Immunogenicity Characterization of the Recombinant gI Protein Fragment from Pseudorabies Virus and an Evaluation of Its Diagnostic Use in Pigs

SIMPLE SUMMARY: Pseudorabies virus (PRV) is a double-stranded linear DNA virus with an envelope, and it can cause acute neurological symptoms and death in piglets, which has caused large economic loss in pig farms. The eradication of PRV in pig farms has always been a goal, and the diagnosis of wild virus infection is particularly important in this process. In this study, we designed specific primers to amplify the fragment of gI with the region of antigen epitopes and the transmembrane regions removed. The recombinant truncated fragment of gI was expressed, and the native gI protein was obtained. After a small-scale ELISA test for clinical serum samples, it was found that purified gI protein may potentially be applied to clinically diagnose PRV infection. ABSTRACT: Serological testing is an important method for the diagnosis of pseudorabies virus (PRV) infection. We aimed to investigate the envelope glycoprotein I (gI) of PRV, a strong immunogen, and its potential as an efficient and low-cost diagnostic reagent. In this study, the DNA of the PRV SC strain was used as the template, and the recombinant fragment of gI (633 bp) was amplified via PCR using synthetic primers, and was then ligated into the pET-30a expression vector. The constructs were transferred into Escherichia coli (E. coli) for prokaryotic expression, and the antigenicity of the expression products was identified by Western blot analysis with pig positive serum against PRV. The recombinant protein was purified by a Ni column, and BALB/c mice were immunized with purified gI protein to obtain anti-gI-positive serum. After PK-15 cells had been infected by PRV for 48 h, the immunogenicity of purified gI protein was identified with a fluorescence immunoassay using anti-gI mouse serum. The recombinant plasmid (pET-30a-gI) was expressed, and the native gI protein was obtained after denaturation by urea and renaturation by dialysis. A small-scale ELISA test containing 1.0 µg/mL of purified gI protein was designed to evaluate pig serum (80 samples), and the results of the ELISA test were compared to those of competitive ELISA (cELISA) tests using IDEXX Kits, which resulted in 97.5% consistency. The results suggested that the truncated gI protein may be a potential diagnostic reagent.