基于海绵共附生放线菌的抗结核活性物质筛选

Screening of anti-tuberculosis active substances from marine sponge associated Actinomycetes

目的:从海绵相关放线菌中高通量筛选具有拮抗分枝杆菌活性的菌株及其代谢物,评估已建立的海洋相关放线菌菌种库中抗分枝杆菌的活性比例,为发现新的拮抗结核病的先导化合物奠定基础.方法:对已建立的海绵相关放线菌菌种库中461株进行发酵,分别制备菌体丙酮提取物和发酵液乙酸乙酯萃取物,以抗耻垢分枝杆菌(Mycobacterium smegmatis,Msm)活性为导向,刃天青为筛选指示剂,于96孔板中测定放线菌菌体与发酵液萃取物的抗菌活性.通过测定供试菌株的16S rDNA序列,对抗Msm菌株进行聚类分析,获得具Msm抗性菌株种群特性.结果:筛选的461株放线菌中394株具有抗Msm活性,阳性率为85.5%,中等活性以上菌株占25.6%,32.2%的菌株菌体与发酵液均有活性,表明该菌种库所选择的评价体系阳性率高,具有抗结核药物及先导化合物的较好开发潜力.对23株具有高抑菌活性菌株16S rDNA分析表明,这些菌株均为链霉菌属.培养液乙酸乙酯萃取物中,菌株编号772,596与DL238抑菌活性最高,MIC为0.24μg/mL,低于目前广泛应用的乙胺丁醇(ethambutol)、链霉素(streptomycin)和吡嗪酰胺(pyrazinamide)等抗结核药物;菌体丙酮提物中,菌株D98活性最高为4.9μg/mL,与卷曲霉素(capreomycin)和卡那霉素(kanamycin)抗结核活性水平相当.结论:本研究结果表明海绵相关放线菌种库有望成为开发新型拮抗结核药物先导物的资源库.

Objective:To high-throughput screen the strains with anti-Mycobacterium activity from established marine sponge associated Actinomycete repository and evaluate the positive percentage.Method:461 Actinomycete strains isolated from marine sponge were fermented.The cells and cultured media were extracted using acetone and ethyl acetate,respectively.The anti-Msm activity of the extracts was measured on 96-well plates using resazurin as an indicator.Additionally,the 16 S rDNA from these strains was analyzed and the strains with anti-Msm activity were clustered.Results:394 strains of the 461 strains showed anti-Msm activity.The positive percentage was 85.5%,of which,25%of strains with above medium activity and 32.2%of strains showed anti-Msm activity in their extracts of microbial cells and culture media.These results indicated that our repository exhibited highly positive percentage for screening the anti-Msm active strains,which is promising for unearthing novel anti-tuberculosis drugs.23 strains are Streptomyces with high anti-Msm activity.The culture medium extracts from strains 772,596 and DL238 showed the highest activity with MIC at 0.24μg/mL,which was lower than that of ethambutol,streptomycin and pyrazinamide.Of the cell extracts,strain D98 had the lowest MIC of 4.9μg/mL,which was similar to that of capreomycin and kanamycin.Conclusion:Marine sponge related Actinomycetes repository is highly promising for exploiting novel anti-tuberculosis drugs.