铜绿假单胞菌外膜耐药和保护原功能蛋白组学的研究

发布时间：2018-07-22 15:37

【摘要】： 铜绿假单胞菌是人类的机会致病菌,极少感染健康个体,但对免疫缺陷者,如烧伤、艾滋病、癌症或囊性纤维化等患者则有较高的感染率。铜绿假单胞菌具有内在的耐药性,该菌的耐药机制以及相关疫苗的开发一直以来都备受研究人员的重视。本文运用蛋白质组学特别是免疫蛋白质组学的方法对铜绿假单胞菌的外膜蛋白进行了研究,对其进行分离和鉴定,在此基础上分析了耐药株外膜蛋白的表达变化以及铜绿假单胞菌外膜免疫原性蛋白。首先,我们用月桂酰肌氨酸钠的方法提取了铜绿假单胞菌的外膜蛋白,对其进行了2-DE,并通过质谱分析鉴定了其中26个外膜蛋白,构建了铜绿假单胞菌外膜蛋白的分子表达图谱,为下一步的研究奠定了基础。然后,通过药物选择获得铜绿假单胞菌5种不同药物的耐药株,进而对耐药株的外膜蛋白质组进行系统比较,共鉴定出8种耐药相关外膜蛋白质,包括红霉素耐药株1种,氯霉素和巴洛沙星耐药株各有2种,头胞曲松钠耐药株有4种,氨苄青霉素耐药株有5种。在这8种变化的蛋白质中,OprD为铜绿假单胞菌对氯霉素、氨苄青霉素、头胞曲松钠及巴洛沙星共同变化的蛋白质,OprF、OmpH1、MexA是铜绿假胞菌对氨苄青霉素和头胞曲松钠共同变化的蛋白质。这种耐药性研究的方法和规模在国内外尚未见报道。最后,我们应用免疫蛋白质组学的方法,即双向电泳与Western blot相结合,继之于MALDI-TOF-MS分析的方法,发现了铜绿假单胞菌外膜蛋白中的8个免疫原性蛋白质。在对这8个蛋白进行克隆表达及纯化后,进行小鼠免疫和攻毒实验,发现了其中3个免疫保护作用较好,可望作为铜绿假单胞菌潜在的疫苗候选靶位。
[Abstract]:Pseudomonas aeruginosa is a opportunistic pathogen in human being and rarely infects healthy individuals, but it has a high infection rate in patients with immune deficiency such as burn, AIDS, cancer or cystic fibrosis. Pseudomonas aeruginosa has inherent drug resistance, the drug resistance mechanism and the development of related vaccines have been paid attention to by researchers. In this paper, the outer membrane proteins of Pseudomonas aeruginosa were studied by proteomics, especially immune proteomics, and the outer membrane proteins of Pseudomonas aeruginosa were isolated and identified. The expression of outer membrane protein and the immunogenicity of Pseudomonas aeruginosa were analyzed. Firstly, the outer membrane protein of Pseudomonas aeruginosa was extracted by sodium lauroyl sarcosine, 2-DE. 26 of them were identified by mass spectrometry, and the molecular expression map of outer membrane protein of Pseudomonas aeruginosa was constructed. It lays a foundation for the next research. Then, five drug resistant strains of Pseudomonas aeruginosa were obtained by drug selection, and then the proteome of the resistant strains were systematically compared. Eight kinds of resistance related outer membrane proteins, including one erythromycin resistant strain, were identified. Chloramphenicol and balfloxacin resistant strains were 2, cephaltriaxone sodium resistant and ampicillin resistant were 4 and 5, respectively. OprD was the protein of Pseudomonas aeruginosa p-chloramphenicol, ampicillin, cephaltriaxone sodium and balofloxacin. OprFN OmpH1MexA was the protein of Pseudomonas aeruginosa to ampicillin and cephaltriaxone sodium. The method and scale of drug resistance research have not been reported at home and abroad. Finally, we found eight immunogenicity proteins in the outer membrane protein of Pseudomonas aeruginosa by two dimensional electrophoresis combined with Western blot, followed by MALDI-TOF-MS. After cloning, expression and purification of the eight proteins, the mice were immunized and purified, and three of them were found to have good immuno-protective effects, which are expected to be potential vaccine candidate targets for Pseudomonas aeruginosa (Pseudomonas aeruginosa).
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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