蠟樣芽胞桿菌對家蠶的毒性及其蛋白質(zhì)組的研究
發(fā)布時間：2019-06-03 19:39
【摘要】：蠟樣芽孢桿菌(Bacillus cereus)為革蘭氏陽性細(xì)菌,需氧或兼性厭氧,具輕微運(yùn)動能力。由于生長環(huán)境,質(zhì)粒的內(nèi)容物以及毒性因子的不同,有些蠟樣芽孢桿菌在畜牧業(yè)和食品行業(yè)會被作為益生菌添加到食品和飼料中,有的菌株則為條件致病菌,對食品健康,生活環(huán)境都存在著潛在的危害。本實(shí)驗(yàn)室前期從中國地方醫(yī)院分離獲得26個菌株,通過鑒定獲得一株致病性強(qiáng)的蠟樣芽孢桿菌。為了評估該菌的毒性,本研究選用家蠶作為模式生物通過過夜培養(yǎng)的細(xì)菌懸液注射到家蠶幼蟲體內(nèi)觀察死亡情況,通過組織切片觀測病理變化,再通過蛋白質(zhì)組學(xué)技術(shù)分析家蠶中腸和脂肪體蛋白變化。為環(huán)境病原微生物對家蠶以及其它昆蟲毒性提供分子理論基礎(chǔ)。本研究得到如下一些結(jié)果:1.個體水平上分析:取5μL蠟樣芽孢桿菌細(xì)菌懸液從家蠶節(jié)間膜部位定量注射至血淋巴中,注射生理鹽水的作為對照,發(fā)現(xiàn)該菌12h內(nèi)可殺死全部五齡幼蟲并伴隨明顯的病癥。感染后,家蠶會出現(xiàn)類似于食物中毒病人“上吐下瀉”的病狀,大部分幼蟲完全停止進(jìn)食,身體變得腫脹。幼蟲身體中上部分膨大變綠。解剖后發(fā)現(xiàn),上部膨大是因?yàn)樯Ｈ~已經(jīng)全部集中到了中腸的上半部分,中腸組織明顯變細(xì),獨(dú)特的褶皺結(jié)構(gòu)松散消失,外壁變薄并部分溶解。12h后,家蠶幼蟲完全死亡,身體已經(jīng)發(fā)生明顯變黑,變軟的現(xiàn)象,身體膨脹僵硬,解剖后的中腸組織結(jié)構(gòu)完全溶解。24 h后觀察,幼蟲身體已經(jīng)全部變黑,上部膨大,身體腐爛病伴隨流出黑色的液體。2.組織水平上分析:通過組織切片方法,對注射蠟樣芽孢桿菌后不同時間段中腸組織發(fā)生的連續(xù)病理變化進(jìn)行鏡檢觀察,觀察到中腸組織細(xì)胞核無明顯變化,柱狀細(xì)胞變小變少,中腸結(jié)構(gòu)明顯的褶皺現(xiàn)象消失,中腸組織的外壁逐漸變薄并出現(xiàn)了斷裂的現(xiàn)象,直至完全溶解。組織切片和個體病變都觀察到中腸組織溶解等明顯病變,說明該菌對家蠶的組織器官會造成巨大的損傷,中腸組織明顯的損傷病變可能是導(dǎo)致家蠶個體死亡的直接原因。3.分子水平上分析:在6h時分別從處理組和對照組的五齡家蠶幼蟲中提取脂肪體總蛋白,利用二維電泳技術(shù)找出表達(dá)量差異較大的蛋白,經(jīng)過質(zhì)譜鑒定和生物學(xué)檢索分析找到了5個家蠶的差異蛋白點(diǎn),經(jīng)過生物學(xué)功能分析得出,下調(diào)的蛋白核糖核酸酶與宿主的代謝能力和神經(jīng)傳導(dǎo)系統(tǒng)相關(guān),上調(diào)的蛋白中低分子量脂蛋白(30 kDa)與宿主中相關(guān)的免疫應(yīng)答反應(yīng),抗炎反應(yīng)以及細(xì)胞凋亡等方面相關(guān);蛋白酶體的主要功能是參與了生物體細(xì)胞的周期調(diào)控以及胞外蛋白的降解過程;14-3-3zeta蛋白是抑制凋亡因子。為了從轉(zhuǎn)錄水平上驗(yàn)證這些基因的表達(dá)量變化情況與蛋白水平是否一致,提取了1、2、4、6、8、10h的家蠶脂肪體組織,經(jīng)qPCR檢測,發(fā)現(xiàn)家蠶4種基因的表達(dá)量在轉(zhuǎn)錄水平和蛋白水平的變化情況一致。4.為了進(jìn)一步研究蠟樣芽孢桿菌對家蠶中腸組織的毒性,從分子水平上,在6 h時分別從處理組和對照組提取中腸總蛋白,同樣是應(yīng)用了二維電泳和質(zhì)譜檢測分析相結(jié)合的方法找到13個家蠶的差異蛋白點(diǎn),經(jīng)生物學(xué)功能分析得出,上調(diào)的蛋白為微管蛋白、乙酰水解酶、谷氨酰胺合成酶和14-3-3zeta蛋白,分別于宿主的吞噬作用、解毒代謝能力、免疫系統(tǒng)和神經(jīng)系統(tǒng)息息相關(guān);下調(diào)的蛋白為空泡膜ATP合成酶、EF-domain蛋白和膜聯(lián)蛋白,分別于神經(jīng)傳導(dǎo)、生物膜和機(jī)體生理活動的穩(wěn)定性相關(guān)聯(lián);經(jīng)qPCR檢測,發(fā)現(xiàn)家蠶11種基因的表達(dá)量在轉(zhuǎn)錄水平和蛋白水平的變化情況一致。本研究分別從個體水平、組織水平上闡述了不同時間段蠟樣芽孢桿菌所引起的家蠶的病理變化,為醫(yī)院中該菌創(chuàng)傷感染的防治提供了理論依據(jù);同時我們找到了家蠶脂肪體和中腸組織中一些表達(dá)量差異較大并與免疫、代謝相關(guān)的蛋白,可以作為今后抗菌藥物研究的預(yù)警基因,為后續(xù)研究奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q93
本文編號：2492171
[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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q93
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