药用地衣中内生菌抗菌活性物质的研究

Lichens, which are the special symbiotic of cyanobacteria and filamentous fungus,are widely used as antibacterial drugs. The development and utilization of the lichens are limited, because most lichens grow very slowly. There are few reports about the chemical constitutions and the biological activities of the endolichenic fungi, although some novel antibiotic secondary metabolites were isolated from the endolichenic fungi. In our studies, we will isolate the antimicrobial secondary metabolites from four endophytic fungi of medicinal lichen, based on the agar diffusion method,study the antibiotic effect of the compounds in vivo and in vitro,and investigate the antibacterial effect and related mechanism of those compounds on MRSA strain by observing the effect of the compounds on the cell wall components and the whole- cell protein profile of MRSA strain. The Synergistic effect of those compounds and β-lactams in vitro against MRSA was performed by microdilution method. Expressions of lytM and lgrA gene were detected by RT- PCR, and the changes of autolysis activity were monitored. Expressions of PBP2a protein were detected by western blot. β-lactamase were extracted by ultrasonic fragmentation method , and inhibition test of those compounds and tazobactam against β-lactamase was performed and analyzed. Finally, we will optimize fermentation conditions of antibacterial strains for medicinal compositions.

虽然地衣这种特殊的菌藻共生体被作为抗菌药物广泛使用，但是因生长非常缓慢，其资源的开发利用被限制。内共生菌存在大量结构新颖的活性物质，而关于地衣中内生菌化学成分及生物活性的报道为数不多。本课题以琼脂扩散法抗菌实验为指导，对四种药用地衣的内生菌中的抗菌活性成分进行分离；对获得的化合物进行抗菌作用的体内外药效学评价，并总结其构效关系；通过研究活性化合物对MRSA细胞壁和总蛋白的影响，初步阐明其抗MRSA菌作用机制；通过对活性化合物与β-内酰胺类药物联用，观察对相关耐药基因lytM、lgrA表达的影响，观察药物对MRSA中PBP2a蛋白，自溶酶，β-内酰胺酶的影响，初步探讨其逆转MSRA耐药作用的分子机制；同时选取具有开发前景的活性菌株，进行发酵和提取工艺的优化，以期得到具有能耗少，发酵单位高、产品质量好，成本低的工艺条件，为利用丰富的地衣内生菌资源奠定基础。

虽然地衣这种特殊的菌藻共生体被作为抗菌药物广泛使用，但是因生长非常缓慢，其资源的开发利用被限制。内共生菌存在大量结构新颖的活性物质，而关于地衣中内生菌化学成分及生物活性的报道为数不多。本课题以琼脂扩散法抗菌实验为指导，对四种药用地衣的内生菌中的抗菌活性成分进行分离得到20多个化合物，其中新化合物5个；对获得的化合物进行抗菌作用的体外药效学评价；同时选取具有开发前景的活性菌株，进行发酵和提取工艺的优化，初步得到具有能耗少，发酵单位高、产品质量好，成本低的工艺条件。但未进行研究活性化合物对MRSA细胞壁和总蛋白的影响，初步阐明其抗MRSA菌作用机制；通过对活性化合物与β-内酰胺类药物联用，观察对相关耐药基因lyt M、lgr A 表达的影响，观察药物对MRSA中PBP2a蛋白，自溶酶，β-内酰胺酶的影响，初步探讨其逆转MSRA耐药作用的分子机制方面的研究工作。