维氏气单胞菌浮游态和生物被膜态差异蛋白的筛选及ΔglpD的构建
发布时间：2018-12-13 07:09
【摘要】：维氏气单胞菌(Aeromonas veronii,AV)是一种人-兽-鱼共患病原菌,由于近年来分离率逐渐升高,已逐渐引起人们的广泛关注与重视。有研究报道称细菌在环境中大多以生物被膜形式存在,该形式是一种同浮游态相对应的生长方式,在一定程度上增强了细菌对外界环境的抵抗力,而且细菌生物被膜态可释放游离菌从而导致感染的迁延不愈和反复发作。若是能够有效预防和控制生物被膜的感染,将会充分降低细菌致病的几率,这在临床感染治疗中有着十分重要的意义。因此,筛选维氏气单胞菌浮游态与生物被膜态间的差异蛋白,在探讨维氏气单胞菌生物被膜形成机理以及抑制生物被膜形成的用药靶点的筛选等方面具有一定意义。本研究以维氏气单胞菌TH0426为研究对象,首先应用96孔微量板定量检测法探究在不同的条件下体外生物被膜形成能力,并对其条件进行优化。结果显示,维氏气单胞菌TH0426在LB、THB以及TSB培养基中培养,生物被膜形成能力差异不显著;培养基pH为6、Nacl浓度6g/L放置于20℃培养箱中培养24h时,生物被膜形成能力较佳。同时运用扫描电子显微镜进一步检测了生物被膜形成情况。在此基础上,应用iTRAQ技术分析浮游态与生物被膜态下维氏气单胞菌TH0426蛋白表达的差异,并应用MRM技术进行验证。结果显示,浮游态细菌与生物被膜态下细菌相比,蛋白表达存在一定的差异,共获得277个差异蛋白,其中上调蛋白130个,下调蛋白147个,同时对这些蛋白进行了初步的生物信息学分析。本研究又挑选5个差异蛋白进行MRM验证;同时随机挑选上调和下调蛋白共20个,应用荧光定量PCR进行验证,结果与iTRAQ结果相同。研究最后选出上调蛋白基因glpD,利用同源重组技术构建维氏气单胞菌glpD基因缺失株,并通过比较野生株、缺失株的生物被膜形成能力:发现glpD基因缺失株的生物被膜的形成能力较野生株有所下降。应用K-B纸片法检测缺失株与野生株耐药情况,变化不显著。研究结果有助于从蛋白水平上深化维氏气单胞菌生物被膜免疫机理及致病机理的研究,并为其在疫苗生产及药物研发等发面提供科学依据。
[Abstract]:Aeromonas Vickers (Aeromonas veronii,AV) is a zoonotic pathogen, which has attracted more and more attention due to the increasing isolation rate in recent years. It has been reported that bacteria mostly exist in the form of biofilm in the environment, which is a growth mode corresponding to the planktonic state, which to some extent enhances the resistance of bacteria to the outside environment. Moreover, bacterial biofilm can release free bacteria and lead to prolonged infection and repeated attacks. If the biofilm infection can be effectively prevented and controlled, the probability of bacterial pathogenicity will be reduced, which is of great significance in the treatment of clinical infection. Therefore, the screening of differential proteins between planktonic and biofilm states of Aeromonas Vickers has a certain significance in exploring the mechanism of biofilm formation of Aeromonas Vickers and the screening of drug targets for inhibiting biofilm formation. In this study, the ability of biofilm formation in vitro under different conditions was investigated by 96 hole microplate quantitative detection method with Aeromonas Vickers TH0426 as the research object, and the conditions of the biofilm formation were optimized. The results showed that the ability of biofilm formation of Aeromonas Vickers TH0426 cultured in LB,THB and TSB medium was not significant. The ability of biofilm formation was better when the culture medium pH was 6g/L with a concentration of 6 渭 mol / L, and the culture medium was cultured in a incubator at 20 鈩，
本文编号：2376128
[Abstract]:Aeromonas Vickers (Aeromonas veronii,AV) is a zoonotic pathogen, which has attracted more and more attention due to the increasing isolation rate in recent years. It has been reported that bacteria mostly exist in the form of biofilm in the environment, which is a growth mode corresponding to the planktonic state, which to some extent enhances the resistance of bacteria to the outside environment. Moreover, bacterial biofilm can release free bacteria and lead to prolonged infection and repeated attacks. If the biofilm infection can be effectively prevented and controlled, the probability of bacterial pathogenicity will be reduced, which is of great significance in the treatment of clinical infection. Therefore, the screening of differential proteins between planktonic and biofilm states of Aeromonas Vickers has a certain significance in exploring the mechanism of biofilm formation of Aeromonas Vickers and the screening of drug targets for inhibiting biofilm formation. In this study, the ability of biofilm formation in vitro under different conditions was investigated by 96 hole microplate quantitative detection method with Aeromonas Vickers TH0426 as the research object, and the conditions of the biofilm formation were optimized. The results showed that the ability of biofilm formation of Aeromonas Vickers TH0426 cultured in LB,THB and TSB medium was not significant. The ability of biofilm formation was better when the culture medium pH was 6g/L with a concentration of 6 渭 mol / L, and the culture medium was cultured in a incubator at 20 鈩，
本文编号：2376128
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