Mycobacterium marinum is an opportunistic pathogen which can infect 150 kinds of aquatic animals. In this paper,the same bacteria strain FZ09 was isolated from the mucus,dorsal fins and gills of Sebastods schlegeli, and was identified as Mycobacterium marinum by physiological, biochemical characterization and heat shock protein 65 DNA sequence analysis. Lipopolysaccharide (LPS ) was extracted from strain FZ09 by phenol-chloroform-methanol method, ethanol-phenol-water method and hot phenol-water method, respectively, and the composition and antigenic differences of the extracted LPS were analyzed by SDS-PAGE, Western blotting and ELISA. The results showed that LPS extracted by three methods shared the same compositions and the main bands had molecular weights of 14,16 and 23 ku. LPS extracted by ethanol- phenol method had the highest purity and concentration, and LPS extracted by traditional hot phenol water method had the most bands and best antigenicity. Western-blotting showed that anti-FZ09 antiserum mainly reacted with 14 ku band of the LPS, which showed a negative reaction after mild treatment with periodate except for LPS extraction by traditional hot phenol-water method that demonstrated weak positive. ELISA revealed that reactivity of anti-FZ09 antiserum and LPS decreased after periodate oxidation and OD405 reduced significantly.%从许氏平鲉粘液、背鳍、鳃等部位分离筛选到同一菌株FZ09,经形态学观察、生理生化特性及热激蛋白65( HSP65)基因序列分析,鉴定菌株FZ09为海分枝杆菌.分别用酚-氯仿-甲醇法、乙醇-酚水法和传统热酚水法提取菌株FZ09的脂多糖(LPS),分析了LPS的组成和抗原性差异.SDS-PAGE结果表明,3种方法提取的LPS条带分布基本相同,主要条带分子量分别为14、16和23ku,其中以乙醇-酚水法提取的LPS含量和纯度最高,热酚水法提取的LPS条带最多,抗原性最好.Western-blotting显示,海分枝杆菌抗血清主要与LPS的14 ku条带发生特异性免疫反应.高碘酸氧化免疫印迹结果表明,仅传统热酚水法提取的LPS在14 ku分子量区域有弱的阳性条带出现,其他方法提取LPS与抗血清的免疫反应呈阴性.高碘酸氧化ELISA反应显示,LPS经高碘酸氧化后与海分枝杆菌FZ09抗血清的反应活性降低,OD405明显减弱.
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