克隆猪主要组织相容性复合体Ⅱ类DR基因座α链和β链及其体外复合体的构建  

Cloning α and β chains of SLA-DR loci and reconstruction of their complex in vitro

作　　者：高凤山[1] 王磊[1] 李新生[1] 李云岗[1] 王慧飞[2] 方勤美[1] 郝慧芳[1] 夏春[1] 

机构地区：[1]中国农业大学动物医学院 [2]中国人民武装警察部队学院消防工程系,河北廊坊065000

出　　处：《中国农业大学学报》2006年第3期21-26,共6页Journal of China Agricultural University

基　　金：国家自然科学基金资助项目(30371098)

摘　　要：为推动猪主要组织相容性复合体(MHC)空间构象的研究,体外构建了猪的MHC-Ⅱ类蛋白复合体(SLA-Ⅱ)。首先克隆长白-达兰杂交商品猪DRA和DRB基因,用富含甘氨酸-丝氨酸的Linker(G4S)3连接DRA和DRB的胞外区,用SOE-PCR得到复合体基因DRA-linker-DRB,插入原核表达载体进行表达;对表达的融合蛋白进行Western-blot鉴定、纯化、蛋白酶切割并对切割后的蛋白进行分离和纯化。对分离的目的蛋白、融合蛋白及麦芽糖结合蛋白(MBP)进行二级结构测定。得到83.4 ku的可溶性融合蛋白MBP-DRA-(G4S)3-DRB,其Western杂交带与表达条带大小一致。切割分离后的目的蛋白大小为40.9 ku。目的蛋白DRA-(G4S)3-DRB二级图谱显示该蛋白呈典型α-螺旋结构,各二级结构元件α螺旋、β折叠、转角和随机卷曲的氨基酸数目分别为80、121、101和80,融合蛋白MBP-DRA-(G4S)3-DR与目的蛋白DRA-(G4S)3-DRB的二级结构各元件的符合率分别达到了95%、96.7%、91.1%和93.0%。结果分析表明得到的复合体构象正确,可用于进一步的B细胞抗原表位的筛选等结构和功能研究。In order to prompt conformational research on swine major histocompatibility complex （MHC）, a swine MHC-Ⅱ protein complex （SLAⅡ ） was constructed in vitro. DRA and DRB were cloned from an outbred pig （changbaidalan）. Subcloned extracellular parts of DRA and DRB were linked together by a linker containing rich glycine-serine （G4S）3, and the whole length of two genes, named DRA-linker-DRB, was amplified by splicing overlap extension PCR （SOE PCR）. DRA-linker-DRB was inserted into pMAL-p2X prokaryotic system and expressed. The expressed fusion protein MBP-DRA （G4S）3-DRB was identified by Western-blot, then purified and cleaved to obtain the protein of interest DRA-（G4S）3-DRB. The secondary structure of this protein was determined in circular dichroism （CD） apparatus. The results indicated that the subsequent protein MBP-DRA-（G4S）3-DRB was soluble, and its molecule weight was 83.4 ku in consistent with the Western-blot. Cleaved by Factor Xa, the protein of interest was separated with a molecule weight of 40.9 ku. The CD spectrum demonstrated that the protein displayed a favorable α-Helix structure,and the contents of α-Helix, β-sheet, turn, and random coil were 80 aa, 121 aa, 101 aa and 80 aa respectively. The i-dentical ratios of α-Helix, β-sheet, turn, and random coil between MBP-DRA-（G4S）3-DRB and DRA-（G4S）3-DRB were 95%, 96.7%, 91.1% and 93.0% respectively. The result also indicated that the reconstructed SLA-Ⅱ complex presented an ideal conformation and could be used to study its structure and function in vitro.

关 键 词：猪 主要组织相容性复合体 基因座 二级结构 

分 类 号：Q518.1[生物学—生物化学] S858.28[农业科学—临床兽医学]