阅读说明：本技术 一种白眉蛇毒血凝酶特征多肽及其在注射用蛇毒血凝酶种属鉴别中的应用 (White-eyebrow snake venom hemocoagulase characteristic polypeptide and application thereof in species identification of snake venom hemocoagulase for injection ) 是由 王聪聪 石峰 咸瑞卿 巩丽萍 杭宝建 迟连利 张群业 杜宏明 于 2020-07-30 设计创作，主要内容包括：本发明提供了一种白眉蛇毒血凝酶特征多肽及其在注射用蛇毒血凝酶种属鉴别中的应用,待测样品及特征肽对照品分别溶解制成供试品及对照品溶液,经二硫苏糖醇及碘乙酰胺还原烷基化处理；碳酸氢氨溶液稀释后,加入酶进行水解；酶解结束后,高速离心,取上清液注入液相色谱-质谱仪进行分析。采用酶解结合液相色谱-串联质谱-多反应监测(LC-MS/MS-MRM)方法,以三电荷935.8→861.4和三电荷935.8→602.3作为检测离子对提取离子流色谱图,对注射用白眉蛇毒血凝酶进行种属鉴别。该方法简便快捷,专属性强,填补了注射用白眉蛇毒血凝酶种属来源鉴定的空白,提高了质量控制水平。(The invention provides a white brow venom hemocoagulase characteristic polypeptide and application thereof in species identification of snake venom hemocoagulase for injection, wherein a sample to be detected and a characteristic peptide reference substance are respectively dissolved to prepare a test sample and a reference substance solution, and reduction alkylation treatment is carried out by dithiothreitol and iodoacetamide; diluting ammonium bicarbonate solution, and adding enzyme for hydrolysis; after enzymolysis, high-speed centrifugation is carried out, and supernate is taken and injected into a liquid chromatogram-mass spectrometer for analysis. The species identification of the injection white brow venom hemocoagulase is carried out by adopting an enzymolysis combined liquid chromatography-tandem mass spectrometry-multiple reaction monitoring (LC-MS/MS-MRM) method and taking three charges of 935.8 → 861.4 and 935.8 → 602.3 as detection ion pairs to extract an ion flow chromatogram. The method is simple, convenient and rapid, has strong specificity, fills the blank of identifying the source of the hemocoagulase of the white-eyebrow snake venom for injection, and improves the quality control level.)

1. A polypeptide with the characteristics of the snake venom hemocoagulase, which is characterized in that the amino acid sequence of the polypeptide with the characteristics of the snake venom hemocoagulase is as follows: LDSPVSNSAHIAPLSLPSSAPSVGSVCR are provided.

2. A use of the hemagglutinin enzyme of Baimei snake venom of claim 1 in identification of hemagglutinin enzyme species for injection.

3. The use of claim 2, wherein the method of using the hemagglutinin characteristic polypeptide of Baimei snake venom in the identification of hemagglutinin enzyme species for injection comprises the following steps:

a: dissolving hemocoagulase for injection and thrombin-like enzyme characteristic polypeptide reference substance of white eyebrow venom respectively to obtain test solution and reference substance solution;

b: respectively taking the test solution and the reference solution, and carrying out reductive alkylation treatment on dithiothreitol and iodoacetamide;

c: after the reductive alkylation is finished, diluting the mixture by using an ammonium bicarbonate solution, and adding enzyme for hydrolysis;

d: after enzymolysis is finished, centrifuging at a high speed, and injecting supernate into a liquid chromatogram-mass spectrometer for analysis;

e: in the chromatogram of the extracted ion current for detecting the ion pair, if the sample solution presents a chromatographic peak with the same retention time as the reference solution, the amino acid sequence LDSPVSNSAHIAPLSLPSSAPSVGSVCR in the sample to be detected is shown, and the sample to be detected is proved to be derived from the Agkistrodon halys albus; otherwise, it is derived from non-white-eyebrow Agkistrodon Halys.

4. The use of claim 3, wherein the pair of detector ions of step E is triply charged 935.8 → 861.4 and triply charged 935.8 → 602.3.

5. The use according to claim 3, wherein the conditions for detecting the liquid phase and mass spectrum in the liquid chromatography-mass spectrometer in step D are as follows:

chromatographic mass spectrum conditions: the chromatographic column is Waters ACQUITY UPLC BEH C18 chromatographic column 50mm × 2.1 mm, 1.7 μm; mobile phase A: 0.1% formic acid solution mobile phase, B: 0.1% formic acid acetonitrile; column temperature: 40 ℃; sample introduction amount: 2 mu L of the solution; flow rate: 0.2mL/min, elution procedure: 0 → 1 min, mobile phase A80%; 1 → 5 min, mobile phase A80% → 10%; 5 → 7 min, mobile phase A10% → 10%;

the mass spectrum condition adopts an electrospray ion source, a positive ion scanning mode and multi-reaction monitoring; the vortex ion spraying temperature is 500 ℃; the ionization voltage is 5.5 kV; collision cell exit voltage 10V; inlet voltage 10V; three charges 935.8 → 861.4 and 935.8 → 602.3 are used as detection ion pairs, the collision voltage is 45V and 40V, and the cluster removing voltage is 135V.

6. The use according to any one of claims 2-5, wherein the hemagglutinin characteristic polypeptide is obtained by identifying hemagglutinin species of snake venom for injection by the following steps:

(1) taking 10 mg of the white brow venom hemocoagulase characteristic polypeptide LDSPVSNSAHIAPLSLPSSAPSVGSVCR, placing in a10 mL measuring flask, dissolving with water, fixing volume, mixing well, and preparing a reference substance stock solution 1 with the concentration of 1 mg/mL; taking 10 mu L of reference substance stock solution 1, dissolving with water, and fixing the volume to 10ml, and preparing reference substance stock solution 2 with the concentration of 1 mu g/ml; taking 600 mu L of the reference substance stock solution 2, and fixing the volume to 10mL by using 25 mmol/L ammonium bicarbonate solution to prepare a reference substance solution of 60 ng/mL; taking 50mg of a sample to be detected, and adding 500 mu L of 25 mmol/L ammonium bicarbonate solution for dissolving to obtain a sample solution;

(2) weighing 400 mu L of each of the test solution and the reference solution, adding 20 mu L of 0.4 mol/L dithiothreitol solution, mixing uniformly, reacting at 60 ℃ for 1 h, adding 40 mu L of 0.4 mol/L iodoacetamide solution, standing in the dark for 30 min, adding 10 mu L of 0.4 mu g/mu L trypsin, and performing enzymolysis at 37 ℃ for 1 h; inactivating at 90 deg.C for 10 min, taking out, cooling to room temperature, centrifuging at 1200 rpm for 10 min, and collecting supernatant and injecting into liquid chromatogram-mass spectrometer for analysis;

(3) in the chromatogram of the extracted ion current for detecting the ion pair, if the sample solution presents a chromatographic peak with the same retention time as the reference solution, the amino acid sequence LDSPVSNSAHIAPLSLPSSAPSVGSVCR in the sample to be detected is shown, and the sample to be detected is proved to be derived from the Agkistrodon halys albus; otherwise, it is derived from non-white-eyebrow Agkistrodon Halys.

Technical Field

The invention relates to a white-eyebrow snake venom hemocoagulase characteristic polypeptide and application thereof in species identification of snake venom hemocoagulase for injection, belonging to the field of species identification of snake venom hemocoagulase products.

Background

The snake venom blood coagulase medicine is hemostatic originated from different snake species, and the main active component of the snake venom blood coagulase medicine is serine proteinase with arginine esterase and amidase activity, which can play an important role in the blood coagulation process and is mainly used for treating hemorrhagic diseases. At present, the commercially available snake venom blood coagulase medicines at home mainly comprise white eyebrow snake venom blood coagulase (bunting) for injection, bovid snake venom blood coagulase (batroxobin) for injection, agkistrodon acutus blood coagulase 'souling' for injection, and snake venom blood coagulase injection (Sulexuan) of Megaku pharmaceutical industry (Hefei) Limited.

The quality standard of the current snake venom blood coagulase medicines mainly utilizes biochemical reaction, chemical chromogenic reaction, a liquid chromatogram-peptide diagram contrast method or a gel electrophoresis method for identification, but the methods do not identify the species source of the biochemical medicines, and the species source of unknown samples can not be identified. Because the structures of the hemagglutinases from different snake species are different, the acting mechanisms are different, and the corresponding pharmacological actions are different, a method for identifying and detecting the hemagglutinases with species specificity is needed to be established, the quality control of the products is enhanced, and the safety of clinical medication is guaranteed.

Disclosure of Invention

In order to solve the technical problems, the invention provides a white-eyebrow snake venom hemocoagulase characteristic polypeptide and application thereof in species identification of snake venom hemocoagulase for injection.

In order to realize the purpose, the invention adopts the following technical scheme:

a polypeptide with the characteristic of the hemocoagulase of the white brow venom has the amino acid sequence as follows: LDSPVSNSAHIAPLSLPSSAPSVGSVCR are provided.

An application of the above polypeptide in identification of species of snake venom hemocoagulase is provided.

Preferably, the application method of the polypeptide with the hemocoagulase characteristics of the white eyebrow venom in the identification of the hemocoagulase species for injection comprises the following steps:

a: dissolving hemocoagulase for injection and thrombin-like enzyme characteristic polypeptide reference substance of white eyebrow venom respectively to obtain test solution and reference substance solution;

b: respectively taking the test solution and the reference solution, and carrying out reductive alkylation treatment on dithiothreitol and iodoacetamide;

c: after the reductive alkylation is finished, diluting the mixture by using an ammonium bicarbonate solution, and adding enzyme for hydrolysis;

d: after enzymolysis is finished, centrifuging at a high speed, and injecting supernate into a liquid chromatogram-mass spectrometer for analysis;

e: in the chromatogram of the extracted ion current for detecting the ion pair, if the sample solution presents a chromatographic peak with the same retention time as the reference solution, the amino acid sequence LDSPVSNSAHIAPLSLPSSAPSVGSVCR in the sample to be detected is shown, and the sample to be detected is proved to be derived from the Agkistrodon halys albus; otherwise, it is derived from non-white-eyebrow Agkistrodon Halys.

Preferably, the detected ion pair in step E is triply charged 935.8 → 861.4 and triply charged 935.8 → 602.3.

Preferably, the application method of the polypeptide with the characteristics of the white-eyebrow snake venom hemocoagulase in the identification of the snake venom hemocoagulase species for injection specifically comprises the following steps:

(1) taking 10 mg of the white brow venom hemocoagulase characteristic polypeptide LDSPVSNSAHIAPLSLPSSAPSVGSVCR, placing in a10 mL measuring flask, dissolving with water, fixing volume, mixing well, and preparing a reference substance stock solution 1 with the concentration of 1 mg/mL; taking 10 mu L of reference substance stock solution 1, dissolving with water, and fixing the volume to 10ml, and preparing reference substance stock solution 2 with the concentration of 1 mu g/ml; taking 600 mu L of the reference substance stock solution 2, and fixing the volume to 10mL by using 25 mmol/L ammonium bicarbonate solution to prepare a reference substance solution of 60 ng/mL; taking 50mg of a sample to be detected, and adding 500 mu L of 25 mmol/L ammonium bicarbonate solution for dissolving to obtain a sample solution;

(2) weighing 400 mu L of each of the test solution and the reference solution, adding 20 mu L of 0.4 mol/L dithiothreitol solution, mixing uniformly, reacting at 60 ℃ for 1 h, adding 40 mu L of 0.4 mol/L iodoacetamide solution, standing in the dark for 30 min, adding 10 mu L of 0.4 mu g/mu L trypsin, and performing enzymolysis at 37 ℃ for 1 h; inactivating at 90 deg.C for 10 min, taking out, cooling to room temperature, centrifuging at 1200 rpm for 10 min, and collecting supernatant and injecting into liquid chromatogram-mass spectrometer for analysis;

(3) in the chromatogram of the extracted ion current for detecting the ion pair, if the sample solution presents a chromatographic peak with the same retention time as the reference solution, the amino acid sequence LDSPVSNSAHIAPLSLPSSAPSVGSVCR in the sample to be detected is shown, and the sample to be detected is proved to be derived from the Agkistrodon halys albus; otherwise, it is derived from non-white-eyebrow Agkistrodon Halys;

preferably, the liquid chromatography-mass spectrometer has the following detection conditions of liquid phase and mass spectrum:

chromatographic mass spectrum conditions: the chromatographic column is Waters ACQUITY UPLC BEH C18 chromatographic column 50mm × 2.1 mm, 1.7 μm; mobile phase A: 0.1% formic acid solution mobile phase, B: 0.1% formic acid acetonitrile; column temperature: 40 ℃; sample introduction amount: 2 mu L of the solution; flow rate: 0.2mL/min, elution procedure: 0 → 1 min, mobile phase A80%; 1 → 5 min, mobile phase A80% → 10%; 5 → 7 min, mobile phase A10% → 10%;

the mass spectrum condition adopts an electrospray ion source, a positive ion scanning mode and multi-reaction monitoring; the vortex ion spraying temperature is 500 ℃; the ionization voltage is 5.5 kV; collision cell exit voltage 10V; inlet voltage 10V; three charges 935.8 → 861.4 and 935.8 → 602.3 are used as detection ion pairs, the collision voltage is 45V and 40V, and the cluster removing voltage is 135V.

The invention has the beneficial effects that:

(1) the invention utilizes NCBI and UniProt to carry out library searching comparison after the existing snake protein library and venom protein library are integrated, and combines a large amount of experimental studies to find out the characteristic polypeptide LDSPVSNSAHPLSLPSSAPSVGSVCR of the agkistrodon saxatilis emelianov venom thrombin, and because the amino acid sequences of other snake venom thrombin such as agkistrodon boviensis, viper, agkistrodon acutus and the like are known to not contain the amino acid sequence of the section, the characteristic polypeptide can be used for specially characterizing the agkistrodon saxatilis emelianov venom thrombin.

(2) The traditional thrombin-like enzyme identification method cannot identify the species, the characteristic polypeptide species identification method provided by the invention is simple, convenient and rapid, has strong specificity, fills the blank of quality standard of the injection white brow venom hemocoagulase, greatly improves the quality control level of the medicine, and is beneficial to ensuring the safety and effectiveness of clinical medication.

Drawings

FIG. 1 shows the secondary mass spectrum and fragment assignment of thrombin-like protein of Agkistrodon saxatilis emelianov;

FIG. 2 is a first-order mass spectrum of thrombin-like protein of Agkistrodon saxatilis emelianov;

FIG. 3 is an extracted ion chromatogram of a blank solution;

FIG. 4 is a chromatogram of a characteristic peptide extracted from a control solution;

FIG. 5 is a chromatogram of ion extraction from hemagglutinin enzyme characteristic peptide of snake venom of white eyebrow for injection;

FIG. 6 is ion chromatogram of the haemocoagulase characteristic peptide extracted from Bothrops atrox for injection.

The specific implementation mode is as follows:

the invention is further illustrated by the following specific examples:

the preparation methods of the relevant reagents and solutions in the following examples are as follows:

(1) reagent: trypsin (Sigma, batch SLBS 8956), agkistrodon saxatilis venom hemagglutination (98.5% pure, aohong, ca), guanidine hydrochloride (VETEC, batch WXBC 4261V), trihydroxyaminomethane (husband, batch 20181206), dithiothreitol (BBI Life Sciences, batch D911BA 0011), iodoacetamide (BBI Life Sciences, batch B326BA 1943), and other reagents were analytically pure.

(2) 25 mmol/L ammonium bicarbonate solution: weighing 79.06 mg of ammonium bicarbonate, and adding 40 mL of water for dissolving to obtain the ammonium bicarbonate.

(3) 0.4 mol/L Dithiothreitol (DTT) solution: weighing 15.42 mg dithiothreitol, and dissolving with 250 μ L water.

(4) 0.4 mol/L iodoacetamide solution (fresh for use): weighing 18.5 mg of iodoacetamide, and dissolving with 250 mu L of water to obtain the iodoacetamide.

(5) 0.4 mg/mL trypsin solution (fresh to use): weighing 8.0 mg of trypsin, and dissolving with 20 mL of water to obtain the finished product.