本文選題：結(jié)核分枝桿菌 + 非結(jié)核分枝桿菌�。� 參考：《重慶醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的:研究分枝桿菌菌體蛋白表達(dá),篩選特征性蛋白,研究結(jié)核分枝桿菌和非結(jié)核分枝桿菌分泌蛋白表達(dá),篩選差異蛋白。 方法:1.培養(yǎng)分枝桿菌標(biāo)準(zhǔn)菌株,滅活菌株,觀察比較滅活效果。2.提取分枝桿菌菌體蛋白,干化學(xué)法測(cè)蛋白濃度。3.表面增強(qiáng)激光解吸電離飛行時(shí)間質(zhì)譜技術(shù)(SELDI-TOF-MS)檢測(cè)分枝桿菌蛋白表達(dá),與非分枝桿菌蛋白指紋圖譜比較,篩選分枝桿菌特征性蛋白。4.重復(fù)測(cè)定20次分枝桿菌菌體蛋白標(biāo)本,評(píng)價(jià)SELDI檢測(cè)分枝桿菌菌體蛋白分子量的重復(fù)性。5.提取結(jié)核分枝桿菌和非結(jié)核分枝桿菌分泌蛋白,干化學(xué)法測(cè)蛋白濃度。6. SELDI-TOF-MS技術(shù)檢測(cè)菌株分泌蛋白表達(dá),Biomaker Wizard Software篩選差異蛋白。7.重復(fù)測(cè)定20次分枝桿菌分泌蛋白標(biāo)本,評(píng)價(jià)SELDI-TOF- MS檢測(cè)分枝桿菌分泌蛋白分子量的重復(fù)性。 結(jié)果:1. 60℃,90 min滅活后有2株菌株7天后生長(zhǎng),而100℃,30 min滅活后所有菌株45天均未見生長(zhǎng)。60℃,90min和100℃,30 min滅活后未生長(zhǎng)的菌株菌體蛋白和分泌蛋白表達(dá)經(jīng)SELDI-TOF-MS檢測(cè)無(wú)差異。2.分枝桿菌菌體蛋白指紋圖譜顯示恥垢分枝桿菌ATCC14468有約20個(gè)蛋白峰,結(jié)核分枝桿菌ATCC25177、土地分枝桿菌ATCC15755、胞內(nèi)分枝桿菌ATCC13950、恥垢分枝桿菌ATCC607共有近40個(gè)蛋白峰。其中4個(gè)蛋白峰在所有分枝桿菌標(biāo)準(zhǔn)菌株均表達(dá)。同時(shí)分析其它細(xì)菌(大腸埃希菌、金黃色葡萄球菌、肺炎克雷伯菌、銅綠假單胞菌等)蛋白指紋圖譜中未見這4個(gè)蛋白峰。3.重復(fù)檢測(cè)20次,分枝桿菌菌體蛋白4個(gè)特征性蛋白峰的分子量變異系數(shù)均小于0.05%。4.分枝桿菌分泌蛋白指紋圖譜顯示結(jié)核分枝桿菌ATCC25177、土地分枝桿菌ATCC15755、恥垢分枝桿菌ATCC14468,胞內(nèi)分枝桿菌ATCC13950、恥垢分枝桿菌ATCC607有近30個(gè)蛋白峰。4個(gè)蛋白峰為分枝桿菌共有,與非結(jié)核分枝桿菌分泌蛋白指紋圖譜比較,1個(gè)蛋白峰為結(jié)核分枝桿菌所特有。同時(shí)分析其它細(xì)菌(大腸埃希菌、金黃色葡萄球菌、肺炎克雷伯菌、銅綠假單胞菌等)蛋白指紋圖譜中未見此蛋白峰。5.重復(fù)檢測(cè)20次,分枝桿菌分泌蛋白峰的分子量變異系數(shù)均小于0.05%。 結(jié)論:1.分枝桿菌菌體蛋白有其特征性蛋白峰,可能有助于分枝桿菌的快速鑒定。2.結(jié)核分枝桿菌和非結(jié)核分枝桿菌分泌蛋白有差異,此差異蛋白的發(fā)現(xiàn)可能有助于結(jié)核分枝桿菌的鑒別。
[Abstract]:Objective: to study the expression of mycobacterium mycobacterial protein, to screen the characteristic protein, to study the expression of secretory protein of Mycobacterium tuberculosis and non-tuberculosis, and to screen the differential protein. Method 1: 1. The standard and inactivated mycobacterium strains were cultured and the inactivation effect was observed and compared. Mycobacterial protein was extracted and the protein concentration was measured by dry chemical method. Surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) was used to detect the expression of Mycobacterium protein, and compared with that of non-mycobacterium protein fingerprint, the characteristic protein of Mycobacterium was screened. To evaluate the reproducibility of SELDI in detecting the molecular weight of Mycobacterium mycoprotein, 20 times of mycobacterial protein samples were determined repeatedly. The secreted proteins of Mycobacterium tuberculosis and Mycobacterium tuberculosis were extracted and the concentration of protein was measured by dry chemical method. Detection of secreting protein expression by SELDI-TOF-MS screening differential protein. 7. 7 by Biomaker Wizard Software. To evaluate the reproducibility of detecting the molecular weight of mycobacterium secretory protein by SELDI-TOF- MS. The result is 1: 1. After inactivation at 60 鈩，

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