Structural Analyses Of Swine MHC Class ? Complexes And Their Binding Epitopes Of IAV And PRRSV

The major histocompatibility complex(MHC)class I molecules play a pivotal role to activate cytotoxic T lymphocytes(CTL)which determine specific cellular immune responses.Different MHC I molecules can selectively present different peptides processed by large multifunctional proteasome(LMP)and transporter associated with antigen processing(TAP)associated protein,causing different immune responses with different individuals.How to screen and identify super MHC I from different species and different individuals and expatiate on its mechanism of structure and function or potential application is a novelty and important research topic.In this research,21 swine MHC I(swine leukocyte antigen,SLA ?)sequences were cloned from spleen of Tibetan wild boars by reverse transcription-polymerase chain reaction(RT-PCR)with allele group-specific PCR primer pairs.The polymorphisms of the 21 sequences together with 28 sequences of heishan pigs,44 sequences of landraces and 86 uploaded sequences in IPD were analyzed.The high variation sites were analyses by Wu-kabat method and the high variation sites which composed PBG were located in the three dimensional structures of SLA I.Many different peptides from PRRSVand IAV were screened by bioinformatic method and refolding assay in vitro,the super SLA I molecules which have the ability to combine with more peptides were selected from Tibetan wild boars,heishan pigs and landraces.Finally,the crystal structure of SLA-3*hs0202 complexes the epitope(HA-KMN9)from Influenza A virus(IAV)hemagglutinin was determined by structural biology technique.Our data showed that within one asymmetric unit,there are two pSLA I molecules(short for M1 and M2),the HA-KMN9 peptide displays different conformations in M1 and M2.However,the distinct HA-KMN9 peptide conformations are not only caused by the side chains in the PBG but also by the skewing of the al and a2 helixes.The RMSD between M1 and M2 is 0.873 A,which is higher than the RMSD of the SLA-1*0401(0.401 A)and BOLA-N*01801(0.267 A)molecules,which are also within one asymmetric unit.This finding indicates that,besides the side chains of the PBG,flexibility of the swine MHC class I carbon backbone might expand the peptide conformation and facilitate the activation of an increased TCR repertory.In addition,peptides from PRRSV were screened by refolding in vitro assay,and the crystal structure of SLA-1*0501 complexing the epitope(GP3-ALL9)from porcine reproductive and respiratory syndrome virus(PRRSV)was determined by structural biology technique.The tetrameric complexes of biotinylated SLA-1*0501-BSP/ALL9 and SLA-1*0501-BSP/RLY9 were produced by mixing biotinylated monomer with PE-labled streptavidin.The immunological activity of GP5-RLY9 peptide was validated using the flow cytometry technology.In conclusion,super SLA I molecules which have the ability to combine with more peptides were selected from Tibetan wild boars and heishan pigs by bioinformatic method and refolding assay in vitro.The crystal structures of of SLA-3*hs0202 with IAV-derived peptides were determined In addition,peptides from PRRSV were screened by refolding in vitro assay,the crystal structure of SLA-1*0501 binding the epitope(GP3-ALL9)from PRRSV was determined by structural biology technique.Finally,the immunological activity of GP5-RLY9 peptide was validated using the flow cytometry technology.These results might promote the development of swine molecular immunology and the disease resistant breeding of swine and virus-derived epitopes based vaccines.