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Specific inhibition compound of SAHN enzyme protein and synthetic method of specific inhibition compound

A compound and specific technology, applied in the direction of organic active ingredients, medical preparations containing active ingredients, organic chemistry, etc., can solve the problem of ineffective antibiotic treatment, and achieve the effect of good antibacterial activity

Active Publication Date: 2013-07-10
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that the strain carries a variety of commonly used antibiotic resistance genes, and the expression products of drug resistance genes can resist aminoglycosides (combined with the ribosomal 30S subunit of bacterial cells), macrolides ( Binding to the ribosomal 50S subunit of bacterial cells) and other antibiotics, resulting in ineffective antibiotic treatment

Method used

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  • Specific inhibition compound of SAHN enzyme protein and synthetic method of specific inhibition compound
  • Specific inhibition compound of SAHN enzyme protein and synthetic method of specific inhibition compound
  • Specific inhibition compound of SAHN enzyme protein and synthetic method of specific inhibition compound

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Effect test

Embodiment 1

[0034] Cloning, expression and purification of Escherichia coli SAHN, SRHH proteins and host SAHH proteins

[0035] 1. Construction of Escherichia coli gene sequence SAHN and SRHH recombinant vectors: obtain the corresponding gene sequences from Genebank, design PCR primers with Primer5.0, and use PCR amplification technology to obtain DNA sequences from E.coli XL1-Blue respectively . The PCR product was connected to the previously constructed pYEMF-T vector, transformed into E.coli K-12DH5α, selected white positive clones, extracted the plasmid after cultivation, separated and identified by agarose electrophoresis after enzyme digestion, and the digested fragments were passed through the MagExtractor (TOYOBO Inc.) Kit Purified, cloned into pCA24N plasmid, transformed into competent cells BL21 (DE3), and screened on chloramphenicol or ampicillin LB plates.

[0036] 2. Construction of human gene sequence SAHH recombinant vector: Human placenta tissue was quick-frozen in liquid...

Embodiment 2

[0039] Establishment of Enzyme Coupled Assay Detection System for SAHN and SAHH

[0040] S-adenosyl homocysteine ​​nuclease (SAHN) enzyme coupling assay detection reaction mechanism is as follows:

[0041]

[0042] The analysis and detection reaction mechanism of S-adenosylhomocysteine ​​hydrolase (SAHH) is as follows:

[0043]

[0044] The enzyme coupling analysis detection system contains 0.1-20μM different concentrations of SAH, 10μM SRHH (SRHH is not added for SAHH enzyme detection), 1.0mM dithiothreitol, 50mM Hepes buffer at pH 7.4 and different concentrations of enzyme inhibitors After shaking and balancing in a water bath at 37°C for 5 minutes, add SAHN or SAHH protein to start the enzyme reaction, react at 37°C for 10 minutes, and use four times the volume of 5,5'-dithio-2,2'-bisnitrobenzoic acid ( DTNB) (133μM)-guanidine hydrochloride (8M) solution to terminate the reaction, 412nm colorimetric measurement of the generated 5-thio-2-nitrobenzoic acid (TNB), and d...

Embodiment 3

[0046] 1. The SAHN sequence was obtained by PCR amplification, and the pEX-sahn expression vector was constructed. After transforming BL21(DE3), IPTG induced expression to obtain the SAHN fusion protein with a histidine tag. Ni 2+ Agarose affinity chromatography column adsorption, imidazole elution, separation and purification to obtain recombinant enzyme protein, as shown in the attached figure figure 1 shown.

[0047] 2. Construct pCA-SRHH expression vector, after transforming BL21(DE3), induce expression with IPTG to obtain SRHH fusion protein with histidine tag, absorb on Ni2+ agarose affinity chromatography column, elute with imidazole, separate and purify to obtain recombinant enzyme protein SRHH, the protein can be used for enzyme coupling assay detection of SAHN enzyme activity, as shown in the accompanying drawing figure 2 shown.

[0048] 3. Linear correlation analysis of SAHN nucleosidase activity detected by enzyme coupling analysis:

[0049] Based on the enzyme...

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Abstract

The invention relates to the technical field of polymer synthesis and in particular relates to a specific inhibition compound of an SAHN enzyme protein and a synthetic method of the specific inhibition compound. The specific inhibition compound of escherichia coli SAHN enzyme protein is obtained in a targeted manner by virtue of an organic synthetic method, so that a novel anti-infective drug pilot compound is developed.

Description

technical field [0001] The present invention relates to the technical field of polymer synthesis, in particular to a specific inhibitory compound of SAHN enzyme protein and a synthesis method thereof. The specific inhibitory compound of Escherichia coli SAHN enzyme protein is obtained in a targeted manner through an organic synthesis method, Development of new anti-infective drugs. Background technique [0002] In the field of infectious diseases, the infection of drug-resistant bacteria is one of the important issues of global concern. In June 2011, an outbreak of enterohaemorrhagic Escherichia coli infection occurred in Germany, and it was later confirmed that Escherichia coli serogroup O104:H4 was the pathogen. The epidemic spread in at least 14 countries in Europe, with more than 4,000 infected patients and confirmed deaths 52 cases, 470 cases of renal failure. Studies have found that the strain carries a variety of commonly used antibiotic resistance genes, and the ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D487/04A61K31/519A61P31/04
CPCY02A50/30
Inventor 谷劲松谭晓军张玉
Owner UNIV OF JINAN
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