文档介绍：结核分枝杆菌Ag85B/IL
【关键词】 Ag85B;IL
Prokaryotic expression, purification and identification of Mycobacterium tuberculosis Ag85B/IL2 fusion protEin and detection of its biological activity
【Abstract】 AIM: To express Mycobacterium tuberculosis Ag85B/ IL2 fusion protEIn in , to purify and identify it, and to detect its biological activity. METHODS: By using geic engineering techniques, id pGEXAg85B/IL2, and expressed Ag85B/IL2GST fusion protein in BL21 under the induction of IPTG. Moreover, al expression conditions concerning IPTG concentration and induction time. After renatured by urea in a concentration gradient, and purified by GSTSepharose affinity chromatography and digested by thrombin, the Ag85B/IL2 fusion protein atography and RPHPLC and identified inal amino acid sequence and IL2 bioactivity easured as atography, anionexchange chromatography and RPHPLC, the desired Ag85B/IL2 fusion protein ed by SF)重悬,加入1 g溶菌酶,冻溶3次,冰浴中超声破菌, 4℃ 8000 r/min离心20 min,收集沉淀. 用2 mol/L尿素反复洗涤包涵体后,溶于变性液(8 mol/L尿素,2 mmol/L β巯基乙醇,10 mmol/L PBS pH ), 以磁力搅拌器轻柔搅拌,放置于4℃冰箱溶解过夜(12 h), 次日以15000 r/min 离心20 min,吸取上清,装入透析袋以4 mol/L尿素,2 mol/L尿素,1 mol/L尿素,和20 mmol/L PBS(pH )逐步透析,透析完成后收集透析袋中蛋白溶液,浓缩后-20℃贮存备用.
2b,柱床体积50 mL. 样品流速10 mL/min,10 mmol/L谷胱甘肽GSH溶液洗脱,收集蛋白峰. 将重组蛋白样品用10 mmol/L PBS (pH )稀释至1 mg/mL, u混匀,室温下作用5 h. 取20 μL