贵州典型生境来源放线菌分离及抗临床耐药菌株\抗肿瘤活性天然产物发现

In recent years, clinically isolated drug-resistant strains has increased dramatically and the increase of incidence of tumors put forward a huge challengenew for antibiotic drug development. There may exist some new new antibiotics actinomycetes in typical ecological environment in guizhou. In this study,potential actinomyces with new antibiotics production capacity will be isolated guided by the results of anti-clinical drug resistant bacteria and anti-tumor acticity. actinomyces with a potential of production of new natrual product will be screening based on the analysis of ultraviolet spectrum andmass spectrometry. Molecular manipulation,ribosome Engineering and growth condition optimization were employed to provoke the produce of new natrual products.Draft genome was sequenced in order to help the structure elucidation of new natrual products and the analysis of their bioactive mechanisms.

近年来，临床上分离耐药菌株急剧增加以及肿瘤发病率的增加给新型抗生素类药物开发提出了挑战。贵州相对封闭的典型生境可能存在着产生新型抗生素的放线菌资源。本研究以抗临床耐药菌和肿瘤为活性导向，对从贵州不同典型生境中分离的链霉菌进行筛选，获得抗耐药菌或抗肿瘤的潜力菌株。以紫外光谱和质谱分析为基础，对潜力菌株小量发酵产物进行筛选，获得可能产生新活性化合物的待开发菌株。以分子操作、核糖体工程和优化培养条件为手段，刺激待开发菌株产生活性次级代谢产物（或提高其产量）。在纯化制备新活性产物的基础上，绘制待开发菌株基因组草图，预测其次级代谢基因簇及产物合成途径和可能的产物结构以指导活性产物结构解析和活性机制分析。以期获得一定数量的抗临床耐药菌株或抗肿瘤的新活性天然产物并初步阐明其活性机制。

采用分离培养基纯培养技术对贵州省特殊生境土壤放线菌进行分离、纯化、鉴定，采用高通量筛选方法 筛选出抗菌抗肿瘤活性菌株；分离鉴定活性菌株的次级代谢产物活性成分，得到zunyimycin系列化合物；利用DNA重组技术构建基因工程菌，诱导蛋白表达使之与zunyimycin C相互作用从而研究zunyimycin C化合物抗MRSA活性机制进行初步分析；分别使用CCK-8和集落形成试验研究zunyimycin C对肺癌细胞的细胞增殖抑制影响。 通过RT-PCR实验研究肺癌细胞相关凋亡基因mRNA表达。通过流式细胞术检测细胞凋亡率，蛋白质表达通过使用特异性抗体蛋白质印迹显现探究zunyimycin C在肺癌细胞信号通路中的作用。