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 ?-Helix structure, and the contents of ?-Helix, ?-sheet, turn, and random coil were 80 aa, 121 aa, 101 aa and 80 aa respectively. The identical ratios of ?-Helix, ?-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.
GAO Fengshan, WANG Lei, LI Xinsheng, LI Yungang, FANG Qinmei, HAO Huifang, XIA Chun, WANG Huifei
. Cloning α and β chains of SLA-DR loci
and reconstruction of their complex[J]. Frontiers of Agriculture in China, 2008
, 2(3)
: 355
-360
.
DOI: 10.1007/s11703-008-0044-0
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