金黄色葡萄球菌转座子突变文库的构建与肠毒素A相关基因的研究
本文关键词:金黄色葡萄球菌转座子突变文库的构建与肠毒素A相关基因的研究 出处:《西北农林科技大学》2017年硕士论文 论文类型:学位论文
【摘要】:金黄色葡萄球菌(Staphylococcus aureus,S.aureus)简称金葡菌,是一种重要的食源性致病菌,其引起食物中毒的主要致病因子是肠毒素。由于进食被金黄色葡萄球菌及其产生的肠毒素所污染的食物而引起的食物中毒被称为金黄色葡萄球菌食物中毒(Staphylococcal food poisoning,SFP),一般在短时间内就会有相应的症状发作,包括恶心、呕吐、腹部绞痛或腹泻等。由于金黄色葡萄球菌无处不在,广泛分布于空气、灰尘和食品加工设备表面等,使得食品极易产生交叉污染,所以尽管严格控制食品加工的各个过程,还是会受到不同程度的污染。其中,食源性动物和食品加工者的手或呼吸道分泌物被视为食品污染金葡菌的主要来源。在常见的金葡菌食物中毒事件中,大部分是由SEA引起的,其次依次是SED、SEC、SEB和SEE。转座子(transposon)是细菌和许多原核与真核生物基因组中一段特异的具有转位特性的独立的DNA序列,可以在细菌的染色体、质粒或噬菌体之间水平转移。研究质粒消除和Tn917转座子的随机插入对食源性金黄色葡萄球菌肠毒素A表达的影响,是本课题的主要研究内容。本研究对48株不同来源并携带肠毒素sea基因的金葡菌先进行质粒消除,再对消除前后菌株进行18种肠毒素基因和29种耐药基因的PCR检测,以及12种临床常用抗生素的药敏检测。随后将质粒消除后金葡菌通过随机插入转座子Tn917对其染色体进行诱变,产生变异体,并建立转座子文库。最后通过ELISA方法筛选肠毒素A表达异常的菌株,同时检测生物膜形成能力以及产溶血毒素异常的突变株,并与原始菌株比较其差异性。这为阐明肠毒素A分子机制和金黄色葡萄球菌引起食物中毒的防控提供了理论依据。主要研究结果如下:(1)对48株不同来源并携带肠毒素sea基因的金葡菌先进行质粒消除,再对消除前后菌株进行18种肠毒素基因和29种耐药基因的PCR检测,以及12种临床常用抗生素的药敏检测。结果表明,质粒消除对肠毒素基因see,耐药基因tet O和tetK,以及抗生素AMP的影响较为显著,对ERY有影响但不显著,说明耐药基因tetO和tetK存在细菌质粒上,以及AMP和ERY的基因也可能在质粒上。金葡菌的肠毒素基因和耐药基因位点比较复杂,同种基因在不同菌株间存在差异,说明其具有无规律性。此外,耐药基因型和耐药表型之间也存在一定差异。(2)将含有转座子Tn917的温敏穿梭质粒转入菌株Y1和Y2中,并在ERY和42℃高温条件下进行诱导转座。在初步建立转座子文库中,我们随机挑取200个单菌落进行抗性筛选,最终得到2株复制子融合菌株,其余均为成功发生转座作用突变株。(3)对转座子突变株进行了SEA产量、生物膜和溶血毒素的测定,最终筛选出了2株不产SEA和溶血毒素的菌株,1株生物膜形成能力较弱的菌株。
[Abstract]:Staphylococcus aureus (Staphylococcus, aureus, S.aureus) S.aureus, is one of the important food borne pathogens, the main pathogenic factor causing food poisoning is caused by enterotoxin. Contaminated by Staphylococcus aureus enterotoxin food and the food poisoning is called staphylococcal food (Staphylococcal food poisoning, SFP poisoning), usually in a short period of time there will be corresponding to the onset of symptoms, including nausea, vomiting, abdominal cramps and diarrhea. Due to Staphylococcus aureus is widely distributed in the air, dust and food processing equipment such as surface, making the food easy to produce cross contamination, so although each process strict control of food processing, will be subject to different degrees of pollution. Among them, animal and food processors hand or respiratory secretions are regarded as food. The main source of infection of Staphylococcus aureus. In common staphylococcal food poisoning incidents, most of which is caused by SEA, followed by SED, SEC, SEB and SEE. transposon (transposon) are DNA sequences of bacteria and many prokaryotic and eukaryotic genomes has a specific translocation features alone the can in the bacterial chromosome, horizontal transfer between plasmids and phages. Study on plasmid elimination and Tn917 transposon random insertion effect on the expression of foodborne Staphylococcus aureus enterotoxin A, is the main content of the research. The study of 48 different strains and carrying enterotoxin sea gene of Staphylococcus aureus first detection of plasmid elimination, 18 enterotoxin genes and 29 kinds of resistance genes to eliminate the strains before and after PCR, and the susceptibility of 12 antibiotics in clinical detection. Then the plasmid elimination of postburn Staphylococcus aureus T by random transposon insertion The n917 mutation, the chromosome generated variants, and the establishment of the transposon library. Finally, using ELISA method to screen the abnormal expression of enterotoxin A strains, the simultaneous detection of biofilm formation ability and hemolytic toxin abnormal mutant, and compared with the original strain of the differences. This provides the theoretical basis for clarifying the molecular mechanism and enterotoxin A Staphylococcus aureus caused food poisoning prevention and control. The main results are as follows: (1) of 48 strains of different sources and carrying the enterotoxin sea gene of Staphylococcus aureus by plasmid elimination, detection of 18 kinds of enterotoxin genes and 29 kinds of resistance genes to eliminate the strains before and after PCR, and the drug sensitivity of 12 kinds of commonly used antibiotics in clinic the detection. The results showed that the plasmid elimination of enterotoxin genes see, O and tetK resistance genes Tet, AMP and the effects of antibiotics on ERY effect is significant, but not significant, the drug resistance gene TetO and tetK are bacterial plasmids, and genes AMP and ERY may also be in the plasmid. The enterotoxin genes and drug resistance gene of Staphylococcus aureus is more complex, there are differences between the different strains in the same gene, indicating that it has no regularity. In addition, there are some differences between the resistant genotype and drug resistance phenotype. (2) containing a temperature sensitive transposon Tn917 shuttle plasmid was transformed into strain Y1 and Y2, and induced by transposition in ERY and 42 DEG C under high temperature conditions. In the initial establishment of the transposon library, we randomly selected 200 single colonies screened 2 strains, the final replicon fusion strains, the rest are the success of transposition. The mutant. (3) the transposon mutant of SEA production, determination of biofilm and hemolytic toxin, screened 2 strains producing SEA and hemolytic toxin, 1 strains of biofilm formation ability of strains.
【学位授予单位】:西北农林科技大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:TS201.3
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