猪CD8β基因的克隆、表达及其结构与功能分析  

Cloning, expression and structure and function analysis of porcine CD8β gene

作　　者：王生富[1] 景志忠[1] 陈国华[1] 房永祥[1] 莫斯科[1] 段凤云[1] 何延华[1] 

机构地区：[1]中国农业科学院兰州兽医研究所甘肃省动物寄生虫病重点实验室/家畜疫病病原生物学国家重点实验室

出　　处：《中国预防兽医学报》2008年第9期737-742,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：“863”项目(2006AA10A203);国家支撑计划项目(2006BAD31B03)

摘　　要：本研究应用RT-PCR技术,从猪胸腺细胞总RNA中扩增、克隆了猪CD8β基因,序列分析表明CD8β含621bp的开放阅读框,编码207个氨基酸,与NCBI/GeneBank已发表的参考基因的核苷酸及推导氨基酸序列的同源性分别为97.8%和96.8%,与人、小鼠和鸡CD8β蛋白的氨基酸同源性分别为75.7%、67.9%和33.3%。根据大肠杆菌密码子偏嗜性改造目的基因,构建了pET28a/PCD8β原核表达系统,并经诱导获得高效表达的分子量为24ku的重组蛋白(rPCD8β),表达量占菌体蛋白总量的30%。利用生物信息学和分子生物学软件对猪CD8β基因编码的蛋白进行结构预测,表明猪CD8β成熟蛋白为跨膜蛋白,其中172aa在胞外区,22aa在跨膜区,10aa在胞内区;蛋白骨架内含有较多的柔性区域,而且分布不均匀,能形成结构松散的球状蛋白;猪CD8β分子V区三维结构与鼠的具有非常相似的空间结构,与人的差别较大,这为猪CD8β蛋白结构与功能的进一步研究奠定了基础。A CD8β gene was amplified by RT-PCR from total RNA of porcine thoracic gland lymphocytes and sequenced. The gene contained a 621 bp open reading frame encoding a 207 amino acids and shared 97.8 % sequence identities at the nucleotide level and 96.8 % at amino acid level with published sequences, The homology at the amino acid level with human, mouse, rat, and chicken was 75.7 %, 67.9 %, 67.8 % and 33.3 %, respectively. The CD8β gene was inserted into expression vector pET28 and expressed in E,coli. SDS-PAGE analysis showed that the expressed protein had a molecular weight of 24.0 ku and accounted for 30.0 % of total mass of bacterial proteins. In addition, structural prediction indicated the porcine CD8β might be a typical transmembrane protein comprising an extracellular region, a transmembrane region and an intracellular region, respectively. It contained many flexible regions and maldistributed, forming a globule protein. The three-dimensional structure of V region of the protein was similar to that of mouse but different from the human.

关 键 词：猪CD8 Β基因 克隆 表达 结构预测 

分 类 号：S852.42[农业科学—基础兽医学]