两株细菌的比较鉴定及非溶血性肠毒素基因的表达

发布时间：2018-04-15 00:41

本文选题：菌种鉴定 + 蛋白质组　；参考：《江苏大学》2017年硕士论文

【摘要】：医院周围环境的细菌污染是当前医疗卫生行业关注的重要问题。本研究选取医院分离的两株对家蚕具有致死性的细菌为研究对象,进行菌种鉴定,比较蛋白质组差异,对致病基因进行PCR鉴定,非溶血性肠毒素进行了原核表达,构建穿梭质粒。主要的研究内容及结果如下:(1)两种致病性不同的细菌的菌种鉴定对从医院分离的具有不同致病性的两株细菌,通过表型、生理生化特征反应以及16S rRNA基因序列构建系统发育树分析。鉴定结果显示细菌65-5为枯草芽孢杆菌(Bacillus subtilis),细菌41-1为广义的蜡样芽孢杆菌(Bacilluscereus sensu lato)。(2)两株细菌的蛋白质组比较研究通过二维电泳串联质谱的方法,比较两种细菌的蛋白质组差异。两种细菌蛋白质图谱中的蛋白点存在较大的差异。选取图谱上的蛋白点,进行质谱鉴定,进一步确定强致病菌41-1为蜡样芽孢杆菌。通过蛋白质组研究细菌分泌的蛋白,蜡样杆菌上清的蛋白中包含有鞭毛蛋白和金属蛋白酶。(3)PCR鉴定蜡样芽孢杆菌中致病基因41-1菌株通过菌种鉴定确定为蜡样芽孢杆菌。蜡样芽孢杆菌是条件致病菌,能产生多种致病性毒素。利用PCR检测鉴定出来的蜡样芽孢杆菌存在的毒素基因,确定该菌株包含非溶血性肠毒素基因nhe A、nhe B、nhe C,不包含溶血性肠毒素基因hbl A、hbl C、hbl D,细胞毒素K基因cyt K和肠毒素Cereulide基因ces。并获得nhe A、nhe B、nhe C三种致病基因的克隆。(4)对三种nhe A、nhe B、nhe C基因的原核表达将克隆获得的三种致病基因nhe A、nhe B、nhe C基因,分别构建成大肠杆菌表达载体 pET-30a(+)-nhe A、pET-30a(+)-nheB 以及 pET-30a(+)-nhe C,三种非溶血性肠毒素亚基成功在大肠杆菌中表达,NheB亚基在原核表达时易于形成多聚体,质谱鉴定结果显示三种蛋白正确表达。用割胶纯化的三种非溶血性肠毒素蛋白作为抗原免疫新西兰大白兔,获得三种目的蛋白的多克隆抗血清。用原核表达相应的重组蛋白验证多克隆抗体的效果,确定三种多克隆抗体成功制备。用制备的多克隆抗体对蜡样芽孢杆菌上清中的蛋白进行了检测,非溶血性肠毒素亚基Nhe A和Nhe B被特异性的鉴定,而未鉴定到非溶血性肠毒素Nhe C亚基。(5)穿梭质粒的构建为了研究三种非溶血性肠毒素亚基的功能和相互作用之间的关系。利用基于AcMNPV bacmid表达系统,将三种非溶血性肠毒素基因克隆到杆状病毒的穿梭载体上,获得对应的穿梭质粒。将穿梭质粒转化到Bacmid的感受态细胞发生转座,成功构造 pACnhe C-nhe-nhe B bacmid,pACnhe C-nhe A bacmid,pACnhe C-nhe B bacmid。
[Abstract]:Bacterial pollution in the surrounding environment of hospitals is an important issue concerned by the medical and health industry.In this study, two strains of bacteria that are lethal to silkworm (Bombyx mori) were selected as the research object. The strains were identified, the proteome difference was compared, the pathogenic gene was identified by PCR, and the non-hemolytic enterotoxin was expressed in prokaryotic.The shuttle plasmid was constructed.The main contents and results of this study are as follows: (1) the identification of two strains of bacteria with different pathogenicity to two strains of bacteria with different pathogenicity isolated from hospitals, through phenotypes,Physiological and biochemical responses and phylogenetic tree analysis of 16s rRNA gene sequence.The results showed that bacteria 65-5 were Bacillus subtilisus and bacteria 41-1 were generalized Bacillus cereus sensu lato.2.The proteome differences between the two strains were compared by two-dimensional electrophoresis tandem mass spectrometry.The protein spots in the protein map of the two bacteria differ greatly.The protein spots on the map were selected and identified by mass spectrometry, and the strong pathogenic bacteria 41-1 was further identified as Bacillus cereus.The protein secreted by bacteria was studied by proteome. The protein of supernatant of Bacillus cereus contained flagellin and metalloproteinase. The pathogenic gene 41-1 of Bacillus cereus was identified as Bacillus cereus by PCR.Bacillus cereus is a conditional pathogen that produces a variety of pathogenic toxins.The toxin gene of Bacillus cereus was identified by PCR detection. It was determined that the strain contained the non-hemolytic enterotoxin gene nhe Anhe Bnhe C, not the hemolytic enterotoxin gene hbl nhe hbl ChblD, the cytotoxin K gene cyt K and the enterotoxin Cereulide gene Ces.The prokaryotic expression of three pathogenicity genes, nhe Anhe Bnhe Bnhe C, was obtained from the prokaryotic expression of three kinds of nhe Anhe Bnhe C genes, and the three pathogenicity genes nhe Anhe Bnhe C gene were cloned, and the three pathogenicity genes nhe Anhe Bnhe C gene were cloned.E. coli expression vectors pET-30a (pET-30a) and pET-30a (pET-30a) and pET-30a (pET-30a) were constructed respectively. Three non-hemolytic enterotoxin subunits were successfully expressed in E. coli.The results of mass spectrometry showed that the three proteins were correctly expressed.Three kinds of non-hemolytic enterotoxin proteins purified by tapping were used as antigens to immunize New Zealand white rabbits and polyclonal antisera of three kinds of target proteins were obtained.The effect of polyclonal antibody was verified by prokaryotic expression of corresponding recombinant protein, and three polyclonal antibodies were successfully prepared.The protein in the supernatant of Bacillus cereus was detected by polyclonal antibody. The non-hemolytic enterotoxin subunits Nhe A and Nhe B were specifically identified.In order to study the relationship between the function and interaction of the three non-hemolytic enterotoxin subunits, the shuttle plasmid of Nhe C subunit of non-hemolytic enterotoxin was not identified.Based on AcMNPV bacmid expression system, three non-hemolytic enterotoxin genes were cloned into the shuttle vector of baculovirus and the corresponding shuttle plasmid was obtained.The shuttle plasmid was transformed into the competent cells of Bacmid, and pACnhe C-nhe-nhe B bacmidhe C-nhe A bacmidhe C-nhe C-nhe B was successfully constructed.
【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】：R446.5

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