Molecular Cloning and Expression of Bovine Viral Diarrhea Virus Nonstructural Protein 3 in Escherichia coli

Abstract

Background: Bovine viral diarrhea (BVD) is an economically important disease of cattle with a worldwide distribution. Diagnosis of BVD relies on laboratory-based detections of its viral causing agent or virus specific antibodies. The most common laboratory method used for this purpose is the ELISA. Bovine viral diarrhea virus (BVDV) nonstructural protein 3 (NS3) is one of the most highly conserved immunogenic proteins of BVDV, thus, it is a proper candidate antigen to detect antibodies against the virus in the sera from infected animals. Objectives: The aim of this study was to synthesize a plasmid construct for high-level expression of NS3 with more solubility in Escherichia coli. Materials and Methods: A segment of BVDV genome encoding the NS3 protein was amplified using RT-PCR and cloned into pMAL-c2X expression vector, under the control of the lac promoter. After sequencing of the amplified gene, the recombinant protein was expressed in E. coli strain BL21 and analyzed by SDS-PAGE and western blotting. Results: The strong promoter of pMAL-c2X vector allowed a high level expression of NS3 as a maltose binding protein-NS3 (MBP-NS3) fusion protein. Expression of the expected fusion protein was confirmed by electrophoresis on SDS-PAGE and immunoblotting, using a BVDV positive bovine serum. Conclusions: Based on our results, it appears that this plasmid construct may be suitable for the production of NS3 recombinant antigen to develop BVDV laboratory diagnostic assays.

Immunoinformatic prediction of the pathogenicity of bovine viral diarrhea virus genotypes: implications for viral virulence determinants, designing novel diagnostic assays and vaccines development

“…However, BVDV-C protein has been shown to induce cellular and humoral immune response in mouse model ( 65) and a B cell epitope that can induce the formation of antibodies was mapped in the C protein of BVDV (66) and other pestiviruses (67). The main inducers of antibody response against BVDV were reported to be the E2 and NS2-3 proteins and to a lesser extent E1 and Erns glycoproteins (8,68,69). Additionally, a low level of anti-NS4B antibody was also detected in calves after natural BVDV infection (70).…”

Production of Monoclonal Antibody Against Recombinant Polypeptide From the Erns Coding Region of the Bovine Viral Diarrhea Virus

“…Production of recombinant MBP-NS3 protein in pMalc2x expression vector, under the control of the lac promoter in E. coli BL-21 strain had been previously produced in our laboratory. 16 For expression of MBP-NS3 protein, a bacterial colony which had no mutation in the NS3 insert was selected and cultured in high volume of ampicillin embedded Luria-Bertani (LB) broth media (Merck, Darmstadt, Germany) containing 20 mM glucose, until the OD 600 reached to 0.5. Then, protein expression was induced by adding isopropyl-β-D-thio-galactoside (IPTG) (Cinnagen, Tehran, Iran) at a final concentration of 1 mM.…”