猪源Bb生物被膜态和浮游态的比较蛋白组学及相关蛋白特性

发布时间：2018-03-30 16:11

本文选题：猪支气管败血波氏杆菌　切入点：生物被膜　出处：《甘肃农业大学》2017年硕士论文

【摘要】：支气管败血波氏杆菌是一种革兰氏阴性菌,能长期或终生感染哺乳动物的呼吸道,引起猪萎缩性鼻炎、犬窝咳、兔鼻塞等呼吸系统疾病。生物被膜能够增强细菌对外界环境的抵抗力,逃避宿主的免疫防御机制;在感染部位不易被清除,导致宿主的持续性感染;而且能够抵制抗生素的作用,使膜内微生物耐药性增加一千倍;据统计约80%以上的细菌性疾病过程中细菌是以生物被膜状态存在的,但是对于生物被膜的形成机制在国内外尚无统一明确的认识,有待进一步深入研究,同时也没有从生物被膜的层面去挖掘更有价值的保护性抗原,因此,通过优化该菌的生物被膜形成条件,确定生物被膜形成的最佳条件并观察其被膜结构,而后应用比较蛋白组学从蛋白水平揭示生物被膜的形成机制,并对相关蛋白的特性进行研究,为生物被膜的形成机制以及疫苗的开发提供科学依据。1猪支气管败血波氏杆菌生物被膜的体外优化及结构观察为确定影响支气管败血波氏杆菌生物被膜的形成条件,本研究采用体外定性与定量两种方法对1株猪源支气管败血波氏杆菌在不同条件下(时间、氯化钠浓度、pH、葡萄糖、蔗糖、乳糖)产生的生物被膜的差异进行了研究,同时对生物被膜的结构进行观察。结果发现24 h为支气管败血波氏杆菌生物被膜成熟稳定期,氯化钠浓度超过0.4%后,生物被膜形成量逐渐下降,培养基pH略偏碱性时有利于支气管败血波氏杆菌生物被膜的生长,乳糖能够促进支气管败血波氏杆菌生物被膜的生成。显微镜观察到24 h后网格状的生物被膜已经形成,并且趋于稳定。2猪支气管败血波氏杆菌生物被膜态和浮游态比较蛋白组学分析本试验通过双向电泳比较了支气管败血波氏杆菌处于生物被膜态和浮游态下全菌蛋白的表达差异,结果发现在生物被膜态的全菌蛋白中,有15个蛋白点表达上调,9个蛋白点表达下调。选取7个上调的蛋白点进行质谱鉴定,发现上调的蛋白点主要有延伸因子、超氧化物歧化酶、分子伴侣等,功能主要体现在应激,调控,代谢方面。该结果从蛋白质水平上深化了对细菌生物被膜形成机制的研究,并为生物被膜的防控和深入研究提供科学依据。3超氧化物歧化酶(SOD)基因的克隆表达及生物学特性研究为探讨猪源支气管败血波氏杆菌超氧化物歧化酶的生物学特性,本研究参照GenBank中RB50株的SOD基因序列设计引物,通过PCR扩增技术获得SOD基因,并将其克隆至pET-28a(+)载体,构建重组质粒pET-SOD,转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达纯化目的蛋白并测定其酶比活性,免疫小鼠,进行攻毒试验检测其免疫保护效率,并研究了多抗对生物被膜的抑制作用;结果发现重组蛋白以上清的形式表达,其酶比活性为1272(u/mgprot),对小鼠的免疫保护效率为20%,SOD多抗对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.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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