【作者】 陈龙；

【导师】 生秀梅；

【作者基本信息】 江苏大学 ， 临床检验诊断学（专业学位）， 2017， 硕士

【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。更多还原

【Abstract】 Objective:mig-14 is a horizontal-transfer acquired gene of salmonella,plays an important role in resistance of bacteria to polymyxin B.Furthermore,MIG-14,the expression product of mig-14,acts as a regulator to regulate a series of genes.However,despite the critical role of mig-14,the expression mechanisms and characteristics of mig-14 remain unknown.This study aims to investigate the expression mechanisms and characteristics of mig-14 in Salmonella enterica serovar Typhi in different environments.Methods:1.The constructions of mutant strains of Salmonella enterica serovar Typhi The homologous recombination,which is mediated by suicide plasmid,was used to knock out Hil D,Him A,Him D,Ydg T,Hha,HNS in S.Typhi,and the Fis and Omp R mutant strains were stored in our laboratory.2.q RT-PCR q RT-PCR was used to analyse the transcriptional level of mig-14 in S.Typhi wild type strains and mutations.Analysed the difference of mig-14 transcriptional levels in S.Typhi derivatives,which were cultured in LB medium,to demonstrate whether mig-14 transcription is repressed by HNS,Hha and Ydg T or not in a non-selected environment;Analysed the difference of mig-14 transcriptional levels in S.Typhi derivatives,which were under polymyxin B exposure,to demonstrate whether mig-14 transcription is activated by regulator Hil D,Him A,Him D,Fis,Omp R or not.3.Western blot to detect the Mig-14 level The Mig-14 antiserum prepared in previous work was used to detect Mig-14 protein levels in S.Typhi strains.4.lacZ fusion experiment The recombinant plasmid p HRP309 was constructed.Amplified the promoter region of mig-14 using PCR;Inserted the promoter region into p HRP309 plasmid,located at the upstream of the β-galactosidase gene-lac Z.The recombinant p HRP309 plasmids were obtained and transferred into strains.According to the difference of the β-galactosidase contained in strains,we can determine the mig-14 transcriptional and expression levels.5.Protein expression and purification Expressed and purificated HNS and Him A(α subunit of IHF)by E.coli BL21 to investigate the relationships between these two proteins and mig-14.6.Electrophoretic mobility shift assay The promoter region of mig-14 was co-incubated with HNS or Him A,and then subjected into electrophoresis on 6% polyacrylamide nondenaturing gels to test the binding between proteins and the promoter DNAs.7.Analysis the influences of IHF,mig-14 to iagA Constructed a him A/mig-14 double gens mutant strain of S.Typhi.Constructed the heterologous expression plasmids of him A and mig-14.Transferred the heterologous expression plasmids into him A/mig-14 double gens mutant strain respectively,analysed the iag A transcriptional level to demonstrate the regulator relationship of IHF and Mig-14 to iag A under early hyperosmotic condition.Results:1.The hil D,him A,him D,hha and ydg T mutant strains of S.Typhi were successfully constructed.Because of the failure to knock out hns gene in S.Typhi after repeated attempts,we constructed a over-expression hns strain by transferring a heterologous expression plasmids of hns into S.Typhi,and sought to demonstrate that hns could repress mig-14 transcription.2.The results of qRT-PCR indicated that the transcription of mig-14 was repressed by silencing protein-HNS/Hha/Ydg T in a non-selected environment.Both of the transcription levels of mig-14 in hha and ydg T mutants were below which in wild type strain,what’s more,the deletion of hha resulted a considerable evaluation than the deletion of ydg T did.Furthermore,over-expression of hns in S.Typhi resulted a significantly reduction of the transcription levels of mig-14.In general,the transcription of mig-14 was repressed by silencing protein-HNS/Hha/Ydg T in a non-selected environment.3.Exposed S.Typhi under polymyxin B to activate the transcription and expression of mig-14.The q RT-PCR results showed that the transcriptional levels of mig-14 were significantly reduced in fis,omp R,hil D,him A and him D mutant strains compared with the wild type strain.The results of lac Z fusion assay also indicated that the promoter activities of mig-14 were also significantly decreased in fis,omp R,hil D,him A and him D mutant strains compared with the wild type strain.4.We sought to use the Mig-14 antiserum which was prepared in previous work to test Mig-14 levels in S.Typhi strains.Unfortunately,we could not find a significantly specific bind at the corresponding site even though we checked it by mig-14 deletion mutant.So we concluded that the Mig-14 antiserum lost the specificity to Mig-14 protein.5.HNS was successfully expressed using plasmid pET-28 a,Him A was expressed utilizing p ET-15 b.Both of the proteins with 6x His Tag were purified using a Ni-colum.6.Both of HNS and Him A could bind to the promoter region of mig-14.7.him A/mig-14 double gens mutant strain of S.Typhi was successfully constructed.The results of q RT-PCR showd that the transcripition level of iag A was significantly increased after transferring a heterologous expression plasmids of him A while heterologous expression plasmids of mig-14 only could not induce the iag A.Conclusion:As a horizontal-transfer acquired gene in S.Typhi,mig-14 is repressed by silencing protein--HNS/Hha/Ydg T in a non-selected environment.When bacteria suffering from polymyxin B,the silencing of mig-14 relieves.In this study,we demonstrated that the regulators Fis,Omp R,IHF and Hil D participated in the activation of mig-14 when S.Typhi under polymyxin B exposure.HNS and Him A,as important regulators,could bound to the promoter region of mig-14.Previously,we found Mig-14 could regulate many genes located at SPI-1 which could be activated by iag A.And now we discovered that IHF could activated iag A and mig-14 in early hyperosmotic stress.More important than all of that,Mig-14 activated iag A depended on IHF.更多还原