CN107827971A - A kind of biologically active polypeptide QSLTLTDVE and its preparation method and application - Google Patents
A kind of biologically active polypeptide QSLTLTDVE and its preparation method and application Download PDFInfo
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- CN107827971A CN107827971A CN201711288372.9A CN201711288372A CN107827971A CN 107827971 A CN107827971 A CN 107827971A CN 201711288372 A CN201711288372 A CN 201711288372A CN 107827971 A CN107827971 A CN 107827971A
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- qsltltdve
- biologically active
- active polypeptide
- polypeptide
- inflammatory
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Classifications
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- 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
- 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
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (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 QSLTLTDVE and its preparation method and application, biologically active polypeptide QSLTLTDVE is Gln Ser Leu Thr Leu Thr Asp Val Glu.By extracorporeal anti-inflammatory activity experiment, internal Antisenility Experiment, demonstrating polypeptide QSLTLTDVE has preferable anti-inflammatory activity and activity of fighting against senium, on the one hand, the biologically active polypeptide QSLTLTDVE 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 the body incidence of disease；On the other hand, the vigor of internal anti-peroxidation enzyme system can be improved, strengthen the function of body resistance external source sexual stimulus, so as to reduce organism aging process, aging and sick probability, exploitation is of great significance with 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 QSLTLTDVE and preparation method thereof and
Using.
Background technology
During cow's milk is through lactobacillus-fermented, a part of protein in cow's milk is metabolized by lactic acid bacteria to be utilized, concurrently
A series of biochemical reactions have been given birth to, protein is changed into polypeptide or free amino acid, is digested or passes through
The absorption and transport of intestinal epithelial cell is directly entered the blood circulation of human body.In these polypeptides, some has special
Physiological function, it is referred to as " biologically active peptide ".
It is particularly important that safe biologically active peptide is found in natural food source.In the last few years, it has been found that some foods
The polypeptides matter in thing source has good bioactivity, such as corn small peptide, Soybean Peptide, cow's milk polypeptide.These polypeptides can
To be obtained by number of ways such as microbial fermentation, digestion enzymolysis, and the polypeptide with bioactivity is by 2~20 mostly
Amino acid residue forms, and molecular weight is less than 6000Da, contains a certain amount of hydrophobic amino acid, aromatic amino acid.
Immune-active peptides are to obtain and prove that one kind biology of its physiologically active is living from breast first after opioid peptides discovery
Property polypeptide.Jolles in 1981 et al. has found first, using trypsin hydrolysis people lactoprotein, can obtain an amino acid sequence
Val-Glu-Pro-Ile-Pro-Tyr hexapeptide is classified as, experiment in vitro proves that the peptide can strengthen Turnover of Mouse Peritoneal Macrophages pair
The phagocytosis of sheep red blood cell (SRBC).Migliore-Samour et al. has found the hexapeptide Thr-Thr-Met-Pro- from casein
Leu-Trp can stimulate phagocytosis and enhancing of the sheep erythrocyte to mouse peritoneal macrophages for kerekou pneumonia primary
The resistance of bacterium, there are 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 anti-inflammatory properties that the phagocytosis of phagocyte is related to 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, promote 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 the body incidence of disease, 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 life-spans of these organisms and reach
As many as 6 times.
Anti-aging peptide in terms of physiological function there is amino acid can not compare excellent as a kind of emerging antidotal agent
Gesture, it can produce promotion or inhibitory action to the enzyme in organism, improve absorption and the profit to mineral matter and other nutrients
With, removing interior free yl, the resistance to oxidation of enhancing body itself, with anti-aging.Therefore, the nutrition and health care of biologically active peptide
Effect has turned into the emphasis of domestic and foreign scholars subject study.Qiu Juan et al. pass through experimental studies have found that, milk-derived bioactive micro peptide
Life span of drosophila melanogaster can effectively be extended, delay its aging, and also there is preferable antioxidation, thus it is speculated that be probably wherein to be rich in coloured glaze
Base peptides.SOD vigor in serum, reduces its lipid in discovery bovine colostrum extract energy conspicuousness raising the elderly's body such as the brightness in week
Peroxide and enhancing body resistance to oxidation, have certain anti-senescence function.
The research on biologically active polypeptide has much at present, for example Chinese patent CN105254738A discloses one kind and come
The milk-derived biologically active polypeptide DELQDKIH of beta-casein is come from, Chinese patent CN105254739A discloses one kind and derived 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.
United States Patent (USP) US20090285936A1 discloses a kind of lactobacillus-fermented product albumen matter beta-casein-H, has
Excellent meals function.Beta-casein-the H of the patented invention is also a part for bovine casein, from bovine casein.
Beta-casein-H belongs to macro-molecular protein disclosed in the patent, and its property digested and assimilated is poor.It which disclose this simultaneously
Protein has excellent meals function, does not disclose whether it has other performances.
The content of the invention
It is an object of the invention to provide a kind of biologically active polypeptide QSLTLTDVE 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 QSLTLTDVE, its amino acid sequence are Gln-Ser-
Leu-Thr-Leu-Thr-Asp-Val-Glu, such as SEQ ID NO：Shown in 1.
Preferably, the biologically active polypeptide is milk-derived.Beta-casein is derive specifically from, and is beta-casein variant
The amino acid residue that B is the 123rd~131.Beta-casein variant B amino acid sequences such as SEQ ID NO：Shown in 3.
The amino acid sequence of beta-casein and corresponding nucleotides sequence are classified as existing technology, encoding ss-casein variant B
The biologically active polypeptide QSLTLTDVE of the nucleotide fragments energy encoding mature of 123rd~131 amino acids residue.
Preferably, the biologically active polypeptide has anti-inflammatory properties and anti-senescence function.
Second aspect of the present invention, there is provided encode the nucleotide fragments of the biologically active polypeptide QSLTLTDVE, its sequence
For：5 '-cag agc ttg act ttg act gat gtt gaa-3 ', such as SEQ ID NO：Shown in 2.
Third aspect present invention, there is provided the preparation method of the biologically active polypeptide QSLTLTDVE, gene can be passed through
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 QSLTLTDVE is preparing the food with anti-inflammatory properties
Application in product, health products, medicine or cosmetics.
Fifth aspect present invention, there is provided the biologically active polypeptide QSLTLTDVE is being prepared with anti-senescence function
Application in food, health products or medicine.
Sixth aspect present invention, there is provided the biologically active polypeptide QSLTLTDVE is being prepared while had anti-inflammatory properties
With the application in the food, health products or medicine of anti-senescence function.
Specifically, biologically active polypeptide QSLTLTDVE of the invention, which can be used for preparing, reduces free radical to skin wound
The harmful medicine of cosmetics, preparation with anti-inflammatory and/or anti-aging；And due to the biologically active polypeptide of the present invention
Product after QSLTLTDVE 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 anti-inflammatory and/or anti-aging.
Seventh aspect present invention, there is provided a kind of anti-inflammatory products, including the biologically active polypeptide QSLTLTDVE or described
Biologically active polypeptide QSLTLTDVE derivative；Described 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 QSLTLTDVE, refers to biologically active polypeptide QSLTLTDVE's
On amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation,
The modification such as esterification or glycosylation, obtained polypeptide derivative.
Eighth aspect present invention, there is provided a kind of anti-aging product, including the biologically active polypeptide QSLTLTDVE or institute
State biologically active polypeptide QSLTLTDVE derivative；Described anti-aging product include antisenility cistanche food, antisenescence health product or
Antiaging agent；The derivative of the biologically active polypeptide QSLTLTDVE, refers to the ammonia in biologically active polypeptide QSLTLTDVE
On base acid side-chain radical, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, ester
The modification such as change 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
Biologically active polypeptide QSLTLTDVE or described biologically active polypeptides QSLTLTDVE derivative；With anti-inflammatory properties and anti-aging
The product of function includes food, health products or medicine；The derivative of the biologically active polypeptide QSLTLTDVE, refers in biology
On active peptides QSLTLTDVE amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, first
Base, acetylation, phosphorylation, esterification or glycosylation etc. are modified, obtained polypeptide derivative.
Biologically active polypeptide QSLTLTDVE's of the present invention has the beneficial effect that：The milk-derived biologically active polypeptide of the present invention
QSLTLTDVE has preferable anti-inflammatory activity and activity of fighting against senium；On the one hand, biologically active polypeptide QSLTLTDVE of the invention
Factor of Macrophage can be promoted, promote the increase of the macrophage nitric oxide amount of inducing, raising body is resisted outer
The ability of boundary's pathogenic infection, reduce the body incidence of disease；On the other hand, it is possible to increase the vigor of internal anti-peroxidation enzyme system, increase
The function of strong body resistance external source sexual stimulus, so as to reduce organism aging process, aging and sick probability, there is anti-inflammatory work(to exploitation
Energy, the food of anti-senescence function, health products and medicine tool are of great significance.
The application polypeptide (such as beta-casein-H in background technology) with prior art structurally and functionally having essence
Difference：Biologically active polypeptide QSLTLTDVE of the present invention is a kind of small molecule bioactive peptide, belongs to core fragment, and the present invention is raw
Thing active peptides QSLTLTDVE has more the property digested and assimilated.Biologically active polypeptide QSLTLTDVE of the present invention has anti-inflammatory simultaneously
And anti-senescence function, there is significant difference with disclosed polypeptide in the prior art on functional activity.
Brief description of the drawings
Fig. 1：Mass chromatography extraction figure (m/z=1005.5126)；
Fig. 2：Mass-to-charge ratio is the second order mses figure of 1005.5126 fragment；
Fig. 3：Mass-to-charge ratio is 1005.5126 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 end points.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except used in embodiment specific method, equipment,
Outside material, according to grasp of the those skilled in the art to prior art and the record of the present invention, it can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Unless otherwise indicated, disclosed in this invention experimental method, detection method, preparation method using this technology lead
Domain conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987 and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
The active peptide QSLTLTDVE's of embodiment 1 is artificial synthesized
First, the synthesis of biologically active peptide
1. RINK resin 3g (substitution value 0.3mmol/g) are weighed in 150ml reactor, with 50ml dichloromethane
(DCM) soak.
After 2.2 hours, resin is washed with nitrogen-dimethylformamide (DMF) of 3 times of resin volumes, is then drained, so weight
It is multiple four times, resin is drained rear stand-by.
3. a certain amount of 20% piperidines (piperidines/DMF=1 is added into reactor:4,v:V), it is placed on decolorization swinging table and shakes
20min is shaken, the Fmoc blocking groups on resin are sloughed with this.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 Gln in right amount and 1- hydroxyls-benzene a pair of horses going side by side triazole (HOBT) is in right amount in 50ml centrifuge tube, addition
20ml DMF is dissolved, and then adds 3ml N, and N DICs (DIC) vibration shakes up 1min, treats that solution is clear
It is added to after clear in reactor, then reactor is placed in 30 DEG C of shaking table and reacted.
After 6.2 hours, with a certain amount of acetic anhydride end socket (acetic anhydride:DIEA:DCM=1:1:2,v:v:V) half an hour, so
Washed four times, drained stand-by with the DMF of 3 times of resin volumes afterwards.
7. a certain amount of 20% piperidines (piperidines/DMF=1 is added into reactor:4, v:V), it is placed on decolorization swinging table and shakes
20min is shaken, the Fmoc blocking groups on resin are sloughed with this.Washed four times with DMF after having taken off protection, then drained.
8. take the detection of a small amount of resin ninhydrin (nine well ninhydrins) method (each two drop of inspection A, inspection B, 100 DEG C of reactions
1min), resin has color, illustrates to be deprotected successfully.
9. weighing second amino acid next in right amount and HOBT being in right amount in 50ml centrifuge tube, 25ml DMF generals are added
It dissolves, and the DIC vibrations for then adding 2.5ml shake up 1min, are added to after solution clarification in reactor, then by reactor
It is placed in 30 DEG C of shaking table and reacts.
After 10.1 hours, take a small amount of resin to detect, (each two drop of inspection A, inspection B, 100 DEG C of reactions are detected with ninhydrin method
1min), if resin is colourless, illustrate that reaction is complete；If resin has color, illustrate that condensation is incomplete, continue to react.
After 11. question response is complete, washs resin four times with DMF, then drain, a certain amount of 20% is added into reactor
Piperidines (piperidines/DMF=1:4,v:V), it is placed on decolorization swinging table and rocks 20min, the Fmoc protection groups on resin is sloughed with this
Group.Washed four times with DMF after having taken off protection, then drain whether detection protection sloughs.
12. amino acid Ser, Leu, Thr, Leu, Thr, Asp, Val and Glu 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 QSLTLTDVE.
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 QSLTLTDVE carries out chromatography and mass spectral analysis, and its mass chromatography extraction figure is as shown in figure 1, extract the two level matter at this peak
As shown in Figures 2 and 3, the polypeptide mass-to-charge ratio that can obtain this peak is 1005.5126Da, and retention time is for spectrogram and az, by crack conditions
49.8min。
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
1005.5126Da fragment sequence is Gln-Ser-Leu-Thr-Leu-Thr-Asp-Val-Glu (QSLTLTDVE), is designated as SEQ
ID NO：1.The fragment and beta-casein variant B the 123rd~131 residue sequence is corresponding, beta-casein amino acid sequence
GenBank numbering be AAA30431.1, sequence is shown in SEQ ID NO：3.
The anti-inflammatory activity experiment of the biologically active peptide of embodiment 2
First, the experiment (ELISA method) of biologically active polypeptide QSLTLTDVE rush Factor of Macrophage
1. experiment reagent and instrument：
Reagent：Experimental animal balb/c mouse (male 6-8 week old), Shanghai Slac Experimental Animal Co., Ltd.；Mouse
Lymphocyte extract 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
QSLTLTDVE；ELISA cell factors Quick kit (TNF-α, IL-1 β and IL-6), the limited public affairs of Wuhan doctor's moral bioengineering
Department.
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 ELIASA Labsystems companies.
2. experimental method：
It is 2 × 10 to add number of cells6/ ml μ l/ the holes of cell suspension 100, it is adherent to add after purification containing peptide
The μ l/ holes of RPMI1640 complete culture solutions (10%FBS) 200, inflammation group added LPS to the μ g/ml of final concentration 10 at 24 hours, even
Continuous culture 48 hours, inflammation group add LPS to final concentration 100ng/ml in 24 hours 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, react 30 minutes.Nitrite ion is added after PBS washings, is reacted 20 minutes.After adding colour developing terminate liquid, using ELIASA in ripple
Absorbance (OD450) is determined under long 450nm.
3. experimental result and analysis：
The measure that the biologically active peptide QSLTLTDVE of table 1 influences on Macrophage Cell factor level
Note：*, compared with negative control, there is significant difference (P ＜ 0.05)；*, compared with negative control group, have significantly
Sex differernce (P ＜ 0.01)
As can be known from Table 1, TNF-α, IL-1 β and IL-6 these three cell factors experimental result in, 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 QSLTLTDVE under finite concentration can promote the Turnover of Mouse Peritoneal Macrophages to activate and discharge TNF-α, IL-1 β,
IL-6, floors of these cell factors under normal macrophages quiescent condition is improved, so as to adjust the immunity of body.
2nd, the measure (Griess methods) of the biologically active polypeptide QSLTLTDVE rush macrophage nitric oxide amount of inducing
1. experiment reagent and instrument：
Reagent：Experimental animal balb/c mouse (male 6-8 week old) Shanghai Communications University is agriculture real with biological institute animal
Test center；The milk-derived biologically active polypeptide QSLTLTDVE that embodiment 1 obtains；LPS, purchased from Sigma companies；Neutral red staining
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 ELIASA Labsystems companies.
2. test method：
It is 2 × 10 to add number of cells6/ ml μ l/ the holes of cell suspension 100, it is adherent to add after purification containing peptide
The μ l/ holes of RPMI1640 complete culture solutions (10%FBS) 200, inflammation group add LPS to the μ g/ml of final concentration 10 in 24h, continuously
After cultivating 48h, the μ l/ holes of nutrient solution supernatant 50 are collected, add Griess reagents 1 and Griess reagents successively in nutrient solution supernatant
2 each 50 μ l/ holes, after reacting at room temperature 10 minutes, absorbance (OD540) is determined under 540nm wavelength.
3. experimental result and analysis：
The biologically active polypeptide QSLTLTDVE of table 2 promotees the measure of the macrophage nitric oxide amount of inducing
| Experiment packet | Normal group | Inflammation group |
| Cell blank | 0.0592±0.00525 | 0.3241±0.0381 |
| QSLTLTDVE 1mg/ml | 0.1318±0.0247** | 0.4975±0.0227** |
| QSLTLTDVE 0.5mg/ml | 0.1285±0.0568** | 0.3276±0.0373** |
| QSLTLTDVE 0.1mg/ml | 0.2588±0.0696** |
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, as shown in Table 2, biologically active polypeptide QSLTLTDVE, concentration difference is added in experimental group
For 1mg/mL and 0.5mg/mL, the one of the promotion macrophage grown under inflammatory conditions is made with LPS for growing under normal circumstances
The nitrogen oxide amount of inducing has facilitation.Compared with cell blank group, there is significant difference (P<0.05).When bioactivity is more
Peptide QSLTLTDVE addition concentration is 0.1mg/mL, is compared in the case where LPS makes inflammatory conditions, also macrophage one can be promoted to aoxidize
The increase of the nitrogen amount of inducing, and there is significant difference (P<0.05).But compared with the cell blank group grown under normal circumstances,
There is no significant difference.Illustrate that biologically active polypeptide QSLTLTDVE has under the conditions of finite concentration and promote the oxygen of macrophage one
Change the increased ability of the nitrogen amount of inducing.
The activity of fighting against senium experiment of the biologically active peptide of embodiment 3
First, experiments of the biologically active polypeptide QSLTLTDVE to internal spleen tissue structure function
1. experiment reagent and instrument：
Reagent：Experimental animal ICR mouse (male 5 week old), Shanghai City Experimental Animal Center；D-gal, Chinese medicines group chemistry
Reagent Co., Ltd；Paraformaldehyde, Chemical Reagent Co., Ltd., Sinopharm Group；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagent
Department；The milk-derived biologically active polypeptide QSLTLTDVE that embodiment 1 obtains.
Instrument and equipment：The ultra-clean water of CM-230 types mole, Shanghai Moller scientific instrument Co., Ltd；Mi Libo Millipore
MILLEX GP0.22 μm filter membranes, Millipore Corp. of the U.S.；GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument centrifuge instruments
Co., Ltd.
2. experimental method：
(1) Animal Aging model modeling
After ICR mouse adaptability is raised one week, it is divided into 4 groups, every group 6.Group 1 is low dosage gavage group, and mouse is daily
D-gal is subcutaneously injected with 500mg/kg dosage nape part, and with the dosage gavage biologically active polypeptide in 1mg/ day
QSLTLTDVE；Group 2 is high dose gavage group, and D-gal is subcutaneously injected with 500mg/kg dosage nape part daily in mouse, and
The day dosage gavage biologically active polypeptide QSLTLTDVE of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is animal
Model group, D-gal is subcutaneously injected with 500mg/kg dosage nape part daily in mouse, and gavage concentration is 0.9% physiology
Salt solution；D-gal injection cycle and the gavage cycle of polypeptide are 42 days.Bedding and padding are changed, and ensure feed and distilled water within every 3 days
Supply.The every five days body weight for weighing a mouse, D-gal parenteral solutions are prepared according to the body 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 4% paraformaldehyde solution prepared in advance, in the form of fixing it.Poly
Formaldehyde powder more indissoluble, micro sodium acid carbonate can be added and adjust pH value to alkalescence, with hydrotropy.Paraformaldehyde solution
Preparing needs to complete in fume hood.
(3) sample detection
The making of histotomy：Mouse spleen sample need at least fix 24 hours in 4% paraformaldehyde solution.Spleen group
The wax stone knitted makes, section entrusts Shanghai Wei Ao bio tech ltd to complete with HE dyeing.
3. experimental result and analysis：
In this experiment, 4 groups of mouse are shared, the mouse normal growth of wherein blank group is not affected by any environmental stimuli, its
3 groups of mouse of remaininging receive D-gal long term injections.Using light microscope, the spleen section of separate groups of mice is seen
Examine, can be found from Fig. 4, contrast the spleen section of each group mouse, for naive mice, the spleen of animal model mouse
Dirty red pulp obscures with white pulp boundary, and atrophy occurs in white pulp, shows that the glycometabolism approach of mouse occurs for long-term D-gal injections
Disorder, cause anti-oxidant enzyme activity to reduce, peroxide accumulation, and then the aging and atrophy of spleen may have been triggered.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 whole D-gal injection cycle, experiment animal sustained is constantly by 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 QSLTLTDVE has certain guarantor to spleen aging of the animal caused by the stimulation by the bad factor with atrophy
Shield acts on.
2nd, the experiment that biologically active polypeptide QSLTLTDVE is acted on intracorporeal organ antioxidant levels
1. experiment reagent and instrument：
Reagent：Experimental animal ICR mouse (male 5 week old), Shanghai City Experimental Animal Center；D-gal, Chinese medicines group chemistry
Reagent Co., Ltd；Paraformaldehyde, Chemical Reagent Co., Ltd., Sinopharm Group；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagent
Department；The milk-derived biologically active polypeptide QSLTLTDVE that embodiment 1 obtains；BCA protein reagent boxes, Nanjing Keygen Biotech's science and technology have
Limit company；MDA MDA kits, Science and Technology Ltd. of Nanjing Keygen Biotech；SOD superoxide dismutase reagents
Bio tech ltd is built up in box, Nanjing；T-AOC antioxidative activities kits, bio tech ltd is built up in Nanjing.
Instrument and equipment：The ultra-clean water of CM-230 types mole, Shanghai Moller scientific instrument Co., Ltd；Mi Libo Millipore
MILLEX GP0.22 μm filter membranes, Millipore Corp. of the U.S.；GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument centrifuge instruments
Co., Ltd.
2. experimental method：
(1) Animal Aging model modeling
After ICR mouse adaptability is raised one week, it is divided into 4 groups, every group 6.Group 1 is low dosage gavage group, and mouse is daily
D-gal is subcutaneously injected with 500mg/kg dosage nape part, and with the dosage gavage biologically active polypeptide in 1mg/ day
QSLTLTDVE；Group 2 is high dose gavage group, and D-gal is subcutaneously injected with 500mg/kg dosage nape part daily in mouse, and
The day dosage gavage biologically active polypeptide QSLTLTDVE of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is animal
Model group, D-gal is subcutaneously injected with 500mg/kg dosage nape part daily in mouse, and gavage concentration is 0.9% physiology
Salt solution；D-gal injection cycle and the gavage cycle of polypeptide are 42 days.Bedding and padding are changed, and ensure feed and distilled water within every 3 days
Supply.The every five days body weight for weighing a mouse, D-gal parenteral solutions are prepared according to the body 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 4% paraformaldehyde solution prepared in advance, in the form of fixing it.Poly
Formaldehyde powder more indissoluble, micro sodium acid carbonate can be added and adjust pH value to alkalescence, with hydrotropy.Paraformaldehyde solution
Preparing needs to complete in fume hood.
(3) sample detection
All internal organs that need to be detected, are ground at low ambient temperatures, and 10% group is diluted to 4 DEG C of sterile PBS solutions
Knit homogenate, under the conditions of 4 DEG C after 4000g centrifugations, take supernatant, discard precipitation, operated according to kit specification, or put
It is to be measured in -80 DEG C of refrigerators.
3. experimental result and analysis：
The change of SOD contents in each group experimental animal mouse Different Organs of table 3
Note：* sign compares with model group, there is significant difference (P<0.05)；* signs compare with model group,
There is significant difference (P<0.01), similarly hereinafter.
As can be known from Table 3, relative to animal model group mouse, the SOD in the liver and kidney of polypeptide gavage group mouse contains
Increase (the P of conspicuousness is presented in amount<0.01).Although mean D-gal of the mouse by long-term, high-dose of polypeptide gavage group
Stimulation, even if D-gal excess injection also without completely destroy Mice Body in SOD enzyme systems, illustrate in injection cycle, test
Animal can cause the reduction of SOD contents in Different Organs in the case of continuously by the stimulation for causing senescence-factor, but together
When take in a certain amount of polypeptide QSLTLTDVE there is certain protective role to the oxidative damage in Mice Body.
The situation of change of MDA contents in each group experimental animal mouse Different Organs of table 4
As can be known from Table 4, the liver MDA content of animal model group mouse is 26.86 ± 7.04nmol/L, with animal model
Group compares, and significant difference (P is presented in the MDA contents in two groups of mouse livers of polypeptide gavage<0.01).Because MDA can be used for
Estimate the accumulation situation of animal body lipid peroxide, therefore it follows that animal model group mouse is causing aging model to be formed
During, due to excess D-gal long term injections, the glycometabolism approach of mouse is got muddled, produce a large amount of free radicals from
And oxidative damage is caused, occur a large amount of MDAs in its liver organization, MDA is as MDA, and it is in animal
The rise of in-vivo content, the reduction of Antioxidant Enzymes vigor in Mice Body can be reflected from side.And polypeptide gavage group Mouse Liver
Dirty MDA contents significantly reduce, illustrate polypeptide QSLTLTDVE intake can effectively protect vital tissue organ from it is bad because
Son, which stimulates, produces substantial amounts of MDA.
T-AOC situation of change in each group experimental animal Mice Body of table 5
As known from Table 5, the liver T-AOC values of animal model group mouse are 0.68 ± 0.21U/mgprot, are compared to mould
Significant difference (P is presented with low dosage gavage group mouse in type group, polypeptide high dose therewith<0.05)；The kidney of naive mice
Dirty T-AOC contents are 0.61 ± 0.25U/mgprot, and compared with animal model group mouse, low dosage gavage group presents aobvious therewith
Write sex differernce (P<0.05), significant difference (P is also presented in high dose gavage group mouse therewith<0.01).This result shows, whole
In individual experimental period, because experiment animal sustained is constantly stimulated by cause senescence-factor, the liver of animal model group mouse,
Renal tissue is destroyed, and causes the reduction of its TAC.Compared with animal model group and blank group, polypeptide gavage group is small
The TAC of mouse major organs maintains a higher level all the time in by the stimulating course for causing senescence-factor,
Illustrate that taking in biologically active polypeptide QSLTLTDVE makes animal body and its major organs have higher self-protection function.
3rd, the biologically active polypeptide QSLTLTDVE experiment acted on immune cell factor in serum
1. experiment reagent and instrument：
Reagent：Experimental animal ICR mouse (male 5 week old), Shanghai City Experimental Animal Center；D-gal, Chinese medicines group chemistry
Reagent Co., Ltd；Paraformaldehyde, Chemical Reagent Co., Ltd., Sinopharm Group；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagent
Department；The milk-derived biologically active polypeptide QSLTLTDVE that embodiment 1 obtains；BCA protein reagent boxes, Nanjing Keygen Biotech's science and technology have
Limit 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
After ICR mouse adaptability is raised one week, it is divided into 4 groups, every group 6.Group 1 is low dosage gavage group, and mouse is daily
D-gal is subcutaneously injected with 500mg/kg dosage nape part, and with the dosage gavage biologically active polypeptide in 1mg/ day
QSLTLTDVE；Group 2 is high dose gavage group, and D-gal is subcutaneously injected with 500mg/kg dosage nape part daily in mouse, and
The day dosage gavage biologically active polypeptide QSLTLTDVE of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is animal
Model group, D-gal is subcutaneously injected with 500mg/kg dosage nape part daily in mouse, and gavage concentration is 0.9% physiology
Salt solution；D-gal injection cycle and the gavage cycle of polypeptide are 42 days.Bedding and padding are changed, and ensure feed and distilled water within every 3 days
Supply.The every five days body weight for weighing a mouse, D-gal parenteral solutions are prepared according to the body 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, after mouse blood is stored at room temperature 1 hour, 15min is centrifuged with 3000g, separates blood
Clearly.Serum keeping is to be checked in -80 DEG C of refrigerators.All operations during disposal experimental animal follow the Ministry of Science and Technology in 2006
Issue《On treating the guiding opinion of experimental animal kindly》.The mouse spleen won directly is soaked in what is prepared in advance
In 4% paraformaldehyde solution, in the form of fixing it.Paraformaldehyde powder more indissoluble, can add micro sodium acid carbonate will
PH value is adjusted to alkalescence, with hydrotropy.The preparation of paraformaldehyde solution needs to complete in fume hood.
(3) sample detection
Indicated according to kit specification, draw standard curve first, standard items powder is prepared with standard dilutions
Into 1000pg/mL solution, then serial dilution is 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/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
The μ L of blood serum sample 100, 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 μ L of hole 100, at 37 DEG C, react 60min.After completion of the reaction, utilize 0.01M's
PBS solution is washed 3 times, adds 100 μ L PBS 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 μ L of hole 90,37 DEG C of lucifuges react 8-
12min.TMB terminate liquids are sequentially added by every hole 0.1ml, now blueness is vertical turns yellow, and OD values are determined in 450nm with ELIASA.
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 each group mice serum of table 6
IL-6 and TNF-α content are respectively in the model group Mice Body being can be found that from table 6, Fig. 5, Fig. 6 in this experiment
168.01 ± 26.38pg/mL, 4.34 ± 0.76pg/mL, the increase (P of conspicuousness is presented compared to normal group<0.01), therefore
It is considered that due to continuously injecting cause senescence-factor, animal model group mouse is caused aging occur in cell factor aspect
The symptom of property inflammation, and 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.Found from Fig. 7 result, IL-2 as a kind of significant cell factor for judging aging model group in this experiment 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 can easily make various modifications to these embodiments, and described herein general
Principle is applied in other embodiment without by performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability
Field technique personnel do not depart from improvement that scope made and modification all should be the present invention's according to the announcement of the present invention
Within protection domain.
Sequence table
<110>Zhejiang Hui Tai life and healths Science and Technology Ltd.；Shanghai Bo Hui bio tech ltd
<120>A kind of biologically active polypeptide QSLTLTDVE and its preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Gln Ser Leu Thr Leu Thr Asp Val Glu
1 5
<210> 2
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cagagcttga ctttgactga tgttgaa 27
<210> 3
<211> 209
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Arg Glu Leu Glu Glu Leu Asn Val Pro Gly Glu Ile Val Glu Ser Leu
1 5 10 15
Ser Ser Ser Glu Glu Ser Ile Thr Arg Ile Asn Lys Lys Ile Glu Lys
20 25 30
Phe Gln Ser Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln Asp Lys
35 40 45
Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly
50 55 60
Pro Ile Pro Asn Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr
65 70 75 80
Pro Val Val Val Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser
85 90 95
Lys Val Lys Glu Ala Met Ala Pro Lys His Lys Glu Met Pro Phe Pro
100 105 110
Lys Tyr Pro Val Glu Pro Phe Thr Glu Arg Gln Ser Leu Thr Leu Thr
115 120 125
Asp Val Glu Asn Leu His Leu Pro Leu Pro Leu Leu Gln Ser Trp Met
130 135 140
His Gln Pro His Gln Pro Leu Pro Pro Thr Val Met Phe Pro Pro Gln
145 150 155 160
Ser Val Leu Ser Leu Ser Gln Ser Lys Val Leu Pro Val Pro Gln Lys
165 170 175
Ala Val Pro Tyr Pro Gln Arg Asp Met Pro Ile Gln Ala Phe Leu Leu
180 185 190
Tyr Gln Glu Pro Val Leu Gly Pro Val Arg Gly Pro Phe Pro Ile Ile
195 200 205
Val
Claims (10)
1. a kind of biologically active polypeptide QSLTLTDVE, it is characterised in that its amino acid sequence is Gln-Ser-Leu-Thr-Leu-
Thr-Asp-Val-Glu。
2. a kind of biologically active polypeptide QSLTLTDVE according to claim 1, it is characterised in that the bioactivity is more
Peptide is milk-derived.
3. encode the nucleotide fragments of biologically active polypeptide QSLTLTDVE described in claim 1, it is characterised in that the nucleosides
The sequence of acid fragment such as SEQ ID NO：Shown in 2.
4. biologically active polypeptide QSLTLTDVE as claimed in claim 1 preparation method, it is characterised in that pass through genetic engineering
Method it is artificial synthesized, or directly obtained from dairy products by the method isolated and purified, or directly prepared by chemical synthesis.
5. biologically active polypeptide QSLTLTDVE as claimed in claim 1 application, it is characterised in that the biologically active polypeptide
Applications of the QSLTLTDVE in the food with anti-inflammatory properties, health products, medicine or cosmetics are prepared.
6. biologically active polypeptide QSLTLTDVE as claimed in claim 1 application, it is characterised in that the biologically active polypeptide
Applications of the QSLTLTDVE in the food with anti-senescence function, health products or medicine is prepared.
7. biologically active polypeptide QSLTLTDVE as claimed in claim 1 application, it is characterised in that the biologically active polypeptide
Applications of the QSLTLTDVE in the food with anti-inflammatory properties and anti-senescence function, health products or medicine is prepared.
8. a kind of anti-inflammatory products, it is characterised in that including biologically active polypeptide QSLTLTDVE or described as claimed in claim 1
Biologically active polypeptide QSLTLTDVE derivative；Described 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 QSLTLTDVE, refers to biologically active polypeptide QSLTLTDVE's
On amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, 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 QSLTLTDVE as claimed in claim 1 or institute
State biologically active polypeptide QSLTLTDVE derivative；Described anti-aging product include antisenility cistanche food, antisenescence health product or
Antiaging agent；The derivative of the biologically active polypeptide QSLTLTDVE, refers to the ammonia in biologically active polypeptide QSLTLTDVE
On base acid side-chain radical, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, ester
Change 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
Active peptides QSLTLTDVE or described biologically active polypeptides QSLTLTDVE derivative；With anti-inflammatory properties and anti-senescence function
Product include food, health products or medicine；The derivative of the biologically active polypeptide QSLTLTDVE, refers in bioactivity
On polypeptide QSLTLTDVE amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, methyl
Change, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1359157A1 (en) * | 2002-04-29 | 2003-11-05 | Société des Produits Nestlé S.A. | Metallo-proteinase inhibitory agent |
| WO2005081628A2 (en) * | 2004-03-01 | 2005-09-09 | Peptera Pharmaceutical Ltd. | Casein derived peptides and therapeutic uses thereof |
| WO2008004794A1 (en) * | 2006-07-03 | 2008-01-10 | Sang Kee Han | New functional fermented milk (yogurt) for use in dieting |
| CN107176995A (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 SKVLPVPEKAVPYPQ and its preparation method and application |
| CN107200780A (en) * | 2017-07-06 | 2017-09-26 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide LVYPFPG and its preparation method and application |
-
2017
- 2017-12-07 CN CN201711288372.9A patent/CN107827971B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1359157A1 (en) * | 2002-04-29 | 2003-11-05 | Société des Produits Nestlé S.A. | Metallo-proteinase inhibitory agent |
| WO2005081628A2 (en) * | 2004-03-01 | 2005-09-09 | Peptera Pharmaceutical Ltd. | Casein derived peptides and therapeutic uses thereof |
| WO2008004794A1 (en) * | 2006-07-03 | 2008-01-10 | Sang Kee Han | New functional fermented milk (yogurt) for use in dieting |
| CN107176995A (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 SKVLPVPEKAVPYPQ and its preparation method and application |
| CN107200780A (en) * | 2017-07-06 | 2017-09-26 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide LVYPFPG and its preparation method and application |
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