CN108017708A - A kind of biologically active polypeptide NPIGSENSEKTTMPL and its preparation method and application - Google Patents
A kind of biologically active polypeptide NPIGSENSEKTTMPL and its preparation method and application Download PDFInfo
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- CN108017708A CN108017708A CN201711319285.5A CN201711319285A CN108017708A CN 108017708 A CN108017708 A CN 108017708A CN 201711319285 A CN201711319285 A CN 201711319285A CN 108017708 A CN108017708 A CN 108017708A
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- Prior art keywords
- npigsensekttmpl
- biologically active
- active polypeptide
- polypeptide
- inflammatory
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- 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
-
- 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
-
- 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)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Birds (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Toxicology (AREA)
- Dermatology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to albumen field, more particularly to a kind of biologically active polypeptide NPIGSENSEKTTMPL and its preparation method and application, its amino acid sequence of biologically active polypeptide NPIGSENSEKTTMPL is Asn Pro Ile Gly Ser Glu Asn Ser Glu Lys Thr Thr Met Pro Leu.By extracorporeal anti-inflammatory activity experiment, internal Antisenility Experiment, demonstrating polypeptide NPIGSENSEKTTMPL has preferable anti-inflammatory activity and activity of fighting against senium, on the one hand, the biologically active polypeptide NPIGSENSEKTTMPL of the present invention can promote Factor of Macrophage, promote the increase of the macrophage nitric oxide amount of inducing, 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 anti-inflammatory properties, 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 NPIGSENSEKTTMPL and its preparation
Methods and applications.
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, has anti-inflammatory properties.Li Su duckweeds et al. find that rat abdominal cavity is huge with newborn source peptide (PGPIPN) the feeding rat of synthesis
The phagocytosis of phagocyte and the relevant anti-inflammatory properties 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, the increase for promoting the macrophage nitric oxide amount of inducing, raising machine
Body resists the ability of extraneous pathogenic infection, reduces body incidence, and will not cause the immunological rejection of body.
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 NPIGSENSEKTTMPL and preparation method thereof and answer
With.
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 NPIGSENSEKTTMPL, its amino acid sequence are Asn-
Pro-Ile-Gly-Ser-Glu-Asn-Ser-Glu-Lys-Thr-Thr-Met-Pro-Leu, such as SEQ ID NO：Shown in 1.
Preferably, the biologically active polypeptide is milk-derived.α s1- caseins are derive specifically from, and are α s1- caseins
The amino acid residue that variation B is the 184th~198.α s1- ss-casein variants B amino acid sequences such as SEQ ID NO：Shown in 3.
The amino acid sequence and corresponding nucleotides sequence of α s1- caseins are classified as existing technology, and coding for alpha s1- caseins become
The biologically active polypeptide NPIGSENSEKTTMPL of the nucleotide fragments energy encoding mature of the 184th~198 amino acids residues of body B.
Preferably, the biologically active polypeptide has anti-inflammatory properties and anti-senescence function.
Second aspect of the present invention, there is provided the nucleotide fragments of the biologically active polypeptide NPIGSENSEKTTMPL are encoded,
Its sequence is：5 '-aat cct att ggc tct gag aac agt gaa aag act act atg cca ctg-3 ', such as
SEQ ID NO：Shown in 2.
Third aspect present invention, there is provided the preparation method of the biologically active polypeptide NPIGSENSEKTTMPL, Ke Yitong
The method for crossing genetic engineering is artificial synthesized, can be directly obtained, can directly led to by the method isolated and purified from dairy products
Cross chemical synthesis preparation.
Fourth aspect present invention, there is provided the biologically active polypeptide NPIGSENSEKTTMPL has anti-inflammatory work(in preparation
Can food, health products, the application in medicine or cosmetics.
Fifth aspect present invention, there is provided the biologically active polypeptide NPIGSENSEKTTMPL has anti-aging in preparation
Application in the food of function, health products or medicine.
Sixth aspect present invention, there is provided the biologically active polypeptide NPIGSENSEKTTMPL is being prepared while had anti-
Application in the food of scorching function and anti-senescence function, health products or medicine.
Specifically, biologically active polypeptide NPIGSENSEKTTMPL of the invention, which can be used for preparing, reduces free radical pair
The cosmetics of skin damage, prepare the medicine with anti-inflammatory and/or anti-aging；And due to the biologically active polypeptide of the present invention
Product after NPIGSENSEKTTMPL is degraded by intestines and stomach still has bioactivity, therefore can be also used for preparing Yoghourt
Deng food, the health products of adjusting immunity, and the oral medicine being used to prepare with anti-inflammatory and/or anti-aging.
Seventh aspect present invention, there is provided a kind of anti-inflammatory products, including the biologically active polypeptide NPIGSENSEKTTMPL
Or the derivative of the biologically active polypeptide NPIGSENSEKTTMPL；The anti-inflammatory products include anti-inflammatory food, anti-inflammatory health
Product, anti-inflammatory drug or anti-inflammatory cosmetics；The derivative of the biologically active polypeptide NPIGSENSEKTTMPL, refers to living in biology
Property polypeptide NPIGSENSEKTTMPL amino acid side groups on, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonyl
Change, 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
The derivative of NPIGSENSEKTTMPL or described biologically active polypeptides NPIGSENSEKTTMPL；The anti-aging product includes
Antisenility cistanche food, antisenescence health product or antiaging agent；The derivative of the biologically active polypeptide NPIGSENSEKTTMPL,
Refer on the amino acid side groups of biologically active polypeptide NPIGSENSEKTTMPL, aminoterminal or c-terminus carry out hydroxylating,
Carboxylated, be carbonylated, methylate, acetylation, phosphorylation, the modification such as esterification or glycosylation, obtained polypeptide derivative.
Ninth aspect present invention, there is provided product a kind of while that there is anti-inflammatory properties and anti-senescence function, including it is described
The derivative of biologically active polypeptide NPIGSENSEKTTMPL or described biologically active polypeptides NPIGSENSEKTTMPL；With anti-inflammatory
The product of function and anti-senescence function includes food, health products or medicine；The biologically active polypeptide NPIGSENSEKTTMPL's
Derivative, refers on the amino acid side groups of biologically active polypeptide NPIGSENSEKTTMPL, aminoterminal or c-terminus carry out
Hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, modification, the obtained polypeptide such as esterification or glycosylation derive
Thing.
Biologically active polypeptide NPIGSENSEKTTMPL's of the present invention has the beneficial effect that：The milk-derived bioactivity of the present invention
Polypeptide NPIGSENSEKTTMPL has preferable anti-inflammatory activity and activity of fighting against senium；On the one hand, biologically active polypeptide of the invention
NPIGSENSEKTTMPL can promote Factor of Macrophage, promote the increase of the macrophage nitric oxide amount of inducing,
The ability that body resists extraneous pathogenic infection is improved, reduces body incidence；On the other hand, it is possible to increase internal anti-peroxidation
The vigor of enzyme system, the function of enhancing body resistance external source sexual stimulus, so that organism aging process, aging and sick probability are reduced, it is right
Exploitation is of great significance with anti-inflammatory properties, the food of anti-senescence function, health products and medicine tool.
Brief description of the drawings
Fig. 1：Mass chromatography extraction figure (m/z=809.3987)；
Fig. 2：Mass-to-charge ratio is the second order ms figure of 809.3987 fragment；
Fig. 3：Mass-to-charge ratio is 809.3987 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 NPIGSENSEKTTMPL'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
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 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,
Then washed four times, drained stand-by with the DMF of 3 times of resin volumes.
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. according to step 9-11 connect successively amino acid Pro, Ile, Gly, Ser, Glu, Asn, Ser, Glu, Lys, Thr,
Thr, Met, Pro and Leu.
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 NPIGSENSEKTTMPL.
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 NPIGSENSEKTTMPL carries out chromatography and mass spectral analysis, its mass chromatography extraction figure is as shown in Figure 1, extract this peak
As shown in Figures 2 and 3, the polypeptide mass-to-charge ratio that can obtain this peak is 809.3987Da for second order ms figure and az, by crack conditions, during reservation
Between be 33.6min.
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
809.3987Da fragment sequence be Asn-Pro-Ile-Gly-Ser-Glu-Asn-Ser-Glu-Lys-Thr-Thr-Met-
Pro-Leu (NPIGSENSEKTTMPL), is denoted as SEQ ID NO：1.The fragment and α s1- ss-casein variants B the 184th~198
Residue sequence it is corresponding, the GenBank of α s1- casamino acid sequences numbering is AAA30428.1, and sequence is shown in SEQ ID
NO：3.
The anti-inflammatory activity experiment of 2 biologically active peptide of embodiment
First, the experiment (ELISA method) of the rush Factor of Macrophage of biologically active polypeptide NPIGSENSEKTTMPL
1. experiment reagent and instrument：
Reagent：Experimental animal balb/c mouse (male 6-8 week old), Shanghai Slac Experimental Animal Co., Ltd.；Mouse
Lymphocyte extracting solution, Shanghai Suo Laibao bio tech ltd；RPMI1640 culture mediums, GIBCO companies；Bovine serum albumin
(bovine serum albumin, BSA) in vain, Genebase companies；The milk-derived biologically active polypeptide that embodiment 1 obtains
NPIGSENSEKTTMPL；ELISA cell factors Quick kit (TNF-α, IL-1 β and IL-6), Wuhan doctor's moral bioengineering
Co., Ltd.
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 150CO2 incubator Heraeus companies；Dragon Wellscan
MK3 microplate reader Labsystems companies.
2. experimental method：
It is 2 × 10 to add number of cells6The 100 μ l/ holes of cell suspension of/ml, it is adherent to add after purification containing peptide
200 μ l/ holes of RPMI1640 complete culture solutions (10%FBS), inflammation group added LPS to 10 μ g/ml of final concentration at 24 hours, even
When continuous culture 48 is small, inflammation group adds LPS to final concentration 100ng/ml when 24 is small before culture terminates.After culture terminates, centrifugation
Collect cell culture supernatant liquid.100 μ l supernatants, 37 DEG C of reactions 90 are added in the ELISA Plate of coated cell factor antibody
After minute, biotin labelled antibodies are added, 37 DEG C are reacted 60 minutes, and after PBS washings, it is compound to add Avidin-peroxidase
Thing, reacts 30 minutes.Nitrite ion is added after PBS washings, is reacted 20 minutes.After adding colour developing terminate liquid, using microplate reader in ripple
Absorbance (OD450) is measured under long 450nm.
3. experimental result and analysis：
The measure that 1 biologically active peptide NPIGSENSEKTTMPL of table influences Macrophage Cell factor level
Note：*, compared with negative control, there is significant difference (P ＜ 0.05)；*, compared with negative control group, has significantly
Sex differernce (P ＜ 0.01)
As can be known from Table 1, in TNF-α, the experimental result of IL-1 β and IL-6 these three cell factors, TNF-α, IL-1 β
In 0.2mg/ml and significant difference (P ＜ 0.01) is appeared above, IL-6 significant difference (P ＜ occurs in 0.5mg/ml
0.01), it was demonstrated that the NPIGSENSEKTTMPL under a certain concentration can promote the Turnover of Mouse Peritoneal Macrophages to activate and discharge TNF-α,
IL-1 β, IL-6, improve floors of these cell factors under normal macrophages quiescent condition, so as to adjust the immune of body
Power.
2nd, the measure (Griess of the rush macrophage nitric oxide amount of inducing of biologically active polypeptide NPIGSENSEKTTMPL
Method)
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 NPIGSENSEKTTMPL that embodiment 1 obtains；LPS, purchased from Sigma companies；It is neutral
Red colouring liquid, green skies biotechnology research institute production.
Instrument and equipment：LRH-250F biochemical cultivation cases Shanghai perseverance Science and Technology Ltd.；On GL-22M high speed freezing centrifuges
Hai Luxiang instrument centrifuges Instrument Ltd.；Hera cell 150CO2 incubator Heraeus companies；Dragon Wellscan
MK3 microplate reader Labsystems companies.
2. test method：
It is 2 × 10 to add number of cells6The 100 μ l/ holes of cell suspension of/ml, it is adherent to add after purification containing peptide
200 μ l/ holes of RPMI1640 complete culture solutions (10%FBS), inflammation group add LPS to 10 μ g/ml of final concentration in 24h, continuously
After cultivating 48h, 50 μ l/ holes of nutrient solution supernatant are collected, add Griess reagents 1 and Griess reagents successively in nutrient solution supernatant
2 each 50 μ l/ holes, room temperature reaction after ten minutes, measure absorbance (OD540) under 540nm wavelength.
3. experimental result and analysis：
2 biologically active polypeptide NPIGSENSEKTTMPL of table promotees the measure of the macrophage nitric oxide amount of inducing
Note：*, compared with negative control, there is significant difference (P ＜ 0.05)；
*, compared with negative control group, there is significant difference (P ＜ 0.01)
Experimental result is shown in Table 2, and as shown in Table 2, addition biologically active polypeptide NPIGSENSEKTTMPL, dense in experimental group
Degree is respectively 1mg/mL and 0.5mg/mL, thin for growing the promotion macrophage made with LPS and grown under inflammatory conditions under normal circumstances
The nitric oxide amount of inducing of born of the same parents has facilitation.Compared with cell blank group, there is significant difference (P<0.05).Work as biology
The addition concentration of active peptides NPIGSENSEKTTMPL is 0.1mg/mL, is compared in the case where LPS makes inflammatory conditions, can also be promoted huge
The increase of the phagocyte nitric oxide amount of inducing, and there is significant difference (P<0.05).It is but thin with growing under normal circumstances
Born of the same parents' blank group is compared, without significant difference.Illustrate that biologically active polypeptide NPIGSENSEKTTMPL has under the conditions of a certain concentration
Have and promote the increased ability of the macrophage nitric oxide amount of inducing.
The activity of fighting against senium experiment of 3 biologically active peptide of embodiment
First, experiments of the biologically active polypeptide NPIGSENSEKTTMPL 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 NPIGSENSEKTTMPL 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
NPIGSENSEKTTMPL；Group 2 is high dose gavage group, and D- is subcutaneously injected with the dosage nape part of 500mg/kg daily in mouse
Gal, and the day dosage gavage biologically active polypeptide NPIGSENSEKTTMPL of 3mg/ only；Group 3 is blank group, and mouse is normal
Growth；Group 4 is animal model group, and D-gal, and gavage concentration is subcutaneously injected with the dosage nape part of 500mg/kg daily in mouse
For 0.9% physiological saline；The injection cycle of D-gal and the gavage cycle of polypeptide are 42 days.Bedding and padding are replaced, and are protected within every 3 days
Demonstrate,prove the supply of feed and distilled water.The every five days weight for weighing a mouse, D-gal parenteral solutions are prepared according to the weight of mouse,
It is sterile to ensure and D-gal parenteral solutions are filtered through 0.22 μm of needle cylinder type filter membrane.
(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 NPIGSENSEKTTMPL has one to spleen aging of the animal caused by being subject to the stimulation of the bad factor with atrophy
Fixed protective effect.
2nd, the experiment that biologically active polypeptide NPIGSENSEKTTMPL 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 NPIGSENSEKTTMPL that embodiment 1 obtains；BCA protein reagent boxes, Nanjing Keygen Biotech
Science and Technology Ltd.；MDA lipid peroxide kits, Science and Technology Ltd. of Nanjing Keygen Biotech；SOD superoxide dismutases
Bio tech ltd is built up in kit, Nanjing；The limited public affairs of biotechnology are built up in T-AOC antioxidative activities kits, Nanjing
Department.
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
NPIGSENSEKTTMPL；Group 2 is high dose gavage group, and D- is subcutaneously injected with the dosage nape part of 500mg/kg daily in mouse
Gal, and the day dosage gavage biologically active polypeptide NPIGSENSEKTTMPL of 3mg/ only；Group 3 is blank group, and mouse is normal
Growth；Group 4 is animal model group, and D-gal, and gavage concentration is subcutaneously injected with the dosage nape part of 500mg/kg daily in mouse
For 0.9% physiological saline；The injection cycle of D-gal and the gavage cycle of polypeptide are 42 days.Bedding and padding are replaced, and are protected within every 3 days
Demonstrate,prove the supply of feed and distilled water.The every five days weight for weighing a mouse, D-gal parenteral solutions are prepared according to the weight of mouse,
It is sterile to ensure and D-gal parenteral solutions are filtered through 0.22 μm of needle cylinder type filter membrane.
(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 NPIGSENSEKTTMPL 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 26.86 ± 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).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 NPIGSENSEKTTMPL intake can effectively protect vital tissue organ from
The bad factor, 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 ± 0.21U/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 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 NPIGSENSEKTTMPL makes animal body and its major organs have higher self-protection work(
Energy.
3rd, the experiment acted on immune cell factor in serum of biologically active polypeptide NPIGSENSEKTTMPL
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 NPIGSENSEKTTMPL that embodiment 1 obtains；BCA protein reagent boxes, Nanjing Keygen Biotech
Science and Technology Ltd.；ELISA cell factors Quick kit (TNF-α, IL-2 and IL-6), Wuhan doctor's moral bioengineering are limited
Company.
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
NPIGSENSEKTTMPL；Group 2 is high dose gavage group, and D- is subcutaneously injected with the dosage nape part of 500mg/kg daily in mouse
Gal, and the day dosage gavage biologically active polypeptide NPIGSENSEKTTMPL of 3mg/ only；Group 3 is blank group, and mouse is normal
Growth；Group 4 is animal model group, and D-gal, and gavage concentration is subcutaneously injected with the dosage nape part of 500mg/kg daily in mouse
For 0.9% physiological saline；The injection cycle of D-gal and the gavage cycle of polypeptide are 42 days.Bedding and padding are replaced, and are protected within every 3 days
Demonstrate,prove the supply of feed and distilled water.The every five days weight for weighing a mouse, D-gal parenteral solutions are prepared according to the weight of mouse,
It is sterile to ensure and D-gal parenteral solutions are filtered through 0.22 μm of needle cylinder type filter membrane.
(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 ± 26.38pg/mL, 4.34 ± 0.76pg/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.
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>Zhejiang Hui Tai life and healths Science and Technology Ltd.；Shanghai Bo Hui bio tech ltd
<120>A kind of biologically active polypeptide NPIGSENSEKTTMPL and its preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Asn Pro Ile Gly Ser Glu Asn Ser Glu Lys Thr Thr Met Pro Leu
1 5 10 15
<210> 2
<211> 45
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
aatcctattg gctctgagaa cagtgaaaag actactatgc cactg 45
<210> 3
<211> 199
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Arg Pro Lys His Pro Ile Lys His Gln Gly Leu Pro Gln Glu Val Leu
1 5 10 15
Asn Glu Asn Leu Leu Arg Phe Phe Val Ala Pro Phe Pro Gln Val Phe
20 25 30
Gly Lys Glu Lys Val Asn Glu Leu Ser Lys Asp Ile Gly Ser Glu Ser
35 40 45
Thr Glu Asp Gln Ala Met Glu Asp Ile Lys Glu Met Glu Ala Glu Ser
50 55 60
Ile Ser Ser Ser Glu Glu Ile Val Pro Asn Ser Val Glu Gln Lys His
65 70 75 80
Ile Gln Lys Glu Asp Val Pro Ser Glu Arg Tyr Leu Gly Tyr Leu Glu
85 90 95
Gln Leu Leu Arg Leu Lys Lys Tyr Lys Val Pro Gln Leu Glu Ile Val
100 105 110
Pro Asn Ser Ala Glu Glu Arg Leu His Ser Met Lys Gln Gly Ile His
115 120 125
Ala Gln Gln Lys Glu Pro Met Ile Gly Val Asn Gln Glu Leu Ala Tyr
130 135 140
Phe Tyr Pro Glu Leu Phe Arg Gln Phe Tyr Gln Leu Asp Ala Tyr Pro
145 150 155 160
Ser Gly Ala Trp Tyr Tyr Val Pro Leu Gly Thr Gln Tyr Thr Asp Ala
165 170 175
Pro Ser Phe Ser Asp Ile Pro Asn Pro Ile Gly Ser Glu Asn Ser Glu
180 185 190
Lys Thr Thr Met Pro Leu Trp
195
Claims (10)
1. a kind of biologically active polypeptide NPIGSENSEKTTMPL, it is characterised in that its amino acid sequence is Asn-Pro-Ile-
Gly-Ser-Glu-Asn-Ser-Glu-Lys-Thr-Thr-Met-Pro-Leu。
A kind of 2. biologically active polypeptide NPIGSENSEKTTMPL according to claim 1, it is characterised in that the biology
Active peptides are milk-derived.
3. encode the nucleotide fragments of biologically active polypeptide NPIGSENSEKTTMPL described in claim 1, it is characterised in that institute
State the sequence such as SEQ ID NO of nucleotide fragments：Shown in 2.
4. the preparation method of biologically active polypeptide NPIGSENSEKTTMPL as claimed in claim 1, it is characterised in that pass through base
Because the method for engineering is artificial synthesized, or directly obtained by the method isolated and purified from dairy products, or directly closed by chemistry
Into preparation.
5. the application of biologically active polypeptide NPIGSENSEKTTMPL as claimed in claim 1, it is characterised in that the biology is living
Property polypeptide NPIGSENSEKTTMPL preparing with the application in the food of anti-inflammatory properties, health products, medicine or cosmetics.
6. the application of biologically active polypeptide NPIGSENSEKTTMPL as claimed in claim 1, it is characterised in that the biology is living
Property polypeptide NPIGSENSEKTTMPL preparing with the application in the food of anti-senescence function, health products or medicine.
7. the application of biologically active polypeptide NPIGSENSEKTTMPL as claimed in claim 1, it is characterised in that the biology is living
Property polypeptide NPIGSENSEKTTMPL in the food with anti-inflammatory properties and anti-senescence function, health products or medicine is prepared should
With.
8. a kind of anti-inflammatory products, it is characterised in that including biologically active polypeptide NPIGSENSEKTTMPL as claimed in claim 1
Or the derivative of the biologically active polypeptide NPIGSENSEKTTMPL；The anti-inflammatory products include anti-inflammatory food, anti-inflammatory health
Product, anti-inflammatory drug or anti-inflammatory cosmetics；The derivative of the biologically active polypeptide NPIGSENSEKTTMPL, refers to living in biology
Property polypeptide NPIGSENSEKTTMPL amino acid side groups on, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonyl
Change, 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 as claimed in claim 1
The derivative of NPIGSENSEKTTMPL or described biologically active polypeptides NPIGSENSEKTTMPL；The anti-aging product includes
Antisenility cistanche food, antisenescence health product or antiaging agent；The derivative of the biologically active polypeptide NPIGSENSEKTTMPL,
Refer on the amino acid side groups of biologically active polypeptide NPIGSENSEKTTMPL, aminoterminal or c-terminus carry out hydroxylating,
Carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
10. a kind of product with anti-inflammatory properties and anti-senescence function, it is characterised in that including biology as claimed in claim 1
The derivative of active peptides NPIGSENSEKTTMPL or described biologically active polypeptides NPIGSENSEKTTMPL；With anti-inflammatory properties
Include food, health products or medicine with the product of anti-senescence function；The derivative of the biologically active polypeptide NPIGSENSEKTTMPL
Thing, refers on the amino acid side groups of biologically active polypeptide NPIGSENSEKTTMPL, aminoterminal or c-terminus carry out hydroxyl
Change, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005081628A2 (en) * | 2004-03-01 | 2005-09-09 | Peptera Pharmaceutical Ltd. | Casein derived peptides and therapeutic uses thereof |
| WO2009138762A2 (en) * | 2008-05-15 | 2009-11-19 | Regen Therapeutics Plc | Therapeutic use of peptides |
-
2017
- 2017-12-12 CN CN201711319285.5A patent/CN108017708A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005081628A2 (en) * | 2004-03-01 | 2005-09-09 | Peptera Pharmaceutical Ltd. | Casein derived peptides and therapeutic uses thereof |
| WO2009138762A2 (en) * | 2008-05-15 | 2009-11-19 | Regen Therapeutics Plc | Therapeutic use of peptides |
Non-Patent Citations (1)
| Title |
|---|
| Y. JIN ET AL.: "Peptide profiling and the bioactivity character of yogurt in the simulated gastrointestinal digestion", 《JOURNAL OF PROTEOMICS》 * |
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