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Agkistrodon halys venom phospholipase A2 marker polypeptide and application thereof

A technology of snake venom phospholipids and pit vipers, applied in the field of chemical analysis and quantitative detection, can solve the problems of poor accuracy, cumbersome determination methods, inability to accurately characterize the quality of snake venom and its extracts, and achieve high detection accuracy

Active Publication Date: 2021-10-15
SHANDONG INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently reported detection methods for snake venom phospholipase A2 are mainly gel electrophoresis and potency assays, which are cumbersome and have poor accuracy, and cannot accurately characterize the quality of snake venom and its extracts.

Method used

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  • Agkistrodon halys venom phospholipase A2 marker polypeptide and application thereof
  • Agkistrodon halys venom phospholipase A2 marker polypeptide and application thereof
  • Agkistrodon halys venom phospholipase A2 marker polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This example aims to screen characteristic peptides, using purified phospholipase A2 as an analyte, using a nanoliter liquid phase-high resolution mass spectrometer, the method focuses on obtaining abundant enzymatic peptides to ensure that the screening range has as high a coverage as possible Rate.

[0039] The preparation of the solution for feature polypeptide screening comprises the following steps:

[0040] Put 5 mg of Agkistrodon venom phospholipase A2 in a volumetric flask with a volume of 10 mL, then dissolve it with ammonium bicarbonate solution with a molar concentration of 25 mmol / L and dilute to 10 mL, and accurately measure 200 μL, Solution a is obtained.

[0041]Add 10 μL of dithiothreitol solution with a concentration of 0.2 mol / L to the solution a, mix well, react for 1 hour under the condition of heating in a constant temperature water bath at 60 °C, and then add ethyl iodide with a concentration of 0.2 mol / L Amide solution 20 μL, protected from ligh...

Embodiment 2

[0046] The "test solution" described in this example is based on the detection of the target characteristic polypeptide WDDYTYSWK screened in Example 1, which maximizes the enzymatic hydrolysis efficiency of the sample and does not need to consider other enzymatic hydrolysis peptides, so the optimized method There is no need to use dithiothreitol and iodoacetamide in Example 1 for treatment.

[0047] Multiple reaction monitoring (MRM) quantitative analysis of Agkistrodon venom phospholipase A2 in samples.

[0048] 1. Instruments and equipment: SCIEX Triple Quad 6500 triple quadrupole mass spectrometer, CP225D electronic balance (Sartorius, Germany), Sigma3-30K refrigerated centrifuge (Sigma, Germany), Milli-QAdvantageA10 ultrapure water instrument (Millipore, USA ).

[0049] 2. Chromatography and mass spectrometry conditions:

[0050] (1) Liquid conditions: Agilent Poroshell 120, SB-C18 column (100 mm×2.1 mm, 1.7 μm); column temperature: 40 ℃; injection volume: 10 μL; flow r...

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Abstract

The invention relates to the technical field of chemical analysis and quantitative detection, in particular to an agkistrodon halys venom phospholipase A2 marker polypeptide and application thereof. The amino acid sequence of the characteristic polypeptide is WDDYTYSWK, the characteristic polypeptide can be used for detecting the content of the agkistrodon halys venom phospholipase A2 in a sample to be detected, and the detection method comprises the following steps: taking the characteristic polypeptide of the agkistrodon halys venom phospholipase A2 with the amino acid sequence being WDDYTYSWK, and preparing a series of concentration reference substance solutions; taking a sample to be detected, performing enzymolysis, and taking supernate after enzymolysis as a test solution; and respectively injecting into a liquid chromatography-mass spectrometer, and selecting double charges 632.4-624.0 as quantitative ions to detect the content of the characteristic polypeptide of the agkistrodon halys venom phospholipase A2 in a sample to be detected, so that the quality control level of an agkistrodon halys venom medicine can be greatly improved, and the method has a very good guiding effect on the quality control of the agkistrodon halys venom, an intermediate and a medicinal preparation thereof.

Description

technical field [0001] The invention relates to the technical field of chemical analysis and quantitative detection, in particular to a characteristic polypeptide of Agkistrodon venom phospholipase A2 and an application thereof. Background technique [0002] Snake venom is a complex mixture secreted by snake venom glands, which contains various proteins, polypeptides, nucleosides, enzymes, metal ions and other small molecular substances. Using snake venom as a raw material, the separation and purification of a single component with unique pharmacological activity is a hot spot in the development of new drugs. At present, it has been obtained from the Ussuri viper ( Gloydius ussuriensis ), short-tailed vipers ( Gloydius brevicaudus ), Harry's Viper ( Gloydius Halys ), Japanese Agkistrodon ( Gloydius blomhoffii ) and other components such as thrombin, defibrase, nerve growth factor, arginine lipase, L-amino acid oxidase, and phospholipase A2 were isolated from the ven...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12Q1/34G01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72
CPCC12N9/18C12Y301/01004C12Q1/34G01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72G01N2030/324
Inventor 王维剑石峰咸瑞卿杭宝建巩丽萍王聪聪张迅杰姜树银
Owner SHANDONG INST FOR FOOD & DRUG CONTROL
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