本文選題：腸炎沙門菌　切入點(diǎn)：SEN2967　出處：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：腸炎沙門菌(Salmonella enterica serovar Entrritidis,S.Enteritidis,SE)是革蘭陰性兼性胞內(nèi)寄生菌,對人類和動物都具有致病性,是重要的人獸共患病原菌。轉(zhuǎn)錄因子(Transcriptionfactor,TF)是一類能夠結(jié)合在特定基因上游特異核苷酸序列上的蛋白,并能調(diào)控該基因的轉(zhuǎn)錄。轉(zhuǎn)錄因子可調(diào)控細(xì)菌毒力因子的表達(dá),參與生理生化代謝過程,因此對沙門菌轉(zhuǎn)錄因子的研究對于沙門菌致病機(jī)制與防控研究都具有重要意義。本實(shí)驗(yàn)室前期研究中利用SCOTS技術(shù)篩選腸炎沙門菌感染禽源巨噬細(xì)胞HD-11的過程中表達(dá)的基因,兩個候選轉(zhuǎn)錄因子SEN2967和SEN3610基因的胞內(nèi)轉(zhuǎn)錄水平顯著高于體外培養(yǎng)狀態(tài)下的表達(dá)水平,但國內(nèi)外對這兩個假定轉(zhuǎn)錄調(diào)控因子的研究未見報(bào)道。本研究通過同源重組的方法成功構(gòu)建了腸炎沙門菌SEN2967和SEN3610基因缺失株及其誘導(dǎo)型表達(dá)株;并通過雙向電泳、iTraQ技術(shù)對二者調(diào)控的基因表達(dá)進(jìn)行了分析;對于篩選獲得的基因,采用EMSA進(jìn)行驗(yàn)證。1腸炎沙門菌C50041轉(zhuǎn)錄因子SEN2967和SEN3610的缺失株與表達(dá)株的構(gòu)建及其生物學(xué)特性鑒定利用熒光定量PCR比較腸炎沙門菌C50041感染HD-11細(xì)胞5 h后與體外培養(yǎng)條件下轉(zhuǎn)錄水平的表達(dá)差異,證實(shí)SEN2967上調(diào)約98倍,SEN3610上調(diào)約15倍。本研究利用λ-red同源重組系統(tǒng)成功構(gòu)建了腸炎沙門菌缺失株C50041△SEN2967、C50041△SEN3610*pBad24及以 pBad24 為載體的誘導(dǎo)型表達(dá)株 C50041△SEN2967(pBad24-SEN29673*Flag)、C50041△SEN3610(pBad24-SEN36103*Flag)。Western Blot 結(jié)果顯示,誘導(dǎo)型表達(dá)株中SEN2967和SEN3610蛋白經(jīng)誘導(dǎo)后均成功表達(dá)。生物學(xué)特性結(jié)果表明,SEN2967和SEN3610的缺失均不影響腸炎沙門菌的生理生化特性;小鼠體內(nèi)競爭實(shí)驗(yàn)表明,SEN2967和SEN3610缺失株的致病力均明顯低于野生株C50041。腸炎沙門菌SEN2967和SEN3610基因缺失株和誘導(dǎo)型表達(dá)株的成功構(gòu)建為后期篩選SEN2967和SEN3610調(diào)控的基因表達(dá)研究提供了相應(yīng)的生物材料。2腸炎沙門菌C50041轉(zhuǎn)錄因子SEN2967和SEN3610的蛋白質(zhì)組學(xué)研究本研究通過雙向電泳分析SEN2967和SEN3610缺失株與表達(dá)株的蛋白表達(dá)水平差異。分別提取SEN2967和SEN3610的缺失株和表達(dá)株的全菌蛋白,在確保上樣量一致的前提下,進(jìn)行雙向凝膠電泳,銀染后掃描獲得2D圖譜,經(jīng)PD Quest軟件分析,檢測出SEN2967的缺失株和表達(dá)株有56個差異蛋白點(diǎn),檢測出SEN3610的缺失株和表達(dá)株有91個差異蛋白點(diǎn)。隨后我們利用iTraQ技術(shù)檢測缺失株與表達(dá)株中表達(dá)差異的蛋白譜,在差異倍數(shù)≥1.5或者≤0.67倍,P值≤0.05的條件下,SEN2967缺失株與誘導(dǎo)型表達(dá)株相比,上調(diào)表達(dá)的蛋白有81個,下調(diào)表達(dá)的蛋白有90個,共計(jì)差異數(shù)量171;SEN3610缺失株與表達(dá)株相比,上調(diào)表達(dá)的蛋白有25個,下調(diào)表達(dá)的蛋白有21個,共計(jì)差異數(shù)量46。這一結(jié)果與雙向電泳均表明SEN2967和SEN3610基因的缺失或誘導(dǎo)表達(dá)可以引起細(xì)菌中其它蛋白表達(dá)的變化。iTraQ結(jié)果進(jìn)一步顯示,在SEN2967和SEN3610缺失株中下調(diào)表達(dá)的蛋白庫中存在與T3SS緊密相關(guān)的蛋白,主要包括SPI-1和SPI-5的效應(yīng)蛋白,以及與針狀結(jié)構(gòu)形成相關(guān)的蛋白。選擇部分與T3SS相關(guān)且表達(dá)量差異明顯的蛋白表達(dá)基因,利用熒光定量PCR檢測轉(zhuǎn)錄水平的表達(dá)差異,結(jié)果表明轉(zhuǎn)錄水平與翻譯水平的表達(dá)并非平行關(guān)系。本研究篩選獲得的蛋白為進(jìn)一步研究SEN2967和SEN3610在腸炎沙門菌轉(zhuǎn)錄調(diào)控過程中參與的信號通路奠定了基礎(chǔ)。3腸炎沙門菌C50041轉(zhuǎn)錄因子SEN3610調(diào)控基因的EMSA驗(yàn)證為了篩選和鑒定轉(zhuǎn)錄因子SEN3610直接調(diào)控的基因,我們利用EMSA實(shí)驗(yàn)來檢測轉(zhuǎn)錄因子和調(diào)控的基因啟動子之間的相互作用。首先利用pMAL-5載體成功構(gòu)建了 SEN3610原核表達(dá)載體,獲得重組菌株、E.coli ER2523(pMAL-c5X-SEN3610)和E.coli ER2523(pMAL-p5X-SEN3610);純化回收MBP-SEN3610融合蛋白約40 mg,并經(jīng)透析后用于EMSA實(shí)驗(yàn)。根據(jù)第二章篩選獲得的基因序列,分別定位其啟動子序列,通過兩級PCR合成5' FAM熒光標(biāo)記的探針。在EMSA反應(yīng)體系下驗(yàn)證SEN3610與各探針的結(jié)合情況。結(jié)果表明,SEN3610與hilA,hilD和pipD的啟動子序列有明顯的結(jié)合現(xiàn)象。這一結(jié)果表明SEN3610可能通過參與調(diào)控hilA和hilD的表達(dá)間接參與調(diào)控T3SS效應(yīng)蛋白的表達(dá)。為深入研究T3SS的表達(dá)調(diào)控奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R378;S852.61

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