Construction method of anti-tuberculosis medicine high-throughput screening model

A tuberculosis, high-throughput technology for use in microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc., capable of addressing issues such as reduced virulence of recombinant Mycobacterium tuberculosis

Inactive Publication Date: 2012-12-26
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

ESAT-6 is an important effector molecule in the ESX-1 secretion system. When individual amino acids of ESAT-6 protein ar...

Method used

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  • Construction method of anti-tuberculosis medicine high-throughput screening model
  • Construction method of anti-tuberculosis medicine high-throughput screening model
  • Construction method of anti-tuberculosis medicine high-throughput screening model

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0020] Example 1 Construction process of high-throughput screening model for anti-tuberculosis drugs

[0021] 1.1 Construction of plasmid pMV261-LUC-CFP-10

[0022] The plasmid pGL4 (purchased from Promega) was used as a template, 5'-TTCCGAATTCATGGAAGATGCCAAAAACATTAAGA-3' was used as an upstream primer, and 5'-CTTCATCTCTGCCATCACGGCGATCTTG-3' was used as a downstream primer to perform PCR amplification to obtain the LUC fragment. As a template, 5′-CAAGATCGCCGTGATGGCAGAGATGAAG-3′ was used as an upstream primer, and 5′-AATTAAGCTTTCAGAAGCCCATTTGCGAGG-3′ was used as a downstream primer to obtain a CFP-10 fragment. As the upstream primer, use 5'-AATTAAGCTTTCAGAAGCCCATTTGCGAGG-3' as the downstream primer to perform PCR amplification to obtain the LUC-CFP-10 fragment, insert this fragment between the restriction sites EcoR I and Hind III on the plasmid pMV261, and obtain the recombinant plasmid , named pMV261-LUC-CFP-10 (the structure of the recombinant plasmid is as follows figure ...

experiment example 1

[0050] The recombinant mycobacterium marinum obtained in embodiment 1 is cultivated to A 600 About 0.8, collect the bacteria by centrifugation, replace with fresh medium and continue to cultivate A 600 About 0.5, centrifugal. Take 198 μl supernatant and add it to a 96-well plate, add 2 μl sample and mix well, take 20 μl in a white microplate plate half an hour later, add 50 μl substrate, and measure Luciferase activity.

experiment example 2

[0052] The recombinant Mycobacterium marinum obtained in Example 1 was placed in 3ml 7H9 liquid medium and cultivated to A 600 About 0.8, centrifugal. The supernatant was collected, added to a MWCO 30000 centrifugal ultrafiltration tube (Amicon Ultra-4ml, Millipore), and centrifuged at 4000 g for 25 minutes to a final volume of 100 μl. The bacterial cells collected by centrifugation were resuspended in PBS, ultrasonically disrupted, and centrifuged at 5000 g for 10 minutes to remove unbroken bacterial cells and cell debris. The above-mentioned concentrated supernatant and the bacterial protein obtained by ultrasonic crushing were subjected to protein quantification (Bradford protein quantification kit was purchased from Shanghai Meiji Biotechnology Co., Ltd.), and all quantified samples were added to SDS-PAGE loading buffer, and shaken to mix. Mix well, cook in a boiling water bath for 10 minutes, load the sample after cooling, and perform Western blotting detection. The ant...

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Abstract

The invention provides a construction method of an anti-tuberculosis medicine high-throughput screening model, characterized by using mycobacterium marinum endogenously expressing an ESX-1 secretion system as a model organism, selecting important virulence factor CFP-10 protein with signal peptides at C end in the ESX-1 secretion system to fuse with luciferase, constructing recombinant mycobacterium marinum exogenously expressing CFP-10 and luciferase fusion protein, adding a pharmaceutical compound to be screened to a nutrient solution containing the recombinant mycobacterium marinum, culturing for a certain time and then taking the supernatant to conduct luciferase activity determination for evaluating the inhibitory activity of the medicine to the ESX-1 secretion system; and simultaneously monitoring the growth of the mycobacterium marinum by determining A600. According to the invention, by developing the analytical method into the high-throughput screening model and screening the ESX-1 secretion system inhibitor, a novel anti-tuberculosis active compound which is capable of reducing the pathogenicity of tuberculosis mycobacterium without inhibiting the growth in vitro and is not easy for inducing drug resistance is obtained.

Description

technical field [0001] The invention relates to a method for constructing a high-throughput screening model for drugs, in particular to a method for constructing a high-throughput screening model for anti-tuberculosis drugs. Background technique [0002] Tuberculosis is an infectious disease with a high mortality rate, which seriously threatens human health. About one-third of the people in the world are infected with Mycobacterium tuberculosis. There are about 20 million tuberculosis patients, about 9 million new patients occur every year, and the death toll is as high as 3 million. As one of the 22 countries with a high incidence of tuberculosis, China ranks second in the world in terms of the number of tuberculosis patients after India. About 250,000 people die from tuberculosis every year, more than twice the total number of deaths from various infectious diseases. In addition, HIV-infected Mycobacterium tuberculosis carriers are 30-50 times more likely to develop activ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/62C12N15/63C07K19/00C12Q1/66G01N21/64C12R1/32
Inventor 岑山贾平平张义李晓宇周金明殷霄
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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