CAJ | 학술논문

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TcdB基因并克隆至穿梭载体pHis1522.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5～10 mg/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.
목적 획득고순도화구유생물활성적중조장독소B(rTcdB).방법 이간난사상아포간균염색체DNA위모판,통과PCR방법확증득도전장TcdB기인병극륭지천사재체pHis1522.구건적질립경직접측서험증무오후,전화거대아포간균원생질체,재목당적유도작용하진행TcdB적표체、순화급생물활성감정.결과 종세균배양액중순화득도rTcdB,농도체도5～10 mg/L,기상대분자질량여천연적TcdB단백접근,생물활성여천연적TcdB단백상사.결론 재거대아포간균중성공표체료구유완정결구화활성적간난사상아포간균TcdB중조단백.
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 mg 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 megaterium.