1. Stick The Separation And Purification Of Aspergillus 3277-3 3277 Metabolites And Their Anti-tb Activity In Vivo And In Vitro Studies (2) Targeted Isocitrate Lyase Withholding Was Mycobacterium Tuberculosis Drug Resistance Screening Study

My work focus on two parts:First is the isolation and purification of anti-tuberculosis Aspergillus clavatus 3277 from fermentation products. We got Aspergillus clavatus 3277 by screening 16,000 microorganism fermentation by a model targeting DNA graseB. A new anti-tuberculosis compound,3277-3 was isolated from the fermentation mycelium of Aspergillus clavatus 3277 by solvent extraction and silica gel column chromatography. The structure was identified by NMR, EI-MS and X-Ray apparatus. The compound was Xanthocillin x dimethyl ether (XDE).3277-3 exhibited strong activity on tuberculosis in vitro:the MIC on Mycobacterium tuberculosis H37Rv was 0.5μg/ml, and The MIC on MDR-330 and XDR-926 was≤0.125μg/ml; and 3277-3 exhibited activity on tuberculosis in vivo in a acute aerosol infection model.3277-3 had a low toxity and the maximum lethal dose>2000mg/kg. Second, we optimized and evaluated the high-throughtout screening model targeting to isocitrate lyase of of Mycobacterium tuberculosis established in our laboratory. we optimized the reaction time, substrate concentration and enzyme quantity; and we evaluated the model by four parameters:S/B, S/N, CV and Z. Then 2,960 samples of compounds were tested, and 9 positive compounds were acquired. In order to test the positive compounds on cellular level, a recombinant plasmid bearing icl-egfp gene was constrcted by using the shuttle vector pMV261. Then it was electroporated into avirulent Mycobacterium smegmatis mc2155. The transformant was induced to express the ICL-EGFP and EGFP protein and then will be used to infect the macrophage cell.