本文選題：致腎盂腎炎大腸桿菌 + 比較蛋白質(zhì)組學��； 參考：《天津醫(yī)科大學》2009年碩士論文

【摘要】： 目的: 研究國內(nèi)分離的致腎盂腎炎大腸桿菌(UPEC)132與國外模型菌株UPECJ96及完成基因組測序的非致病性大腸桿菌K-12 MG1655的蛋白質(zhì)組的差異。 方法: 1.對UPEC 132、UPEC J96和E.coli K-12 MG1655進行革蘭染色、生化反應、papC PCR檢測,鑒定菌株。 2.采用比濁法進行細菌計數(shù)并繪制各菌株的生長曲線。以此為依據(jù),分別培養(yǎng)至對數(shù)生長前期(6h)和后期(12h),離心收獲菌體,超聲破菌制備雙向凝膠電泳蛋白樣品,Bradford法測蛋白濃度。 3.優(yōu)化細菌培養(yǎng)和雙向凝膠電泳的試驗條件:(1)將UPEC132不同培養(yǎng)時間收獲的菌體或不同pH梯度下進行的雙向凝膠電泳圖譜進行比較分析;(2)根據(jù)E.coli K-12 MG1655的基因組注釋結(jié)果,使用pI/Mr Prediction Tool(http:∥www.expasy.org/tools/pi_tool.html)計算,構建其理論雙向凝膠電泳圖譜,預測其全部蛋白點的分布情況;(3)用pH 4-7的IPG膠條對E.coli K-12MG1655的胞內(nèi)蛋白樣品進行雙向凝膠電泳,與其理論雙向電泳圖譜進行比較,計算分辨率。 4.依據(jù)優(yōu)化的條件,分別對三株菌的蛋白樣品進行雙向凝膠電泳,電泳后經(jīng)考馬斯亮藍染色,采集圖像,使用PD Quest7.0軟件對圖像進行配比分析。以UPEC 132為核心,選取其特異的及上調(diào)表達(表達量差異2倍以上)的蛋白點進行凝膠內(nèi)胰蛋白酶消化萃取和MALDI-TOF-MS質(zhì)譜分析。 結(jié)果: 1.UPEC132、UPEC J96以及E.coli K.12 MG1655菌株的鑒定 1)三株菌均為革蘭陰性桿菌,生化反應符合大腸桿菌。 2)papC PCR結(jié)果顯示:UPEC132和J96均出現(xiàn)預期328bp的陽性擴增帶,而E.coli K-12 MG1655沒有顯示此擴增帶,可確定前二者為UPEC菌株。 2.三株菌生長曲線相似,經(jīng)過短暫的遲緩期后進入對數(shù)生長期(2-12h)。 3.三株菌培養(yǎng)6h收獲菌體制備胞內(nèi)蛋白電泳樣品,其蛋白濃度分別為:10.63mg/ml、11.93mg/ml以及11.55mg/ml,培養(yǎng)12h制備的胞內(nèi)蛋白樣品其蛋白濃度分別為11.06mg/ml、12.84mg/ml及12.52mg/ml,結(jié)果符合要求。 4.雙向凝膠電泳試驗條件的優(yōu)化分析 1)UPEC132培養(yǎng)6h和12h雙向凝膠電泳圖譜分布相似,經(jīng)PD Quest軟件分析,前者分辨出472個蛋白點,后者為513個蛋白點,后者蛋白點較前者增加了8.69%。 2)UPEC132經(jīng)pH 3-10的IPG膠條分離獲得的雙向電泳圖譜中蛋白點集中于pH 4-7區(qū)間,堿性區(qū)域蛋白點稀疏;而用pH 4-7的IPG膠條分離的雙向電泳圖譜中蛋白點分布均勻,分辨率較好。經(jīng)軟件分析表明,二者蛋白點數(shù)分別為268和513,后者分離效果明顯優(yōu)于前者。 3)E.coli K-12 MG1655全菌蛋白的理論雙向凝膠電泳圖譜顯示:62.9%的理論蛋白點都集中于pH 4-7之間,27.1%的理論蛋白點分命于pH 8-10之間,只有5.3%的理論蛋白點位于pH 7-8之間。 4)使用pH 4-7的IPG膠條分離獲得E.coli K-12 MG1655胞內(nèi)蛋白的雙向凝膠電泳圖譜,與上述理論圖譜相比較已有13.2%(356個)的蛋白點顯現(xiàn)。據(jù)報道,在當前試驗條件下雙向凝膠電泳分辨率尚不超過理論值的10%,故本試驗的分辨率較為理想。 5.雙向凝膠電泳結(jié)果顯示:UPEC132平均識別466±11個蛋白點,J96平均識別382±12個蛋白點,E.coli K-12 MG1655平均識別338±15個蛋白點。三株菌共有的蛋白點為298個(59.7%),UPEC132和E.coli K-12 MG1655共有的蛋白點為33個(6.6%),UPEC132和J96共有的蛋白點為56個(11.2%);UPEC132特異的蛋白點為89個(17.8%),J96特異的蛋白點為22個(4.4%),MG1655特異的蛋白點只有1個(0.2%)。 6.經(jīng)MALDI-TOF-MS分析,22個差異蛋白點得到鑒定,其中15個為UPEC132特異或明顯上調(diào)表達的蛋白點,7個為UPEC132與J96均上調(diào)表達的蛋白點,涉及毒力因子、物質(zhì)代謝轉(zhuǎn)運、蛋白合成調(diào)節(jié)、生物氧化及未知功能等。 結(jié)論: 本研究獲得了UPEC132、UPEC J96以及E.coli K-12 MG1655胞內(nèi)蛋白的雙向凝膠電泳圖譜,通過對三者之間蛋白表達的差異分析,發(fā)現(xiàn)了UPEC菌株與非致病性大腸桿菌間的蛋白質(zhì)組有差異,UPEC132與J96蛋白質(zhì)組間也有顯著不同,表明UPEC菌株存在多態(tài)性和國內(nèi)外菌株的差異,為致腎盂腎炎大腸桿菌的致病機制等深入研究奠定了基礎。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R378.21

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本文編號：1981359