摘要
目的从2000多株放线菌发酵液中高通量筛选特异性端粒酶抑制剂。方法经对持有的3种端粒酶抑制剂筛选模型即3株酵母菌PCR验证后,将其接种于含3-AT、组氨酸缺乏的酵母培养液,然后分别调整其菌液浓度和筛选样品剂量,以备高通量筛选。将酵母菌液移于96孔板,加入待测样品,振摇培养并定时经多功能微孔板分析仪进行定量分析,选取抑菌率大于0.45的样品进行复筛。结果经验证所持有的3种端粒酶抑制剂筛选模型均可用于下一步的筛选,并选取酵母菌液OD595nm 0.04做为起始筛选浓度,选取放线菌发酵液剂量为5ml,通过对放线菌发酵液进行初筛和复筛,发现了14株放线菌发酵液的抑菌效率超过0.45。结论该研究首次高通量筛选端粒酶抑制剂,方法简便快捷,筛选结果稳定可靠,并筛选到14株放线菌发酵液可能具有抗肿瘤活性,为进一步研究和开发抗肿瘤新药奠定基础。
Aim To discover specific telomerase inhib- itors by high-throughput screening in the more than 2 000 strains of actinomycetes fermentation broth. Methods After verifying the three kinds of screening models for telomerase inhibitor by yeast PCR,the three yeast strains were inoculated overnight into yeast liquid culture which was lacking in histidine and contained 3-AT. Then concentration of yeast strains in culture liquid and dosage of screening sample were regulated for better adjustment to the high-throughput screening. At last the adjusted liquid culture containing yeast strain piped to 96-well plates,drug samples and control article were also added,then the yeast was incubated in the thermostat oscillator. At the same time OD595nm of the yeast liquid was measured every 12 h by using Multifunctional Microplate Analyzer,and the samples whose inhibitory efficiency was over 0. 45 were selected for secondary screening. Results The high-throughput screening method related to yeast three-hybrid system was effective in screening samples that had potential telomerase inhibitory ability. The initial concentration of yeast liquid OD595nm was 0. 04 and the best dosage of samples was 5 ml. 14 active samples,whose inhibitory efficiency was over 0. 45,were founded after primary and secondary screening. Conclusions High-throughput screening method related to yeast three-hybrid system is simple and stable in discovering telomerase inhibitors,and 14 new antitumor compounds are identified,which lays the foundation in the development of new anti-tumor agents.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2010年第1期40-43,共4页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No306724487)
关键词
放线菌发酵液
高通量筛选
端粒酶抑制剂
酵母三杂交系统
96孔板
抗肿瘤活性
actinomycetes fermentation broth high-throughput screening telomerase inhibitors yeast threehybrid system 96-well plates anti-tumor activity
