本文選題：致腎盂腎炎大腸桿菌 + R049基因��； 參考：《天津醫(yī)科大學(xué)》2009年博士論文

【摘要】： 【目的】 從國(guó)內(nèi)臨床分離的致腎盂腎炎大腸桿菌(uropathogenic Escherichia coli，UPEC)132中發(fā)現(xiàn)與致病性相關(guān)的新基因，并初步了解其特性，為進(jìn)一步研究UPEC致病機(jī)制奠定基礎(chǔ)。 【方法】 1．采用PCR方法對(duì)UPEC132特異性編碼序列R049片段(789bp)在UPEC菌株和正常人糞便分離大腸桿菌菌株中的分布進(jìn)行分析。R049片段陽(yáng)性的菌株經(jīng)脈沖場(chǎng)凝膠電泳后轉(zhuǎn)膜進(jìn)行Southern雜交，確認(rèn)R049片段在受試菌株染色體的分布特征。 2．采用基因組步移技術(shù)，以R049片段(789bp)為核心雙向延伸，獲得其5’端和3’端側(cè)翼序列，經(jīng)生物信息學(xué)分析尋找可能的開(kāi)放閱讀框R049-ORF，并提交GenBank注冊(cè)。 3．構(gòu)建原核表達(dá)系統(tǒng)E．coli BL21(DE3)／pET32a-R049，SDS-PAGE分析目的蛋白表達(dá)情況，鎳親和層析純化后的R049重組蛋白免疫小鼠獲得抗血清，ELISA測(cè)定血清效價(jià)。以此抗血清對(duì)UPEC132全菌裂解物以及提取的UPEC132內(nèi)膜與外膜蛋白進(jìn)行Western blot分析。 4．觀察UPEC132對(duì)人膀胱移行上皮細(xì)胞(EJ)的黏附，并且用激光共聚焦顯微鏡檢測(cè)綠色熒光蛋白標(biāo)記的UPEC132是否能夠侵入EJ細(xì)胞內(nèi)部。采用22KHuman Genome Array基因芯片檢測(cè)UPEC132感染的EJ細(xì)胞的基因表達(dá)譜變化，分析感染過(guò)程中涉及的信號(hào)轉(zhuǎn)導(dǎo)途徑。相比之下，構(gòu)建R049-ORF真核表達(dá)質(zhì)粒，，將其轉(zhuǎn)染EJ細(xì)胞，檢測(cè)基因表達(dá)譜的變化，分析差異表達(dá)基因的功能。 5．以大腸桿菌無(wú)菌毛株K-12 p678-54構(gòu)建表達(dá)R049-ORF的重組菌E．coliK-12 p678-54／pACYC184-R049，該重組菌與E．coli K-12p678-54分別經(jīng)尿道逆行感染BALB／c小鼠，通過(guò)小鼠尿液與腎組織細(xì)菌計(jì)數(shù)和腎臟病理學(xué)檢查觀察兩種菌株對(duì)動(dòng)物致病性的差異。同時(shí)以R049重組蛋白免疫BALB／c小鼠，對(duì)免疫組小鼠和未免疫組小鼠經(jīng)尿道逆行感染UPEC132，通過(guò)上述檢測(cè)分析R049-ORF編碼蛋白對(duì)同源性菌株攻擊的免疫保護(hù)作用。 【結(jié)果】 1．R049片段(789bp)在20株UPEC和40株正常人糞便分離的大腸桿菌中陽(yáng)性率分別為40％(8／20株)和7．5％(3／40株)，兩者有顯著性差異(P＜0．01)。11株R049片段陽(yáng)性的菌株經(jīng)脈沖場(chǎng)凝膠電泳和Southernblot分析，其中有6株UPEC陽(yáng)性雜交帶均為350kb，與國(guó)外模型菌株UPEC J96陽(yáng)性雜交帶不同，提示R049片段在國(guó)內(nèi)UPEC中具有共同聚集性分布的特征。 2．對(duì)R049片段(789bp)雙向延伸，獲得其5’端1502bp和3’端1207bp，經(jīng)生物信息學(xué)分析獲得一個(gè)可能的R049-ORF(1311bp)，經(jīng)BLAST搜索未發(fā)現(xiàn)與之相似序列，GenBank注冊(cè)號(hào)為EF488001。 3．R049重組蛋白表達(dá)量占菌體總蛋白26．2％，表達(dá)產(chǎn)物以包涵體形式存在，鎳親和層析純化后其純度大于95％。純化的R049重組蛋白制備的抗血清，效價(jià)達(dá)1：102400以上。經(jīng)Western blot檢測(cè)，該抗血清與UPEC132全菌裂解物和UPEC132的外膜蛋白發(fā)生特異性結(jié)合，陽(yáng)性條帶與R049-ORF編碼蛋白預(yù)測(cè)分子量(47 KD)相一致，表明R049-ORF系編碼UPEC132外膜蛋白的基因。 4．UPEC132對(duì)EJ細(xì)胞黏附率為(73．20±5．26)％，共聚焦顯微鏡觀察發(fā)現(xiàn)該菌能夠侵入EJ細(xì)胞內(nèi)。UPEC132感染的EJ細(xì)胞有29個(gè)差異表達(dá)基因(1個(gè)下調(diào)，28個(gè)上調(diào))，主要涉及細(xì)胞增殖與生長(zhǎng)、炎癥反應(yīng)和細(xì)胞凋亡，分屬M(fèi)APK信號(hào)途徑、Toll樣受體信號(hào)途徑和誘導(dǎo)細(xì)胞凋亡的FNF受體途徑。R049-ORF轉(zhuǎn)染的EJ細(xì)胞IL-6基因上調(diào)大于2倍，與炎癥反應(yīng)有關(guān)。 5．分別以重組菌E．coli K-12 p678-54／pACYC184-R049與E．coli K-12p678-54感染小鼠，尿液與腎組織細(xì)菌計(jì)數(shù)和腎臟病理學(xué)改變無(wú)明顯差異。R049重組蛋白免疫小鼠的血清抗體效價(jià)為1：25600-1：51200，免疫組小鼠尿液和腎組織細(xì)菌計(jì)數(shù)均顯著小于未免疫組(P＜0．01，P＜0．05)，但兩組各有部分小鼠腎臟出現(xiàn)嚴(yán)重炎癥反應(yīng)，無(wú)明顯差異。提示R049基因未表現(xiàn)出致病性，但R049基因編碼蛋白對(duì)同源性菌株的攻擊具有一定的免疫保護(hù)作用。 【結(jié)論】 1．UPEC132新基因R049特異性地與UPEC菌株相關(guān)。 2．R049-ORF(1311bp)編碼UPEC132的外膜蛋白(47KD)，經(jīng)NCBI數(shù)據(jù)庫(kù)查詢和BLAST比對(duì)尚無(wú)相同報(bào)道。 3．R049-ORF轉(zhuǎn)染EJ細(xì)胞，IL-6表達(dá)上調(diào)，R049基因可能涉及炎癥反應(yīng)。 4．動(dòng)物實(shí)驗(yàn)結(jié)果顯示R049編碼蛋白對(duì)同源菌株的攻擊有一定的保護(hù)作用。
[Abstract]:[Objective]
A new gene related to pathogenicity in uropathogenic Escherichia coli (UPEC) 132, which is clinically isolated from China, is found and its characteristics are preliminarily understood, which lays the foundation for further research on the pathogenesis of UPEC.
[method]
1. the distribution of UPEC132 specific coding sequence R049 fragment (789bp) in UPEC strain and normal human feces isolated Escherichia coli strains were analyzed by PCR method, and the.R049 fragment positive strain meridian flushing field gel electrophoresis was used for Southern hybridization, and the distribution characteristics of R049 fragment in the tested strains were confirmed.
2. using the genomic step technique, the R049 fragment (789bp) was used as the core, and the 5 'end and the 3' side flanking sequences were obtained. Through bioinformatics analysis, the possible open reading frame R049-ORF was found, and GenBank registration was submitted.
3. the prokaryotic expression system E.coli BL21 (DE3) / pET32a-R049 was constructed, the expression of the target protein was analyzed by SDS-PAGE. The antiserum was obtained by the R049 recombinant protein purified by the nickel affinity chromatography, and the serum titer was determined by ELISA. The antiserum was used for Western blot fraction of UPEC132 whole bacteria lysate and the extracted UPEC132 intima and epicardium protein. Analysis.
4. the adhesion of UPEC132 to human bladder transitional epithelial cells (EJ) was observed, and whether the UPEC132 labeled by green fluorescent protein could invade the EJ cells was detected by laser confocal microscopy. The gene expression profiles of EJ cells infected with UPEC132 were detected by 22KHuman Genome Array gene chip, and the signals involved in the infection process were analyzed. In contrast, R049-ORF eukaryotic expression plasmid was constructed and transfected into EJ cells to detect the changes of gene expression profile and analyze the function of differentially expressed genes.
5. the recombinant strain E.coliK-12 p678-54 / pACYC184-R049 expressing R049-ORF was constructed with Escherichia coli K-12 p678-54. The recombinant bacteria and E.coli K-12p678-54 were retrograde infecting BALB / c mice via urethra, and the difference between the bacteria count of the urine and kidney tissue of mice and the pathological examination of kidney to observe the difference between the pathogenicity of the animals and the pathogenicity of the two strains. At the same time, BALB / c mice were immunized with R049 recombinant protein, and UPEC132 was infected by transurethral retrograde infection of mice and unimmunized groups in the immune group and unimmunized group. The immune protective effect of R049-ORF encoded protein on the homologous strain was analyzed by the above detection.
[results]
The positive rates of 1.R049 fragment (789bp) in Escherichia coli isolated from 20 UPEC and 40 normal human feces were 40% (8 / 20 strains) and 7.5% (3 / 40) respectively. There were significant differences (P < 0.01) in.11 strain positive strains of.11 strain via pulse field gel electrophoresis and Southernblot segregation, of which 6 strains of UPEC positive hybrid bands were 350kb. The foreign model strains UPEC J96 positive hybridization zones are different, suggesting that R049 fragments are clustered in domestic UPEC.
2. R049 fragment (789bp) is extended bi-directional, and its 5 'end 1502bp and 3' end 1207bp are obtained. A possible R049-ORF (1311bp) is obtained by bioinformatics analysis. The similar sequence is not found by BLAST search. The GenBank registration number is EF488001.
The expression of 3.R049 recombinant protein accounted for 26.2% of the total body protein, and the expression product existed in the form of inclusion body. After purification, the purity of the recombinant protein was greater than that of the recombinant protein of the 95%. purified R049. The titer was above 1:102400. The antiserum was detected by Western blot, and the antiserum was associated with the outer membrane protein of UPEC132 whole bacteria lysate and UPEC132. Specific binding and positive bands were consistent with the predicted molecular weight (47 KD) of the R049-ORF encoded protein, indicating that the R049-ORF gene encodes the outer membrane protein of UPEC132.
The adhesion rate of 4.UPEC132 to EJ cells was (73.20 + 5.26)%. Confocal microscopy showed that the bacteria could invade.UPEC132 infected EJ cells with 29 differentially expressed genes (1 down-regulation, 28 up-regulated), mainly involving cell proliferation and growth, inflammatory reaction and cell apoptosis, belonging to MAPK signal pathway, Toll like receptor signal path. The IL-6 gene of.R049-ORF transfected EJ cell transfected with FNF receptor pathway and apoptosis inducing cell was more than 2 times higher than that of the transfected cells.
5. the mice were infected with recombinant bacteria E.coli K-12 p678-54 / pACYC184-R049 and E.coli K-12p678-54 respectively. There was no significant difference between urine and renal tissue bacteria count and renal pathological changes. The serum antibody titer of.R049 recombinant protein immunized mice was 1:25600-1:51200, and the count of urine and kidney tissue in the immune group was significantly less than that of the immune group. The unimmunized group (P < 0.01, P < 0.05), but there were some serious inflammatory reactions in the kidneys of the two groups, suggesting that the R049 gene did not show pathogenicity, but the R049 gene encoded protein had a certain protective effect on the attack of homologous strains.
[Conclusion]
The new 1.UPEC132 gene R049 is specifically related to UPEC strain.
The outer membrane protein (47KD) encoded by 2.R049-ORF (1311bp) encoding UPEC132 has not been reported by NCBI database query and BLAST comparison.
3.R049-ORF transfected EJ cells, IL-6 expression was up-regulated, R049 gene may involve inflammatory reaction.
4. animal experiments showed that R049 encoding protein had some protective effects on homologous strains.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R692.7;R378

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