Method for screening Mycobacterium tuberculosis drug-resistant protein

A Mycobacterium tuberculosis and protein technology, which is applied in the fields of biochemical equipment and methods, microbial determination/inspection, material inspection products, etc.

Inactive Publication Date: 2007-04-11
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no comparative analysis of whole-cell constituent proteins of M.tb clinical drug-resistant strains

Method used

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  • Method for screening Mycobacterium tuberculosis drug-resistant protein
  • Method for screening Mycobacterium tuberculosis drug-resistant protein
  • Method for screening Mycobacterium tuberculosis drug-resistant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The cultivation of embodiment 1 Mycobacterium tuberculosis and drug susceptibility test

[0037] Mycobacterium tuberculosis standard strain Mycobacterium tuberculosis H37Rv (ATCC93009), clinical rifampicin (ie for 3-{[(4-methyl-1-piperazinyl)imino]methyl}-rifamycin, rifampicin , RFP, Sigma R 3501) single-drug-resistant strains (5 strains), and clinical all-sensitive strains (9 strains) were provided by the Tuberculosis Laboratory of Shanghai Pulmonary Hospital. The strains were inoculated into Middlebrook 7H9Broth (DIFCO, Bection-Dickinson) added with 10% ADC (DIFCO, 0.5% bovine serum albumin, 0.2% glucose, 140mmol / LNaCl) and 0.5% glycerol, and placed in a static incubator at 37°C. Place and cultivate for 15-20 days, when the bacterial concentration reaches to 1×10 8 ~2×10 8 or optical density (OD 600 ) was collected between 0.8 and 1.

[0038] Heat in a water bath at 80°C for 2 hours to kill bacteria. After centrifugation at 5000rpm at 4°C, the supernatant was dis...

Embodiment 2

[0040] The extraction of embodiment 2 thalline protein

[0041] Centrifuge at 6000g at 4°C for 15 minutes to precipitate 20 mL of cells in logarithmic growth phase. The pelleted bacteria were washed twice with PBS buffer at pH 7.4. Dissolve the bacteria with 0.5mL sonication buffer. Sonicate in an ice bath to disrupt and lyse bacteria for 15 minutes, with an amplitude of 70%, 200W for 1 minute, and stop for 20 seconds. Slowly add sample lysate and place at room temperature for 30 minutes, shaking at intervals. Centrifuge at 10,000 r / min at 20°C for 15 minutes, collect the supernatant, and freeze at -80°C. Protein concentration was measured by Bradford method.

Embodiment 3

[0042] The extraction of embodiment 3 basic protein

[0043] Centrifuge at 6000g at 4°C for 15 minutes to precipitate 20 mL of cells in logarithmic growth phase. Centrifuge at 4000r / min at 4°C for 15 minutes to precipitate the bacteria, and wash the precipitated bacteria twice with PBS buffer solution of pH 7.4. Resuspend the pelleted bacteria with 1 mL of LPBS and transfer to a 1.5 mL EP tube. Centrifuge at 6000r / min for 15 minutes at 4°C to pellet the bacteria. Dissolve the bacteria with 0.5mL sonication buffer. Sonicate in an ice bath to disrupt and lyse bacteria for 15 minutes, with an amplitude of 70%, 200W for 1 minute, and stop for 20 seconds. Fix with pre-cooled methanol at 4°C for 40 minutes (change methanol once in the middle), slowly add 1 / 4 volume of pre-cooled 1.0N H 2 SO 4 , stirring continuously for 30 minutes. The suspension was centrifuged at 12000×g for 20 minutes at 4°C, and the supernatant was retained. Break up the pellet and resuspend in cold 0.4N ...

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Abstract

The present invention belongs to the field of screening antituberculotic target, vaccine antigen and detecting target in molecular biological technology, and is especially process of screening protein resisting rifampicin as antituberculotic. The process includes the first culturing drug resistant strain, the subsequent separating protein of drug resistant strain from protein of sensitive strain, comparing protein of drug resistant strain and protein of sensitive strain to determine the drug resistant protein, and final separating and identifying drug resistant protein. The process of the present invention can separate and identify drug resistant protein of Mycobacterium tuberculosis to provide new way for further understanding the drug resisting mechanism of Mycobacterium tuberculosis, fast clinical detection of drug resistant strain, and developing vaccine and medicine.

Description

technical field [0001] The invention belongs to the field of molecular biology screening of novel anti-tuberculosis drug targets, vaccine antigens and detection targets. Specifically, the invention provides a method for screening rifampicin-resistant proteins in Mycobacterium tuberculosis through proteomic technology . The invention also provides the application of the method in drug screening, vaccine preparation and the like. Background technique [0002] Tuberculosis still poses a huge threat to mankind. Nearly 32% of the world's population has been infected with Mycobacterium tuberculosis. About 2.7 million people die from tuberculosis every year, an average of more than 7,000 people die every day. The World Health Organization declared tuberculosis a global health emergency in 1993. [0003] my country is one of the 22 countries with a high burden of tuberculosis in the world, and the number of tuberculosis patients ranks second in the world. According to the resul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/48C12Q1/04
Inventor 王洪海王庆忠张鹭乐军张旻徐颖祝秉东王九龄郄亚卿
Owner FUDAN UNIV
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