CAJ | 학술논문

目的研究一种有效去除内毒素并保留菌体抗原蛋白的纯化方法,以期为口腔中间普氏菌(Prevotella intermedia,Pi)蛋白菌苗研制提供参考.方法利用辛基苯基聚氧乙烯醚(Triton X-114)处理饱和硫酸铵沉淀的细菌蛋白成分,经4、37、25 ℃等不同温度分别处理,重复处理步骤并将所获得的最终蛋白产物通过蛋白质印迹法、鲎实验和小鼠动物实验分别在体内和体外实验中检测菌体蛋白含量及内毒素去除水平.结果利用Triton X-114萃取法处理Pi 3个循环后,蛋白质印迹法、鲎实验证明超过99.99％的细菌内毒素被剔除,同时超过90％的细菌主要蛋白成分得以保留；体外细胞刺激实验及小鼠动物实验均显示处理组产物刺激细胞因子的表达水平均显著低于未处理组(P＜0.05).结论用Triton X-114萃取法去除内毒素的方法去除Pi内毒素效果得到验证,为其菌苗大规模制备工艺提供了技术选择.
목적 연구일충유효거제내독소병보류균체항원단백적순화방법,이기위구강중간보씨균(Prevotella intermedia,Pi)단백균묘연제제공삼고.방법 이용신기분기취양을희미(Triton X-114)처리포화류산안침정적세균단백성분,경4、37、25 ℃등불동온도분별처리,중복처리보취병장소획득적최종단백산물통과단백질인적법、후실험화소서동물실험분별재체내화체외실험중검측균체단백함량급내독소거제수평.결과 이용Triton X-114췌취법처리Pi 3개순배후,단백질인적법、후실험증명초과99.99％적세균내독소피척제,동시초과90％적세균주요단백성분득이보류；체외세포자격실험급소서동물실험균현시처리조산물자격세포인자적표체수평균현저저우미처리조(P＜0.05).결론 용Triton X-114췌취법거제내독소적방법거제Pi내독소효과득도험증,위기균묘대규모제비공예제공료기술선택.
Objective To investigate an effective purification method for removing endotoxin from Prevotella intermedia.Methods The main protein ingredients of bacteria prepared from ammonium sulfate precipitation were further treated with octyl phenyl polyoxyethylene ether(Triton X-114),and then processed at 4 ℃,37 ℃ and 25 ℃.The obtained aqueous phase after at least two more cycle repeated operations was assayed for endotoxin by Western blotting,LAL-clotting method,in vitro cell stimulation and in vivo animal experiments.Results Western blotting and LAL-clotting method demonstrated that the reduction in endotoxin level was greater than 99.99％ and recovery of the proteins after endotoxin removal was greater than 90％ with Triton X-114 treatment for 3 cycles.The cytokines expression level was lower in both in vitro cell stimulation and in vivo animal experiments than in untreated group(P ＜0.05).Conclusions The extraction method provides a new choice for endotoxin removal from large volumes of the oral Prevotella intermedia.