【摘要】： 銅綠假單胞菌是人類的機會致病菌,極少感染健康個體,但對免疫缺陷者,如燒傷、艾滋病、癌癥或囊性纖維化等患者則有較高的感染率。銅綠假單胞菌具有內(nèi)在的耐藥性,該菌的耐藥機制以及相關(guān)疫苗的開發(fā)一直以來都備受研究人員的重視。 本文運用蛋白質(zhì)組學(xué)特別是免疫蛋白質(zhì)組學(xué)的方法對銅綠假單胞菌的外膜蛋白進(jìn)行了研究,對其進(jìn)行分離和鑒定,在此基礎(chǔ)上分析了耐藥株外膜蛋白的表達(dá)變化以及銅綠假單胞菌外膜免疫原性蛋白。 首先,我們用月桂酰肌氨酸鈉的方法提取了銅綠假單胞菌的外膜蛋白,對其進(jìn)行了2-DE,并通過質(zhì)譜分析鑒定了其中26個外膜蛋白,構(gòu)建了銅綠假單胞菌外膜蛋白的分子表達(dá)圖譜,為下一步的研究奠定了基礎(chǔ)。 然后,通過藥物選擇獲得銅綠假單胞菌5種不同藥物的耐藥株,進(jìn)而對耐藥株的外膜蛋白質(zhì)組進(jìn)行系統(tǒng)比較,共鑒定出8種耐藥相關(guān)外膜蛋白質(zhì),包括紅霉素耐藥株1種,氯霉素和巴洛沙星耐藥株各有2種,頭胞曲松鈉耐藥株有4種,氨芐青霉素耐藥株有5種。在這8種變化的蛋白質(zhì)中,OprD為銅綠假單胞菌對氯霉素、氨芐青霉素、頭胞曲松鈉及巴洛沙星共同變化的蛋白質(zhì),OprF、OmpH1、MexA是銅綠假胞菌對氨芐青霉素和頭胞曲松鈉共同變化的蛋白質(zhì)。這種耐藥性研究的方法和規(guī)模在國內(nèi)外尚未見報道。 最后,我們應(yīng)用免疫蛋白質(zhì)組學(xué)的方法,即雙向電泳與Western blot相結(jié)合,繼之于MALDI-TOF-MS分析的方法,發(fā)現(xiàn)了銅綠假單胞菌外膜蛋白中的8個免疫原性蛋白質(zhì)。在對這8個蛋白進(jìn)行克隆表達(dá)及純化后,進(jìn)行小鼠免疫和攻毒實驗,發(fā)現(xiàn)了其中3個免疫保護(hù)作用較好,可望作為銅綠假單胞菌潛在的疫苗候選靶位。
[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).
【學(xué)位授予單位】：廈門大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R378

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王美珍;陳昌福;劉振興;譚晶晶;高宇;李革雷;;嗜水氣單胞菌對四環(huán)素類和氟喹諾酮類藥物的耐藥性研究[J];華中農(nóng)業(yè)大學(xué)學(xué)報;2011年01期

相關(guān)碩士學(xué)位論文 前1條

1 王美珍;耐藥嗜水氣單胞菌的外膜結(jié)構(gòu)和免疫原性變化的初步研究[D];華中農(nóng)業(yè)大學(xué);2011年

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本文編號：2137932