抗结核海洋放线菌的筛选及菌株HY286生物活性研究

doi:10.13560/j.cnki.biotech.bull.1985.2017-0388

摘要/Abstract

摘要： 海洋放线菌是研究抗结核药物及其先导化合物的重要来源。以耻垢分枝杆菌（Mycobacterium smegmatis,Msm）作为指示菌,采用平板对峙法,从漳州浅滩泥土分离的77株放线菌中筛选出9株对Msm具有抑制作用的抗性放线菌,其中1株对Msm具有高效抑制作用的拮抗放线菌HY286。通过对菌株HY286的菌落形态观察、生理生化鉴定及16S rDNA序列分析,初步鉴定其为一株马杜拉属放线菌。通过测定菌株HY286发酵产物的抗耻垢分枝杆菌MIC值、抗菌活性和细胞毒活性。结果表明,该菌株产生化合物的抗耻垢分枝杆菌MIC值为200 μg/mL,具有较好的抗结核分枝杆菌的潜力,且对枯草芽孢杆菌、短小芽孢杆菌也具有较好的抑制作用,对HeLa和HepG2的抑制率分别为89.3%和94.2%。

关键词: 抗结核, 马杜拉属, 海洋放线菌, 筛选, 生物活性

Abstract: Marine actinomycetes are recognized as an important resource of screening new anti-tuberculosis drugs and their precursor compounds. Nine marine actinomycetes strains with efficient antagonism to Mycobacterium smegmatis（Msm）were screened by plate confrontation method from 77 actinomycetes in shallow soil,Zhangzhou. One strain HY286 with the highest inhibitory effect was identified as Actinomadura genus by colony morphological,physiological and biochemical characteristics,and 16S rDNA sequence analysis. The MIC of fermentation crude extract of strain HY286 was 200 μg/mL,indicating that it had a significant inhibitory effect on Msm. The fermentation crude extract of strain HY286 also showed inhibitory effects on Bacillus subtilis and Bacillus pumilus,and the inhibition rate to HeLa and HepG2 was 89.3% and 94.2%,respectively.

Key words: anti-tuberculosis, Actinomadura, marine actinomycetes, screening, bioactivity

瞿佳, 赵玲侠, 陈锐, 路鹏鹏, 孙晓宇, 沈卫荣. 抗结核海洋放线菌的筛选及菌株HY286生物活性研究[J]. 生物技术通报, 2017, 33(11): 194-199.

QU Jia, ZHAO Ling-xia, CHEN Rui, LU Peng-peng, SUN Xiao-yu, SHEN Wei-rong. Screening of Anti-Tuberculosis Marine Actinomycetes and the Bioactivity of Strain HY286[J]. Biotechnology Bulletin, 2017, 33(11): 194-199.

参考文献

[1] Kim PS, Hafner R. Viewpoint：Challenges and opportunities in tuberculosis research[J]. Journal of Infectious Disease, 2012, 205（suppl. 2）：347-352.
[2] Amaral L, Molnar J. Potential therapy of multidrug-resistant and extremely drug-resistant tuberculosis with thioridazine[J]. In Vivo, 2012, 26（2）：231-236.
[3] Villemagne B, Crauste C, Flipo M, et al. Tuberculosis：the drug development pipeline at a glance[J]. European Journal of Medicinal Chemistry, 2012, 51（5）：1-16.
[4] Fenical W, Jensen PR. Developing a new resource for drug discovery：marine actinomycete bacteria[J]. Nature Chemical Biology, 2006, 2（12）：666-673.
[5] 焦炳华, 穆军, 许强芝, 等. 海洋微生物来源新抗生素的研究[J]. 抗感染药学, 2004, 1（1）：1-9.
[6] Feling RH, Buchanan GO, Mincer TJ, et al. Salinosporamide A：a highly cytotoxic proteasome inhibitor from a novel microbial source, a marine bacterium of the new genus Salinospora[J]. Angewandte Chemie International Edition, 2003, 42（3）：355-357.
[7] Bister B, Bischoff D, et al. Abyssomicin C-A polycyclic antibiotic from a marine Verrucosispora strain as an inhibitor of the p-aminob-enzoic acid/tetrahydrofolate biosynthesis pathway[J]. Angewandte Chemie International Edition, 2004, 43（19）：2574-2576.
[8] Kwonhc HC, Kauffman CA, Jensen PR, et al. Fenical W. Marinomycins A-D antitumour antibiotics of a new structure class from a marine actinomycete of the recently discovered genus “Marinispora”[J]. Journal of the American Chemical Society, 2006, 128（5）：1622-1632.
[9] Olano C, Mendez C, Salas JA, et al. Antitumor compounds from marine actinomycetes[J]. Marine Drugs, 2009, 7（2）：210-248.
[10] Rahman H, Austin B, Mitchell WJ, et al. Novel anti-infective compounds from marine bacteria[J]. Marine Drugs, 2010, 8（3）：498-518.
[11] Fattorusso E, Taglialatela SO. Marine antimalarials[J]. Marine Drugs, 2009, 7（2）：130-152.
[12] Stanley SA, et al. Identification of novel inhibitors of M. tuberculo-sis growth using whole cell based high-throughput screening[J]. ACS Chemical Biology, 2012, 7（8）：1377-1384.
[13] 李仲兴. 耻垢分枝杆菌群感染及其菌种鉴定[J]. 中华检验医学杂志, 2006, 29（5）：475-477.
[14] 关艳, 杨延辉, 等. 以耻垢分枝杆菌为模式菌快速筛选新型抗结核药物[J]. 中国新药杂志, 2014, 23（10）：1175-1187.
[15] 东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京：科学出版社, 2001：162-364.
[16] 萨姆布鲁克, 拉塞尔著;黄培堂, 等译. 分子克隆实验指南[M]. 第3版, 北京：科学出版社, 2008.
[17] Tamura K, Dulley J, Nei M, et al. MEGA4：molecular evolutionary genetics analysis（MEGA）software verison 4. 0[J]. Molecular Biology Evolution, 2007, 24（8）：1596-1599.
[18] 鄂征. 组织培养与分子细胞学技术[M]. 北京：北京出版社, 1995.
[19] 刘全永, 胡江春, 薜德林, 等. 海洋微生物生物活性物质研究[J]. 应用生态学报, 2002, 13（7）：901-905.
[20] Goodfellow M, Fiedler HP. A guide to successful bioprospecting：informed by actinobacterial systematics[J]. Antonie Van Leeuwenhoek, 2010, 98（2）：119-142.
[21] 王宏梅, 赵心清. 可培养海洋放线菌生物多样性研究进展[J]. 微生物学通报, 2007, 34（5）：996-1000.
[22] Ergan B, Coplu L, Alp A, et al. Mycobacterium smegmatis pneumonia[J]. Respirology, 2004, 9（2）：283-295.
[23] Lechevalier HA, Lechevalier MP, Gerber NN, et al. The actinomycetes[M]Jena：Gustav-Fische, 1970.
[24] Stackebrandt E, Woese CR. Towards a phylogeny of the actinomycetes and related organisms[J]. Current Microbiology, 1981, 5（4）：197-202.
[25] Panchanathan M, Kyong-Hwa K, Kannan S, et al. Marine actinobacteria：An important source of bioactive natural products[J]. Environmental Toxicology and Pharmacology, 2014, 38（1）：172-188.
[26] Lahouuma A, Aouichea N, Bourasab C, et al. Antifungal activity of a Saharan strain of Actinomadura sp. ACD1 against toxigenic fungi and other pathogenic microorganisms[J]. Journal of Medical Mycology, 2016, 26（3）：193-200.
[27] Mazzei E, Iorio M, Maffioli, et al. Characterization of madurastatin C1, a novel siderophore from Actinomadura sp.[J]. The Journal of Antibiotics, 2012, 65（5）：267-269.
[28] Motoki T, Keiichiro M, Shams T, et al. JBIR-65, a new diterpene, isolated from a sponge-derived Actinomadura sp. SpB081030SC-15[J]. Cheminform, 2010, 41（49）：401.
[29] Han XX, Cui CB, Gu QQ, et al. ZHD-0501, a novel naturally occurring staurosporine analog from Actinomadum sp.[J]. Tetrahedron Letters, 2015, 46（36）：6137-6140.
[30] Harada K, Tomita K, Fujii K, et al. Isolation and structural characterization of siderophores, madurastatins, produced by a pathogenic Actinomadura madurae[J]. Journal of Antibiotics, 2004, 57（2）：125-135.
[31] Dirlam JP, Belton AM, Bordner J, et al. CP-82, 996, a novel diglycoside polyether antibiotic related to monensin and produced by Actinomadura sp.[J]. Journal of Industrial Microbiology, 1990, 6（2）：135-177.
[32] Badji B, Zitouni A, Mathieu F, et al. Antimicrobial compounds produced by Actinomadura sp. AC104 isolated from an Algerian Saharan soil[J]. Canadian Journal of Microbiology, 2006, 52（4）：373-382.
33] Maskey RP, Li FC, Qin S, Fiebig HH, Laatsch H. Chandrananimy-cins A ~ C：Production of novel anti-cancer antibiotics from a marine Actinomadura sp. isolate M048 by variation of medium composition and growth conditions[J]. Journal of Antibiotics, 2003, 56（7）：373-382.
[34] Chakapong I, Aibrohim D, Sumalee S, et al. 3-Oxyanthranilic acid derivatives from Actinomadura sp. BCC27169[J]. Cheminform, 2014, 45（36）：102.