Construction Of SLA-I Complex And Screening Of PRRSV M Protein CD8+ T Cell Epitopes

Porcine leukocyte antigen(SLA)gene is highly polymorphic,and is closely related to the immune response caused by infectious diseases and vaccines.The classical SLA class Ⅰ molecules are expressed on the surface of all nucleated cells.SLA class Ⅰ molecules are highly polymorphic α1 and α2 regions constitute the virus antigen peptide binding groove,capable of presenting antigen peptide to CD8+ cytotoxic T lymphocytes(CTLs),and activate to kill the infectious target cells,so as to induce has to produce specific cellular immune response.Because the SLA-Ⅰ and virus T cell epitope binding is the first step of CD8+ T lymphocyte clearance of infected cells,and SLA has strict restriction on the epitopes,so the study of SLA class Ⅰ molecules to clarify SLA-Ⅰ polymorphism and virus CTLs epitope interaction molecular mechanism has important scientific significance.The study of SPF SLA-1 gene and Landrace lines were amplified by using RT-PCR SBT method,a total of 5 alleles of SLA-1 gene,its polymorphism is mainly concentrated in the exon second and third,compared the α1 and α2 functional domains encoded by exon2 and 3,found that 5 SLA-1 allele in the peptide binding groove 6 pockets(A-F)33 key amino acids,there are 10 completely conserved amino acid sites were 5(L),7(Y),59(Y),81(L),84(Y),123(Y),146(K),159(Y),160(L),171(Y),the remaining 24 loci had different degrees of mutation.The mutation may affect the size of the peptide binding pocket,which results in the difference of the binding ability of different SLA-1 alleles to the same T lymphocyte epitope.Porcine reproductive and respiratory syndrome virus(PRRSV)is a serious obstacle to the development of pig breeding industry in the world.Studies have shown that PRRSV infection can induce humoral and cellular immune responses,which may play a more important role in the clearance of PRRSV infection.CTL is one of the important immune cells to control the infection of virus.The specific CTL response of Ⅰ class MHC antigen is the main mechanism of virus replication and proliferation,and virus clearance.M protein,N protein and GPS protein are 3 important structural protein PRRSV,which M protein is the matrix protein of PRRSV,is the most conserved structural proteins,it has strong immunogenicity and can induce specific cellular immune response,and plays an important role in cellular immunity.Therefore,in this study,we used PRRSV M protein as the research object to predict the 10 candidate T cell epitopes with potential binding ability of SLA class Ⅰ molecules.By splicing overlap extension PCR(SOE-PCR)complex technology was constructed in Landrace 5 SLA-1 genes extracellular domain and β2m,with the predicted 10 candidate CTLs epitopes in series,using the prokaryotic expression system for expression,purification and renaturation,the establishment of ELⅠSA detection method for screening PRRSV M protein SLA-1 etc.the allele CTL restricted CD8+ epitope candidates.5 complexes with no epitope pET-SLA-1*X-Linker-β2m were successfully constructed,which contained the epitope pET-epitope-SLA-1*X-Linker-β2m complex.Ⅰt was found that the predicted PRRSV M protein T cell epitopes could be specifically combined with different SLA-1 alleles.M1,M2,M5 and M8 epitopes that specifically bind to SLA-1*07:02,M4,M6,M7 and M9 epitopes specifically associated with SLA-1*08:01,and M2,M4,M6 and M10 epitopes specifically associated with SLA-1*13:01.