结核分枝杆菌蛋白标志物的筛选

发布时间：2018-05-08 22:18

  本文选题：结核分枝杆菌 + 非结核分枝杆菌　； 参考：《重庆医科大学》2009年硕士论文

【摘要】： 目的:研究分枝杆菌菌体蛋白表达,筛选特征性蛋白,研究结核分枝杆菌和非结核分枝杆菌分泌蛋白表达,筛选差异蛋白。 方法:1.培养分枝杆菌标准菌株,灭活菌株,观察比较灭活效果。2.提取分枝杆菌菌体蛋白,干化学法测蛋白浓度。3.表面增强激光解吸电离飞行时间质谱技术(SELDI-TOF-MS)检测分枝杆菌蛋白表达,与非分枝杆菌蛋白指纹图谱比较,筛选分枝杆菌特征性蛋白。4.重复测定20次分枝杆菌菌体蛋白标本,评价SELDI检测分枝杆菌菌体蛋白分子量的重复性。5.提取结核分枝杆菌和非结核分枝杆菌分泌蛋白,干化学法测蛋白浓度。6. SELDI-TOF-MS技术检测菌株分泌蛋白表达,Biomaker Wizard Software筛选差异蛋白。7.重复测定20次分枝杆菌分泌蛋白标本,评价SELDI-TOF- MS检测分枝杆菌分泌蛋白分子量的重复性。 结果:1. 60℃,90 min灭活后有2株菌株7天后生长,而100℃,30 min灭活后所有菌株45天均未见生长。60℃,90min和100℃,30 min灭活后未生长的菌株菌体蛋白和分泌蛋白表达经SELDI-TOF-MS检测无差异。2.分枝杆菌菌体蛋白指纹图谱显示耻垢分枝杆菌ATCC14468有约20个蛋白峰,结核分枝杆菌ATCC25177、土地分枝杆菌ATCC15755、胞内分枝杆菌ATCC13950、耻垢分枝杆菌ATCC607共有近40个蛋白峰。其中4个蛋白峰在所有分枝杆菌标准菌株均表达。同时分析其它细菌(大肠埃希菌、金黄色葡萄球菌、肺炎克雷伯菌、铜绿假单胞菌等)蛋白指纹图谱中未见这4个蛋白峰。3.重复检测20次,分枝杆菌菌体蛋白4个特征性蛋白峰的分子量变异系数均小于0.05%。4.分枝杆菌分泌蛋白指纹图谱显示结核分枝杆菌ATCC25177、土地分枝杆菌ATCC15755、耻垢分枝杆菌ATCC14468,胞内分枝杆菌ATCC13950、耻垢分枝杆菌ATCC607有近30个蛋白峰。4个蛋白峰为分枝杆菌共有,与非结核分枝杆菌分泌蛋白指纹图谱比较,1个蛋白峰为结核分枝杆菌所特有。同时分析其它细菌(大肠埃希菌、金黄色葡萄球菌、肺炎克雷伯菌、铜绿假单胞菌等)蛋白指纹图谱中未见此蛋白峰。5.重复检测20次,分枝杆菌分泌蛋白峰的分子量变异系数均小于0.05%。 结论:1.分枝杆菌菌体蛋白有其特征性蛋白峰,可能有助于分枝杆菌的快速鉴定。2.结核分枝杆菌和非结核分枝杆菌分泌蛋白有差异,此差异蛋白的发现可能有助于结核分枝杆菌的鉴别。
[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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