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Prokaryotic expression of nucleoprotein gene of Transmissible gastroenteritis virus and development of ELISA based on the expressed protein

Published online by Cambridge University Press:  29 January 2010

Song Zhen-Hui ,
Guo Wan-Zhu  and
Zhang Ying-Jun
Song Zhen-Hui*
Affiliation:
Animal Medical Science, Southwest University, Chongqing 402460, China
Guo Wan-Zhu
Affiliation:
Animal Biology Technology Center, Sichuan Agricultural University, Yaan 625014, China
Zhang Ying-Jun
Affiliation:
Library of Southwest University, Chongqing 402460, China
*
*Corresponding author. E-mail: [email protected]
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Abstract

The recombinant PET-N plasmid, which includes the N gene of the Transmissible gastroenteritis virus (TGEV), was transformed into Escherichia coli BL21 (DE3), and induced at 37°C with 1.0 mmol/l IPTG (isopropyl β-d-1-thiogalactopyranoside). An indirect enzyme-linked immunosorbent assay (ELISA) for detecting the TGEV nucleocapsid protein antibody was developed after the reactogenicity of the recombinant protein was demonstrated by Western blot. The operating conditions for the ELISA, an antigen concentration of 15 μg/ml, serum dilution of 1:40, blocking solution of 0.5% fetal bovine serum (FBS), serum sample incubation for 90 min, a concentration of horseradish peroxidase (HRP)-spa of 1:5000, incubated for 60 min, incubation of the substrate at room temperature for 10 min, and a cutoff value for the ELISA OD450⩾0.35, were carried out using a checkerboard titration method. The sensitivity and specificity of this method relative to the Svanova TGEV/PRCV antibody diagnosis kit were 93.5% and 93.8%, respectively.

Keywords

recombinant N proteinTransmissible gastroenteritis virus antibodyindirect ELISA
Type
Research Papers
Information
Chinese Journal of Agricultural Biotechnology , Volume 6 , Issue 3 , December 2009 , pp. 249 - 252
Copyright
Copyright © China Agricultural University 2009

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References

Briton, P, Carnemes, RS, Page, KW, Garwes, DJ and Parra, F (1988) Sequence of the nucleoprotein gene from a virulent British field isolate of transmissible gastroenteritis virus and its expression in Saccharomyces cerevisiae. Molecular Microbiology 2: 89–99.CrossRefGoogle Scholar
Carman, S, Josephson, G, McEwen, B, et al. (2002) 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. Journal of Veterinary Diagnostic Investigation 14: 97–105.CrossRefGoogle ScholarPubMed
Kim, SH, Kim, IJ, Pyo, HM, Tark, DS, Song, JY and Hyun, BH (2007) Multiplex real-time RT-PCR for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus. Journal of Virological Methods 146: 172–177.CrossRefGoogle ScholarPubMed
Liu, C, Kokuho, T, Kubota, T, et al. (2001) A serodiagnosis ELISA using recombinant antigen of swine transmissible gastroenteritis virus nucleoprotein. Journal of Veterinary Medical Science 63: 1253–1256.CrossRefGoogle Scholar
Sambrook, J, Fritsch, EF and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.Google Scholar
Song, DS, Kang, BK, Oh, JS, et al. (2006) Multiplex reverse transcription-PCR for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus and porcine group A rotavirus. Journal of Veterinary Diagnostic Investigation 18: 278–281.CrossRefGoogle ScholarPubMed