CN108822193A - A kind of biologically active polypeptide VMTMLDK and its preparation method and application - Google Patents
A kind of biologically active polypeptide VMTMLDK and its preparation method and application Download PDFInfo
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- CN108822193A CN108822193A CN201810715249.9A CN201810715249A CN108822193A CN 108822193 A CN108822193 A CN 108822193A CN 201810715249 A CN201810715249 A CN 201810715249A CN 108822193 A CN108822193 A CN 108822193A
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- vmtmldk
- biologically active
- active polypeptide
- polypeptide
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Classifications
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
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- Chemical & Material Sciences (AREA)
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- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
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- Birds (AREA)
- Mycology (AREA)
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- 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 fields, and in particular to a kind of biologically active polypeptide VMTMLDK and its preparation method and application, its amino acid sequence of biologically active polypeptide VMTMLDK are Val-Met-Thr-Met-Leu-Asp-Lys.By the experiment of ion vitro immunization function point analysis, internal Antisenility Experiment, polypeptide VMTMLDK is demonstrated with preferable immunoloregulation function and activity of fighting against senium, on the one hand, biologically active polypeptide VMTMLDK of the invention can enhance the in-vitro multiplication ability of lymphocyte and macrophage, the ability that body resists extraneous pathogenic infection is improved, body disease incidence is reduced；On the other hand, it can be improved the vigor of internal anti-peroxidation enzyme system, enhance body and resist the function of external source sexual stimulus, to reduce organism aging process, aging and sick probability, there is immunoloregulation function, the food of anti-senescence function, health care product and drug to have a very important significance exploitation.
Description
Technical field
The present invention relates to albumen fields, more particularly, to a kind of biologically active polypeptide VMTMLDK and preparation method thereof and answer
With.
Background technique
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, so that protein is become 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.There is also the eggs of itself some synthesis for lactic acid bacteria thallus
White matter polypeptide fragment is utilized for bacterial growth.In these polypeptides, some is referred to as " raw with special physiological function
Object 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 object 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 enzymatic hydrolysis, and biologically active polypeptide is by 2~20 mostly
Amino acid residue composition, molecular weight are less than 6000Da, contain 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 cream for the first time after opioid peptides discovery
Property polypeptide.Jolles in 1981 et al. has found for the first time, using trypsin hydrolysis people lactoprotein, an available amino acid sequence
It is classified as the hexapeptide of Val-Glu-Pro-Ile-Pro-Tyr, experiment in vitro proves that the peptide can enhance Turnover of Mouse Peritoneal Macrophages pair
The phagocytosis of sheep red blood cell (SRBC).Migliore-Samour et al. has found the hexapeptide Thr-Thr-Met-Pro- from casein
Leu-Trp can stimulate sheep erythrocyte to the phagocytosis of mouse peritoneal macrophages and enhancing for kerekou pneumonia primary
The resistance of bacterium.Newborn source immunomodulatory peptides (PGPIPN) the feeding rat of Li Su duckweed et al. synthesis finds rat peritoneal macrophages
Phagocytosis and the relevant immunoloregulation function of red blood cell have significant enhancing.
Studies have shown that immune-active peptides can not only enhance immunity of organisms, the proliferation of body lymphocyte, enhancing are stimulated
The phagocytic function of macrophage promotes the release of cell factor, improves the ability that body resists extraneous pathogenic infection, reduces machine
Body disease incidence, and the immunological rejection of body will not be caused.
Aging is a natural phenomena, and process is often accompanied by the variation of antioxidant levels, organ-tissue, immune factor,
Complicated variation, such as the trend that 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 pass through utilize some model organisms, as mouse,
The term single gene mutating experiment of drosophila and caenorhabditis elegan etc. finds that some genes can dramatically increase service life 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 can generate promotion or inhibiting effect to the intracorporal enzyme of biology, improve absorption and benefit to minerals and other nutrients
With removing interior free yl enhances the resistance to oxidation of body itself, to delay senescence.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. warp 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 may be wherein 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.
Have much about the research of biologically active polypeptide at present, for example Chinese patent CN105254738A discloses one kind and comes
Derived from the milk-derived biologically active polypeptide DELQDKIH of beta-casein, Chinese patent CN105254739A discloses one kind and derives from
Milk-derived the biologically active polypeptide GTQYTD, Chinese patent CN105254740A of α s1- casein disclose a kind of from α s2-
The milk-derived biologically active polypeptide NQFYQKF of casein.
Summary of the invention
The purpose of the present invention is to provide a kind of biologically active polypeptide VMTMLDK and its preparation method and application.
The purpose of the present invention can be achieved through the following technical solutions：
First aspect present invention provides a kind of biologically active polypeptide VMTMLDK, amino acid sequence Val-Met-Thr-
Met-Leu-Asp-Lys, such as SEQ ID NO：Shown in 1.
Preferably, the biologically active polypeptide derives from Lactobacillus helveticus mycoprotein.It is derive specifically from LBH_1801 |
m.863 LBH_1801|g.863 ORF LBH_1801|g.863 LBH_1801|m.863 type:complete len:181
(+)LBH_1801:659-1201 (+), and the 85th~91, albumen amino acid residue thus.LBH_1801|m.863
LBH_1801|g.863 ORF LBH_1801|g.863 LBH_1801|m.863 type:complete len:181(+)LBH_
1801:659-1201 (+) protein amino acid sequence such as SEQ ID NO：Shown in 3.
LBH_1801|m.863 LBH_1801|g.863 ORF LBH_1801|g.863 LBH_1801|m.863 type:
complete len:181(+)LBH_1801:The amino acid sequence and corresponding nucleotides sequence of 659-1201 (+) albumen are classified as
Existing technology encodes the biologically active polypeptide of the nucleotide fragments energy encoding mature of this 85th~91 amino acids residue of albumen
VMTMLDK。
Preferably, the biologically active polypeptide has immunoloregulation function and anti-senescence function.
Second aspect of the present invention provides the nucleotide fragments for encoding the biologically active polypeptide VMTMLDK, sequence
For：5 '-taa tga cga tgc ttg ata aat-3 ', such as SEQ ID NO：Shown in 2.
Third aspect present invention provides the preparation method of the biologically active polypeptide VMTMLDK, can pass through gene work
The method of journey is artificial synthesized, can be directly obtained from dairy products by the method isolated and purified, can directly be closed by chemistry
At preparation.
Fourth aspect present invention, providing the biologically active polypeptide VMTMLDK in preparation has immunoloregulation function
Application in food, health care product, drug or cosmetics.
Fifth aspect present invention provides the food that the biologically active polypeptide VMTMLDK has anti-senescence function in preparation
Application in product, health care product or drug.
Sixth aspect present invention provides the biologically active polypeptide VMTMLDK in preparation while having immunological regulation function
It can be with the application in the food, health care product or drug of anti-senescence function.
Specifically, biologically active polypeptide VMTMLDK of the invention, which can be used for preparing, reduces free radical to skin damage
Cosmetics, preparation there is the drug of immunological regulation and/or anti-aging；And due to biologically active polypeptide of the invention
Product after VMTMLDK is degraded by gastrointestinal tract still has bioactivity, therefore can be also used for preparing the food such as Yoghourt, adjust
Save the health care product of immunity, and the oral drug being used to prepare with immunological regulation and/or anti-aging.
Seventh aspect present invention provides a kind of immunological regulation product, including the biologically active polypeptide VMTMLDK or institute
State the derivative of biologically active polypeptide VMTMLDK；The immunological regulation product includes immunological regulation food, immunological regulation health care
Product, immunoregulation medicament or immunological regulation cosmetics；The derivative of the biologically active polypeptide VMTMLDK refers to living in biology
Property polypeptide VMTMLDK amino acid side groups on, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, methyl
The modification such as change, acetylation, phosphorylation, esterification or glycosylation, obtained polypeptide derivative.
Eighth aspect present invention provides a kind of anti-aging product, including the biologically active polypeptide VMTMLDK or described
The derivative of biologically active polypeptide VMTMLDK；The anti-aging product includes antisenility cistanche food, antisenescence health product or anti-ageing
Old drug；The derivative of the biologically active polypeptide VMTMLDK, refers to the amino acid side chain in biologically active polypeptide VMTMLDK
On group, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosyl
The modification such as change, obtained polypeptide derivative.
Ninth aspect present invention provides product that is a kind of while having immunoloregulation function and anti-senescence function, including
The derivative of the biologically active polypeptide VMTMLDK or the biologically active polypeptide VMTMLDK；With immunoloregulation function and resist
The product of aging function includes food, health care product or drug；The derivative of the biologically active polypeptide VMTMLDK, refers in life
On the amino acid side groups of object active peptides VMTMLDK, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, first
The modification such as base, acetylation, phosphorylation, esterification or glycosylation, obtained polypeptide derivative.
Biologically active polypeptide VMTMLDK's of the present invention has the beneficial effect that：Biologically active polypeptide VMTMLDK of the invention has
It is preferable to adjust immunity of organisms activity and activity of fighting against senium；On the one hand, biologically active polypeptide VMTMLDK of the invention can increase
The in-vitro multiplication ability of strong lymphocyte and macrophage improves the ability that body resists extraneous pathogenic infection, reduces body
Disease incidence；On the other hand, can be improved the vigor of internal anti-peroxidation enzyme system, enhancing body resists the function of external source sexual stimulus,
To reduce organism aging process, aging and sick probability, there are the dairy products of immunoloregulation function and anti-senescence function to exploitation
It is had a very important significance with health care product.
Detailed description of the invention
Fig. 1：Mass chromatography extracts figure (m/z=853.4145)；
Fig. 2：The second order ms figure for the segment that mass-to-charge ratio is 853.4145；
Fig. 3：Polypeptide az, by crack conditions that mass-to-charge ratio is 853.4145；
Fig. 4：Each group experimental animal mouse spleen situation of change；
It a) is low dosage stomach-filling group mouse spleen organization chart；It (b) is high dose stomach-filling group mouse spleen organization chart；(c) it is
Naive mice spleen tissue；It (d) is animal model group mouse spleen organization chart；
Fig. 5：Each group mice serum IL-6 changes table；
Fig. 6：Each group mice serum TNF-α changes table；
Fig. 7：Each group mice serum IL-2 changes table；
Fig. 8：Each group mice serum IL-10 changes table.
Specific embodiment
Before further describing specific embodiments of the present invention, 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 term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989 and 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.
1 active peptide VMTMLDK's of embodiment is artificial synthesized
One, the synthesis of biologically active peptide
1. RINK resin 3g (degree of substitution 0.3mmol/g) is weighed in the reactor of 150ml, with the methylene chloride of 50ml
(DCM) it impregnates.
After 2.2 hours, resin is washed with nitrogen-dimethylformamide (DMF) of 3 times of resin volumes, is then drained, so weight
It is four times multiple, it is stand-by after resin is drained.
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 group on resin is sloughed with this.It is washed four times after having taken off protection with the DMF of 3 times of resin volumes,
Then it drains.
4. taking 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. it weighs amino acid Val in right amount and 1- hydroxyl-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 the N of 3ml is then added, N diisopropylcarbodiimide (DIC) oscillation shakes up 1min, clear to solution
It is added in reactor after clear, then reactor is placed in 30 DEG C of shaking table and is 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
It is washed four times, is 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 group on resin is sloughed with this.It is washed four times after having taken off protection with DMF, is then drained.
8. taking 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 general of 25ml is added
It is dissolved, and the DIC oscillation that 2.5ml is then added shakes up 1min, is added in reactor after solution clarification, then by reactor
It is placed in 30 DEG C of shaking table and reacts.
After 10.1 hours, a small amount of resin is taken to detect, with ninhydrin method detection (each two drop of inspection A, inspection B, 100 DEG C of reactions
1min), if resin be it is colourless, illustrate fully reacting；If resin has color, illustrate condensation not exclusively, the reaction was continued.
11. after complete reaction, being washed resin four times with DMF, then drains, 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 protecting group on resin is sloughed with this
Group.It is washed four times after having taken off protection with DMF, then drains whether detection protection sloughs.
12. successively connecting amino acid Met, Thr, Met, Leu, Asp and Lys according to step 9-11.
13. sloughing protection after connecting the last one amino acid, is washed four times with DMF, 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
It is cut down from resin (every gram of resin adds 10ml cutting liquid), and with ice ether (cutting liquid：Ether=1:9,v:V) centrifugation is heavy
Drop four times.
So far, artificial synthesized biologically active peptide VMTMLDK.
Two, the confirmation of biologically active peptide
1) UPLC is analyzed
UPLC condition is as follows：
Instrument：Waters ACQUITY UPLC ultra high efficiency liquid phase-electron spray-level four bars-time of-flight mass spectrometer
Chromatographic column specification：BEH C18 chromatographic column
Flow velocity：0.4mL/min
Temperature：50℃
Ultraviolet detection wavelength：210nm
Sample volume：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
Remove solvent stream (L/hr)：700.0
Collision energy (eV)：4.0
Sweep time (sec)：0.25
Interior sweep time (sec)：0.02
According to the above analysis method, using ultra high efficiency liquid phase-electron spray-level four bars-flight time mass spectrum, to bioactivity
Peptide VMTMLDK carries out chromatography and mass spectral analysis, and mass chromatography extraction figure is as shown in Figure 1, extract the second order ms at this peak
Figure and az, by crack conditions are as shown in Figures 2 and 3, and the polypeptide mass-to-charge ratio that can obtain this peak is 853.4145Da, and retention time is
47.7min。
3) result
From the figure 3, it may be seen that the case where being broken according to az, by, by Mascot software analytical calculation, obtains mass-to-charge ratio
The fragment sequence of 853.4145Da is Val-Met-Thr-Met-Leu-Asp-Lys (VMTMLDK), is denoted as SEQ ID NO：1.It should
Segment and LBH_1801 | m.863 LBH_1801 | g.863 ORF LBH_1801 | g.863 LBH_1801 | m.863 type:
complete len:181(+)LBH_1801:The residue sequence of the 85th~91,659-1201 (+) albumen is corresponding, and sequence is shown in
SEQ ID NO：3.
The adjusting immunity of organisms activity experiment of 2 biologically active peptide of embodiment
One, the vitro lymphocyte proliferation capacity experimental of mtt assay measurement biologically active polypeptide VMTMLDK
1. experimental material and instrument：
Reagent and material：Experimental animal balb/c mouse (male 6-8 week old, Shanghai Communications University's agricultural and biological institute
Animal experimental center)；The biologically active polypeptide VMTMLDK that embodiment 1 obtains；Mouse lymphocyte extracting solution (is purchased from Suo Laibao
Company)；RPMI1640 culture medium (is purchased from GIBCO company)；3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromide
(MTT is purchased from Amresco company)；ConA (ConA is purchased from Sigma company)；(BSA is purchased from bovine serum albumin(BSA)
Genebase company)；Pepsin (is purchased from Sigma company)；Pancreatin (Corolase PP is purchased from AB company).
Instrument and equipment：LRH-250F biochemical cultivation case, Shanghai perseverance Science and Technology Ltd.；GL-22M high speed freezing centrifuge,
Shanghai Lu Xiang instrument centrifuge Instrument Ltd.；150 CO2 incubator of Hera cell, Heraeus company；Dragon
Wellscan MK3 microplate reader, Labsystems company；ALPHA 1-2-LD vacuum freeze drier, Christ company；Superelevation
Effect liquid phase chromatogram-quadrupole rod time of-flight mass spectrometer, waters company.
2. experimental method：
Mouse spleen is taken under aseptic condition, mouse lymphocyte is extracted with lymphocyte extracting solution, carries out Yuan Dynasty's culture.With
Cell density is adjusted to 2.5 × 10 by complete RPMI1640 culture solution6A/mL.It is sequentially added in 96 porocyte culture plates：
100 μ L mouse lymphocyte suspensions, 100 μ L RPMI1640 complete culture solutions, 20 μ L ConAs, 100 μ L samples.In addition,
Blank control group (PBS of pH7.2~7.4,3mol/L) and negative control group (500 μ g/mL BSA) are set, research shows that its is right
It is not influenced in vitro lymphocyte proliferation.Every group of 3 parallel laboratory test samples.In 5%CO2After cultivating 68h in 37 DEG C of incubators,
20 μ L MTT are added in every hole under aseptic condition, continue to cultivate 4h, carefully discard supernatant liquid, and 100 μ L dimethyl sulfoxides are added in every hole,
37 DEG C of biochemical cultivation cases hatch 10min, shake up, measure light absorption value at 570nm with microplate reader.
Vitro lymphocyte proliferation ability indicates that calculation method is as follows with stimulus index：
In formula：A1For blank control at the 570nm under light absorption value；A2For negative control group at the 570nm under extinction
Value, A3For experimental group at the 570nm under light absorption value.
3. experimental result and analysis：
Influence of the 1 biologically active polypeptide VMTMLDK of table to vitro lymphocyte proliferation
| Experimental group | Stimulus index SI |
| Negative control group | 1 |
| VMTMLDK | 1.188±0.061* |
Note：* labelled notation is to have significant difference (P < 0.05) compared with negative control.
Experimental result is shown in Table 1.As shown in Table 1, in the condition that the mass concentration of biologically active peptide VMTMLDK is 100 μ g/mL
Under, the stimulus index of biologically active peptide VMTMLDK is greater than BSA, illustrates that VMTMLDK can stimulate external mouse lymph to a certain extent
The proliferation of cell.And the stimulus index of VMTMLDK reached 1.188 and negative control group have significant difference (P<0.05).
Therefore, it can be assumed that active peptides VMTMLDK has the ability for remarkably promoting mouse lymphocyte proliferation, it can be used as one kind
Health care product or additive are edible, can be improved the immunity of animal and human body.
Two, the macrophages in vitro proliferative capacity experiment of mtt assay measurement biologically active polypeptide VMTMLDK
1) experiment reagent and instrument
Reagent：Experimental animal balb/c mouse (male 6-8 week old) Shanghai Communications University's agricultural and biological institute animal are real
Test center；The biologically active polypeptide VMTMLDK that embodiment 1 obtains；3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide
Bromide (MTT) Amresco company；LPS (lipopolysaccharides) Sigma company；Bovine serum albumin(BSA) (Bovine Serum Albumin,
BSA) Genebase company；Three lysates, the aqueous solution containing 10%SDS, 5% isobutanol and 0.012mol/L HCl.
Instrument and equipment：LRH-250F biochemical cultivation case Shanghai perseverance Science and Technology Ltd.；On GL-22M high speed freezing centrifuge
Hai Luxiang instrument centrifuge Instrument Ltd.；Hera cell 150 CO2Incubator Heraeus company；Dragon Wellscan
MK3 microplate reader Labsystems company.
2) test method：
Balb/c mouse peritoneal injects 2% (w/w) the sterilizing starch solution of 2ml, and continuous injection three days, last time is injected
The neck that breaks after 24 hours is put to death.Skin of abdomen is peelled off, draws 4 DEG C of phosphate buffer (PBS) repeated flushing abdominal cavities with syringe, from
After heart pipe collects flushing liquor, supernatant is abandoned in centrifugation (1000rpm, 4 DEG C) after ten minutes, (is contained with 4 DEG C of RPMI1640 complete culture solutions
It 10%FBS) washes twice, cell viability examination is done in the dyeing of 0.2% trypan blue solution, confirms collected vibrant macrophage
Account for 95% or more.After cell counting board reading, adjustment cell concentration to suitable concentration.
It will blow and beat to the cell suspension to suspend completely and 96 porocyte culture plates, 37 DEG C, 5%CO be added with suitable volumes2
After being cultivated 4 hours under environment, inhales and abandon liquid in hole, carefully clean cell culture plate well with 37 DEG C of RPMI1640 complete culture solutions
Bottom washes away not adherent cell and cell fragment, obtains adherent peritoneal macrophage after purification.0.2ml is added in every hole
RPMI1640 complete medium, experiment small peptide sample and LPS are added after being dissolved in culture medium in advance, start cell culture.
After obtaining adherent peritoneal macrophage after purification, the every hole of experimental group adds dissolved with biologically active polypeptide VMTMLDK
200 hole μ l/ of RPMI1640 complete culture solution (10%FBS) of (1mg/ml), continuously cultivates 48h；The every hole solubilization of negative control group
Solution has 200 hole μ l/ of RPMI1640 complete culture solution (10%FBS) of BSA (500 μ g/mL)；It is complete that blank group adds RPMI1640
200 hole μ l/ of culture solution (10%FBS), continuously cultivates 48h.Also, experimental group, negative control group and blank group are set just respectively again
Often group and inflammation group；LPS to final concentration of 100ng/ml is added when culture is arrived for 24 hours for inflammation group；LPS is not added in normal group；And
20 hole μ l/ 5%MTT is added in 44h for normal group and inflammation group；Cell culture reaches, and the three molten of 100 holes μ l/ is added after 48h
Solution liquid after dissolution overnight, surveys the absorbance value (OD570) in each hole, growth refers to terminate culture at wavelength 570nm with microplate reader
The calculation formula of number (Growth Indices) is as follows：
Wherein, blank culture solution is the RPMI1640 complete culture solution containing 10%FBS.
3) experimental result and analysis
The influence that 2 biologically active polypeptide VMTMLDK of table is proliferated macrophages in vitro
| Experimental group | Normal group GI | Inflammation group GI |
| Negative control group | 1 | 1 |
| VMTMLDK(1mg/ml) | 1.1073±0.0529** | 1.120±0.0599** |
Note：* it indicates compared with negative control, there is significant difference (P < 0.05)；* indicate compared with negative control group,
There is significant difference (P < 0.01)
Experimental result is shown in Table 2, as shown in Table 2, under conditions of adding 1mg/ml biologically active polypeptide VMTMLDK, normally
The macrophage of group and inflammation group has proliferation.And compared with negative control group, there is significant difference (P < 0.01).It says
Gelatine/biological activity polypeptide VMTMLDK has significant proliferation function to macrophages in vitro.
The activity of fighting against senium of 3 biologically active peptide of embodiment is tested
One, experiment of the biologically active polypeptide VMTMLDK 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 biologically active polypeptide VMTMLDK that embodiment 1 obtains.
Instrument and equipment：The ultra-clean water of CM-230 type mole, Shanghai Moller scientific instrument Co., Ltd；Mi Libo Millipore
MILLEX GP0.22 μm filter membrane, Millipore Corp. of the U.S.；GL-22M high speed freezing centrifuge, Shanghai Lu Xiang instrument centrifuge instrument
Co., Ltd.
2. experimental method：
(1) Animal Aging model modeling
After a week by ICR mouse adaptive feeding, it is divided into 4 groups, every group 6.Group 1 is low dosage stomach-filling group, and mouse is daily
D-gal is subcutaneously injected with the dosage the nape of the neck of 500mg/kg, and with the dosage stomach-filling biologically active polypeptide in 1mg/ day
VMTMLDK；Group 2 is high dose stomach-filling group, and D-gal is subcutaneously injected daily with the dosage the nape of the neck of 500mg/kg in mouse, and
The day dosage stomach-filling biologically active polypeptide VMTMLDK of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is animal mould
D-gal, and the physiology salt that stomach-filling concentration is 0.9% is subcutaneously injected daily with the dosage the nape of the neck of 500mg/kg in type group, mouse
Water；The injection cycle of D-gal and the stomach-filling period of polypeptide are 42 days.Every 3 days replacement paddings, and guarantee feed and distilled water
Supply.The weight for weighing a mouse for every five days prepares D-gal injection according to the weight of mouse, and D-gal injection passes through
0.22 μm of needle cylinder type filter membrane filtering, it is sterile to guarantee.
(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, it is obtained dirty
Device is placed in the 1.5mL centrifuge tube to sterilize in advance, and all organ samples are maintained in -80 DEG C of refrigerators standby inspection.Disposition is real
All operations tested during animal follow Ministry of Science and Technology's publication in 2006《Directiveness meaning about kind treatment experimental animal
See》.The mouse spleen won directly is soaked in preparatory prepared 4% paraformaldehyde solution, to fix in the form of it.Poly
Formaldehyde powder more indissoluble can be added micro sodium bicarbonate and pH value is adjusted to alkalinity, with hydrotropy.Paraformaldehyde solution
It prepares and needs to complete in draught cupboard.
(3) sample detection
The production of histotomy：Mouse spleen sample need at least fix 24 hours in 4% paraformaldehyde solution.Spleen group
Wax stone production, slice and HE the dyeing commission Shanghai Wei Ao Biotechnology Co., Ltd knitted complete.
3. experimental result and analysis：
In this experiment, 4 groups of mouse are shared, wherein the mouse normal growth of blank group is not affected by any environmental stimuli,
3 groups of mouse of remaininging receive the long term injections of D-gal.Using optical microscopy, the spleen section of separate groups of mice is seen
It examines, can be found from Fig. 4, compare the spleen section of each group mouse, for naive mice, the spleen of animal model mouse
Dirty red pulp and white pulp boundary are fuzzy, and atrophy occurs in white pulp, show that the glycometabolism approach of mouse occurs for long-term D-gal injection
Disorder causes anti-oxidant enzyme activity to reduce, peroxide accumulation, and then may cause the aging and atrophy of spleen.And stomach-filling 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 explanation, in the injection cycle of entire D-gal, experiment animal sustained is constantly by cause senescence-factor
Stimulation, lead to the aging and atrophy of spleen.Therefore from the changes in microstructure the case where from the point of view of, the biology invented in this experiment
Active peptides VMTMLDK is to animal because the spleen aging caused by the stimulation by the bad factor has certain protection with atrophy
Effect.
Two, the experiment that biologically active polypeptide VMTMLDK 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 biologically active polypeptide VMTMLDK that embodiment 1 obtains；BCA protein reagent box, Science and Technology Ltd. of Nanjing Keygen Biotech；
MDA lipid peroxide kit, Science and Technology Ltd. of Nanjing Keygen Biotech；SOD superoxide dismutase kit, Nanjing is built
At Biotechnology Co., Ltd；T-AOC antioxidative activities kit, Biotechnology Co., Ltd is built up in Nanjing.
Instrument and equipment：The ultra-clean water of CM-230 type mole, Shanghai Moller scientific instrument Co., Ltd；Mi Libo Millipore
MILLEX GP0.22 μm filter membrane, Millipore Corp. of the U.S.；GL-22M high speed freezing centrifuge, Shanghai Lu Xiang instrument centrifuge instrument
Co., Ltd.
2. experimental method：
(1) Animal Aging model modeling
After a week by ICR mouse adaptive feeding, it is divided into 4 groups, every group 6.Group 1 is low dosage stomach-filling group, and mouse is daily
D-gal is subcutaneously injected with the dosage the nape of the neck of 500mg/kg, and with the dosage stomach-filling biologically active polypeptide in 1mg/ day
VMTMLDK；Group 2 is high dose stomach-filling group, and D-gal is subcutaneously injected daily with the dosage the nape of the neck of 500mg/kg in mouse, and
The day dosage stomach-filling biologically active polypeptide VMTMLDK of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is animal mould
D-gal, and the physiology salt that stomach-filling concentration is 0.9% is subcutaneously injected daily with the dosage the nape of the neck of 500mg/kg in type group, mouse
Water；The injection cycle of D-gal and the stomach-filling period of polypeptide are 42 days.Every 3 days replacement paddings, and guarantee feed and distilled water
Supply.The weight for weighing a mouse for every five days prepares D-gal injection according to the weight of mouse, and D-gal injection passes through
0.22 μm of needle cylinder type filter membrane filtering, it is sterile to guarantee.
(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, it is obtained dirty
Device is placed in the 1.5mL centrifuge tube to sterilize in advance, and all organ samples are maintained in -80 DEG C of refrigerators standby inspection.Disposition is real
All operations tested during animal follow Ministry of Science and Technology's publication in 2006《Directiveness meaning about kind treatment experimental animal
See》.The mouse spleen won directly is soaked in preparatory prepared 4% paraformaldehyde solution, to fix in the form of it.Poly
Formaldehyde powder more indissoluble can be added micro sodium bicarbonate and pH value is adjusted to alkalinity, with hydrotropy.Paraformaldehyde solution
It prepares and needs to complete in draught cupboard.
(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
Homogenate is knitted, under the conditions of 4 DEG C after 4000g centrifugation, supernatant is taken, discards precipitating, operated according to kit specification, or set
It is to be measured in -80 DEG C of refrigerators.
3. experimental result and analysis：
The variation of SOD content in 3 each group experimental animal mouse Different Organs of table
Note：* mark is compared with model group, there is significant difference (P<0.05)；* mark is 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 stomach-filling group mouse contains
Increase (the P of conspicuousness is presented in amount<0.01).Although meaning D-gal of the mouse of polypeptide stomach-filling group by long-term, high-dose
Stimulation, the excess injection of D-gal without completely destroy Mice Body in SOD enzyme system, illustrate in injection cycle, experiment
Animal will lead to the reduction of SOD content in Different Organs, but same continuously by the stimulation for causing senescence-factor
When take in a certain amount of polypeptide VMTMLDK to the intracorporal oxidative damage of mouse have certain protective role.
The situation of change of MDA content 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 25.86 ± 7.08nmol/L, with animal model
Group compares, and significant difference (P is presented in the MDA content in two groups of mouse livers of polypeptide stomach-filling<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
In the process, due to the long term injections of excessive D-gal, the glycometabolism approach of mouse is made to get muddled, generate a large amount of free radicals from
And oxidative damage is caused, occur a large amount of lipid peroxide in liver organization, MDA is as lipid peroxide, in animal
The raising of in-vivo content can reflect the reduction of Antioxidant Enzymes vigor in Mice Body from side.And polypeptide stomach-filling group Mouse Liver
The significant decrease of dirty MDA content illustrates that the intake of polypeptide VMTMLDK can be effectively protected vital tissue organ from the bad factor
Stimulation generates a large amount 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 value of animal model group mouse is 0.69 ± 0.19U/mgprot, is compared to mould
Significant difference (P is presented in type group, polypeptide high dose and low dosage stomach-filling group mouse therewith<0.05)；The kidney of model group mouse
Dirty T-AOC content is 0.71 ± 0.19U/mgprot, and compared with animal model group mouse, low dosage stomach-filling group presents aobvious therewith
Write sex differernce (P<0.05), significant difference (P is also presented in high dose stomach-filling group mouse therewith<0.01).This result shows that, whole
In a experimental period, due to experiment animal sustained constantly by cause senescence-factor stimulation, the liver of animal model group mouse,
Renal tissue is destroyed, and the reduction of its total antioxidant capacity is caused.Compared with animal model group and blank group, polypeptide stomach-filling group is small
The total antioxidant capacity of mouse major organs maintains a higher level in by the stimulating course for causing senescence-factor always,
Illustrate that taking in biologically active polypeptide VMTMLDK makes animal body and its major organs self-protection function with higher.
Three, the experiment that immune cell factor in serum is acted on of biologically active polypeptide VMTMLDK
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 biologically active polypeptide VMTMLDK that embodiment 1 obtains；BCA protein reagent box, Science and Technology Ltd. of Nanjing Keygen Biotech；
ELISA cell factor Quick kit (TNF-α, IL-2, IL-6 and IL-10), Wuhan Boster Biological Technology Co., Ltd..
Instrument and equipment：The ultra-clean water of CM-230 type mole, Shanghai Moller scientific instrument Co., Ltd；Mi Libo Millipore
MILLEX GP0.22 μm filter membrane, Millipore Corp. of the U.S.；GL-22M high speed freezing centrifuge, Shanghai Lu Xiang instrument centrifuge instrument
Co., Ltd.
2. experimental method：
(1) Animal Aging model modeling
After a week by ICR mouse adaptive feeding, it is divided into 4 groups, every group 6.Group 1 is low dosage stomach-filling group, and mouse is daily
D-gal is subcutaneously injected with the dosage the nape of the neck of 500mg/kg, and with the dosage stomach-filling biologically active polypeptide in 1mg/ day
VMTMLDK；Group 2 is high dose stomach-filling group, and D-gal is subcutaneously injected daily with the dosage the nape of the neck of 500mg/kg in mouse, and
The day dosage stomach-filling biologically active polypeptide VMTMLDK of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is animal mould
D-gal, and the physiology salt that stomach-filling concentration is 0.9% is subcutaneously injected daily with the dosage the nape of the neck of 500mg/kg in type group, mouse
Water；The injection cycle of D-gal and the stomach-filling period of polypeptide are 42 days.Every 3 days replacement paddings, and guarantee feed and distilled water
Supply.The weight for weighing a mouse for every five days prepares D-gal injection according to the weight of mouse, and D-gal injection passes through
0.22 μm of needle cylinder type filter membrane filtering, it is sterile to guarantee.
(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 disposition experimental animal follow the Ministry of Science and Technology in 2006
Publication《Guiding opinion about kind treatment experimental animal》.The mouse spleen won directly is soaked in prepared in advance
In 4% paraformaldehyde solution, to fix in the form of it.Paraformaldehyde powder more indissoluble, micro sodium bicarbonate can be added will
PH value is adjusted to alkalinity, with hydrotropy.The preparation needs of paraformaldehyde solution are completed in draught cupboard.
(3) sample detection
It is indicated according to kit specification, first drafting standard curve, standard items powder is prepared with standard dilutions
At the solution of 1000pg/mL, then serial dilution is 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL,
15.6pg/mL waiting 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, be added in same ELISA Plate (need to press when detecting calculating again if blood serum sample is inadequate, after can suitably diluting
Ratio conversion).ELISA Plate is covered, places it under 37 DEG C of environment and is incubated for 90min.After completion of the reaction, it carefully gets 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, reacts 60min.After completion of the reaction, utilize 0.01M's
PBS solution is washed 3 times, and the PBS of 100 μ L is added in each every hole, is impregnated 1min hypsokinesis and is removed solution, and 3 times repeatedly.It will be preheated
ABC working solution is 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
Impregnate 1min or so.The TMB developing solution for balancing 30min at 37 DEG C is sequentially added by every 90 μ L of hole, 37 DEG C are protected from light 8-
12min.TMB terminate liquid is sequentially added by every hole 0.1ml, blue is vertical at this time turns yellow, measures OD value in 450nm with microplate reader.
Known concentration is done by the standard protein of cell factor to be serially diluted, draws out standard curve after measuring OD value, according to standard song
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 out of model group Mice Body that can be found that in this experiment in table 6, Fig. 5, Fig. 6
168.03 ± 25.38pg/mL, 4.44 ± 0.86pg/mL, compared to the increase (P that conspicuousness is presented in normal group<0.01), therefore
It is considered that causing animal model group mouse aging occur in cell factor level due to continuously injecting cause senescence-factor
The symptom of property inflammation, and the IL-6 of the mice serum of polypeptide stomach-filling group is effectively controlled with TNF-α content.According to cell because
The experimental result of son, the serum levels of inflammatory cytokines IL-6 of polypeptide stomach-filling group mouse, the secretion level of TNF-α are below animal mould
Type group, from the point of view of oxidative damage angle, mouse because free radical attack, Peroxidation Product accumulation due to caused by oxidative damage may obtain
Inhibition to a certain extent；From the perspective of inflammation, mouse inflammation because of caused by oxidation has obtained effective inhibition；From declining
From the point of view of old angle, a series of geriatric diseases caused by mouse aging by caused by long term injections D-gal are possible to obtain
Control.
From Fig. 7's, as a result, it has been found that, IL-2 is as a kind of significant cell factor model in this experiment for judging aging
Do not occur conspicuousness effect in group and stomach-filling group, thus it is speculated that, it may be possible to as the model and naturally-aged employed in this experiment
Still difference or modeling period falls short of, therefore this index is caused not change.Meanwhile from Fig. 8's as a result, it has been found that, this
Biologically active polypeptide VMTMLDK fails to have an impact the secretion of mouse IL-10 in the period in experiment, in various mice serums
In the detection of IL-10 content, -10 content of serum IL of only normal growth group mouse is maintained at relatively low level, remaining
Each group mice serum IL-10 content, which is presented, to be increased, it may be possible to biologically active polypeptide VMTMLDK to the secretion access of IL-10 without
It influences.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Sequence table
<110>Zhejiang Hui Tai life and health Science and Technology Ltd.；Shanghai Bo Hui Biotechnology Co., Ltd
<120>A kind of biologically active polypeptide VMTMLDK and its preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Val Met Thr Met Leu Asp Lys
1 5
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
taatgacgat gcttgataaa t 21
<210> 3
<211> 180
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Met His Lys Leu Val Leu Lys Gly Ile Arg Arg Asn Lys Arg Lys Tyr
1 5 10 15
Ser Ser Tyr Lys Gly Thr Ile Gly Lys Ile Ala Pro Asn Leu Ile His
20 25 30
Arg Asp Phe Phe Ala Pro Met Pro Asn Met Lys Trp Tyr Thr Asp Ile
35 40 45
Thr Glu Phe Arg Leu Asn Gly Glu Lys Leu Tyr Leu Ser Pro Ile Leu
50 55 60
Asp Gly Cys Gly Gly Asp Ile Val Ser Tyr Ser Ile Ser Arg His Pro
65 70 75 80
Asp Met Asp Leu Val Met Thr Met Leu Asp Lys Ser Phe Val Lys Glu
85 90 95
Thr Thr Leu Asn Asn Cys Thr Phe His Thr Asp Gln Gly Cys Gln Tyr
100 105 110
Gln Ser Ser Gln Tyr Gln Arg Ala Leu Lys Leu Gln Gly Ile Thr Gln
115 120 125
Ser Met Ser Arg Lys Gly Asn Ser Met Asp Asp Gly Leu Met Glu Asn
130 135 140
Phe Phe Gly Leu Leu Lys Thr Glu Met Phe Tyr Asp Gln Glu Tyr Lys
145 150 155 160
Tyr His Ser Leu Gln Glu Leu Thr Gln Ala Ile Lys Glu Tyr Ile Thr
165 170 175
Arg Gly Ala Ser
180
Claims (10)
1. a kind of biologically active polypeptide VMTMLDK, which is characterized in that its amino acid sequence is Val-Met-Thr-Met-Leu-
Asp-Lys。
2. a kind of biologically active polypeptide VMTMLDK according to claim 1, which is characterized in that the biologically active polypeptide
From Lactobacillus helveticus mycoprotein.
3. encoding the nucleotide fragments of biologically active polypeptide VMTMLDK described in claim 1, which is characterized in that the nucleotide
The sequence of segment such as SEQ ID NO：Shown in 2.
4. the preparation method of biologically active polypeptide VMTMLDK as described in claim 1, which is characterized in that pass through genetic engineering
Method is artificial synthesized or Lactobacillus helveticus thallus is directly obtained by the method that clasmatosis isolates and purifies, or directly passing through
Be synthetically prepared.
5. the application of biologically active polypeptide VMTMLDK as described in claim 1, which is characterized in that the biologically active polypeptide
Application of the VMTMLDK in food, health care product, drug or the cosmetics that preparation has immunoloregulation function.
6. the application of biologically active polypeptide VMTMLDK as described in claim 1, which is characterized in that the biologically active polypeptide
Application of the VMTMLDK in the food, health care product or drug that preparation has anti-senescence function.
7. the application of biologically active polypeptide VMTMLDK as described in claim 1, which is characterized in that the biologically active polypeptide
Application of the VMTMLDK in the food, health care product or drug that preparation has immunoloregulation function and anti-senescence function.
8. a kind of immunological regulation product, which is characterized in that including biologically active polypeptide VMTMLDK as described in claim 1 or institute
State the derivative of biologically active polypeptide VMTMLDK；The immunological regulation product includes immunological regulation food, immunological regulation health care
Product, immunoregulation medicament or immunological regulation cosmetics；The derivative of the biologically active polypeptide VMTMLDK refers to living in biology
Property polypeptide VMTMLDK amino acid side groups on, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, methyl
Change, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
9. a kind of anti-aging product, which is characterized in that including biologically active polypeptide VMTMLDK or described as described in claim 1
The derivative of biologically active polypeptide VMTMLDK；The anti-aging product includes antisenility cistanche food, antisenescence health product or anti-ageing
Old drug；The derivative of the biologically active polypeptide VMTMLDK, refers to the amino acid side chain in biologically active polypeptide VMTMLDK
On group, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosyl
Change modification, obtained polypeptide derivative.
10. a kind of product with immunoloregulation function and anti-senescence function, which is characterized in that including as described in claim 1
The derivative of biologically active polypeptide VMTMLDK or the biologically active polypeptide VMTMLDK；With immunoloregulation function and anti-aging
The product of function includes food, health care product or drug；The derivative of the biologically active polypeptide VMTMLDK refers to living in biology
Property polypeptide VMTMLDK amino acid side groups on, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, methyl
Change, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
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| CN112745381A (en) * | 2021-01-22 | 2021-05-04 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide with amino acid structure NIRNIGKTLVTR, and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009121176A1 (en) * | 2008-03-31 | 2009-10-08 | The University Of British Columbia | Insulin-induced gene (insig) peptide compositions and methods for cytoprotection |
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2018
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009121176A1 (en) * | 2008-03-31 | 2009-10-08 | The University Of British Columbia | Insulin-induced gene (insig) peptide compositions and methods for cytoprotection |
Non-Patent Citations (2)
| Title |
|---|
| IEHUI,ZHOU 等: "Immunomodulating effects of casein-derived peptides QEPVL and QEPV on lymphocytes in vitro and in vivo", 《FOOD & FUNCTION》 * |
| 钱蕙佶等: "瑞士乳杆菌胞内生物活性肽的分离鉴定", 《中国乳品工业》 * |
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| CN112745381A (en) * | 2021-01-22 | 2021-05-04 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide with amino acid structure NIRNIGKTLVTR, and preparation method and application thereof |
| CN112745381B (en) * | 2021-01-22 | 2022-05-31 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide with amino acid structure NIRNIGKTLVTR, and preparation method and application thereof |
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