受轉(zhuǎn)化調(diào)控的肺炎鏈球菌體內(nèi)誘導(dǎo)基因的篩選及其表達(dá)產(chǎn)物定位
發(fā)布時(shí)間：2019-05-16 09:23
【摘要】： 目的 肺炎鏈球菌(streptococcus pneumoniae, S.pn)是一種常見的革蘭陽性條件致病菌,是引起肺炎、腦膜炎和中耳炎的主要病因。由于S.pn抗生素耐藥率的增加和目前疫苗的缺陷性,使得其感染的后果廣泛而嚴(yán)重,要從根本上解決S.pn的防治問題,就要深入了解其致病的分子機(jī)制。 轉(zhuǎn)化是指細(xì)菌形成感受態(tài),攝取外源性DNA的過程,細(xì)菌的自然轉(zhuǎn)化是許多重要生理現(xiàn)象的起始和源頭。S.pn自然轉(zhuǎn)化效率最高,大量證據(jù)表明轉(zhuǎn)化可導(dǎo)致細(xì)菌的毒力和耐藥性改變,然而目前人們并不清楚轉(zhuǎn)化如何調(diào)控其毒力。 細(xì)菌進(jìn)入宿主體內(nèi)后必須表達(dá)一些使自身毒力增強(qiáng)或?qū)λ拗鲹p傷加劇的基因,因此細(xì)菌在宿主體內(nèi)表達(dá)的基因往往就是與致病緊密相關(guān)的毒力基因。本課題組前期研究通過體內(nèi)表達(dá)技術(shù)(in vivo expression technology, IVET)和差異熒光誘導(dǎo)技術(shù)( differential fluorescence induction, DFI),篩選到了多個(gè)S.pn體內(nèi)誘導(dǎo)基因,其中部分基因功能未知。這些功能未知基因在體內(nèi)表達(dá)增加,與毒力密切相關(guān)。那么這些基因在宿主體內(nèi)是否受轉(zhuǎn)化調(diào)控與S.pn的條件致病相關(guān)呢?我們擬通過構(gòu)建轉(zhuǎn)化缺陷菌,來篩選受轉(zhuǎn)化調(diào)控的S.pn體內(nèi)誘導(dǎo)基因,為深入研究S.pn的致病機(jī)制奠定基礎(chǔ)。 為進(jìn)一步研究這些基因的功能,我們選擇受轉(zhuǎn)化調(diào)控的基因spd_0873和dnaJ進(jìn)行其表達(dá)產(chǎn)物的定位分析。為判斷亞細(xì)胞組分分離效果,我們選擇一個(gè)已知在細(xì)胞質(zhì)中恒定表達(dá)的蛋白CodY做對照。首先原核表達(dá)重組蛋白,制備多克隆抗體,然后通過western blot和流式細(xì)胞術(shù)分別對這2種蛋白進(jìn)行亞細(xì)胞定位分析。 方法 1.構(gòu)建轉(zhuǎn)化缺陷菌株comE是影響S.pn感受態(tài)形成的關(guān)鍵基因,失活基因comE使細(xì)菌不能發(fā)生轉(zhuǎn)化,因此通過插入失活構(gòu)建的D39 comE缺陷菌(D39△comE),即為轉(zhuǎn)化缺陷菌。 2.篩選受轉(zhuǎn)化調(diào)控的肺炎鏈球菌體內(nèi)誘導(dǎo)基因?qū)39 comE缺陷菌與D39野生菌同時(shí)腹腔注射小鼠后,用RT-PCR分析野生菌和缺陷菌中13個(gè)體內(nèi)誘導(dǎo)基因的mRNA表達(dá)差異,探討這些基因是否受轉(zhuǎn)化調(diào)控。 3.原核表達(dá)Spd_0873、CodY通過構(gòu)建PW28/0873、PW28/codY重組表達(dá)載體,表達(dá)并純化Spd_0873、CodY。 4.多克隆抗體的制備利用純化后的Spd_0873、CodY蛋白,分別免疫新西蘭大白兔和BALB/c小鼠,得到Spd_0873、CodY的多克隆抗體。 5.亞細(xì)胞組分分離及亞細(xì)胞定位分離D39的上清蛋白、細(xì)胞壁、原生質(zhì)體,通過western blot和流式細(xì)胞術(shù)分析Spd_0873、DnaJ(其多克隆抗體已由本實(shí)驗(yàn)室制備)的亞細(xì)胞定位。 結(jié)果 1.通過PCR和測序驗(yàn)證,D39 comE缺陷菌株構(gòu)建成功。 2. 8個(gè)體內(nèi)誘導(dǎo)基因在缺陷菌株和野生菌株中mRNA表達(dá)水平具有統(tǒng)計(jì)學(xué)差異(P0.05),其中spd_0300、spd_0414、spd_0622、spd_1663、spd_1719、spd_0235、spd_0873受轉(zhuǎn)化上調(diào),spd_1672受轉(zhuǎn)化下調(diào)。 3.通過測序和酶切鑒定PW28/0873、PW28/codY重組表達(dá)載體構(gòu)建成功,表達(dá)并純化得到Spd_0873、CodY。 4.經(jīng)過免疫大白兔和BALB/c小鼠,得到Spd_0873兔多克隆抗體、CodY鼠多克隆抗體。 5.流式細(xì)胞術(shù)分析得出蛋白Spd_0873和DnaJ在S.pn表面均有表達(dá),western blot鑒定DnaJ在細(xì)胞壁和原生質(zhì)體均有表達(dá)。 結(jié)論 1.篩選出受轉(zhuǎn)化上調(diào)的體內(nèi)誘導(dǎo)基因spd_0300、spd_0414、spd_0622、spd_1663、spd_1719、spd_0235、spd_0873及下調(diào)的基因spd_1672,它們可能參與生長調(diào)節(jié)、溫度感應(yīng)、糖脂代謝等環(huán)節(jié),表明細(xì)菌轉(zhuǎn)化可通過調(diào)節(jié)某些體內(nèi)誘導(dǎo)基因的表達(dá)來增強(qiáng)細(xì)菌的毒力。 2. Spd_0873蛋白在S.pn表面有表達(dá),是否在細(xì)菌其他部位也有表達(dá)需進(jìn)一步研究;DnaJ定位在S.pn的細(xì)胞壁和原生質(zhì)體,為下一步的功能研究提供了初步依據(jù)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378.14
本文編號：2478183
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378.14
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