致肾盂肾炎大肠杆菌新基因R049的鉴定和特性的研究

发布时间：2018-05-16 14:26

本文选题：致肾盂肾炎大肠杆菌 + R049基因　；参考：《天津医科大学》2009年博士论文

【摘要】： 【目的】从国内临床分离的致肾盂肾炎大肠杆菌(uropathogenic Escherichia coli，UPEC)132中发现与致病性相关的新基因，并初步了解其特性，为进一步研究UPEC致病机制奠定基础。【方法】 1．采用PCR方法对UPEC132特异性编码序列R049片段(789bp)在UPEC菌株和正常人粪便分离大肠杆菌菌株中的分布进行分析。R049片段阳性的菌株经脉冲场凝胶电泳后转膜进行Southern杂交，确认R049片段在受试菌株染色体的分布特征。 2．采用基因组步移技术，以R049片段(789bp)为核心双向延伸，获得其5’端和3’端侧翼序列，经生物信息学分析寻找可能的开放阅读框R049-ORF，并提交GenBank注册。 3．构建原核表达系统E．coli BL21(DE3)／pET32a-R049，SDS-PAGE分析目的蛋白表达情况，镍亲和层析纯化后的R049重组蛋白免疫小鼠获得抗血清，ELISA测定血清效价。以此抗血清对UPEC132全菌裂解物以及提取的UPEC132内膜与外膜蛋白进行Western blot分析。 4．观察UPEC132对人膀胱移行上皮细胞(EJ)的黏附，并且用激光共聚焦显微镜检测绿色荧光蛋白标记的UPEC132是否能够侵入EJ细胞内部。采用22KHuman Genome Array基因芯片检测UPEC132感染的EJ细胞的基因表达谱变化，分析感染过程中涉及的信号转导途径。相比之下，构建R049-ORF真核表达质粒，，将其转染EJ细胞，检测基因表达谱的变化，分析差异表达基因的功能。 5．以大肠杆菌无菌毛株K-12 p678-54构建表达R049-ORF的重组菌E．coliK-12 p678-54／pACYC184-R049，该重组菌与E．coli K-12p678-54分别经尿道逆行感染BALB／c小鼠，通过小鼠尿液与肾组织细菌计数和肾脏病理学检查观察两种菌株对动物致病性的差异。同时以R049重组蛋白免疫BALB／c小鼠，对免疫组小鼠和未免疫组小鼠经尿道逆行感染UPEC132，通过上述检测分析R049-ORF编码蛋白对同源性菌株攻击的免疫保护作用。【结果】 1．R049片段(789bp)在20株UPEC和40株正常人粪便分离的大肠杆菌中阳性率分别为40％(8／20株)和7．5％(3／40株)，两者有显著性差异(P＜0．01)。11株R049片段阳性的菌株经脉冲场凝胶电泳和Southernblot分析，其中有6株UPEC阳性杂交带均为350kb，与国外模型菌株UPEC J96阳性杂交带不同，提示R049片段在国内UPEC中具有共同聚集性分布的特征。 2．对R049片段(789bp)双向延伸，获得其5’端1502bp和3’端1207bp，经生物信息学分析获得一个可能的R049-ORF(1311bp)，经BLAST搜索未发现与之相似序列，GenBank注册号为EF488001。 3．R049重组蛋白表达量占菌体总蛋白26．2％，表达产物以包涵体形式存在，镍亲和层析纯化后其纯度大于95％。纯化的R049重组蛋白制备的抗血清，效价达1：102400以上。经Western blot检测，该抗血清与UPEC132全菌裂解物和UPEC132的外膜蛋白发生特异性结合，阳性条带与R049-ORF编码蛋白预测分子量(47 KD)相一致，表明R049-ORF系编码UPEC132外膜蛋白的基因。 4．UPEC132对EJ细胞黏附率为(73．20±5．26)％，共聚焦显微镜观察发现该菌能够侵入EJ细胞内。UPEC132感染的EJ细胞有29个差异表达基因(1个下调，28个上调)，主要涉及细胞增殖与生长、炎症反应和细胞凋亡，分属MAPK信号途径、Toll样受体信号途径和诱导细胞凋亡的FNF受体途径。R049-ORF转染的EJ细胞IL-6基因上调大于2倍，与炎症反应有关。 5．分别以重组菌E．coli K-12 p678-54／pACYC184-R049与E．coli K-12p678-54感染小鼠，尿液与肾组织细菌计数和肾脏病理学改变无明显差异。R049重组蛋白免疫小鼠的血清抗体效价为1：25600-1：51200，免疫组小鼠尿液和肾组织细菌计数均显著小于未免疫组(P＜0．01，P＜0．05)，但两组各有部分小鼠肾脏出现严重炎症反应，无明显差异。提示R049基因未表现出致病性，但R049基因编码蛋白对同源性菌株的攻击具有一定的免疫保护作用。【结论】 1．UPEC132新基因R049特异性地与UPEC菌株相关。 2．R049-ORF(1311bp)编码UPEC132的外膜蛋白(47KD)，经NCBI数据库查询和BLAST比对尚无相同报道。 3．R049-ORF转染EJ细胞，IL-6表达上调，R049基因可能涉及炎症反应。 4．动物实验结果显示R049编码蛋白对同源菌株的攻击有一定的保护作用。
[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.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R692.7;R378

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