The Study On Targeting FtsZ's Bacteria Cell-Division Inhibitor Screening Model

FtsZ is a necessary protein in E.coli which plays an important role for cell division and now it is a hot antimicrobial target for new drug's screening. This research is based on FtsZ which has GTPase activity, and establishmed a model which can screen cell division inhibitors target FtsZ. The screening samples were fermentation products from endophytic bacteria. Our point was to find the sample which can inhibit the GTPase activity of FtsZ, and it may be the cell division inhibitors.1,First, amplified ftsZ gene from E.coli DH5a using PCR, then ftsZ was cloned into pET30a which was an E.coli expression vector. Transformed the plasmid into Escherichia coli BL21 (DE3) to express FtsZ with His tag. And we got white powder-like FtsZ protein by ultrtasonic disruption, Ni-NTA efficient affinity chromatography, dialysis, and vacuum freeze-drying2,Established FtsZ's GTPase activity measurement method: PMA-Malachite green spectrophotemetry. And used this method to detect FtsZ's enzymatic properties. The results showed that when we use GTP as substrate, the soluble FtsZ's optimum pH was 7.5, suitable temperature was 37℃, Km = 9.9×10-5 mol/1, optimal storage conditions of the freeze-dried protein powder was saved in -70℃.3,We established a vitro high throughput screening method with the best reaction conditions of FtsZ in 96 well plate, and used berberine as a positive control.4,We separated 1876 endophytic fungus from 256 species of medicinal plants, and prepared 1874 fermentation samples, we used high throughput screening method to screen these fermentation samples, the result was 282 samples can inhibit the GTPase of FtsZ, and further used liquid culture and microscopic examination to rescreening 282 samples, the result was that No. 127-B-2 and 140-A-10 can inhibit cell divisiong, we found they can elong the E.coli.