无活性放线菌野生株抗肿瘤活性突变株的自发突变抗性筛选和化学诱变抗性筛选

Screening for bioactive mutants with antitumor activity from an actinomycetic wild-type strain without antitumor activity by antibiotic-resistant mutation technique and by coupled with chemical mutagen-induced mutation

目的由无活性放线菌野生株转化获取活性突变株,为药源活性产物研究拓展新菌株资源。方法以海洋来源无活性放线菌野生株L35-1为出发菌株,通过自发突变抗性筛选以及化学诱变结合抗性筛选,筛选获得抗性突变株。采用MTT法检测突变株发酵样品对K562细胞的抑制活性。结果通过自发突变抗性筛选,得到新霉素抗性突变株114株、链霉素抗性突变株68株,其中7株新霉素抗性突变株和3株链霉素抗性突变株样品对K562细胞有抑制作用,在100μg/ml浓度下抑制率大于20%。通过化学诱变结合抗性筛选,得到硫酸二乙酯诱变链霉素抗性突变株41株、硫酸二乙酯诱变新霉素抗性突变株32株、亚硝基胍诱变新霉素抗性突变株46株,其中1株硫酸二乙酯诱变链霉素抗性突变株和1株亚硝基胍诱变新霉素抗性突变株有抗肿瘤活性,100μg/ml样品对K562细胞的抑制率大于20%。结论上述结果初步表明,无活性放线菌野生株可通过抗性筛选转化为活性突变株,因此有可能成为筛选获取药源活性新菌株的重要原始资源。

Objective To obtain antibiotic-resistant mutants producing metabolites with antitumor activity from wild-type actinomycete strains without antitumor activity.Methods An actinomycete strain L35-1 was used as an initial strain for obtaining antibiotic-resistant mutants,which is a marine-derived wild-type strain without antitumor activity with an inhibition rate of 2.8% at the 1000 μg/ml of high sample concentration on K562 cells.The antibiotic-resistant mutants both from auto-mutagenesis and chemical mutagen-induced mutagenesis were selected by single colony isolation on antibiotic-containing plates according to the method for obtaining drug-resistant mutants in ribosome engineering.The antitumor activity was assayed by the MTT method using K562 cells for the mutants with aqueous acetone extracts of the whole broth of their fermentation.Results A total of 114 neomycin-resistant （ner） and 68 streptomycin-resistant （str） mutants,all from auto-mutagenesis,was obtained on drug-containing plates.Among them,the 7 ner and 3 str mutants appeared to be bioactive with an inhibition rate above 20% at the 100 μg/ml sample concentration on K562 cells.On the other hand,41 str and 32 ner mutants from DES-induced mutagenesis and 46 ner mutants from NTG-induced mutagenesis were obtained by mutagen-induced mutation coupled with the single colony isolation on antibiotic-containing plates,among which,one str mutant from DES-induced mutagenesis and one ner mutant from NTG-induced mutagenesis were bioactive with an inhibition rate over 20% at the 100 μg/ml sample concentration on K562 cells.Conclusions The present result has revealed that the wild-type actinomycete strains without bioactivity might become a great source initial strains to obtain bioactive mutants by drug-resistant mutation technique.