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目的:表达、纯化重组卡介苗热休克蛋白70(BCGHSP70)并去除其中的内毒素。方法:采用10L发酵罐经IPTG诱导表达含有重组表达质粒pET28a/HSP70的BL21(DE3)工程菌(pET28a/HSP70/BL21),SDS-PAGE检测BCGHSP70的表达。超声破碎菌体,裂菌后上清依次经过NiSepharose4B亲和层析、TritonX-114洗涤、SuperdexG-25凝胶过滤层析、Q-SepharoseFF离子交换层析进行纯化。SDS-PAGE鉴定纯化蛋白,Lowry法检测蛋白浓度,37℃水浴孵育0～4h检测纯化蛋白的稳定性,鲎试剂法检测内毒素含量。结果:工程菌pET28a/HSP70/BL21在发酵表达3h后获得96g湿菌,其中相对分子质量约为70000的蛋白约占菌体总蛋白量的29.6%;表达产物纯化后在相对分子质量约70000处可见特异性条带,该分子质量蛋白约占总蛋白量的96.5%;纯化后蛋白浓度约1.2g.L-1;37℃水浴中放置4h后相对分子质量为70000的蛋白约占总蛋白量的95.1%;纯化产物内毒素含量<0.01EU.μg-1。结论:成功表达、纯化了重组BCGHSP70,并有效地去除了其中的内毒素。
Objective: To express and purify the recombinant BCG heat shock protein 70 (BCGHSP70) and remove endotoxin. Methods: BL21 (DE3) engineered bacteria (pET28a / HSP70 / BL21) containing recombinant plasmid pET28a / HSP70 were induced by IPTG in 10 L fermenter. The expression of BCG HSP70 was detected by SDS-PAGE. The sonication medium and sonication supernatant were purified by NiSepharose 4B affinity chromatography, Triton X-114 washing, Superdex G-25 gel filtration chromatography and Q-Sepharose FF ion exchange chromatography. The purified protein was identified by SDS-PAGE. Lowry method was used to detect the protein concentration. The stability of the purified protein was detected by incubation for 0-4h in a 37 ° C water bath. The endotoxin content was determined by the reagent method. Results: 96h wet bacteria were obtained from the engineered bacteria pET28a / HSP70 / BL21 after fermentation for 3h, in which the protein with the relative molecular mass of about 70,000 accounted for about 29.6% of the total bacterial biomass. After purification, the expressed product was purified at a relative molecular mass of about 70,000 The molecular weight of the protein was about 96.5% of the total protein, and the concentration of the purified protein was about 1.2gL-1. The protein with a relative molecular mass of 70,000 was about 95.1 %; The purified product endotoxin content <0.01EU.μg-1. Conclusion: The recombinant BCGHSP70 was successfully expressed and purified, and endotoxin was effectively removed.