【作者】 褚亚东；

【导师】 马郁芳；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2017， 硕士

【摘要】 结核病(Tuberculosis),是由结核分枝杆菌(Mycobacterium tuberculosis)引起的空气传播性疾病。2014年全球约150万人死于结核病,约960万结核病新病例。且耐多药结核病的威胁越来越大,约3.3%新病例和20%曾接受治疗的病例患耐多药结核病,且多年来处于此水平。但近40年来只有一种新药物—贝达喹啉被批准。筛选新的抗结核药物迫在眉睫。放线菌作为重要的活性产物来源,其在抗结核药领域也贡献了大量一线、二线抗结核药物,如利福平、利福定、链霉素、阿米卡星、卷曲霉素等。海洋放线菌由于其独特的生活环境,活性物质也频频被人们报道尤其是海绵共附生放线菌,是筛选高活性产物的良好资源。本论文基于中国科学院大连化学物理研究所海洋生物工程组多年来从大连黄渤海、南海和南极的海绵样品中分离的放线菌组成的海洋放线菌资源库为基础,利用耻垢分枝杆菌作为活性筛选模式菌,以Alamar Blue Test法测定放线菌两种活性提取物(菌体提取物,发酵液提取物)抗耻垢分枝杆菌的活性,并结合菌株的16S r DNA信息构建海洋放线菌抗分枝杆菌活性菌株库,最后对两株菌株进行大量发酵和活性物质纯化。本论文意在建立专门用于抗结核病药物筛选的放线菌菌株库和组分库,为今后的抗结核病药物的发现提供有效的资源和借鉴。研究结果如下:(1)测定了185株海绵共附生放线菌两种提取物EF1(菌体湿重:水:丙酮1:2:2萃取后干燥制得),EF2(发酵液:乙酸乙酯1:2萃取后干燥制得)抗耻垢分枝杆菌的活性。通过Alamar Blue Test法,在96孔板中以耻垢分枝杆菌为模式菌,以刃天青为指示剂,分别测定EF1、EF2抗耻垢分枝杆菌最小抑菌浓度MIC值。其阳性率达到43.2%,高活性菌株比例为9.7%。在18株高活菌株中(EF1:MIC低于9.8μg/m L,EF2:MIC低于1.95μg/m L),有三株菌的发酵液提取物EF2的MIC值为0.24μg/m L,与临床抗结核药乙胺丁醇相同。这些高活菌株可作为新型抗结核药物筛选的资源。(2)提取放线菌基因组DNA后采用引物8F、1492R对放线菌16S r DNA进行PCR扩增并测序。用Molecular Evolutionary Genetics Analysis软件包(MEGA v6)分析放线菌16S r DNA的测序信息,构建系统进化树。发现高活性菌株都属于链霉菌属,且有一定的聚集性。将高活性菌株的16S r DNA序列信息通过NCBI的Gen Bank数据库比对分析,并用相似度前10的菌株名称进行检索,筛选其中抗生素有关报道及活性物质种类,发现其活性物质种类分布广泛,包括大环内酯类、多肽类、核苷类、氨基糖苷类、多烯类、杂环类、吡喃酮类等。这对抗结核病药物筛选放线菌菌株库和组分库的构建提供了重要的信息。(3)对菌株706进行了培养基及培养时间优化,最终确定养条件为低盐低葡萄糖培养基(酵母浸粉4 g/L、麦芽提取物10 g/L),培养时间5天。经发酵罐培养后,其EF1经过两次固相萃取纯化后,其MIC值从156μg/m L下降到7.8μg/m L。菌株D238经发酵罐培养后发酵液提取物EF2通过液相色谱柱纯化后,其MIC值从0.78μg/m L下降到0.37μg/m L。菌株706和菌株D238的活性组分还不够纯,需要继续纯化后做结构分析。更多还原

【Abstract】 Tuberculosis(TB)is an airborne disease caused by Mycobacterium tuberculosis.In2014,1.5 million people died from TB,and 9.6 million people were infected as new cases with TB.The threat of multidrug-resistant TB(MDR-TB)is growing,globally,an estimated 3.3% of new cases and 20% of previously treated cases have MDR-TB;these levels have remained virtually unchanged in recent years.However,in the last 40 years only one new drug-Beidaquinoline was approved.Screening new anti-tuberculosis drugs is imminent.Actinomycetes,as important source of active products,have also contributed a large number of first-line and second-line anti-tuberculosis drugs such as Rifampicin,Rifaxin,Streptomycin,Amikacin and Capreomycin.Marine actinomycetes is good resource for screening highly active products as a result of its unique living environment and frequently reported active substances.This thesis was based on the marine actinomycete resource library composed of actinomycetes isolated from the sponge samples of Dalian Yellow Sea,Bo-hai Sea,South China Sea and Antarctica for many years by Marine Bio Engineering Group of Dalian Institute of Chemical Physics,Chinese Academy of Sciences.Mycobacterium smegmatis was use as a model of Mycobacterium tuberculosis to screen active substances.The anti-mycobacterial activity of active extracts from actinomycetes(EF1:bacterial extract,,EF2: fermentation broth extract)was determine by Alamar Blue Test.A marine actinomycetes library with anti-mycobacterial activity was build and 16 S r DNA information of strains was analyzed.Finally,two strains of marine actinomycetes were fermented and the active components were purified.In this paper,it is intended to establish a marine actinomycete strain library and active component library specifically for the screening anti-tuberculosis drugs,which will provide effective resources for anti-tuberculosis drugs discovery in the future Result:(1)The two kinds of extracts(EF1,EF2)from 185 starins epiphytic actinomycetes with sponge showed activity against M.smegmatis.Mycobacterium smegmatis,a model bacterium of M.tuberculosis,grown in 96-well plates was used to detemine the minimum inhibitory concentration(MIC)value by Alamar Blue Test method.The positive rate with inhibition was 43.2%(80/185),the proportion of highly active strains was 9.7%(18/185).In the 18 highly active strains(MIC of EF1 was less than 9.8 μg/m L and MIC of EF2 was less than 1.95 μg/m L),the MIC value of EF2 extracted from 3 strains was 0.24μg/m L,which was the same as ethambutol.These highly active strains can be used as a resource for screening new anti-tuberculosis drugs.(2)The genomic DNA of actinomycetes was extracted,then the 16 S r DNA of actinomycetes was PCR amplified using universal primer 8F and 1492 R and sequenced by Takara Biotechnology(Dalian).The phylogenetic tree was constructed by using 16 S r DNA sequence information.The result showed that highly active strains belonged to Streptomyces and had some aggregation.The 16 S r DNA sequence information of the highly active strains was analyzed by Gen Bank database of NCBI,and the name of the bacterial strain with top 10 similarity was used to search for the informations about antibiotics and the type of the active substance.The species of active substances are widely distributed through the activity of the same or similar species,including Macrolides,Peptides,Nucleosides,Aminoglycosides,Polyenes,Heterocycles and Pyrones.(3)The culture medium and culture time of strain 706 were optimized.The final conditions were as follows: low salt and low glucose medium(yeast leaching powder 4g / L,malt extract 10 g / L),culture time of 5 days.The MIC of extract EF1 of strain706 which was cultured in fermenter was decreased from 156 μg / m L to 7.8 μg / m L after two solid phase extraction.The MIC of extract EF2 of strain D238 which was cultured in fermenter was decreased from 0.78 μg / m L to 0.37 μg / m L after purification by liquid chromatography.The active components of strain 706 and strain D238 were insufficient pure,therefore,they need to be further purified for structural analysis.更多还原