Expression Of The Multiepitope Peptide Of GP4 And GP5 Of PRRSV And Its Immune Response Elicited In Mice

Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease of swine caused by porcine reproductive and respiratory syndrome virus (PRRSV). This disease is characterized by reproductive failure in sows and respiratory illness in piglets. In the study, the gene fragments of two preponderant epitopes of GP5 and GP4 of PRRSV BJ-4 were amplified and fused by means of the gene splicing using overlap extension (gene SOEing) PCR technique in order to construct two multiepitopes fusion genes. The immune responses elicited by the eukaryotic recombinant plasmids expressing the two multiepitopes fusion genes and prokaryotic expressed multiepitopes peptides were analyzed in mice.The epitope areas of GP5 aa29~60 (named E5) and GP4 aa33~70 (named E4) were amplified and fused by gene SOEing PCR technique. Two new genes (E54-2 and E54-4) encoding the multiepitope peptides were generated. The two new genes were then cloned into the eukaryotic vector pcDNA3.1 to construct two eukaryotic recombinant plasmids, pcDNA3-E54-2 and pcDNA3-E54-4. The fusion gene Ub-E54-2, obtained by fusing the ubiquiten gene of porcine with the E54-2 gene using gene SOEing PCR, was inserted into pcDNA3.1 to construct eukaryotic recombinant plasmid pcDNA3Ub-E54-2. Indirect immunofluorescence assay was carried out following COS7 cells transfected by all three eukaryotic recombinant plasmids respectively and P815 cells transfected by the two former recombinant plasmids. The results indicated that the multiepitope peptides E54-2 and E54-4 could be instantaneously and steadily expressed in mammalian cells.The E54-2 and E54-4 genes were cloned into the prokaryotic expression vector pGEX-6P-l respectively to construct two prokaryotic recombinant expression plasmids, pGEX-6P-E54-2 and pGEX-6P-E54-4. The two prokaryotic recombinant expression plasmids were expressed in Escherichia coli BL21 (DE3). The recombinant fusion proteins GST-E54-2 and GST-E54-4 (expressed by pGEX-6P-E54-2 and pGEX-6P-E54-4, respectively) could amount to 40% and 30% of the total mass of bacterial proteins. The recombinant proteins GST-E54-2 and GST-E54-4 were purified through denaturation and refolding of inclusion bodies and electro-elution assay. Western-Blotting analysis indicated that the purified proteins GST-E54-2 and GST-E54-4 could be recognized by the anti-PRRSV serum of porcine, anti-GP4 and anti-GP5 serum of mice.The mice were immunized with the three eukaryotic recombinant plasmids, respectively. It was shown that the three plasmids were able to induce the humoral immune responses. The specific anti-GP4, anti-GP5 and anti-E54-2 peptide antibodies could be deteced. At day 28 post primary immunization (PPI), the antibodies against GP5 in groups immunized with pcDNA3-E54-2 and pcDNA3-E54-4 were significantly higher than that of the control group (p<0.05). There was a significant difference in response levels of antibodies against GP5 between pcDNA3-E54-2 group and pcDNA3-E54-4 group at day 49 PPI (p<0.05). At day 28 PPI, the antibodies against GP4 showed significant differences between the control group and pcDNA3-E54-2 or pcDNA3-E54-4 groups (p<0.05). After three weeks following the fourth immunization, the titre of viral neutralizing (VN)antibodies in groups immunized with pcDNA3-E54-2 or pcDNA3-E54-4 were 1:16, 1:28, respectively.Mice were immunized with the purified fusion proteins (GST-E54-2 and GST-E54-4), and genetically engineered bacteria expressing the fusion proteins GST-E54-2 and GST-E54-4, respectively. The results of ELISA analysis exhibited that the anti-GP4 and anti-GP5 antibody titres in the E54-2 bacteria group and purified protein group were 1:1,600 and 1:3,200 respectively, anti-GP4 antibody titres of the E54-4 bacteria and purified protein group were 1:3,200, anti-GP5 antibody titres of the E54-4 bacteria and purified protein group were 1:12,800 and 1:6,400 respectively one week after the third immunization. The data of VN antibodies detection demonstrated that the titre of VN antibodies of the E54-2 bacteria group was 1:22.2, and that of puri...