Heterologous expression of fused genes encoding the glycoprotein 5 from PRRSV: a way for producing functional protein in prokaryotic microorganism

Abstract

Based on the bioinformatics analysis of the gene encoding glycoprotein 5 (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) isolate HH08, two gene fragments were amplified by polymerase chain reaction (PCR), deleting the signal peptide and transmembrane sequences in GP5 gene. Both gene fragments were designated GP5a and GP5b, respectively. They were ligated with a linker and cloned into prokaryotic expression vector, pET-30a. Expression of the protein of interest was induced by isopropyl beta-d-1-thiogalactopyranoside. The purified protein was used as an immunogen to elicit antibody in rabbit. The immunoreactivity of the protein was determined using ELISA and Western blot. Biologically active GP5 and anti-GP5 antibody inhibited cell infection by PRRSV. Moreover, the antibody produced in this study was capable of detecting the cell infection by PRRSV and distinguishing this virus from other viruses.

Fig. 1

Molecular characteristics of GP5. The molecular characteristics of the GP5 were analyzed and indicated using DNAstar software (USA).

Fig. 2

SDS-PAGE analysis on the GP5. The bacteria bearing GP5 gene or empty vector were induced by IPTG and then the proteins were subjected to SDS-PAGE. Lane 1: Un-induced bacteria; Lane 2: Induced bacteria bearing the empty vector; Lanes 3–9: Bacteria bearing the GP5 gene induced at 1–7 h post-IPTG induction; Lane M: Protein molecular weight marker

Fig. 3

Immunoblotting. The bacteria bearing GP5 gene or empty vector were induced by IPTG and then the protein was subjected to Western blot analysis using the antibody against PRRSV. Lane 1: Bacteria bearing GP5 gene; Lane 2: Control bacteria bearing empty vector

Fig. 4

ELISA. The PRRSV, TGEV, PEDV, IBV and PRV were used as coating antigens. The purified GP5 and BSA were used as control antigens. Polyclonal antibody against the GP5 protein was used as the primary antibody in the indirect ELISA. The concentration of viruses and proteins was 20 μg/ml and 100 μg/ml, respectively. Number 1–6 in the horizontal axis is PRRSV, TGEV, PEDV, IBV, PRV, GP5 and BSA, respectively. The vertical axis is the OD₄₉₀ value.

Fig. 5

Inhibitory effect of GP5 and anti-GP5 serum on PRRSV infection invitro. Marc-145 cells were pre-incubated serially diluted GP5 protein (5⁻¹ dilution is equal to 12 μg protein) and then the cells were infected with PRRSV. The inhibition rate of the protein to virus infection is shown in Panel A. In parallel, PRRSV was pre-treated with serially diluted anti-GP5 antibody, and then the viruses were used to infect Marc-145 cells. The inhibition rate of the antibody to virus infection is shown in Panel B. Values along the horizontal axis indicate protein or antibody dilution and the infection inhibition rates are shown in vertical axis.

Fig. 6

Immunofluorescence analysis. The polyclonal antibody against the GP5 was used as primary antibody to detect PRRSV-infected Marc-145 cells. Panel A: Virus-infected cells; Panel B: Mock-infected cells. The nuclei were stained with propidium iodide.