CN104311651A - Method for preparing fluorescent single-labeled cobra neurotoxin - Google Patents
Method for preparing fluorescent single-labeled cobra neurotoxin Download PDFInfo
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- CN104311651A CN104311651A CN201410566748.8A CN201410566748A CN104311651A CN 104311651 A CN104311651 A CN 104311651A CN 201410566748 A CN201410566748 A CN 201410566748A CN 104311651 A CN104311651 A CN 104311651A
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- cobratoxin
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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Abstract
The invention relates to a method for preparing a fluorescent single-labeled cobra neurotoxin. The method comprises the following steps: on the basis of taking FITC (fluorescein isothiocyanate) as a derivatizing agent, carrying out a derivatization reaction on cobra neurotoxin (Neurotoxin, NT) and FITC at a molar ratio of 1:(1-1.2) in a buffer solution with the pH of 8.4-8.6 at a temperature of 20-40 DEG C for 8-10h, and separating and purifying a reaction solution to obtain a fluorescent single-labeled cobra neurotoxin. The preparation method provided by the invention is simple and rapid, the prepared FITC-NT has higher purity (more than 90%), a technical support is provided for improving the safety of an NT preparation and studying the intracorporal process of NT and a preparation thereof, and a reference is provided for the preparation and separation and purification study on a fluorescent single-labeled product of a macromolecular biological polypeptide.
Description
(1) technical field
The present invention relates to a kind of method preparing the Cobratoxin of fluorescence list mark, especially a kind of semi-preparative liquid chromatography prepares the method for the Cobratoxin of fluorescence list flag F ITC.
(2) background technology
Cobratoxin (Neurotoxin, NT)---a kind of water-soluble macromolecule polypeptide, is the short-chain neurotoxin be separated from Chinese cobra venom, is made up of 62 amino-acid residues, containing 3 lysine residues in its structure.Find in the practice of permanent tcm clinical, Chinese medicine snake venom has good analgesic activity.The traditional Chinese medical science is thought: snake venom can the meridian dredging, wind-damp dispelling, has the effect improved the health.Modern medicine study shows, snake venom not only analgesic effect is obvious, and without gastrointestinal side effect, without tolerance and additive, its potency is than the morphine height decades of times of Isodose, and analgesic effect is more lasting than morphine, is a kind of novel substituting analgesic.Now existing intramuscularly agent listing, is mainly used in treatment pain caused by cancer and various chronic, intractable pain etc.Neurotoxin clinical administration dosage is less, and toxicity is large, mainly act on maincenter, but its mechanism of action not yet can be illustrated at present completely.
In recent years, deepen continuously along with to the research of NT, increasing experimental study needs to carry out mark spike to NT, and the foundation of its micro-quantitative method runs into larger difficulty.In vivo analysis many employings radio isotope of protein and peptide drugs and fluorescein-labelled.Relative to the high cost of radio isotope in non-rodent (as dog etc.) research, high pollution, the use of fluorescein is cheaper, safety, and little to the harm of human body and environment.Lsothiocyanates (fluorescein isothiocyanate, FITC) is a kind of conventional cationic fluorescent marker, its mark principle mainly with the NH on biomacromolecule
2-group combines, and in the present invention, the amino that FITC can hold with the lysine residue on Cobratoxin peptide chain and N reacts, and form thiocarbamide key, the reaction related to is shown below:
Have report display, fluorescein isothiocyanate (fluorescein isothiocyanate, FITC) mark with
125it is high that I marks tracer method consistence, and FITC fluorescein can be used as radio isotope
125the surrogate markers probe of I, for the quantitative analysis in experimental animals.FITC marks high performance liquid chromatography due to can marker site containing multiple FITC in NT structure, and causes marked product may be multiple labeling mixture, therefore its application is restricted.
(3) summary of the invention
The present invention seeks to overcome the defect that above-mentioned existing method exists, for reducing the impact of marker on pharmaceutical properties and experimenter, the preparation method of the Cobratoxin (FITC-NT) providing a kind of FITC fluorescence list to mark as far as possible.
The technical solution used in the present invention is:
A kind of method preparing the Cobratoxin of fluorescence list mark, described method comprises: with thiocyanic acid fluorescein (fluorescein isothiocyanate, FITC) be derivating agent, Cobratoxin (Neurotoxin, NT) be 1:1-1.2 with the ratio of thiocyanic acid fluorescein mole dosage, be carry out derivative reaction 8-10h in the borate buffer of 8.4-8.6 at 20-40 DEG C, at pH, reaction solution obtains the Cobratoxin of fluorescence list mark through separation and purification.
Concrete, described reaction solution separation and purification adopts semi-preparative liquid chromatography to be separated, and chromatographic condition is: adopt reverse Féraud door Jupiter 5u C18300A (10.0 × 250mm) chromatographic column; Moving phase: mobile phase A to be mass concentration be 0.1% the TFA aqueous solution, Mobile phase B to be mass concentration be 0.1% TFA acetonitrile solution, with 3mLmin
-1flow velocity carries out gradient elution; Chromatographic instrument is equipped with fluorimetric detector, and fluoroscopic examination excitation wavelength is 445nm, and emission wavelength is 525nm; Sample size is 200 μ L; Column temperature is 35 DEG C.
Concrete, Parameters of gradient elution is as follows: 0min, 5% (v/v, B account for A+B volume percent) B; 15-30min, 23%B; 35min, 90%B; 40min, 90%B; 42min, 5%B.
Preferably, described derivatize step is as follows: get in the borate buffer and acetone that Cobratoxin and thiocyanic acid fluorescein be dissolved in appropriate pH 8.4 respectively, Cobratoxin is 1:1 with the ratio of thiocyanic acid fluorescein mole dosage, after at room temperature reacting 8h, reaction solution carries out liquid chromatography separation, collect peak, 19.651min place, vacuum lyophilization, obtain the Cobratoxin of described fluorescence list mark.
Beneficial effect of the present invention is mainly reflected in: preparation method of the present invention is specially easy, quick, prepare the FITC-NT purity higher (more than 90%) of gained, technical support is provided, for the preparation of the fluorescence list marked product of macromole biological polypeptide and separation and purification research provide reference for improving the security of neurotoxin preparation, the research physiological disposition of neurotoxin and preparation thereof.
(4) accompanying drawing explanation
Fig. 1 is the impact of pH on derivatization reaction, and in figure, ordinate zou is fluorescence intensity;
Fig. 2 be temperature, time on the impact of derivatization reaction, in figure, ordinate zou is fluorescence intensity;
Fig. 3 is the impact of FITC and NT ratio on derivatization reaction, and in figure, ordinate zou is fluorescence intensity;
Fig. 4 is partly preparing liquid phase (A) and analyzing liquid phase (B) color atlas of FITC-NT;
Fig. 5 is the list mark FITC-NT product mass spectra figure of preparation.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: the optimization of each parameter
1 instrument and reagent
1.1 instrument
Agilent 1200 high performance liquid chromatograph (Agilent company of the U.S.);
Labconco freeze drier (Labconco company of the U.S.);
Matrix Assisted Laser Desorption ionization time-of-flight mass spectrometry instrument, MALDI-TOF/TOF-4800 (Applied Biosystems company of the U.S.);
Five Easy TM pH meter (Mettler toledo company of Switzerland);
CP225D type electronic analytical balance (German Sartorius company);
DF-101S heat collecting type thermostatically heating magnetic agitation (Henan Yu Hua Instrument Ltd.).
1.2 reagent
Cobratoxin (purity >97%), Yunnan dragon and phoenix paddy Bioceuticals Inc.;
Thiocyanic acid fluorescein, Sigma, the U.S.;
Acetonitrile (chromatographically pure), Honeywell Burdick & Jackson, the U.S.;
Alpha-cyano-4-hydroxycinnamic acid (α-Cyano-4-hydroxycinnamic Acid, CHCA), Sigma, the U.S.);
All the other reagent are domestic analytical pure.
2 methods and result
2.1 chromatographic condition
2.1.1 semi-preparative liquid chromatography condition
Chromatographic column: Féraud door Jupiter 5u C18 300A (10.0 × 250mm); Mobile phase A: the 0.1%TFA aqueous solution, Mobile phase B: 0.1%TFA acetonitrile solution; Gradient elution: 0min, 5%B; 15-30min, 23%B; 35min, 90%B; 40min, 90%B; 42min, 5%B; Flow velocity: 3mLmin
-1; Excitation wavelength: 445nm, emission wavelength 525nm; Sample size: 200 μ L; Column temperature: 35 DEG C.
2.1.2 liquid phase chromatogram condition is analyzed
Chromatographic column: Féraud door Aeris PEPTIDE XB-C18 (3.6 μm, 4.6mm × 150mm); Mobile phase A: 0.1%TFA-water, Mobile phase B: 0.1%TFA-acetonitrile; Gradient elution: 0min, 10%B; 20min, 35%B; 25min, 90%B; 30min, 90%B; 32min, 10%B; Flow velocity: 1mLmin
-1; Excitation wavelength: 445nm, emission wavelength 525nm; Sample size: 20 μ L; Column temperature: 35 DEG C.
The optimization of the mono-labeled derivative reaction conditions of 2.2 FITC-NT
Get NT and FITC and be dissolved in the borate buffer of pH 7.5-10.0 and the acetone of 1/10 liquor capacity respectively, make 1mgmL
-1solution.Appropriate FITC solution is added in NT solution, is placed in the reaction of constant temperature blender with magnetic force lucifuge.Reaction solution is analyzed by chromatographic condition sample introduction under " 2.1.2 " item.
2.2.1 the impact of pH of buffer
Within the scope of pH 7.5-10.0, investigate borate buffer pH to the impact of derivatization reaction.The results are shown in Figure 1, as seen from the figure, between pH 7.5-8.4 scope, the fluorescence intensity raising derivative along with pH increases gradually; When pH is 8.6-10.0, the fluorescence intensity of derived products obviously declines, and therefore selects pH to be the pH of 8.4-8.6 as derivatization reaction, more preferably carries out for 8.4 times at pH.
2.2.2 the impact in temperature, reaction times
Within the scope of 20-40 DEG C, investigate temperature of reaction to the impact of derivatization reaction.The results are shown in Figure 2, as seen from the figure, temperature is when 20-40 DEG C, and the derived products fluorescence intensity of NT is substantially constant.Therefore select the 20-40 DEG C of temperature as derivatization reaction, more preferably at room temperature carry out.
Within the scope of 2-12h, investigate the reaction times to the impact of derivatization reaction.The results are shown in Figure 2, as seen from the figure, when the reaction times is shorter, increasing derived products fluorescence intensity in time increases; Extend to 8h derived products fluorescence intensity with the reaction times and reach maximum, continue to extend reaction times fluorescence intensity and decrease, therefore select the derivative time to be 8-10h, be more preferably 8h.
2.2.3 the impact of reactant ratio
Within the scope of NT and FITC ratio 0.6-2.0, investigate reactant ratio (mol ratio) to the impact of derivatization reaction.The results are shown in Figure 3, as seen from the figure, FITC and NT ratio is between 0.6-1.0 scope, and the fluorescence intensity raising derivative along with FITC ratio increases gradually; When FITC and NT ratio is 1.2-2.0, the fluorescence intensity of derived products slightly declines, and therefore selects FITC and NT ratio to be 1:1-1.2, is more preferably 1:1.
The preparation of embodiment 2:FITC-NT
Get NT and FITC to be dissolved in respectively in the borate buffer of pH 8.4 and the acetone of 1/10 liquor capacity, make 1mgmL
-1solution.Add in NT solution by appropriate FITC solution, control FITC and NT mol ratio are 1:1, are placed in constant temperature blender with magnetic force lucifuge reaction 8h under room temperature.
By FITC-NT reaction solution by chromatographic condition repeatedly sample introduction under embodiment 1 " 2.1.1 " item, collect peak, 19.651min place, after vacuum lyophilization, obtain the FITC-NT of single mark.Flight time mass spectrum mensuration NT molecular weight is 6939.2Da, FITC-NT molecular weight is 7325.4Da.
By chromatographic condition under embodiment 1 " 2.1.2 " item, to reach the sample size of column overload, the wash-out duration of 2.5 times of main peak retention time carries out purity detecting to preparing gained FITC-NT.The purity recording obtained FITC-NT by areas of peak normalization method is 99.29%.Fig. 5 is preparation single mark FITC-NT product mass spectra figure, two peaks just in time differ the molecular weight of a FITC as seen from the figure, visible according to the inventive method, successfully prepare the Cobratoxin (FITC-NT) of FITC fluorescence list mark, there is better application prospect.
Claims (3)
1. prepare the method for the Cobratoxin of fluorescence list mark for one kind, described method comprises: with thiocyanic acid fluorescein (fluorescein isothiocyanate, FITC) be derivating agent, Cobratoxin (Neurotoxin, NT) be 1:1-1.2 with the ratio of thiocyanic acid fluorescein mole dosage, be carry out derivative reaction 8-10h in the borate buffer of 8.4-8.6 at 20-40 DEG C, at pH, reaction solution obtains the Cobratoxin of fluorescence list mark through separation and purification.
2. the method for claim 1, it is characterized in that described reaction solution separation and purification adopts semi-preparative liquid chromatography to be separated, chromatographic condition is: adopt reverse Féraud door Jupiter 5u C18300A (10.0 × 250mm) chromatographic column; Moving phase: mobile phase A to be mass concentration be 0.1% the TFA aqueous solution, Mobile phase B to be mass concentration be 0.1% TFA acetonitrile solution, carry out gradient elution with 3mLmin-1 flow velocity; Chromatographic instrument is equipped with fluorimetric detector, and fluoroscopic examination excitation wavelength is 445nm, and emission wavelength is 525nm; Sample size is 200 μ L; Column temperature is 35 DEG C.
3. method as claimed in claim 2, it is characterized in that described derivatize step is as follows: get in the borate buffer and acetone that Cobratoxin and thiocyanic acid fluorescein be dissolved in pH 8.4 respectively, Cobratoxin is 1:1 with the ratio of thiocyanic acid fluorescein mole dosage, after at room temperature reacting 8h, reaction solution carries out liquid chromatography separation, collect peak, 19.651min place, vacuum lyophilization, obtain the Cobratoxin of described fluorescence list mark.
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| CN201410566748.8A CN104311651A (en) | 2014-10-22 | 2014-10-22 | Method for preparing fluorescent single-labeled cobra neurotoxin |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106814049A (en) * | 2016-12-05 | 2017-06-09 | 浙江中医药大学 | A kind of Capillary Electrophoresis vivo detection method of Cobratoxin |
| CN108524916A (en) * | 2018-05-31 | 2018-09-14 | 浙江中医药大学 | A kind of preparation method for the soluble micropin carrying Neurotoxin From Naja Naja Atra |
| CN111320673A (en) * | 2020-03-26 | 2020-06-23 | 应连心 | FITC-labeled pasireotide derivative and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1281978A (en) * | 2000-07-13 | 2001-01-31 | 厦门大学 | Anti-alpha-fetoprotein antibody labelled by fluorescein and its preparation method |
-
2014
- 2014-10-22 CN CN201410566748.8A patent/CN104311651A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1281978A (en) * | 2000-07-13 | 2001-01-31 | 厦门大学 | Anti-alpha-fetoprotein antibody labelled by fluorescein and its preparation method |
Non-Patent Citations (4)
| Title |
|---|
| HU YI-QIN ET AL.: "Study of FITC-Neurotoxin loaded Angiopep-2 Modified Nanoparticles", 《2014年中国药物制剂大会》 * |
| HU YI-QIN ET AL.: "Study on FITC-Neurotoxin loaded Angiopep-2 Modified Nanoparticles", 《2014年中国药物制剂大会》 * |
| MICHAEL J. LITTLE ET AL.: "Single-label fluorescent derivatization of peptides", 《ANALYTICA CHIMICA ACTA》 * |
| 林丽丽的呢过: "眼镜蛇毒神经毒素的125I标记及其在大鼠体内的分布", 《海峡药学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106814049A (en) * | 2016-12-05 | 2017-06-09 | 浙江中医药大学 | A kind of Capillary Electrophoresis vivo detection method of Cobratoxin |
| CN108524916A (en) * | 2018-05-31 | 2018-09-14 | 浙江中医药大学 | A kind of preparation method for the soluble micropin carrying Neurotoxin From Naja Naja Atra |
| CN111320673A (en) * | 2020-03-26 | 2020-06-23 | 应连心 | FITC-labeled pasireotide derivative and preparation method and application thereof |
| CN111320673B (en) * | 2020-03-26 | 2023-10-24 | 应连心 | FITC-labeled pasireotide derivative as well as preparation method and application thereof |
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