艰难梭状杆菌肠毒素B在巨大芽孢杆菌中的表达和鉴定  

Expression and identification of recombinant Clostridium difficile toxin B using Bacillus megaterium system

作　　者：杨桂林[1] 刘威龙[1] 姚红艳[1] 周伯平[1] 冯汉平 

机构地区：[1]深圳市第三人民医院感染科,深圳 518020 [2]美国Tufts大学Cummings兽医学院

出　　处：《中华传染病杂志》2011年第1期1-5,共5页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金资助项目（NSF30972603）;美国国际发明专利（w02009/139919A2）

摘　　要：目的获得高纯度和具有生物活性的重组肠毒素B（rTcdB）。方法以艰难梭状芽孢杆菌染色体DNA为模板，通过PCR方法扩增得到全长TedB基因并克隆至穿梭载体pHisl522。构建的质粒经直接测序验证无误后，转化巨大芽孢杆菌原生质体，在木糖的诱导作用下进行TcdB的表达、纯化及生物活性鉴定。结果从细菌培养液中纯化得到rTcdB，浓度达到5～10mg／L，其相对分子质量与天然的TcdB蛋白接近，生物活性与天然的TcdB蛋白相似。结论在巨大芽孢杆菌中成功表达了具有完整结构和活性的艰难梭状芽孢杆菌TcdB重组蛋白。Objective To express and purify recombinant and biologically active Clostridium difficile toxin B （rTcdB）. Methods The genes of TcdB were amplified by polymerase chain reaction （PCR） using chromosomal DNA from a toxigenic strain, and cloned into a shuttle vector pHis1522. The sequences of TcdB genes in the vector were verified by DNA sequencing. The construction was transformed into Bacillus megaterium protoplasts and the protein expression was driven by a xylose promoter. The purified protein was tested for biological activity. Results rTcdB was successfully purified from bacterial crude extracts. Approximately 5--10 nag of highly purified recombinant toxin was obtained from one liter of bacterial culture. The expressed rTcdB had molecular mass similar to the native toxin, and its biological activity was proved to be similar to its native counterpart after an extensive examination. Conclusion rTcdB with biological activities is successfully expressed in Bacillus megateriurn.

关 键 词：梭菌 难辨 芽胞杆菌属 肠毒素类 基因表达 质粒 重组蛋白质类 

分 类 号：R378[医药卫生—病原生物学]