Application of 3 [(4 - ((Z)[(2Z) - 4 oxo 2 (phenylimino) - 1, 3 - thiazolidine 5 idene] methyl) phenoxy) methyl] benzoic acid in agent for restraining staphylococcal bacteria
A phenylimino, staphylococcus technology, applied in antibacterial drugs, medical preparations containing active ingredients, organic active ingredients, etc., can solve problems such as easy development of chronic infection and severe infection problems
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preparation Embodiment 1
[0013] Preparation Example 1: Construction of Prokaryotic Expression Plasmid of Staphylococcus Epidermidis Tryptophanyl-tRNA Synthetase
[0014] Staphylococcus epidermidis tryptophanyl-tRNA synthetase gene (NC_004461, locus tag: SE0685) acquisition method is as follows:
[0015] Design an NcoI restriction site at the 5′ end of the sense strand primer, and an NheI restriction site at the 5′ end of the antisense strand primer:
[0016] sense:
[0017] 5'-CC CCA TGG AAA CTT TAT TCT CAG GA-3’
[0018] anti-sense:
[0019] 5’-CG GCT AGC TTA TCT TTT TCG ACC TAA GCC C-3’
[0020] The underlined sequence is the restriction site of Nco I, and the sequence in italics is the restriction site of Nhe I.
[0021] The coding gene of Staphylococcus epidermidis tryptophanyl-tRNA synthetase was amplified by PCR using the above primers. Through the digestion reaction of restriction endonuclease (Nco I and Nhe I) and the ligation reaction of T4 DNA ligase, the above gene was connected into...
preparation Embodiment 2
[0022] Preparation Example 2: Expression and Purification of Tryptophanyl-tRNA Synthetase Protein
[0023] Host bacteria: Escherichia coli BL21-DE3
[0024] 1) Pick a single clone colony and inoculate into 5ml LB medium, 37℃×250rpm to OD 600 =1~2;
[0025] 2) Inoculate 10 μl to 5ml LBK medium, add 1M IPTG to a final concentration of 1mM, and induce expression for 4 hours;
[0026] 3) Centrifuge the bacterial cells, resuspend them in PBS, and ultrasonically disrupt them, centrifuge at 12,000 rpm for 5 minutes at 4°C, and take the supernatant; use Invitrogen ProBond TM Purify the protein with Purification System; determine the distribution of the target protein by 12% SDS-PAGE protein electrophoresis analysis. Proteins were quantified by the Bradford method.
Embodiment 1
[0027] Example 1: The effect of compound FDDDC03 on the activity of Staphylococcus epidermidis tryptophanyl-tRNA synthetase and the activity of human tryptophanyl-tRNA synthetase (using two methods for detection):
[0028] 1) Detection of the reduction of the substrate ATP - Kinase-Glo TM Luminescent Kinase Assay Kit (Promega, USA): 2 μg of recombinantly expressed and purified tryptophanyl-tRNA synthetase from Staphylococcus epidermidis and 3 μg of human tryptophanyl-tRNA synthetase were reacted with serially diluted compound FDDDC03 at 25°C for 20 minutes, Then add 100 μM ATP (total reaction volume 50 μL), after 20 minutes at 25 ° C, add the reaction mixture to a 96-well microtiter plate, 50 μL per well, add Kinase-Glo TM 50 μL of Reagent per well was allowed to stand at room temperature for 10 minutes, and the chemiluminescence value was read to indicate the amount of ATP remaining in the reaction. In the experiment, the group without compound was set as the control group....
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