数字农科院2.0

Construction of multiple recombinant SLA-I proteins by linking heavy chains and light chains in vitro and analyzing their secondary and 3-dimensional structures

文献类型： 外文期刊

作者： Gao, Feng-shan;Bai, Jing;Xu, Chong-bo;Zhang, Qiang;Li, Yanmin

作者机构：

关键词： Circular dichroism spectroscopy;Expression;Homology modeling;Swine leukocyte antigen

期刊名称： GENE

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年卷期：

页码：

收录情况： SCI

摘要： Six breeds of swine were used to study the structure of swine leukocyte antigen class I (SLA-I). SLA-I complexes were produced by linking SLA-2 genes and β 2m genes via a linker encoding a 15 amino acid glycine-rich sequence, (G4S)3, using splicing overlap extension (SOE)-PCR in vitro. The six recombinant SLA-2-linker-β 2m genes were each inserted into p2X vectors and their expression induced in Escherichia coli TB1. The expressed proteins were detected by SDS-PAGE and western blotting. The maltose binding protein (MBP)-SLA-I fusion proteins were purified by amylose affinity chromatography followed by cleavage with factor Xa and separation of the SLA-I protein monomers from the MBP using a DEAE Ceramic Hyper D F column. The purified SLA-I monomers were detected by circular dichroism (CD) spectroscopy and the 3-dimensional (3D) structure of the constructed single-chain SLA-I molecules were analyzed by homology modeling. Recombinant SLA-2-Linker-β 2m was successfully amplified from all six breeds of swine by SOE-PCR and expressed as fusion proteins of 84.1kDa in pMAL-p2X, followed by confirmation by western blotting. After purification and cleavage of the MBP-SLA-I fusion proteins, SLA-I monomeric proteins of 41.6kDa were separated. CD spectroscopy demonstrated that the SLA-I monomers had an α-helical structure, and the average α-helix, β-sheet, turn and random coil contents were 21.6%, 37.9%, 15.0% and 25.5%, respectively. Homology modeling of recombinant single-chain SLA-I molecules showed that the heavy chain and light chain constituted SLA-I complex with an open antigenic peptide-binding groove. It was concluded that the expressed SLA-I proteins in pMAL-p2X folded correctly and could be used to bind and screen nonameric peptides in vitro.

分类号： R394

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