伤寒沙门菌mig-14基因表达特性研究

发布时间：2018-06-12 01:10

本文选题：伤寒沙门菌 + mig-14　；参考：《江苏大学》2017年硕士论文

【摘要】：目的:mig-14是沙门菌属通过水平转移获得的外来毒力基因,其产物蛋白Mig-14在沙门菌属拮抗多粘菌素B杀伤的过程中扮演着重要的角色。并且作为一种调节因子,Mig-14可调控多种基因表达。但是,对于mig-14的表达机制与特性仍然未明。本研究旨在探索不同环境中伤寒沙门菌中mig-14基因的表达特性。方法:1.伤寒沙门菌缺失变异株的制备使用自杀质粒法,利用同源重组的原理,分别制备伤寒沙门菌调节因子Hil D、Him A、Him D、Ydg T、Hha、HNS的缺失变异株,Fis及Omp R缺失变异株由实验室保存。2.q RT-PCR分析mig-14转录水平采用实时定量PCR技术,观察伤寒沙门菌野生株及各缺陷变异株中mig-14基因的转录水平。普通LB(Luria-Bertani培养基)培养条件下,比较伤寒沙门菌野生株及ydg T、hha、hns缺失变异株中mig-14基因的转录水平,从而判断mig-14作为伤寒沙门菌水平转移获得的外来基因,其转录是否被沉默蛋白(Ydg T、Hha、HNS)抑制;多粘菌素B(polymyxin,PB)刺激条件下,比较伤寒沙门菌野生株及hil D、him A、him D、fis、omp R缺失变异株中mig-14的转录水平,从而判断mig-14在PB存在的情况下,其转录是否由调节因子Hild、Him A、Him D、Fis、Omp R激活。3.Western blot免疫印迹法检测Mig-14表达水平实验室存有Mig-14兔多克隆抗体,收集伤寒沙门菌及各缺失变异株全菌蛋白,以Mig-14兔多克隆抗体进行Western blot免疫印迹实验从而分析Mig-14在各菌株中的表达情况。4.lacZ融合实验构建mig-14启动子区域的lacZ基因融合质粒pHRP309。PCR扩增mig-14启动子区域,与含β-半乳糖苷酶基因(lac Z)的质粒载体p HRP309连接构建mig-14启动子-lac Z融合质粒。将融合质粒导入伤寒沙门菌野生株和各缺失变异株中,细菌胞内的β-半乳糖苷酶活性即可表示mig-14的转录表达水平。裂解细菌,收集上清,测定β-半乳糖苷酶活性,以Miller Units表示。5.蛋白表达及纯化利用大肠杆菌BL21系统,分别表达纯化沉默蛋白HNS以及宿主整合因子IHF的α亚基Him A,以进一步分析调节因子对mig-14的调控作用。6.凝胶阻滞实验(EMSA)通过体外凝胶阻滞实验(EMSA)分析调节因子HNS、Him A对mig-14表达的调节机制。7.分析IHF、Mig-14对iagA的影响构建伤寒沙门菌的himA/mig-14双基因缺失变异株,构建him A和mig-14的p BAD33异源表达载体,双缺失变异株分别回补him A和mig-14后,观察高渗早期iag A基因的转录情况。结果:1.伤寒沙门菌基因缺失变异株的制备成功制备伤寒沙门菌hil D、him A、him D、hha及ydg T的缺失变异株,并经序列鉴定分析验证;伤寒沙门菌hns的缺失变异株构建失败,遂通过构建伤寒沙门菌hns高表达株反向分析HNS对mig-14的抑制。2.普通LB培养条件下沉默蛋白抑制mig-14转录利用实时定量PCR(q RT-PCR)比较伤寒沙门菌野生株及hha、ydg T缺失变异株及hns高表达株中的mig-14的转录水平。结果显示hha、ydg T缺失变异株中mig-14转录水平明显高于伤寒沙门菌野生株,且缺失了hha后mig-14转录水平上升明显高于ydg T缺失变异株。另外,伤寒沙门菌GIFU10007的hns基因无法敲除,故通过构建hns高表达菌株来反映HNS对mig-14的抑制作用。结果显示,伤寒沙门菌hns高表达菌株的mig-14转录水平明显低于野生株。这表明,沉默蛋白HNS、Hha、Ydg T抑制外来基因mig-14的转录表达。3.PB刺激下野生株及各缺失变异株中mig-14转录水平PB可刺激mig-14表达。实时定量PCR及lac Z融合实验结果均显示:在PB刺激下,伤寒沙门菌的hil D、him A、him D、fis、omp R缺失变异株同野生株相比,mig-14转录表达水平均明显下降。结果表明,在PB刺激下,伤寒沙门菌调节因子Hil D、Fis、IHF(Him A/Him D)、Omp R均参与激活mig-14。4.Western blot免疫印迹法检测Mig-14表达水平利用实验室前期工作制备的Mig-14兔多克隆抗体,检测各菌株中Mig-14表达水平。结果发现伤寒沙门菌mig-14缺失变异株及野生株在目的条带处(Mig-14大小约36.5 KD)无明显差异条带,故认为Mig-14兔多克隆抗体失效。5.成功表达伤寒沙门菌HNS蛋白及IHF的α亚基Him A成功构建p ET28a-HNS、p ET15b-Him A及p ET15b-Him D表达载体,利用大肠杆菌BL21表达系统,成功表达并纯化得到HNS及Him A蛋白(Him D形成包涵体,尝试多次后均无法得到有效活性形式)。6.凝胶阻滞实验(EMSA)结果显示,HNS及Him A都能与mig-14基因的启动子区域结合,形成DNA-蛋白复合物,延缓DNA迁移速率。这也意味着,调节因子HNS及Him A能直接作用于mig-14的启动子区域发挥调节作用。7.高渗早期宿主整合因子(IHF)激活mig-14表达可增加iagA的转录成功构建伤寒沙门菌him A/mig-14双缺失变异株。分别回补him A和mig-14后发现,在高渗早期,回补him A后,iag A转录水平同双缺陷相比明显上升;而回补mig-14后,iag A转录水平同双缺陷相比并无明显统计学差异。结果表明,高渗早期,IHF激活mig-14与iag A,Mig-14也能激活iag A,但Mig-14发挥激活iag A的作用依赖IHF的存在。结论:mig-14作为伤寒沙门菌水平转移获得的外来基因,其表达受沉默蛋白HNS、Hha、Ydg T抑制;多粘菌素B刺激下,调节因子Hil D、Fis、IHF(Him A/Him D)、Omp R均参与激活mig-14的转录表达。沉默蛋白HNS及宿主整合因子(IHF)的α亚基Him A能直接与mig-14的启动子结合,调节mig-14转录。本实验室前期工作发现Mig-14在高渗早期大量表达并能影响SPI-1上基因的表达,本次工作发现Mig-14在高渗早期对iag A及下游致病岛1的调节作用依赖于宿主整合因子(IHF)。这些证据初步揭示了Mig-14作为一种新的调节因子在复杂的细菌调节网络中的角色。
[Abstract]:Objective: mig-14 is a foreign virulence gene obtained by the level transfer of Salmonella, and its product protein Mig-14 plays an important role in the process of inhibiting the killing of polymyxin B by Salmonella. And as an regulatory factor, Mig-14 can regulate the expression of multiple genes. However, the mechanism and characteristics of mig-14 are still unknown. The aim of this study is to explore the expression characteristics of mig-14 gene in Salmonella typhi in different environments. Methods: the preparation of the deletion mutant of 1. Salmonella typhi was prepared by using the suicide plasmid method. Using the principle of homologous recombination, Hil D, Him A, Him D, Ydg T, Hha, HNS mutant strain were prepared respectively. The transcriptional level of the mig-14 gene in the wild Salmonella typhi strain and the defective variants was observed by real-time quantitative PCR technique, and the transcriptional level of the mig-14 gene in the wild Salmonella typhi and the defective variants was observed by.2.q RT-PCR analysis. The transcriptional level of the mig-14 genes in the wild strains of Salmonella typhi and YDG T, HHA, HNS deletion mutants were compared under the normal LB (Luria-Bertani medium) culture. To judge whether the transcription of mig-14 as an alien gene of Salmonella typhi was suppressed by Ydg T (Hha, HNS), and the transcriptional level of the wild strains of Salmonella typhi and HIL D and him A were compared under the conditions of polymyxin (PB). In the case, whether the transcriptional factor Hild, Him A, Him D, Fis, Omp R activated.3.Western blot to detect the Mig-14 rabbit polyclonal antibody in the Mig-14 expression laboratory, and collect the total bacterial protein of Salmonella typhimurium and the missing mutant strain. The expression of -14 in each strain.4.lacZ fusion experiment constructed the lacZ gene fusion plasmid pHRP309.PCR of the mig-14 promoter region to amplify the mig-14 promoter region and construct the mig-14 promoter -lac fusion plasmid with the plasmid carrier P HRP309 containing the beta galactosidase gene (LAC Z). The fusion plasmid was introduced into the wild Salmonella typhi strain and the wild strain. Among the missing variants, the activity of beta galactosidase in bacterial cell could indicate the transcriptional expression level of mig-14. Lysate bacteria, collect supernatant, determine the activity of beta galactosidase, express the expression of.5. protein by Miller Units and use the BL21 system of Escherichia coli to express the purified silencing protein HNS and the host integrator IHF respectively. Subunit Him A, in order to further analyze the regulatory role of regulatory factors on mig-14,.6. gel block experiment (EMSA) through in vitro gel block test (EMSA) analysis of regulatory factor HNS, Him A's regulation mechanism for mig-14 expression.7. analysis IHF. 14 P BAD33 heterologous expression vector, double deletion mutants were added to him A and mig-14 to observe the transcriptional situation of IAG A gene in early hypertonic. Results: the preparation of the gene deletion mutant of Salmonella typhi was successfully prepared to prepare HIL D, him A, him him A, and the sequence identification and validation; Salmonella typhi. The failure of the deletion mutant of HNS was constructed by constructing the HNS high expression strain of Salmonella typhimurium in reverse analysis of the inhibition of mig-14 by HNS and the inhibition of mig-14 transcriptional utilization of mig-14 transcriptional using real-time quantitative PCR (Q RT-PCR) compared with the wild strains of Salmonella typhi and HHA, YDG deletion mutant and high expression strain of the transcriptional water The results showed that the mig-14 transcriptional level in the HHA YDG T deletion mutant was significantly higher than that of the wild Salmonella typhi strain, and the mig-14 transcriptional level increased significantly higher than that of the YDG T deletion mutant. In addition, the HNS gene of the GIFU10007 Salmonella typhi could not be knocked out, so the inhibitory effect of HNS to HNS was reflected by the construction of the HNS high expression strain. The results showed that the mig-14 transcriptional level of the HNS high expression strain of Salmonella typhi was significantly lower than that of the wild strain. This indicated that the silencing protein HNS, Hha, Ydg T inhibited the transcription of the foreign gene mig-14, and the mig-14 transcriptional level PB stimulated mig-14 expression in the wild strain and the missing variant strains. Under the stimulation of PB, the HIL D, him A, him D, FIS, OMP R deletion mutants of the Salmonella typhi were significantly lower than those of the wild strains. The Mig-14 rabbit polyclonal antibody prepared by the early laboratory work was used to detect the Mig-14 expression level in each strain. The results showed that the mig-14 deletion mutant and the wild strain of Salmonella typhi had no distinct difference bands at the destination strip (Mig-14 size about 36.5 KD). Therefore, the Mig-14 rabbit polyclonal antibody failed.5. to successfully express the HNS of Salmonella typhimurium. The protein and the alpha subunit Him A of IHF successfully construct P ET28a-HNS, P ET15b-Him A and P ET15b-Him D expression vector, and use the Escherichia coli BL21 expression system. Combining with the promoter region of the mig-14 gene to form a DNA- protein complex and delay the migration rate of DNA, it also means that the regulatory factor HNS and Him A can directly act on the promoter region of mig-14 and play a regulatory role in the early stage of.7. hyperosmotic host integration factor (IHF) activation mig-14, which can increase iagA transcriptional success for the construction of Salmonella typhi him A/mig-14 double deletion mutants. After supplementing him A and mig-14, the transcriptional level of IAG A increased obviously in the early stage of hypertonic, after the recharge of him A, while the IAG A transcription level was not significantly different from those of the double defects. But the role of Mig-14 to activate IAG A depends on the existence of IHF. Conclusion: mig-14 is a foreign gene obtained by the horizontal transfer of Salmonella typhi, its expression is inhibited by the silent protein HNS, Hha, Ydg T; under the stimulation of the polymyxin B, the regulatory factor Hil D is involved in the activation of the transcriptional expression. The alpha subunit Him A of the combination factor (IHF) can directly bind to the promoter of mig-14 and regulate mig-14 transcription. Earlier work in our laboratory found that Mig-14 was expressed in early hypertonic and could affect the expression of genes on SPI-1. This work found that the regulation of Mig-14 on IAG A and downstream pathogeny island 1 in early hypertonic is dependent on host integration factor (IHF). These evidence preliminarily reveals the role of Mig-14 as a new regulatory factor in complex bacterial regulatory networks.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378

【参考文献】