Studies Of The Molecular Characters And Secondary Structures Of Chicken BF2 And β2m And Their Reconstructed Complex Identifying Virus Epitopes

Chicken Major histocompatibility complex (MHC) class I molecules (BF) locates on 16 chromosome and has two loci- encoding class I heavy chains : BF1 and BF2. The BF1 is a minor locus and less expressed, whereas the BF2 is a major locus and dominantly expressed in the class 1 molecule. The BF associated with β2-microglobulin (β2m) could bind antigen peptides and present them to cytotoxin T lymphocyte cell (CTL) to trigger the cellular immune response. The aims of this study are to elucidate the molecular characters of BF2 and β2m genes in three Chinese eugenic endemic breeds and illustrate the conformational structures of chicken BF2 and β2m proteins; and moreover, to reconstruct the BF2-β2m complex being used to bind antigen peptides and constitute the identification system of virus-derived T cell epitopes in vitro. Twenty-four BF2 genes and ten β2m genes from Chinese Sanhuang {Sanhuang, SH), Wuji (silky, SI), and Zhenzhu (Numida Meleagris, NM) chicken breeds were cloned, and the amino acid substitution ratio of BF2 polypeptide binding domain (PBD) were investigated. The chicken (SH) BF2 , β2m mature peptide genes were cloned by PCR and expressed by using the pMAL-p2X soluble expression plasmid in E. coli. TB1 strain. The purified fusion MBP-BF2, MBP- P 2m proteins and monomer MBP were obtained through the processing of first and further purification by Amylose-affinity resin column and DEAE-Sepharose ion exchange chromatography, cleavage by Factor Xa protease, separation and purification by DEAE-Sepharose ion exchange chromatography. The two-dimensional (2D) and secondary structures elements contents of fusion MBP-BF2, MBP- P 2m proteins and monomer MBP were estimated by circular dichroism (CD) spectrum. The three-dimensional structures (3D) of BF2 and P 2m proteins were estimated by homology modeling.Subsequently, the recombination BF2-linker- β2m gene constructed by BF2 linked β2m mature peptide genes via a 15-amino acid glycine-rich linker [linker, (G4S) 3] by SOE-PCR (splicing by overlap extension-PCR) method were cloned and expressed by using the pMAL-p2X soluble expression plasmid in E. coli. TB1 strain. The fusion recombination MBP-BF2-linker- P 2m protein and monomer recombination BF2-linker- P 2m protein were purified and separated by the methods described above, and the 2D as well as secondary structures elements contents of which were estimated by CD. Whereafter, the fusion recombination MBP-BF2-1inker- P 2m protein bound two synthesized nonameric peptides representing the T-cell epitopes which was corresponding to avian influenza virus (AIV) subtypes H5N1 and H9N2 hemagglutinin (HA) (named A1V-HA5-TP1 and AIV-HA9-TP2, respectively) were carried out in vitro with a mol ratio of 1:10 , and incubated at 37°C for 12h. The acid treatment was adopted to release the bound peptide AIV-HA5-TP1 and AIV-HA9-TP2 from the BF2-linker-β 2m-peptide complex. Peptides in acid solution were concentrated, desalted and changed medium solution on Sep-Pak C18 cartridges and were subsequently lyophilized to completely dryness. The lyophilized peptides were dissolved in matrix solution and detected by mass spectrometer, and the molecular weight and amino acids sequences of peptides were illustrated by primary and secondaryspectra, respectively.The amino acids identical ratios of BF2 alleles among three chicken breeds were from 75.5%~ 99.2 % . Comparison of the amino acids sequences of BF2*SH, BF2*SI and BF2*NM alleles with human HLA-A2 showed that seven, seven and six key amino acids of the peptide binding sites in HLA-A2 (YYYRTKWY) were also conserved in three chicken breeds, respectively. The high amino acids substitution ratios were observed in PBD of overall BF2 alleles, but relative conservation involved in the CD8+ interaction sites and fi2m contact sites. Based on the amino acid identical ratio standard established by ourselves, all BF2 alleles from the three chicken lines and Leghorn chicken line could be divided into eleven gene groups (within an allelic group the members shared > 91% amino acid identity with e...