单增李斯特菌ΔInlAB、ΔInlABC突变株的构建及部分生物学特性研究
发布时间：2018-07-24 19:55
【摘要】：单核细胞增生李斯特菌(Listeria monocytogenes,LM)是重要的人畜共患传染病李斯特菌病的主要病原菌,人类和动物感染发病后主要表现为脑膜炎、胃肠道症状、败血症及孕畜(孕妇)流产等临床症状,发病死亡率可达到30%。该菌在自然环境中分布十分广泛,且具有较强的环境适应性,可在低温、高盐、酸碱等不利的环境条件下生长繁殖。LM作为胞内寄生致病菌可穿越宿主的肠道屏障、母婴胎盘屏障、血脑屏障,入侵宿主机体后能在吞噬细胞和非吞噬细胞内生长和繁殖。这些都与LM的内化素蛋白有直接的关系,内化素蛋白家族中的Inl A、Inl B为LM所特有,位于菌体表面,在LM黏附、侵袭宿主细胞的过程中扮演着重要角色,其介导的侵袭细胞并内化至吞噬小体是LM建立感染的前提;Inl C作为内化素家族中唯一的分泌型蛋白,在真核细胞表面未见有能与之相结合的受体,但在细菌感染的晚期,其表达量较高。这些重要的毒力因子对LM的毒力调控及生长代谢的影响,目前知之甚少。本研究以4b血清型中的食品分离株LM681及临床绵羊脑炎分离株LM90为亲本株,在LM681△Inl A和LM90△Inl A菌株基础上构建LM681△Inl A△Inl B、LM681△Inl A△Inl B△Inl C和LM90△Inl A△Inl B菌株,通过比较亲本株与突变株及各突变株之间在小鼠致病性和体外生物学特性的差异,分析Inl A、Inl B、Inl C毒力基因对LM的影响,为Inl A、Inl B、Inl C的进一步研究提供条件和奠定基础。主要研究内容和结果如下:1、LM681△Inl AB、LM681△Inl ABC、LM90△Inl AB突变株的构建与鉴定:以LM681和LM90菌株的基因组为模板,扩增Inl B基因和Inl C基因的上下游同源臂,使用同源重组的方法构建内化素基因缺失株;最终成功构建LM681△Inl AB、LM90△Inl AB、LM681△Inl ABC突变株。2、Inl A、Inl B、Inl C毒力基因的缺失对LM毒力的影响:亲本株LM681和各突变株LM681△Inl A、LM681△Inl B、LM681△Inl AB、LM681△Inl ABC在同等条件下培养后:1)小白鼠腹腔接种菌液,饲养观察10d,统计死亡率测定LD50;2)小白鼠腹腔接种菌液后,24h、48h、72h每个时间段无菌摘取小白鼠肝脏、脾脏、脑组织,CFU计数,统计各组织的载菌量;3)各菌株划线接种于7%绵羊血BHI平板培养基上,37℃条件下培养24h,观察各菌株溶血情况。试验结果:与亲本株LM681相比,LM681△Inl ABC的毒力降低了3个数量级,LM681△Inl AB的毒力降低了2个数量级,LM681△Inl A和LM681△Inl B的毒力均下降1个数量级;各突变株的肝脏、脾脏、脑组织载菌量减少;亲本株LM681和各突变株的在7%绵羊血BHI培养基上均呈β溶血。结果表明: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在肝脏和脑组织中的载菌量,Inl A的缺失降低了LM在脾脏中的增殖,Inl C能够协同Inl A和Inl B介导LM在肝脏、脾脏和脑组织中的增殖;Inl A、Inl B、Inl C毒力基因的缺失不影响LM的溶血性。3、Inl A、Inl B、Inl C毒力基因的缺失对LM体外生物学特性的影响:通过测定亲本株LM681和各突变株LM681△Inl A、LM681△Inl B、LM681△Inl AB、LM681△Inl ABC在37℃条件下不同p H值BHI培养基中的生长浓度,比对Inl A、Inl B、Inl C毒力基因的缺失对LM体外生长能力及酸碱耐受性的影响;同等条件下通过生化反应实验,比对Inl A、Inl B、Inl C毒力基因的缺失对LM代谢能力的影响;通过对15种临床常用敏感药物的耐药性检测,比对Inl A、Inl B、Inl C毒力基因的缺失对LM药物敏感性的影响。结果显示:Inl A、Inl B、Inl C毒力基因的缺失均能降低LM的体外生长能力,但不影响LM的耐酸耐碱性,不影响LM的代谢能力,不改变LM对药物的敏感性。所有以上结果表明:Inl A、Inl B、Inl C基因缺失降低了LM的毒力,LM的内化作用是以不同的内化素之间相互作用的复杂的网络结构来调节。研究结果可为深入研究内化素在LM的致病机制中的作用提供了重要的参考依据。
[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.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
本文编号：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.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
【参考文献】
相关期刊论文 前1条
1 冯莹颖;张强;黄兰红;秦龙娟;罗勤;;InlA和InlB介导单核细胞增生李斯特菌入侵宿主细胞分子机制的研究进展[J];微生物学通报;2009年12期
，本文编号：2142482
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