Polypeptide probe pair for measuring transpeptidase alpha activity, method for measuring transpeptidase alpha activity and application of both

A technology of transpeptidase and transpeptidase reaction, which is applied in chemical instruments and methods, biochemical equipment and methods, and the determination/inspection of microorganisms. specific effect

Active Publication Date: 2020-07-24
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The first object of the present invention is to provide a pair of polypeptide probes for measuring the activity of transpeptidase A, so as to alleviate the need for labeling, Technical problems of tedious preparation and limited sensitivity

Method used

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  • Polypeptide probe pair for measuring transpeptidase alpha activity, method for measuring transpeptidase alpha activity and application of both
  • Polypeptide probe pair for measuring transpeptidase alpha activity, method for measuring transpeptidase alpha activity and application of both
  • Polypeptide probe pair for measuring transpeptidase alpha activity, method for measuring transpeptidase alpha activity and application of both

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Effect test

Embodiment 1

[0073] Embodiment 1 is used for measuring the design of the polypeptide probe pair of transpeptidase A activity

[0074] This embodiment provides a pair of polypeptide probes for measuring the activity of transpeptidase A, including a first polypeptide probe and a second polypeptide probe, and the two designed polypeptide probes are named aPP- N and aPP-C, wherein the amino acid sequence of aPP-N is: CCPSQPTYPGLPATGG (SEQ ID NO.1), and the amino acid sequence of aPP-C is: GGVEDLIRFYDNLQQYLNVCC (SEQ ID NO.2). Each probe contains three different functional regions: among them, LPATGG and GG will participate in the transpeptidase A-mediated transpeptidation reaction; CC is the site combined with arsenic dye to read the activity of transpeptidase A ; PSQPTYPG and VEDLIRFYDNLQQYLNV will bind to each other so that the two cysteine-cysteine ​​doublets are spatially adjacent. The transpeptidation reaction mediated by transpeptidase A will form a new chimeric polypeptide whose amino a...

Embodiment 2

[0075] Embodiment 2 is used for measuring the performance measurement of the polypeptide probe pair of transpeptidase A activity

[0076] In order to determine whether transpeptidase A can catalyze aPP-N and aPP-C provided in Example 1 of the present invention to form CC-aPP-CC, this example adds 10 micromoles per liter of aPP-N, 40 micromoles per liter of a PP-C and 0.1 micrograms of transpeptidase A to 20 microliters of transpeptidation reaction buffer (50mM Tris·HCl, pH 7.5, 150mM NaCl, 5mM CaCl 2 and 1mM DTT) and incubated at 37°C for two hours. Subsequently, the ten-fold diluted reaction product was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and the results were as follows: Figure 1A As shown, the transpeptidation reaction will generate a product with a molecular weight of 3920.85 Daltons, which is consistent with the theoretical molecular weight of CC-aPP-CC (3920.51 Daltons), indicating that transpeptidase A can recognize ...

Embodiment 3

[0077] Embodiment 3 is used for measuring the activity of the polypeptide probe of transpeptidase A activity in vitro quantitative determination of transpeptidase A

[0078] This example further verifies that aPP-N and aPP-C can specifically measure the activity of transpeptidase A through experiments. In this example, the linking effects of transpeptidase A, T4 DNA ligase and ubiquitin ligase on aPP-N and aPP-C were determined in the transpeptidation reaction buffer. The specific experimental operation is as follows: 10 micromoles per liter of aPP-N and 10 micromoles per liter of aPP-C were mixed with 0.1 micrograms of transpeptidase A, 0.1 micrograms of T4 DNA ligase and 0.1 micrograms of ubiquitin ligase in Incubate at 37 °C for two hours, then add the reaction mixture to 80 µl containing 0.4 µmol FlAsH-EDT per liter 2 Double arsenic dye labeling buffer (100mM Tris·Cl, pH 7.4, 75mM NaCl, 1mM EDTA, 1mM DTT, 0.05mM BAL), and incubated at room temperature for 30 minutes in th...

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Abstract

Relating to the technical field of biological detection, the invention provides a polypeptide probe pair for determination of transpeptidase A activity, a determination method for the transpeptidase Aactivity and application of the two. The polypeptide probe pair for determination of the transpeptidase A activity provided by the invention comprises a first polypeptide probe and a second polypeptide probe that include a reaction zone, a complementary zone and a dye combination zone, has the advantages of strong specificity, good accuracy and high sensitivity, and can achieve the purpose of determining the transpeptidase A activity without modification or labeling treatment. The determination method for the transpeptidase A activity provided by the invention includes: mixing the polypeptideprobe pair for determination of the transpeptidase A activity with a to-be-detected sample in a transpeptidation reaction buffer solution and performing incubation, then using a biarsenical dye to label the incubated reaction product, combining the incubated reaction product with the biarsenical dye to form a compound, and judging the transpeptidase A activity according to the fluorescence levelof the compound. The method is simple and convenient to operate.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a pair of polypeptide probes for measuring the activity of transpeptidase A, a method for measuring the activity of transpeptidase A and the application of the two. Background technique [0002] Bacterial surface proteins play an important role in the infection process of pathogenic bacteria (such as adhesion, immune escape, biofilm formation), and are indispensable pathogenic factors that determine the severity of pathogenic bacterial infection (Nat.Rev. Microbiol.,2014,12,49 -62). In Gram-positive bacteria, transpeptidation reactions catalyzed by transpeptidases are required for proper anchoring of surface proteins to the cell wall. Usually, surface proteins are synthesized in the form of precursors, the N-terminus of the precursor protein contains a secretion signal peptide, the C-terminus contains an LPXTG (X represents any amino acid) domain, a hydrophobic regi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K14/00C12Q1/48G01N21/64
CPCC07K7/08C07K14/00C12Q1/48G01N21/6486
Inventor 杨用许爱君梁岩
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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