受转化调控的肺炎链球菌体内诱导基因的筛选及其表达产物定位

发布时间：2019-05-16 09:23

【摘要】： 目的肺炎链球菌(streptococcus pneumoniae, S.pn)是一种常见的革兰阳性条件致病菌,是引起肺炎、脑膜炎和中耳炎的主要病因。由于S.pn抗生素耐药率的增加和目前疫苗的缺陷性,使得其感染的后果广泛而严重,要从根本上解决S.pn的防治问题,就要深入了解其致病的分子机制。转化是指细菌形成感受态,摄取外源性DNA的过程,细菌的自然转化是许多重要生理现象的起始和源头。S.pn自然转化效率最高,大量证据表明转化可导致细菌的毒力和耐药性改变,然而目前人们并不清楚转化如何调控其毒力。细菌进入宿主体内后必须表达一些使自身毒力增强或对宿主损伤加剧的基因,因此细菌在宿主体内表达的基因往往就是与致病紧密相关的毒力基因。本课题组前期研究通过体内表达技术(in vivo expression technology, IVET)和差异荧光诱导技术( differential fluorescence induction, DFI),筛选到了多个S.pn体内诱导基因,其中部分基因功能未知。这些功能未知基因在体内表达增加,与毒力密切相关。那么这些基因在宿主体内是否受转化调控与S.pn的条件致病相关呢?我们拟通过构建转化缺陷菌,来筛选受转化调控的S.pn体内诱导基因,为深入研究S.pn的致病机制奠定基础。为进一步研究这些基因的功能,我们选择受转化调控的基因spd_0873和dnaJ进行其表达产物的定位分析。为判断亚细胞组分分离效果,我们选择一个已知在细胞质中恒定表达的蛋白CodY做对照。首先原核表达重组蛋白,制备多克隆抗体,然后通过western blot和流式细胞术分别对这2种蛋白进行亚细胞定位分析。方法 1.构建转化缺陷菌株comE是影响S.pn感受态形成的关键基因,失活基因comE使细菌不能发生转化,因此通过插入失活构建的D39 comE缺陷菌(D39△comE),即为转化缺陷菌。 2.筛选受转化调控的肺炎链球菌体内诱导基因将D39 comE缺陷菌与D39野生菌同时腹腔注射小鼠后,用RT-PCR分析野生菌和缺陷菌中13个体内诱导基因的mRNA表达差异,探讨这些基因是否受转化调控。 3.原核表达Spd_0873、CodY通过构建PW28/0873、PW28/codY重组表达载体,表达并纯化Spd_0873、CodY。 4.多克隆抗体的制备利用纯化后的Spd_0873、CodY蛋白,分别免疫新西兰大白兔和BALB/c小鼠,得到Spd_0873、CodY的多克隆抗体。 5.亚细胞组分分离及亚细胞定位分离D39的上清蛋白、细胞壁、原生质体,通过western blot和流式细胞术分析Spd_0873、DnaJ(其多克隆抗体已由本实验室制备)的亚细胞定位。结果 1.通过PCR和测序验证,D39 comE缺陷菌株构建成功。 2. 8个体内诱导基因在缺陷菌株和野生菌株中mRNA表达水平具有统计学差异(P0.05),其中spd_0300、spd_0414、spd_0622、spd_1663、spd_1719、spd_0235、spd_0873受转化上调,spd_1672受转化下调。 3.通过测序和酶切鉴定PW28/0873、PW28/codY重组表达载体构建成功,表达并纯化得到Spd_0873、CodY。 4.经过免疫大白兔和BALB/c小鼠,得到Spd_0873兔多克隆抗体、CodY鼠多克隆抗体。 5.流式细胞术分析得出蛋白Spd_0873和DnaJ在S.pn表面均有表达,western blot鉴定DnaJ在细胞壁和原生质体均有表达。结论 1.筛选出受转化上调的体内诱导基因spd_0300、spd_0414、spd_0622、spd_1663、spd_1719、spd_0235、spd_0873及下调的基因spd_1672,它们可能参与生长调节、温度感应、糖脂代谢等环节,表明细菌转化可通过调节某些体内诱导基因的表达来增强细菌的毒力。 2. Spd_0873蛋白在S.pn表面有表达,是否在细菌其他部位也有表达需进一步研究;DnaJ定位在S.pn的细胞壁和原生质体,为下一步的功能研究提供了初步依据。
[Abstract]:Purpose Streptococcus pneumoniae (S. pn) is a common Gram-positive condition pathogen, which is the main cause of pneumonia, meningitis and otitis media. In order to solve the problem of the prevention and treatment of S. pn, it is necessary to understand the cause of S. pn. Submechanism. Transformation refers to the process of forming a competent state of bacteria and taking exogenous DNA. The natural transformation of bacteria is the beginning of many important physiological phenomena and the natural transformation efficiency of the source .S.pn is the highest, and a large amount of evidence indicates that the transformation can lead to the toxicity of bacteria. The change in force and resistance, however, is not well known at present How to control its virulence. After the bacteria enter the host, it is necessary to express a number of genes that increase their virulence or to increase the damage to the host, so that the genes that are expressed in the host are often the same as those expressed in the host. A number of S. pn-induced genes were screened by in vivo expression technology (IVET) and differential fluorescence induction (DFI). in which part of the gene function is unknown. The unknown genes of these functions are in vivo In addition, it is closely related to the virulence, and whether these genes are regulated and controlled by the transformation in the host We intend to screen the S. pn in vivo induced by the transformation and control the S. pn in order to study the S. In order to further study the function of these genes, we select the genes spd _ 0873 and d, which are subject to the transformation and control. naJ carries out the location analysis of its expression product. In order to determine the effect of the separation of subcellular components, we select one known to be in the cytoplasm Prokaryotic expression of recombinant protein, preparation of polyclonal antibody, Western blot and flow cytometry, respectively That's right. Method 1. Construction of a transformed defective strain comE is a key gene that affects the formation of S. pn competent state. And after the mice were intraperitoneally injected with D39comE and D39 wild bacteria,13 of the wild bacteria and the defective bacteria were analyzed by RT-PCR. 3. Prokaryotic expression Spd _ 0873, Cogeneration by constructing PW28/0873, PW2 8/ codeY recombinant expression vector, expression and purification of Spd _ 0873, Cod.4. polyclonal antibody preparation and purification of Spd _ 0873, CodY protein, respectively. Rabbit and BALB/ c mice were used to obtain the polyclonal antibody of Spd _ 0873 and CodY.5. The isolation of the subcellular components and the isolation of the subcellular components and the isolation of the supernatant from the D39, the cell wall, the protoplast, the western blot and the flow. endocytosis Analysis of Spd _ 0873, DnaJ (its polyclonal antibody has been prepared by this laboratory) Results 1. The construction of the D39comE deficient strain was successful by PCR and sequencing.2. The expression of the mRNA in the 8 in-vivo induced gene in the defective strain and the wild strain was statistically different (P0.05), with spd _ 0300, spd _ 0414, spd _ 0622, spd _ 1663, spd _ 1719, spd _ 0235, spd _ 0873 was up-regulated, and spd _ 1672 was downregulated.3. by sequencing And the expression and purification of the PW28/ coding recombinant expression vector are successful, expressed and purified to obtain Spd _ 0. 873, CoCoY.4. The polyclonal antibody of Spd _ 0873 was obtained by immunizing the white rabbits and the BALB/ c mice. endocytosis The expression of protein Spd _ 0873 and DnaJ on the surface of S. pn was analyzed, and the expression of DnaJ in cell wall and protoplast was identified by western blot. Conclusion 1. The in vivo induced gene spd _ 0300, spd _ 0414, spd _ 0622, sp. d _ 1663, spd _ 1719, spd _ 0235, spd _ 08 73 and down-regulated genes spd _ 1672, which may be involved in growth regulation, temperature sensing, glycolipid metabolism, etc., indicate that bacterial transformation can enhance the virulence of bacteria by regulating the expression of the induced genes in some bodies.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378.14

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