Shotgun proteomic profiling of Yersinia ruckeri strains

Project goal is to identify proteomic profiles of Yersinia ruckeri, the causative agent of enteric redmouth disease in fish. Four strains (SP-05, CSF007-82, 7959-11 and YRNC-10) of Y. ruckeri were isolated from disease rainbow trout, Oncorhynchus mykiss. Strains, SP-05 and CSF007-82, belong to serotype 1 and biotype 1 (motile and lipase positive), while strains 7959-11 and YRNC-10 belong to serotype 1 and biotype 2 (non-motile and lipase negative) and belong to serotype 1. A single colony of each strain was inoculated into tryptic soy broth (Casein peptone, dipotassium hydrogen phosphate, glucose, sodium chloride, soya peptone, Sigma) and grown at 22 °C with shaking (150 rpm). These starter cultures were then diluted with fresh sterile tryptic soy broth to an optical density (OD 600) of 0.10 ± 0.05. Five hundred microlitres of the diluted starter cultures were inoculated in duplicates, into 25 ml of tryptic soy broth. Cultures were grown overnight at 22 °C with shaking (150 rpm) until the late log phase. Cells were harvested by centrifugation, then washed three times with PBS.

本项目旨在鉴定耶尔森氏菌（Yersinia ruckeri）的蛋白质组学特征，该菌是鱼类肠红嘴病的致病菌。本研究从患病虹鳟（Oncorhynchus mykiss）中分离得到4株耶尔森氏菌，编号依次为SP-05、CSF007-82、7959-11及YRNC-10。其中SP-05与CSF007-82属于血清型1、生物型1（具有运动性且脂酶阳性）；7959-11与YRNC-10属于血清型1、生物型2（无运动性且脂酶阴性）。挑取各菌株的单菌落接种至胰蛋白胨大豆肉汤（Tryptic Soy Broth，TSB；成分：酪蛋白胨、磷酸氢二钾、葡萄糖、氯化钠、大豆蛋白胨，购自Sigma公司）中，于22℃、150 rpm条件下振荡培养。随后用新鲜无菌的胰蛋白胨大豆肉汤将上述种子培养液稀释至光密度（OD600）为0.10±0.05。取500微升稀释后的种子培养液，以双复孔形式接种至25 ml胰蛋白胨大豆肉汤中，于22℃、150 rpm条件下振荡培养过夜，至对数生长晚期。通过离心收集菌体，并用磷酸盐缓冲液（Phosphate Buffered Saline，PBS）洗涤三次。