Abstract
Bovine herpesvirus-1 (BHV-1) causes Infectious bovine rhinotracheitis/Pustular vulvovaginitis in cattle. Glycoprotein D (gD) of BHV-1 represents a major component of the viral envelope and is a dominant immunogen. gD encoding gene was expressed in baculovirus-insect cell system. Viral genomic DNA extracted from BHV-1 grown on Madin-Darby Bovine Kidney (MDBK) cell monolayer was used as a template for PCR amplification of gD gene (1255 bp). Gel purified gD gene was used for directional cloning into pENTR/SD/D Directional TOPO vector to produce entry clone. Recombinant plasmids were screened by PCR and restriction enzyme (RE) digestion for gD gene insert. Endotoxin free purified plasmids were then subjected to LR recombination reaction with baculovirus linear DNA. LR recombination mix was transfected into Sf-9 cells and observed for appearance of cytopathic effects (CPE). Recombinant virus was serially passaged for 3 more generations and the 4th passage viral stock was used to infect fresh Sf-9 cells for gene expression study. Recombinant gD protein was immunoprecipitated and when subjected to SDS-PAGE and western blot analysis protein band of ~70 kDa was detected consistently. The recombinant gD protein was further weakly confirmed by dot-ELISA indicating its limited potential as a coating antigen in gD-based diagnostic ELISA. Key words: Bovine herpesvirus-1 (BHV-1), Madin-Darby Bovine Kidney (MDBK) cells, Sf-9 cells, pENTR TOPO vector, Baculovirus mediated gene expression, glycoprotein D, eukaryotic expression,
Highlights
Bovine herpesvirus type-1 (BHV-1) causes various diseases worldwide (Biswas et al, 2013; Levings and Roth, 2013)
All three glycoproteins are used in vaccine production and induce higher titer neutralizing antibodies with glycoprotein D (gD) producing the highest titer (Dummer et al, 2014; Blanc et al, 2012). gD is reported to be potential candidate for production of subunit vaccines (Majumder et al, 2014) and its expression is limited to the alpha herpesvirus, the largest subfamily of herpesvirus (Connolly et al, 2001; Grauwet et al, 2014)
Screening clones for gD gene insert was done by touch down PCR for presence of gD gene insert where PCR product of 1255 bp could successfully be amplified from recombinant plasmids
Summary
Bovine herpesvirus type-1 (BHV-1) causes various diseases worldwide (Biswas et al, 2013; Levings and Roth, 2013). GD abundance in the viral envelope and its role in initial stage of replication define it as a good target of the host immune response (Drummer et al, 2014) making it immunodominant and a good candidate for vaccine (Blanc et al, 2012; Majumder et al, 2014). These biological properties make the three glycoproteins powerful antigens in the study of BHV-1 immune response (Abdelmagid et al, 1998). Keeping these points in view, this study was aimed at cloning for eukaryotic expression of BHV-1 gD and characterization of the expressed recombinant protein
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