铜绿假单胞菌生物膜形成相关的蛋白质组学研究

发布时间：2018-05-26 10:03

本文选题：铜绿假单胞菌 + 蛋白质组学　；参考：《广西医科大学》2007年硕士论文

【摘要】： 目的:找出铜绿假单胞菌野生型PAO1与黏液型PDO300菌株、PAO1lasI rhlI基因缺陷型菌株、PAO1 lasR rhlR基因缺陷型菌株之间的差异蛋白质,探讨这些蛋白质与铜绿假单胞菌生物膜形成的关系。方法:(1)将同一份样品配制成不同蛋白浓度的标本分别上样检测,对检测所得蛋白质图谱进行分析。(2)根据最佳检测蛋白浓度,将黏液型PDO300、野生型PAO1、PAO1 lasI rhlI基因缺陷型和PAO1 lasR rhlR基因缺陷型菌株的菌体蛋白分别采用CM10、IMAC3-Cu这两种蛋白质芯片,运用SELDI技术进行测定,筛选最佳蛋白质芯片。(3)利用SELDI技术对黏液型PDO300、野生型PAO1、PAO1 lasI rhlI基因缺陷型和PAO1 lasR rhlR基因缺陷型菌株的菌体蛋白分别进行检测,并采用Biomarker Wizard软件进行分析。结果:(1)检测蛋白浓度为1.5μg/μl时,进行SELDI检测可发现较多的蛋白峰。(2)同样检测蛋白浓度时,CM10芯片捕获的蛋白峰数量比IMAC3-Cu芯片多。(3)铜绿假单胞菌野生型PAO1与黏液型PDO300菌株的菌体蛋白比较,有16个蛋白峰存在差异(P＜0.01)。与野生型PAO1菌株相比,PDO300菌株中有10个蛋白质高表达,分子量分别为4,074Da、3,817Da、8,923Da、8,148Da、3,332Da、4,521Da、7,621Da、11,594Da、7,831Da和13,751Da;有6个蛋白质低表达,分子量分别为6,199Da、6,899Da、12,379Da、6,115Da、6,763Da和8,576Da。(4)铜绿假单胞菌PAO1 lasI rhlI基因缺陷型与野生型PAO1菌株相比有1个蛋白质低表达(P＜0.05),分子量为6,910Da。(5)铜绿假单胞菌PAO1 lasR rhlR基因缺陷型与野生型PAO1菌株之间有4个蛋白峰存在差异(P＜0.05)。在PAO1 lasR rhlR基因缺陷型菌株中有2个蛋白质高表达,分子量分别为8,924Da和8,790Da:有2个蛋白质低表达,分子量分别为6,735Da和6,986Da。结论:(1)运用SELDI技术对细菌菌体蛋白进行分析的最佳检测蛋白浓度为1.5μg/μl。 (2)与IMAC3-Cu芯片相比,CM10芯片更适于细菌菌体蛋白质组学的研究。 (3)铜绿假单胞菌野生型PAO1与黏液型PDO300的菌体蛋白有16个蛋白质表达明显不同,这些差异蛋白可能与铜绿假单胞菌的表型和生物膜形成有关。 (4)铜绿假单胞菌野生型PAO1与PAO1 lasI rhlI基因缺陷型菌株之间有1个蛋白质表达不同,野生型PAO1与PAO1 lasR rhlR基因缺陷型菌株之间有4个蛋白质表达不同,这些蛋白质可能与QS系统调控生物膜形成有关。 (5)用蛋白质芯片和SELDI技术对铜绿假单胞菌进行蛋白质组学研究,方法简便、敏感性高,可发现一系列传统研究手段难以测得的低分子量或低丰度的蛋白质。
[Abstract]:Objective: to find out the differential proteins between wild-type PAO1 of Pseudomonas aeruginosa and PAO1lasI rhlI gene deficient strain of PAO1lasI rhlI gene, and to explore the relationship between these proteins and biofilm formation of Pseudomonas aeruginosa. Methods 1) the same sample was prepared into samples with different protein concentrations, and the protein profiles were analyzed. 2) according to the optimum protein concentration, The bacterial proteins of myxotypic PDO300, wild type PAO1, PAO1 lasI rhlI gene deficient type and PAO1 lasR rhlR gene deficient type strain were determined by SELDI technique by using CM10 / IMAC3-Cu protein chip, respectively. Screening the best protein microarray, using SELDI technique to detect myxotypic PDO300, wild type PAO1 PAO1 lasI rhlI gene deficient type and PAO1 lasR rhlR gene deficient type strain respectively, and to analyze them by Biomarker Wizard software. Results when the concentration of protein was 1.5 渭 g / 渭 l, The number of protein peaks captured by CM10 microarray was more than that of IMAC3-Cu chip by SELDI detection. There were 16 protein peaks of Pseudomonas aeruginosa wild-type PAO1 compared with myxic-type PDO300 strains (P < 0.01). Compared with wild-type PAO1 strains, 10 proteins were highly expressed, and their molecular weights were 4074 Da3817Dan8923Daan8148Da31332Dax4521Daf7621Da7594Da77831Da and 13751Da.There were 6 proteins that were low expressed. The molecular weight of the PAO1 lasI rhlI gene deficiency type of Pseudomonas aeruginosa was 6199 Daxin6899Daf12379Daji6115Da6763Da and 8576Da.Y4) there was a protein low expression between the PAO1 lasI rhlI gene deficient type of Pseudomonas aeruginosa and the wild type PAO1 strain (P < 0.05), and the molecular weight was 6910 Da. 5) between the PAO1 lasR rhlR gene deficiency type and the wild type PAO1 strain of Pseudomonas aeruginosa. There were four protein peaks (P < 0.05). Two proteins were highly expressed in PAO1 lasR rhlR gene deficient strains with molecular weight of 8924Da and 8790 Da, and two proteins were low expressed, with molecular weight of 6735Da and 6986 Da, respectively. ConclusionThe optimal concentration of SELDI for the analysis of bacterial protein is 1.5 渭 g / 渭 l. Compared with IMAC3-Cu chip, CM10 chip is more suitable for proteomics of bacteria. (3) the expression of 16 proteins in wild-type PAO1 and myxotypic PDO300 of Pseudomonas aeruginosa was significantly different, which might be related to the phenotype and biofilm formation of Pseudomonas aeruginosa. 1 protein expression was different between wild-type PAO1 and PAO1 lasI rhlI gene deficient strains of Pseudomonas aeruginosa, and 4 proteins were different between wild-type PAO1 and PAO1 lasR rhlR gene deficient strains. These proteins may be related to QS system regulating biofilm formation. The proteomics of Pseudomonas aeruginosa was studied by using protein chip and SELDI technique. The method was simple and sensitive. A series of low molecular weight or low abundance proteins could be found by traditional methods.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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