Reconstruction of a swine SLA-I protein complex and determination of binding nonameric peptides derived from the foot-and-mouth disease virus
Reconstruction of a swine SLA-I protein complex and determination of binding nonameric peptides derived from the foot-and-mouth disease virus
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DOI：
10.1016/j.vetimm.2006.06.002
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发表时间：
2006-10-15
影响因子：
1.8
通讯作者：
Xia, Chun
中科院分区：
文献类型：
--
作者：
Gao, Feng-Shan;Fang, Qin-Mei;Xia, Chun
迄今为止，尚无实验系统可用于鉴定猪体内的病毒 T 细胞表位。为了重建识别短抗原肽的系统，使用剪接重叠延伸 PCR (SOE-PCR)，通过 (G4S)3（编码 15 个氨基酸的富含甘氨酸序列 (G4S)3 的接头）将猪 SLA-2 基因与 β M-2 基因连接。麦芽糖结合蛋白(MBP)-SLA-2-(G4S)3-β M-2融合蛋白在pMAL-p2X/大肠杆菌TBI系统中表达和纯化。纯化的MBP-SLA-2-(G4S)3-β M-2蛋白被Xa因子蛋白酶切割，并通过DEAE-Sepharose层析进一步纯化。通过圆二色性 (CD) 光谱测定 SLA-2-(G4S)3-β M-2 蛋白的构象。此外，重折叠的 SLA-2-(G4S)3-beta M-2 蛋白用于结合源自口蹄疫病毒 (FMDV) O 亚型 VP1 的三种非聚肽。通过质谱法检测SLA-2-(G4S)3-β(2)m相关肽。肽的分子量和氨基酸序列分别通过一级和二级光谱确认。结果表明，SLA-2-(G4S)3-β(2)m为41.6 kDa，CD估计其α螺旋、β折叠、转角和无规卷曲分别为78个氨基酸、149个氨基酸、67个氨基酸和93个氨基酸。 SLA-2-(G4S)3-beta(2)m 蛋白能够结合源自 FMDV VP1 区域的九聚肽：26-34 (RRQHTDVSF) 和 157-165 (RTLPTSFNY)。实验系统证明，重建的 SLA-2-(G4S)3-beta(2)m 蛋白复合物可用于鉴定非聚肽，包括猪的 T 细胞表位。 (c) 2006 Elsevier B.V. 保留所有权利。
No experimental system to date is available to identify viral T-cell epitopes in swine. In order to reconstruct the system for identification of short antigenic peptides, the swine SLA-2 gene was linked to the beta M-2 gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich sequence (G4S)3, using splicing overlap extension-PCR (SOE-PCR). The maltose binding protein (MBP)-SLA-2-(G4S)3-beta M-2 fusion protein was expressed and purified in a pMAL-p2X/Escherichia coli TB I system. The purified MBP-SLA-2-(G4S)3-beta M-2 protein was cleaved by factor Xa protease, and further purified by DEAE-Sepharose chromatography. The conformation of the SLA-2-(G4S)3-beta M-2 protein was determined by circular dichroism (CD) spectrum. In addition, the refolded SLA-2-(G4S)3-beta M-2 protein was used to bind three nonameric peptides derived from the foot-and-mouth disease virus (FMDV) O subtype VP1. The SLA-2-(G4S)3-beta(2)m-associated peptides were detected by mass spectrometry. The molecular weights and amino acid sequences of the peptides were confirmed by primary and secondary spectra, respectively. The results indicate that the SLA-2-(G4S)3-beta(2)m was 41.6 kDa, and its alpha-helix, beta-sheet, turn, and random coil by CD estimation were 78 aa, 149 aa, 67 aa, and 93 aa, respectively. SLA-2-(G4S)3-beta(2)m protein was able to bind the nonameric peptides derived from the FMDV VP1 region: 26-34 (RRQHTDVSF) and 157-165 (RTLPTSFNY). The experimental system demonstrated that the reconstructed SLA-2-(G4S)3-beta(2)m protein complex can be used to identify nonameric peptides, including T-cell epitopes in swine. (c) 2006 Elsevier B.V. All rights reserved.