Reconstruction of a chicken BF2 protein complex and identification of binding nonamer peptides derived from avian influenza virus hemagglutinin.
In order to reconstruct the system for identification of short antigenic peptides, the chicken BF2 gene of Chinese Sanhuang (SH) chicken line was linked to the beta(2)m gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich peptide, by splicing overlap extension PCR (SOE-PCR). The MBP-BF2-(G4S)3-beta(2)m fusion protein was expressed and purified in a pMAL-p2X/E. coli TB1 system. The purified MBP-BF2-(G4S)3-beta(2)m protein was cleaved by Factor Xa protease, and further purified by DEAE-Sepharose chromatography. The conformation of the BF2-(G4S)3-beta(2)m protein was determined by circular dichroism (CD). In addition, the refolded BF2-(G4S)3-beta2m protein was used to bind three predicted nonameric peptides derived from the hemagglutinins of the avian influenza virus (AIV) H5N1 and H9N2 subtypes. The BF2-(G4S)3-beta2m-associated peptides were detected by mass spectrometry. The molecular weights and amino acid sequences of the peptides were confirmed by primary and tandem mass spectrometry analysis, respectively. The results indicate that the secondary structures and predicted three-dimensional crystal structure of BF2-(G4S)3-beta(2)m are similar to those of the monomers of chicken BF2 and beta(2)m. The BF2-(G4S)3-beta(2)m protein could bind two of the three predicted nonamer peptides derived from AIV hemagglutinin. The experimental system demonstrated that the reconstructed BF2-(G4S)3-beta(2)m protein complex can be used to identify nonamer peptides, including T-cell epitopes in chicken.
Yan
Ruo Qian
RQ
Henan Center for Animal Disease Control & Prevention, Animal Husbandry Bureau of Henan Province, Zhengzhou 450008, Henan Province, China. yrq1688@126.com
Wu
Zhi Ming
ZM
Fang
Qin Mei
QM
Zhang
Zhi Ling
ZL
Zhang
Jian
J
Li
Xin Sheng
XS
Hao
Hui Fang
HF
Xia
Chun
C
D000818:Animals; D002645:Chickens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005786:Gene Expression Regulation; D005805:Genes, MHC Class I / Q000235:genetics* / Q000276:immunology; D006388:Hemagglutinins / Q000737:chemistry / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D053124:Influenza A Virus, H5N1 Subtype / Q000737:chemistry / Q000378:metabolism*; D053127:Influenza A Virus, H9N2 Subtype / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins
D006388:Hemagglutinins; D007143:Immunoglobulin Heavy Chains; D011994:Recombinant Proteins