致肾盂肾炎大肠杆菌132的比较蛋白质组学研究

发布时间：2018-06-05 08:35

本文选题：致肾盂肾炎大肠杆菌 + 比较蛋白质组学　；参考：《天津医科大学》2009年硕士论文

【摘要】： 目的: 研究国内分离的致肾盂肾炎大肠杆菌(UPEC)132与国外模型菌株UPECJ96及完成基因组测序的非致病性大肠杆菌K-12 MG1655的蛋白质组的差异。方法: 1.对UPEC 132、UPEC J96和E.coli K-12 MG1655进行革兰染色、生化反应、papC PCR检测,鉴定菌株。 2.采用比浊法进行细菌计数并绘制各菌株的生长曲线。以此为依据,分别培养至对数生长前期(6h)和后期(12h),离心收获菌体,超声破菌制备双向凝胶电泳蛋白样品,Bradford法测蛋白浓度。 3.优化细菌培养和双向凝胶电泳的试验条件:(1)将UPEC132不同培养时间收获的菌体或不同pH梯度下进行的双向凝胶电泳图谱进行比较分析;(2)根据E.coli K-12 MG1655的基因组注释结果,使用pI/Mr Prediction Tool(http:∥www.expasy.org/tools/pi_tool.html)计算,构建其理论双向凝胶电泳图谱,预测其全部蛋白点的分布情况;(3)用pH 4-7的IPG胶条对E.coli K-12MG1655的胞内蛋白样品进行双向凝胶电泳,与其理论双向电泳图谱进行比较,计算分辨率。 4.依据优化的条件,分别对三株菌的蛋白样品进行双向凝胶电泳,电泳后经考马斯亮蓝染色,采集图像,使用PD Quest7.0软件对图像进行配比分析。以UPEC 132为核心,选取其特异的及上调表达(表达量差异2倍以上)的蛋白点进行凝胶内胰蛋白酶消化萃取和MALDI-TOF-MS质谱分析。结果: 1.UPEC132、UPEC J96以及E.coli K.12 MG1655菌株的鉴定 1)三株菌均为革兰阴性杆菌,生化反应符合大肠杆菌。 2)papC PCR结果显示:UPEC132和J96均出现预期328bp的阳性扩增带,而E.coli K-12 MG1655没有显示此扩增带,可确定前二者为UPEC菌株。 2.三株菌生长曲线相似,经过短暂的迟缓期后进入对数生长期(2-12h)。 3.三株菌培养6h收获菌体制备胞内蛋白电泳样品,其蛋白浓度分别为:10.63mg/ml、11.93mg/ml以及11.55mg/ml,培养12h制备的胞内蛋白样品其蛋白浓度分别为11.06mg/ml、12.84mg/ml及12.52mg/ml,结果符合要求。 4.双向凝胶电泳试验条件的优化分析 1)UPEC132培养6h和12h双向凝胶电泳图谱分布相似,经PD Quest软件分析,前者分辨出472个蛋白点,后者为513个蛋白点,后者蛋白点较前者增加了8.69%。 2)UPEC132经pH 3-10的IPG胶条分离获得的双向电泳图谱中蛋白点集中于pH 4-7区间,碱性区域蛋白点稀疏;而用pH 4-7的IPG胶条分离的双向电泳图谱中蛋白点分布均匀,分辨率较好。经软件分析表明,二者蛋白点数分别为268和513,后者分离效果明显优于前者。 3)E.coli K-12 MG1655全菌蛋白的理论双向凝胶电泳图谱显示:62.9%的理论蛋白点都集中于pH 4-7之间,27.1%的理论蛋白点分命于pH 8-10之间,只有5.3%的理论蛋白点位于pH 7-8之间。 4)使用pH 4-7的IPG胶条分离获得E.coli K-12 MG1655胞内蛋白的双向凝胶电泳图谱,与上述理论图谱相比较已有13.2%(356个)的蛋白点显现。据报道,在当前试验条件下双向凝胶电泳分辨率尚不超过理论值的10%,故本试验的分辨率较为理想。 5.双向凝胶电泳结果显示:UPEC132平均识别466±11个蛋白点,J96平均识别382±12个蛋白点,E.coli K-12 MG1655平均识别338±15个蛋白点。三株菌共有的蛋白点为298个(59.7%),UPEC132和E.coli K-12 MG1655共有的蛋白点为33个(6.6%),UPEC132和J96共有的蛋白点为56个(11.2%);UPEC132特异的蛋白点为89个(17.8%),J96特异的蛋白点为22个(4.4%),MG1655特异的蛋白点只有1个(0.2%)。 6.经MALDI-TOF-MS分析,22个差异蛋白点得到鉴定,其中15个为UPEC132特异或明显上调表达的蛋白点,7个为UPEC132与J96均上调表达的蛋白点,涉及毒力因子、物质代谢转运、蛋白合成调节、生物氧化及未知功能等。结论: 本研究获得了UPEC132、UPEC J96以及E.coli K-12 MG1655胞内蛋白的双向凝胶电泳图谱,通过对三者之间蛋白表达的差异分析,发现了UPEC菌株与非致病性大肠杆菌间的蛋白质组有差异,UPEC132与J96蛋白质组间也有显著不同,表明UPEC菌株存在多态性和国内外菌株的差异,为致肾盂肾炎大肠杆菌的致病机制等深入研究奠定了基础。
[Abstract]:Objective:
To study the differences between the domestic isolated Escherichia coli (UPEC) 132 and the foreign model strain UPECJ96 and the genome sequencing of the non pathogenic Escherichia coli K-12 MG1655.
Method:
1. UPEC 132, UPEC J96 and E.coli K-12 MG1655 were tested for Gram stain, biochemical reaction, papC PCR, and strain identification.
2. the bacterial count was counted by turbidimetric method and the growth curve of each strain was plotted. On this basis, the bacteria were cultured to the early stage of logarithmic growth (6h) and later (12h), the centrifuge harvested bacteria were harvested, the bi-directional gel electrophoresis protein samples were prepared by ultrasonic breaking bacteria, and the protein concentration was measured by the Bradford method.
3. optimize the test conditions for bacterial culture and two-dimensional gel electrophoresis: (1) compare and analyze the bi-directional gel electrophoresis Atlas of UPEC132 harvested cells at different incubation times or under different pH gradients; (2) pI/Mr Prediction Tool (http: www.expasy.org/tools/pi_tool.html) is used according to the results of the genome annotation of E.coli K-12 MG1655. The theoretical bi-directional gel electrophoresis atlas was constructed to predict the distribution of all the protein points. (3) the two-dimensional gel electrophoresis of the intracellular protein samples of E.coli K-12MG1655 was carried out with the IPG strip of pH 4-7, and the resolution was calculated by comparing its theoretical two-dimensional electrophoresis atlas.
4. according to the optimized conditions, the protein samples of three strains of bacteria were separated by two-dimensional gel electrophoresis. After electrophoresis, the images were collected by komomo blue, and the images were collected by PD Quest7.0 software. UPEC 132 was used as the core, and the specific and up expression (more than 2 times of the expression difference) were selected for the intra gel trypsin. Enzyme digestion extraction and MALDI-TOF-MS mass spectrometry analysis.
Result:
Identification of 1.UPEC132, UPEC J96 and E.coli K.12 MG1655 strains
1) three strains were gram negative bacilli, and the biochemical reaction accorded with E. coli.
2) papC PCR results showed that both UPEC132 and J96 showed positive amplification bands of expected 328bp, while E.coli K-12 MG1655 did not show the amplified band, and the first two were UPEC strains.
2. the growth curves of three strains were similar, and entered a logarithmic growth phase (2-12h) after a short lag period.
3. three strains of bacteria culture 6h harvested bacteria to prepare the intracellular protein electrophoresis samples. The protein concentration is 10.63mg/ml, 11.93mg/ml and 11.55mg/ml respectively. The protein concentration of the intracellular protein samples prepared by 12h is 11.06mg/ml, 12.84mg/ml and 12.52mg/ml, respectively.
Optimal analysis of 4. bi-directional gel electrophoresis test conditions
1) the distribution of 6h and 12h bi-directional gel electrophoresis profiles in UPEC132 culture was similar. After the analysis of PD Quest software, the former identified 472 protein points, the latter was 513 protein points, and the latter increased 8.69%. compared with the former.
2) the protein points in the two-dimensional electrophoresis atlas obtained by pH 3-10 IPG gel strip concentration are concentrated in the pH 4-7 interval, and the protein points in the alkaline region are sparse, while the protein points in the two-dimensional electrophoresis map separated by pH 4-7 of pH 4-7 are evenly distributed and the resolution is better. The software analysis shows that the number of two protein points is 268 and 513 respectively, and the latter separation effect is clear. The former is better than the former.
3) the theoretical bi-directional gel electrophoresis Atlas of E.coli K-12 MG1655 whole bacteria protein showed that 62.9% of the theoretical protein points were concentrated between pH 4-7, 27.1% of the theoretical protein points were divided between pH 8-10, and only 5.3% of the theoretical protein points were located between pH 7-8.
4) the bi-directional gel electrophoresis Atlas of E.coli K-12 MG1655 intracellular protein was obtained by using the IPG strip of pH 4-7. Compared with the above theoretical map, 13.2% (356) protein spots appeared. It is reported that the resolution of two-dimensional gel electrophoresis is not more than 10% of the theoretical value under the current test conditions, so the resolution of this experiment is ideal.
The results of 5. bi-directional gel electrophoresis showed that UPEC132 identified 466 + 11 protein points with an average identification of 382 + 12 protein points, and E.coli K-12 MG1655 identified 338 + 15 protein points on average. The total protein points of three strains were 298 (59.7%), UPEC132 and E.coli K-12 MG1655 shared 33 spots (6.6%), UPEC132 and J96 common protein points were 56. (11.2%) UPEC132 specific protein spots were 89 (17.8%), J96 specific protein spots were 22 (4.4%), and MG1655 specific protein spots were only 1 (0.2%).
6. by MALDI-TOF-MS analysis, 22 differential protein points were identified, of which 15 were UPEC132 specific or obviously up-regulated protein points, and 7 were both UPEC132 and J96 up-regulated protein points, involving toxicity factor, material metabolism, protein synthesis regulation, biological oxidation and unknown function.
Conclusion:
This study obtained the bi-directional gel electrophoresis Atlas of UPEC132, UPEC J96 and E.coli K-12 MG1655 intracellular proteins. Through the analysis of the protein expression differences between the three, it was found that the protein groups between the UPEC strain and the non pathogenic Escherichia coli were different, and the UPEC132 and the J96 proteome were also significantly different, indicating that the UPEC strain was polymorphic. The differences between domestic and foreign strains laid the foundation for further research on pathogenic mechanism of pyelonephritis and E. coli.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R378.21

【参考文献】