Preparation And Functional Detection Of Hebao Pig SLA--2/peptide Tetramer

Major histocomaptibility complex(MHC) is a tightly linked cluster of genes in vertebrate, whose products are the cell surface molecules that plays roles in intercellular recognition and in discrimination between self and nonself. The product ofMHC is involved a key molecules for the antigen presenting and T cell activation. The MHC and the encoding antigen of the MHC have different nomenclature in the various species of animals. In pigs, the MHC is referred to as the swine leukocyte antigen(SLA) complex.SLA I teramer was used for detecting and screening of the CTL special for antigen, which was served as a tool for studying the function of the cellular immunity in body, evaluating specialfor immunotherapy, exploring the mechanism of disease.The genes of the intracellular domain SLA-2-BSPfrom six kinds and sβ2m were subcloned, consisting the three foreign breeds of Landrace pigs(YC), Yorkshire pigs(DCY), Topigs pigs(TPK) and the three local breeds of Hebao pigs(HB),Laiwu pigs(LWH), Yantai pigs(YTH). Subsequently, recombinant SLA-2-BSP and sβ2m proteins were highly expressed in pET-21a(+)/E.coli. and pET-28a(+)/E.coli. system. And recombinant proteins werepurified.The T cell epitope peptide of VP1 and RNA polymerase 3D protein from Foot-and-mouth disease virus waspredictedby bioinformatics method, and was screened predicted specific epitope by ELISPOT assay. Then, the candidate epitopes was co-binding refolding with the heavy chain and light chain of the purified recombinant protein in vitro, identifyingthe specific binding with SLA-2-BSP, sβ2m and candidate epitope by gel filtration assay,and separating SLA-2-BSP/peptide monomer protein. On this basis, the tetramer was generated by the Streptavidin reaction labeled FITC with biotined SLA-2-BSP/peptide monomer, and testing by FACS.In summary, the peptide epitope of the foot-and-mouth disease virus VP1 was successfully identified by ELISPOT assay, And the SLA-2-BSP/peptide tetramer of Hebao pigs with one of the identified peptides was constructed, and the VP1 antigen specific CTL epitopes were determined by FACS. Tetramer technology platform was established in the pig primarily, and these assays laid foundation for further research specific CTL of FMDV immune response and screening T cell epitopes in pig.