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

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.

Keywords: ELISA; Ni-NTA chromatography; envelope glycoprotein I; immunogenicity; pseudorabies virus.

Figure 1

Prediction of possible epitopes and transmembrane regions for gI squence of PRV. (A) Possible epitopes for gI squence were predicted by BepiPred online server. (B) Possible transmembrane regions for gI squence were predicted by TMHMM-2.0 online server.

Figure 2

Electrophoresis analysis of PCR products and recombinant plasmid identification of the PRV-gI gene. (A) Electrophoretic analysis of PCR products of the PRV-gI gene. Lane 1: PCR product of the gI gene, a target fragment of approximately 633 bp; Lanes 2 and 3: control group; Lane 4: DNA molecular quality standards. (B) Recombinant plasmid pBlue-gI enzyme digestion identification. Lanes 1, 2 and 3: Bam I and Sal I double enzyme cleavage product of pBlue-gI; Lane 4: DNA molecular quality standards. (C) Recombinant plasmid pET-30(a)-gI enzyme digestion identification. Lanes 1, 2, 3, 4: Strip analysis using repeated enzyme digestion electrophoresis; Lane 5: DNA molecular quality standards.

Figure 3

Expression and identification of truncated gI protein. (A) SDS-PAGE gel results of target proteins expressed in E. coli after 0.3 and 1.0 mM IPTG induction for 7 h at 37 °C. pET-30(a): inducible expression band of empty vector; pET-30(a)-gI: the induced expression of an approximately 38 kDa band of recombinant expression plasmid. (B) Western blot analysis was conducted with pig standard positive serum against PRV as the primary antibody; pET-30(a): inducible expression band of empty vector; pET-30(a)-gI: pig standard positive serum against PRV as the primary antibody. (C) predicted secondary structure of the monomeric form of the truncated gI peptides generated using Protean software.

Figure 4

Analysis and purification of the location of the target protein. (A) Analysis of target protein supernatant and precipitate via SDS-PAGE gels. Soluble: solubility is expressed in supernatant; inclusion: a protein in the form of an inclusion body. (B) Protein purified via nickel column. Purified: purified gI proteins.

Figure 5

Identification of mouse pAb reacting with PRV via indirect immunofluorescence. PRV infected PK-15 cells for 48 h, and pig anti-PRV-positive serum (A) and mouse anti-gI-positive serum (B) were used as primary antibodies to detect PRV via IFA; (C,D) were uninfected PK-15 cells as control, and scale bars represent 200 μm.

Figure 6

Distribution of the OD values obtained from clinical pig serum samples. Forty PRV-negative and forty PRV-positive pig serum samples using PRV-based IDEXX cELISA (A), S/N = Sample A (650)/controls, and (B) using gI indirect ELISA.