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目的:从海泥样品中分离培养并筛选获取野生型与突变型药源微生物抗肿瘤活性菌株。方法:通过单菌落挑选与划线培养,分离纯化野生菌株;利用核糖体工程抗性筛选技术,筛选获得抗生素抗性突变株;用K562细胞,采用MTT法测试发酵样品的抗肿瘤活性。结果:从天津塘沽驴驹河渤海湾潮间带海泥样品中分离得到放线菌126株、真菌29株,其中,在100μg/ml样品浓度下抑制率大于20%的放线菌17株、真菌3株,在1000μg/ml样品浓度下抑制率小于5.5%的无活性放线菌19株。以1000μg/ml样品浓度下抑制率为4.8%的无活性放线菌野生株L8-5x为原始菌,经抗性筛选获得链霉素抗性突变株78株,其中在100μg/ml样品浓度下抑制率大于25%的活性突变株2株。结论:所获野生型与突变型抗肿瘤活性菌株为后续新药筛选提供了药源微生物活性菌株。由无活性菌株转化获取活性突变株的研究结果表明,已分离得到的大量无活性菌株也是可转化获取药源活性突变株的很好资源。
OBJECTIVE: To isolate and culture antimicrobial strains of wild-type and mutant-type microorganisms from sea mud samples. Methods: The wild strains were isolated and purified by single colony selection and scribing culture. The antibiotic resistant mutant strains were screened by using ribosome engineering resistance screening technology. The antitumor activity of the fermented samples was tested by MTT assay using K562 cells. Results: A total of 126 actinomycetes and 29 fungi were isolated from the intertidal sea mud samples from the Donkey Juhe intertidal zone in Tanggu, Tianjin. Among them, 17 actinomycetes with the inhibitory rate greater than 20% at the concentration of 100μg / ml, 3 strains of fungi, 19 strains of actinomycetes with the inhibition rate of less than 5.5% at 1000μg / ml sample concentration. At the concentration of 1000μg / ml sample concentration inhibition of 4.8% of the actinomycetes wild-type strain L8-5x as the original strain, resistant strains were screened 78 strains of streptomycin-resistant, which at 100μg / ml sample concentration Inhibition rate greater than 25% of the 2 active mutant strains. Conclusion: The obtained wild-type and mutant antitumor activity strains provide drug-source microbial strains for the follow-up screening of new drugs. The results obtained from the transformation of inactive strains to obtain active mutants showed that a large number of inactive strains that have been isolated are also good resources for transforming and obtaining active mutant strains.