Production of monoclonal antibody against recombinant NS3 protein of bovine viral diarrhea virus (NADL strain)

Abstract

This study was conducted to investigate the prevalence of subclinical mastitis caused by Staphylococcus spp. in ewes in West-Azerbaijan province of Iran. Molecular characterization of isolated Staphylococcus spp. from diseased ewes were performed using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and DNA sequencing of glyceraldehyde-3-phosphate dehydrogenase (gap) gene. Also, antibiotic resistance of staphylococcal isolates against different antibiotics was investigated. A total number of 900 milk samples from 450 native ewes in their mid-lactation period were examined by the California mastitis test (CMT). The CMT positive samples were cultured and bacteria were isolated from 86 (9.50%) glands and 74 (16.40%) ewes. The prevalence of subclinical mastitis in the examined ewes was 16.40%. Microbiological analysis of milk samples revealed that 27 out of 74 sheep with subclinical mastitis were infected with Staphylococcus spp. Amplification of gap gene of 27 Staphylococcus isolates generated a single amplicon of 933 bp in size confirming that isolates were belonged to Staphylococcus genus. Digestion of PCR products by AluI endonuclease generated different RFLP patterns for each species. Nucleotide sequencing of gap gene followed by phylogenetic analysis showed that the most dominant Staphylococcus species were S. epidermidis, S. xylosus and S. chromogenes. Staphylococcal isolates showed the highest resistance to penicillin and ampicillin. In conclusion, Staphylococcus species, except for the southern parts of the province, play an important role in the development of subclinical mastitis in sheep in West-Azerbaijan province of Iran. Also, chloramphenicol, ciprofloxacin and neomycin are the most effective antibiotics for treatment of this disease.

Keywords: Bovine viral diarrhea; Monoclonal antibody; NS3; Recombinant antigen.

Fig. 1

SDS-PAGE analysis of bacteria expressing MBP-NS3 fusion protein before (lane 4) and after (lane 5) induction by IPTG. The expression of a protein of about 117 kDa, corresponding to MBP-NS3 is shown in the lane 5. Lane 2 and 3 indicate a bacterium expressing MBP before and after induction by IPTG, respectively. The molecular weight marker is shown in Lane 1

Fig. 2

SDS-PAGE analysis of purified MBP-NS3 fusion protein by amylose-resin column. Lane 1 shows purified nucleo-protein of influenza virus (~97 KDa) and purified MBP (~50 KDa) from control colony containing only pMAL-c2X as protein weight marker. Lane 2 and 3 indicate purified preparation of MBP-NS3 fusion protein (~117 KDa

Fig. 3

Reactivity of supernatants of subclones producing anti-NS3 MAbs to recombinant NS3 antigen by Western blotting. Lanes 1 and 5 belong to immunized and non-immunized mice sera as negative and positive controls, respectively. Lanes 2, 3 and 4 represent supernatant of subclones containing anti-NS3 MAbs

Fig. 4

Reactivity of supernatants of subclones producing anti-NS3 MAbs to natural NS3 antigen by Western blotting. Lanes 1 shows purified nucleoprotein of influenza virus (~97 KDa) and purified MBP (~50 KDa) as protein weight marker. Lanes 2 indicates supernatant of an unrelated hybridoma. Lanes 3-6 represent supernatant of subclones containing anti-NS3 MAbs