單增李斯特菌ΔInlAB、ΔInlABC突變株的構(gòu)建及部分生物學(xué)特性研究
發(fā)布時間：2018-07-24 19:55
【摘要】：單核細胞增生李斯特菌(Listeria monocytogenes,LM)是重要的人畜共患傳染病李斯特菌病的主要病原菌,人類和動物感染發(fā)病后主要表現(xiàn)為腦膜炎、胃腸道癥狀、敗血癥及孕畜(孕婦)流產(chǎn)等臨床癥狀,發(fā)病死亡率可達到30%。該菌在自然環(huán)境中分布十分廣泛,且具有較強的環(huán)境適應(yīng)性,可在低溫、高鹽、酸堿等不利的環(huán)境條件下生長繁殖。LM作為胞內(nèi)寄生致病菌可穿越宿主的腸道屏障、母嬰胎盤屏障、血腦屏障,入侵宿主機體后能在吞噬細胞和非吞噬細胞內(nèi)生長和繁殖。這些都與LM的內(nèi)化素蛋白有直接的關(guān)系,內(nèi)化素蛋白家族中的Inl A、Inl B為LM所特有,位于菌體表面,在LM黏附、侵襲宿主細胞的過程中扮演著重要角色,其介導(dǎo)的侵襲細胞并內(nèi)化至吞噬小體是LM建立感染的前提;Inl C作為內(nèi)化素家族中唯一的分泌型蛋白,在真核細胞表面未見有能與之相結(jié)合的受體,但在細菌感染的晚期,其表達量較高。這些重要的毒力因子對LM的毒力調(diào)控及生長代謝的影響,目前知之甚少。本研究以4b血清型中的食品分離株LM681及臨床綿羊腦炎分離株LM90為親本株,在LM681△Inl A和LM90△Inl A菌株基礎(chǔ)上構(gòu)建LM681△Inl A△Inl B、LM681△Inl A△Inl B△Inl C和LM90△Inl A△Inl B菌株,通過比較親本株與突變株及各突變株之間在小鼠致病性和體外生物學(xué)特性的差異,分析Inl A、Inl B、Inl C毒力基因?qū)M的影響,為Inl A、Inl B、Inl C的進一步研究提供條件和奠定基礎(chǔ)。主要研究內(nèi)容和結(jié)果如下:1、LM681△Inl AB、LM681△Inl ABC、LM90△Inl AB突變株的構(gòu)建與鑒定:以LM681和LM90菌株的基因組為模板,擴增Inl B基因和Inl C基因的上下游同源臂,使用同源重組的方法構(gòu)建內(nèi)化素基因缺失株;最終成功構(gòu)建LM681△Inl AB、LM90△Inl AB、LM681△Inl ABC突變株。2、Inl A、Inl B、Inl C毒力基因的缺失對LM毒力的影響:親本株LM681和各突變株LM681△Inl A、LM681△Inl B、LM681△Inl AB、LM681△Inl ABC在同等條件下培養(yǎng)后:1)小白鼠腹腔接種菌液,飼養(yǎng)觀察10d,統(tǒng)計死亡率測定LD50;2)小白鼠腹腔接種菌液后,24h、48h、72h每個時間段無菌摘取小白鼠肝臟、脾臟、腦組織,CFU計數(shù),統(tǒng)計各組織的載菌量;3)各菌株劃線接種于7%綿羊血BHI平板培養(yǎng)基上,37℃條件下培養(yǎng)24h,觀察各菌株溶血情況。試驗結(jié)果:與親本株LM681相比,LM681△Inl ABC的毒力降低了3個數(shù)量級,LM681△Inl AB的毒力降低了2個數(shù)量級,LM681△Inl A和LM681△Inl B的毒力均下降1個數(shù)量級;各突變株的肝臟、脾臟、腦組織載菌量減少;親本株LM681和各突變株的在7%綿羊血BHI培養(yǎng)基上均呈β溶血。結(jié)果表明:Inl A、Inl B、Inl C毒力基因的缺失能降低LM的致病性;Inl B的缺失降低了LM在肝臟和腦組織中的載菌量,Inl A的缺失降低了LM在脾臟中的增殖,Inl C能夠協(xié)同Inl A和Inl B介導(dǎo)LM在肝臟、脾臟和腦組織中的增殖;Inl A、Inl B、Inl C毒力基因的缺失不影響LM的溶血性。3、Inl A、Inl B、Inl C毒力基因的缺失對LM體外生物學(xué)特性的影響:通過測定親本株LM681和各突變株LM681△Inl A、LM681△Inl B、LM681△Inl AB、LM681△Inl ABC在37℃條件下不同p H值BHI培養(yǎng)基中的生長濃度,比對Inl A、Inl B、Inl C毒力基因的缺失對LM體外生長能力及酸堿耐受性的影響;同等條件下通過生化反應(yīng)實驗,比對Inl A、Inl B、Inl C毒力基因的缺失對LM代謝能力的影響;通過對15種臨床常用敏感藥物的耐藥性檢測,比對Inl A、Inl B、Inl C毒力基因的缺失對LM藥物敏感性的影響。結(jié)果顯示:Inl A、Inl B、Inl C毒力基因的缺失均能降低LM的體外生長能力,但不影響LM的耐酸耐堿性,不影響LM的代謝能力,不改變LM對藥物的敏感性。所有以上結(jié)果表明:Inl A、Inl B、Inl C基因缺失降低了LM的毒力,LM的內(nèi)化作用是以不同的內(nèi)化素之間相互作用的復(fù)雜的網(wǎng)絡(luò)結(jié)構(gòu)來調(diào)節(jié)。研究結(jié)果可為深入研究內(nèi)化素在LM的致病機制中的作用提供了重要的參考依據(jù)。
[Abstract]:Mononuclear cell proliferation List Rand (Listeria monocytogenes, LM) is an important pathogen of Lester's disease of zoonosis and infectious disease. After human and animal infection, the main manifestations are meningitis, gastrointestinal symptoms, septicemia and pregnant animals (pregnant women). The mortality rate can reach 30%. in the natural environment. It is widely distributed and has strong environmental adaptability. It can grow and propagate.LM as a parasitic pathogenic bacterium at low temperature, high salt and acid base. As a parasitic pathogenic bacterium, it can pass through the intestinal barrier of host, mother to baby placenta barrier, blood brain barrier, and invade host organism to grow and reproduce in phagocytic and non phagocytic cells after invading host organism. These are all with LM The endogenous hormone protein has a direct relationship. The Inl A and Inl B in the protein family are specific to LM and are located on the surface of the bacteria. They play an important role in the process of LM adhesion and invasion of host cells. Their mediated invasion of cells and internalizing to the phagocytic body is the premise for LM to establish infection; Inl C is the only secretory egg in the family of the hormone family. In white, there is no receptor on the surface of eukaryotic cells, but its expression is high in the late stage of bacterial infection. These important virulence factors have little knowledge on the effect of LM on the regulation of virulence and growth and metabolism. This study uses the food isolate LM681 in the 4B serotype and the clinical sheep encephalitis isolate LM90 as the parent strain. Based on the strains of LM681 Delta Inl A and LM90 Delta Inl A, we construct LM681 Delta Inl A Delta Inl B, and analyze the difference between the virulence and the biological characteristics of the mutant strain and the mutant strain and the mutant strain. B, Inl C provides the basis for further research. The main research contents and results are as follows: 1, LM681 Delta Inl AB, LM681 Delta Inl ABC, LM90 Delta Inl AB mutant. In the end, LM681 Delta Inl AB, LM90 Delta Inl AB, LM681 Delta Inl ABC mutant.2, Inl A, have been successfully constructed. Observation 10d, statistical mortality measurement LD50; 2) after inoculation of bacteria in the abdominal cavity of mice, 24h, 48h, 72h were taken asepsis to extract the liver, spleen, brain tissue, CFU count, statistics of the bacteria carrying amount of each tissue; 3) the strains were inoculated on the 7% sheep blood BHI flat culture medium, and cultured under the condition of 37 degrees C, the haemolysis of each strain was observed. Results: compared with the parent strain LM681, the virulence of LM681 Delta Inl ABC decreased by 3 orders of magnitude, the virulence of LM681 Delta Inl AB decreased by 2 orders of magnitude, and the toxicity of LM681 Delta Inl A and LM681 Delta Inl decreased by 1 orders of magnitude; the liver, spleen, and brain tissue of each mutant strain decreased, and the parent strain and the mutants were in 7% sheep blood culture medium. The results showed that the absence of Inl A, Inl B, Inl C toxicity gene reduced the pathogenicity of LM, and the deletion of Inl B reduced the amount of bacteria carrying LM in the liver and brain tissue, and the absence of Inl A reduced the proliferation of the spleen in the spleen. The loss of virulence genes does not affect the LM's hemolytic.3, Inl A, Inl B, and the deletion of the Inl C virulence gene. The effect of the deletion of NL C virulence gene on the growth ability and acid-base tolerance of LM in vitro; under the same condition, the effect of the deletion of the Inl A, Inl B, Inl C virulence gene on the metabolic ability of LM was compared with that of the Inl A, and the drug resistance of the 15 commonly used sensitive drugs was compared to the Inl A The results showed that the loss of Inl A, Inl B, Inl C toxicity gene can reduce the growth ability of LM in vitro, but does not affect the acid and alkali resistance of LM, does not affect the metabolic ability of LM, and does not change the sensitivity of LM to the drug. The complex network structure of the interaction of different endogenous hormones can be adjusted. The results can provide an important reference for the in-depth study of the role of the hormone in the pathogenesis of LM.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61
本文編號：2142482
[Abstract]:Mononuclear cell proliferation List Rand (Listeria monocytogenes, LM) is an important pathogen of Lester's disease of zoonosis and infectious disease. After human and animal infection, the main manifestations are meningitis, gastrointestinal symptoms, septicemia and pregnant animals (pregnant women). The mortality rate can reach 30%. in the natural environment. It is widely distributed and has strong environmental adaptability. It can grow and propagate.LM as a parasitic pathogenic bacterium at low temperature, high salt and acid base. As a parasitic pathogenic bacterium, it can pass through the intestinal barrier of host, mother to baby placenta barrier, blood brain barrier, and invade host organism to grow and reproduce in phagocytic and non phagocytic cells after invading host organism. These are all with LM The endogenous hormone protein has a direct relationship. The Inl A and Inl B in the protein family are specific to LM and are located on the surface of the bacteria. They play an important role in the process of LM adhesion and invasion of host cells. Their mediated invasion of cells and internalizing to the phagocytic body is the premise for LM to establish infection; Inl C is the only secretory egg in the family of the hormone family. In white, there is no receptor on the surface of eukaryotic cells, but its expression is high in the late stage of bacterial infection. These important virulence factors have little knowledge on the effect of LM on the regulation of virulence and growth and metabolism. This study uses the food isolate LM681 in the 4B serotype and the clinical sheep encephalitis isolate LM90 as the parent strain. Based on the strains of LM681 Delta Inl A and LM90 Delta Inl A, we construct LM681 Delta Inl A Delta Inl B, and analyze the difference between the virulence and the biological characteristics of the mutant strain and the mutant strain and the mutant strain. B, Inl C provides the basis for further research. The main research contents and results are as follows: 1, LM681 Delta Inl AB, LM681 Delta Inl ABC, LM90 Delta Inl AB mutant. In the end, LM681 Delta Inl AB, LM90 Delta Inl AB, LM681 Delta Inl ABC mutant.2, Inl A, have been successfully constructed. Observation 10d, statistical mortality measurement LD50; 2) after inoculation of bacteria in the abdominal cavity of mice, 24h, 48h, 72h were taken asepsis to extract the liver, spleen, brain tissue, CFU count, statistics of the bacteria carrying amount of each tissue; 3) the strains were inoculated on the 7% sheep blood BHI flat culture medium, and cultured under the condition of 37 degrees C, the haemolysis of each strain was observed. Results: compared with the parent strain LM681, the virulence of LM681 Delta Inl ABC decreased by 3 orders of magnitude, the virulence of LM681 Delta Inl AB decreased by 2 orders of magnitude, and the toxicity of LM681 Delta Inl A and LM681 Delta Inl decreased by 1 orders of magnitude; the liver, spleen, and brain tissue of each mutant strain decreased, and the parent strain and the mutants were in 7% sheep blood culture medium. The results showed that the absence of Inl A, Inl B, Inl C toxicity gene reduced the pathogenicity of LM, and the deletion of Inl B reduced the amount of bacteria carrying LM in the liver and brain tissue, and the absence of Inl A reduced the proliferation of the spleen in the spleen. The loss of virulence genes does not affect the LM's hemolytic.3, Inl A, Inl B, and the deletion of the Inl C virulence gene. The effect of the deletion of NL C virulence gene on the growth ability and acid-base tolerance of LM in vitro; under the same condition, the effect of the deletion of the Inl A, Inl B, Inl C virulence gene on the metabolic ability of LM was compared with that of the Inl A, and the drug resistance of the 15 commonly used sensitive drugs was compared to the Inl A The results showed that the loss of Inl A, Inl B, Inl C toxicity gene can reduce the growth ability of LM in vitro, but does not affect the acid and alkali resistance of LM, does not affect the metabolic ability of LM, and does not change the sensitivity of LM to the drug. The complex network structure of the interaction of different endogenous hormones can be adjusted. The results can provide an important reference for the in-depth study of the role of the hormone in the pathogenesis of LM.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61
【參考文獻】
相關(guān)期刊論文 前1條
1 馮瑩穎;張強;黃蘭紅;秦龍娟;羅勤;;InlA和InlB介導(dǎo)單核細胞增生李斯特菌入侵宿主細胞分子機制的研究進展[J];微生物學(xué)通報;2009年12期
，本文編號：2142482
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