發(fā)布時(shí)間：2018-03-30 16:11

  本文選題：豬支氣管敗血波氏桿菌　切入點(diǎn)：生物被膜　出處：《甘肅農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：支氣管敗血波氏桿菌是一種革蘭氏陰性菌,能長期或終生感染哺乳動(dòng)物的呼吸道,引起豬萎縮性鼻炎、犬窩咳、兔鼻塞等呼吸系統(tǒng)疾病。生物被膜能夠增強(qiáng)細(xì)菌對外界環(huán)境的抵抗力,逃避宿主的免疫防御機(jī)制;在感染部位不易被清除,導(dǎo)致宿主的持續(xù)性感染;而且能夠抵制抗生素的作用,使膜內(nèi)微生物耐藥性增加一千倍;據(jù)統(tǒng)計(jì)約80%以上的細(xì)菌性疾病過程中細(xì)菌是以生物被膜狀態(tài)存在的,但是對于生物被膜的形成機(jī)制在國內(nèi)外尚無統(tǒng)一明確的認(rèn)識(shí),有待進(jìn)一步深入研究,同時(shí)也沒有從生物被膜的層面去挖掘更有價(jià)值的保護(hù)性抗原,因此,通過優(yōu)化該菌的生物被膜形成條件,確定生物被膜形成的最佳條件并觀察其被膜結(jié)構(gòu),而后應(yīng)用比較蛋白組學(xué)從蛋白水平揭示生物被膜的形成機(jī)制,并對相關(guān)蛋白的特性進(jìn)行研究,為生物被膜的形成機(jī)制以及疫苗的開發(fā)提供科學(xué)依據(jù)。1豬支氣管敗血波氏桿菌生物被膜的體外優(yōu)化及結(jié)構(gòu)觀察為確定影響支氣管敗血波氏桿菌生物被膜的形成條件,本研究采用體外定性與定量兩種方法對1株豬源支氣管敗血波氏桿菌在不同條件下(時(shí)間、氯化鈉濃度、pH、葡萄糖、蔗糖、乳糖)產(chǎn)生的生物被膜的差異進(jìn)行了研究,同時(shí)對生物被膜的結(jié)構(gòu)進(jìn)行觀察。結(jié)果發(fā)現(xiàn)24 h為支氣管敗血波氏桿菌生物被膜成熟穩(wěn)定期,氯化鈉濃度超過0.4%后,生物被膜形成量逐漸下降,培養(yǎng)基pH略偏堿性時(shí)有利于支氣管敗血波氏桿菌生物被膜的生長,乳糖能夠促進(jìn)支氣管敗血波氏桿菌生物被膜的生成。顯微鏡觀察到24 h后網(wǎng)格狀的生物被膜已經(jīng)形成,并且趨于穩(wěn)定。2豬支氣管敗血波氏桿菌生物被膜態(tài)和浮游態(tài)比較蛋白組學(xué)分析本試驗(yàn)通過雙向電泳比較了支氣管敗血波氏桿菌處于生物被膜態(tài)和浮游態(tài)下全菌蛋白的表達(dá)差異,結(jié)果發(fā)現(xiàn)在生物被膜態(tài)的全菌蛋白中,有15個(gè)蛋白點(diǎn)表達(dá)上調(diào),9個(gè)蛋白點(diǎn)表達(dá)下調(diào)。選取7個(gè)上調(diào)的蛋白點(diǎn)進(jìn)行質(zhì)譜鑒定,發(fā)現(xiàn)上調(diào)的蛋白點(diǎn)主要有延伸因子、超氧化物歧化酶、分子伴侶等,功能主要體現(xiàn)在應(yīng)激,調(diào)控,代謝方面。該結(jié)果從蛋白質(zhì)水平上深化了對細(xì)菌生物被膜形成機(jī)制的研究,并為生物被膜的防控和深入研究提供科學(xué)依據(jù)。3超氧化物歧化酶(SOD)基因的克隆表達(dá)及生物學(xué)特性研究為探討豬源支氣管敗血波氏桿菌超氧化物歧化酶的生物學(xué)特性,本研究參照GenBank中RB50株的SOD基因序列設(shè)計(jì)引物,通過PCR擴(kuò)增技術(shù)獲得SOD基因,并將其克隆至pET-28a(+)載體,構(gòu)建重組質(zhì)粒pET-SOD,轉(zhuǎn)化大腸桿菌BL21(DE3)感受態(tài)細(xì)胞,經(jīng)IPTG誘導(dǎo)表達(dá)純化目的蛋白并測定其酶比活性,免疫小鼠,進(jìn)行攻毒試驗(yàn)檢測其免疫保護(hù)效率,并研究了多抗對生物被膜的抑制作用;結(jié)果發(fā)現(xiàn)重組蛋白以上清的形式表達(dá),其酶比活性為1272(u/mgprot),對小鼠的免疫保護(hù)效率為20%,SOD多抗對生物被膜具有一定的抑制作用。
[Abstract]:Bacillus bronchius is a gram-negative bacterium that can infect the respiratory tract of mammals for a long time or for life, causing atrophic rhinitis in pigs, rotting cough in dogs. Rabbit nasal obstruction and other respiratory diseases. The biofilm can enhance the resistance of bacteria to the external environment and escape the host's immune defense mechanism. Moreover, it can resist the effect of antibiotics and increase the drug resistance of microbes in the membrane by a factor of 1,000. According to statistics, about 80% of the bacteria in the process of bacterial diseases exist in the state of biofilm. However, the formation mechanism of biofilm has not been fully understood at home and abroad, which needs to be further studied. At the same time, it has not excavated more valuable protective antigens from the level of biofilm. By optimizing the conditions of biofilm formation, the optimum conditions of biofilm formation were determined and the structure of biofilm was observed. The mechanism of biofilm formation was revealed from protein level by comparative proteomics. And the characteristics of the related proteins were studied. To provide the scientific basis for the formation mechanism of biofilm and the development of vaccine. 1 optimizing the biofilm and observing the structure of the biofilm of Bacillus bronchiae in vitro in order to determine the conditions that affect the formation of the biofilm of Bacillus bronchiae. In this study, in vitro qualitative and quantitative methods were used to study the difference of biofilm produced by a porcine strain of Bordetella bronchitis under different conditions (time, pH of sodium chloride, glucose, sucrose, lactose). At the same time, the structure of biofilm was observed. The results showed that the biofilm was mature and stable at 24 h, and when the concentration of sodium chloride exceeded 0.4%, the amount of biofilm formation decreased gradually. When pH was slightly alkaline, the biofilm of Bacillus bronchius was increased by lactose, and the biofilm of Bacillus bronchius was formed 24 hours after the growth of the biofilm was observed by microscope, and lactose was able to promote the formation of biofilm. In this experiment, we compared the expression of whole bacterial proteins in the biofilm and zooplankton of B. bronchiae by two dimensional electrophoresis, which were in the biofilm state and in the planktonic state of B. bronchiae, and tended to stabilize the expression of the whole bacterial protein in the biofilm state and the planktonic state of B. bronchitis. The results showed that 15 protein spots were up-regulated and 9 protein spots were down-regulated in the whole bacterial proteins in the biofilm state. Seven up-regulated protein spots were identified by mass spectrometry. Superoxide dismutase (SOD), molecular chaperone, etc., whose functions are mainly reflected in stress, regulation and metabolism. This result deepens the study on the formation mechanism of bacterial biofilm at protein level. The cloning, expression and biological characteristics of SOD gene were studied in order to study the biological characteristics of the superoxide dismutase (SOD) of Bos bronchitis derived from pigs, and provide scientific basis for the prevention and control of biofilm and further study on the biological characteristics of SOD gene. In this study, we designed primers according to the SOD gene sequence of RB50 strain in GenBank, obtained SOD gene by PCR amplification technique, cloned it into pET-28a () vector, constructed recombinant plasmid pET-SOD, transformed Escherichia coli BL21DE3) receptive cells. The purified protein was induced by IPTG and its specific enzyme activity was determined. The mice were immunized. The immunoprotection efficiency of the purified protein was tested by virus attack test, and the inhibitory effect of polyclonal antibody on the biofilm was studied, the results showed that the recombinant protein supernatant was expressed in the form of supernatant. The specific enzyme activity was 1272 渭 / mg protterium, and the immunoprotection efficiency was 20%. The SOD polyclonal antibody had a certain inhibitory effect on the biofilm.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.61

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