Pyrazinamide hydrolase, and encoding gene and application thereof

A technology of pyrazinamide and coding gene, which is applied in the fields of hydrolase, application, genetic engineering, etc., can solve the problem that pyrazinamide hydrolase has not been characterized, and achieve the effect of high activity and good pH tolerance

Active Publication Date: 2017-09-15
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biochemical characterization of certain bacterial pyrazinamide hydrolases has been studied and the Mycobacterium tuberculosis pyrazinamide hydrolase MtPncA has been well characterized, but pyrazinamide hydrolysis in Pseudonocardia carboxydivorans Enzymes have not been characterized

Method used

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  • Pyrazinamide hydrolase, and encoding gene and application thereof
  • Pyrazinamide hydrolase, and encoding gene and application thereof
  • Pyrazinamide hydrolase, and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Preparation and function of protein and gene

[0038] 1. Extraction of total DNA from Pseudonocardia carboxydivorans:

[0039] Use Pseudonocardia carboxydivorans to take 20 grams of its fresh wet bacteria and suspend it in 10 ml of 50 mM Tris buffer (pH 8.0), add a small amount of lysozyme and 8 ml of 0.25 mM EDTA (pH 8.0), mix well and place at 37°C for 20 minutes; Then add 2 ml of 10% SDS, place at 55°C for 5 minutes, and extract with equal volumes of phenol and chloroform respectively; take the last supernatant solution, add 2 times the volume of absolute ethanol, and recover the DNA. Wash twice with 70% absolute ethanol; the precipitate is dried in vacuum and dissolved in 0.5 ml TE buffer (pH 8.0, 10 mM Tris, 1 mM EDTA).

[0040] 2. Synthesize the nucleotide sequence shown in SEQ ID NO. 2

[0041] According to the nucleotide sequence shown in SEQ ID NO. 2, the primer pairs are designed as follows:

[0042] Forward primer: 5′-CGC GGATCC ATGGCACGAGCGTTGATCGTCGTC-3...

Embodiment 2

[0064] Example 2. Using pyrazinamide as a substrate to verify protein function

[0065] Enzyme activity unit is defined as the amount of enzyme required to catalyze the production of 1 μmol pyrazinic acid in 1 minute.

[0066] (1) Optimal temperature

[0067] The PncA-Pse pure enzyme solution in step 5 of Example 1 was diluted with 50 mM Tris-HCl buffer of pH 8.5, and the enzyme activity was measured with the diluted enzyme solution. The diluted enzyme solution is recorded as the diluted enzyme solution.

[0068] Solution A consists of 50 mM Tris-HCl buffer and pyrazinamide solution at pH 8.5; the concentration of pyrazinamide in solution A is 100 μM.

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Abstract

The invention discloses a pyrazinamide hydrolase, and an encoding gene and an application thereof. The hydrolase has an amino acid sequence represented as the SEQ ID No.1; and the encoding gene is a DNA molecule which has a nucleotide sequence represented as the SEQ ID No.2. The pyrazinamide hydrolase is high in enzyme activity and has good pH tolerance. The invention demonstrates a direction of therapy on patients, which has pyrazinamide resistance in vivo, by combining pyrazinamide with the pyrazinamide hydrolase.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a pyrazinamide hydrolase and its coding gene and application. Background technique [0002] Pyrazinamide is one of the first-line drugs recommended by the World Health Organization for the treatment of tuberculosis. The drug itself does not have antibacterial activity, but is hydrolyzed into pyrazinic acid in the body of pathogenic bacteria, thereby continuously killing Mycobacterium tuberculosis existing in the population in an acidic pH environment, which can shorten the treatment time from 9-12 months to 6 Months. Although the importance of pyrazinamide in the treatment of tuberculosis has been recognized, its mechanism of action may be the least understood of all anti-tuberculosis drugs. [0003] At present, the emergence of more and more drug-resistant tuberculosis strains has posed a major challenge to the antibacterial activity of pyrazinamide. Pyrazinamide is a prodrug ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/55C12P17/12A61K38/50A61P31/06
CPCA61K38/50C12N9/80C12P17/12
Inventor 张桂敏张景轩严闯马延和
Owner HUBEI UNIV
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