The Research On Leghorn Chicken MHC- â…  Reconstruction In Vitro And Identification Of Avian Influenza Virus T Cell Epitopes

In order to promote studies related to the structure and function of the chicken MHC I molecules, the chicken MHCⅠmolecules was construct by prokaryotic expression vectors, recombinant MHCⅠandβ2m proteins was highly expressed in pET-21a(+)/E. coli. System and four T cell epitomes like peptides bond to the MHCⅠwas identified. The method is a sensitive tool to evaluate the foundation for the preparation of MHC-Ⅰ-Peptide complex. The research paved the way for study on the relationship between the configuration of chicken MHCⅠand its functions and on its role in antigen identificiation and immunity response.In this experiment, genes of the chicken MHCⅠαchain(BF2) andβ2 microglobulin(β2m) were amplified by reverse transcription polymerase chain reaction(RT-PCR) from the total RNA of peripheral blood monouclear cells (PBMCs) from the Leghom. The result of sequence determined suggested that the full length of Chicken BF2 gene is consisted of 1035bp, which includes an open reading frame and encodes 345 amino acid residues. The full length of Chickenβ2m gene is consisted of 360 bp, which includes an open reading frame and encodes 120 amino acid residues. The nucleotide sequence of homology to the chicken MHC-Ⅰheavy chain gene was between 93.0% and 97.6%, and Chickenβ2m gene was 100% in the Genbank. Comparison of the nucleotides sequence of chicken MHC-Ⅰmolecules with those from human, cattle, goose, duck, horse and house mouse showed that the amino acids homologies of both chicken BF2 andβ2m were between 32.6%-82.3% and between 24.2%-100.0%. Based on the gene sequence comparison and phylogenetic tree analysis, the genes difference was found in chicken MHC-Ⅰheavy chain andβ2m gene, and the closer the genetic relationship, the higher the homology.The software SignalP 3.0 was used to predict the signal peptide of chicken BF2 andβ2m gene. The PCR products of both chicken BF2 andβ2m (absent signal peptide sequence at the 5'end) were subcloned into expression vector pET-21a(+)/E. coli. System and named as pET-21-BF2 and pET-21-β2m. High levels of chicken BF2 andβ2m proteins were expressed efficiently in prokaryotic expresion system with the recombinant vectors pET-21-BF2 and pET-21-β2m. The results of the BF2 andβ2m fusion protein were about 31 kD and 11 kD and were successfully expressed and purified by SDS-PAGE. High quality of the target protein was recovered from the inclusion body fraction.Peptide-MHC-Ⅰbinding was predicted using 2 algorithms: BIMAS and SYFPEITHI on web. To predict the HLA-B restricted CTL epitopes of antigen NP, associated with Avian influenza virus (AIV), so as to provide appropriate CTL epitope for AIV. The peptides were ranked for each algorithm and sorted by a cumulative score. Four candidate epitope peptides were synthesized. MHC I andβ2m were refolded in refolding buffer with synthetic peptide originated from Avian influenza virus to generate the MHCⅠ/peptide complex monomer. The result of Non-denaturing SDS-PAGE indicated that the MHCⅠcould bind to the peptides P80, P219 and P251. Three T cell like epitopes derived from NP protein of AIV were identified in this study using refolding. MHCⅠ/peptide complex monomer, and the NP antigen specific CTL epitopes were determined by non-denaturing SDS-PAGE. These assays paved the way for further research on specific CTL of the AIV immune response and screening T cell epitopes in chicken.