Production of Recombinant N Protein of Infectious Bronchitis Virus Using the Baculovirus Expression System and Its Assessment as a Diagnostic Antigen
The avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is recognized as an important avian pathogen, and new viral variants are a continuous threat to the poultry industry worldwide. Sensitive diagnostics and efficacious vaccines are necessary to combat IBV infections in chickens. The aim of this study was to produce recombinant N protein of IBV in the baculovirus system to use in ELISA diagnostic tests in order to enable the assessment of the sero-prevalence and risk of IBV infections in chickens in Turkey. For this, the gene encoding the N protein of the Beaudette strain of IBV was expressed using a recombinant baculovirus expression system. The recombinant N protein was purified using Ni-NTA affinity chromatography. An estimated 50-kDa recombinant protein corresponding to the expected molecular weight of IBV N including the 6xHis tag was detected using an anti-His monoclonal antibody. Specific immunoreactivity of the recombinant protein was confirmed by Western blot using antiserum obtained from vaccinated and naturally infected chicken from Turkey as well as using a monoclonal antibody raised against the N protein of the IBV Massachusetts strain. The results obtained with the in-house ELISA had high agreement with a commercial ELISA. Immunoreactivity analysis using antisera in Western blotting and the in-house ELISA suggests that the recombinant IBV N protein could be broadly cross-reactive with antisera produced against different IBV strains. We conclude that the recombinant baculovirus expressed IBV N protein could serve as a useful diagnostic antigen for detection of IBV infections in chickens by ELISA.
KeywordsInfectious bronchitis virus Baculovirus Expression Recombinant N protein
Infectious bronchitis virus
Infectious bronchitis virus N
Polymerase chain reaction
Enzyme-linked immunosorbent assay
We would like to thank to TUBITAK: Scientific and Technological Research Council of Turkey (Project No: 113O411) for funding; and the DHS Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD). Also thanks to Professor Paul Britton, Professor Dan Heller, Dr. Jack deWit, Dr. Loneke Vervelde, Dr. Tina Dalgaard, Dr. Jacob Pitcovski, Dr. Isabella Monne, Dr. Siamak Zohari, Dr. Helen Verheije, and Dr. Mariette Ducatez and all researchers for sharing the information on IBV during the COST action FA 1207.
This study was funded by TUBITAK: Scientific and Technological Research Council of Turkey (Project No: 113O411), Developing Scholars Program (DSP), Kansas State University, Office of Undergraduate Research and Creative Inquiry; DHS Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD).
Compliance with Ethical Standards
National and international ethical rules were followed during this study.
Conflict of Interests
The authors declare that they have no conflicts of interest.
- 7.Faburay, B., Wilson, W., McVey, D. S., Drolet, B. S., Welngartl, H., Madden, D., Young, A., Ma, V., & Richt, J. A. (2013). Rift Valley fever virus structural and nonstructural proteins: Recombinant protein expression and immunoreactivity against antisera from sheep. Vector Borne and Zoonotic Diseases, 13(9), 619–629.CrossRefGoogle Scholar
- 14.Liu, W. X., Hu, S., Qiao, Z. J., Chen, W. Y., Liu, L. T., Wang, F. K., Hua, R. H., Bu, Z. G., & Li, X. R. (2011). Expression, purification, and improved antigenic specificity of a truncated recombinant bp26 protein of Brucella melitensis M5-90: a potential antigen for differential serodiagnosis of brucellosis in sheep and goats. Biotechnology and Applied Biochemistry, 58(1), 32–38.CrossRefGoogle Scholar
- 15.Lugovskaya, N. N., Scherbakov, A. V., Yakovleva, A. S., Tsyvanyuk, M. A., Mudrak, N. S., Drygin, V. V., & Borisov, A. V. (2006). Detection of antibodies to avian infectious bronchitis virus by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay. J. Virol. Methods., 135(2), 292–296.CrossRefGoogle Scholar
- 16.Meir, R., Krispel, S., Simanov, L., Eliahu, D., Maharat, O., & Pitcovski, J. (2012). Immune responses to mucosal vaccination by the recombinant A1 and N proteins of infectious bronchitis virus. Viral Immunology, 25(1), 55–62.Google Scholar
- 18.Pradhan, S. K., Kamble, N. M., Pillai, A. S., Galkwad, S. S., Khulape, S. A., Reddy, M. R., Mohan, C. M., Kataria, J. M., & Dey, S. (2014). Recombinant nucleocapsid protein based single serum dilution ELISA for the detection of antibodies to infectious bronchitis virus in poultry. Journal of Virological Methods, 209, 1–6.CrossRefGoogle Scholar
- 23.Yilmaz, H., Altan, E., Cizmecigil, U. Y., Gurel, A., Ozturk, G. Y., Bamac, O. E., Aydin, O., Britton, P., Monne, I., Cetinkaya, B., Morgan, K. L., Faburay, B., Richt, J. A., & Turan, N. (2016). Phylogeny and S1 gene variation of infectious bronchitis virus detected in broilers and layers in Turkey. Avian Diseases, 60(3), 596–602.CrossRefGoogle Scholar