Reconstruction of a swine SLA-I protein complex and determination of binding nonameric peptides derived from the foot-and-mouth disease virus.
No experimental system to date is available to identify viral T-cell epitopes in swine. In order to reconstruct the system for identification of short antigenic peptides, the swine SLA-2 gene was linked to the beta(2)m gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich sequence (G4S)3, using splicing overlap extension-PCR (SOE-PCR). The maltose binding protein (MBP)-SLA-2-(G4S)3-beta(2)m fusion protein was expressed and purified in a pMAL-p2X/Escherichia coli TB1 system. The purified MBP-SLA-2-(G4S)3-beta(2)m protein was cleaved by factor Xa protease, and further purified by DEAE-Sepharose chromatography. The conformation of the SLA-2-(G4S)3-beta(2)m protein was determined by circular dichroism (CD) spectrum. In addition, the refolded SLA-2-(G4S)3-beta(2)m protein was used to bind three nonameric peptides derived from the foot-and-mouth disease virus (FMDV) O subtype VP1. The SLA-2-(G4S)3-beta(2)m-associated peptides were detected by mass spectrometry. The molecular weights and amino acid sequences of the peptides were confirmed by primary and secondary spectra, respectively. The results indicate that the SLA-2-(G4S)3-beta(2)m was 41.6kDa, and its alpha-helix, beta-sheet, turn, and random coil by CD estimation were 78 aa, 149 aa, 67 aa, and 93 aa, respectively. SLA-2-(G4S)3-beta(2)m protein was able to bind the nonameric peptides derived from the FMDV VP1 region: 26-34 (RRQHTDVSF) and 157-165 (RTLPTSFNY). The experimental system demonstrated that the reconstructed SLA-2-(G4S)3-beta(2)m protein complex can be used to identify nonameric peptides, including T-cell epitopes in swine.
Gao
Feng-Shan
FS
Department of Microbiology and Immunology, College of Veterinary Medicine, China Agricultural University, Beijing 100094, China.
Fang
Qin-Mei
QM
Li
Yun-Gang
YG
Li
Xin-Sheng
XS
Hao
Hui-Fang
HF
Xia
Chun
C
D000818:Animals; D015153:Blotting, Western; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology; D002942:Circular Dichroism; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000276:immunology; D029544:Foot-and-Mouth Disease Virus / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D000949:Histocompatibility Antigens Class II; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D013552:Swine; D013556:Swine, Miniature; D001613:beta 2-Microglobulin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D036022:Capsid Proteins; D004279:DNA, Viral; D018984:Epitopes, T-Lymphocyte; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C442501:VP1 protein, Foot-and-mouth disease virus; D001613:beta 2-Microglobulin; C438668:swine leukocyte antigen