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Mutational SHV beta-lactamase, gene order and uses thereof

A technology of lactamase and gene sequence, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of bacterial drug resistance, irregular abuse of antibiotics, etc., and achieve the effect of improving pertinence

Inactive Publication Date: 2008-03-12
SHANGHAI BIOCHIP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the long-term and extensive use of antibiotics, especially the irregular misuse of antibiotics, the problem of bacterial resistance has become increasingly serious

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 Obtaining of SHV gene mutation site

[0027] Primers SHV-F (5'-CAAGGATGTATTGTGGTTATGCGT-3', SEQ ID NO: 7) and SHV-R (5'-GTTAGCGTTGCCAGTGCTCGAT-3', SEQ ID NO: 8) were used to amplify the full-length SHV gene. PCR amplification was carried out with 30μl reaction system, the reaction system was 0.3mM dNTP, 10mM Tris-HCl; 50mM KCl, 2mMMgCl2, 20% Q solution (Qiagen), 0.2μM SHV-F and SHV-R, 80ng genomic DNA, 0.8U Ex Taq enzyme (Takara). Cycle parameters: denaturation at 94°C for 5 min; denaturation at 94°C for 40 s, annealing at 58°C for 1 min, extension at 72°C for 1 min, a total of 40 cycles; final extension at 72°C for 5 min. PCR products were purified with QIAquick PCRPurification Kit (Qiagen, Cat. No. 28106) for sequencing.

[0028] The determined sequence was compared with the wild-type SHV (SHV-1) gene sequence (SEQ ID NO: 1), so as to obtain the sequence difference, and a total of 4 mutations were obtained, which are: 223 G→A (A79T), That is, the mutatio...

Embodiment 2

[0029] Example 2 Preparation of Detection Kit

[0030] A detection kit for detecting SHV gene mutations is prepared, which contains the following primer pairs that can amplify the 223rd, 289th, 628th, or 596th mutation:

[0031] Forward primer: 5'-TGTGGTTATGCGTTATATTCGCCT-3' (SEQ ID NO: 9)

[0032] Reverse primer: 5'-GGCGTATCCCGCAGATAAATCA-3' (SEQ ID NO: 10)

[0033] PCR amplification was carried out with 30 μl reaction system, the reaction system was 0.3mM dNTP, 10mM Tris-HCl; 50mM KCl, 2mM MgCl2, 20% Q solution (Qiagen), 0.2μM forward primer and reverse primer, 80ng genomic DNA, 0.8 U Ex Taq enzyme (Takara). Cycle parameters: denaturation at 94°C for 5 min; denaturation at 94°C for 40 s, annealing at 57°C for 1 min, extension at 72°C for 50 min, a total of 40 cycles; final extension at 72°C for 5 min. PCR products were purified with QIAquick PCR Purification Kit (Qiagen, Cat. No. 28106) for sequencing.

[0034] The amplified product is sequenced and compared with the wil...

Embodiment 3

[0036] The mutated SHV type β-lactamase gene sequence of the present invention can be used to detect drug-resistant bacteria through a gene chip method.

[0037] 1. Preparation of gene chip

[0038] (1) Probe dissolution

[0039] Each probe was diluted with TE solution to a final concentration of 10 mM. Mix the probe with a concentration of 10mM and the PBS solution with a concentration of 200mM in a medium volume of a 384-well plate, seal the 384-well plate with an adhesive sheet, shake at room temperature for 2 minutes, centrifuge, and store at -20°C for sample application .

[0040] (2) Spotting

[0041] The pre-designed and synthesized probes are loaded onto the solid-phase carrier substrates made of glass slides, silicon wafers, etc. through contact spotting or inkjet spotting. The film base adopts Cell Associates CSS-100 aldehyde base film base, and the Ominigrid 100 model spotting instrument of GeneMachine Company is applied at a humidity of 65-75% (based on the Ful...

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PUM

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Abstract

The invention discloses a varied SHV type beta-lactamase, which comes from bacteria and comprises a variant from the following: the 79th alanine residue in amino-acid sequence shown in sequence 2 in the sequence table is substituted by threonine residue, or 101th aspartic acid residue is substituted by histidine residue, or 214th aspartic acid residue is substituted by alanine residue, or 203th serine is substituted by leucine residue. The invention also discloses the gene sequence of the varied SHV type beta-lactamase and the application of the beta-lactamase. The varied SHV type beta-lactamase of the invention is of activity from super-wide spectrum beta-lactamase, provides useful information for developing and preparing anti-medicine gene inspection chip of beta-lactamase, guides the research and development as well as preparation of anti-medicine bacteria antibiotic, and effectively improve the direction-targeting in the use of antibiotic.

Description

technical field [0001] The present invention relates to the mutation of bacterial β-lactamase, in particular to a mutant SHV ​​type β-lactamase, its gene sequence and application. Background technique [0002] Since the invention of penicillin in 1929, antibiotics have played a huge role in the prevention and treatment of bacterial diseases. However, due to the long-term and extensive use of antibiotics, especially the irregular abuse of antibiotics, the problem of bacterial resistance has become increasingly serious. The most important mechanism of bacterial drug resistance is the production of inactivating enzymes, such as β-lactamases and aminoglycoside inactivating enzymes. β-lactamase can covalently bind to the carboxyl group of the β-lactam ring, hydrolyze its amide bond, and inactivate penicillins and / or cephalosporins. The types of β-lactamase are relatively complex, and more than 300 kinds have been found so far, and due to the extensive use of extended-spectrum β...

Claims

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Application Information

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IPC IPC(8): C12N9/86C12N15/55C12N1/21C12Q1/68C12Q1/04A61K38/50
Inventor 盛海辉肖华胜
Owner SHANGHAI BIOCHIP
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