猪链球菌金属依赖转录调节基因mtsR研究

发布时间：2018-04-10 15:31

本文选题：猪链球菌2型 + 缺失突变株　；参考：《西南交通大学》2012年硕士论文

【摘要】：猪链球菌(Streptococcus suis, SS)为革兰阳性溶血性兼性厌氧球菌,根据其荚膜多糖抗原性的差异,分为35个血清型,其中以猪链球菌2型(SS2)中国分离株毒力最强、危害最严重、流行最广,是一种重要的人兽共患病病原体。我国江苏和四川部分地区曾经在1998年和2005年暴发SS2感染的大面积流行,导致上万头生猪死亡,上百人感染发病,几十人死亡,受到全球关注,猪链球菌2型感染已成为成为了公共卫生防疫的重点。虽然近年来我国对猪链球菌2型的毒力因子及其有关发病机制的研究取得了进展,但该菌生理生化、遗传特性、毒力和致病机理方面还有很多未解之谜,猪链球菌2型中国分离菌株如何获得必需的金属元素铁及其调控机理就是其中之一。为此,本研究通过细菌形态学观察、革兰氏染色镜检和16SrDNA序列同源性分析等方法与技术,从四川成都附近某养猪场分离获得一株与NCBI数据库中05ZYH33(猪链球菌2型中国分离毒株)同源性达到99%的猪链球菌菌株SS86；对该分离菌株的七个主要毒力因子CPS, MRP, EF, Sly, GDH, FBPS和ORF2的基因PCR检测结果表明,本实验分离菌株与目前报道的2型猪链球菌的多数致病株毒力因子分布情况一致；将分离菌株培养在添加浓度为250gM的铁专一螯合剂4,4'-联吡啶(Dipyridyl, DPD)的链球菌THYB培养基中,分离菌株SS86几乎不能生长,在含DPD螯合剂的THYB培养基中,分别添加1.5μM浓度的Fe2+,1.0μM浓度的Fe3+和30μM浓度的血红蛋白时,菌株SS86可恢复到正常生长水平,表明铁元素为猪链球菌SS86生长繁殖所必需；为了今后进一步弄清猪链球菌2型中国分离株铁元素吸收和调节机理,本实验以分离株SS86为出发菌株,采用基因敲除缺失突变/温度敏感自杀性质粒/电转化方法,构建了金属依赖转录调节基因mtsR缺失突变株。用RT-PCR检测mtsR基因缺失对猪链球菌金属离子吸收系统Fhu、Feo、Mut和Mts操纵子的金属离子绑定相关基因fhuA、feoA、mutA和mtsA以及铁吸收调节基因fur的表达影响,研究结果显示,野生菌株和mtsR基因缺失突变株的铁吸收操纵子金属元素绑定基因及调节基因fur表达存在差异,研究结果确认了猪链球菌SS86菌株MtsR影响猪链球菌铁吸收转运相关基因表达。本研究结果不仅让我们对猪链球菌中国分离株的毒力基因分布、铁元素对菌株生长繁殖影响以及金属依赖转录调节基因mtsR对金属元素吸收操纵子金属元素绑定基因及调节基因fur表达的调节有了较为系统的了解,同时也为我们今后进一步深入研究猪链球菌2型菌株金属离子吸收调控机理以及揭示猪链球菌致病机理奠定了基础。
[Abstract]:Streptococcus suis (SS2) is a Gram-positive hemolytic facultative anaerobic cocci. According to the antigenicity of its capsule polysaccharides, it is divided into 35 serotypes. Among them, Streptococcus suis type 2 (SS2) is the most virulent, most harmful and prevalent strain in China.Is an important zoonotic pathogen.In some parts of Jiangsu and Sichuan provinces in China, an outbreak of SS2 infection occurred in 1998 and 2005, resulting in the deaths of tens of thousands of live pigs, hundreds of people infected with the disease and dozens of deaths.Streptococcus suis type 2 infection has become the focus of public health epidemic prevention.Although recent progress has been made in the study of virulence factors of Streptococcus suis type 2 and its pathogenesis in China, there are still many unsolved questions about the physiological and biochemical characteristics, genetic characteristics, virulence and pathogenicity of Streptococcus suis.One of them is how to obtain essential metal element iron and its regulation mechanism of Streptococcus suis type 2 Chinese isolate.Therefore, the methods and techniques of bacterial morphology observation, Gram staining and homology analysis of 16SrDNA sequence were used in this study.A strain of Streptococcus suis SS86 with 99% homology with 05ZYH33in NCBI database was isolated from a pig farm near Chengdu, Sichuan Province.The results of PCR analysis of GDH, FBPS and ORF2 showed that,The distribution of virulence factors of most pathogenic strains of Streptococcus suis type 2 was consistent with that reported at present, and the isolated strains were cultured in the THYB medium of Streptococcus suis, which was supplemented with iron specific chelator (250gM), 4x4, dipyridyl, dipyridyl, dipyridyl, dipyridyl, dipyridyl, dipyridyl, and dipyridyl.The isolated strain SS86 could hardly grow. In the THYB medium containing DPD chelator, the strain SS86 could return to normal growth level when adding 1.5 渭 M Fe2 1.0 渭 M Fe3 and 30 渭 M hemoglobin, respectively.The results showed that iron element was necessary for the growth and reproduction of Streptococcus suis SS86, and in order to further understand the mechanism of iron absorption and regulation of Streptococcus suis type 2 Chinese isolate, the isolated strain SS86 was used as the starting strain.A metal-dependent transcription regulatory gene (mtsR) deletion mutant was constructed by gene knockout deletion / temperature-sensitive suicide plasmid / electrotransformation.RT-PCR was used to detect the effect of mtsR gene deletion on the expression of metal ion-binding genes FhuAfeo AmomutA and mtsA in the metal ion absorption system of Streptococcus suis and Mts operon, and the iron absorption regulatory gene fur.There were differences in the expression of iron absorption operon metal element binding gene and regulatory gene fur between wild strain and mtsR gene deletion mutant strain. The results confirmed that SS86 strain MtsR affected the expression of iron absorption transporter related genes of Streptococcus suis.The results of this study not only allowed us to distribute virulence genes in Chinese isolates of Streptococcus suis,The effect of iron on the growth and reproduction of the strain and the regulation of metal-dependent transcription regulator gene mtsR on the metal element absorption operon metal element binding gene and the regulation gene fur expression have been systematically understood.It also lays a foundation for further study on the regulation mechanism of metal ion absorption of Streptococcus suis type 2 strain and for revealing the pathogenic mechanism of Streptococcus suis in the future.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346;S855.1

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