布鲁菌BP26基因的克隆、表达及纯化  

Gene Cloning,Expression and Purification of BP26 Protein of B. abortus

作　　者：闫广谋[1] 王兴龙[2] 张辉[1] 刘锴[1] 庞盼娇[1] 王学理[1] 高健勇[1] 李晓燕[2] 张付贤[1] 王俊霞[1] 

机构地区：[1]吉林大学兽医畜牧学院,长春130062 [2]军事医学科学院军事兽医研究所5室,长春130062

出　　处：《中国生物制品学杂志》2007年第12期887-889,共3页Chinese Journal of Biologicals

基　　金：军事医学科学院新学科培育项目.

摘　　要：目的克隆布鲁氏菌BP26基因,并在大肠杆菌中高效表达及纯化BP26蛋白。方法提取布鲁氏菌基因组DNA,用PCR扩增出BP26基因,并克隆到pMD18-T载体上,测序正确后,再亚克隆至pET-28a(+)载体上,转化到大肠杆菌BL21(DE3)中,用IPTG诱导表达,经组氨酸结合树脂柱纯化,并对纯化后的表达产物进行Western blot鉴定。结果重组质粒pETBP26经Nde I与Sal I酶切后,可见大小分别为5400和700bp的片段,与理论值相符。转化大肠杆菌BL21(DE3)后,37℃诱导4h,经SDS-PAGE分析,高效表达出带有His-tag标签的融合蛋白BP26,纯化后的蛋白纯度达96%,经Western blot分析,目的蛋白具有抗原特异性。结论已成功克隆了BP26基因,且表达并纯化了布鲁氏菌BP26蛋白。Objective To clone the BP26 gene of B. abortus, highly express in E. coli and purify the expressed product. Methods Extract the genomic DNA of B. abortus ,amplify BP26 gene by PCR and clone to pMD18-T simple vector. Identify the constructed recombinant plasmid pMDBP26 by sequencing, then subclone to vector pET-28a（ ＋ ）. Transform the constructed recombinant plasmid pETBP26 to E. coli BL21 （DE3） for expression under induction of IPTG. Purify the expressed product by histidine-binding resin column chromatography and identify by Western blot. Results Two gene fragments at lengths of 5 400 and 700 bp respectively were obtained by digestion of recombinant plasmid pETBP26 with Nde I ＋ Sal I, which were consistent with the theoretical value. After the transformed E. coli BL21 （DE3）was induced at 37 ℃ for 4 h,SDS-PAGE proved that fusion protein BP26 with His-tag label was highly expressed.The fusion protein reached a purity of 96% after purification and showed antigenic specificity as proved by Western blot. Conclusion BP26 gene was successfully cloned and expressed, and the expressed prpduct was purified.

关 键 词：布鲁菌 BP26蛋白 基因克隆 原核表达 纯化 

分 类 号：Q786[生物学—分子生物学]