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A kind of pyrazinamide hydrolase and its coding gene and application

A pyrazinamide, coding gene technology, applied in hydrolase, application, genetic engineering and other directions, 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: 2019-12-24
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A kind of pyrazinamide hydrolase and its coding gene and application
  • A kind of pyrazinamide hydrolase and its coding gene and application
  • A kind of pyrazinamide hydrolase and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, preparation and function of protein and gene

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

[0039]Take 20 grams of fresh wet cells from Pseudonocardia carboxydivorans, suspend 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 and place at 37 ° C for 20 min; Then add 2 ml of SDS with a mass fraction of 10%, place at 55°C for 5 min, and extract once with equal volumes of phenol and chloroform respectively; take the last supernatant solution, add 2 times the volume of absolute ethanol, recover DNA, and use Washed twice with 70% absolute ethanol; the precipitate was vacuum-dried 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 pair is designed as follows:

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

Embodiment 2

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

[0065] The enzyme activity unit is defined as the amount of enzyme required to catalyze the production of 1 μmol of pyrazinoic acid within 1 min.

[0066] (1) Optimum temperature

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

[0068] Composition of solution A: composed of 50 mM Tris-HCl buffer solution with pH 8.5 and pyrazinamide solution; 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 in particular relates to a pyrazinamide hydrolase, 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 has no antibacterial activity, but is hydrolyzed into pyrazinoic acid in the pathogenic bacteria, so that it can continuously kill Mycobacterium tuberculosis existing in the crowd in an acidic pH environment, which can shorten the treatment time from 9-12 months to 6 months. months. Despite the recognized importance of pyrazinamide in the treatment of tuberculosis, its mechanism of action is perhaps the least understood of all anti-TB drugs. [0003] Currently, the emergence of more and more drug-resistant tuberculosis strains has posed a major challenge to the antibacterial activity of pyrazinamide, a prodrug with no significant antibacterial a...

Claims

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Application Information

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Patent Type & Authority Patents(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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