绵羊朊蛋白PrP^C51-216蛋白酶K抵抗片段的克隆原核表达及纯化  

Cloning prokaryotic expression and purification of prion PrP^C51-216 from ovis aries

作　　者：张奥[1] 余琦[1,2] 周向梅[1] 刘爱林[3] 赵德明[1] 

机构地区：[1]中国农业大学国家动物传染性海绵状脑病实验室,北京海淀100193 [2]北京市畜牧兽医总站,北京朝阳100107 [3]河北省邯郸市动物疫病预防控制中心,河北邯郸056005

出　　处：《中国兽医杂志》2010年第3期21-23,共3页Chinese Journal of Veterinary Medicine

基　　金：科技部星火计划(2006EA125015);国家科技支撑计划(2006BAD06A13-1)

摘　　要：根据绵羊朊蛋白基因(prion protein nucleic acid,PrNP)序列,截去PrNP部分N端信号肽(150 bp)和C端GPI锚定位点(123 bp)形成PrNPT-08,设计特异性引物,以绵羊全血提取DNA为模板,扩增PrNPT-08序列。与表达载体pET30a(+)连接,转化入BL21(DE3)感受态细胞。用IPTG诱导表达,其产物经SDS-PAGE电泳、纯化、Western blotting验证。结果表明,所插入的克隆片段含有498个核苷酸,共编码166个氨基酸,序列对比分析表明,与GenBank中绵羊朊蛋白基因序列同源性为99.6%,氨基酸序列同源性为98.8%。PrNP T-08基因在大肠杆菌得到高效表达,表达产物为相对分子量为27 kDa的融合蛋白,并能被PrP单克隆抗体AH6所识别。该蛋白成功表达为研究朊蛋白生理生化功能和结构转变提供了基础生物学材料,同时为朊蛋白疾病诊断中所需单克隆抗体的制备提供基本的试验材料。An ovine PrNP truncated fragment designed as PrNPT-08,was formed by removal of both N-terminal signal（150 bp）and C-terminal GPI sequences（123 bp）.Using a pair of primers designed according to the ovine prion protein sequence in GenBank and the DNA template of ovine blood,the PrNPT-08 DNA segment.PrNPT-08 was subcloned into the expression vector pET30a（＋）,and the recombinant plasmid pET30a（＋）-PrNPT-08 was transformed into BL21（DE3） competent cell of E.coli.Induced by IPTG and the production was examined by SDS-PAGE,purification and western blot.The results showed that the subcloned fragment was 498 bp and encodes 166 amino acids,and the sequence analysis indicated that gene sequence similarity was up to 99.6% between the sequence we constructed and that in GenBank,amino acids similarity was up to 98.8%.PrNP T-08 was highly expressed,and the fusion protein can be recognized by monoclone antibody AH6.The successful expression of the protein provides us the biological material to investigate into the physiological-biochemical functions and structure changes of prion,and prepare the monoclonal antibody for the diagnosis of prion disease.

关 键 词：绵羊 朊蛋白基因 克隆 原核表达 

分 类 号：S852.659.7[农业科学—基础兽医学]