Abstract

摘要：

试验构建了pET28a-TGEV-N重组表达质粒，完成TGEV N蛋白的表达和纯化，通过SDS-PAGE和Western blotting分析，表明表达产物N蛋白可被猪TGEV阳性血清识别。用TGEV N蛋白作为诊断抗原，建立了检测TGEV血清抗体的间接ELISA检测方法，该方法批内、批间重复试验变异系数均小于5%；检测血清的特异性为100%，假阳性率为0，假阴性率为5%，与7种常见猪病血清无交叉反应。针对临床血清，用该方法检测结果与中和试验结果相比，符合率为100%。该方法能够快速、有效地检出TGEV抗体，重复性和特异性好，为标准化诊断试剂盒的开发奠定了基础。

关键词: 猪传染性胃肠炎病毒; N蛋白; 原核表达; 间接ELISA

Abstract:

In the study,the recombinant expression plasmid pET28a-TGEV-N was constructed,and expression and purification of the TGEV N protein was completed.Through SDS-PAGE and Western blotting analysis,the N protein could be identified by transmissible gastroenteritis virus (TGEV) positive serum.Using the TGEV N protein as diagnosis antigen,we established an indirect ELISA method for detecting TGEV serum antibodies.The coefficients of variation of intro-batch and inter-batch duplicability test were less than 5%.The specificity was 100%,and the rate of false positive and the false negative rate were 0 and 5%,respectively.No cross reactions with seven porcine diseases positive serum were detected.Using this method,the clinical serum samples were detected.The results showed 100% coincidence rate compared with neutralization test.The method could detect TGEV antibody quickly and effectively,and had good repeatability and specificity,which laid the foundation for the development of standardized diagnostic kits.

Key words: transmissible gastroenteritis virus (TGEV); N protein; prokaryotic expression; indirect ELISA

中图分类号:

S852.65⁺1

郭宏伟, 郑鸣, 乔宏兴, 马辉, 赵绪永, 王永芬. 猪传染性胃肠炎病毒N蛋白原核表达和间接ELISA检测方法的建立[J]. 《中国畜牧兽医》, 2016, 43(9): 2296-2301.

GUO Hong-wei, ZHENG Ming, QIAO Hong-xing, MA Hui, ZHAO Xu-yong, WANG Yong-fen. Prokaryotic Expression and Development of an Indirect ELISA Detection Method of Porcine Transmissible Gastroenteritis Virus N Protein[J]. , 2016, 43(9): 2296-2301.

参考文献

[1] 魏海芳,王鸿忠.青海省部分地区PEDV、TGEV和RV感染情况调查与分析[J].动物医学进展,2013,34(9):133-136.
[2] 刘云波,赵洪翠,王志成,等.猪病毒性腹泻分子流行病学调查[J].中国畜牧兽医,2013,40(2):204-207.
[3] 孙振钊,赵伟男,王靖飞.猪腹泻病例中PEDV、TGEV及PRV的感染调查[J].动物医学进展,2014,35(5):132-135.
[4] Laude H,Rasschaert D,Delmas B,et al.Molecular biology of transmissible gastroenteritis virus[J].Vet Virol,1990,23:147-154.
[5] Spaan W,Cavanagh D,Horizinek M C.Coronaviruses:Structure and gene expression[J].Journal of General Virology,1988,69:2939-2952.
[6] Liu C,Kokuho T,Kubot A T,et al.A serodiagnostic ELISA using recombinant antigen of swine transmissible gastroenteritis virus nucleoprotein[J].J Vet Med,2001,63(11):1253-1256.
[7] Motokawa K.Comparison of the amino acid sequence and phylogenetic analysis of the peplomer,integral membrane and nucleocapsid protein of feline coronavirus[J].Microbiology and Immunology,1996,40(6):425-433.
[8] Wurm T,Chen H,Hoddgson T,et al.Localization to the nueleolus is a cornrnon feature of coronavirus nucleoproteins and the protein may disrupt host cell division[J].J Virol,2001,75(19):9345-9356.
[9] Kapke P A,Brian D A.Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene[J].Virology,1986,151:41-49.
[10] Bridgen A,Duarte M,Tobler K,et al.Sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhea virus confirms that this virus is a coronavirus related to human coronavirus 229E and porcine transmissible gastroenteritis virus[J]. J Gen Virol, 1993,74:1795-1804.
[11] Jean Y H,Kang M I,Hwang E K,et al.Detection of transmissible gastroenteritis virus in tisse by peroxidase/antiperoxidase method[J].Research Reports of the Rural Development Administration(Livestock Veterinary),Korea,1987,29:48-53.
[12] Van Nieuwstadt A P,Cornelissen J B,Vreeswijk J.Solid phase immune electron microscopy for diagnosis of transmissible gastroenteritis in pigs[J].Research in Veterinary Science,1988,44(3):286-294.
[13] Witte K H.Micro-color test for assay of transmissible gastroenteritis virus-neutralizing antibodies[J].Arch Gesamte Virusforsch,1971,33(1):171-176.
[14] Sirinarumitr T,Paul P S,Halbur P G,et al.An overview of immunological and genetic methods for detecting swine coronaviruses,transmissible gastroenteritis virus,and porcine respiratory coronavirus in tissues[J].Springer US,1997,412:37-46.
[15] Paton D,Ibata G,Sands J,et al.Detection of transmissible gastroenteritis virus by RT-PCR and differentiation from porcine respiratory coronavirus[J].J Virol Methods, 1997,66(2):303-309.
[16] Nelson L D,Kelling C L.Enzyme-linked immunosorbent assay for detection of transmissible gastroenteritis virus antibody in swine sera[J].Am J Vet Res, 1984,45(8):1645-1657.
[17] 王继科,马思奇,王明,等.猪流行性腹泻与猪传染性胃肠炎病毒的电镜和免疫电镜观察[J].中国预防兽医学报,1999,3:191-194.
[18] Carman S,Josephson G,McEwen B,et al.Field validation of commercial blocking ELISA to differentiate antibody to transmissible gastroenteritis virus(TGEV) and porcine respiratory coronavirus and to identify TGEV-infected swine herds[J].J Vet Diagn Invest, 2002,14(2):97-105.
[19] Martin A J M,Balbin M,Garwes D J,et al.Antigenic structure of ransmissible gastroenteritis virus nucleoprotein[J].Virology,1992,188:168-174.
[20] Sestak K,K Meister R K,Hayes J R,et al.Active immunity and T cell populations with baculovirus expressed transmissible gastroenteritis virus structural protein[J].Veterinary Immunology and Immunopathology, 1999,70:203-221.
[21] Jacobs L,Vander Zeijst B A M,Horzinek M C,et al.Characterization and translation of TGEV mRNAs[J].J Virol,1986,57:1010-1015.
[22] 孙东波,冯力,时洪艳,等,猪传染性胃肠炎病毒重组N蛋白抗原间接ELISA抗体检测方法的建立[J].中国预防兽医学报,2006,28(5):572-580.

猪传染性胃肠炎病毒N蛋白原核表达和间接ELISA检测方法的建立

Prokaryotic Expression and Development of an Indirect ELISA Detection Method of Porcine Transmissible Gastroenteritis Virus N Protein

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