CN107903315A - A kind of biologically active polypeptide NEINQFYQ and its preparation method and application - Google Patents
A kind of biologically active polypeptide NEINQFYQ and its preparation method and application Download PDFInfo
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- CN107903315A CN107903315A CN201711125056.XA CN201711125056A CN107903315A CN 107903315 A CN107903315 A CN 107903315A CN 201711125056 A CN201711125056 A CN 201711125056A CN 107903315 A CN107903315 A CN 107903315A
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- Prior art keywords
- neinqfyq
- biologically active
- active polypeptide
- polypeptide
- mouse
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Classifications
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- C—CHEMISTRY; METALLURGY
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- 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
-
- A—HUMAN NECESSITIES
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- 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
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- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
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- Engineering & Computer Science (AREA)
- Birds (AREA)
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- Toxicology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Dermatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to albumen field, and in particular to its amino acid sequence of a kind of biologically active polypeptide NEINQFYQ and its preparation method and application, biologically active polypeptide NEINQFYQ is Asn Glu Ile Asn Gln Phe Tyr Gln.By the experiment of ion vitro immunization function point analysis, internal Antisenility Experiment, demonstrating polypeptide NEINQFYQ has preferable immunoloregulation function and activity of fighting against senium, on the one hand, the biologically active polypeptide NEINQFYQ 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 body incidence；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 NEINQFYQ and preparation method thereof and answers
With.
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 is with special
Physiological function, is referred to as " biologically active peptide ".
It is particularly important that safe biologically active peptide is found in natural food source.In recent 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 biologically active polypeptide 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 physiological activity 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
The hexapeptide of Val-Glu-Pro-Ile-Pro-Tyr 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 rat peritoneal macrophages with newborn source immunomodulatory peptides (PGPIPN) the feeding rat of synthesis
Phagocytosis and the relevant immunoloregulation function of red blood cell have 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
Body incidence, 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 service lifes of these organisms and reach
As many as 6 times.
Anti-aging peptide in terms of physiological function there is amino acid cannot 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, to slow down aging.Therefore, the nutrition and health care of biologically active peptide
Effect has become 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 comes
The milk-derived biologically active polypeptide DELQDKIH of beta-casein is come from, Chinese patent CN105254739A discloses one kind and derives from
The milk-derived biologically active polypeptide GTQYTD of α s1- caseins, Chinese patent CN105254740A, which are disclosed, a kind of derives from α s2-
The milk-derived biologically active polypeptide NQFYQKF of casein.
The content of the invention
It is an object of the invention to provide a kind of biologically active polypeptide NEINQFYQ 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 NEINQFYQ, its amino acid sequence are Asn-Glu-
Ile-Asn-Gln-Phe-Tyr-Gln, such as SEQ ID NO：Shown in 1.
Preferably, the biologically active polypeptide is milk-derived.α s2- caseins are derive specifically from, and are α s2- caseins
The amino acid residue that modification A is the 98th~105.α s2- ss-casein variants A amino acid sequences such as SEQ ID NO：Shown in 3.
The amino acid sequence and corresponding nucleotides sequence of α s2- caseins are classified as existing technology, and coding for alpha s2- caseins become
The biologically active polypeptide NEINQFYQ of the nucleotide fragments energy encoding mature of the 98th~105 amino acids residues of body A.
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 NEINQFYQ, its sequence
For：5 '-aat gaa atc aat cag ttt tat cag-3 ', such as SEQ ID NO：Shown in 2.
Third aspect present invention, there is provided the preparation method of the biologically active polypeptide NEINQFYQ, can pass through gene
The method of engineering is artificial synthesized, can be directly obtained from dairy products by the method isolated and purified, can directly pass through chemistry
It is synthetically prepared.
Fourth aspect present invention, there is provided the biologically active polypeptide NEINQFYQ is being prepared with immunoloregulation function
Application in food, health products, medicine or cosmetics.
Fifth aspect present invention, there is provided the biologically active polypeptide NEINQFYQ is preparing the food with anti-senescence function
Application in product, health products or medicine.
Sixth aspect present invention, there is provided the biologically active polypeptide NEINQFYQ is being prepared while had immunological regulation work(
Can be with the application in the food, health products or medicine of anti-senescence function.
Specifically, biologically active polypeptide NEINQFYQ of the invention, which can be used for preparing, reduces free radical to skin damage
Cosmetics, prepare the medicine with immunological regulation and/or anti-aging；And due to the biologically active polypeptide of the present invention
Product after NEINQFYQ is degraded by intestines and stomach still has bioactivity, thus can be also used for preparing the food such as Yoghourt,
Adjust the health products of 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 NEINQFYQ or
The derivative of the biologically active polypeptide NEINQFYQ；The immunological regulation product includes immunological regulation food, immunological regulation
Health products, immunoregulation medicament or immunological regulation cosmetics；The derivative of the biologically active polypeptide NEINQFYQ, refers in life
On the amino acid side groups of thing active peptides NEINQFYQ, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation,
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 NEINQFYQ or institute
State the derivative of biologically active polypeptide NEINQFYQ；The anti-aging product include antisenility cistanche food, antisenescence health product or
Antiaging agent；The derivative of the biologically active polypeptide NEINQFYQ, refers to the amino in biologically active polypeptide NEINQFYQ
On sour side-chain radical, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification
Or the modification such as glycosylation, 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 NEINQFYQ or described biologically active polypeptides NEINQFYQ；With immunoloregulation function and
The product of anti-senescence function includes food, health products or medicine；The derivative of the biologically active polypeptide NEINQFYQ, refers to
On the amino acid side groups of biologically active polypeptide NEINQFYQ, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonyl
Change, methylate, acetylation, phosphorylation, the modification such as esterification or glycosylation, obtained polypeptide derivative.
Biologically active polypeptide NEINQFYQ's of the present invention has the beneficial effect that：The milk-derived biologically active polypeptide of the present invention
NEINQFYQ has preferable adjusting immunity of organisms activity and activity of fighting against senium；On the one hand, biologically active polypeptide of the invention
NEINQFYQ can strengthen the in-vitro multiplication ability of lymphocyte and macrophage, improve body and resist extraneous pathogenic infection
Ability, reduces body incidence；On the other hand, it is possible to increase the vigor of internal anti-peroxidation enzyme system, enhancing body resistance external source
The function of sexual stimulus, so as to reduce organism aging process, aging and sick probability, has immunoloregulation function and anti-aging to exploitation
Dairy products and the health products tool of function are of great significance.
Brief description of the drawings
Fig. 1：Mass chromatography extraction figure (m/z=528.2476)；
Fig. 2：Mass-to-charge ratio is the second order ms figure of 528.2476 fragment；
Fig. 3：Mass-to-charge ratio is 528.2476 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) it is
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.
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 endpoints.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical with the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment,
Outside material, according to grasp of the those skilled in the art to the 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 stated, disclosed in this invention experimental method, detection method, preparation method using this technology lead
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, 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.
1 active peptide NEINQFYQ's of embodiment is artificial synthesized
First, the synthesis of biologically active peptide
1. RINK resin 3g (substitution value 0.3mmol/g) are weighed in the reactor of 150ml, with the dichloromethane of 50ml
(DCM) soak.
2.2 it is small when after, wash resin with nitrogen-dimethylformamide (DMF) of 3 times of resin volumes, then drain, so weight
It is four times multiple, 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 Asn in right amount and 1- hydroxyls-benzene a pair of horses going side by side triazole (HOBT) is in right amount in the centrifuge tube of 50ml, addition
The DMF of 20ml is dissolved, and then adds the N of 3ml, and N diisopropylcarbodiimide (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 is reacted.
6.2 it is small when after, 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 the centrifuge tube of 50ml, the DMF generals of 25ml are added
It is dissolved, 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.
10.1 it is small when after, take a small amount of resin to detect, (each two drop of inspection A, inspection B, 100 DEG C of reactions detected with ninhydrin method
1min), if resin is colourless, illustrate that the reaction was complete；If resin has color, illustrate that condensation is incomplete, the reaction was continued.
11. after complete reaction, washing 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, Ile, Asn, Gln, Phe, Tyr and Gln 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) by polypeptide
Cut down from resin (every gram of resin adds 10ml cutting liquids), and with ice ether (cutting liquid：Ether=1:9,v:V) centrifugation is heavy
Drop four times.
So far, artificial synthesized biologically active peptide NEINQFYQ.
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 NEINQFYQ carries out chromatography and mass spectral analysis, its mass chromatography extraction figure is as shown in Figure 1, extract the second order ms at this peak
As shown in Figures 2 and 3, the polypeptide mass-to-charge ratio that can obtain this peak is 528.2476Da, and retention time is for figure and az, by crack conditions
37.9min。
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
The fragment sequence of 528.2476Da is Asn-Glu-Ile-Asn-Gln-Phe-Tyr-Gln (NEINQFYQ), is denoted as SEQ ID
NO：1.The residue sequence of the fragment and α s2- ss-casein variants A the 98th~105 is corresponding, α s2- casamino acid sequences
GenBank numbering be AAA30479.1, sequence is shown in SEQ ID NO：3.
The adjusting immunity of organisms activity experiment of 2 biologically active peptide of embodiment
First, the vitro lymphocyte proliferation capacity experimental of mtt assay measure biologically active polypeptide NEINQFYQ
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 NEINQFYQ that embodiment 1 obtains；Mouse lymphocyte extracting solution (is purchased 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 microplate reader, 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 extracting solution, carries out Yuan Dynasty's culture.With
Cell density is adjusted to 2.5 × 10 by complete RPMI1640 nutrient solutions6A/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.In addition,
Blank control group (PBS of pH7.2~7.4,3mol/L) and negative control group (500 μ g/mL BSA) are set, and research shows that its is right
Do not influenced in vitro lymphocyte proliferation.Every group of 3 parallel laboratory test samples.In 5%CO2After 68h being cultivated in 37 DEG C of incubators,
20 μ L MTT are added under aseptic condition per hole, continue to cultivate 4h, careful abandoning supernatant, 100 μ L dimethyl sulfoxide (DMSO)s are added per hole,
37 DEG C of biochemical cultivation cases hatch 10min, shake up, light absorption value is measured at 570nm with microplate reader.
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 1 biologically active polypeptide NEINQFYQ of table to vitro lymphocyte proliferation
| Experiment packet | Stimulus index SI |
| Negative control group | 1 |
| NEINQFYQ | 1.161±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, the bar for being 100 μ g/mL in the mass concentration of biologically active peptide NEINQFYQ
Under part, the stimulus index of milk-derived biologically active peptide NEINQFYQ is more than BSA, illustrates that NEINQFYQ can stimulate body to a certain extent
The propagation of outer mouse lymphocyte.And the stimulus index of NEINQFYQ has reached 1.161, and negative control group has significance difference
Different (P<0.05).Therefore, it can be assumed that active peptides NEINQFYQ has the ability for remarkably promoting mouse lymphocyte 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, the macrophages in vitro multiplication capacity experiment of mtt assay measure biologically active polypeptide NEINQFYQ
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 NEINQFYQ that embodiment 1 obtains；3- (4,5- dimethylthiazole -2) -2,5- hexichol
Base 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 microplate reader Labsystems companies.
2) test method：
2% (w/w) the sterilizing starch solutions of balb/c mouse peritoneals injection 2ml, continuous injection three days, last time is injected
24 it is small when after break neck 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, from
After heart pipe collects flushing liquor, supernatant is abandoned in centrifugation (1000rpm, 4 DEG C) after ten 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 concentration.
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 when culture 4 is small 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 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 LPLP per hole
The 200 μ l/ holes of RPMI1640 complete culture solutions (10%FBS) of (1mg/ml), continuously cultivate 48h；Negative control group is per hole solubilization
Solution has the 200 μ l/ holes of RPMI1640 complete culture solutions (10%FBS) of BSA (500 μ g/mL)；Blank group addition RPMI1640 is complete
200 μ l/ holes of nutrient solution (10%FBS), continuously cultivate 48h.Also, experimental group, negative control group and blank group are set just respectively again
Often 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
Normal group and inflammation group add 20 μ l/ holes of 5%MTT in 44h；Cell culture, which reaches, adds the three molten of 100 μ l/ holes after 48h
Solution liquid is to terminate culture, after dissolving overnight, surveys the absorbance (OD570) in each hole with microplate reader under wavelength 570nm, growth refers to
The calculation formula of number (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 2 biologically active polypeptide NEINQFYQ of table breeds macrophages in vitro
| Experiment packet | Normal group GI | Inflammation group GI |
| Negative control group | 1 | 1 |
| NEINQFYQ(1mg/ml) | 1.0862±0.0733** | 1.143±0.0246** |
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 NEINQFYQ is added, normally
The macrophage of group and inflammation group has propagation.And compared with negative control group, there is significant difference (P ＜ 0.01).Say
Gelatine/biological activity polypeptide NEINQFYQ has significant proliferation function to macrophages in vitro.
The activity of fighting against senium experiment of 3 biologically active peptide of embodiment
First, experiments of the biologically active polypeptide NEINQFYQ 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, Sinopharm Chemical Reagent Co., Ltd.；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagent
Department；The milk-derived biologically active polypeptide NEINQFYQ 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
By the raising of ICR mouse adaptability after a 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 the dosage nape part of 500mg/kg, and with the dosage gavage biologically active polypeptide in 1mg/ day
NEINQFYQ；Group 2 is high dose gavage group, and D-gal is subcutaneously injected with the dosage nape part of 500mg/kg daily in mouse, and
The day dosage gavage biologically active polypeptide NEINQFYQ of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is animal mould
D-gal is subcutaneously injected with the dosage nape part of 500mg/kg daily in type group, mouse, and the physiology salt that gavage concentration is 0.9%
Water；The injection cycle of D-gal and the gavage cycle of polypeptide are 42 days.Replace bedding and padding within every 3 days, and ensure feed and distilled water
Supply.The every five days weight for weighing a mouse, D-gal parenteral solutions are prepared according to the weight of mouse, and D-gal parenteral solutions pass through
0.22 μm of needle cylinder type filter membrane filtering, 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 advance prepared 4% paraformaldehyde solution, in the form of fixing it.Poly
Formaldehyde powder more indissoluble, can add micro sodium acid carbonate 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 be fixed in 4% paraformaldehyde solution 24 it is small when.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 the long term injections of D-gal.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 is obscured with white pulp boundary, and atrophy occurs in white pulp, shows that the glycometabolism approach of mouse occurs for long-term D-gal injections
Disorder, causes anti-oxidant enzyme activity to reduce, peroxide accumulation, and then may trigger the aging and atrophy of spleen.And gavage is more
Then white pulp atrophy degree is lighter relative to animal model group mouse for the spleen tissue of the mouse of peptide group, and the boundary of red pulp and white pulp
It is more clearly demarcated.This result illustrates, in the injection cycle of whole D-gal, experiment animal sustained is constantly subject to cause senescence-factor
Stimulation, cause the aging and atrophy of spleen.Therefore from the point of view of the situation of changes in microstructure, the biology invented in this experiment
Active peptides NEINQFYQ has certain guarantor to spleen aging of the animal caused by being subject to the stimulation of the bad factor with atrophy
Shield acts on.
2nd, the experiment that biologically active polypeptide NEINQFYQ acts 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, Sinopharm Chemical Reagent Co., Ltd.；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagent
Department；The milk-derived biologically active polypeptide NEINQFYQ that embodiment 1 obtains；BCA protein reagent boxes, Nanjing Keygen Biotech's science and technology are limited
Company；MDA lipid peroxide kits, Science and Technology Ltd. of Nanjing Keygen Biotech；SOD superoxide dismutase kits,
Bio tech ltd is built up in 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
By the raising of ICR mouse adaptability after a 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 the dosage nape part of 500mg/kg, and with the dosage gavage biologically active polypeptide in 1mg/ day
NEINQFYQ；Group 2 is high dose gavage group, and D-gal is subcutaneously injected with the dosage nape part of 500mg/kg daily in mouse, and
The day dosage gavage biologically active polypeptide NEINQFYQ of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is animal mould
D-gal is subcutaneously injected with the dosage nape part of 500mg/kg daily in type group, mouse, and the physiology salt that gavage concentration is 0.9%
Water；The injection cycle of D-gal and the gavage cycle of polypeptide are 42 days.Replace bedding and padding within every 3 days, and ensure feed and distilled water
Supply.The every five days weight for weighing a mouse, D-gal parenteral solutions are prepared according to the weight of mouse, and D-gal parenteral solutions pass through
0.22 μm of needle cylinder type filter membrane filtering, 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 advance prepared 4% paraformaldehyde solution, in the form of fixing it.Poly
Formaldehyde powder more indissoluble, can add micro sodium acid carbonate 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, grind 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 3 each group experimental animal mouse Different Organs of table
Note：* sign is compared with model group, there is significant difference (P<0.05)；* signs are compared 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).Mean although the mouse of polypeptide gavage group is subject to the D-gal of long-term, high-dose
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 being continuously subject to cause the stimulation of senescence-factor, but together
When take in a certain amount of polypeptide NEINQFYQ there is certain protective role to the oxidative damage in Mice Body.
The situation of change of MDA contents in 4 each group experimental animal mouse Different Organs of table
As can be known from Table 4, the liver MDA content of animal model group mouse is 26.84 ± 7.12nmol/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).Since 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 the long term injections of excessive D-gal, 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 lipid peroxide in its liver organization, MDA is as lipid peroxide, it is in animal
The rise of in-vivo content, can reflect the reduction of Antioxidant Enzymes vigor in Mice Body from side.And polypeptide gavage group Mouse Liver
Dirty MDA contents significantly reduce, illustrate polypeptide NEINQFYQ intake can effectively protect vital tissue organ from it is bad because
Son, which stimulates, produces substantial amounts of lipid peroxide.
The situation of change of T-AOC in 5 each group experimental animal Mice Body of table
As known from Table 5, the liver T-AOC values of animal model group mouse are 0.68 ± 0.28U/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 ± 0.23U/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 that, whole
In a experimental period, since experiment animal sustained is constantly stimulated be subject to cause senescence-factor, the liver of animal model group mouse,
Renal tissue is destroyed, and causes the reduction of its total antioxidant capacity.Compared with animal model group and blank group, polypeptide gavage group is small
The total antioxidant capacity of mouse major organs maintains a higher level all the time in the stimulating course for being subject to cause senescence-factor,
Illustrate that taking in biologically active polypeptide NEINQFYQ makes animal body and its major organs have higher self-protection function.
3rd, the experiment acted on immune cell factor in serum of biologically active polypeptide NEINQFYQ
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, Sinopharm Chemical Reagent Co., Ltd.；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagent
Department；The milk-derived biologically active polypeptide NEINQFYQ that embodiment 1 obtains；BCA protein reagent boxes, Nanjing Keygen Biotech's science and technology are limited
Company；ELISA cell factors Quick kit (TNF-α, IL-2 and IL-6), Wuhan Boster Biological Technology 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
By the raising of ICR mouse adaptability after a 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 the dosage nape part of 500mg/kg, and with the dosage gavage biologically active polypeptide in 1mg/ day
NEINQFYQ；Group 2 is high dose gavage group, and D-gal is subcutaneously injected with the dosage nape part of 500mg/kg daily in mouse, and
The day dosage gavage biologically active polypeptide NEINQFYQ of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is animal mould
D-gal is subcutaneously injected with the dosage nape part of 500mg/kg daily in type group, mouse, and the physiology salt that gavage concentration is 0.9%
Water；The injection cycle of D-gal and the gavage cycle of polypeptide are 42 days.Replace bedding and padding within every 3 days, and ensure feed and distilled water
Supply.The every five days weight for weighing a mouse, D-gal parenteral solutions are prepared according to the weight of mouse, and D-gal parenteral solutions pass through
0.22 μm of needle cylinder type filter membrane filtering, 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, mouse blood be stored at room temperature 1 it is small when after, with 3000g centrifuge 15min, separate 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 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 the solution of 1000pg/mL, then serial dilution is 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL,
15.6pg/mL wait various concentrations.Each concentration gradient solution pipettes 100 μ L in the ELISA Plate of coated antibody.Draw mouse
100 μ L of blood serum sample, add in same ELISA Plate (need to press when detecting calculating again if blood serum sample is inadequate, after can suitably diluting
Ratio is converted).ELISA Plate is covered, is placed under 37 DEG C of environment and is incubated 90min.After completion of the reaction, carefully get rid of in ELISA Plate
Liquid, and ELISA Plate is placed on blotting paper, it is careful to pat, remove surplus liquid.By preheated biotin antiantibody work
Make liquid to sequentially add in each hole of ELISA Plate by every 100 μ L of hole, at 37 DEG C, react 60min.After completion of the reaction, utilize 0.01M's
PBS solution is washed 3 times, adds the PBS of 100 μ L in every hole every time, and solution is removed in immersion 1min hypsokinesis, 3 times repeatedly.Will be preheated
ABC working solutions are sequentially added by every hole 0.1ml, 37 DEG C of reaction 30min.After completion of the reaction, 5 times are washed with 0.01M PBS, every time
Soak 1min or so.The TMB nitrite ions for balancing 30min at 37 DEG C are sequentially added by every 90 μ L of hole, 37 DEG C of lucifuges react 8-
12min.TMB terminate liquids are sequentially added by every hole 0.1ml, blueness is vertical at this time turns yellow, and OD values are measured in 450nm with microplate reader.
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, it is bent according to standard
Line can extrapolate the content of cell factor in sample.
3. experimental result and analysis：
The situation of change of cell factor in 6 each group mice serum of table
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 ± 26.31pg/mL, 4.34 ± 0.56pg/mL, compared to the increase (P that conspicuousness is presented in 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 the IL-6 of the mice serum of polypeptide gavage group is effectively controlled with TNF-α content.According to cell because
The experimental result of son, serum levels of inflammatory cytokines IL-6, the secretion level of TNF-α of polypeptide gavage group mouse are below animal mould
Type 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
Suppression to a certain extent；From the perspective of inflammation, mouse inflammation because of caused by oxidation has obtained effective suppression；From declining
From the point of view of old angle, a series of geriatric diseases caused by mouse aging caused by long term injections D-gal are possible to obtain
Control.From Fig. 7's it turns out that, IL-2 as a kind of significant cell factor model group in this experiment for judging aging with
Do not occur conspicuousness effect in gavage group, thus it is speculated that, it may be possible to due to the model employed in this experiment and naturally-aged still
Difference, or modeling cycle fall short of, therefore cause this index not change.
Above example illustrates that biologically active polypeptide NEINQFYQ of the present invention has immunoloregulation function and anti-aging work(
Energy.
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 easily can make these embodiments various modifications, 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 disclose according to the present invention, do not depart from improvement that scope made and modification all should be the present invention's
Within protection domain.
Sequence table
<110>Shanghai Communications University；Zhejiang Hui Tai life and healths Science and Technology Ltd.
<120>A kind of biologically active polypeptide NEINQFYQ and its preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Asn Glu Ile Asn Gln Phe Tyr Gln
1 5
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
aatgaaatca atcagtttta tcag 24
<210> 3
<211> 222
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Met Lys Phe Phe Ile Phe Thr Cys Leu Leu Ala Val Ala Leu Ala Lys
1 5 10 15
Asn Thr Met Glu His Val Ser Ser Ser Glu Glu Ser Ile Ile Ser Gln
20 25 30
Glu Thr Tyr Lys Gln Glu Lys Asn Met Ala Ile Asn Pro Ser Lys Glu
35 40 45
Asn Leu Cys Ser Thr Phe Cys Lys Glu Val Val Arg Asn Ala Asn Glu
50 55 60
Glu Glu Tyr Ser Ile Gly Ser Ser Ser Glu Glu Ser Ala Glu Val Ala
65 70 75 80
Thr Glu Glu Val Lys Ile Thr Val Asp Asp Lys His Tyr Gln Lys Ala
85 90 95
Leu Asn Glu Ile Asn Gln Phe Tyr Gln Lys Phe Pro Gln Tyr Leu Gln
100 105 110
Tyr Leu Tyr Gln Gly Pro Ile Val Leu Asn Pro Trp Asp Gln Val Lys
115 120 125
Arg Asn Ala Val Pro Ile Thr Pro Thr Leu Asn Arg Glu Gln Leu Ser
130 135 140
Thr Ser Glu Glu Asn Ser Lys Lys Thr Val Asp Met Glu Ser Thr Glu
145 150 155 160
Val Phe Thr Lys Lys Thr Lys Leu Thr Glu Glu Glu Lys Asn Arg Leu
165 170 175
Asn Phe Leu Lys Lys Ile Ser Gln Arg Tyr Gln Lys Phe Ala Leu Pro
180 185 190
Gln Tyr Leu Lys Thr Val Tyr Gln His Gln Lys Ala Met Lys Pro Trp
195 200 205
Ile Gln Pro Lys Thr Lys Val Ile Pro Tyr Val Arg Tyr Leu
210 215 220
Claims (10)
1. a kind of biologically active polypeptide NEINQFYQ, it is characterised in that its amino acid sequence is Asn-Glu-Ile-Asn-Gln-
Phe-Tyr-Gln。
A kind of 2. biologically active polypeptide NEINQFYQ according to claim 1, it is characterised in that the biologically active polypeptide
For milk-derived.
3. encode the nucleotide fragments of biologically active polypeptide NEINQFYQ described in claim 1, it is characterised in that the nucleotide
The sequence of fragment such as SEQ ID NO：Shown in 2.
4. the preparation method of biologically active polypeptide NEINQFYQ as claimed in claim 1, it is characterised in that pass through genetic engineering
Method is artificial synthesized, or is directly obtained from dairy products by the method isolated and purified, or is directly prepared by chemical synthesis.
5. the application of biologically active polypeptide NEINQFYQ as claimed in claim 1, it is characterised in that the biologically active polypeptide
Applications of the NEINQFYQ in the food with immunoloregulation function, health products, medicine or cosmetics are prepared.
6. the application of biologically active polypeptide NEINQFYQ as claimed in claim 1, it is characterised in that the biologically active polypeptide
Applications of the NEINQFYQ in the food with anti-senescence function, health products or medicine is prepared.
7. the application of biologically active polypeptide NEINQFYQ as claimed in claim 1, it is characterised in that the biologically active polypeptide
Applications of the NEINQFYQ in the food with immunoloregulation function and anti-senescence function, health products or medicine is prepared.
8. a kind of immunological regulation product, it is characterised in that including biologically active polypeptide NEINQFYQ as claimed in claim 1 or institute
State the derivative of biologically active polypeptide NEINQFYQ；The immunological regulation product includes immunological regulation food, immunological regulation is protected
Strong product, immunoregulation medicament or immunological regulation cosmetics；The derivative of the biologically active polypeptide NEINQFYQ, refers in biology
On the amino acid side groups of active peptides NEINQFYQ, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, first
Base, 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 NEINQFYQ or described as claimed in claim 1
The derivative of biologically active polypeptide NEINQFYQ；The anti-aging product includes antisenility cistanche food, antisenescence health product or anti-
Senescence drug；The derivative of the biologically active polypeptide NEINQFYQ, refers to the amino acid in biologically active polypeptide NEINQFYQ
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 immunoloregulation function and anti-senescence function, it is characterised in that including as claimed in claim 1
The derivative of biologically active polypeptide NEINQFYQ or described biologically active polypeptides NEINQFYQ；With immunoloregulation function and anti-ageing
The product of old function includes food, health products or medicine；The derivative of the biologically active polypeptide NEINQFYQ, refers in biology
On the amino acid side groups of active peptides NEINQFYQ, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, first
Base, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
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Cited By (1)
| 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 |
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|---|---|---|---|---|
| CN104710524A (en) * | 2014-12-19 | 2015-06-17 | 上海交通大学 | Bovine alpha s2-casein source bioactive peptides preparation and application thereof |
| CN105254740A (en) * | 2015-10-16 | 2016-01-20 | 上海交通大学 | Bioactive polypeptide NQFYQKF as well as preparation and application thereof |
| CN105254750A (en) * | 2015-10-16 | 2016-01-20 | 上海交通大学 | Bioactive polypeptide FGYSGAFKCL as well as preparation and application thereof |
| CN107177000A (en) * | 2017-07-06 | 2017-09-19 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide DRAAHVKQVL and its preparation method and application |
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2017
- 2017-11-14 CN CN201711125056.XA patent/CN107903315A/en not_active Withdrawn
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104710524A (en) * | 2014-12-19 | 2015-06-17 | 上海交通大学 | Bovine alpha s2-casein source bioactive peptides preparation and application thereof |
| CN105254740A (en) * | 2015-10-16 | 2016-01-20 | 上海交通大学 | Bioactive polypeptide NQFYQKF as well as preparation and application thereof |
| CN105254750A (en) * | 2015-10-16 | 2016-01-20 | 上海交通大学 | Bioactive polypeptide FGYSGAFKCL as well as preparation and application thereof |
| CN107177000A (en) * | 2017-07-06 | 2017-09-19 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide DRAAHVKQVL and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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