一商品猪SLA-Ⅰ分子复合体的体外构建

Construction of the SLA-I Protein Complex from an Outbred Pig

【目的】研究中国北方杂交商品猪SLA-Ⅰ类分子结构特点,并为进一步的多肽结合等功能研究奠定基础,需要在体外构建正确折叠的SLA-I类分子复合体。【方法】以长白-达兰商品猪为研究材料,克隆SLA-2和β2m。然后采用剪接重叠延伸PCR(splicingoverlapextentionPCR,SOEPCR)法,将SLA-2的胞外区和β2m的成熟肽部分通过一富含甘氨酸/丝氨酸的Linker(G4S)3连接,形成SLA-2-Linker-β2m全长,并插入原核表达质粒pMAL-p2X,转化E.coliTB1,IPTG诱导表达。表达得到的融合蛋白分别经过Western-blot、纯化及FactorXa切割,分离纯化单体蛋白。圆二色谱(circulardichroismspectrum,CD)测定蛋白的二级结构。【结果】SDS-PAGE和Western-blot均证实融合蛋白MBP-SLA-Ⅰ大小为84.1kD。SLA-I单体蛋白大小为41.6kD。圆二色谱分析单体蛋白和融合蛋白二级结构元件α-螺旋、β-折叠、转角和随机卷曲的符合率分别达到了100%、90.6%、88.5%和96.9%。【结论】结果表明重构的SLA-I复合体具有正确的二级结构,可以用于体外多肽结合等研究。

【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 α-helix, β-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.