Characterization Of The Epitopes For Structural Proteins Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS. It causes an important disease in pigs characterized by reproductive failure in sows and gilts, pneumonia and an increase in perinatal mortality, resulting in great economic losses to the swine industry. In order to provide valuable data for further exploring the immunogenicity and function of structural proteins of PRRSV and designing epitoe vaccine for PRRSV, the purpose of the research was to characterize the epitopes on structural proteins of PRRSV BJ-4 by means of phage display technique, ELISA and gene expression etc.1 .The epitopes and secondary structures of the structural proteins of PRRSV BJ-4 were forecasted by molecular biology software GoldenKey. Thirteen putative epitopes showing characteristics of antigenic epitope were found from the analysis information. Using PCR, the nucleotide acid fragments encoding these putative epitopes were amplified, then cloned into the expression vector MISKE.The positive recombinant phage displying the epitopes were found out by using PCR, sequencing and the determination of phage plaque titer.2.The putative epitopes that displayed on phages were identified by indirect ELISA using swine antisera against PRRSV and mouse antisera to recombinant structural protein of PRRSV. Seven putative epitopes could be recognized by antisera. GP2110 (aa110-aal36) on GP2a could be recognized by mouse antisera to recombinant GP2a; GP381 (aa81-aa91) that is present on GP3 showed a reactivity with swine antisera agaist PRRSV and mouse antisera to recombinant GP3; GP4 presented two epitopes including GP453 (aa53-aa67) and GP4107 (aal07-aa115) that reacted with both swine antisera to PRRSV and mouse antisera to recombinant GP4; GP530 (aa30-aa39), GP5161 (aal61-aa169) and GP5190 (aal90-aa200) were exhibited on GP5, that could be reacted with swine antisera to PRRSV and mouse antisera to recombinant GP5. Three epitope, including GP2110, GP453 and GP530, showed a similarity with those described previously. The four epitopes ,GP381, GP4107, GP5161 and GP5190 were found to be new epitopes on structural protein of PRRSV BJ-4.3. The monoclonal antibodies (mAb) specific for the Nucleoprotein of PRRSV were used to select a phage random heptapeptide library. Through three rounds of screening, seventeen clones were selected and used in competitive test. The mAb GE3 could specifically inhibit eight out of seventeen clones from binding to swine antisera. Based on the amin acid sequences deduced from the foreign sequence inserted in the phage, it was indicated that all clones shared the core sequence - P/EKPHF, that was similar to aa50~aa55 domain of N protein of PRRSV. All the clones could react with swine antiseraagainst PRRSV and mouse antisera to PRRSV, suggesting aa50~aa55 domain on N protein might be the antigenic site recognized by mAb GE3.4. The gene fragment encoding the epitope recognized by mAb GE3 was cloned into the expression vector pGEX-4T-2. SDS-PAGE analysis revealed that the GST-epitope fusion protein could be highly expressed in BL21. By using Western-blot, the fusion protein could not be react with antiserum to PRRSV, whereas it presented a reactivity with swine antisera against PRRSV and mAb GE3 by ELISA The results implied that the epitope might be one conformational epitope that was mainly composed of aa50~aa55 domain on N protein.