CN107746428A - A kind of biologically active polypeptide DERFFSDKIA and its preparation method and application - Google Patents
A kind of biologically active polypeptide DERFFSDKIA and its preparation method and application Download PDFInfo
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- CN107746428A CN107746428A CN201711250900.1A CN201711250900A CN107746428A CN 107746428 A CN107746428 A CN 107746428A CN 201711250900 A CN201711250900 A CN 201711250900A CN 107746428 A CN107746428 A CN 107746428A
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- Prior art keywords
- derffsdkia
- biologically active
- active polypeptide
- polypeptide
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Landscapes
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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 DERFFSDKIA and its preparation method and application, biologically active polypeptide DERFFSDKIA is Asp Glu Arg Phe Phe Ser Asp Lys Ile Ala.By the experiment of ion vitro immunization function point analysis, internal Antisenility Experiment, demonstrating polypeptide DERFFSDKIA has preferable immunoloregulation function and activity of fighting against senium, on the one hand, the biologically active polypeptide DERFFSDKIA of the present invention can strengthen the in-vitro multiplication ability of lymphocyte and macrophage, the ability that body resists extraneous pathogenic infection is improved, reduces the body incidence of disease；On the other hand, the vigor of internal anti-peroxidation enzyme system can be improved, strengthen the function of body resistance external source sexual stimulus, so as to reduce organism aging process, aging and sick probability, exploitation is of great significance with immunoloregulation function, the food of anti-senescence 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 DERFFSDKIA and preparation method thereof and
Using.
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, it is referred to as " biologically active peptide ".
It is particularly important that safe biologically active peptide is found in natural food source.In the last few years, it has been found that some foods
The polypeptides matter in thing source has good bioactivity, such as corn small peptide, Soybean Peptide, cow's milk polypeptide.These polypeptides can
To be obtained by number of ways such as microbial fermentation, digestion enzymolysis, and the polypeptide with bioactivity is by 2~20 mostly
Amino acid residue forms, and molecular weight is less than 6000Da, contains a certain amount of hydrophobic amino acid, aromatic amino acid.
Immune-active peptides are to obtain and prove that one kind biology of its physiologically active is living from breast first after opioid peptides discovery
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 phagocytosis and enhancing of the sheep erythrocyte to mouse peritoneal macrophages 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, promote the release of cell factor, improve 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.
Aging is a natural phenomena, and process is often accompanied by the change of antioxidant levels, organ-tissue, immune factor, its
The change of complexity, the trend that such as proinflammatory cytokine IL-6, IL-4, TNF-α presentation increase, IL-6 occur for middle cell factor
It is all considered to play an important role in the generating process of geriatric disease with TNF-a.With science of heredity and molecular biology
Development, the research of biological decay mechanism achieve gratifying progress.Researcher by using some model organisms, as mouse,
The term single gene mutating experiment of drosophila and C. Elegans Automatic Screening etc., it is found that some genes can dramatically increase life-spans of these organisms and reach
As many as 6 times.
Anti-aging peptide in terms of physiological function there is amino acid can not compare excellent as a kind of emerging antidotal agent
Gesture, it can produce promotion or inhibitory action to the enzyme in organism, improve absorption and the profit to mineral matter and other nutrients
With, removing interior free yl, the resistance to oxidation of enhancing body itself, with anti-aging.Therefore, the nutrition and health care of biologically active peptide
Effect has turned into the emphasis of domestic and foreign scholars subject study.Qiu Juan et al. pass through experimental studies have found that, milk-derived bioactive micro peptide
Life span of drosophila melanogaster can effectively be extended, delay its aging, and also there is preferable antioxidation, thus it is speculated that be probably wherein to be rich in coloured glaze
Base peptides.SOD vigor in serum, reduces its lipid in discovery bovine colostrum extract energy conspicuousness raising the elderly's body such as the brightness in week
Peroxide and enhancing body resistance to oxidation, have certain anti-senescence function.
The research on biologically active polypeptide has much at present, for example Chinese patent CN105254738A discloses one kind and come
Milk-derived the biologically active polypeptide DELQDKIH, Chinese patent CN105254739A for coming from beta-casein disclose a kind of source
In the milk-derived biologically active polypeptide GTQYTD of α s1- caseins, Chinese patent CN105254740A, which is disclosed, a kind of derives from α
The milk-derived biologically active polypeptide NQFYQKF of s2- caseins.
Chinese patent CN1566152A discloses a kind of immune-active peptides with antibacterial activity, and immune-active peptides are named as
" it is rich in tyrosine polypeptide (Try-rich polypeptide, be abbreviated as Trpi).Its amino acid sequence is： Cys-Glu-Lys-
Asp-Glu-Arg-Phe-Phe-Ser-Asp-Lys-Ile-Ala-Lys-Tyr-Ile-Pro-Ile-Gln-Tyr-V al-Leu-
Ser-Arg-Tyr-Pro-Ser-Tyr-Gly-Leu-Asn-Tyr-Tyr-Gln-Gln-Lys-Pro-Val-Ala-Leu-Il e-
Asn-Asn-Gln-Phe-Leu-Pro-Tyr-Pro-Tyr-Tyr-Ala-Lys.Polypeptide is bovine casein in above patent
A part, belong to macromolecular gluco, from bovine casein.Because it is macromolecular substances, its property digested and assimilated is poor.
Which disclose macromolecular bioactivity has immunocompetence, but is not described whether it has other performances.
The content of the invention
It is an object of the invention to provide a kind of biologically active polypeptide DERFFSDKIA and its preparation method and application.
The purpose of the present invention can be achieved through the following technical solutions：
First aspect present invention, there is provided a kind of biologically active polypeptide DERFFSDKIA, its amino acid sequence are Asp-Glu-
Arg-Phe-Phe-Ser-Asp-Lys-Ile-Ala, such as SEQ ID NO：Shown in 1.
Preferably, the biologically active polypeptide is milk-derived.κ-casein is derive specifically from, and is κ-ss-casein variants
The amino acid residue that A is the 35th~44.κ-ss-casein variants A amino acid sequences such as SEQ ID NO：Shown in 3.
The amino acid sequence of κ-casein and corresponding nucleotides sequence are classified as existing technology, encode κ-ss-casein variants A
The biologically active polypeptide DERFFSDKIA of the nucleotide fragments energy encoding mature of 35th~44 amino acids residue.
Preferably, the biologically active polypeptide has immunoloregulation function and anti-senescence function.
Second aspect of the present invention, there is provided encode the nucleotide fragments of the biologically active polypeptide DERFFSDKIA, its sequence
It is classified as：5 '-gat gaa aga ttc ttc agt gac aaa ata gcc-3 ', such as SEQ ID NO：Shown in 2.
Third aspect present invention, there is provided the preparation method of the biologically active polypeptide DERFFSDKIA, base can be passed through
Because the method for engineering is artificial synthesized, can be directly obtained from dairy products by the method isolated and purified, can direct passing through
Learn synthetically prepared.
Fourth aspect present invention, there is provided the biologically active polypeptide DERFFSDKIA has immunoloregulation function in preparation
Food, health products, the application in medicine or cosmetics.
Fifth aspect present invention, there is provided the biologically active polypeptide DERFFSDKIA is being prepared with anti-senescence function
Application in food, health products or medicine.
Sixth aspect present invention, there is provided the biologically active polypeptide DERFFSDKIA is being prepared while had immunological regulation
Application in the food of function and anti-senescence function, health products or medicine.
Specifically, biologically active polypeptide DERFFSDKIA of the invention, which can be used for preparing, reduces free radical to skin wound
The harmful medicine of cosmetics, preparation with immunological regulation and/or anti-aging；And due to the biologically active polypeptide of the present invention
Product after DERFFSDKIA is degraded by intestines and stomach still has bioactivity, therefore can be also used for preparing the food such as Yoghourt
Product, the health products for adjusting immunity, and the oral medicine being used to prepare with immunological regulation and/or anti-aging.
Seventh aspect present invention, there is provided a kind of immunological regulation product, including the biologically active polypeptide DERFFSDKIA
Or the derivative of the biologically active polypeptide DERFFSDKIA；Described immunological regulation product includes immunological regulation food, is immunized
Adjust health products, immunoregulation medicament or immunological regulation cosmetics；The derivative of the biologically active polypeptide DERFFSDKIA, it is
Refer on biologically active polypeptide DERFFSDKIA amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated,
Be carbonylated, methylate, acetylation, phosphorylation, the modification such as esterification or glycosylation, obtained polypeptide derivative.
Eighth aspect present invention, there is provided a kind of anti-aging product, including the biologically active polypeptide DERFFSDKIA or
The derivative of the biologically active polypeptide DERFFSDKIA；Described anti-aging product includes antisenility cistanche food, anti-ageing healthcare
Product or antiaging agent；The derivative of the biologically active polypeptide DERFFSDKIA, refers in biologically active polypeptide
On DERFFSDKIA amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, second
Acylation, phosphorylation, esterification or glycosylation etc. are modified, obtained polypeptide derivative.
Ninth aspect present invention, there is provided product a kind of while that there is immunoloregulation function and anti-senescence function, including
The derivative of the biologically active polypeptide DERFFSDKIA or described biologically active polypeptides DERFFSDKIA；With immunological regulation work(
Food, health products or medicine can be included with the product of anti-senescence function；The derivative of the biologically active polypeptide DERFFSDKIA,
Refer on biologically active polypeptide DERFFSDKIA amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxyl
Change, be carbonylated, methylating, acetylation, phosphorylation, the modification such as esterification or glycosylation, obtained polypeptide derivative.
Biologically active polypeptide DERFFSDKIA's of the present invention has the beneficial effect that：The milk-derived biologically active polypeptide of the present invention
DERFFSDKIA has preferably regulation immunity of organisms activity and activity of fighting against senium；On the one hand, bioactivity of the invention is more
PEPD ERFFSDKIA can strengthen the in-vitro multiplication ability of lymphocyte and macrophage, improve body and resist extraneous cause of disease body-sensing
The ability of dye, reduce the body incidence of disease；On the other hand, it is possible to increase the vigor of internal anti-peroxidation enzyme system, enhancing body resistance
The function of external source sexual stimulus, so as to reduce organism aging process, aging and sick probability, exploitation with immunoloregulation function and is resisted
Dairy products and the health products tool of aging function are of great significance.
The application polypeptide structurally and functionally with prior art (tyrosine peptide T rpi is rich in such as background technology))
It is essentially different：Biologically active polypeptide DERFFSDKIA of the present invention is a kind of small molecule bioactive fragment, belongs to core
Fragment；Biologically active polypeptide DERFFSDKIA of the present invention has more the property digested and assimilated.Biologically active polypeptide of the present invention simultaneously
DERFFSDKIA has immunoloregulation function and anti-senescence function, has on functional activity with disclosed polypeptide in the prior art
Significant difference.
Brief description of the drawings
Fig. 1：Mass chromatography extraction figure (m/z=614.3037)；
Fig. 2：Mass-to-charge ratio is the second order mses figure of 614.3037 fragment；
Fig. 3：Mass-to-charge ratio is 614.3037 polypeptide az, by crack conditions；
Fig. 4：Each group experimental animal mouse spleen situation of change；
(a) it is low dosage gavage group mouse spleen organization chart；(b) it is high dose gavage group mouse spleen organization chart； (c)
For naive mice spleen tissue；(d) it is animal model group mouse spleen organization chart
Fig. 5：Each group mice serum IL-6 changes table；
Fig. 6：Each group mice serum TNF-α changes table；
Fig. 7：Each group mice serum IL-2 changes table；
Fig. 8：Each group mice serum IL-10 changes table.
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 be selected 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 used in embodiment specific method, equipment,
Outside material, according to grasp of the those skilled in the art to 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, 1987and 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 DERFFSDKIA'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.
2. after 2 hours, resin is washed with nitrogen-dimethylformamide (DMF) of 3 times of resin volumes, is then drained, so
It is repeated 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.Washed four times with the DMF of 3 times of resin volumes after having taken off protection,
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. weigh amino acid Asp in right amount and 1- hydroxyls-benzene a pair of horses going side by side triazole (HOBT) is in right amount in 50ml centrifuge tube, addition
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.Washed four times with DMF after having taken off protection, 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 in right amount and HOBT being in right amount in 50ml centrifuge tube, 25ml DMF generals are added
It dissolves, and the DIC vibrations for then adding 2.5ml shake up 1min, are added to after solution clarification in reactor, then by reactor
It is placed in 30 DEG C of shaking table and reacts.
10. after 1 hour, taking 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.
After 11. question response is complete, washs resin four times with DMF, 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.Washed four times with DMF after having taken off protection, then drain whether detection protection sloughs.
12. amino acid Glu, Arg, Phe, Phe, Ser, Asp, Lys, Ile and Ala are connected successively according to step 9-11.
13. after last amino acid is connected, protection is sloughed, is washed four times with DMF, is then taken out resin with methanol
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 more
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 DERFFSDKIA.
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
PEPD ERFFSDKIA carries out chromatography and mass spectral analysis, and its mass chromatography extraction figure is as shown in figure 1, extract the two level matter at this peak
As shown in Figures 2 and 3, the polypeptide mass-to-charge ratio that can obtain this peak is 614.3037Da, and retention time is for spectrogram and az, by crack conditions
35.5min。
3) result
From the figure 3, it may be seen that situation about being broken according to az, by, calculates by Mascot software analysis, obtains mass-to-charge ratio
614.3037Da fragment sequence is Asp-Glu-Arg-Phe-Phe-Ser-Asp-Lys-Ile-Ala (DERFFSDKIA), note
For SEQ ID NO：1.The fragment is corresponding with κ-ss-casein variants A the 35th~44 residue sequence, κ-casamino acid
The GenBank numberings of sequence are AAA30433.1, and sequence is shown in SEQ ID NO：3.
The regulation immunity of organisms activity experiment of the biologically active peptide of embodiment 2
First, mtt assay measure biologically active polypeptide DERFFSDKIA vitro lymphocyte proliferation capacity experimental
1. experiment material and instrument：
Reagent and material：(male 6-8 week old, Shanghai Communications University are agriculture with biological institute for experimental animal balb/c mouse
Animal experimental center)；The milk-derived biologically active polypeptide DERFFSDKIA that embodiment 1 obtains；(the purchase of mouse lymphocyte extract solution
From Suo Laibao companies)；RPMI1640 culture mediums (are purchased from GIBCO companies)；3- (4,5- dimethylthiazole -2) -2,5- diphenyl four
Nitrogen azoles 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 and equipment：LRH-250F biochemical cultivation cases, Shanghai perseverance Science and Technology Ltd.；GL-22M high speed freezing centrifuges,
Shanghai Lu Xiang instrument centrifuges Instrument Ltd.；Hera cell 150CO2 incubators, Heraeus companies；Dragon
Wellscan MK3 ELIASAs, Labsystems companies；ALPHA 1-2-LD vacuum freeze driers, Christ companies；Superelevation
Effect liquid phase chromatogram-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, carries out Yuan Dynasty's culture.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, continues to cultivate 4h, careful abandoning supernatant, 100 μ L dimethyl is added 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：
Influences of the biologically active polypeptide DERFFSDKIA of table 1 to vitro lymphocyte proliferation
| Experiment packet | Stimulus index SI |
| Negative control group | 1 |
| DERFFSDKIA | 1.178±0.071* |
Note：* labelled notation is compared with negative control, there is significant difference (P ＜ 0.05).
Experimental result is shown in Table 1.As shown in Table 1, it is 100 μ g/mL's in biologically active peptide DERFFSDKIA mass concentration
Under the conditions of, milk-derived biologically active peptide DERFFSDKIA stimulus index is more than BSA, illustrates DERFFSDKIA to a certain extent
The propagation of external mouse lymphocyte can be stimulated.And DERFFSDKIA stimulus index has reached 1.178, and negative control
Group has significant difference (P<0.05).Therefore, it can be assumed that active peptides DERFFSDKIA, which has, remarkably promotes mouse lymph
The ability of cell propagation, a kind of health products or additive can be used as to eat, it is possible to increase the immunity of animal and human body.
2nd, mtt assay measure biologically active polypeptide DERFFSDKIA macrophages in vitro multiplication capacity experiment 1) experiment examination
Agent and instrument
Reagent：Experimental animal balb/c mouse (male 6-8 week old) Shanghai Communications University is agriculture real with biological institute animal
Test center；The milk-derived biologically active polypeptide DERFFSDKIA that embodiment 1 obtains；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, the water containing 10%SDS, 5% isobutanol and 0.012mol/L HCl
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 150CO2Incubator 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,
After centrifuge tube collects flushing liquor, 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, confirms 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 to suspend completely and added 96 porocyte culture plates, 37 DEG C, 5%CO with suitable volumes2
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, not adherent cell and cell fragment are washed away, obtain adherent peritoneal macrophage after purification.0.2ml is added per hole
RPMI1640 complete mediums, experiment add after being dissolved in culture medium in advance with small peptide sample and LPS, start cell culture.
After obtaining adherent peritoneal macrophage after purification, experimental group adds dissolved with biologically active polypeptide per hole
DERFFSDKIA (1mg/ml) the μ l/ holes of RPMI1640 complete culture solutions (10%FBS) 200, continuously cultivate 48h；Negative control
Group adds the μ l/ holes of RPMI1640 complete culture solutions (10%FBS) 200 dissolved with BSA (500 μ g/mL) per hole；Blank group is added
The μ l/ holes of RPMI1640 complete culture solutions (10%FBS) 200, continuously cultivate 48h.Also, experimental group, negative control group and blank
Group sets normal group and inflammation group respectively again；Inflammation group adds LPS to final concentration of 100ng/ml when 24h is arrived in culture；Normal group
It is not added with LPS；And normal group and inflammation group add the μ l/ holes of 5%MTT 20 in 44h；Cell culture adds 100 μ after reaching 48h
Three lysates in l/ holes are to terminate culture, after dissolving overnight, survey the absorbance in each hole with ELIASA under wavelength 570nm
(OD570), the calculation formula of growth index (Growth Indices) is as follows：
Wherein, blank nutrient solution is the RPMI1640 complete culture solutions containing 10%FBS.
3) experimental result and analysis
The influence that the biologically active polypeptide DERFFSDKIA of table 2 breeds to macrophages in vitro
| Experiment packet | Normal group GI | Inflammation group GI |
| Negative control group | 1 | 1 |
| DERFFSDKIA(1mg/ml) | 1.0873±0.0539** | 1.128±0.0604** |
Note：* represent compared with negative control, there is significant difference (P ＜ 0.05)；* represent compared with negative control group,
There is significant difference (P ＜ 0.01)
Experimental result is shown in Table 2, as shown in Table 2, under conditions of 1mg/ml biologically active polypeptides DERFFSDKIA is added,
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 DERFFSDKIA has significant proliferation function to macrophages in vitro.
The activity of fighting against senium experiment of the biologically active peptide of embodiment 3
First, experiments of the biologically active polypeptide DERFFSDKIA to internal spleen tissue structure function
1. experiment reagent and instrument：
Reagent：Experimental animal ICR mouse (male 5 week old), Shanghai City Experimental Animal Center；D-gal, Chinese medicines group chemistry
Reagent Co., Ltd；Paraformaldehyde, Chemical Reagent Co., Ltd., Sinopharm Group；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagent
Department；The milk-derived biologically active polypeptide DERFFSDKIA that embodiment 1 obtains.
Instrument and equipment：The ultra-clean water of CM-230 types mole, Shanghai Moller scientific instrument Co., Ltd；Mi Libo Millipore
MILLEX GP0.22 μm filter membranes, Millipore Corp. of the U.S.；GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument centrifuge instruments
Co., Ltd.
2. experimental method：
(1) Animal Aging model modeling
After ICR mouse adaptability is raised one week, it is divided into 4 groups, every group 6.Group 1 is low dosage gavage group, and mouse is daily
D-gal is subcutaneously injected with 500mg/kg dosage nape part, and with the dosage gavage biologically active polypeptide in 1mg/ day
DERFFSDKIA；Group 2 is high dose gavage group, and D-gal is subcutaneously injected with 500mg/kg dosage nape part daily in mouse, and
The day dosage gavage biologically active polypeptide DERFFSDKIA of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is
Thing model group, D-gal is subcutaneously injected with 500mg/kg dosage nape part daily in mouse, and gavage concentration is 0.9% life
Manage salt solution；D-gal injection cycle and the gavage cycle of polypeptide are 42 days.Change bedding and padding within every 3 days, and ensure feed and distillation
The supply of water.The every five days body weight for weighing a mouse, D-gal parenteral solutions, and D-gal parenteral solutions are prepared according to the body weight of mouse
Filtered through 0.22 μm of needle cylinder type filter membrane, it is sterile to ensure.
(2) animal viscera is obtained
After the completion of experimental period, to pluck the blood that eyeball blood taking method obtains mouse, disconnected neck puts to death mouse after obtaining blood, and
The body of mouse is placed on low temperature ice box afterwards, and wins brain, spleen, liver and the kidney of mouse rapidly, what is obtained is dirty
Device is placed in the 1.5mL centrifuge tubes that sterilize in advance, and all organ samples are maintained in -80 DEG C of refrigerators standby inspection.Disposal is real
All operations tested during animal follow Ministry of Science and Technology's issue in 2006《Directiveness meaning on kind treatment experimental animal
See》.The mouse spleen won directly is soaked in 4% paraformaldehyde solution prepared in advance, in the form of fixing it.Poly
Formaldehyde powder more indissoluble, micro sodium acid carbonate can be added and adjust pH value to alkalescence, with hydrotropy.Paraformaldehyde solution
Preparing needs to complete in fume hood.
(3) sample detection
The making of histotomy：Mouse spleen sample need at least fix 24 hours in 4% paraformaldehyde solution.Spleen group
The wax stone knitted makes, section entrusts Shanghai Wei Ao bio tech ltd to complete with HE dyeing.
3. experimental result and analysis：
In this experiment, 4 groups of mouse are shared, the mouse normal growth of wherein blank group is not affected by any environmental stimuli, its
3 groups of mouse of remaininging receive D-gal long term injections.Using light microscope, the spleen section of separate groups of mice is seen
Examine, can be found from Fig. 4, contrast the spleen section of each group mouse, for naive mice, the spleen of animal model mouse
Dirty red pulp obscures with white pulp boundary, and atrophy occurs in white pulp, shows that long-term D-gal injections send out the glycometabolism approach of mouse
It is raw disorderly, cause anti-oxidant enzyme activity to reduce, peroxide accumulation, and then the aging and atrophy of spleen may have been triggered.And gavage
Then white pulp atrophy degree is lighter relative to animal model group mouse for the spleen tissue of the mouse of polypeptide group, and the boundary of red pulp and white pulp
Limit is more clearly demarcated.This result illustrate, in whole D-gal injection cycle, experiment animal sustained constantly by cause aging because
The stimulation of son, causes the aging and atrophy of spleen.Therefore from the point of view of the situation of changes in microstructure, the life invented in this experiment
Thing active peptides DERFFSDKIA has necessarily to spleen aging of the animal caused by the stimulation by the bad factor with atrophy
Protective effect.
2nd, the experiment that biologically active polypeptide DERFFSDKIA is acted on intracorporeal organ antioxidant levels
1. experiment reagent and instrument：
Reagent：Experimental animal ICR mouse (male 5 week old), Shanghai City Experimental Animal Center；D-gal, Chinese medicines group chemistry
Reagent Co., Ltd；Paraformaldehyde, Chemical Reagent Co., Ltd., Sinopharm Group；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagent
Department；The milk-derived biologically active polypeptide DERFFSDKIA that embodiment 1 obtains；BCA protein reagent boxes, Nanjing Keygen Biotech's science and technology
Co., Ltd；MDA MDA kits, Science and Technology Ltd. of Nanjing Keygen Biotech；SOD superoxide dismutase reagents
Bio tech ltd is built up in box, Nanjing；T-AOC antioxidative activities kits, bio tech ltd is built up in Nanjing.
Instrument and equipment：The ultra-clean water of CM-230 types mole, Shanghai Moller scientific instrument Co., Ltd；Mi Libo Millipore
MILLEX GP0.22 μm filter membranes, Millipore Corp. of the U.S.；GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument centrifuge instruments
Co., Ltd.
2. experimental method：
(1) Animal Aging model modeling
After ICR mouse adaptability is raised one week, it is divided into 4 groups, every group 6.Group 1 is low dosage gavage group, and mouse is daily
D-gal is subcutaneously injected with 500mg/kg dosage nape part, and with the dosage gavage biologically active polypeptide in 1mg/ day
DERFFSDKIA；Group 2 is high dose gavage group, and D-gal is subcutaneously injected with 500mg/kg dosage nape part daily in mouse, and
The day dosage gavage biologically active polypeptide DERFFSDKIA of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is
Thing model group, D-gal is subcutaneously injected with 500mg/kg dosage nape part daily in mouse, and gavage concentration is 0.9% life
Manage salt solution；D-gal injection cycle and the gavage cycle of polypeptide are 42 days.Change bedding and padding within every 3 days, and ensure feed and distillation
The supply of water.The every five days body weight for weighing a mouse, D-gal parenteral solutions, and D-gal parenteral solutions are prepared according to the body weight of mouse
Filtered through 0.22 μm of needle cylinder type filter membrane, it is sterile to ensure.
(2) animal viscera is obtained
After the completion of experimental period, to pluck the blood that eyeball blood taking method obtains mouse, disconnected neck puts to death mouse after obtaining blood, and
The body of mouse is placed on low temperature ice box afterwards, and wins brain, spleen, liver and the kidney of mouse rapidly, what is obtained is dirty
Device is placed in the 1.5mL centrifuge tubes that sterilize in advance, and all organ samples are maintained in -80 DEG C of refrigerators standby inspection.Disposal is real
All operations tested during animal follow Ministry of Science and Technology's issue in 2006《Directiveness meaning on kind treatment experimental animal
See》.The mouse spleen won directly is soaked in 4% paraformaldehyde solution prepared in advance, in the form of fixing it.Poly
Formaldehyde powder more indissoluble, micro sodium acid carbonate can be added and adjust pH value to alkalescence, with hydrotropy.Paraformaldehyde solution
Preparing needs to complete in fume hood.
(3) sample detection
All internal organs that need to be detected, are ground at low ambient temperatures, and 10% group is diluted to 4 DEG C of sterile PBS solutions
Knit homogenate, under the conditions of 4 DEG C after 4000g centrifugations, take supernatant, discard precipitation, operated according to kit specification, or put
It is to be measured in -80 DEG C of refrigerators.
3. experimental result and analysis：
The change of SOD contents in each group experimental animal mouse Different Organs of table 3
Note：* sign compares with model group, there is significant difference (P<0.05)；* signs compare with model group,
There is significant difference (P<0.01), similarly hereinafter.
As can be known from Table 3, relative to animal model group mouse, the SOD in the liver and kidney of polypeptide gavage group mouse contains
Increase (the P of conspicuousness is presented in amount<0.01).Although mean D-gal of the mouse by long-term, high-dose of polypeptide gavage group
Stimulation, even if D-gal excess injection also without completely destroy Mice Body in SOD enzyme systems, illustrate in injection cycle, test
Animal can cause the reduction of SOD contents in Different Organs in the case of continuously by the stimulation for causing senescence-factor, but together
When take in a certain amount of polypeptide DERFFSDKIA there is certain protective role to the oxidative damage in Mice Body.
The situation of change of MDA contents in each group experimental animal mouse Different Organs of table 4
As can be known from Table 4, the liver MDA content of animal model group mouse is 26.86 ± 7.04nmol/L, with animal model
Group compares, and significant difference (P is presented in the MDA contents in two groups of mouse livers of polypeptide gavage<0.01).Because MDA can be used for
Estimate the accumulation situation of animal body lipid peroxide, therefore it follows that animal model group mouse is causing aging model to be formed
During, due to excess D-gal long term injections, the glycometabolism approach of mouse is got muddled, produce a large amount of free radicals from
And oxidative damage is caused, occur a large amount of MDAs in its liver organization, MDA is as MDA, and it is in animal
The rise of in-vivo content, the reduction of Antioxidant Enzymes vigor in Mice Body can be reflected from side.And polypeptide gavage group Mouse Liver
Dirty MDA contents significantly reduce, and illustrate polypeptide DERFFSDKIA intake and can effectively protect vital tissue organ from bad
The factor, which stimulates, produces substantial amounts of MDA.
T-AOC situation of change in each group experimental animal Mice Body of table 5
As known from Table 5, the liver T-AOC values of animal model group mouse are 0.68 ± 0.21U/mgprot, are compared to mould
Significant difference (P is presented with low dosage gavage group mouse in type group, polypeptide high dose therewith<0.05)；The kidney of naive mice
Dirty T-AOC contents are 0.61 ± 0.25U/mgprot, and compared with animal model group mouse, low dosage gavage group presents aobvious therewith
Write sex differernce (P<0.05), significant difference (P is also presented in high dose gavage group mouse therewith<0.01).This result shows, whole
In individual experimental period, because experiment animal sustained is constantly stimulated by cause senescence-factor, the liver of animal model group mouse,
Renal tissue is destroyed, and causes the reduction of its TAC.Compared with animal model group and blank group, polypeptide gavage group is small
The TAC of mouse major organs maintains a higher level all the time in by the stimulating course for causing senescence-factor,
Illustrate that taking in biologically active polypeptide DERFFSDKIA makes animal body and its major organs have higher self-protection function.
3rd, the biologically active polypeptide DERFFSDKIA experiment acted on immune cell factor in serum
1. experiment reagent and instrument：
Reagent：Experimental animal ICR mouse (male 5 week old), Shanghai City Experimental Animal Center；D-gal, Chinese medicines group chemistry
Reagent Co., Ltd；Paraformaldehyde, Chemical Reagent Co., Ltd., Sinopharm Group；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagent
Department；The milk-derived biologically active polypeptide DERFFSDKIA that embodiment 1 obtains；BCA protein reagent boxes, Nanjing Keygen Biotech's science and technology
Co., Ltd；ELISA cell factors Quick kit (TNF-α, IL-2, IL-6 and IL-10), Wuhan doctor's moral bioengineering
Co., Ltd.
Instrument and equipment：The ultra-clean water of CM-230 types mole, Shanghai Moller scientific instrument Co., Ltd；Mi Libo Millipore
MILLEX GP0.22 μm filter membranes, Millipore Corp. of the U.S.；GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument centrifuge instruments
Co., Ltd.
2. experimental method：
(1) Animal Aging model modeling
After ICR mouse adaptability is raised one week, it is divided into 4 groups, every group 6.Group 1 is low dosage gavage group, and mouse is daily
D-gal is subcutaneously injected with 500mg/kg dosage nape part, and with the dosage gavage biologically active polypeptide in 1mg/ day
DERFFSDKIA；Group 2 is high dose gavage group, and D-gal is subcutaneously injected with 500mg/kg dosage nape part daily in mouse, and
The day dosage gavage biologically active polypeptide DERFFSDKIA of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is
Thing model group, D-gal is subcutaneously injected with 500mg/kg dosage nape part daily in mouse, and gavage concentration is 0.9% life
Manage salt solution；D-gal injection cycle and the gavage cycle of polypeptide are 42 days.Change bedding and padding within every 3 days, and ensure feed and distillation
The supply of water.The every five days body weight for weighing a mouse, D-gal parenteral solutions, and D-gal parenteral solutions are prepared according to the body weight of mouse
Filtered through 0.22 μm of needle cylinder type filter membrane, it is sterile to ensure.
(2) animal viscera and serum are obtained
After the completion of experimental period, to pluck the blood that eyeball blood taking method obtains mouse, disconnected neck puts to death mouse after obtaining blood, and
The body of mouse is placed on low temperature ice box afterwards, after mouse blood is stored at room temperature 1 hour, 15min is centrifuged with 3000g, separates blood
Clearly.Serum keeping is to be checked in -80 DEG C of refrigerators.All operations during disposal experimental animal follow the Ministry of Science and Technology in 2006
Issue《On treating the guiding opinion of experimental animal kindly》.The mouse spleen won directly is soaked in what is prepared in advance
In 4% paraformaldehyde solution, in the form of fixing it.Paraformaldehyde powder more indissoluble, can add micro sodium acid carbonate will
PH value is adjusted to alkalescence, with hydrotropy.The preparation of paraformaldehyde solution needs to complete in fume hood.
(3) sample detection
Indicated according to kit specification, draw standard curve first, standard items powder is prepared with standard dilutions
Into 1000pg/mL solution, then serial dilution is 500pg/mL, 250pg/mL, 125pg/mL, 62.5 pg/mL, 31.3pg/
The various concentrations such as mL, 15.6pg/mL.Each concentration gradient solution pipettes 100 μ L in the ELISA Plate of coated antibody.Draw small
The μ L of mouse blood serum sample 100, add in same ELISA Plate (needs when detecting calculating again if blood serum sample is inadequate, after can suitably diluting
Convert in proportion).ELISA Plate is covered, is placed under 37 DEG C of environment and is incubated 90min.After completion of the reaction, ELISA Plate is carefully got rid of
Interior liquid, and ELISA Plate is placed on blotting paper, it is careful to pat, remove surplus liquid.By preheated biotin antiantibody
Working solution is sequentially added in each hole of ELISA Plate by every μ L of hole 100, at 37 DEG C, reacts 60min.After completion of the reaction, 0.01M is utilized
PBS solution wash 3 times, every time per the PBS that 100 μ L are added in hole, soak 1min hypsokinesis and remove solution, 3 times repeatedly.Will preheating
Good ABC working solutions are sequentially added by every hole 0.1ml, 37 DEG C of reaction 30min.After completion of the reaction, washed 5 times with 0.01M PBS,
Immersion 1min or so every time.The TMB nitrite ions for balancing 30min at 37 DEG C, 37 DEG C of lucifuge reactions are sequentially added by every μ L of hole 90
8-12min.TMB terminate liquids are sequentially added by every hole 0.1ml, now blueness is vertical turns yellow, and OD is determined in 450nm with ELIASA
Value.Concentration known is done by the standard protein of cell factor to be serially diluted, and standard curve is drawn out after measuring OD values, according to standard
Curve can extrapolate the content of cell factor in sample.
3. experimental result and analysis：
The situation of change of cell factor in each group mice serum of table 6
IL-6 and TNF-α content are respectively in the model group Mice Body being can be found that from table 6, Fig. 5, Fig. 6 in this experiment
168.01 ± 26.38pg/mL, 4.34 ± 0.76pg/mL, the increase (P of conspicuousness is presented compared to normal group<0.01), therefore
It is considered that due to continuously injecting cause senescence-factor, animal model group mouse is caused aging occur in cell factor aspect
The symptom of property inflammation, and IL-6 and the TNF- alpha content of the mice serum of polypeptide gavage group are effectively controlled.According to cell
The experimental result of the factor, serum levels of inflammatory cytokines IL-6, the secretion level of TNF-α of polypeptide gavage group mouse are below animal
Model group, from the point of view of oxidative damage angle, mouse because free radical attack, Peroxidation Product accumulation and caused by oxidative damage may
Obtain a certain degree of suppression；From the perspective of inflammation, mouse inflammation because of caused by oxidation has obtained effective suppression；From
From the point of view of aging angle, a series of geriatric diseases caused by mouse aging caused by long term injections D-gal are possible to
To control.
Found from Fig. 7 result, a kind of models of the IL-2 as significant cell factor for judging aging in this experiment
Group in gavage group with not occurring conspicuousness effect, thus it is speculated that, it may be possible to due to the model and naturally-aged employed in this experiment
Still difference, or modeling cycle fall short of, therefore cause this index not change.Meanwhile found from Fig. 8 result, at this
The secretion that biologically active polypeptide DERFFSDKIA fails to mouse IL-10 in the cycle in experiment has an impact, in various mouse blood
In the detection of clear IL-10 contents, the content of serum IL -10 of only normal growth group mouse is maintained at relatively low level, its
Remaining each group mice serum IL-10 contents, which are presented, to be increased, it may be possible to which secretions of the biologically active polypeptide DERFFSDKIA to IL-10 is led to
Road is without influence.
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 by performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability
Field technique personnel do not depart from improvement that scope made and modification all should be the present invention's according to the announcement of the present invention
Within protection domain.
Sequence table
<110>Zhejiang panda dairy industry Group Plc；Zhejiang Hui Tai life and healths Science and Technology Ltd.
<120>A kind of biologically active polypeptide DERFFSDKIA and its preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Asp Glu Arg Phe Phe Ser Asp Lys Ile Ala
1 5 10
<210> 2
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gatgaaagat tcttcagtga caaaatagcc 30
<210> 3
<211> 190
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Met Met Lys Ser Phe Phe Leu Val Val Thr Ile Leu Ala Leu Thr Leu
1 5 10 15
Pro Phe Leu Gly Ala Gln Glu Gln Asn Gln Glu Gln Pro Ile Arg Cys
20 25 30
Glu Lys Asp Glu Arg Phe Phe Ser Asp Lys Ile Ala Lys Tyr Ile Pro
35 40 45
Ile Gln Tyr Val Leu Ser Arg Tyr Pro Ser Tyr Gly Leu Asn Tyr Tyr
50 55 60
Gln Gln Lys Pro Val Ala Leu Ile Asn Asn Gln Phe Leu Pro Tyr Pro
65 70 75 80
Tyr Tyr Ala Lys Pro Ala Ala Val Arg Ser Pro Ala Gln Ile Leu Gln
85 90 95
Trp Gln Val Leu Ser Asn Thr Val Pro Ala Lys Ser Cys Gln Ala Gln
100 105 110
Pro Thr Thr Met Ala Arg His Pro His Pro His Leu Ser Phe Met Ala
115 120 125
Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile Asn
130 135 140
Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val
145 150 155 160
Glu Ser Thr Val Ala Thr Leu Glu Asp Ser Pro Glu Val Ile Glu Ser
165 170 175
Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val
180 185 190
Claims (10)
1. a kind of biologically active polypeptide DERFFSDKIA, it is characterised in that its amino acid sequence is Asp-Glu-Arg-Phe-
Phe-Ser-Asp-Lys-Ile-Ala。
2. a kind of biologically active polypeptide DERFFSDKIA according to claim 1, it is characterised in that the bioactivity is more
Peptide is milk-derived.
3. encode the nucleotide fragments of biologically active polypeptide DERFFSDKIA described in claim 1, it is characterised in that the nucleosides
The sequence of acid fragment such as SEQ ID NO：Shown in 2.
4. biologically active polypeptide DERFFSDKIA as claimed in claim 1 preparation method, it is characterised in that pass through genetic engineering
Method it is artificial synthesized, or directly obtained from dairy products by the method isolated and purified, or directly prepared by chemical synthesis.
5. biologically active polypeptide DERFFSDKIA as claimed in claim 1 application, it is characterised in that the biologically active polypeptide
Applications of the DERFFSDKIA in the food with immunoloregulation function, health products, medicine or cosmetics are prepared.
6. biologically active polypeptide DERFFSDKIA as claimed in claim 1 application, it is characterised in that the biologically active polypeptide
Applications of the DERFFSDKIA in the food with anti-senescence function, health products or medicine is prepared.
7. biologically active polypeptide DERFFSDKIA as claimed in claim 1 application, it is characterised in that the biologically active polypeptide
Applications of the DERFFSDKIA in the food with immunoloregulation function and anti-senescence function, health products or medicine is prepared.
A kind of 8. immunological regulation product, it is characterised in that including biologically active polypeptide DERFFSDKIA as claimed in claim 1 or
The derivative of the biologically active polypeptide DERFFSDKIA；Described immunological regulation product includes immunological regulation food, immune tune
Save health products, immunoregulation medicament or immunological regulation cosmetics；The derivative of the biologically active polypeptide DERFFSDKIA, refers to
On biologically active polypeptide DERFFSDKIA amino acid side groups, aminoterminal or c-terminus progress hydroxylating, carboxylated, carbonyl
Base, methylate, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
9. a kind of anti-aging product, it is characterised in that including biologically active polypeptide DERFFSDKIA as claimed in claim 1 or institute
State biologically active polypeptide DERFFSDKIA derivative；Described anti-aging product includes antisenility cistanche food, antisenescence health product
Or antiaging agent；The derivative of the biologically active polypeptide DERFFSDKIA, refers in biologically active polypeptide DERFFSDKIA
Amino acid side groups on, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphoric acid
Change, esterification or glycosylation modified, obtained polypeptide derivative.
10. a kind of product with immunoloregulation function and anti-senescence function, it is characterised in that including as claimed in claim 1
Biologically active polypeptide DERFFSDKIA or described biologically active polypeptides DERFFSDKIA derivative；With immunoloregulation function and
The product of anti-senescence function includes food, health products or medicine；The derivative of the biologically active polypeptide DERFFSDKIA, refers to
On biologically active polypeptide DERFFSDKIA amino acid side groups, aminoterminal or c-terminus progress hydroxylating, carboxylated, carbonyl
Base, methylate, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
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Cited By (4)
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| CN112759635A (en) * | 2021-01-21 | 2021-05-07 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide with amino acid structure VAKVTGGAASKL, and preparation method and application thereof |
| CN112812171A (en) * | 2021-01-22 | 2021-05-18 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide with amino acid structure VVRKPLNKEGKKP, and preparation method and application thereof |
| CN113875992A (en) * | 2021-03-03 | 2022-01-04 | 陕西医赛尔生物科技有限公司 | Polypeptide composition preparation with function of improving immunity and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112661830A (en) * | 2021-01-21 | 2021-04-16 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide with amino acid structure AIRNDEELNKLLGR, and preparation method and application thereof |
| CN112759635A (en) * | 2021-01-21 | 2021-05-07 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide with amino acid structure VAKVTGGAASKL, and preparation method and application thereof |
| CN112812171A (en) * | 2021-01-22 | 2021-05-18 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide with amino acid structure VVRKPLNKEGKKP, and preparation method and application thereof |
| CN112812171B (en) * | 2021-01-22 | 2022-04-08 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide with amino acid structure VVRKPLNKEGKKP, and preparation method and application thereof |
| CN113875992A (en) * | 2021-03-03 | 2022-01-04 | 陕西医赛尔生物科技有限公司 | Polypeptide composition preparation with function of improving immunity and application thereof |
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Address after: 325800 LITE-ON East Road, Ling Xi Town, Cangnan County, Zhejiang 650-668 Applicant after: Panda Dairy Group Limited by Share Ltd Applicant after: Zhejiang peptide life health science and Technology Co Ltd Address before: 325800 LITE-ON East Road, Ling Xi Town, Cangnan County, Wenzhou, Zhejiang 650-668 Applicant before: ZHEJIANG PANDA DAIRY CORPORATION Applicant before: Zhejiang peptide life health science and Technology Co Ltd |
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Application publication date: 20180302 |