海绵附生放线菌抗结核分枝杆菌活性物质的筛选及分离

Screening and separation of anti-tuberculosis active substances from Actinomycete co-evolution with Spongilla

目的从海绵共附生的放线菌代谢物质中筛选出具有抗结核分枝杆菌活性的新型物质。方法采用平板稀释法从南海海域采集的海绵中分离共附生放线菌菌株,使用微孔板稀释法评估放线菌代谢物抗结核分枝杆菌H37Rv能力。根据分子量和极性,使用高效液相色谱(HPLC)对初筛具有较高抑菌活性的菌株培养上清进行分离,并分别从分离各组分中进一步筛选、鉴定具有抑制结核杆菌生长作用的活性物质。提取该菌株的基因组DNA,采用通用引物对其16S rRNA行PCR扩增,并对扩增产物进行测序比对。结果分离出的416株放线菌中,6株具有抗结核分枝杆菌功能,其中编号为NH128菌株的培养上清中含有抑制H37Rv生长的活性物质且作用较为明显;对其进行分离后的组分F3具有较高的抑菌活性;进一步通过HPLC和微孔板稀释法鉴定出,F3的亚组分S3含有高抑菌活性的物质,其MIC50约为3μg/m L。对NH128 16S rRNA序列比对结果显示,其与Aclinomycetales(R03 AY944262)同源性为99%。结论南海海域海绵附生放线菌能够产生较高的抗结核分枝杆菌活性物质;NH128菌株可能为放线菌菌属Aclinomycetales(R03 AY944262)的变种。

Objective To screen and separate anti-tuberculosis active substances from Actinomycete metabolite co-evolutionary with Spongilla. Methods The monoclonal colony of different kinds of actinomycete co-evolutionary with Spongilla from the South China Sea were separated by the plate dilute method,and the anti-tuberculosis ability of the cultured supernatant was detected. Aaccording to molecular weight and polarity,the anti-tuberculosis active substances of strains were selected out and cultured for further separation by HPLC. Meanwhile,the genome DNA of NH128 strains were extracted and the 16 S rRNA were amplified by PCR for sequencing. Results Of the 416 strains,6 positive strains,in which the supernatant of NH128 strain showed a significant anti-tuberculosis H37 Rv activity,were selected out. After the further separation,the component F3 and its sub-fraction S3,whose MIC50 component was about3 μg / m L,of the supernatant of NH128 strain showed strong anti-tuberculosis activities. The NH128 strain was highly homologous with Streptomyces Aclinomycetales bacterium（ R03 AY944262） in 99%. Conclusion Actinomycete coevolutionary with spongilla in South China Sea have some anti-tuberculosis substances,and NH128 strain may be the variant of Aclinomycetales bacterium（ R03 AY944262）.