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Baculovirus Mediated Expression of Bovine Herpesvirus-1 Glycoprotein-C in Insect Cells

Baculovirus Mediated Expression of Bovine Herpesvirus-1 Glycoprotein-C in Insect Cells Bovine herpesvirus-1 (BHV-1) glycoprotein C (gC) is an immunodominant protein that is believed to play a role in initial viral attachment. In the present study a recombinant baculovirus was constructed by incorporating BHV-1 gC coding gene to characterize the expression of the glycoprotein in infected Spodoptera frugiperda cells. The BHV-1 complete gC gene was cloned into pENTR/SD/D Directional TOPO vector (entry clone). The purified entry clone plasmid was subjected to LR recombination with the linear baculovirus DNA which was then transfected into S. frugiperda cells. The recombinant baculovirus carrying the gC coding gene was selected against ganciclovirus in the S. frugiperda cells (first passage), serially passaged for two more generations and the third passage viral stock was used to infect fresh S. frugiperda cells for gene expression study. The reactivity of polyclonal antibody with the S. frugiperda cell expressed gC was detected by immunoperoxidase test. The recombinant gC was purified by Ni-NTA column chromatography and immunoprecipitation. Protein bands of molecular weight 54 and 28 kDa were detected consistently with both monoclonal and polyclonal antibodies, when the recombinant protein was subjected to SDS-PAGE and Western blot analysis. Expression of gC gene in the infected S. frugiperda cells was further confirmed by dot-ELISA indicating its potential use as a coating antigen in an indirect ELISA for diagnosis of BHV-1, the causative agent of infectious bovine rhinotracheitis/infectious pustular vulvovaginitis. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Proceedings of the National Academy of Sciences, India Section B: Biological Sciences Springer Journals

Baculovirus Mediated Expression of Bovine Herpesvirus-1 Glycoprotein-C in Insect Cells

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References (19)

Publisher
Springer Journals
Copyright
Copyright © 2014 by The National Academy of Sciences, India
Subject
Life Sciences; Life Sciences, general; Behavioural Sciences; Plant Biochemistry; Nucleic Acid Chemistry
ISSN
0369-8211
eISSN
2250-1746
DOI
10.1007/s40011-013-0302-7
Publisher site
See Article on Publisher Site

Abstract

Bovine herpesvirus-1 (BHV-1) glycoprotein C (gC) is an immunodominant protein that is believed to play a role in initial viral attachment. In the present study a recombinant baculovirus was constructed by incorporating BHV-1 gC coding gene to characterize the expression of the glycoprotein in infected Spodoptera frugiperda cells. The BHV-1 complete gC gene was cloned into pENTR/SD/D Directional TOPO vector (entry clone). The purified entry clone plasmid was subjected to LR recombination with the linear baculovirus DNA which was then transfected into S. frugiperda cells. The recombinant baculovirus carrying the gC coding gene was selected against ganciclovirus in the S. frugiperda cells (first passage), serially passaged for two more generations and the third passage viral stock was used to infect fresh S. frugiperda cells for gene expression study. The reactivity of polyclonal antibody with the S. frugiperda cell expressed gC was detected by immunoperoxidase test. The recombinant gC was purified by Ni-NTA column chromatography and immunoprecipitation. Protein bands of molecular weight 54 and 28 kDa were detected consistently with both monoclonal and polyclonal antibodies, when the recombinant protein was subjected to SDS-PAGE and Western blot analysis. Expression of gC gene in the infected S. frugiperda cells was further confirmed by dot-ELISA indicating its potential use as a coating antigen in an indirect ELISA for diagnosis of BHV-1, the causative agent of infectious bovine rhinotracheitis/infectious pustular vulvovaginitis.

Journal

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences – Springer Journals

Published: Jan 28, 2014

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