副溶血弧菌生物膜及LacZ报告基因融合实验技术平台的建立

发布时间：2018-04-09 01:30

本文选题：VP　切入点：生物膜表型　出处：《四川农业大学》2012年硕士论文

【摘要】：背景：副溶血弧菌(Vibrio parahaemolyticus, VP)是一种革兰阴性嗜盐弧菌。广泛分布于海洋和其他咸水环境中,多数海产品均天然携带本菌,人们食入未煮熟或烹饪不当的产品,会引起胃肠炎或食物中毒,其流行具有季节性,一般温暖的月份(5月-10月)易爆发。该菌最早是从1950年日本大阪的海产品食物中毒事件体检中筛选出来,此事件中由该菌引发上的食物中毒占40-60%。近几年由VP引起急性胃肠炎占世界之首,在我国,特别是一些沿海城市,中毒事件在数量上超过沙门氏菌,是食源性病原菌之首。其主要症状表现为：腹泻、下腹绞痛、反胃、呕吐,治疗不及时会引起败血病及组织感染,严重者甚至死亡。因此,为降低食源性疾病的风险,有必要对副溶血弧菌的致病机制进行深入研究。副溶血弧菌在生存和感染的过程中都能形成生物膜,同霍乱弧菌(Vibrio cholerae),哈氏弧菌(Vibrio harveyi)及创伤弧菌(Vibrio vulnificus)一样,生物膜在其适应环境和广泛传播过程中发挥着重要作用。生物膜的形成和消散是一个动态过程,这过程存在一系列基因表达的变化,而主要调控这些基因表达的就是密度感应(Quorum Sensing,QS)系统,VP的QS系统调控子蛋白OpaR与哈氏弧菌的LuxR在氨基酸序列上有96%的同源性,因此二者的QS系统应该具有相似的作用机制。虽然表型实验及表达谱分析表明OpaR能调控VP荚膜多糖基因、胞外多糖基因、c-di-GMP分子代谢及鞭毛基因等生物膜形成相关基因的表达,但是OpaR对这些基因的转录调控机制并未被阐明,因此有必要做进一步的研究。目的：本研究目的期望建立副溶血弧菌RIMD2210633生物膜表型分析实验方案和LacZ实验的技术平台。为进一步研究转录调控子OpaR的转录调控机制,以及副溶血弧菌后其他调控子的功能奠定基础。方法：(1)生物膜表型实验方案：生物膜形成过程中可能会受很多因素的影响,包括温度、pH、盐度、营养状况、培养方式及培养基等,通过对这些因素的比较和分析,建立了3种不同的生物膜表型分析方法(菌落表面褶皱、生物膜基质的刚果红染色、生物膜的结晶紫染色)。并通过对VP突变株(ΔopaR、ΔtoxR)与野生株(J5421)的生物膜测定分析,来验证这三个生物膜表型试验各自的技术平台的稳定性。 (2)LacZ报告基因融合实验技术：PCR扩增靶基因的整个启动子区序列,并将其直接克隆入pHRP309质粒中,构建重组质粒。将VPA1513重组质粒转入VP野生株(WT)和OpaR突变株(AopaR)中,而后通过比较二者β-半乳糖苷酶活性的差异,确定OpaR对VPA1513的调控关系,以检验实验平台的稳定性。结果：30℃比37℃更适合VP生长而形成生物膜,在两种温度下,200rpm振荡培养12h就到平台期了；VP在营养丰富的HI平板比营养次之的LB、M、APW#3平板的菌落褶皱更明显；盐浓度越低(为0.5%)褶皱越明显；静置培养更适合褶皱表型,100rpm摇动培养方式更适合生物膜形成的定量测定；生物膜的形成在中性和偏碱环境中的差别不大；培养时间对刚果红染色的影响没有菌落表面褶皱那么明显,但都有培养时间越长表型越明显的趋势；三种生物膜表型分析试验都证明了OpaR负调控和ToxR正调控生物膜形成。用载体pHRP309成功构建出9个与VP生物膜或毒力相关基因的的LacZ(?)重组质粒；并通过测定VP突变株AopaR与野生株(J5421)β-半乳糖苷酶活性的差异,证明了OpaR对VPA1513的转录具有抑制作用。结论：成功建立了菌落表面褶皱、生物膜基质的刚果红染色、生物膜的结晶紫染色三种VP生物膜相关的表型试验和LacZ报告基因融合实验的稳定的技术平台。发现了温度、盐浓度、培养时间、培养方式和营养状况对生物膜形成有影响,且偏碱环境不会影响生物膜的形成。本研究给后续生物膜表型和其他转录调控机制的研究奠定了基础。
[Abstract]:Background: Vibrio parahaemolyticus (Vibrio parahaemolyticus VP) is a gram-negative halophilic Vibrio. Widely distributed in marine and other saltwater environment, most of the seafood were carrying the natural bacteria, people undercooked or improper cooking products, can cause gastroenteritis or food poisoning, which is seasonal, general the warm months (May -10 months) explosive. The strain was first screened from medical poisoning incident in Japan in 1950 Osaka seafood food, this incident caused by the bacteria on food poisoning accounted for 40-60%. in recent years caused by VP of acute gastroenteritis accounted for first in the world, in our country, especially in some coastal areas city, poisoning more than Salmonella in quantity, is the first food borne pathogens. The main symptoms are diarrhea, abdominal cramps, nausea, vomiting, not timely treatment will cause septicemia and tissue infection, severe and even Death. Therefore, in order to reduce the risk of food borne diseases, it is necessary to study the pathogenesis of Vibrio parahaemolyticus.
Vibrio parahaemolyticus could form biofilm in the process of survival and infection with Vibrio cholerae (Vibrio, cholerae), Vibrio harveyi (Vibrio harveyi) and Vibrio vulnificus (Vibrio vulnificus), biofilm plays an important role in its adaptation to environment and wide spread process. The formation and dissipation of biofilm is a a dynamic process, this process has a series of changes in the expression of genes, which mainly regulates the expression of these genes is induction density (Quorum Sensing, QS) system, has 96% homology in amino acid sequence of the VP QS system regulator protein OpaR and Vibrio harveyi LuxR, so QS system two. Should have a similar mechanism. Although the expression and spectrum analysis show that OpaR can regulate VP capsular polysaccharide gene phenotype experiments, the extracellular polysaccharide gene, expression of related gene c-di-GMP molecular metabolism and flagellar gene biological membrane, but OpaR The transcriptional regulation mechanism of these genes has not been clarified, so it is necessary to do further research.
Objective: the aim of this study is to establish the experimental program of RIMD2210633 biofilm phenotypic analysis of Vibrio parahaemolyticus and the technological platform of LacZ experiment, in order to further study the transcriptional regulation mechanism of transcriptional regulator OpaR and the function of other regulators of Vibrio parahaemolyticus.
Methods: (1) experimental scheme of biofilm phenotype: biofilm formation may be affected by many factors in the process of pH, including temperature, salinity, nutrient status, culture method and culture medium, through the comparison and analysis of these factors, we establish 3 different biofilm phenotype (colony surface analysis method fold, biofilms stained with Congo red and crystal violet staining) and biofilm. The VP mutant (delta opaR, Delta toxR) and wild-type (J5421) determination of the biological membrane technology platform to verify the stability of the three biofilm phenotype test respectively.
(2) LacZ reporter gene fusion experiments: PCR sequence was amplified by the entire promoter region of target genes, and directly cloned into pHRP309 plasmid, to construct recombinant plasmid VPA1513. The recombinant plasmid was transformed into VP wild strain (WT) and OpaR mutant (AopaR), then the difference of beta galactosidase activity comparison of the two, to determine the regulation between OpaR to VPA1513, to test the stability of the experimental platform.
Results: 30 degrees centigrade than 37 DEG C is more suitable for VP growth and biofilm formation, at the two temperatures, 200rpm 12h oscillating culture to the platform period; VP HI tablet in nutrient rich nutrition than the LB, M, APW#3 were more obvious fold flat; the lower the concentration of salt (0.5%) fold more obvious; static culture is more suitable for the quantitative determination of 100RPM phenotype fold, shaking more suitable for biofilm formation in culture; formed in neutral and alkaline environment is not very different biofilm; effect of culture time on the Congo red staining of the colony surface folds not so obvious, but have a longer phenotype more training obvious; three kinds of biofilm phenotype tests have proved that the OpaR negative regulation and ToxR positive regulation of biofilm formation.
9 recombinant plasmids with VP biofilm or virulence related genes were successfully constructed by vector pHRP309. The difference between VP mutant AopaR and wild J5421 (J5421) beta galactosidase activity was proved by OpaR, which proved that OpaR had inhibitory effect on VPA1513 transcription.
Conclusion: we have successfully established the colony surface folds, Congo red staining of the biofilm matrix, crystal violet staining of the biofilm stable technology platform of three kinds of VP biofilm phenotype correlation test and LacZ reporter gene fusion experiments. It is found that the temperature, salt concentration, culture time, culture and nutrition effect of biofilm the formation, and the alkaline environment will not affect biofilm formation. This study laid the foundation for the subsequent biofilm phenotype and transcriptional regulation.

【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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