Model for screening 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD) inhibitor and novel application of IspD inhibitor domiphen bromide
A use, mycobacterial technology, applied in the field of chemical drugs and drug screening, can solve the problem of no anti-tuberculosis drug of dumiphene
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Embodiment 1
[0045] Example 1: Cloning of ispD
[0046] Mycobacterium tuberculosis H 37 Rv (gifted by Professor Zhang Jianyuan, Institute of Tuberculosis) genome as a template, respectively using F1: 5'-GTTCATC CATATG GTCAGGGAAGCGGGCGAAGTAGTTGCG-3' (SEQ ID NO: 1) and R1: 5'-GTCTTAT CTCGAG CCCGCGCACTATAGCTTGGGCCAGC-3' (SEQ ID NO: 2) was used as a primer to amplify the ispD gene sequence by PCR; DNA agarose gel electrophoresis was used to detect the amplification result of the target gene, and then the 720bp target fragment waspD( image 3 ).
[0047] PCR reaction system:
[0048]
[0049]
[0050] PCR amplification conditions: pre-denaturation at 95°C for 5 min, then denaturation at 95°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 2 min, 32 reaction cycles, and finally extension at 72°C for 10 min.
[0051] The obtained target gene ispD was ligated with the plasmid pET28a, and the ligated product was transformed into competent cells of Escherichia coli DH5α. P...
Embodiment 2
[0053] Embodiment 2: Expression of IspD protein
[0054] The Escherichia coli B121 (DE3) that can efficiently express the Mycobacterium tuberculosis IspD protein prepared in the frozen example 1 was streak-inoculated in the LB containing 100 μg / ml kanamycin (Kan) and 37 μg / ml chloramphenicol (Ch1). Plate, 37°C, cultivate overnight, pick a single colony and inoculate in LB liquid medium containing 100 μg / ml Kan and 37 μg / ml Ch1, 200 r.p.m., 37°C, cultivate overnight; inoculate the overnight culture at 1:50 In fresh LB liquid medium containing 100 μg / ml Kan and 37 μg / ml Ch1, culture at 200 r.p.m., 37°C until cell OD 600 ≈0.5; IPTG was added to the culture to a final concentration of 1 mmol / L, and 50% yeast extract was added to a final concentration of 0.5%. 16°C, 200r.p.m., cultured for 12h, to induce the expression of IspD protein.
Embodiment 3
[0055] Embodiment 3: Purification and detection of IsDD protein
[0056] The Escherichia coli BL21 (DE3) plysS that can efficiently express the Mycobacterium tuberculosis IspD protein is cultivated according to the conditions of Example 2, and the IspD protein is induced to be expressed; 10000 × g is centrifuged for 10 min to collect the thalline; , 3s / 8s, 99 times of sonication to disrupt bacterial cells, and centrifuge at 14000×g for 1h at 4°C to collect the supernatant; use prime system, under the condition of pH 8.0, the supernatant was applied to a Hi-trap affinity column (GE Company, 17-5247-01), gradient elution was performed with a buffer, and the eluate was collected; the collected samples were added to 15ml ultrafiltration centrifuge tube (10kDa, MilliPore Company), centrifuge at 5000g for 15min at 4°C until the sample volume is less than 2.5ml; use PD-10 desalting column and desalting buffer to desalt the ultrafiltered sample. Store at -80°C.
[0057] The purif...
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