蜡样芽胞杆菌对家蚕的毒性及其蛋白质组的研究

发布时间：2019-06-03 19:39

【摘要】：蜡样芽孢杆菌(Bacillus cereus)为革兰氏阳性细菌,需氧或兼性厌氧,具轻微运动能力。由于生长环境,质粒的内容物以及毒性因子的不同,有些蜡样芽孢杆菌在畜牧业和食品行业会被作为益生菌添加到食品和饲料中,有的菌株则为条件致病菌,对食品健康,生活环境都存在着潜在的危害。本实验室前期从中国地方医院分离获得26个菌株,通过鉴定获得一株致病性强的蜡样芽孢杆菌。为了评估该菌的毒性,本研究选用家蚕作为模式生物通过过夜培养的细菌悬液注射到家蚕幼虫体内观察死亡情况,通过组织切片观测病理变化,再通过蛋白质组学技术分析家蚕中肠和脂肪体蛋白变化。为环境病原微生物对家蚕以及其它昆虫毒性提供分子理论基础。本研究得到如下一些结果:1.个体水平上分析:取5μL蜡样芽孢杆菌细菌悬液从家蚕节间膜部位定量注射至血淋巴中,注射生理盐水的作为对照,发现该菌12h内可杀死全部五龄幼虫并伴随明显的病症。感染后,家蚕会出现类似于食物中毒病人“上吐下泻”的病状,大部分幼虫完全停止进食,身体变得肿胀。幼虫身体中上部分膨大变绿。解剖后发现,上部膨大是因为桑叶已经全部集中到了中肠的上半部分,中肠组织明显变细,独特的褶皱结构松散消失,外壁变薄并部分溶解。12h后,家蚕幼虫完全死亡,身体已经发生明显变黑,变软的现象,身体膨胀僵硬,解剖后的中肠组织结构完全溶解。24 h后观察,幼虫身体已经全部变黑,上部膨大,身体腐烂病伴随流出黑色的液体。2.组织水平上分析:通过组织切片方法,对注射蜡样芽孢杆菌后不同时间段中肠组织发生的连续病理变化进行镜检观察,观察到中肠组织细胞核无明显变化,柱状细胞变小变少,中肠结构明显的褶皱现象消失,中肠组织的外壁逐渐变薄并出现了断裂的现象,直至完全溶解。组织切片和个体病变都观察到中肠组织溶解等明显病变,说明该菌对家蚕的组织器官会造成巨大的损伤,中肠组织明显的损伤病变可能是导致家蚕个体死亡的直接原因。3.分子水平上分析:在6h时分别从处理组和对照组的五龄家蚕幼虫中提取脂肪体总蛋白,利用二维电泳技术找出表达量差异较大的蛋白,经过质谱鉴定和生物学检索分析找到了5个家蚕的差异蛋白点,经过生物学功能分析得出,下调的蛋白核糖核酸酶与宿主的代谢能力和神经传导系统相关,上调的蛋白中低分子量脂蛋白(30 kDa)与宿主中相关的免疫应答反应,抗炎反应以及细胞凋亡等方面相关;蛋白酶体的主要功能是参与了生物体细胞的周期调控以及胞外蛋白的降解过程;14-3-3zeta蛋白是抑制凋亡因子。为了从转录水平上验证这些基因的表达量变化情况与蛋白水平是否一致,提取了1、2、4、6、8、10h的家蚕脂肪体组织,经qPCR检测,发现家蚕4种基因的表达量在转录水平和蛋白水平的变化情况一致。4.为了进一步研究蜡样芽孢杆菌对家蚕中肠组织的毒性,从分子水平上,在6 h时分别从处理组和对照组提取中肠总蛋白,同样是应用了二维电泳和质谱检测分析相结合的方法找到13个家蚕的差异蛋白点,经生物学功能分析得出,上调的蛋白为微管蛋白、乙酰水解酶、谷氨酰胺合成酶和14-3-3zeta蛋白,分别于宿主的吞噬作用、解毒代谢能力、免疫系统和神经系统息息相关;下调的蛋白为空泡膜ATP合成酶、EF-domain蛋白和膜联蛋白,分别于神经传导、生物膜和机体生理活动的稳定性相关联;经qPCR检测,发现家蚕11种基因的表达量在转录水平和蛋白水平的变化情况一致。本研究分别从个体水平、组织水平上阐述了不同时间段蜡样芽孢杆菌所引起的家蚕的病理变化,为医院中该菌创伤感染的防治提供了理论依据;同时我们找到了家蚕脂肪体和中肠组织中一些表达量差异较大并与免疫、代谢相关的蛋白,可以作为今后抗菌药物研究的预警基因,为后续研究奠定基础。
[Abstract]:Bacillus cereus is gram-positive bacteria, aerobic or facultative anaerobic, with slight motion capability. Due to the different growth environment, the contents of the plasmid and the toxicity factors, some bacillus cereus can be added to the food and the feed as the probiotics in the animal husbandry and food industry, and some strains are the condition pathogenic bacteria, and the food is healthy and the living environment has potential hazards. In this lab,26 strains were isolated from Chinese local hospital, and a highly pathogenic bacillus cereus was obtained by identification. In order to assess the toxicity of the bacteria, this study chose the silkworm as the model organism to observe the death in the larvae of the silkworm through the overnight culture of the bacterial suspension, observe the pathological changes by the tissue sections, and then analyze the changes of the intestinal and fat body proteins in the silkworm by the proteomics technology. And provides a molecular theoretical basis for the toxicity of the environment pathogenic microorganism to the silkworm and other insects. The results of this study are as follows:1. Individual level analysis: A 5. mu.L of Bacillus cereus bacterial suspension was injected into the blood lymph node from the position of the internode membrane of the silkworm, and the normal saline was injected as a control. It was found that the bacteria could kill all the five-instar larvae within 12 h and accompanied by a clear condition. After the infection, the silkworm will show a condition similar to the "vomiting and diarrhea" of the food poisoning patient, most of the larvae are completely stopped, and the body becomes swollen. The upper part of the larva's body is enlarged and green. after the dissection, the upper part of the silkworm is expanded because the mulberry leaves are all concentrated in the upper part of the middle intestine, the middle-intestinal tissue is obviously thinner, the unique fold structure is loose and disappears, the outer wall is thinned and partially dissolved, After 24 h, the body of the larvae was completely blackened, the upper part expanded, and the body putrefaction was accompanied by a black liquid. tissue level analysis: the continuous pathological changes of the intestinal tissue in different time periods after injection of the bacillus cereus were observed by means of a tissue slice method, and the cell nucleus of the middle intestine was observed to have no obvious change, and the columnar cells become smaller and less, The obvious fold phenomenon of the middle-intestine structure disappeared, and the outer wall of the middle-intestinal tissue gradually became thinner and the phenomenon of the fracture appeared until completely dissolved. The pathological changes of the intestinal tissue were observed in both the tissue sections and the individual lesions. It is indicated that the bacteria can cause great damage to the tissue organs of the silkworm, and the obvious damage to the intestinal tissue may be a direct cause of the death of the individual in the silkworm. molecular level analysis: at 6 hours, the total protein of the fat body was extracted from the five-instar silkworm larvae of the treatment group and the control group, and the protein with great difference in the expression quantity was found by the agarose gel electrophoresis technology, and the difference protein points of the five silkworm were found through the mass spectrum identification and the biological search analysis, After biological function analysis, the down-regulated protein ribonuclease is related to the metabolic capacity of the host and the neural conduction system, and the up-regulated protein low-molecular-weight lipoprotein (30 kDa) is related to the related immune response, anti-inflammatory reaction and cell apoptosis in the host; The primary function of the proteasome is to participate in the cycle regulation of the organism cells and the degradation of the extracellular proteins; the 14-3-3 zeta protein is an inhibitor of apoptosis. In order to verify the change of the expression of these genes from the level of transcription and whether the level of the protein is consistent with the level of the protein, the silkworm fat body tissues of 1,2,4,6,8 and 10 h were extracted, and the expression of the four genes of the silkworm was found to be consistent with the change of the level of transcription and the level of the protein by qPCR. In order to further study the toxicity of Bacillus cereus to the intestinal tissue in the silkworm, the total protein from the treatment group and the control group was extracted from the treatment group and the control group at the time of 6 h, and the difference protein point of 13 silkworm was found by using the method of the combination of the fingerprint electrophoresis and the mass spectrum detection analysis. According to the biological function analysis, the up-regulated protein is a microtubule protein, a glyohydrolase, a oryzamide synthetase and a 14-3-3zeta protein, which are related to the phagocytosis of the host, the detoxification and metabolic capacity, the immune system and the nervous system, and the down-regulated protein is a vacuolated membrane ATP synthase, The expression of EF-domain and membrane-linked proteins was associated with the stability of nerve conduction, biofilms and the physiological activities of the body, and the expression of 11 kinds of genes in the silkworm was found to be consistent with the change of the level of transcription and the level of the protein by qPCR. In this study, the pathological changes of the silkworm caused by Bacillus cereus were described in different time periods from the individual level and the tissue level, which provided a theoretical basis for the prevention and treatment of the bacterial wound infection in the hospital. At the same time, we found that some of the expression levels of the silkworm fat body and the middle intestine tissue were large, and the protein related to immunity and metabolism could be used as the early warning gene of the research on the future antibacterial drugs and laid the foundation for the follow-up study.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q93

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