| Porcine reproductive and respiratory syndrome (PRRS) is a highly contact and infectious disease with worldwide distribution, and has caused severe economic losses to the swine industry, which is characterized both reproductive failure in sows and a respiratory illness tract in nursery-age pigs. The variation of PRRSV genome enhances the difficulty of prevention of PRRSV. Since 1996, the virulent PRRSV variant strain has caused inestimable expenses to our country.The prevention of PRRSV mainly relies on vaccination. How to evaluate the immunity effect rapidly and correctly occupies important meanings in realistic production. This study adopted artificial synthetic neutralization B epitope of GP5 protein of PRRSV(S38HIQLIYNLC47)as coating antigen. An indirect synthetic peptide ELISA assay was constructed to detected antibodies against this neutralization epitope. The result shows that the optimal concentration of synthetic peptide antigen was 2ug/mL. The sealing buffer was 10% FBS, and the sealing time was 37℃for 2h. The serum sample was diluted by 1:200 and incubated for 60 min at 37℃.The dilution of conjugate was 1:5000, and the reaction time was 1h. The TMB substrate was added and incubated at 37℃for 12min before terminated with stopping solution. The study owned high specificity that it had no cross reaction with antibodies to other 4 porcine diseases. Using this method to detect the antibodies to PRRSV of pigs vaccinated with PRRS vaccine and challenged with PRRS isolate virus, neutralizing antibodies could be detected in 80% choosen serum samples. Make statistical analysis of the results, when the serum antibody titers of 1:200 dilution using this method greater than or equal to 0.156, the ones could counteract following virus challenge.The immunoprotection of traditional PRRS vaccines can't reach the request. It is very stringent to explore PRRS new generation vaccines. This study referred to the sequence of virulent PRRSV variation named JXA1 isolate, selected potential B-cell linear epitopes as follows: No. 61 to 105 Aa of GP3 protein, No. 38 to 45 and 187 to 200 Aa of GP5 protein, No. 151 to 174 Aa of M protein which then serially connected with a artificial synthetic adjuvanticity T-cell epitope, constructed multi-epitopes serially connected prokaryotic expression vector which then transformed into the competent cells of E.coli BL21 (DE3). The recombinant bacterium were induced by IPTG, the fusion proteins 2GP3-2GP5-2M,4GP3,4G5 and 4M were analyzed by SDS-PAGE and Western-blotting. Resluts show that the relative molecular mass of the fusion proteins were measured to be 41kDa,39kDa,31kDa,31kDa as anticipated. The purified fusion proteins were obtained by Ni-sepharose affinity chromatography and appreciated favourable activity.Associated the purified fusion proteins 2GP3-2GP5-2M,4GP3,4G5,4M with a a kind of mast cell activators (C48/80) and conventional oil adjuvant, immunized 5-week-old clean mouse, blood samples were collected one week interval from priming immunization. The specific antibodies were detected by indirect ELISA and neutralizing antibodies were determined on Marc-145 cells. The results show that each group can be detected high-level antibody titers by ELISA, the antibody levels reach the top at 5th week of each group. Group immunized with mixture of three simple-epitope proteins combined with adjuvant C48/80 induced the highest antibody titer. The neutralizing antibody titer of group immunized with 4GP5 and C48/80 was 1:4. which could provide foundation and demonstration for the molecular epitope-vaccine investigation of PRRSV.
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