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Cell model for screening inhibitor of mycobacterium tuberculosis ribosomal protein L12 and L10 and application thereof

A blocker, L12 technology, applied in the fields of biology and pharmacy

Active Publication Date: 2014-11-12
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

[0006]Although the interaction between L12 and L10 has been verified by the co-crystal method, is it possible to verify the interaction between L12 and L10 of Mycobacterium tuberculosis in a biological model? Whether the interaction can be used for screening new anti-tuberculosis drugs still needs further research

Method used

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  • Cell model for screening inhibitor of mycobacterium tuberculosis ribosomal protein L12 and L10 and application thereof
  • Cell model for screening inhibitor of mycobacterium tuberculosis ribosomal protein L12 and L10 and application thereof
  • Cell model for screening inhibitor of mycobacterium tuberculosis ribosomal protein L12 and L10 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Construction of yeast two-hybrid system

[0062] 1. Preparation of rplL (L12 encoding gene), rplJ (L10 encoding gene) genes

[0063] Using the total cDNA of Mycobacterium tuberculosis H37Rv (preserved in this laboratory; commercially available strains can also be used to prepare the total cDNA of Mycobacterium tuberculosis H37Rv using methods well known to those skilled in the art, such as cDNA extraction kits) as a template, design primers 1 and 2, primers 3 and 4 , using TaKaRa's high-fidelity PrimeSTAR HS DNA polymerase to obtain rplL (the gene encoding L12) and rplJ (the gene encoding L10) by PCR. The specific cloning method and primers are as follows:

[0064] 1) Primers for amplifying rplL (the gene encoding L12):

[0065] Primer 1: 5'-TC CATATG GCAAAGCTCTCCACCGACG-3' (SEQ ID NO: 5) (the underlined part is the Nde I restriction site)

[0066] Primer 2: 5'-GC GGATCC ACACGCTGGGCAGAGCTAC-3' (SEQ ID NO: 6) (the underlined part is the restriction si...

Embodiment 2

[0096] Example 2: Detection of β-galactosidase activity inhibition of positive compounds in primary screening

[0097] 1. Cultivate the screening model AH109 (AD-L12+BD-L10) and the screening negative control AH109 (AD-T+BD-53) in the defective medium SD / -Trp-Leu-His-Ade overnight at 30°C until logarithmic growth phase.

[0098] 2. Transfer 50 μl of overnight cultured cells to 5 ml of defective medium SD / -Leu / -Trp at a ratio of 1:100, and at the same time, add corresponding concentrations (final concentrations of 50, 10, 5, 1, 0.5, 0.1 μg / ml) of the compound, continue to culture.

[0099] 3. After 24 hours, the cells were collected for quantitative detection of β-galactosidase activity. The method is described in Example 1 with reference to. Obtain β-gal units.

[0100] 4. Take the β-gal units (0) value obtained by the screening model AH109 (AD-L12+BD-L10) without positive compounds and the negative control AH109 (AD-T+BD-53) as 100%, and add β-gal units (50, 10, 5, 1,...

Embodiment 3

[0105] Embodiment 3: the anti-tuberculosis activity detection of positive compound

[0106] Based on 96-well plate, 200μl culture system, 7H9 medium, Mycobacterium tuberculosis standard strain H37Rv concentration: 10 6 CFU / ml. The initial concentration is 40μg / ml, double dilution, become the concentration of 40μg / ml, 20μg / ml, 10μg / ml, 5μg / ml, 2.5μg / ml, 1.25μg / ml, 0.625μg / ml, 0.312μg / ml for MIC experiment ml, 0.156 μg / ml. Incubate at 37°C for 7 days, add 20 μl of 10×Alamar blue and 50 μl of 5% Tween80 mixture, incubate at 37°C for another 24 hours, and use a multi-functional microplate reader to measure and obtain MIC data quantitatively. The measured MIC value of T766 was 0.312 μg / ml, and that of T054 was 1.25 μg / ml. The results indicated that both compounds were effective in inhibiting Mycobacterium tuberculosis.

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Abstract

Provided are a cell model and screening method for screening inhibitors of the tubercle bacillus. Provided is a cell which expresses the tubercle bacillus proteins L12 and L10, wherein the L12 encoding gene and the transcription activation domain of its transcription factor are located in one expression vector, and the L10 encoding gene and the DNA binding domain of its transcription factor are located in another expression vector. The present invention also relates to the use of the compound shown in formulae I or II in preparing a blocker of the interaction between proteins L12 and L10 of tubercle bacillus, an anti-tubercle bacillus drug, or a drug for treating and / or preventing tuberculosis.

Description

technical field [0001] The invention belongs to the fields of biology and pharmacy, and relates to a screening model and application of a blocking agent for the interaction between tuberculosis ribosomal protein L12 and L10. Background technique [0002] In recent years, tuberculosis has made a comeback around the world, and the drug resistance of tuberculosis has become the most serious problem in the current treatment of tuberculosis. Screening, discovering and verifying the molecular targets of anti-tuberculosis drugs, and implementing breakthroughs from targets are the key to obtaining new anti-tuberculosis drugs with high efficiency and low toxicity, and solving the problem of tuberculosis treatment, especially drug-resistant tuberculosis treatment. [0003] Ribosome is the site of protein synthesis in cells, and the maintenance of cell life depends on the normal exercise of protein function, so ribosome has become an important target for drug development (Huntington K...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12Q1/18C12Q1/02C12Q1/34A61K31/5415A61P31/06
CPCG01N33/5695G01N2333/35A61K31/03G01N2500/10G01N2500/02C12N15/1055A61P31/06
Inventor 司书毅林媛李妍王彦昶蒋建东肖春玲洪斌高娜娜
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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