Study On Multi-Epitope Vaccine Of Porcine Reproductive And Respiratory Syndrome (PRRS)

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of reproductive and respiratory syndrome, a newly recognized pig disease of economic importance, characterized by reproductive failure (abortions, stillbirths, mummies and weak-born piglets) and respiratory problems affecting pigs of all ages. Although live PRRSV vaccines provide protection against homologous virus challenge, the genetic diversity of field PRRSV isolates is very high, and vaccine effect against heterologous virus challenge will be quested. Also, live PRRSV vaccines have been observed to revert to virulence. And the safer, killed vaccines have so far proved less effective. PRRSV has recently been noticed and considered to be one of the most important swine diseases. At present, we still lack safe and credible PRRSV vaccine.Based on the analysis of epitopes of PRRSV and a large number of reference. A synthetic gene encoding a polypeptide composed of antigenic epitopes of the PRRSV proteins was constructed artificially. The polypeptid comprises a mosaic of four antigenic epitopes from the protein encoded by ORF5, four antigenic epitopes from the protein encoded by ORF7. The spacers between the epitopes is crucial for the epitope-induced protection. the spacers residues GGA were determined to support epitopes. To get recombinant baculovirus transposition rBacmid8EP, Which are the recombinant baculovirus particles containing PRRSV 8EP gene. We transformed baculovirus pFBH-8EP into competent cells of DH10BAC.The recombinant transposition were obtained by identification of PCR amplification with the primers of M13 and the primers specific to 8EP.Then the recombinant transpositions were used to transfect Sf9 cells and the recombinant baculovirus were got. SDS-PAGE analysis showed that 8EP has been expressed in sf9 cells infected with the recombinant baculovirus. Then we digested 8EP into two fragment with restriction endonuclease Pvuâ…¡. The two fragment were resubcloned into pGEX-6P-1. After IPTG induced, the protein was expressed in Escherichia coli fused with glutathione S-transferase. The recombinant fused protein was identified by the Western-blot with polyclonal antibodies againstPRRSV.This study provided some basic data for further development of PRRSV epitope-based gene immunization,multi-epitope vaccine and subunit vaccine.