CN103554221B - The preparation method of one group of venin-derived bioactive peptide - Google Patents
The preparation method of one group of venin-derived bioactive peptide Download PDFInfo
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Abstract
The invention belongs to biomedicine field, relate to one group of venin-derived preparation method of bioactive peptide and the application at anti-tumor aspect thereof.Be specifically related to the peptides that a kind of chemically preparation N end is Pyrrolidonecarboxylic acid, and this kind of peptide compounds is in the concrete purposes in antitumor field.The invention still further relates to the chemical synthesis process of pyroGlu-Lys-Trp (pyroGlu-Lys-Trp), pyroGlu-Asn-Trp (pyroGlu-Asn-Trp) or pyroGlu-Gln-Trp (pyroGlu-Gln-Trp), and this tripeptide compound is in the application in antitumor field.
Description
The application is the applying date is on April 29th, 2010, and application number is 201010158898.7, and denomination of invention is the divisional application of one group of venin-derived preparation method of bioactive peptide and the Chinese invention patent application in the application of anti-tumor aspect thereof.
Technical field
The present invention relates to biomedicine field, be specifically related to the peptides that a kind of chemically preparation N end is Pyrrolidonecarboxylic acid, and this kind of peptide compounds is in the concrete purposes in antitumor field.
Background technology
Snake venom is the natural mixture secreted by the poison gland of poisonous snake, comprises albumen, polypeptide and enzyme, has the biological activitys such as anti-freezing, analgesia, promotion nerve growth and inhibition tumor cell growth, is one of important natural compounds storehouse of research and development new drug.Studies on Anticancer Components in snake venom mainly contains enzyme and other proteins and peptides factors of the element that dissociates, cytotoxin and energy cell death inducing.CN101177452A discloses the method that preparation from docking pallas pit viper has the element that dissociates of inhibition tumor cell Infiltration and metastasis activity; CN101270159A discloses a kind of antitumor cell toxin, and molecular weight is 6kDa; CN101429525A discloses the method for the snake venom metalloprotease inhibitors of expressing anti metastasis, and this inhibitor molecules amount is 46kDa; CN1781551A discloses a kind of sv-Cystatin, and can attack by inhibition tumor cell, its molecular weight is about 100kDa.This kind of high molecular weight protein, while its anti-tumor activity of performance, also shows strong immunogenicity and easily by protease hydrolysis, the shortcomings such as the transformation period is short, so constrain its clinical application.Therefore, be necessary to utilize snake resource, the small molecule, anti-tumor drug in exploitation snake venom.
Kai-Fa Hang etc. once reported and found the little peptide with suppression metal proteinase activity of 3 molecular weight below 1000 in trimeresurus stejnegeri snake venom, see: Kai-Fa Hang, Chin-Chun Hang, Shih-Hsiung Wu etc., Characterization of Three Endogenous Peptide Inhibitors for Multiple Metalloproteinases with Fibrinogenolytic Activity from the Venom of Taiwan Habu (
trimeresursu mucrosquamatus), Biochemical And Biophysical Research Communications.1998,248:562-566.These three the little peptides with suppression metal proteinase activity are tripeptides, and its N-terminal is all Pyrrolidonecarboxylic acids.CN101058572A discloses the method being separated from Agkistrodon acutus and having and suppress the little peptide of tumor cell of liver proliferation activity, and its molecular weight is 429, and one of them in three polypeptide reported with above-mentioned Kai-Fa Hang etc. is same substance.The research and development of the current small peptide to snake venom small molecular bioactive peptide, particularly below 3KDa also far away from high molecular weight protein deeply generally.Micromolecule active polypeptide content in thick poison is very low simultaneously, thus from a small amount of thick malicious sample, extracts bioactive peptide and does not often reach research consumption, although can obtain the bioactive peptide of q.s from a large amount of thick toxogen material, obviously increases research and development cost.In view of small-molecular peptides compounds is only by several to dozens of Amino acid profiles, sequence is easier to determine, thus utilizes Solid-phase synthesis peptides technology can be easy to prepare a large amount of bioactive peptide, solves the difficult problem that extracted amount is few from thick poison.But it be often Pyrrolidonecarboxylic acid that the N of some snake venom bioactive peptide holds, specifically see Liang Ningsheng, the step-down component-angiotensin-convertion enzyme inhibitor in snake venom, Chinese " snake will " impurity, 1993,5 (3) 2-5.Pyrrolidonecarboxylic acid is that L-glutamic acid is by being formed after cyclisation, the reaction of this step is what to be completed by Pyrrolidonecarboxylic acid enzyme catalysis in the poison gland of snake venom, but from the poison gland of snake, does not extract Pyrrolidonecarboxylic acid enzyme temporarily at present or utilize other means that polypeptide N is held the report of L-glutamic acid cyclisation.Because existing solid phase synthesis technique is merely able to provide N to hold to be the polypeptide of L-glutamic acid usually, thus need to hold L-glutamic acid to carry out cyclisation process, to reach the activity of expection to N.
Summary of the invention
One aspect of the present invention provides a kind ofly prepares the method for oligopeptides that N end is Pyrrolidonecarboxylic acid, and the method comprises:
Method (one): N is held as the oligopeptides sample dissolution of L-glutamic acid is in the damping fluid of pH value 2 ~ 6, adds the CuCl of catalytic amount
2, in 60 ~ 95 DEG C of heating, will namely obtain the oligopeptides that corresponding N end is Pyrrolidonecarboxylic acid after solution lyophilize.Or
Method (two): the oligopeptides sample homogeneous heating under solid phase by N end being L-glutamic acid, obtains the oligopeptides that corresponding N end is Pyrrolidonecarboxylic acid.Wherein, the mode of homogeneous heating such as: then heated at the metal sheet upper berth lamellar that heat conduction is good by sample, can take other similar heater meanses, as microwave heating, being paved into thin layer illumination heating etc. for reaching this object.
In aforesaid method, wherein oligopeptides refers to that amino acid number is less than the polypeptide equaling 10, and preferred amino acid number is the oligopeptides of 3.
In aforesaid method (), the pH of damping fluid is preferably 3.5 ~ 4.5, and damping fluid can be Ammonium formate buffer, ammonium acetate buffer or phosphate buffered saline buffer etc., and described Ammonium formate buffer refers to the buffered soln that ammonium formiate and formic acid are formed; Described ammonium acetate buffer refers to the buffered soln that ammonium acetate and acetic acid are formed; Described phosphate buffered saline buffer refers to that negatively charged ion is PO
4 3-, HPO
4 2-, H
2pO
4 -, positively charged ion is H
+, Na
+or/and K
+the buffered soln formed.Preferred use phosphate buffer soln, more preferably the sylvite buffered soln receiving salt buffer solution or phosphoric acid of phosphoric acid.In damping fluid, sample concentration is 0.1 ~ 10mg/ml, is preferably 0.5 ~ 3mg/ml.The CuCl of catalytic amount
2can add in solid form, also can be configured to solution and drip, be preferably arranged to the CuCl of 0.05-0.2mol/l
2drip 1 ~ 10.Heating temperature is preferably 60 ~ 90 DEG C, more preferably 80 ~ 90 DEG C, heat-up time 1 ~ 4h, preferably 1.5 ~ 3h.
In aforesaid method (two), preferably at 100 ~ 150 DEG C of heating 0.5 ~ 4h, more preferably at 120 ~ 130 DEG C of heating 1 ~ 2h.
Another aspect of the present invention there are provided the preparation method of following three tripeptide compounds." tripeptide compound " alleged by the present invention, what namely refer in following three concrete tripeptides if no special instructions is one or all: pyroGlu-Lys-Trp (pyroGlu-Lys-Trp), pyroGlu-Asn-Trp (pyroGlu-Asn-Trp), pyroGlu-Gln-Trp (pyroGlu-Gln-Trp).Described tripeptide compound is the oligopeptides extracted from snake venom that prior art is disclosed.Described " raw material tripeptides " is glutamy lysyl tryptophane (Glu-Lys-Trp), glutamy asparaginyl tryptophane (Glu-Asn-Trp), glutamy glutaminyl tryptophane (Glu-Gln-Trp).
The preparation method of above-mentioned three tripeptide compounds comprises:
Method (one): N is held as the raw material tripeptides sample dissolution of L-glutamic acid is in the damping fluid of pH value 2 ~ 6, adds the CuCl of catalytic amount
2, in 60 ~ 95 DEG C of heating, will namely obtain the tripeptide compound that corresponding N end is Pyrrolidonecarboxylic acid after solution lyophilize.Or
Method (two): the raw material tripeptides sample homogeneous heating under solid phase by N end being L-glutamic acid, obtains the tripeptide compound that corresponding N end is Pyrrolidonecarboxylic acid.Wherein, the mode of homogeneous heating such as: then heated at the metal sheet upper berth lamellar that heat conduction is good by sample, can take other similar heater meanses, as microwave heating, being paved into thin layer illumination heating etc. for reaching this object.
In aforesaid method (), the pH of damping fluid is preferably 3.5 ~ 4.5, and damping fluid can be Ammonium formate buffer, ammonium acetate buffer or phosphate buffered saline buffer etc., and described Ammonium formate buffer refers to the buffered soln that ammonium formiate and formic acid are formed; Described ammonium acetate buffer refers to the buffered soln that ammonium acetate and acetic acid are formed; Described phosphate buffered saline buffer refers to that negatively charged ion is PO
4 3-, HPO
4 2-, H
2pO
4 -, positively charged ion is H
+, Na
+or/and K
+the buffered soln formed.Preferred use phosphate buffer soln, more preferably the sylvite buffered soln receiving salt buffer solution or phosphoric acid of phosphoric acid.In damping fluid, sample concentration is 0.1 ~ 10mg/ml, is preferably 0.5 ~ 3mg/ml.The CuCl of catalytic amount
2can add in solid form, also can be configured to solution and drip, be preferably arranged to the CuCl of 0.05-0.2mol/l
2drip 1 ~ 10.Heating temperature is preferably 60 ~ 90 DEG C, more preferably 80 ~ 90 DEG C, heat-up time 1 ~ 4h, preferably 1.5 ~ 3h.
In aforesaid method (two), preferably at 100 ~ 150 DEG C of heating 0.5 ~ 4h, more preferably at 120 ~ 130 DEG C of heating 1 ~ 2h.
The present invention only relates to oligopeptides that N-terminal is L-glutamic acid or raw material tripeptides and changes into as corresponding N-terminal is oligopeptides or the tripeptide compound of Pyrrolidonecarboxylic acid through Pyrrolidonecarboxylic acid, does not relate to the formation of peptide bond.Described N-terminal is that the oligopeptides of L-glutamic acid or raw material tripeptides can be obtained by existing peptide synthesis technology, as solid phase synthesis, liquid phase synthesis, enzymic synthesis etc., also can be obtained by natural product extraction.
The structural identification of tripeptide compound of the present invention: the relative molecular mass of three tripeptide compounds adopting present method to prepare through mass spectroscopy is respectively: 443,429 and 443.Ultra-violet absorption spectrum scanning result shows, and three samples all have maximum absorption at 280nm.Carry out ninhydrin reaction detection respectively to three samples, result does not all develop the color, prompting sample N end-NH
2close, namely N end is Pyrrolidonecarboxylic acid.Amino acid sequencing shows: the amino acid that three samples hold N to hold from C is respectively Trp and Lys, Trp and Asn or Trp and Gln.Three tripeptide compounds that to sum up data analysis obtains are respectively: pyroGlu-Lys-Trp, pyroGlu-Asn-Trp, pyroGlu-Gln-Trp.
Preparation method of the present invention can also comprise following purification step, and described purification step comprises reaction product is carried out reverse phase chromatography with preparation HPLC further.The inverted medium that chromatography column wherein used can be commonly used for this area is the chromatography column of filler; Preferred silica gel is skeleton, and surface bond has the chromatography column of C18 or C8 reversed phase chromatographic medium; Most preferably silica gel is skeleton, the chromatography column of surface bond C18 reversed phase chromatographic medium.The type of elution that purifying adopts is preferably gradient elution.Flow is preferably made up of trifluoroacetic acid (TFA) aqueous solution and trifluoroacetic acid (TFA) acetonitrile solution, more preferably the concentration of trifluoroacetic acid (TFA) aqueous solution is 0.05-0.2%, and the concentration of trifluoroacetic acid (TFA) acetonitrile solution is the moving phase of 0.05-0.2%; Most preferably the concentration of trifluoroacetic acid (TFA) aqueous solution is 0.1%, and the concentration of trifluoroacetic acid (TFA) acetonitrile solution is the moving phase of 0.1%.
Preparation method of the present invention raw material simple and easy to do, used is easy to obtain, with low cost.And product that the method obtains is easy to subsequent disposal, the product that purity is very high can be obtained.
Another aspect of the invention is to provide described oligopeptides preparing the application in antitumor drug.Contriver is screened by the test of tumor cell in vitro Proliferation Ability, finds that pyroGlu-Lys-Trp, pyroGlu-Asn-Trp and pyroGlu-Gln-Trp tripeptide compound prepared by the inventive method all has stronger inhibit activities to gastric adenocarcinoma cells, breast cancer cell and human liver cancer cell.
Accompanying drawing explanation
Fig. 1: pyroGlu-Lys-Trp tripeptides mass spectrum
Fig. 2: pyroGlu-Asn-Trp tripeptides mass spectrum
Fig. 3: pyroGlu-Gln-Trp tripeptides mass spectrum
Fig. 4: pyroGlu-Asn-Trp high resolution mass spec figure
The RP-HPLC collection of illustrative plates of Fig. 5: pyroGlu-Lys-Trp tripeptides
The RP-HPLC collection of illustrative plates of Fig. 6: pyroGlu-Asn-Trp tripeptides
The RP-HPLC collection of illustrative plates of Fig. 7: pyroGlu-Gln-Trp tripeptides.
Embodiment
embodiment 1:
n end is the polypeptide sample of L-glutamic acidsynthesis
Take Fmoc-Trp-Wang Resin 0.6779g(0.59mmol/g) be placed in the reactor of Peptide synthesizer, add 10mlDMF and mix 1 hour, make resin fully swelling.Add 25%PIP(DMF) solution 10ml, after mixing 30min, with DMF washing, can carry out linked reaction after washing terminates.0.7591g Fmoc-Lys (Trt)-OH is added in mixing reactor, 0.64mol/L HOBT(DMF) solution 2.5 ml, 0.53mol/L DIC(DMF) solution 3 ml and DMF 3ml reacts, temperature of reaction is room temperature, with ninhydrin reaction determination reaction end, obtain Fmoc-Lys (Trt)-Trp-Wang Resin.
After linked reaction terminates; wash with 10ml DMF; add the DMF solution 10ml of 25% PIP; carry out deprotection 30min; reaction terminates rear DMF and washs; 0.6831g Fmoc-Glu (OtBU)-OH is added to reactor; and 0.64mol/L HOBT(DMF) solution 2.5 ml; 0.53mol/L DIC(DMF) solution 3 ml and DMF 3ml reacts; temperature of reaction is room temperature; with ninhydrin reaction determination reaction end, finally obtain Fmoc-Glu (OtBU)-Lys (Trt)-Trp-Wang Resin.
The resin peptide of vacuum-drying synthesis, adds lytic reagent and carries out cracking.The proportioning of lytic reagent is: TFA/ thioanisole/EDT/ phenol/water/TIS=82/3/5/5/4/1, and stirring at room temperature reacts 3 hours, after cracking terminates, carry out suction filtration with quartz funnel, resin adds a small amount of TFA and washs, and merges suction filtration liquid, to in cracking suction filtration liquid, ice ether is added, precipitated polypeptide by 10 times amount, centrifugal, abandon supernatant, precipitation adds diethyl ether and washs centrifugal 6 times, and vacuum-drying, weigh to obtain Glu-Lys-Trp 0.40241g.
With 0.7011g Fmoc-Trp-Wang Resin(0.59mmol/g), 0.9426g Fmoc-Asn (Trt)-OH and 0.704gFmoc-Glu (OtBU)-OH for raw material, with method synthesis can obtain Glu-Asn-Trp 0.18803g.
With 0.7124g Fmoc-Trp-Wang Resin(0.59mmol/g), 1.027g Fmoc-Gln (Trt)-OH and 0.715gFmoc-Glu (OtBU)-OH for raw material, with method synthesis can obtain Glu-Gln-Trp 0.22254g.
embodiment 2
It is in the sodium phosphate buffer of 2 that Glu-Lys-Trp tripeptides embodiment 1 obtained is dissolved in pH, and Quality control concentration is 1mg/ml, adds the CuCl of 0.1mol/l
2three.In 60 DEG C of heating in water bath 4h, namely obtain pyroGlu-Lys-Trp by after solution lyophilize.Product uses Q-Tof mirco(Waters company) mass spectrograph carries out mass spectroscopy, sees accompanying drawing 1.
embodiment 3
The Glu-Gln-Trp that embodiment 1 obtains being dissolved in pH is in the sodium phosphate buffer of 6, and Quality control concentration is 3mg/ml, adds the CuCl of 0.1mol/l
2three.In 85 DEG C of heating in water bath 2h, namely obtain pyroGlu-Gln-Trp by after solution lyophilize.Product uses Q-Tof mirco(Waters company) mass spectrograph carries out mass spectroscopy, sees accompanying drawing 3.
embodiment 4
The Glu-Asn-Trp that embodiment 1 obtains being dissolved in pH is that in 4 potassium phosphate salt damping fluids, Quality control concentration is 10mg/ml, adds the CuCl of 0.2mol/l
2three.In 90 DEG C of heating in water bath 2h, namely obtain pyroGlu-Asn-Trp by after solution lyophilize.The end product that purity is greater than 98% can be obtained after being further purified.
Purification condition:
Chromatography column: Waters company Sunfire C18 OBD (10 μm); 1.9 × 150mm
Column volume (CV): 42.5 ml
Flow velocity: 10.0 ml/min
The acetonitrile solution of flow composition A:0.2%TFA aqueous solution B:0.2% TFA
Determined wavelength: 215nm
Gradient elution also collects elutriant, utilizes RPLC to carry out peak purity analysis, according to area normalization method, the elutriant of purity more than 98% is pressed peak and merges.By sample lyophilize after underpressure distillation removing acetonitrile.RP-HPLC (RP-HPLC) color atlas is shown in accompanying drawing 6.
Product uses Q-Tof mirco(Waters company) mass spectrograph carries out mass spectroscopy, sees accompanying drawing 2.
Product uses Q-Tof mirco(Waters company) mass spectrograph carries out high resolution mass spec analysis, sees accompanying drawing 4.
embodiment 5
The Glu-Lys-Trp that embodiment 1 obtains being dissolved in pH is in the ammonium acetate buffer of 3.5, and Quality control concentration is 0.5mg/ml, regulates, adds the CuCl of 0.1mol/l
2three.In 80 DEG C of heating in water bath 2h, namely obtain pyroGlu-Lys-Trp by after solution lyophilize.The end product that purity is greater than 98% can be obtained after being further purified.
Purification condition:
Chromatography column: Waters company Sunfire C18 OBD (10 μm); 1.9 × 150mm
Column volume (CV): 42.5 ml
Flow velocity: 10.0 ml/min
The acetonitrile solution of flow composition A:0.1%TFA aqueous solution B:0.1% TFA
Determined wavelength: 215nm
Gradient elution also collects elutriant, utilizes RPLC to carry out peak purity analysis, according to area normalization method, the elutriant of purity more than 98% is pressed peak and merges.By sample lyophilize after underpressure distillation removing acetonitrile.RP-HPLC (RP-HPLC) color atlas is shown in accompanying drawing 5.
embodiment 6
The Glu-Gln-Trp that embodiment 1 obtains being dissolved in pH is in the Ammonium formate buffer of 4.5, and Quality control concentration is 8mg/ml, adds the CuCl of 0.1mol/l
2three.In 75 DEG C of heating in water bath 3h, namely obtain pyroGlu-Gln-Trp by after solution lyophilize.The end product that purity is greater than 98% can be obtained after being further purified.
Purification condition:
Chromatography column: Waters company Sunfire C18 OBD (10 μm); 1.9 × 150mm
Column volume (CV): 42.5 ml
Flow velocity: 10.0 ml/min
The acetonitrile solution of flow composition A:0.1%TFA aqueous solution B:0.1% TFA
Determined wavelength: 215nm
Gradient elution also collects elutriant, utilizes RPLC to carry out peak purity analysis, according to area normalization method, the elutriant of purity more than 98% is pressed peak and merges.By sample lyophilize after underpressure distillation removing acetonitrile.RP-HPLC (RP-HPLC) color atlas is shown in accompanying drawing 7.
embodiment 7
The Glu-Lys-Trp that embodiment 1 obtains is paved into thin layer, in 100 DEG C of heating 1h, impels Pyrrolidonecarboxylic acid, obtain pyroGlu-Lys-Trp.After reaction, sample can obtain the Pyrrolidonecarboxylic acid sample that purity is greater than 98% after being further purified.
embodiment 8
The Glu-Asn-Trp that embodiment 1 obtains is paved into thin layer, in 150 DEG C of heating 1h, impels Pyrrolidonecarboxylic acid, obtain pyroGlu-Asn-Trp.After reaction, sample can obtain the Pyrrolidonecarboxylic acid sample that purity is greater than 98% after being further purified.
Purification condition:
Chromatography column: Waters company Sunfire C18 OBD (10 μm); 1.9 × 150mm
Column volume (CV): 42.5 ml
Flow velocity: 10.0 ml/min
The acetonitrile solution of flow composition A:0.2%TFA aqueous solution B:0.2% TFA
Determined wavelength: 215nm
Gradient elution also collects elutriant, utilizes RPLC to carry out peak purity analysis, according to area normalization method, the elutriant of purity more than 98% is pressed peak and merges.By sample lyophilize after underpressure distillation removing acetonitrile.
embodiment 9
The Glu-Gln-Trp that embodiment 1 obtains is paved into thin layer, in 125 DEG C of heating 1h, impels Pyrrolidonecarboxylic acid, obtain pyroGlu-Gln-Trp.After reaction, sample can obtain the Pyrrolidonecarboxylic acid sample that purity is greater than 98% after being further purified.
Purification condition:
Chromatography column: Waters company Sunfire C18 OBD (10 μm); 1.9 × 150mm
Column volume (CV): 42.5 ml
Flow velocity: 10.0 ml/min
The acetonitrile solution of flow composition A:0.05%TFA aqueous solution B:0.05% TFA
Determined wavelength: 215nm
Gradient elution also collects elutriant, utilizes RPLC to carry out peak purity analysis, according to area normalization method, the elutriant of purity more than 98% is pressed peak and merges.By sample lyophilize after underpressure distillation removing acetonitrile.
embodiment 10:tumor cell in vitro proliferation inhibition activity screens
Experimental technique: people's low differentiation gastric adenocarcinoma cells BGC-823, the breast cancer cell MDA-MB-231, the human liver cancer cells Hep G2 that are in cell log vegetative period are inoculated in 96 well culture plates by certain cell concentration (2500-4000/ hole) respectively, the tripeptides sample of burnt cyclisation prepared by above embodiment is added respectively after cultivating 24h, cell at 37 DEG C, 5%CO
2continue cultivation under condition after 48 hours, continue cultivation after adding MTT 4 hours, dissolve with DMSO, detect under microplate reader 490nm.Concrete data are in table one.
Table one: pyroGlu-Lys-Trp is to the inhibiting rate of people's low differentiation gastric adenocarcinoma cells BGC-823, breast cancer cell MDA-MB-231, Proliferation of Human Hepatoma Cell SMMC-7721
Table two: pyroGlu-Asn-Trp is to the inhibiting rate of people's low differentiation gastric adenocarcinoma cells BGC-823, breast cancer cell MDA-MB-231, Proliferation of Human Hepatoma Cell SMMC-7721
Table three: pyroGlu-Gln-Trp is to the inhibiting rate of people's low differentiation gastric adenocarcinoma cells BGC-823, breast cancer cell MDA-MB-231, Proliferation of Human Hepatoma Cell SMMC-7721
From above result: pyroGlu-Lys-Trp, pyroGlu-Asn-Trp and pyroGlu-Gln-Trp tripeptide compound prepared by the inventive method all has stronger inhibit activities to gastric adenocarcinoma cells BGC-823, breast cancer cell MDA-MB-231 and human liver cancer cells Hep G2.
Claims (5)
1. a preparation method for tripeptide compound, being characterized as N end as the raw material tripeptides of L-glutamic acid is dissolved in pH value is 2 ~ 6, and concentration is in the damping fluid of 0.1 ~ 10mg/ml, adds the CuCl of catalytic amount
2in 60 ~ 95 DEG C of heating 1 ~ 4 hour, the tripeptide compound that corresponding N end is Pyrrolidonecarboxylic acid will be namely obtained after solution lyophilize, wherein tripeptide compound refers to pyroGlu-Lys-Trp, pyroGlu-Asn-Trp, pyroGlu-Gln-Trp, and damping fluid is Ammonium formate buffer, ammonium acetate buffer or phosphate buffered saline buffer.
2. method according to claim 1, wherein damping fluid is phosphate buffered saline buffer.
3. method according to claim 2, is characterised in that phosphate buffered saline buffer is the sodium salt damping fluid of phosphoric acid or the sylvite damping fluid of phosphoric acid.
4. method according to claim 1, is characterised in that the pH value of damping fluid is 3.5 ~ 4.5.
5. the arbitrary described method of claims 1 to 3, is characterised in that in damping fluid, sample concentration is 0.5 ~ 3mg/ml.
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| CN201310493979.6A CN103554221B (en) | 2010-04-29 | 2010-04-29 | The preparation method of one group of venin-derived bioactive peptide |
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| CN108866148A (en) * | 2018-07-12 | 2018-11-23 | 吉首大学 | A kind of detection method for the drug inhibiting human breast cancer cell proliferation |
| CN112725398B (en) * | 2019-10-29 | 2022-07-01 | 中国食品发酵工业研究院有限公司 | Pea peptide with auxiliary hypoglycemic function and preparation method thereof |
| CN113804637A (en) * | 2020-06-16 | 2021-12-17 | 中国人民解放军海军特色医学中心 | Sea snake venom identification method and application thereof |
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| US5225400A (en) * | 1988-07-29 | 1993-07-06 | Ellem Industria Farmaceutica S.R.L. | Immunostimulating peptides, a process for their preparation and pharmaceutical compositions containing them |
| IT1226552B (en) * | 1988-07-29 | 1991-01-24 | Ellem Ind Farmaceutica | IMMUNOSTIMULANT PEPTIDES. |
| CN101314617B (en) * | 2008-07-18 | 2011-05-04 | 南方医科大学 | Novel snake venom polypeptide, preparation and application thereof |
| CN101698656B (en) * | 2009-11-05 | 2011-12-14 | 青岛成功精细化工有限公司 | Method for catalytic synthesis of L-pyrroldone carboxylic acid and salt thereof |
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