Aminoglycoside Antibiotics Bifunctional Enzyme Aac (6 ')-aph (2 ") Cloning And Expression Of Its Inhibitor Screening Model For The Establishment

Aminoglycosides (AGs) are among the most commonly used antibiotics in the treatment of bacteria infection. Because of their broad antimicrobial spectrum, sound effect, stability and other advantages, AGs have gained them a considerable market share. However, the emergence and prevalence of multidrug drug resistance have made the use of AGs limited. The main mechanism of bacterial resistance to AGs is caused by inactivation by intracellular bifunction enzymes which are widely distributed among bacterial pathogens. Researching and developing the screening model of AAC(6')-APH(2") inhibitors possesses some significance for new drug discovery.In the first part, a pair of detection primers were used to detect and confirm the aac(6')-aph(2") harboring in 6 MRSAs. Referred to the aac(6')-aph(2") entire sequence published in GeneBank, a pair of overexpression primer were designed and used to amplify aac(6')-aph(2") from the genome DNA of MRSAs. The purified aac(6')-aph(2") fragment was ligated into pET28a to yield an overexpression plasmid pYG1163. Then the new plasmid, pYG1163 was transformed into the E.Coli BL21(DE3)to form the recombinant strain SIPI-GE1102 which can be capable to overexpress AAC(6')-APH(2") in soluble form. Then the optimized IPTG concentration and induction temperature were determined as 0.1mmol/L and 30℃respectively. The activity of purified enzyme AAC(6')-APH(2") from recombinant strain SIPI-GE1102 was teseted in vitro by disk diffusion assay and Mass Spectrum and the results indicated that the enzyme possessed the expected activity.In the second part, we investigated the reaction conditions of AAC(6')-APH(2") in vitro and then established the AAC(6')-APH(2") inhibitor screening model in vitro, in which B. subtilis was employed as the assay strain, indolicidin as positive inhibitor control and kanamycin B as the target drug. The stability and reproducibility of this model was confirmed by using the known inhibitor as control. The process for samples to be screened was that dissolved 1500 extracts preserved in our laboratory from endophytic fungi broth in water with 2% DMSO and selected 762 samples having no activity to Bacillus subtilis to screen AAC (6 )-APH (2) enzyme inhibitors. After the first round screening, 18 samples showed inhibitory activity to the AAC(6')-APH(2") and in the second round screening, only one sample remained its inhibitory to the AAC(6')-APH(2").The AAC(6')-APH(2") inhibitor screening model established in this paper provided a new method to screening AGs modification enzymes inhibitor.