肠炎沙门菌转录因子SEN2967和SEN3610调控基因的筛选与鉴定

发布时间：2018-03-30 23:19

本文选题：肠炎沙门菌　切入点：SEN2967　出处：《扬州大学》2017年硕士论文

【摘要】：肠炎沙门菌(Salmonella enterica serovar Entrritidis,S.Enteritidis,SE)是革兰阴性兼性胞内寄生菌,对人类和动物都具有致病性,是重要的人兽共患病原菌。转录因子(Transcriptionfactor,TF)是一类能够结合在特定基因上游特异核苷酸序列上的蛋白,并能调控该基因的转录。转录因子可调控细菌毒力因子的表达,参与生理生化代谢过程,因此对沙门菌转录因子的研究对于沙门菌致病机制与防控研究都具有重要意义。本实验室前期研究中利用SCOTS技术筛选肠炎沙门菌感染禽源巨噬细胞HD-11的过程中表达的基因,两个候选转录因子SEN2967和SEN3610基因的胞内转录水平显著高于体外培养状态下的表达水平,但国内外对这两个假定转录调控因子的研究未见报道。本研究通过同源重组的方法成功构建了肠炎沙门菌SEN2967和SEN3610基因缺失株及其诱导型表达株;并通过双向电泳、iTraQ技术对二者调控的基因表达进行了分析;对于筛选获得的基因,采用EMSA进行验证。1肠炎沙门菌C50041转录因子SEN2967和SEN3610的缺失株与表达株的构建及其生物学特性鉴定利用荧光定量PCR比较肠炎沙门菌C50041感染HD-11细胞5 h后与体外培养条件下转录水平的表达差异,证实SEN2967上调约98倍,SEN3610上调约15倍。本研究利用λ-red同源重组系统成功构建了肠炎沙门菌缺失株C50041△SEN2967、C50041△SEN3610*pBad24及以 pBad24 为载体的诱导型表达株 C50041△SEN2967(pBad24-SEN29673*Flag)、C50041△SEN3610(pBad24-SEN36103*Flag)。Western Blot 结果显示,诱导型表达株中SEN2967和SEN3610蛋白经诱导后均成功表达。生物学特性结果表明,SEN2967和SEN3610的缺失均不影响肠炎沙门菌的生理生化特性;小鼠体内竞争实验表明,SEN2967和SEN3610缺失株的致病力均明显低于野生株C50041。肠炎沙门菌SEN2967和SEN3610基因缺失株和诱导型表达株的成功构建为后期筛选SEN2967和SEN3610调控的基因表达研究提供了相应的生物材料。2肠炎沙门菌C50041转录因子SEN2967和SEN3610的蛋白质组学研究本研究通过双向电泳分析SEN2967和SEN3610缺失株与表达株的蛋白表达水平差异。分别提取SEN2967和SEN3610的缺失株和表达株的全菌蛋白,在确保上样量一致的前提下,进行双向凝胶电泳,银染后扫描获得2D图谱,经PD Quest软件分析,检测出SEN2967的缺失株和表达株有56个差异蛋白点,检测出SEN3610的缺失株和表达株有91个差异蛋白点。随后我们利用iTraQ技术检测缺失株与表达株中表达差异的蛋白谱,在差异倍数≥1.5或者≤0.67倍,P值≤0.05的条件下,SEN2967缺失株与诱导型表达株相比,上调表达的蛋白有81个,下调表达的蛋白有90个,共计差异数量171;SEN3610缺失株与表达株相比,上调表达的蛋白有25个,下调表达的蛋白有21个,共计差异数量46。这一结果与双向电泳均表明SEN2967和SEN3610基因的缺失或诱导表达可以引起细菌中其它蛋白表达的变化。iTraQ结果进一步显示,在SEN2967和SEN3610缺失株中下调表达的蛋白库中存在与T3SS紧密相关的蛋白,主要包括SPI-1和SPI-5的效应蛋白,以及与针状结构形成相关的蛋白。选择部分与T3SS相关且表达量差异明显的蛋白表达基因,利用荧光定量PCR检测转录水平的表达差异,结果表明转录水平与翻译水平的表达并非平行关系。本研究筛选获得的蛋白为进一步研究SEN2967和SEN3610在肠炎沙门菌转录调控过程中参与的信号通路奠定了基础。3肠炎沙门菌C50041转录因子SEN3610调控基因的EMSA验证为了筛选和鉴定转录因子SEN3610直接调控的基因,我们利用EMSA实验来检测转录因子和调控的基因启动子之间的相互作用。首先利用pMAL-5载体成功构建了 SEN3610原核表达载体,获得重组菌株、E.coli ER2523(pMAL-c5X-SEN3610)和E.coli ER2523(pMAL-p5X-SEN3610);纯化回收MBP-SEN3610融合蛋白约40 mg,并经透析后用于EMSA实验。根据第二章筛选获得的基因序列,分别定位其启动子序列,通过两级PCR合成5' FAM荧光标记的探针。在EMSA反应体系下验证SEN3610与各探针的结合情况。结果表明,SEN3610与hilA,hilD和pipD的启动子序列有明显的结合现象。这一结果表明SEN3610可能通过参与调控hilA和hilD的表达间接参与调控T3SS效应蛋白的表达。为深入研究T3SS的表达调控奠定了基础。
[Abstract]:Salmonella enteritidis (Salmonella enterica serovar Entrritidis, S.Enteritidis, SE) is a gram-negative bacteria and facultative intracellular, had pathogenicity to human and animal, is an important zoonotic pathogen. Transcription factors (Transcriptionfactor, TF) is a kind of combination of specific genes in specific nucleotide sequence upstream of the protein, and the transcriptional regulation of this gene. The transcription factor expression bacterial virulence factors, involved in the physiological and biochemical metabolism, so it is important to study the Salmonella transcription factor for research on the pathogenic mechanism of prevention and control of Salmonella. The expression of our previous study by SCOTS screening of Salmonella enteritidis infection of avian macrophage HD-11 process the gene transcription level two candidate transcription factor SEN2967 and SEN3610 genes were significantly higher than that of in vitro intracellular expression level under the state, but At home and abroad on the two putative transcription factor has not been reported. In this study, through the method of homologous recombination to construct Salmonella enteritidis SEN2967 and SEN3610 gene deletion strains and its inducible expression strain; and through two-dimensional electrophoresis, gene expression regulation of iTraQ technology on the two was analyzed for screening genes; EMSA is used to verify the.1 of Salmonella enteritidis C50041 transcription factor SEN2967 and SEN3610 mutant and the construction and identification of their biological characteristics comparison of Salmonella enteritidis C50041 infection of HD-11 cells after 5 h cultured in vitro and expression of transcription conditions by using fluorescence quantitative PCR expression strains, confirmed that SEN2967 was increased by about 98 times, about 15 times. The up regulation of SEN3610 study on the use of lambda -red homologous recombination system successfully constructed deletion mutant of Salmonella enteritidis C50041 Delta SEN2967, Delta SEN3610*pBad24 and C50041 using pBad24 as carrier Inducible expression strain C50041 (pBad24-SEN29673*Flag), SEN2967 C50041 SEN3610 (pBad24-SEN36103*Flag).Western Blot results showed that after induction the successful expression of inducible expression of SEN2967 and SEN3610 protein in plant biology. The results show that the deletion of SEN2967 and SEN3610 had no effect on physiological and biochemical characteristics of Salmonella enteritidis; experiments show that competition in mice. SEN2967 and SEN3610 mutants were significantly lower than those of the pathogenicity of Salmonella enteritidis SEN2967 and SEN3610 C50041. gene deletion strains and inducible expression strains of wild strains constructed for later screening SEN2967 and SEN3610 gene expression of proteome provides corresponding biological materials.2 Salmonella enteritidis C50041 transcription factor SEN2967 and SEN3610 of the study through two-dimensional electrophoresis SEN2967 and SEN3610 mutant strains and expression of protein expression level difference analysis. SEN2967 and SEN3610 mutant strains of bacteria and the expression of protein extraction, in the premise to ensure the amount on the same sample, by two-dimensional gel electrophoresis. After silver staining 2D scanned map, analyzed by PD Quest software, the detection of SEN2967 mutant strains and expression of 56 different protein points, the detection of SEN3610 the expression of mutant strains and 91 different protein points. Then we use iTraQ technology to detect missing lines and expression lines differentially expressed proteins in the spectrum, fold difference is more than 1.5 or less than or equal to 0.67 times the value of P is less than 0.05 under the condition of SEN2967 mutant and inducible expression strain by up-regulated protein 81, the down regulated proteins of 90, a total number of 171 different SEN3610 mutant strains and expression; compared with up-regulated protein 25, down regulate the expression of protein 21, a total number of 46. difference compared with the results of two-dimensional electrophoresis showed that SEN2967 and SEN36 are Deletion of 10 gene expression induced by.ITraQ or can cause change of expression of other proteins in bacteria results further show that the fall is closely related with the expression of T3SS protein in the library in SEN2967 and SEN3610 mutants, including the effect of protein SPI-1 and SPI-5, as well as related protein and needle like structure formation. Select the part with T3SS and the expression amount of obvious differences in gene expression by detecting differentially expressed transcription level of fluorescence quantitative PCR, the results showed that the expression level of transcription and translation is not the parallel relationship. This study screened the protein for further study of SEN2967 and SEN3610 signaling pathways involved in transcriptional regulation of Salmonella enteritidis in the process of laying the EMSA verification.3 Salmonella enteritidis C50041 transcription factor SEN3610 gene for screening and identification of transcription factor genes directly regulated by SEN3610, We use EMSA test to detect transcription factors and gene promoter interactions between. The pMAL-5 vector was successfully constructed a prokaryotic expression vector SEN3610, recombinant strains, E.coli ER2523 (pMAL-c5X-SEN3610) and E.coli ER2523 (pMAL-p5X-SEN3610); purified MBP-SEN3610 fusion protein was about 40 mg, and after dialysis for EMSA experiment according to the second chapter. Screening of gene sequences were obtained, respectively. The location of the promoter sequence, through the probe two PCR synthesis of 5'fluorescent labeled FAM. Combination of SEN3610 and verify the probe in the EMSA reaction system. The results show that the SEN3610 and hilA promoter sequences of hilD and pipD are combined with this phenomenon. Results show that by regulating the expression hilA and hilD SEN3610 may be involved in the regulation of expression of T3SS protein in the indirect effect. Laid the foundation for the further study on regulation of expression of T3SS.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378;S852.61

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