CN107236031A - A kind of biologically active polypeptide PMIGVNQELAY and its preparation method and application - Google Patents
A kind of biologically active polypeptide PMIGVNQELAY and its preparation method and application Download PDFInfo
- Publication number
- CN107236031A CN107236031A CN201710546491.3A CN201710546491A CN107236031A CN 107236031 A CN107236031 A CN 107236031A CN 201710546491 A CN201710546491 A CN 201710546491A CN 107236031 A CN107236031 A CN 107236031A
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- China
- Prior art keywords
- pmigvnqelay
- biologically active
- active polypeptide
- polypeptide
- derivative
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The present invention relates to albumen field, and in particular to its amino acid sequence of a kind of biologically active polypeptide PMIGVNQELAY and its preparation method and application, biologically active polypeptide PMIGVNQELAY is Pro Met Ile Gly Val Asn Gln Glu Leu Ala Tyr.Tested by antioxidation in vitro, ion vitro immunization function promotes experiment, demonstrating polypeptide PMIGVNQELAY has preferable antioxidation biology activity and immunoloregulation function, on the one hand there is preferable antioxidation activity, free radical that can be in removing machine body improves the quality of life；On the other hand, the biologically active polypeptide PMIGVNQELAY of the present invention can strengthen the in-vitro multiplication ability of lymphocyte and macrophage, promote macrophage phagocytosis dimethyl diaminophenazine chloride ability, improve the ability that body resists extraneous pathogenic infection, the body incidence of disease is reduced, exploitation is of great significance with immunoloregulation function, the food of anti-oxidation function, health products and medicine tool.
Description
Technical field
The present invention relates to albumen field, more particularly, to a kind of biologically active polypeptide PMIGVNQELAY and preparation method thereof
And application.
Background technology
During cow's milk is through lactobacillus-fermented, a part of protein in cow's milk is metabolized by lactic acid bacteria to be utilized, concurrently
A series of biochemical reactions have been given birth to, protein is changed into polypeptide or free amino acid, is digested or passes through
The absorption and transport of intestinal epithelial cell is directly entered the blood circulation of human body.In these polypeptides, some has special
Physiological function, is referred to as " biologically active peptide ".
Oxidation reaction and oxidative metabolism are all vital for food and human body, and free radical and active oxygen cause
A series of oxidation reaction.When excessive free radical is formed, they can exceed protective enzyme such as superoxide dismutase, peroxide
Change the protective effect of hydrogen enzyme, so as to cause a series of side effect such as lipid oxidation, Apoptosis to produce.The oxidation of this class is anti-
Should, the shelf-life of the food containing fat is not only influenceed, also the health to human body causes certain harm, such as rheumatic arthritis, sugar
Urinate disease, artery sclerosis etc..In addition, Collins et al. researchs in 2005 find that oxidative damage of the generation of cancer also with DNA has
Close.
Some artificial synthesized antioxidant such as butylated hydroxy anisoles (BHA), 2,6- di-t-butyl -4- methylbenzenes in early days
Phenol (BHT) is applied in food, as the antioxidant of lipid, but these artificial synthesized additives have latent for human body
Risk.Therefore, safe antioxidant is found in natural food source particularly important.In the last few years, it has been found that one
The polypeptides matter of a little food sources has good antioxidation, such as corn small peptide, Soybean Peptide, cow's milk polypeptide.These
Polypeptide can be obtained by number of ways such as microbial fermentation, digestion enzymolysis, and the polypeptide with antioxidation activity is mostly
It is made up of 2~20 amino acid residues, molecular weight is less than 6000Da, contains a certain amount of hydrophobic amino acid, aromatic series amino
Acid.
Immune-active peptides are to obtain and prove that the class biology of its physiologically active is living from breast first after opioid peptides is found
Property polypeptide.Jolles in 1981 et al. has found first, using trypsin hydrolysis people lactoprotein, can obtain an amino acid sequence
Val-Glu-Pro-Ile-Pro-Tyr hexapeptide is classified as, experiment in vitro proves that the peptide can strengthen Turnover of Mouse Peritoneal Macrophages pair
The phagocytosis of sheep red blood cell (SRBC).Migliore-Samour et al. has found the hexapeptide Thr-Thr-Met-Pro- from casein
Leu-Trp can stimulate sheep erythrocyte to the phagocytosis of mouse peritoneal macrophages and strengthen for kerekou pneumonia primary
The resistance of bacterium.Li Su duckweeds et al. find that rat abdominal cavity macrophage is thin with newborn source immunomodulatory peptides (PGPIPN) the feeding rat of synthesis
The immunoloregulation function that the phagocytosis of born of the same parents is related to red blood cell has significant enhancing.
Research shows that immune-active peptides can not only strengthen immunity of organisms, stimulates the propagation of body lymphocyte, enhancing
The phagocytic function of macrophage, promotes the release of cell factor, improves the ability that body resists extraneous pathogenic infection, reduce machine
The body incidence of disease, and the immunological rejection of body will not be caused.
The research on biologically active polypeptide has much at present, such as Chinese patent CN105254738A discloses a kind of next
Milk-derived the biologically active polypeptide DELQDKIH, Chinese patent CN105254739A for coming from beta-casein disclose a kind of source
Disclose a kind of from α in milk-derived the biologically active polypeptide GTQYTD, Chinese patent CN105254740A of α s1- caseins
The milk-derived biologically active polypeptide NQFYQKF of s2- caseins.
The content of the invention
It is an object of the invention to provide a kind of biologically active polypeptide PMIGVNQELAY and its preparation method and application.
The purpose of the present invention can be achieved through the following technical solutions：
First aspect present invention is there is provided a kind of biologically active polypeptide PMIGVNQELAY, and its amino acid sequence is Pro-
Met-Ile-Gly-Val-Asn-Gln-Glu-Leu-Ala-Tyr, such as SEQ ID NO：Shown in 1.
Preferably, the biologically active polypeptide is milk-derived.α s1- caseins are derive specifically from, and are α s1- junket eggs
The amino acid residue that Bai BiantiD is the 149th~159.α s1- ss-casein variants D amino acid sequences such as SEQ ID NO：Shown in 3.
The amino acid sequence and corresponding nucleotides sequence of α s1- caseins are classified as existing technology, and coding for alpha s1- caseins become
The biologically active polypeptide PMIGVNQELAY of the nucleotide fragments energy encoding mature of the amino acids residues of body D the 149th~159.
Preferably, the biologically active polypeptide has anti-oxidation function and immunoloregulation function.
There is provided the nucleotide fragments for encoding the biologically active polypeptide PMIGVNQELAY, its sequence for second aspect of the present invention
It is classified as：5 '-cct atg ata gga gtg aat cag gaa ctg gcc tac-3 ', such as SEQ ID NO：Shown in 2.
Third aspect present invention can pass through base there is provided the preparation method of the biologically active polypeptide PMIGVNQELAY
It because the method for engineering is artificial synthesized, can be directly obtained from dairy products by the method isolated and purified, can directly pass through and change
Learn synthetically prepared.
Fourth aspect present invention is being prepared with anti-oxidation function there is provided the biologically active polypeptide PMIGVNQELAY
Food, health products, medicine or cosmetics in application.
Fifth aspect present invention is being prepared with immunological regulation work(there is provided the biologically active polypeptide PMIGVNQELAY
Application in food, health products or the medicine of energy.
Sixth aspect present invention is being prepared while having anti-oxidant there is provided the biologically active polypeptide PMIGVNQELAY
Application in food, health products or the medicine of function and immunoloregulation function.
Specifically, biologically active polypeptide PMIGVNQELAY of the invention, which can be used for preparing, reduces free radical to skin
The cosmetics of injury, preparation have anti-oxidant and/or regulation immunity of organisms medicine；And due to the bioactivity of the present invention
Product after polypeptide PMIGVNQELAY is degraded by intestines and stomach still has bioactivity, therefore can be also used for preparing Yoghourt
There is medicine that is anti-oxidant and/or adjusting immunity of organisms Deng food, the health products of regulation immunity, and the oral preparation that is used for
Thing.
There is provided a kind of oxidation resistant product, including the biologically active polypeptide PMIGVNQELAY for seventh aspect present invention
Or the derivative of the biologically active polypeptide PMIGVNQELAY；Described oxidation resistant product includes antioxidant food, anti-oxidant guarantor
Strong product, anti-oxidation medicine or antioxidation cosmetic product；The derivative of the biologically active polypeptide PMIGVNQELAY, refers in biology
On active peptides PMIGVNQELAY amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation,
Methylate, acetylation, phosphorylation, esterification or glycosylation etc. modification, obtained polypeptide derivative.
There is provided a kind of immunological regulation product, including the biologically active polypeptide for eighth aspect present invention
PMIGVNQELAY or described biologically active polypeptides PMIGVNQELAY derivative；Described immunological regulation product includes immune tune
Go on a diet product, immunological regulation health products or immunoregulation medicament；The derivative of the biologically active polypeptide PMIGVNQELAY, refers to
On biologically active polypeptide PMIGVNQELAY amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated,
Be carbonylated, methylate, acetylation, phosphorylation, esterification or glycosylation etc. modification, obtained polypeptide derivative.
Ninth aspect present invention there is provided a kind of while have the product of anti-oxidation function and immunoloregulation function, including
The derivative of the biologically active polypeptide PMIGVNQELAY or described biologically active polypeptides PMIGVNQELAY；With anti-oxidant work(
Food, health products or medicine can be included with the product of immunoloregulation function；The derivative of the biologically active polypeptide PMIGVNQELAY
Thing, refer on biologically active polypeptide PMIGVNQELAY amino acid side groups, aminoterminal or c-terminus carry out hydroxylating,
Carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification or glycosylation etc. modification, obtained polypeptide derivative.
Biologically active polypeptide PMIGVNQELAY's of the present invention has the beneficial effect that：The milk-derived biologically active polypeptide of the present invention
PMIGVNQELAY has preferable antioxidation activity and regulation immunity of organisms activity；On the one hand can be in removing machine body from
By base, injury of the free radical to human body is reduced；On the other hand, biologically active polypeptide PMIGVNQELAY of the invention can also be adjusted
Immunity of organisms is saved, strengthens the multiplication capacity of lymphocyte, the ability that body resists extraneous pathogenic infection is improved, body is reduced
The incidence of disease, and the immunological rejection of body will not be caused, the breast to developing with anti-oxidation function and adjusting immunologic function
Product and health products tool are of great significance.
Brief description of the drawings
Fig. 1：Mass chromatography extracts figure (m/z=617.81)；
Fig. 2：Mass-to-charge ratio is the first mass spectrometric figure of 617.81 fragment；
Fig. 3：Mass-to-charge ratio is 617.81 polypeptide az, by crack conditions；
Fig. 4：[DPPH] methanol standard curve；
Fig. 5：FeSO4Standard curve.
Embodiment
Before specific embodiments of the present invention are further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range
Any one numerical value can select between point and two end points.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except the specific method used in embodiment, equipment,
Outside material, according to those skilled in the art to the grasp of prior art and the record of the present invention, it can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Unless otherwise indicated, disclosed in this invention experimental method, detection method, preparation method using this technology lead
Domain conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆ Sons, New York, 1987 and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
The active peptide PMIGVNQELAY's of embodiment 1 is artificial synthesized
First, the synthesis of biologically active peptide
1. RINK resin 3g (substitution value 0.3mmol/g) are weighed in 150ml reactor, with 50ml dichloromethane
(DCM) soak.
After 2.2 hours, resin is washed with nitrogen-dimethylformamide (DMF) of 3 times of resin volumes, is then drained, so weight
It is multiple four times, resin is drained rear stand-by.
3. a certain amount of 20% piperidines (piperidines/DMF=1 is added into reactor:4,v:V), it is placed on decolorization swinging table and shakes
20min is shaken, the Fmoc blocking groups on resin are sloughed with this.Take off after protection and to have been washed four times with the DMF of 3 times of resin volumes,
Then drain.
4. take the detection of a small amount of resin ninhydrin (nine well ninhydrins) method (each two drop of inspection A, inspection B, 100 DEG C of reactions
1min), resin has color, illustrates to be deprotected successfully.
5. weighing, amino acid Pro is appropriate and the parallel triazole (HOBT) of 1- hydroxyls-benzene is appropriate in 50ml centrifuge tube, adds
20ml DMF is dissolved, and then adds 3ml N, and N DICs (DIC) vibration shakes up 1min, treats that solution is clear
It is added to after clear in reactor, then reactor is placed in 30 DEG C of shaking table and reacted.
After 6.2 hours, with a certain amount of acetic anhydride end socket (acetic anhydride:DIEA:DCM=1:1:2,v:v:V) half an hour, so
Washed four times, drained stand-by with the DMF of 3 times of resin volumes afterwards.
7. a certain amount of 20% piperidines (piperidines/DMF=1 is added into reactor:4, v:V), it is placed on decolorization swinging table and shakes
20min is shaken, the Fmoc blocking groups on resin are sloughed with this.Take off and washed after protection with DMF four times, then drained.
8. take the detection of a small amount of resin ninhydrin (nine well ninhydrins) method (each two drop of inspection A, inspection B, 100 DEG C of reactions
1min), resin has color, illustrates to be deprotected successfully.
9. weighing, second amino acid next is appropriate and HOBT is appropriate in 50ml centrifuge tube, and the DMF for adding 25ml will
It dissolves, and the DIC vibrations for then adding 2.5ml shake up 1min, are added to after after solution clarification in reactor, then by reactor
It is placed in 30 DEG C of shaking table and reacts.
After 10.1 hours, take a small amount of resin to detect, (each two drop of inspection A, inspection B, 100 DEG C of reactions are detected with ninhydrin method
1min), if resin is colourless, illustrate that reaction is complete；If resin has color, illustrate that condensation is incomplete, continue to react.
11. after question response is complete, resin is washed with DMF four times, then drain, a certain amount of 20% is added into reactor
Piperidines (piperidines/DMF=1:4,v:V), it is placed on decolorization swinging table and rocks 20min, the Fmoc protection groups on resin is sloughed with this
Group.Take off and washed after protection with DMF four times, then drained whether detection protection sloughs.
12. according to step 9-11 connect successively amino acid Met, Ile, Gly, Val, Asn, Gln, Glu, Leu, Ala and
Tyr。
13. after last amino acid is connected, sloughing protection, washed four times, then taken out resin with methanol with DMF
It is dry.Then with 95 cutting liquid (trifluoroacetic acids:1,2 dithioglycols:3, isopropyl base silane:Water=95:2:2:1, v:v:V) will be many
Peptide is cut down (every gram of resin adds 10ml cutting liquids) from resin, and with ice ether (cutting liquid：Ether=1:9,v:V) centrifuge
Sedimentation four times.
So far, artificial synthesized biologically active peptide PMIGVNQELAY.
Referring in right amount described in above step 5, step 9 calculates a theoretical use according to target synthetic quantity and yield
Amount, is multiplied by a coefficient (being 1.1 in the present embodiment), the actual amount finally obtained on the basis of theoretical amount.
Because the synthetic quantity of each target peptide is different, and yield is also different, so the amount of the amino acid weighed every time
It is just different.
The synthetic quantity of such as target peptide is 10 grams, and the yield (rate of recovery) of synthetic peptide is 90%, then the theory of amino acid
The calculation formula for the amount of weighing is：
The theory amount of weighing=10 gram * amino acid moleculars amount/target peptide molecular weight/90%. of amino acid
The result so calculated is the theory amount of weighing of amino acid, in order to ensure to obtain in actual building-up process
10 grams of polypeptide, amino acid will be weighed a little more, and the amino acid amount of weighing is typically all the 1.1 of the theoretical amount of weighing during practical operation
Times.
Similarly, the parallel triazole (HOBT) of 1- hydroxyls-benzene, as an intermediary during Peptide systhesis, is also that theory is weighed
1.1 times of amount.
Due to amino acid used in biologically active peptide building-up process and the theoretical amount of the parallel triazole (HOBT) of 1- hydroxyls-benzene
It is that those skilled in the art can calculate according to the demand of target synthetic peptide and obtain, therefore, during practical operation, in theory use
A coefficient is multiplied by the basis of amount can just synthesize target peptide, in the synthesis step and synthesis technique with reference to the present embodiment
On conditioned basic, the biologically active peptide obtained in the present embodiment can be just synthesized according to the Conventional wisdom of those skilled in the art.
2nd, the confirmation of biologically active peptide
1) UPLC is analyzed
UPLC conditions are as follows：
Instrument：Waters ACQUITY UPLC ultra high efficiency liquid phase-electron spray-level Four bar-time of-flight mass spectrometer
Chromatographic column specification：BEH C18 chromatographic columns
Flow velocity：0.4mL/min
Temperature：50℃
Ultraviolet detection wavelength：210nm
Sample size：2μL
Gradient condition：A liquid：Water containing 0.1% formic acid (v/v), B liquid：Acetonitrile containing 0.1% formic acid (v/v)
2) mass spectral analysis
Mass Spectrometry Conditions are as follows：
Ionic means：ES+
Mass range (m/z)：100-1000
Capillary voltage (Capillary) (kV)：3.0
Sampling spiroid (V)：35.0
Ion source temperature (DEG C)：115
Remove solvent temperature (DEG C)：350
Go solvent stream (L/hr)：700.0
Collision energy (eV)：4.0
Sweep time (sec)：0.25
Interior sweep time (sec)：0.02
According to above analysis method, using ultra high efficiency liquid phase-electron spray-level Four bar-flight time mass spectrum, to bioactivity
Peptide PMIGVNQELAY carries out chromatography and mass spectral analysis, and its mass chromatography extraction figure is as shown in figure 1, extract the one-level at this peak
With second order mses figure as shown in Figures 2 and 3, the polypeptide mass-to-charge ratio that can obtain this peak is 617.81Da, and retention time is 13.38min.
3) result
From the figure 3, it may be seen that situation about being broken according to az, by, calculates by Progenesis QI software analysis, obtains matter lotus
Fragment sequence than 617.81Da is Pro-Met-Ile-Gly-Val-Asn-Gln-Glu-Leu-Ala-Tyr
(PMIGVNQELAY) SEQ ID NO, are designated as：1.The fragment and α s1- ss-casein variants D the 149th~159 residue sequence
Corresponding, the GenBank numberings of α s1- casamino acid sequences are AAA30428.1, and sequence is shown in SEQ ID NO：3.
The antioxidation activity experiment of the biologically active peptide of embodiment 2
Using removing free radical method (DPPH methods) and TAC method (Ferric Reducing Ability
Power FRAP methods), the biologically active polypeptide PMIGVNQELAY obtained to embodiment 1 antioxidation activity is tested.
1st, [DPPH] method determines biologically active peptide PMIGVNQELAY antioxidation activity in vitro
1) experiment reagent and instrument
Reagent：1,1- diphenyl -2- trinitrophenyl-hydrazines (1,1-Diphenyl-2-picrylhydrazyl [DPPH]),
Japanese Wako companies production；Methanol, Shanghai traditional Chinese medicines company provides；The milk-derived biologically active polypeptide that embodiment 1 is obtained
PMIGVNQELAY。
Key instrument：Sunrise ELIASAs, Austrian Tecan Products；96 porocyte culture plates, the U.S.
Millipore companies manufacture；Assay balance, Meitelei-tolido Products.
2) experimental method
(1) 1mmol/L [DPPH] methanol solution
0.349mg [DPPH] is weighed with assay balance to be dissolved in 1mL methanol solutions, prepares obtained 1mmol/L
[DPPH] methanol solution, tinfoil is kept in dark place, i.e., with i.e. use.
(2) measure of [DPPH] methanol standard curve
100 μ L [DPPH] methanol standard curve samples are separately added into by table 1 in 96 orifice plates, 90min is stored at room temperature,
Light absorption value is detected at 517nm with ELIASA.
[DPPH] methanol of table 1 calibration curve solution is prepared
According to experimental result, using Excel matched curves and regression equation is calculated, as a result see Fig. 4 (regression equations：Y=-
0.192x+0.2271, R2=0.9991).The linear relationship of [DPPH] methanol standard curve is good, and coefficient correlation is 0.999,
Show that [DPPH] methanol standard curve preci-sion and accuracy meets detection and required.In terms of result, absorbance with
[DPPH] content is in inverse relation, and [DPPH] content is fewer, and light absorption value is higher, i.e. the ability of sample removing free radical is got over
By force.
(3) [DPPH] method determines biologically active peptide PMIGVNQELAY antioxidation activity
1) sample sets：80 μ L concentration are added in 96 orifice plates for 1mmol/L [DPPH] methanol solution, by table 2 respectively to add
Enter the testing sample (PMIGVNQELAY), positive control 1 (2.5mg/mL Trolox), positive control 2 of 20 μ L various concentrations
(0.025mg/mL Trolox), and negative control (phytic acid)；
2) blank group：On same 96 orifice plate, to add 80 μ L concentration as 1mmol/L [DPPH] methanol solutions and 20 μ L
The sample of deionized water does blank control.
After detected sample sample-adding is finished, 90min is stored at room temperature, light absorption value is detected at 517nm with ELIASA.Under
Formula calculates free radical scavenging activity, and experimental result is shown in Table 2.
Formula：
Table 2 [DPPH] method determines the antioxidation activity result of biologically active polypeptide
From table 2 it can be seen that having under the same conditions as the 2.5mg/mL of positive control Trolox most strong clear
Except the ability of free radical, free radicals all in solution can be almost removed, are secondly 0.025mg/mL Trolox, phytic acid, work
Property polypeptide.It is 30.27% that polypeptide PMIGVNQELAY, which removes [DPPH] free radical rate, and with PMIGVNQELAY concentration
Reduction, Scavenging ability weakens.
2nd, FARP methods determine biologically active peptide PMIGVNQELAY antioxidant activity in vitro
1) experiment reagent and instrument
TAC detection kit (Ferric Reducing Ability of Plasma FRAP methods), is purchased from
The green skies biotechnology company in Shanghai；FeSO4Solution (10mmol/L), watermiscible vitamin E (Trolox solution) (10mmol/
L), the milk-derived biologically active polypeptide PMIGVNQELAY that embodiment 1 is obtained.
Key instrument：Sunrise ELIASAs, Austrian Tecan Products；96 porocyte culture plates, the U.S.
Millipore companies manufacture；Assay balance, Meitelei-tolido Products；HWS26 type electric-heated thermostatic water baths, Shanghai
One permanent Science and Technology Ltd.'s manufacture.
2) experimental method
(1) preparation of FRAP working solutions
According to TAC detection kit, TPTZ 7.5mL dilutions, the μ L solution of TPTZ 750, detection are buffered
The μ L of liquid 750 are well mixed, and are incubated in 37 DEG C of water-baths, are finished in 2 hours h.
(2)FeSO4The making of standard curve curve is determined
180 μ LFRAP working solutions are first added in 96 orifice plates, 5 μ L FeSO are added by table 34Calibration curve solution, is gently mixed
Even, 37 DEG C are incubated after 3-5min, and light absorption value is determined at 593nm with ELIASA.
Table 3FeSO4The solution of standard curve determination is prepared
FeSO4Concentration is in good proportional relation, FeSO with light absorption value4Concentration is higher, and light absorption value is higher.FeSO of the present invention4
Standard curve result is shown in Fig. 5, and the linear relationship of standard curve is good, and coefficient correlation is 0.998, FeSO4The precision of standard curve
Degree and the degree of accuracy meet detection and required, available for follow-up calculating.
(3) FRAP methods determine biologically active polypeptide PMIGVNQELAY oxidation resistance
First added in 96 orifice plates in 180 μ L FRAP working solutions, blank control wells and add 5 μ L ddH2O, sample detection hole
5 μ L phytic acid are added in 5 μ L testing samples of interior addition, positive control, are gently mixed, 37 DEG C are incubated after 3-5min, are existed with ELIASA
Light absorption value is determined at 593nm.TAC representation is with FeSO4The concentration of standard liquid is represented.Count according to the following formula
Free radical scavenging activity is calculated, experimental result is shown in Table 4.
Table 4FARP methods determine biologically active polypeptide PMIGVNQELAY TAC result
By TAC method (Ferric Reducing Ability Power FRAP methods) to polypeptide
PMIGVNQELAY external total antioxidant activity is determined, it is found that biologically active polypeptide PMIGVNQELAY has preferable
Reduction-oxidation material ability；In the case of concentration is 4mg/mL, polypeptide PMIGVNQELAY total antioxidation level reaches
0.0222mmol/g；Illustrate biologically active polypeptide PMIGVNQELAY TAC, higher than having under comparable sodium
The phytic acid of weak antioxidation activity, with conspicuousness (p>0.05) difference.Therefore, the biologically active polypeptide of invention can be assert
PMIGVNQELAY has significant oxidation resistance.
The promotion immunity of organisms activity experiment of the biologically active peptide of embodiment 3
First, mtt assay determines biologically active polypeptide PMIGVNQELAY vitro lymphocyte proliferation capacity experimental
1) experiment material and instrument：
Reagent and material：Experimental animal balb/c mouse (male 6-8 week old, Shanghai Communications University's agricultural and biological institute
Animal experimental center)；The milk-derived biologically active polypeptide PMIGVNQELAY that embodiment 1 is obtained；Mouse lymphocyte extract solution
(being purchased from Suo Laibao companies)；RPMI1640 culture mediums (are purchased from GIBCO companies)；3- (4,5- dimethylthiazole -2) -2,5- hexichol
Base tetrazole bromide (MTT, purchased from Amresco companies)；ConA (ConA, purchased from Sigma companies)；Bovine serum albumin(BSA)
(BSA, purchased from Genebase companies)；Pepsin (is purchased from Sigma companies)；Pancreatin (Corolase PP, purchased from AB companies).
Instrument：LRH-250F biochemical cultivation cases, Shanghai perseverance Science and Technology Ltd.；GL-22M high speed freezing centrifuges, Shanghai
Lu Xiang instrument centrifuges Instrument Ltd.；The CO2 incubators of Hera cell 150, Heraeus companies； Dragon Wellscan
MK3 ELIASAs, Labsystems companies；ALPHA 1-2-LD vacuum freeze driers, Christ companies；Ultra high efficiency liquid phase color
Spectrum-quadrupole rod time of-flight mass spectrometer, waters companies.
2) experimental method：
Mouse spleen is taken under aseptic condition, mouse lymphocyte is extracted with lymphocyte extract solution, Yuan Dynasty's culture is carried out.With
Cell density is adjusted to 2.5 × 10 by complete RPMI1640 nutrient solutions6Individual/mL.Sequentially added in 96 porocyte culture plates：
100 μ L mouse lymphocyte suspensions, 100 μ L RPMI1640 complete culture solutions, 20 μ L ConAs, 100 μ L samples.Separately
Outside, blank control group (pH7.2~7.4,3mol/L PBS) and negative control group (500 μ g/mL BSA) are set, and research shows
It does not influence for vitro lymphocyte proliferation.Every group of 3 parallel laboratory test samples.In 5%CO268h is cultivated in 37 DEG C of incubators
Afterwards, 20 μ L MTT are added under aseptic condition per hole, continue to cultivate 4h, careful abandoning supernatant adds 100 μ L dimethyl per hole
Sulfoxide, 37 DEG C of biochemical cultivation cases hatch 10min, shake up, light absorption value is determined at 570nm with ELIASA.
Vitro lymphocyte proliferation ability represents that computational methods are as follows with stimulus index：
In formula：A1For blank control at the 570nm under light absorption value；A2For negative control group at the 570nm under extinction
Value, A3For experimental group at the 570nm under light absorption value.
3) experimental result and analysis
Experimental result is shown in Table 5.As shown in Table 5, it is 100 μ g/mL in biologically active peptide PMIGVNQELAY mass concentration
Under conditions of, milk-derived biologically active peptide PMIGVNQELAY stimulus index is more than BSA, illustrates PMIGVNQELAY to a certain degree
It is upper to stimulate the propagation of external mouse lymphocyte.And PMIGVNQELAY stimulus index has reached 1.191, and feminine gender is right
There is significant difference (P according to group<0.05).It therefore, it can assert that active peptides PMIGVNQELAY drenches with mouse is remarkably promoted
The ability of bar cell propagation, can be edible as a kind of health products or additive, it is possible to increase the immunity of animal and human body.
Influences of the biologically active polypeptide PMIGVNQELAY of table 5 to vitro lymphocyte proliferation
Note：* labelled notation has significant difference (P ＜ 0.05) to be compared with negative control.
2nd, mtt assay determines biologically active polypeptide PMIGVNQELAY macrophages in vitro multiplication capacity experiment
1) experiment reagent and instrument
Reagent：Experimental animal balb/c mouse (male 6-8 week old) Shanghai Communications University agricultural and biological institute animal reality
Test center；The milk-derived biologically active polypeptide PMIGVNQELAY that embodiment 1 is obtained；3- (4,5- dimethylthiazole -2) -2,5- bis-
Phenyl tetrazole bromide (MTT) Amresco companies；LPS (lipopolysaccharides) Sigma companies；Bovine serum albumin(BSA) (Bovine
Serum Albumin, BSA) Genebase companies；Three lysates, containing 10%SDS, 5% isobutanol and 0.012mol/L
The HCl aqueous solution.
Instrument and equipment：LRH-250F biochemical cultivation cases Shanghai perseverance Science and Technology Ltd.；On GL-22M high speed freezing centrifuges
Hai Luxiang instrument centrifuges Instrument Ltd.；Hera cell 150 CO2Incubator Heraeus companies； Dragon
Wellscan MK3 ELIASA Labsystems companies.
2) test method：
Balb/c mouse peritoneal injections 2ml 2% (w/w) sterilizing starch solutions, continuous injection three days, last time is injected
The neck that breaks after 24 hours is put to death.Skin of abdomen is peelled off, 4 DEG C of phosphate buffers (PBS) is drawn with syringe and rinses abdominal cavity repeatedly,
Centrifuge tube is collected after flushing liquor, and centrifugation (1000rpm, 4 DEG C) abandons supernatant after 10 minutes, (is contained with 4 DEG C of RPMI1640 complete culture solutions
10%FBS) wash twice, cell viability examination is done in the dyeing of 0.2% trypan blue solution, confirm the vibrant macrophage collected
Account for more than 95%.After cell counting count board reading, adjustment cell concentration to suitable concn.
It will blow and beat to the cell suspension suspended completely with suitable volumes 96 porocyte culture plates of addition, 37 DEG C, 5%CO2
After being cultivated 4 hours under environment, liquid in hole is abandoned in suction, and cell culture plate well is carefully cleaned with 37 DEG C of RPMI1640 complete culture solutions
Bottom, washes away not adherent cell and cell fragment, obtains adherent peritoneal macrophage after purification.0.2ml is added per hole
RPMI1640 complete mediums, experiment is dissolved in after culture medium and added in advance with small peptide sample and LPS, starts cell culture.
Obtain after adherent peritoneal macrophage after purification, experimental group adds per hole is dissolved with biologically active polypeptide LPLP
The μ l/ holes of RPMI1640 complete culture solutions (10%FBS) 200 of (1mg/ml), continuously cultivate 48h；Negative control group is per hole solubilization
Solution has BSA (500 μ g/mL) the μ l/ holes of RPMI1640 complete culture solutions (10%FBS) 200；Blank group addition RPMI1640 is complete
The μ l/ holes of full nutrient solution (10%FBS) 200, continuously cultivate 48h.Also, experimental group, negative control group and blank group are set respectively again
Normal group and inflammation group；Inflammation group adds LPS to final concentration of 100ng/ml when 24h is arrived in culture；Normal group is not added with LPS；And
And normal group and inflammation group add the μ l/ holes of 5%MTT 20 in 44h；Cell culture, which reaches, adds the three of 100 μ l/ holes after 48h
Lysate is to terminate culture, after dissolving overnight, surveys the absorbance (OD570) in each hole, growth with ELIASA under wavelength 570nm
The calculation formula of index (Growth Indices) is as follows：
Wherein, blank nutrient solution is the RPMI1640 complete culture solutions containing 10%FBS.
3) experimental result and analysis
Experimental result is shown in Table 6, as shown in Table 6, under conditions of addition 1mg/ml biologically active polypeptides PMIGVNQELAY,
The macrophage of normal group and inflammation group has propagation.And compared with negative control group, there are significant difference (P ＜
0.01).Illustrate that biologically active polypeptide PMIGVNQELAY has significant proliferation function to macrophages in vitro.
The influence that the biologically active polypeptide PMIGVNQELAY of table 6 breeds to macrophages in vitro
| Experiment packet | Normal group GI | Inflammation group GI |
| Negative control group | 1 | 1 |
| PMIGVNQELAY(1mg/ml) | 1.0936±0.0327** | 1.163±0.0564** |
Note：* represent to be compared with negative control, there is significant difference (P ＜ 0.05)；* represents to be compared with negative control group,
There is significant difference (P ＜ 0.01)
3rd, biologically active polypeptide PMIGVNQELAY rush macrophage phagocytosis dimethyl diaminophenazine chloride capacity experimental
1) experiment reagent and instrument
Reagent：Experimental animal balb/c mouse (male 6-8 week old) Shanghai Communications University agricultural and biological institute animal reality
Test center；The milk-derived biologically active polypeptide PMIGVNQELAY that embodiment 1 is obtained；LPS, purchased from Sigma companies；Dimethyl diaminophenazine chloride contaminates
Color liquid, green skies biotechnology research institute production.
Instrument and equipment：LRH-250F biochemical cultivation cases Shanghai perseverance Science and Technology Ltd.；On GL-22M high speed freezing centrifuges
Hai Luxiang instrument centrifuges Instrument Ltd.；The CO2 incubator Heraeus companies of Hera cell 150； Dragon
Wellscan MK3 ELIASA Labsystems companies.
2) test method：
It is 2 × 10 to add number of cells6/ ml μ l/ the holes of cell suspension 100, adherent add after purification contains active peptide
PMIGVNQELAY (1mg/ml) the μ l/ holes of RPMI1640 complete culture solutions (10%FBS) 200 are experimental group, and addition is without activity
What the μ l/ holes of RPMI1640 complete culture solutions (10%FBS) 200 of peptide were cultivated is set to blank group；And experimental group and blank
Group adds LPS to the μ g/ml of final concentration 10 when 24h is arrived in culture；Continue to cultivate to 48h, cell culture fluid is abandoned in suction.PBS
37 DEG C of μ l/ holes of dimethyl diaminophenazine chloride dye liquor 80 are added after bottom hole, is inhaled after 10 minutes and abandons dye liquor, with PBS twice after, added per hole
150 μ l cell pyrolysis liquid (glacial acetic acid：Absolute ethyl alcohol=1:1, v/v).After 4 DEG C of dissolvings overnight, extinction is determined under wavelength 540nm
Angle value (OD540).
3) experimental result and analysis：
The biologically active polypeptide PMIGVNQELAY of table 7 promotees the measure that macrophage swallows dimethyl diaminophenazine chloride ability
| Experiment packet | Inflammation group absorbance (OD540) |
| Blank group | 0.1081±0.0374 |
| Experimental group | 0.1423±0.0474** |
Note：*, compared with negative control, there is significant difference (P ＜ 0.05)
*, is compared with negative control group, there is significant difference (P ＜ 0.01)
Experimental result is shown in Table 7, compared with cell blank, addition 1mg/ml biologically active polypeptides PMIGVNQELAY inflammation
Disease group macrophage phagocytosis dimethyl diaminophenazine chloride ability substantially increases, and is compared with cell blank group, with significant difference (P ＜
0.01).Illustrate that biologically active polypeptide PMIGVNQELAY swallows dimethyl diaminophenazine chloride in the case where there is inflammation generation to macrophages in vitro
Ability has significant facilitation.
The above-mentioned description to embodiment is understood that for ease of those skilled in the art and using invention.
Person skilled in the art obviously can easily make various modifications to these embodiments, and described herein general
Principle is applied in other embodiment without passing through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability
Field technique personnel are according to the announcement of the present invention, and not departing from improvement and modification that scope made all should be the present invention's
Within protection domain.
<110>Zhejiang Hui Tai life and healths Science and Technology Ltd.
<120>A kind of biologically active polypeptide PMIGVNQELAY and its preparation method and application
<160>3
<170> PatentIn version 3.3
<210> 1
<211> 11
<212> PRT
<213>Artificial sequence
<220>
<223>Biologically active polypeptide
<400> 1
Pro Met Ile Gly Val Asn Gln Glu Leu Ala Tyr
5 10
<210> 2
<211> 33
<212> DNA
<213>Artificial sequence
<220>
<223>Biologically active polypeptide coded sequence
<400> 2
cctatgatag gagtgaatca ggaactggcc tac 33
<210> 3
<211> 214
<212> PRT
<213>Artificial sequence
<220>
<223>α s1- ss-casein variants D amino acid sequences
<400> 3
<400> 3
Met Lys Leu Leu Ile Leu Thr Cys Leu Val Ala Val Ala Leu Ala
5 10 15
Arg Pro Lys His Pro Ile Lys His Gln Gly Leu Pro Gln Glu Val
20 25 30
Leu Asn Glu Asn Leu Leu Arg Phe Phe Val Ala Pro Phe Pro Glu
35 40 45
Val Phe Gly Lys Glu Lys Val Asn Glu Leu Ser Lys Asp Ile Gly
50 55 60
Ser Glu Ser Thr Glu Asp Gln Ala Met Glu Asp Ile Lys Gln Met
65 70 75
Glu Ala Glu Ser Ile Ser Ser Ser Glu Glu Ile Val Pro Asn Ser
80 85 90
Val Glu Gln Lys His Ile Gln Lys Glu Asp Val Pro Ser Glu Arg
95 100 105
Tyr Leu Gly Tyr Leu Glu Gln Leu Leu Arg Leu Lys Lys Tyr Lys
110 115 120
Val Pro Gln Leu Glu Ile Val Pro Asn Ser Ala Glu Glu Arg Leu
125 130 135
His Ser Met Lys Glu Gly Ile His Ala Gln Gln Lys Glu Pro Met
140 145 150
Ile Gly Val Asn Gln Glu Leu Ala Tyr Phe Tyr Pro Glu Leu Phe
155 160 165
Arg Gln Phe Tyr Gln Leu Asp Ala Tyr Pro Ser Gly Ala Trp Tyr
170 175 180
Tyr Val Pro Leu Gly Thr Gln Tyr Thr Asp Ala Pro Ser Phe Ser
185 190 195
Asp Ile Pro Asn Pro Ile Gly Ser Glu Asn Ser Glu Lys Thr Thr
200 205 210
Met Pro Leu Trp
Claims (10)
1. a kind of biologically active polypeptide PMIGVNQELAY, it is characterised in that its amino acid sequence is Pro-Met-Ile-Gly-
Val-Asn-Gln-Glu-Leu-Ala-Tyr。
2. a kind of biologically active polypeptide PMIGVNQELAY according to claim 1, it is characterised in that the bioactivity
Polypeptide is milk-derived.
3. encode the nucleotide fragments of biologically active polypeptide PMIGVNQELAY described in claim 1, it is characterised in that the core
The sequence of acid fragments such as SEQ ID NO：Shown in 2.
4. biologically active polypeptide PMIGVNQELAY as claimed in claim 1 preparation method, it is characterised in that pass through gene work
The method of journey is artificial synthesized, or is directly obtained from dairy products by the method isolated and purified, or directly passes through chemical synthesis system
It is standby.
5. biologically active polypeptide PMIGVNQELAY as claimed in claim 1 application, it is characterised in that the bioactivity is more
Applications of the peptide PMIGVNQELAY in food, health products, medicine or cosmetics with anti-oxidation function are prepared.
6. biologically active polypeptide PMIGVNQELAY as claimed in claim 1 application, it is characterised in that the bioactivity is more
Applications of the peptide PMIGVNQELAY in food, health products or medicine with immunoloregulation function is prepared.
7. biologically active polypeptide PMIGVNQELAY as claimed in claim 1 application, it is characterised in that the bioactivity is more
Applications of the peptide PMIGVNQELAY in food, health products or medicine with anti-oxidation function and immunoloregulation function is prepared.
8. a kind of oxidation resistant product, it is characterised in that including biologically active polypeptide PMIGVNQELAY as claimed in claim 1 or
The derivative of the biologically active polypeptide PMIGVNQELAY；Described oxidation resistant product includes antioxidant food, anti-oxidation health
Product, anti-oxidation medicine or antioxidation cosmetic product；The derivative of the biologically active polypeptide PMIGVNQELAY, refers to living in biology
Property polypeptide PMIGVNQELAY amino acid side groups on, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, first
Base, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
9. a kind of immunological regulation product, it is characterised in that including biologically active polypeptide PMIGVNQELAY as claimed in claim 1
Or the derivative of the biologically active polypeptide PMIGVNQELAY；
Described immunological regulation product includes immunological regulation food, immunological regulation health products or immunoregulation medicament；
The derivative of the biologically active polypeptide PMIGVNQELAY, refers to the amino acid in biologically active polypeptide PMIGVNQELAY
On side-chain radical, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification or
Polypeptide derivative that is glycosylation modified, obtaining.
10. a kind of product with anti-oxidation function and immunoloregulation function, it is characterised in that including as claimed in claim 1
Biologically active polypeptide PMIGVNQELAY or described biologically active polypeptides PMIGVNQELAY derivative；With anti-oxidation function and
The product of immunoloregulation function includes food, health products or medicine；The derivative of the biologically active polypeptide PMIGVNQELAY,
Refer on biologically active polypeptide PMIGVNQELAY amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxyl
Change, be carbonylated, methylating, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
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Denomination of invention: A bioactive polypeptide pmigvnqelay and its preparation method and Application Effective date of registration: 20210630 Granted publication date: 20200424 Pledgee: Zhejiang Cangnan rural commercial bank Limited by Share Ltd. Pledgor: ZHEJIANG HUITAI LIFE HEALTH TECHNOLOGY Co.,Ltd. Registration number: Y2021330000661 |