牛胸腺素原α基因的原核表达与分析鉴定  

Expression and identification of recombinant bovine prothymosin αprotein in Escherichia coli

作　　者：李小庆[1] 陈国华[1] 房永祥[1] 何延华[1] 冯海燕[1] 景志忠[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部兽医公共卫生重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2011年第12期1242-1247,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(30871884);"十二五"国家高技术研究发展计划(863)项目(2011AA10A211)

摘　　要：为了把牛胸腺素原α(ProTα)开发为免疫增强剂,从重组克隆载体pMD18-T-ProTα上扩增ProTα基因,分别构建和筛选出了pGEX-4T-1-ProTα、pET-30a(+)-ProTα的BL21、BL21codon plus和Rosetta Blue(DE3)plys重组原核表达菌株,采用不同的条件组合对其进行诱导表达。结果,重组菌BL21codon plus-pET-30a(+)-ProTα表达出约24ku和30ku两个蛋白条带,且在37℃、IPTG浓度为0.5mmol/L、诱导3h时表达量最大,重组蛋白主要以可溶性形式存在;而pGEX-4T-1-ProTα重组菌在诸多条件下都未表达出目的蛋白。纯化产物的Western-blot分析显示,重组蛋白可与兔抗人ProTα多抗发生特异性免疫反应,表明重组蛋白具有免疫学活性结构。研究结果为重组ProTα蛋白的结构与功能研究奠定了基础。In order to develop ProTα as immune adjuvant,bovine ProTα gene was amplified from the recombinant clone vector pMD18-T and ProTα constructed before,then the recombinant expression vectors pGEX-4T-1-ProTα and pET-30a（＋）-ProTα were constructed and screened[host bacteria BL21,BL21 codon plus,Rosetta Blue（DE3） plys included].After being induced with IPTG under differential situations,SDS-PAGE analysis showed that the recombinant proteins with the molecular weight of 24 ku and 30 ku,respectively,were expressed by the recombinant BL21 codon plus-pET-30a（＋）-ProTα.The optimal temperature,concentration of IPTG,induction time for the recombinant BL21 codon plus-pET-30a（＋）-ProTα were 37 ℃,0.5 mmol/L,and 3 hours,and the recombinant proteins mainly existed in soluble form.However,the recombinant protein was not expressed in recombinant pGEX-4T-1-ProTα under any differential situations.After purification of expressed products,the special immunological reaction was observed between the expressed proteins and the rabbit polyclonal antibody against amino acids 1 to 50 at the N-terminus of PTα precursor of human origin by western-blot analysis,which revealed that the recombinant proteins probably possessed the structure of immunological activity.Therefore,the present work would provide basis for further research of structure and function of recombinant bovine ProTα protein.

关 键 词：胸腺素原α 原核表达 优化 鉴定 

分 类 号：S852.42[农业科学—基础兽医学]