[Bactericidal Activity of Constructed Recombinant Fusion Protein Pheromonicin-CT].

OBJECTIVE: To construct engineering peptide pheromonicin-Clostrzaum tretant krn-ui), and to test its bactericidal activity. METHODS: We amplified the gene of variable regions from hybridoma cells which secreted monoclonal antibody (mAb) against antigen in the membrane of Clostridium tetani and linked the small antibody mimetic to the channel-forming domain of colicin Ia to create Ph-CT. The Ph-CT was purified by CM sepharose ion-exchange column. Its in vitro antibacterial activity was evaluated by colony culture with different doses of Ph-CT (final concentration 2, 4, 8, and 16 microg/mL,respectively). Then we inoculated culture medium with CT strains and different doses of Ph-CT (final concentration of 4 and 16 microg/mL). The in vivo antibacterial activity of Ph-CT was evaluated by cumulative survival of mice. After 16 hours' anaerobic culture, the mice was treated with filtered CT medium or CT medium. RESULTS: We constructed Ph-CT successfully. CT colonies appeared in the CT medium treated with Ph-CT (2, 4 microg/mL), while no colony appeared in the CT medium treated with Ph-CT (8, 16 microg/mL). All mice survived when they were injected with filtered CT medium treated with Ph-CT (4, 16 microg/mL) and CT medium treated with Ph-CT (16 microg/mL). Three (50%) mice survived when they were injected with CT medium treated with Ph-CT (4 microg/mL). All mice in the control groups died after CT infections. CONCLUSION: Ph-CT may be of value as antibiotics against Clostridium tetani.

An engineered multidomain fungicidal peptide against plant fungal pathogens.

Fungal pathogens represent major problems for human health and agriculture. As eukaryotic organisms, fungi share some important features with mammalian cells. Therefore, current anti-fungal antibiotics often can not distinguish between fungi and mammalian cells, resulting in serious side effects in mammalian cells. Accordingly, there is strong impetus to develop antifungal alternatives that are both safe and effective. The E1 family of colicin are channel-forming bacteriocins produced by Escherichia coli, which are bactericidal only to E. coli and related species. To target the channel-forming domain of colicin to fungal cell membrane, we engineered a sexual mating pheromone of Candida albicans, α-factor pheromone to colicin Ia. A peptide was constructed consisting of an α mating pheromone of C. albicans fused to the channel-forming domain of colicin Ia to create a new fusion protein, pheromonicin-CA (PMC-CA). Indirect immunolabeling showed that the PMC-CA bound to fungal cells and inhibited growth in the laboratory and field. In the field, the protective activity of pheromonicin against rice blast disease was significantly greater, on a molar basis, than that of triazoles, tricyclazole or isoprothiolane. These results suggest that fusion peptides may be of value as fungicidal agents under agricultural conditions.