辛基苯基聚氧乙烯醚萃取法去除口腔中间普氏菌内毒素的研究  

Removal of oral Prevotella intermedia endotoxin by octyl phenyl polyoxyethylene ether extraction method

作　　者：王爱武[1] 刘岩[1] 胡孔新[2] 程茜[1] 

机构地区：[1]首都医科大学附属北京朝阳医院口腔科,100020 [2]中国检验检疫科学研究院卫生检疫研究所

出　　处：《中华口腔医学杂志》2012年第12期725-729,共5页Chinese Journal of Stomatology

基　　金：国家自然科学基金（30500455）;“十二五”国家科技重大专项（2011ZX10004-001）

摘　　要：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（TritonX-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TritonX-114萃取法处理Pi3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TritonX-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。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.

关 键 词：普里沃菌 中间 内毒素类 免疫印迹法 酶联免疫吸附测定 

分 类 号：O657.3[理学—分析化学]