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Screening model for lspd inhibitors and new application of ispd inhibitor dumiphene

A technology for screening compounds and reagents, which is applied in the field of chemical drugs and drug screening, and can solve problems such as the lack of Dumiphene anti-tuberculosis drugs

Active Publication Date: 2011-12-14
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports of IspD inhibitors at present, and it is possible to find anti-tuberculosis drugs with a new mechanism of action by screening IspD inhibitors of Mycobacterium tuberculosis
There are no reports of dumiphene as an antituberculosis drug

Method used

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  • Screening model for lspd inhibitors and new application of ispd inhibitor dumiphene
  • Screening model for lspd inhibitors and new application of ispd inhibitor dumiphene
  • Screening model for lspd inhibitors and new application of ispd inhibitor dumiphene

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to a reagent set for screening a 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD) inhibitor. The reagent set comprises methylerythritol phosphate (MEP), cytidine triphosphate (CTP), IspD, enzyme reaction buffer solution, an ion reagent and a reagent for determining pyrophosphoric acid (PPi). The invention also relates to a method for screening the IspD inhibitor. By the method, domiphen bromide with anti-tuberculosis activity is screened from more than 3,000 compounds. The invention also relates to application of the domiphen bromide to the in-vivo / in-vitro inhibition of Mycobacterium tuberculosis and the preparation of anti-tuberculosis medicines.

Description

technical field [0001] The invention relates to the field of chemical drugs and drug screening, in particular, the invention relates to a screening model of mycobacterium tuberculosis enzyme IspD inhibitor, and the use of IspD inhibitor dumiphene for preparing anti-tuberculosis treatment drugs. Background technique [0002] Tuberculosis (Tuberculosis, hereinafter referred to as "TB") is a chronic infectious disease caused by Mycobacterium tuberculosis infection, which seriously endangers human life and health. Now one-third of the world's population has been infected with Mycobacterium tuberculosis, and there are 9.27 million new cases of tuberculosis and 3 million deaths every year. It has become the three major infectious diseases together with AIDS and malaria. my country is one of the 22 countries with a high burden of tuberculosis in the world. There are 550 million people infected with TB, and the number of patients is as high as 5.5 million, ranking second in the worl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48G01N21/31A61K31/14A61P31/06
Inventor 肖春玲高鹏杨延辉熊小椒关艳甘茂罗郝雪秦刘忆霜
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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