关键词: 复合体, 圆二色谱, SLA-I

Abstract:

【Objective】 To investigate the structure of swine leukocyte antigen class I (SLA-I) molecules from outbred pigs in north of China and lay a basis for studying their function such as peptide-binding, the correctly refolded SLA-I need to be reconstructed in vitro. 【Method】 Firstly, SLA-2 genes from an outbred pig were cloned. Then the extracellular part of SLA-2 was linked to the mature peptide of β2m gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich sequence, using splicing overlap extension-PCR (SOE-PCR). The reconstructed gene SLA-2-linker-β2m (SLA-I) was inserted into pMAL-p2X and expressed in E. coli TB1 system. The fusion protein was processed followed by Western-blot, purifying and cleaving by Factor Xa and then to separate the monomer protein from MBP. The secondary structure of the monomer and fusion protein was determined by circular dichroism (CD) spectrum. 【Result】 The results of SDS-PAGE and western-blot all proved that the MBP-SLA-I was 84.1 kD, and the monomer protein SLA-I was 41.6 kD. The a-helix, b-sheet, turn, and random coil of the fusion protein and the monomer protein shared high homology ratio at 100%，90.6%，88.5% and 96.9%, respectively, detected by CD estimation. 【Conclusion】 The results indicated that the complex protein SLA-I had correct secondary structure and could be used to have further research, such as peptide binding in vitro.

Key words: complex, circular dichroism spectrum, SLA-I