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Reconstruction of a swine SLA-I protein complex and determination of binding nonameric peptides derived from the foot-and-mouth disease virus
- Gao, F.S., Fang, Q.M., Li, Y.G., Li, X.S., Hao, H.F., Xia, C.
- Veterinary immunology and immunopathology 2006 v.113 no.3-4 pp. 328-338
- protein secondary structure, T-lymphocytes, recombinant fusion proteins, polymerase chain reaction, peptides, amino acid sequences, binding proteins, swine, viral proteins, Foot-and-mouth disease virus, mass spectrometry, molecular weight, viral antigens
- 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 β2m 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-β2m fusion protein was expressed and purified in a pMAL-p2X/Escherichia coli TB1 system. The purified MBP-SLA-2-(G4S)3-β2m protein was cleaved by factor Xa protease, and further purified by DEAE-Sepharose chromatography. The conformation of the SLA-2-(G4S)3-β2m protein was determined by circular dichroism (CD) spectrum. In addition, the refolded SLA-2-(G4S)3-β2m 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-β2m-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-β2m was 41.6 kDa, and its α-helix, β-sheet, turn, and random coil by CD estimation were 78 aa, 149 aa, 67 aa, and 93 aa, respectively. SLA-2-(G4S)3-β2m 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-β2m protein complex can be used to identify nonameric peptides, including T-cell epitopes in swine.