【作者】 王灿；

【导师】 张兴群；

【作者基本信息】 东华大学 ， 有机化学， 2009， 硕士

【摘要】 随着抗生素及其他抗菌药物的广泛应用,病原微生物的耐药问题日趋严重,如耐甲氧西林金黄色葡萄球菌(MRSA)的分离率不断升高,已成为感染的重要致病菌之一。因此,开发新型抗生素迫在眉睫。陆地微生物曾是寻找抗生素的主要来源,但近年来从陆地微生物中发现新抗生素的几率越来越低,人们必须寻找新的药源。海洋环境的特殊性使海洋微生物具有独特的遗传背景与代谢途径,能产生结构新颖、作用独特的代谢产物,成为筛选新型抗生素的重要来源。因此,海洋微生物以其代谢产物的化学多样性、独特的生理活性和潜在的药物开发前景引起了科学家的广泛关注。对东海海域不同地区采集的不同样品(包括海洋沉积物和海洋动物)用平板稀释法分离海洋微生物,实验表明培养基的组成和培养条件对海洋微生物的生长及活性物质的产生都有很大的影响。从三个海底沉积物样品中分离细菌466株,真菌53株,放线菌8株；从深海鲨鱼鱼鳃和胃肠道部位分离得到可培养微生物36株,来自鱼鳃5株,来自胃肠道31株,其中真菌7株,占19%；放线菌1株,占3%；细菌28株,占78%。以枯草芽孢杆菌(Bacillus subtilis)、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coliC)、铜绿假单胞菌(Pseudomonas aeruginose)、产气肠杆菌(Enterobacter aerogenes)、白色念珠菌(Candida albicans)作为指示菌,对鲨鱼胃肠道中的菌株采用牛津杯法筛选产抗菌物质的微生物。对分离自鲨鱼鳃及胃肠道的菌株进行形态、生理生化实验、16SrRNA分子鉴定和系统发育分析,为进一步研制海洋抗菌活性物质提供一些基本参数和活性菌株。本研究以非核糖体多肽酶系作为研究对象,以NRPSs A结构域保守序列设计简并引物,对鲨鱼胃肠道细菌进行了NRPSs的筛选,希望能获得NRPSs基因片段,为今后开发海洋来源的非核糖体多肽类物质打下基础。从样品中分离得到了一株具有抗菌活性高、抗菌谱广的细菌,并对菌株和其代谢抗菌产物进行了研究。研究证实,该菌株对革兰氏阴性菌、革兰氏阳性菌、真菌都有一定的拮抗作用,特别是对产气肠杆菌、金黄色葡萄球菌、白色念珠菌抗菌作用较强。对细菌Y9所产抗菌活性物质理化性质初步研究表明,该抗菌活性成分热稳定性较强,酸性、中性条件下稳定,碱性条件下不稳定,非蛋白类物质,易溶于甲醇、乙醇。更多还原

【Abstract】 With the wide use of antibiotics and other antibacterial agents, bacteria resistant to many antibiotics is increasing seriously, for example, Methicillin-resistant Staphylococcus aureus (MRSA) which is resistant to most antibiotics is increasing rapidly and has become the vital pathogenic bacteria in the clinic. A renewed effort should be made to seek new antibiotics. Microorganism resources in land once were the main source for screening for antibiotic. However, with the research going on, it is becoming more and more dificult to find new antibiotics. It is necessary to seek for the new medicine source. Since the specific living environment, marine microorganisms have unique heredity background and metabolite pathways, and synthesize some distinct antibiotics in structures. Therefor it has significance for antibiotics screening from marine microorganism. And the marine microorganisms have attracted considerable attention because of chemical unique, rich physiological properties and thus potential as important drugs of their metabolites.Many kinds of samples, in cluding sea mud and sea vertebrata were collected from East Sea from different region. It is greatly influenced by the composition of medium and culture condition on the production of the metabolites with antimicrobial activity and the growth of marine microorganism.446 strains marine bacteria,8 strains marine actinomycetes and 53 strains marine fungi were isolated from the sea mud. Total 36 culturable microorganisms,28 marine bacteria,1 strains marine actinomycetes and 7 strains marine fungi were isolated from the gills, stomachs or intestines of deep-sea shark,of which 5 strains were from gills and 31 strains were from stomachs or intestines.Among them,there were 7 strains of fungi (19%),1 strains of actinomycetes (3%) and 28 strains of bacteria (78%). Their antibiotic activities against six microorganisms (Bacillus subtilis、Staphylococcus aureus、Escherichia coli、Pseudomonas aeruginose、Enterobacter aerogenes、Candida albicans) were assayed Oxford Cup method.The isolated strains from the gills, stomachs or intestines of deep-sea shark is identified through morphological, physiological and biochemical experiment,16S rRNA sequence analysis and phylogenetic analysis. It will provide some basic information and some bioactive strains for the discovery of the metabolites with antimicrobial activity from sea.In this study, a pair of degenerate oligonucleotide primers, disigned for amplification A domain in NRPSs, had been employed to screen NRPSs gene fragments from the gills, stomachs or intestines of deep-sea shark. Our purpose was to develop a method to access NRPSs gene diversity in sea. This would open up the possibilities of the secondary metabolites and as the basis for a search for attractive antibiotic biosynthesis genes that could be used in the heterogenous expression and combinatorial biosynthesis. Furthermore, the amplified NRPSs gene fragments can also be used as homologous hybridization probes to detect the clones harbored NRPSs gene clusters.One strain is screened from the gills,stomachs or intestines of deep-sea shark. The metabolites produced by the strain have high antimicrobial activity and broad antimicrobial spectrum. The strain and its metabolites are researched. The results show that the culture has a broad antimicrobial spectrum against some gram-negative and gram-positive bacteria, and moulds, especially against Enterobacter aerogenes, Staphylococcus aureus and Candida albicans. The physical and chemical properies of the metabolites with antimicrobial activity of Y9, the results showed it has good stability against temperature and acidity, but not alkali. The metabolites with antimicrobial activity are identified which is not a kind of protein, and it can be dissolved in methyl alcohol and ethanol.更多还原