Expression of Bovine Viral Diarrhea Virus Envelope Glycoprotein E2 in YeastPichia pastorisand its Application to an ELISA for Detection of BVDV Neutralizing Antibodies in Cattle

Abstract

The aim of this article is to express envelope glycoprotein E2 of bovine viral diarrhea virus (BVDV) in yeast Pichia pastoris and its utility as a diagnostic antigen in ELISA. The BVDV E2 gene was cloned into the pPICZαA vector followed by integration into the Pichia pastoris strain X-33 genome for methanol-induced expression. SDS-PAGE and Western blot results showed that the recombinant BVDV E2 protein (72 kDa) was expressed and secreted into the medium at a concentration of 40 mg/L of culture under optimized conditions. An indirect ELISA was then developed by using the yeast-expressed E2 protein. Preliminary testing of 300 field cattle serum samples showed that the E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02% compared to the reference virus neutralization test. The concordance between the E2 ELISA and VNT was 91.67%. This study demonstrates feasibility of BVDV E2 protein expression in yeast Pichia pastoris for the first time and its efficacy as an antigen in ELISA for detecting BVDV neutralizing antibodies in cattle.

Development and Application of a Rapid Test for Detection of Bovine Viral Diarrhea Virus-specific Antibodies

“…Various systems are currently available for exogenous gene expression, including E. coli expression systems, mammalian expression systems (Thomas et al 2009), Drosophila melanogaster systems (Robiolo and Schauer 2007), baculovirus expression systems (Pecora et al 2012;Zoth and Taboga 2006), Saccharomyces cerevisiae yeast systems (Patterson et al 2012), alfalfa plants (Aguirreburualde et al 2013), yeast Pichia pastoris expression systems (Behera et al 2015) and insect cell expression systems. However, the E. coli expression system remains the preferred choice for expressing foreign proteins due to its well-defined genetic background, rapid reproduction, cost-effectiveness, high expression levels, and ease of operation (Lee and Lee 2003).…”

The recombinant Erns and truncated E2-based indirect enzyme-linked immunosorbent assays to distinguishably test specific antibodies against classical swine fever virus and bovine viral diarrhea virus

“…Currently, an indirect ELISA for specific detection of antibodies against CSFV based on the truncated E2 of CSFV (containing amino acids 690—879 in E2) expressed in mammalian cells has been reported, which did not cross-react serologically with anti-BVDV sera [ 17 ]. Previous reports demonstrated that the CSFV and BVDV proteins prepared from both prokaryotic and eukaryotic expression systems were used as the antigens for diagnostic ELISAs [ 8 , 17 , 34 , 39 ]. Although the biological activities of E rns and E2 glycoproteins expressed in E. coli may not be justifiable in the absence of post-translational modification, its use in the diagnostics may have the advantage of being economic, productive and fast.…”

Peptide enzyme‐linked immunosorbent assay (pELISA) as a possible alternative to the neutralization test for evaluating the immune response to IBV vaccine

“…Several well-known serological techniques, including immunofluorescence [9] and enzyme-linked immunosorbent assay (ELISA) [10], have been tested to potentially replace the neutralization test for other viruses. Recent studies have shown that rabies virus and bovine viral diarrhea virus glycoprotein serology ELISAs can measure the titers of neutralizing antibodies in sera from vaccinated humans and cattle, respectively [11,12]. The correlation of antibody titers between indirect ELISA and neutralization tests has also been studied in Zika virus and human papillomavirus [13,14].…”