Construction of prokaryotic system for expression of porcine circovirus type 2 ORF-2 gene fragment

Abstract

Porcine circovirus-associated diseases (PCVDs) are among the most significant challenges for pig farming in developed countries. Porcine circovirus type 2 (PCV-2) is considered the main etiological agent of postweaning multisystemic wasting syndrome in piglets. Mass PCVD occurrence has been reported in most regions of the world, that results in serious economic consequences. Optimal PCVD prevention is known to be achieved through a set of veterinary and sanitary measures in combination with vaccination. High evolutionary virus variability facilitating new genotype and strain emergence requires development of new candidate recombinant vaccines against PCV-2 infection. The study was aimed at construction of prokaryotic system for PCV-2 ORF-2 gene fragment expression and its functionality assessment. A genetic insert constructed from the most immunogenic type-specific PCV-2 epitopes based on genotype 2a, 2b, 2d strain and isolate consensus sequence was cloned into the expression vector pET-22b(+) that was incorporated into the Escherichia coli strain Rosetta 2(DE3). The transformants were selected based on the marker gene of ampicillin resistance on a selective medium. Target gene expression was induced by adding of isopropyl-β-D-1-thiogalactopyranoside at different concentrations. As a result, Escherichia coli Rosetta 2(DE3)/pET-22b-ORF-2 strain, a producer of capsid protein fragment (92–233 amino acid residues), was constructed. It was found that in the presence of 1 mM isopropyl-β-D-1-thiogalactopyranoside, the expression level of soluble truncated rCap was 35–40 mg/L 6 hours after induction. The expression product was tested for its specificity with indirect ELISA using whole-virion PCV-2-hyperimmunized porcine serum. It was shown that the positivity coefficient of producer strain cell lysates averaged to 4.34 (p < 0.005). The recombinant rCap protein is suitable for serological diagnosis and is also of interest as a vaccine component, which is the goal of our further studies.

Keywords

• porcine circovirus type 2
• postweaning multisystemic wasting sindrome
• orf-2
• prokaryotic expression system