多粘类芽孢杆菌中抗菌物质的分离纯化及沙门氏菌毒性因子启动子库的构建与应用

发布时间：2018-01-09 00:08

本文关键词：多粘类芽孢杆菌中抗菌物质的分离纯化及沙门氏菌毒性因子启动子库的构建与应用　出处：《西北大学》2011年硕士论文　论文类型：学位论文

【摘要】：金黄色葡萄球菌是一种常见的病原菌,目前已对多种抗生素产生了抗药性。寻找新的抗金黄色葡萄球菌的物质,已成为医学和微生物学的一个重要课题。本实验室前期研究发现多粘类芽胞杆菌分泌物对金黄色葡萄球菌有抑制作用。多粘类芽孢杆菌(Paenibacillus polymyxa)是类芽孢杆菌属中的一种革兰氏阳性菌,由于其产生的多种次生代谢产物具有抑菌、防腐作用,近年在植物病虫害防治和抗生素分离研究方面受到了很大关注。本实验从多粘类芽孢杆菌J株培养物中,通过柱层析、高效液相等方法分离纯化出了一种活性物质,其对耐药性的金黄色葡萄球菌(MRSA)有明显的抑杀作用。再通过后续的质谱、核磁共振等实验确定出了该抑菌物质主要是两种分子量为414.5的非多肽类、容易相互转化的类似物。另一方面,在抗生素的后期分离纯化中,由于抗菌物质在溶液中含量低,对其抗菌活性跟踪极其困难。为了解决该问题,构建了一个沙门氏菌的毒性因子启动子-LuxCDABE报告子库。构建好的报告子菌株库,通过药物刺激其Lux发光情况的变化,可以用来检测低浓度的药物存在或者研究药物对启动子对应的毒性因子的影响。这种检测具有高灵敏、高通量、实时快速、重复性好的特点。研究挑选了沙门氏菌(Salmonella typhimurium LT2)的47个毒性因子,并扩展出了它们各自的启动子区域,将其连接到了一个质粒载体pCS26上,该质粒含有未带启动子的发光报告基因LuxCDABE。连接后的启动子-LuxCDABE报告子质粒,经多种方法验证后最终转回到了沙门氏菌中。构建好的启动子-LuxCDABE报告子可以用来检测浓度很低的多粘类芽孢杆菌抗性提取物：经柱层析纯化后的抗菌物稀释1000倍左右,对构建好的四株菌的发光有明显的减弱作用,但却不影响沙门氏菌的生长。由于多粘类芽孢杆菌抗性物质在高效液相单峰回收液中含量极低,常规的抑菌圈检测法很难满足需求,操作复杂。高灵敏度的启动子-LuxCDABE发光检测法可以很灵敏地检测出该物质的存在。此外,该毒性因子启动子库可以用来研究沙门氏菌的致病因子,而致病因子又是新的抗菌靶位。
[Abstract]:Staphylococcus aureus is a common pathogen, has a variety of antibiotic resistant Staphylococcus aureus. Looking for new material, has become an important topic in medicine and microbiology. Our previous studies showed that polymexa Bacillus secretion has inhibitory effect on Staphylococcus aureus. Paenibacilluspolymyxa (Paenibacillus polymyxa) is a kind of gram positive bacteria Paenibacillus species, due to a variety of secondary metabolites which has antibacterial, antiseptic effect, in recent years in plant pest control and antibiotic separation research has attracted much attention.
This experiment from Paenibacillus polymyxa strain J in culture, through column chromatography, HPLC separation purification method of a kind of active substance, the drug resistance of Staphylococcus aureus (MRSA) has obvious anti-tumor effects. Then through the subsequent mass spectrometry, nuclear magnetic resonance experiment determined the antibacterial substances are mainly two kinds of molecular weight of 414.5 non peptide analogues, easily transformed into each other.
On the other hand, in the separation of antibiotics in purification of antibacterial substances in solution, because the content is low, extremely difficult for antibacterial activity tracking. In order to solve the problem, constructs a virulence factor of Salmonella -LuxCDABE promoter report libraries. The report sub strains library, through the changes of light stimulation of Lux, can be used to detect low concentrations of drug effects of drugs on promoter or corresponding virulence factors. This detection with high sensitivity, high throughput, fast real-time, good reproducibility.
Study on selection of Salmonella (Salmonella typhimurium LT2) 47 virulence factors, and expand their promoter region, connect it to a plasmid pCS26, the plasmid containing luminescent reporter gene LuxCDABE. not with promoter after connecting -LuxCDABE promoter report plasmids by many method validation after the final turn back to the Salmonella.
Construction of promoter -LuxCDABE can be used to report good paenibacilluspolymyxa resistance detection of very low concentration of extract by column chromatography purification of antibacterial substances after diluted 1000 times, the constructed four strains light weaken obviously, but it did not affect the growth of Salmonella. The content of polymyxa resistant substance in HPLC peak recovery liquid is very low, the antibacterial circle detection routine is difficult to meet the demand, complex operation and high sensitivity. The promoter -LuxCDABE luminescence assay can sensitively detect the existence of matter. In addition, the pathogenic factor of virulence factor promoter library can be used to study on the Salmonella bacteria, and pathogenic factors and antimicrobial new target sites.

【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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