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.