Avian Myeloblast Virus Reverse Transcriptase 활성 저해 세균의 분리

Abstract

To screen antiretroviral compounds from microbial metabolites, 300 soil microorganism strains focusing Actinomycete family were collected and purified in Jeju. Those microbial isolates were cultivated and their culture filtrates were used as the samples for screening antiretroviral compounds. Inhibitory activity of microbial culture filtrate against avian myeloblast virus(AMV) reverse transcriptase (RT) was determined. About 87% of the 300 isolates showed AMV RT inhibitory activity in the primary screening test. Those isolates that showed higher than 60% inhibition of RT activity were chosen and their inhibitory activities were confirmed repeatedly using their cell-free culture filtrate. The inhibition rates of BLK268, BLK115, BLK418 and BLK338 were 98.7± 0.3%, 97.9± 0.2%, 97.9± 2.7%, and 95.9± 1.6%, respectively. The isolates BLK311, BLK90, BLK462-2, BLK248-2, CB249-2, and BLK107 showed morer than 80% inhibition, and BLK4, BLK86, BLK123, and BLK39 higher than 70% inhibition of AMV RT activity. Among those isolates, six strains including BLK39, BLK90, BLK107, BLK115, BLK268 and BLK418 were chosen for further study. In order to confirm the dependence of the inhibitory activity on microbial metabolite concentration, the cell-free culture filtrates were diluted several folds and added to the RT activity assay system. The results demonstrated that RT inhibition rate increased in general as the dilution rate decreased. The heat stability of active microbial metabolites were also investigated. When the culture filtrates were preincubated for 1 hour at 30℃ or less, no decrease in the inhibitory activity was observed. But most of the isolates lost the inhibitory activity rapidly at temperatures higher than 50℃, whereas the isolate BLK90 maintained relatively high inhibitory activity at above 50℃. The unknown isolates were identified through 16S rRNA gene analysis. The nucleotide sequences of 16S rDNA from BLK39, BLK90, BLK107, BLK115, BLK268 and BLK418 were determined and compared with those of other known microbial species. The isolates BLK 39, BLK115, BLK268 and BLK418 showed more than 96% sequence homology with Streptomyces species. Meanwhile, the 16S rDNAs of isolate BLK90 and BLK107 were similar to those of gram negative bacteria which belong to the genus Erwinia or Pantoea. Morphological features of these bacteria were consistent with the results of rDNA sequence analysis. Further research about inhibition mechanism and chemical nature of those bacterial metabolites are suggested.