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Agkistrodon halas hemocoagulase characteristic polypeptide and application thereof in species identification of hemocoagulase for injection

A technology for hemagglutinin and injection, which is applied in the direction of enzymes, peptidases, hydrolytic enzymes, etc., can solve the problems that the source of unknown samples cannot be confirmed and the source of biochemical drugs can be identified, so as to improve the level of drug quality control and guarantee Safety and efficacy, strong specific effect

Active Publication Date: 2020-11-06
SHANDONG INST FOR FOOD & DRUG CONTROL +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current quality standards for snake venom hemocoagulase drugs mainly use biochemical reactions, chemical chromogenic reactions, liquid chromatography-peptide mapping methods or gel electrophoresis methods to identify, but none of these methods have identified the species of this type of biochemical drugs. Source identification, for unknown samples can not be sure of its species origin

Method used

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  • Agkistrodon halas hemocoagulase characteristic polypeptide and application thereof in species identification of hemocoagulase for injection
  • Agkistrodon halas hemocoagulase characteristic polypeptide and application thereof in species identification of hemocoagulase for injection
  • Agkistrodon halas hemocoagulase characteristic polypeptide and application thereof in species identification of hemocoagulase for injection

Examples

Experimental program
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Effect test

preparation example Construction

[0032] The preparation method of relevant reagent reagent and solution in the following examples is as follows:

[0033] (1) Reagents: trypsin (Sigma, lot number SLBS8956), haemagglutination of Agkistrodon venom (Jinzhou Aohong Pharmaceutical Co., Ltd., purity 98.5%), guanidine hydrochloride (VETEC, lot number WXBC4261V), trihydroxyaminomethane (Shanghai Stock Exchange , batch number 20181206), dithiothreitol (BBI Life Sciences, batch number D911BA0011), iodoacetamide (BBI Life Sciences, batch number B326BA1943), and other reagents were of analytical grade.

[0034] (2) 25 mmol / L ammonium bicarbonate solution: Weigh 79.06 mg of ammonium bicarbonate, add 40 mL of water to dissolve, and obtain.

[0035] (3) 0.4 mol / L dithiothreitol (DTT) solution: Weigh 15.42 mg of dithiothreitol, add 250 μL of water to dissolve, and get ready.

[0036] (4) 0.4 mol / L iodoacetamide solution (newly prepared for immediate use): Weigh 18.5 mg iodoacetamide, add 250 μL water to dissolve, and get rea...

Embodiment 1

[0038] Example 1 Screening and determination of characteristic polypeptides.

[0039] 1. Instruments and equipment

[0040] Thermo Fusion high-resolution mass spectrometer (Thermo Fisher Scientific, USA), EASY-nLC 1000 nanoliter liquid chromatograph (Thermo Fisher Scientific, USA), CP225D electronic balance (Sartorius, Germany), Sigma 3-30 K refrigerated centrifuge ( Sigma, Germany), Milli-QAdvantage A10 ultrapure water meter (Millipore, USA).

[0041] 2. Chromatography and mass spectrometry conditions

[0042] Chromatographic conditions: Chromatographic column: 0.2 mm × 3.5 cm (5 μm particle size) ReproSil-Pur C18-AQ Trap column made in the laboratory for desalting and enrichment, using 75 μm × 25 cm (3 μm particle size) ReproSil made in the laboratory -PurC18-AQ nanoliter analytical column separation. Mobile phase A was 2% acetonitrile in 0.1% formic acid in water; mobile phase B was 98% acetonitrile in 0.1% formic acid in water. The flow rate of the nanoliter separation...

Embodiment 2

[0050] 1. Instruments and equipment

[0051] SCIEX Triple Quad 6500 triple quadrupole mass spectrometer, CP225D electronic balance (Sartorius, Germany), Sigma 3-30 K refrigerated centrifuge (Sigma, Germany), Milli-QAdvantage A10 ultrapure water instrument (Millipore, USA).

[0052] 2. Chromatography and mass spectrometry conditions

[0053] Liquid conditions: the chromatographic column is Waters ACQUITY UPLC BEH C 18 Chromatographic column (50mm×2.1 mm, 1.7μm); mobile phase A: 0.1% formic acid solution mobile phase, B: 0.1% formic acid acetonitrile; gradient elution, elution program: 0→1 min, mobile phase A 80%; 1→5 min, mobile phase A 80%→10%; 5→7 min, mobile phase A 10%→10%; column temperature: 40°C; injection volume: 2 μL; flow rate: 0.2 mL / min.

[0054] Mass spectrometry conditions: Electrospray ion source, positive ion scanning mode, multiple reaction monitoring; vortex ion spray temperature 500°C; ionization voltage 5.5 kV; collision cell outlet voltage 10 V; inlet vol...

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Abstract

The invention provides an agkistrodon halas hemocoagulase characteristic polypeptide and an application thereof in species identification of hemocoagulase for injection. A sample to be detected and acharacteristic peptide reference substance are respectively dissolved to prepare a test solution and a reference substance solution, and reduction alkylation treatment is carried out by dithiothreitoland iodoacetamide; an ammonium bicarbonate solution is diluted, and an enzyme for hydrolysis added; and after enzymolysis is finished, high-speed centrifugation is performed, and supernatant is takenand injected into a liquid chromatography-mass spectrometer for analysis. According to the method, enzymolysis is combined with a liquid chromatography-tandem mass spectrometry-multi-reaction monitoring (LC-MS / MS-MRM) method, three charges 935.8-861.4 and three charges 935.8-602.3 are used as detection ion pairs to extract an ion flow chromatogram, and species identification is carried out on theagkistrodon halas hemocoagulase for injection. The method is simple, convenient, rapid and high in specificity, fills in the blank of identification of the species source of the agkistrodon halas hemocoagulase for injection, and improves the quality control level.

Description

technical field [0001] The invention relates to a characteristic polypeptide of white-browed snake venom hemagglutinase and its application in identifying the species of snake venom hemagglutinase, belonging to the field of species identification of snake venom hemagglutinase products. Background technique [0002] Snake venom hemagglutinase drugs are a class of hemostatic drugs derived from different snake species, and its main active ingredient is snake venom hemagglutinase, a serine protease with arginine esterase and amidase activities, which can be used in the coagulation process Play an important role, mainly for the treatment of bleeding disorders. At present, the snake venom hemocoagulase drugs approved for marketing in China mainly include Snake Venom Hemagglutase for Injection (Bangting), Agkistrodon lanceolata Hemagglutase for Injection (Baquting), and Agkistrodon Akistrodon Hemocoagulase for Injection "Su". Ling", the snake venom hemocoagulase injection (Sulejua...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/74G01N30/02G01N30/06G01N30/34G01N30/72G01N30/88
CPCC12N9/6418C12Y304/21005G01N30/02G01N30/06G01N30/34G01N30/7266G01N30/88G01N2030/8831G01N2030/8818C12Y304/21074C12N9/6429G01N2560/00G01N30/7206C12Q1/37
Inventor 王聪聪石峰咸瑞卿巩丽萍杭宝建迟连利张群业杜宏明
Owner SHANDONG INST FOR FOOD & DRUG CONTROL
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