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Alternative TitleProteome of bacterium and its major infected fish in China: extracellular and outer-membrane proteins of Flavobacterium columnare and gill mucus of Ctenopharyngodon idellus
Thesis Advisor聂品
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword柱状黄杆菌 毒力因子 胞外蛋白 外膜蛋白 草鱼 鳃粘液 蛋白质组
Abstract柱状黄杆菌 (Flavobacterium columnare) 引起的柱形病是一种全球性的危害多种鱼类的细菌性鱼病,在我国流行广且危害严重的细菌性烂鳃病的病原就是柱状黄杆菌。本研究以柱状黄杆菌和草鱼 (Ctenopharyngodon idellus) 为对象,以蛋白质组学技术为手段,用比较蛋白质组学的方法研究了柱状黄杆菌强毒株和弱毒株差异表达的胞外蛋白,研究了柱状黄杆菌外膜蛋白和草鱼鳃粘液的蛋白质组。 浸泡攻毒实验表明F. columnare G4R3 和G18分别是强毒株和弱毒株,对数生长后期的G4R3 和G18 胞外蛋白的双向电泳图谱显示出34个差异表达的蛋白点,利用串联质谱分析,7个蛋白点代表滑动蛋白K,腺酐甲硫氨酸合成酶和假定的膜蛋白等3个蛋白,它们可能是柱状黄杆菌的毒力因子。 柱状黄杆菌的外膜蛋白 (outer membrane proteins, OMPs) 经SDS-PAGE初步分离,用鸟枪法蛋白质组学分析方法,鉴定出757个蛋白,其中有110个蛋白得到可信鉴定。在这757个蛋白中,有30个与本实验室以前利用抗OMPs抗体筛选到的编码与膜相关的抗原性蛋白相同,如lipid A disaccharide syntheatase, outer membrane receptor proteins, alpha-amylase, glucosamine N-aclrytransferase, K+-transporter, aminopeptidase 和 aspartate 1-decarboxylase等。 草鱼鳃粘液经过凝胶过滤层析结合传统的双向凝胶电泳,共分离了406个蛋白点,通过质谱分析,鉴定了其中的99个蛋白点。其中,凝集素样蛋白、转铁蛋白、蛋白酶体、组蛋白样蛋白等在鱼类的免疫反应中可能起着重要作用。 综上所述,本研究用比较蛋白质组学的方法鉴定了胞外蛋白中3个可能的毒力相关蛋白。用鸟枪法蛋白质组学方法获得了柱状黄杆菌外膜蛋白的全蛋白表达谱,用双向电泳结合质谱分析鉴定了草鱼鳃粘液中的99个蛋白点。为认识柱状黄杆菌的毒力因子及进一步研究 OMPs 的功能以及筛选保护性抗原、毒力相关的蛋白以及疫苗的研制奠定了基础,为了解鱼类鳃粘液的组成成分以及鱼类粘膜免疫的组成与作用奠定了基础。
Other AbstractThe bacterium Flavobacterium columnare is a world-wide pathogen causing columnaris disease and a pathogen in China causing bacterial gill-rot disease, a serious disease in several major cultured fish species including the grass carp Ctenopharyngodon idellus. The present study aimed to characterize the proteomic profiles of outer membrane proteins (OMPs) of F. columnare and gill mucus of grass carp. In addition, a comparative proteomic analysis was carried out to analyze extracellular proteins (ECPs) of virulent and avirulent strains of the bacterium. Strains G4R3 and G18 of F. columnare were assessed to be virulent and avirulent, respectively, by immersion challenge. To identify the virulence factors of F. columnare, comparative proteomic analysis by two-dimensional polyacrylamide gel electrophoresis (2-DE) in combination with mass spectrometry and database search was conducted to analyze extracellular proteins of strains G4R3 and G18 at the late exponential growth phase. 34 differential expression spots were revealed by image analysis of 2-DE gels and further analyzed using in-gel tryptic digestion, peptide mass fingerprinting and tandem mass spectrometry. Seven spots representing three proteins, namely GldK, s-adenosylmethionine synthetase and putative membrane protein were identified as possible virulence-related factors in the virulent strain G4R3. Outer membrane proteins of F. columnare were subjected to fractionation by SDS-PAGE, and then shotgun proteomic analysis. A total of 757 proteins were recognized, among which 110 proteins were identified with high-confidence. 30 proteins, which were previously characterized as products of genes encoding OMPs of F. columnare by screening the expression library of the bacterium, were also present in the 757 proteins. The traditional two-dimensional ployacrylamide gel electrophoresis was employed, in combination with gel filtration chromatography as a third dimension, to separate gill mucus of healthy grass carp. It was revealed that 406 spots were present with isoelectric points between about 4 and 7, among which 99 spots were identified using mass spectrometric analysis, such as lectin-like proteins, transferrin, proteasome, histone-like proteins. These identified proteins may be involved in immune response of the fish.
Document Type学位论文
Recommended Citation
GB/T 7714
刘国勇. 柱状黄杆菌胞外和外膜蛋白及草鱼鳃粘液的蛋白质组学研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2006.
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