红树木榄生境中可培养细菌物种多样性及其体外抗乙肝病毒活性研究

Species diversity and anti-hepatitis B virus activity of culturable bacteria isolated from the habitat of Bruguiera gymnorhiza

关键词:  木榄, 可培养细菌, 物种多样性, 代谢产物, 抗乙肝病毒活性

Key words:  Bruguiera gymnorhiza, culturable bacteria, species diversity, metabolites, anti-HBV activity

摘要:

红树生境中微生物菌群丰富,其次生代谢产物结构新颖,是挖掘新型药物的重要来源。该研究利用纯培养技术和16S rRNA分子生物学技术确定细菌种属,并进行物种多样性分析; 以HepG2.2.15细胞株为模型,通过MTT和PCR技术测试细菌代谢产物的抗乙肝病毒活性; 使用LC-HRMS技术对活性菌株代谢产物进行初步分析,初步评价木榄沉积物、根、叶以及胚轴的可培养细菌多样性以及细菌代谢物生物活性,寻找抗乙肝病毒的药源菌株。结果表明:(1)共获得细菌59种,分属于4门5纲14目23科36属,其中芽孢杆菌为优势菌属; 菌株GXIMD07402、GXIMD07665、GXIMD07384分别为Pseudooceanicola属、Thioclava属和Aestuariibaculum属的潜在新种。(2)抗乙肝病毒活性结果显示GXIMD07366、GXIMD07616、GXIMD07384 、GXIMD07550、GXIMD07445X提取物能显著降低HepG2.2.15细胞上清液中HBV DNA水平(P<0.05),抑制率分别为51%、47%、63%、52%、47%。(3)初步鉴定强活性菌株GXIMD07384的4个主要代谢产物有adenosine、cyclo(L-Pro-L-OMet)、acremine G和7,8-dimethylbenzo[g]pteridine-2,4(1H,3H)-dione。综上认为,木榄生境中可培养细菌物种多样性丰富且含有能产生抗乙肝病毒活性化合物的菌株。该研究结果为后续海洋微生物资源的应用提供了基础。

Abstract:

Mangrove habitat is rich in microbial flora and its secondary metabolites have novel structure, which is an important source for mining new drugs. This study preliminarily evaluated the culturable bacterial diversity of sediments, roots, leaves and hypocotyls of Bruguiera gymnorhiza and the biological activity of bacterial metabolites, and looked for anti-HBV drug source strains. Pure culture technique and 16S rRNA molecular biology technique were employed to determine the species of bacteria and analyze the species diversity. Using HepG2.2.15 cell line as a model, the anti-HBV activity of bacterial metabolites was tested by MTT and PCR techniques. The secondary metabolites of active bacteria were preliminary analyzed by LC-HRMS technique. The results were as follows:(1)A total of 59 species of bacteria were obtained, belonging to 4 phyla, 5 classes, 14 orders, 23 families, and 36 genera, among which Bacillus was the dominant genus. Strains GXIMD07402, GXIMD07665 and GXIMD07384 were potential new species of Pseudooceanicola, Thioclava and Aestuariibaculum, respectively.(2)The results of anti-HBV activity showed that GXIMD07366, GXIMD07616, GXIMD07384, GXIMD07550 and GXIMD07445X could significantly reduce the level of HBV DNA in the supernatant of HepG2.2.15 cells(P<0.05), and the inhibition rates were 51%, 47%, 63%, 52% and 47%.(3)Four main secondary metabolites of the highly active strain GXIMD07384 were preliminarily identified as Adenosine, Cyclo(L-Pro-L-OMet), Acremine G and 7,8-dimethylbenzo[g]pteridine -2,4(1H,3H)-dione. The results of the study confirm that the species diversity of culturable bacteria in the habitat of Bruguiera gymnorhiza is rich, and it contains strains that can produce anti-HBV active compounds, which provide a basis for the subsequent application of marine microbial resources.