重组卡介苗热休克蛋白70的纯化及内毒素去除的效果评价

Purification of recombinant BCG heat shock protein 70 and evaluation of effect on endotoxin removal

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 （BCG HSP70）, and remove endotoxin from it. Methods E. coll. BL21 （DE3） containing the recombinant plasmid of pET28a/HSP70 （pET28a/HSP70/BL21） was induced with IPTG in 10 L fermentor, and the expression of BCG HSP70 was detected by SDS-PAGE. Then the bacteria were disrupted by sonication. BCG HSP70 was purified by successive applications of Nikel-affinity chromatography on Sepharose 4B, TritonX-114 washing, gel filtration on Sephadex G-25 and ion-exchange chromatography on Q-Sepharose FF. The purified BCG HSP70 was identified by SDS- PAGE. The concentration of the purified BCG HSP70 was detected by Lowry assay. And the purified BCG HSP70 was incubated in the 37 ℃ water bath for 0 to 4 h to analyze its stability. Endotoxin in the purified proteins was determined by Limulus amebocyte lysate assay. Results 96 g wet weight of bacterial pellet was obtained after 3 h of induction approximatel 70 000 was incubated in of pET28a/HS y accounted for purified to 96. the 37 ℃ water P70/BL21 in the fermentor, and the protein with relative molecular mass of about 29.6% of the total bacterial protein. The protein with relative 5% purity, and the concentration of the purified protein was 1.2 bath for 4 h, the BCG HSP70 accounted for approximately 95.1% of molecular g·L^-1. 70 000 mass of When the total protein. Endotoxin in the purified protein was less than 0.01 EU ·μg^- 1. Conclusion The recombinant BCG HSP70 is expressed and purified successfully, the endotoxin in the purified BCG HSP70 is removed effectively.