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In order to investigate the prompt conformations of swine major histocompatibility complex (MHC), a swine MHC-II protein complex (SLA-II) was reconstructed in vitro. DRA and DRB were cloned from a crossbred commercial pig (Changbai-Dalan). Subcloned extracellular parts of DRA and DRB were linked together by a linker containing rich glycine/serine (G4S)3, and the whole length of two genes, named DRA-linker-DRB, was amplified by splicing overlap extension PCR (SOE PCR). DRA-linker-DRB was inserted into pMAL-p2X prokaryotic system and expressed. The expressed fusion protein MBP-DRA-(G4S)3-DRB was identified by Western-blot, purified and cleaved to obtain the protein of interest DRA-(G4S)3-DRB. The secondary structure of this protein was determined in circular dichroism (CD) apparatus. The results indicated that the subsequent protein MBP-DRA-(G4S)3-DRB was soluble and its molecule weight was 83.4 ku in consistent with the Western-blot. Cleaved by Factor Xa, the protein of interest was separated with a molecular weight of 40.9 ku. The CD spectrum demonstrated that the protein displayed a favorable 伪-Helix structure, and the contents of 伪-Helix, 尾-sheet, turn, and random coil were 80 aa, 121 aa, 101 aa and 80 aa respectively. The identical ratios of 伪-Helix, 尾-sheet, turn, and random coil between MBP-DRA-(G4S)3-DRB and DRA-(G4S)3-DRB were 95%, 96.7%, 91.1% and 93.0%, respectively. The results also suggested that the reconstructed SLA-II complex presented an ideal conformation and can be used for studying its structure and function in vitro. |
