巴马小型猪SLA-2和β_2m复合体蛋白的构建及二级结构测定分析

Cloning of SLA-2 and β_2m from Bama Miniature Pig and Construction of the Protein Complex

以巴马小型猪为研究材料,克隆SLA-2和β2m基因。然后采用剪接重叠延伸PCR(Splicing overlap exten-tion PCR,SOE PCR)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。将SLA-2-Linker-β2m在pMAL-p2X系统上表达,其融合表达蛋白分别经过West-ern-blot、纯化及Factor Xa切割,分离纯化单体蛋白。圆二色谱(Circular dichroism spectrum,CD)测定蛋白的二级结构。结果显示,重构表达的复合体融合蛋白MBP-SLA-2-(G4S)3-β2m大小为84.1 ku。切割后去除MBP的单体蛋白大小为41.6 ku。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%、97.3%、97.1%和97.9%,揭示重构的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。

The SLA-2 and β2m genes were cloned from a Bama miniature pig. 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 was inserted to pMAL-p2X and expressed in E. coli TB1 system. The fusion protein was processed 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. The results indicated that the MBP-SLA-2-（G4S）3-β2m was 84. lku, and the monomer protein SLA-2-（G4S）3-β2m was 41.6 ku. The α-helix, β-sheet, turn, and random coil of the fusion protein and the monomer protein shared high homology ratio at 100%,97.3%,97.1% and 97.9%, respectively, detected by CD estimation. The results indicated that the complex protein SLA-2-（G4S）3-β2m had correct secondary structure and could be used to further research of peptide binding in vitro.