Screening Of T-cell Epitopes In Structural Protein Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus is a highly contagious disease,it can cause reproductive disorders of sows and severe respiratory diseases of pigs of all ages,causing serious economic losses to the global pig industry.At present,the existing traditional vaccines are very important in the prevention and control of PRRS,but their effectiveness and safety are limited.For example,the inactivated vaccine is safe but its protection effect is not good,and the attenuated live vaccine is more effective than the inactivated vaccine but its safety needs to be improved.Therefore,it is necessary to try new antiviral strategies.A large number of research data show that PRRSV infection in pigs can cause cellular immune response and humoral immune response,and cellular immunity seems to play a more important role in the clearance of PRRSV infected cells.Major histocompatibility complex-Ⅰ,which specifically binds to restricted T cell epitopes,is the main mechanism of activating cellular immune response,controlling virus replication and eliminating virus.At present,the largest pig breeding strain in China is ternary hybrid pig,but there are few studies on SLA-Ⅰ molecules of this strain,which is very unfavorable for developing subunit vaccines suitable for this strain of pigs.Therefore,in this study,the functional alleles SLA-1,SLA-2 and SLA-3 of ternary hybrid pig SLA-I were used as the research objects to screen T cell epitopes of PRRSV structural protein,which can provide reference for the development of subunit vaccines that can improve cellular immunity.The research contents mainly include the following aspects:1.Analysis of SLA-I allele characteristics.By designing specific primers for SLA-1,SLA-2 and SLA-3,13 SLA-I allele sequences were cloned from peripheral blood lymphocytes of five three-way hybrid pigs,including 4 SLA-1,5 SLA-2 and 4 SLA-3.The characteristics were analyzed by biological information software,and the results showed that there was no population difference between SLA-I allele of ternary hybrid pigs and other strains of pigs.According to the high variability of amino acids in the extracellular region of SLA-I allele,it was mapped into SLA-1 3D structure,and it was further determined that the amino acid polymorphism constituting PBG was the main reason affecting the presentation characteristics of SLA-I allele.2.Acquisition of T cell epitopes of PRRSV structural proteins.T-cell epitopes of structural protein of PRRSV-JXA1 strain were screened by combinatorial prediction strategy.The results showed that 599 high affinity SLA-I restricted T-cell epitopes with Percentile Rank<0.5 were screened by IEDB online software,and 50 candidate T-cell epitopes were finally obtained by MHCcluster online tool based on combined amino acid preference clustering.3.Identification of T cell epitopesIn order to screen stable and highly sensitive T cell epitopes,PBMCs which can effectively induce IFN-γ secretion from peripheral blood at different immunization/challenge periods were selected by ELIspot test,and then positive T cell epitopes were screened.The preliminary judgment of SLA-I restrictive epitopes and conservative analysis of the obtained positive T cell epitopes showed that the obtained positive T cell epitopes were highly conserved in HP-PRRSV-like,and five of them were highly conserved in PRRSV-2 epidemic strains.In summary,13 SLA-I alleles of three-way hybrid pigs were obtained in this study,and their characteristics were analyzed.It was further determined that the amino acid polymorphism that constitutes PBG is the main reason affecting the SLA-I allele presentation characteristics.50 SLA-I restricted T-cell epitopes were obtained by combined prediction strategy,and positive T-cell epitopes with good conservation were obtained by ELIspot test,which provided a basis for the study of cellular immunity after PRRSV infection and the development of subunit vaccines to improve cellular immunity.