Studies Of The Expression In E.coli And Structures Of Yuanyang Duck Mhcⅰand β2m And Screening Their Restricted T Cell Epitope From Virus

Major histocompatibility complex (MHC) classⅠmolecules (MHCⅠ) could bind antigen peptides and present them to cytotoxin T lymphocyte cell (CTL) to trigger the cellular immune response.In order to illustrate the structures of Yuanyang Duck MHCⅠprotein and establish the platform of identifying the virus antigen T cell epitopes use Duck- MHCⅠ-β2m complex in vitro,we carried out the following studies:1 The extracellular domain region of Yuanyang Duck MHCⅠand mature peptides gene of Yuanyang Duckβ2m were amplifled using RNA from the spleen tissue by RT-PCR method respectively.As result,900 bp PCR fragment of Yuanyang Duck MHCⅠand 440 bp PCR fragment of Yuanyang Duckβ2m werw obtained.Sequencing and sequence analysis results is that cloned Yuanyang Duck MHCⅠαchain gene is 909 bp, which encodes 303 amino acid residues,including a complete Yuanyang duck MHC-Iαchain mature peptide (303 aa) of the extracellular domain region,which contains 92 amino acids of theα1 region,89 amino acids of theα2 region, and 90 amino acids of theα3 region.Moreover,theα2 andα3 region contain two cysteine,and a potential N-glycosylation sites.The results of homology analysis showed that the duck MHCⅠαchain mature peptide of the extracellular domain region has higher homology with human MHC classⅠmolecules than fish,frogs, chickens and other lower vertebrates.Yuanyang Duckβ2m gene has 100% homology with Beijing duck MHCⅠGenBank sequence (AB246408),which proved theβ2m gene is highly conservative.What is more,theβ2m mature peptides has two cysteines at 25 and 80 position ,in the vicinity of cysteine 80 position has a sequence of "YTCRVDH",which is to match the feature-based sequence "YxCxVxH" of immunoglobulin superfamily.2 The fragments of Yuanyang Duck MHCⅠαchain extracellular domain region andβ2m mature peptide were efficiently expressed using pET-28a(+)/E.coli expression system.SDS-PAGE and Bandscan software analysis shown that the Yuanyang duck MHC-Iαchain extracellular domain region andβ2m mature peptide is 31.0 KDa,11.4 KDa and accounts 29.8%,36.5% of the total bacterial protein respectively.Secondary structure analysis shown that Yuanyang duck MHC-Iαchain extracellular domain of mature peptide has a typicalα-helix structure,β-sheet was the least of which is about 13%;a-helix andβ-turn content are both of 24%;other loose structure is the most abundant,which is about 40%;The components ofβ2m mature peptide is relative equilibrium,a-helix,β-turn and other loose structure account about 26%,β-sheet was smaller,is about 21%.As the homology modeling result, the Yuanyang duck MHC-Iαextracellular domain region andβ2m mature peptide has a similar 3D structures to human and mouse's,but has a small differences of the specific location and number whith the a-helix,β-turn, β-sheet and other loose structures.3 We predicted 3 epitopes of Yuanyang Duck MHCⅠrestricted duck-origin avian influenza virus using ultra-motif,quantizatione-motif and other methods.The 3 epitopes are NP71-79 (FDERRNRYL),NP217-215 (IAYERMCNI) and NP380-388 (LERSRYWAI).In summary,in this study we revealled the characteristics Yuanyang Duck MHCⅠandβ2m gene,illustrated 2D and 3D strucures and composition of Yuanyang Duck MHCⅠαchain extracellular domain region andβ2m mature peptide.And based on these,we predicted 3 epitopes of Yuanyang Duck MHCⅠrestricted duck-origin avian influenza virus,which might be a powerful tool to establish the platform of identifying the virus antigen T cell epitopes use Duck- MHCⅠ-β2m complex in vitro.