肺炎克雷伯菌Ⅲ型菌毛的提取与鉴定

将表达Ⅲ型菌毛的肺炎克雷伯菌临床分离株Kp7在改良Minka液体培养基上传代培养,使之充分菌毛化,大量收集菌毛生长良好的细菌,利用热洗脱法分离提取菌毛,经硫酸铵沉淀、透析后进行蔗糖密度梯度离心,收集20%~30%梯度中的蛋白带,经SDS-PAGE电泳可见1条大小在20.5 ku的蛋白条带。提纯菌毛保留了甘露糖抵抗血凝特性,并与用Kp7全菌免疫家兔制备的高免血清在Western blot中反应呈阳性,证实其条带为Ⅲ型菌毛主要结构蛋白。

Extraction and identification of type 3 fimbriae of Klebsiella pneumoniae

A Klebsiella pneumoniae isolate Kp7 that possesses type 3 fimbriae was cultured and passaged in modified Minka liquid medium for sufficient formation of fimbriae.The fimbriae well-expressed bacteria were collected for fimbriae preparation by using heat extraction(HE) method and then to be precipitated with ammonium sulphate and dialyzed.The precipitate was performed by surcose density gradient centrifugation and the fraction containing fimbriae between 20%~30% of sucrose gradients were collected.The purified protein preparation showed a single fragment with an approximate molecular weight of 20.5 ku in the gel after SDS-PAGE,and displayed a ability to bind the erythrocyte in a mannose-resistant fashion as well as showed a positive reaction in Western Blot assay when acting with the rabbit antiserum against whole cell of the isolate Kp7.These results revealed that the preparation was the major structural protein of type 3 fimbriae of K.pneumoniae.