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Porcine contagious pleuropneumonia (porcine contagious pleuropneumonia, PCP) from Bacillus pleuropneumoniae (Actinobacillus pleuropneumoniae, APP) caused serious economic losses to the pig industry world. UDP-acetylglucosamine (UDP-N-acetylglucosamine, UDP-GlcNAc) is synthesized by the pathogen cell wall carbohydrates must precursors, such as peptidoglycan, lipid A, teichoic acids, etc., by the bifunctional enzyme N-acetyl glucose amine -1 - uridine phosphate acyl transferase (N-acetylglucosamine-1-phosphate uridyltransferase, glmU) Catalyst 1 - glucosamine phosphate (glucosamine-1-phosphate, GlcN-1-P), acetyl coenzyme A and UTP after the two-step reaction generation. If you interfere with UDP-GlcNAc synthesis path will be a serious impediment to the growth of bacteria and affect its virulence, so GlmU is an important drug target. Enzymatic kinetic analysis of GlmU protein is very necessary in order to study activity of the compounds of GlmU pilot. This thesis pleural pneumonia Actinomyces bacillus GlmU protein, major achievements are as follows: 1. GlmU gene cloning expression and purification according to the NCBI Genebank provide the pleura pneumonia put line bacillus serum type 3 JL03 strain whole genome sequence design cited material. As a template to the pleuropneumonia actinobacillus JL03 genome the PCR methods and amplified the glmU gene, was cloned into the pET-28a expression vector and highly expressed in E. coli BL21 (DE3) strain, expression products SDS-PAGE and Western blot analysis, the results showed that the recombinant protein with the expected molecular weight of the same size (50 KD), the successful purification of the protein has laid a good foundation for further study. The 2. GlmU uridylyl transferase enzymatic kinetic analysis using malachite green ammonium molybdate colorimetric method the measured initial velocity the GlmU uridylyl transferase enzyme, to determine its optimal cationic surfactant is a polyvinyl alcohol (PVA ), the best detection wavelength of 630 nm, the optimum reaction temperature of 37 ° C, the best reaction time of 5 min, the best reaction pH of 7.6, and its enzymatic kinetic constants were determined under the optimal reaction conditions: substrate GlcNAc-1-P, the Km value for glmU is 0.05478 mM, the maximum rate Vmax is .00370 mMmin-1, for the substrate UTP, glmU The Km value of .06608 mM, Kui Vmax, the maximum speed of .00371 mM min-13. preliminary screening use of GlmU inhibitors of the enzyme assay system to evaluate 44 computer virtual screening of lead compounds, 8 better inhibitors, inhibition constant IC50 determination of each of these eight lead compound, laid the foundation for further study of the mechanism of the inhibition and the development of new drugs.
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