CN108794586A - A kind of biologically active polypeptide LDENDIIL and its preparation method and application - Google Patents
A kind of biologically active polypeptide LDENDIIL and its preparation method and application Download PDFInfo
- Publication number
- CN108794586A CN108794586A CN201810701237.0A CN201810701237A CN108794586A CN 108794586 A CN108794586 A CN 108794586A CN 201810701237 A CN201810701237 A CN 201810701237A CN 108794586 A CN108794586 A CN 108794586A
- Authority
- CN
- China
- Prior art keywords
- ldendiil
- biologically active
- active polypeptide
- polypeptide
- inflammatory
- Prior art date
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- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 230000037380 skin damage Effects 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009747 swallowing Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000035922 thirst Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 150000003852 triazoles Chemical class 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 108010030651 valyl-glutamyl-prolyl-isoleucyl-prolyl-tyrosine Proteins 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
- 235000021247 β-casein Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- 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)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The present invention relates to albumen fields, and in particular to its amino acid sequence of a kind of biologically active polypeptide LDENDIIL and its preparation method and application, biologically active polypeptide LDENDIIL is Leu-Asp-Glu-Asn-Asp-Ile-Ile-Leu.By extracorporeal anti-inflammatory activity experiment, internal Antisenility Experiment, demonstrating polypeptide LDENDIIL has preferable anti-inflammatory activity and activity of fighting against senium, on the one hand, the biologically active polypeptide LDENDIIL 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, body incidence is reduced；On the other hand, the vigor of internal anti-peroxidation enzyme system can be improved, enhance the function that body resists external source sexual stimulus, to reduce organism aging process, aging and sick probability, there is anti-inflammatory properties, the food of anti-senescence function, health products 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 LDENDIIL and preparation method thereof and answer
With.
Background technology
During cow's milk is through lactobacillus-fermented, a part of protein in cow's milk is metabolized by lactic acid bacteria to be utilized, concurrently
A series of biochemical reactions have been given birth to, 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 some itself synthesis for lactic acid bacteria thalline
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 enzymolysis, and biologically active polypeptide is by 2~20 mostly
Amino acid residue forms, and molecular weight is less than 6000Da, contains a certain amount of hydrophobic amino acid, aromatic amino acid.
Immune-active peptides are to obtain and prove that one kind biology of its physiological activity is living from breast 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, can obtain an 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 has anti-inflammatory properties.Newborn source peptide (PGPIPN) the feeding rat of Li Su duckweeds et al. synthesis finds that rat abdominal cavity is huge
The phagocytosis of phagocyte and the relevant anti-inflammatory properties 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, the increase for promoting the macrophage nitric oxide amount of inducing, raising machine
Body resists the ability of extraneous pathogenic infection, reduces body incidence, and will not cause the immunological rejection of body.
Aging is a natural phenomena, and process is often accompanied by the variation of antioxidant levels, organ-tissue, immune factor,
Complicated variation occurs for middle cell factor, such as the trend that proinflammatory cytokine IL-6, IL-4, TNF-α presentation increase, IL-6
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 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 as a kind of emerging antidotal agent, in terms of physiological function have amino acid cannot compare it is excellent
Gesture can generate promotion or inhibiting effect to the enzyme in organism, improve the absorption to minerals and other nutrients and profit
With removing interior free yl enhances the resistance to oxidation of body itself, to slow down aging.Therefore, the nutrition and health care of biologically active peptide
Effect has become the emphasis of domestic and foreign scholars' subject study.Qiu Juan et al. pass through experimental studies have found that, milk-derived bioactive micro peptide
Life span of drosophila melanogaster can effectively be extended, delay its aging, and also there is preferable antioxidation, thus it is speculated that may be 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.
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- caseins disclose a kind of from α s2-
The milk-derived biologically active polypeptide NQFYQKF of casein.
Invention content
The purpose of the present invention is to provide a kind of biologically active polypeptide LDENDIIL 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 LDENDIIL, amino acid sequence Leu-Asp-
Glu-Asn-Asp-Ile-Ile-Leu, such as SEQ ID NO：Shown in 1.
Preferably, the biologically active polypeptide derives from Lactobacillus helveticus mycoprotein.It is derive specifically from LBH_1162 |
m.1083LBH_1162|g.1083ORF LBH_1162|g.1083LBH_1162|m.1083type:complete len:269
(+)LBH_1162:1-807 (+) albumen, and the amino acid residue of the 200th~207, albumen thus.LBH_1162|
m.1083LBH_1162|g.1083ORF LBH_1162|g.1083LBH_1162|m.1083type:complete len:269
(+)LBH_1162:1-807 (+) protein amino acid sequence such as SEQ ID NO：Shown in 3.
LBH_1162|m.1083LBH_1162|g.1083ORF LBH_1162|g.1083LBH_1162|m.1083type:
complete len:269(+)LBH_1162:The amino acid sequence and corresponding nucleotides sequence of 1-807 (+) albumen are classified as both
There is technology, encodes the biologically active polypeptide of the nucleotide fragments energy encoding mature of this 200th~207 amino acids residue of albumen
LDENDIIL。
Preferably, the biologically active polypeptide has anti-inflammatory properties and anti-senescence function.
Second aspect of the present invention provides the nucleotide fragments for encoding the biologically active polypeptide LDENDIIL, sequence
For：5 '-gct cga cga aaa tga tat tat ttt-3 ', such as SEQ ID NO：Shown in 2.
Third aspect present invention provides the preparation method of the biologically active polypeptide LDENDIIL, can pass through gene
The method of engineering is artificial synthesized, can be directly obtained from Lactobacillus helveticus thalline by the method that clasmatosis isolates and purifies, can
Directly to be prepared by chemical synthesis.
Fourth aspect present invention provides the biologically active polypeptide LDENDIIL and is preparing the food with anti-inflammatory properties
Application in product, health products, drug or cosmetics.
Fifth aspect present invention provides the biologically active polypeptide LDENDIIL and is preparing the food with anti-senescence function
Application in product, health products or drug.
Sixth aspect present invention, provide the biologically active polypeptide LDENDIIL prepare and meanwhile have anti-inflammatory properties with
Application in the food of anti-senescence function, health products or drug.
Specifically, the biologically active polypeptide LDENDIIL of the present invention, which can be used for preparing, reduces free radical to skin damage
Cosmetics, prepare have anti-inflammatory and/or anti-aging drug；And since the biologically active polypeptide LDENDIIL of the present invention is logical
The product crossed after gastrointestinal tract degradation still has bioactivity, therefore can be also used for preparing the food such as Yoghourt, adjusts immunity
Health products, and oral be used to prepare with anti-inflammatory and/or anti-aging drug.
Seventh aspect present invention provides a kind of anti-inflammatory products, including the biologically active polypeptide LDENDIIL or described
The derivative of biologically active polypeptide LDENDIIL；The anti-inflammatory products include anti-inflammatory food, anti-inflammatory health product, anti-inflammatory drug or
Anti-inflammatory cosmetics；The derivative of the biologically active polypeptide LDENDIIL refers to the amino in biologically active polypeptide LDENDIIL
On sour side-chain radical, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification
Or the modifications such as glycosylation, obtained polypeptide derivative.
Eighth aspect present invention provides a kind of anti-aging product, including the biologically active polypeptide LDENDIIL or institute
State the derivative of biologically active polypeptide LDENDIIL；The anti-aging product include antisenility cistanche food, antisenescence health product or
Antiaging agent；The derivative of the biologically active polypeptide LDENDIIL refers to the amino in biologically active polypeptide LDENDIIL
On sour side-chain radical, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification
Or the modifications such as glycosylation, obtained polypeptide derivative.
Ninth aspect present invention provides product that is a kind of while having anti-inflammatory properties and anti-senescence function, including described
The derivative of biologically active polypeptide LDENDIIL or described biologically active polypeptides LDENDIIL；With anti-inflammatory properties and anti-aging work(
The product of energy includes food, health products or drug；The derivative of the biologically active polypeptide LDENDIIL, refers in bioactivity
On the amino acid side groups of polypeptide LDENDIIL, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate,
The modifications such as acetylation, phosphorylation, esterification or glycosylation, obtained polypeptide derivative.
Biologically active polypeptide LDENDIIL's of the present invention has the beneficial effect that：The biologically active polypeptide LDENDIIL tools of the present invention
There are preferable anti-inflammatory activity and activity of fighting against senium；On the one hand, biologically active polypeptide LDENDIIL of the invention can promote macrophage
Cell secretion of cytokines promotes the increase of the macrophage nitric oxide amount of inducing, and improves body and resists extraneous pathogenic infection
Ability, reduce body incidence；On the other hand, the vigor of internal anti-peroxidation enzyme system can be improved, enhancing body is resisted outer
The function of source sexual stimulus, to reduce organism aging process, aging and sick probability, to exploitation with anti-inflammatory properties, anti-aging work(
Food, health products and the drug of energy have a very important significance.
Description of the drawings
Fig. 1：Mass chromatography extraction figure (m/z=944.5001)；
Fig. 2：The second order ms figure for the segment that mass-to-charge ratio is 944.5001；
Fig. 3：Polypeptide az, by crack conditions that mass-to-charge ratio is 944.5001；
Fig. 4：IL-4 standard curves；
Fig. 5：Influences of the biologically active polypeptide LDENDIIL to cell factor IL-4 secretory volumes；
Fig. 6：Influences of the various concentration LDENDIIL to caenorhabditis elegan fecundity；
Fig. 7：Nematode growth conditions in the L4 phases under different condition of culture；
Fig. 8：The influence that biologically active polypeptide LDENDIIL grows caenorhabditis elegan body；
Fig. 9：Influences of the biologically active polypeptide LDENDIIL to caenorhabditis elegan under oxidative stress.
Specific implementation mode
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 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 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, in the present invention all technologies for using and
Scientific terminology is identical as the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment,
Outside material, the record according to those skilled in the art to the grasp of the prior art and the present invention can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method 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 field.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
1 active peptide LDENDIIL's of embodiment is artificial synthesized
One, the synthesis of biologically active peptide
1. RINK resin 3g (degree of substitution 0.3mmol/g) are weighed in the reactor of 150ml, with the dichloromethane 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 for use 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 groups on resin are sloughed with this.It is washed four times with the DMF of 3 times of resin volumes after having taken off protection,
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 Leu in right amount and 1- hydroxyls-benzene a pair of horses going side by side triazole (HOBT) is in right amount in the centrifuge tube of 50ml, addition
The DMF of 20ml is dissolved, and the N of 3ml is then added, and N diisopropylcarbodiimide (DIC) oscillation shakes up 1min, waits for that solution is clear
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 for use 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.It is washed four times with DMF after having taken off protection, 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 generals of 25ml are added
It is dissolved, and the DIC oscillations that 2.5ml is then added shake up 1min, are 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 is colourless, illustrate that the reaction was complete；If resin has color, illustrate that condensation is incomplete, the reaction was continued.
11. after complete reaction, washing resin four times with DMF, then 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 groups on resin are sloughed with this
Group.It is washed four times with DMF after having taken off protection, then drains whether detection protection sloughs.
12. connecting amino acid Asp, Glu, Asn, Asp, Ile, Ile and Leu successively according to step 9-11.
13. after connecting the last one amino acid, 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
(every gram of resin adds 10ml cutting liquids) is cut down from resin, and ice ether (cutting liquid is used in combination：Ether=1:9,v:V) centrifugation is heavy
Drop four times.
So far, artificial synthesized biologically active peptide LDENDIIL.
Two, the confirmation of biologically active peptide
1) UPLC is analyzed
UPLC conditions are as follows：
Instrument：Waters ACQUITY UPLC ultra high efficiencies liquid phase-electron spray-level four bars-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 the above analysis method, using ultra high efficiency liquid phase-electron spray-level four bars-flight time mass spectrum, to bioactivity
Peptide LDENDIIL 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 944.5001Da, and retention time is
76.2min。
3) result
From the figure 3, it may be seen that the case where being broken according to az, by, analyzes by Mascot softwares and calculates, obtain mass-to-charge ratio
The fragment sequence of 944.5001Da is Leu-Asp-Glu-Asn-Asp-Ile-Ile-Leu (LDENDIIL), is denoted as SEQ ID
NO：1.The segment and LBH_1162 | m.1083LBH_1162 | g.1083ORF LBH_1162 | g.1083LBH_1162 |
m.1083type:complete len:269(+)LBH_1162:The residue sequence of 200-207,1-807 (+) albumen is opposite
It answers, sequence is shown in SEQ ID NO：3.
The anti-inflammatory activity of 2 biologically active peptide of embodiment is tested
One, the experiment (ELISA method) of the rush Factor of Macrophage of biologically active polypeptide LDENDIIL
1. experiment reagent and instrument：
Reagent：Experimental animal balb/c mouse (male 6-8 week old), Shanghai Slac Experimental Animal Co., Ltd.；Mouse
Lymphocyte extracting solution, the Shanghai bio tech ltd Suo Laibao；RPMI1640 culture mediums, GIBCO companies；Bovine serum albumin
(bovine serum albumin, BSA) in vain, Genebase companies；The biologically active polypeptide LDENDIIL that embodiment 1 obtains；
ELISA cell factors Quick kit (IL-4), Wuhan Boster Biological Technology Co., Ltd..
Instrument and equipment：The ultra-clean water of CM-230 types mole, Shanghai Moller scientific instrument Co., Ltd；LRH-250F biochemical cultures
Case, Shanghai perseverance Science and Technology Ltd.；GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument centrifuges Instrument Ltd.；Hera
cell 150 CO2Incubator, Heraeus companies；Dragon Wellscan MK3 microplate reader, Labsystems companies.
2. experimental method：
(1) preparation of standard curve
Make IL-4 standard curves：By a concentration of 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/
The IL-4 standard items of mL, 15.6pg/mL, 7.8pg/mL are sequentially added into ELISA Plate hole, add biotin labeling resist it is small
Mouse IL-4 antibody (ELISA cell factors Quick kit), ELISA Plate is plus lid, 37 DEG C of reaction 90min.Get rid of liquid in ELISA Plate
Body sequentially adds Avidin-peroxydase complex (ELISA cell factors Quick kit) 0.1mL per hole.37 DEG C of reactions
60min.0.01M PBS are washed 3 times, and 0.1mL ABC working solutions, 37 DEG C of reaction 30min are added per hole.0.01M PBS washings 5
It is secondary, 90ul TMB developing solutions are added per hole, 37 DEG C are protected from light 25min.0.1mL TMB terminate liquids are added per hole, use microplate reader
Light absorption value is measured in 450nm.The IL-4 detection standard curves of making are as shown in Figure 4.IL-4 standard curves are with a concentration of cross
Coordinate (unit pg/mL), the light absorption value under 450nm are ordinate, carry out a regression fit, obtain standard curve Y=
0.0038X+0.1224, R2=0.9979.Wherein X represents IL-4 concentration, and unit pg/mL, Y represent the light absorption value under OD450.
(2) the rush Factor of Macrophage detection of polypeptide LDENDIIL
Aseptically take mouse spleen lymphocyte, adjustment cell concentration to 5 × 105/ mL is inoculated in 96 orifice plates, real
It tests a group addition biologically active polypeptide LDENDIIL to be cultivated, the final concentration of adjustment biologically active polypeptide LDENDIIL is respectively
100,50,10 μ g/mL, with the measurement for carrying out cell factor IL-4 after lymphocyte co-incubation 36 hours.Blank group is not added with life
Object active peptides LDENDIIL, culture 36h is as a contrast.
3. experimental result and analysis：
Experimental result is shown in Fig. 5, and compared with blank control group, with the increase of peptide concentration, the secretory volume of IL-4 gradually increases
Add；When polypeptide addition concentration reaches 50 and 100 μ g/mL, IL-4 secretory volumes are noticeably greater than blank group；It can be seen that biologically active polypeptide
LDENDIIL, which has, promotes lymphopoietic function, and by the adjusting to IL-4 cytokine secretions, plays to machine
The adjustment effect of body humoral immunity.
Two, the measurement (Griess methods) of the rush macrophage nitric oxide amount of inducing of biologically active polypeptide LDENDIIL
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 reality
Test center；The biologically active polypeptide LDENDIIL that embodiment 1 obtains；LPS is purchased from Sigma companies；Neutral red staining solution, green cloud
Its biotechnology research institute produces.
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.；150 CO2 incubator Heraeus companies of Hera cell；Dragon Wellscan
MK3 microplate reader Labsystems companies.
2. test method：
It is 2 × 10 that number of cells, which is added,6100 holes μ l/ of cell suspension of/ml, it is adherent to be added after purification containing peptide
LPS is added to 10 μ g/ml of final concentration, continuously in 200 holes μ l/ of RPMI1640 complete culture solutions (10%FBS), inflammation group when for 24 hours
After cultivating 48h, 50 holes μ l/ of culture solution supernatant are collected, add Griess reagents 1 and Griess reagents successively in culture solution supernatant
2 each holes 50 μ l/, room temperature reaction after ten minutes, measure absorbance value (OD540) under 540nm wavelength.
3. experimental result and analysis：
1 biologically active polypeptide LDENDIIL of table promotees the measurement of the macrophage nitric oxide amount of inducing
| Experiment packet | Normal group | Inflammation group |
| Cell blank | 0.0603±0.0352 | 0.3321±0.0351 |
| LDENDIIL 1mg/ml | 0.1432±0.0332** | 0.4846±0.0394** |
| LDENDIIL 0.5mg/ml | 0.1298±0.0357** | 0.3832±0.0703** |
| LDENDIIL 0.1mg/ml | 0.2835±0.0153** |
Note：*, compared with negative control, significant difference (P < 0.05)；
*, compared with negative control group, significant difference (P < 0.01)
Experimental result is shown in Table 1, as shown in Table 1, biologically active polypeptide LDENDIIL is added in experimental group, concentration is respectively
1mg/mL and 0.5mg/mL makes an oxygen of the promotion macrophage grown under inflammatory conditions for growing under normal circumstances with LPS
Changing the nitrogen amount of inducing has facilitation.Compared with cell blank group, there is significant difference (P<0.05).Work as biologically active polypeptide
A concentration of 0.1mg/mL of addition of LDENDIIL, compares in the case where LPS makes inflammatory conditions, also macrophage nitric oxide can be promoted to lure
The increase of raw amount, and there is significant difference (P<0.05).But compared with the cell blank group grown under normal circumstances, do not have
Significant difference.Illustrating that biologically active polypeptide LDENDIIL has under the conditions of a certain concentration promotes macrophage nitric oxide to lure
It is raw to measure increased ability.
The activity of fighting against senium of 3 biologically active peptide of embodiment is tested
One, the experiment that biologically active polypeptide LDENDIIL influences caenorhabditis elegan fecundity
1. experiment reagent and instrument：
Reagent：Caenorhabditis elegan (Caenorhabditis elegans), attached Chinese and Western binding institute of Fudan University；Large intestine
Bacterial strain E.coli OP50, attached Chinese and Western binding institute of Fudan University；Agar powder, Sinopharm Chemical Reagent Co., Ltd.；Ferment
Female powder, Sinopharm Chemical Reagent Co., Ltd.；The biologically active polypeptide LDENDIIL that embodiment 1 obtains.
Instrument and equipment：Power health RO15 pure water systems, Li Kang biologic medicals Science and Technology Ltd.；G136T types Zealway intelligence
High-temperature sterilization pot, Xiamen Zhi Wei instruments Science and Technology Ltd.；THZ-32 type Desk type constant-temperatureoscillator oscillators, Shanghai precision experimental facilities have
Limit company；TDL-40B centrifuges, Anting Scientific Instrument Factory, Shanghai；Lu Xiang instrument GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument
Instrument Ltd.；Win fast BJ-CD SERIES Biohazard Safety Equipments, Shanghai Bo Xun Industrial Co., Ltd.s；Nikko is inverted electronic display
Micro mirror, Nikon Corp..
2. experimental method：
(1) prepared by NGM tablets
Take strain Escherichia coli in LB plate streakings, picking single bacterium is fallen in 10ml LB liquid mediums, 37 DEG C,
200rpm, shaken cultivation for 24 hours, until OD600=0.4 for be inoculated with NGM tablets feed nematode.100 μ L bacterium solutions are taken to be applied to 60mm
NGM tablets notice that bacterium solution edge should be apart from plate edge 0.5cm or so.The NGM tablets having been coated with are in room temperature (21-25 DEG C) mistake
It can be used after night.
(2) nematode culture
Nematode used in this experiment is hermaphroditic, Standard culture conditions (20 DEG C of temperature, humidity 40%~
60%) growth is cultivated under.
(3) the synchronization processing of nematode
1) sodium perchlorate bleaching
Prepare pregnant worm growth plate (80% or more insect is in reproduction period i.e. in plate) 2-3 plates, takes 5ml M9 wash buffers 2
It is secondary, buffer solution is sucked in 15ml centrifuge tubes, 1000r/min centrifuges 3min, discards supernatant.5ml is added and newly matches synchronization bleaching
Liquid acutely vibrates 2.5min, adult polypide is corroded at room temperature.Centrifugation, discards supernatant.Ensure total processing time no more than
5min prevents damage worm's ovum.It adds M9 buffer solutions precipitation is resuspended, centrifuges, discard supernatant after mixing, repeat this process 3 times.
2) it prescribes a time limit oviposition method
Several nematodes in the egg-laying season of picking are in same tablet, and the particular number of picking is with required synchronization nematode
Number is foundation.Under general condition, an egg-laying season nematode can lay eggs 6 or so within 1h.After 0.5h being cultivated in tablet,
Choose nematode in tablet, then the ovum in tablet is in same growth period.
(4) index determining
This experiment is using caenorhabditis elegan as animal model, and treated that L4 phase nematodes arrive respective concentration for picking synchronization
In NGM plates.Each concentration at least 8 nematodes, each NGM plates are transferred to one, are denoted as 0 day, move to daily later in new plate until
Nematode reproduction is no longer laid eggs substantially, is counted to the total laying of nematode before it enters the egg-laying season.
3. experimental result and analysis：
Experimental result is as shown in fig. 6, compared with the not blank group of feeding polypeptide LDENDIIL, feeding different quality concentration
Experimental group in, eggs on average number has different degrees of increase.When a concentration of 300mg/L of the polypeptide LDENDIIL of feeding,
Nematode eggs on average number has highly significant difference (P compared with blank group<0.01), and its a concentration of 400mg/L,
When 500mg/L, significant difference (P is but only presented compared with blank group<0.05), this has also further demonstrated that 300mg/L is mixed
It closes galanin peptide LDENDIIL and acts on optium concentration, as the raising of peptide concentration can't inhibit nematode reproduction, but its effect is imitated
Fruit weakens.In conclusion polypeptide LDENDIIL can be obviously improved the fecundity of nematode under a certain concentration.Meanwhile this is tested
The result shows that polypeptide LDENDIIL300mg/L is optimum concentration.But with the increase of concentration, the fecundity of nematode is but no longer
It significantly improves.
Two, the experiment that biologically active polypeptide LDENDIIL influences caenorhabditis elegan body length
1. experiment reagent and instrument：
Reagent：Caenorhabditis elegan (Caenorhabditis elegans), attached Chinese and Western binding institute of Fudan University；Large intestine
Bacterial strain E.coli OP50, attached Chinese and Western binding institute of Fudan University；Agar powder, Sinopharm Chemical Reagent Co., Ltd.；Ferment
Female powder, Sinopharm Chemical Reagent Co., Ltd.；The biologically active polypeptide LDENDIIL that embodiment 1 obtains.
Instrument and equipment：Power health RO15 pure water systems, Li Kang biologic medicals Science and Technology Ltd.；G136T types Zealway intelligence
High-temperature sterilization pot, Xiamen Zhi Wei instruments Science and Technology Ltd.；THZ-32 type Desk type constant-temperatureoscillator oscillators, Shanghai precision experimental facilities have
Limit company；TDL-40B centrifuges, Anting Scientific Instrument Factory, Shanghai；Lu Xiang instrument GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument
Instrument Ltd.；Win fast BJ-CD SERIES Biohazard Safety Equipments, Shanghai Bo Xun Industrial Co., Ltd.s；Nikko is inverted electronic display
Micro mirror, Nikon Corp..
2. experimental method：
(1) prepared by NGM tablets
Take strain Escherichia coli in LB plate streakings, picking single bacterium is fallen in 10ml LB liquid mediums, 37 DEG C,
200rpm, shaken cultivation for 24 hours, until OD600=0.4 for be inoculated with NGM tablets feed nematode.100 μ L bacterium solutions are taken to be applied to 60mm
NGM tablets notice that bacterium solution edge should be apart from plate edge 0.5cm or so.The NGM tablets having been coated with are in room temperature (21-25 DEG C) mistake
It can be used after night.
(2) nematode culture
Nematode used in this experiment is hermaphroditic, Standard culture conditions (20 DEG C of temperature, humidity 40%~
60%) growth is cultivated under.
(3) the synchronization processing of nematode
1) sodium perchlorate bleaching
Prepare pregnant worm growth plate (80% or more insect is in reproduction period i.e. in plate) 2-3 plates, takes 5ml M9 wash buffers 2
It is secondary, buffer solution is sucked in 15ml centrifuge tubes, 1000r/min centrifuges 3min, discards supernatant.5ml is added and newly matches synchronization bleaching
Liquid acutely vibrates 2.5min, adult polypide is corroded at room temperature.Centrifugation, discards supernatant.Ensure total processing time no more than
5min prevents damage worm's ovum.It adds M9 buffer solutions precipitation is resuspended, centrifuges, discard supernatant after mixing, repeat this process 3 times.
2) it prescribes a time limit oviposition method
Several nematodes in the egg-laying season of picking are in same tablet, and the particular number of picking is with required synchronization nematode
Number is foundation.Under general condition, an egg-laying season nematode can lay eggs 6 or so within 1h.After 0.5h being cultivated in tablet,
Choose nematode in tablet, then the ovum in tablet is in same growth period.
(4) index determining
Experiment packet：Blank group and polypeptide group.Difference group nematode, the difference between in the same period lower body is grown, Ke Yi
Reflect the influence that the active material develops nematode growth to a certain extent.The nematode of each group synchronization culture is growing to
When L2 phases (culture 2 days or so), 40 nematodes of picking to respective NGM tablets respectively, continuous 2 days, 3 days, 4 days, 5 days, 6 days, 8
It, observe growth state with inverted microscope within 10 days, measure and record that its body is long, and every group takes its average value.
3. experimental result and analysis：
Under 20 DEG C of condition of culture, since the L2 phases (the 2nd day) of nematode growth from, L3 phases (the 3rd day), L4 phases
It in (the 4th day), the adult stage (the 6th day), is observed continuously 8 days, until the 10th day of nematode growth, measure nematode under each time point
Body is long.Can be seen that each group nematode its body length in the L4 phases in conjunction with Fig. 7 and Fig. 8 is 1000 μm or so, no significant difference.Together
When, from the long change curve of nematode body it can also be seen that, the long change curve of experimental group body also almost with the long change curve of blank group body
It coincides, at nematode L3 phases (the 3rd day), although nematode is averaged, slightly different for body length, does not present statistically aobvious
Write sex differernce.Experiment shows that the concentration of polypeptide LDENDIIL can't influence the growth of nematode.Meanwhile it is also seen that nematode exists
The L3 phases (the 3rd day) are growth stages the most quick to L4 phases (the 4th day).
Three, the Acute oxidative of biologically active polypeptide LDENDIIL stress survival experiment
1. experiment reagent and instrument：
Reagent：Caenorhabditis elegan (Caenorhabditis elegans), attached Chinese and Western binding institute of Fudan University；Large intestine
Bacterial strain E.coli OP50, attached Chinese and Western binding institute of Fudan University；Agar powder, Sinopharm Chemical Reagent Co., Ltd.；Ferment
Female powder, Sinopharm Chemical Reagent Co., Ltd.；30% hydrogenperoxide steam generator, Sinopharm Chemical Reagent Co., Ltd.；Implement
The biologically active polypeptide LDENDIIL that example 1 obtains.
Instrument and equipment：Power health RO15 pure water systems, Li Kang biologic medicals Science and Technology Ltd.；G136T types Zealway intelligence
High-temperature sterilization pot, Xiamen Zhi Wei instruments Science and Technology Ltd.；THZ-32 type Desk type constant-temperatureoscillator oscillators, Shanghai precision experimental facilities have
Limit company；TDL-40B centrifuges, Anting Scientific Instrument Factory, Shanghai；Lu Xiang instrument GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument
Instrument Ltd.；Win fast BJ-CD SERIES Biohazard Safety Equipments, Shanghai Bo Xun Industrial Co., Ltd.s；Nikko is inverted electronic display
Micro mirror, Nikon Corp..
2. experimental method：
(1) prepared by NGM tablets
Take strain Escherichia coli in LB plate streakings, picking single bacterium is fallen in 10ml LB liquid mediums, 37 DEG C,
200rpm, shaken cultivation for 24 hours, until OD600=0.4 for be inoculated with NGM tablets feed nematode.100 μ L bacterium solutions are taken to be applied to 60mm
NGM tablets notice that bacterium solution edge should be apart from plate edge 0.5cm or so.The NGM tablets having been coated with are in room temperature (21-25 DEG C) mistake
It can be used after night.
(2) nematode culture
Nematode used in this experiment is hermaphroditic, Standard culture conditions (20 DEG C of temperature, humidity 40%~
60%) growth is cultivated under.
(3) the synchronization processing of nematode
1) sodium perchlorate bleaching
Prepare pregnant worm growth plate (80% or more insect is in reproduction period i.e. in plate) 2-3 plates, takes 5ml M9 wash buffers 2
It is secondary, buffer solution is sucked in 15ml centrifuge tubes, 1000r/min centrifuges 3min, discards supernatant.5ml is added and newly matches synchronization bleaching
Liquid acutely vibrates 2.5min, adult polypide is corroded at room temperature.Centrifugation, discards supernatant.Ensure total processing time no more than
5min prevents damage worm's ovum.It adds M9 buffer solutions precipitation is resuspended, centrifuges, discard supernatant after mixing, repeat this process 3 times.
2) it prescribes a time limit oviposition method
Several nematodes in the egg-laying season of picking are in same tablet, and the particular number of picking is with required synchronization nematode
Number is foundation.Under general condition, an egg-laying season nematode can lay eggs 6 or so within 1h.After 0.5h being cultivated in tablet,
Choose nematode in tablet, then the ovum in tablet is in same growth period.
(4) index determining
Experiment packet：Blank group and polypeptide group.By synchronization, treated that L4 phase nematodes are placed in corresponding NGM plates, experiment
In H containing 20mM2O2NGM tablets in carry out, be no less than 10 per plate quantity, count that nematode is dead, viable count per half an hour,
Nematode death criterion：Without mobile and swallowing act, still without any reaction after touching.Rejecting standard：1. fleeing to tablet wall
Or it covers and thirst；2. worm's ovum is hatched in vivo forms bag sample worm：3. piercing in agar.
3. experimental result and analysis：
Influences of the 2 biologically active polypeptide LDENDIIL of table to nematode under oxidative stress
As can be seen from Table 2, there is conspicuousness to improve (P < 0.05) for experimental group nematode average life span under oxidative stress,
Extremely significant difference (P is presented in polypeptide LDENDIIL groups<0.05).The each group half death time also accordingly has to a certain extent
Extended, hybrid peptide group is presented conspicuousness and improves (P compared with other experimental groups<0.05).As shown in figure 9, in oxidative stress item
Under part, experimental group survival rate is obviously higher than blank group survival rate.Under the conditions of oxidative stress, the survival rate of nematode obtains this explanation
To significantly improving, it may be possible to since polypeptide LDENDIIL can effectively help nematode to resist oxidative damage, remove and generate in vivo
Free radical and the accumulation for reducing peroxide, rather than by enhancing the realization of its heat hardiness.The extension of organism life-span is certain
It is the resistance due to improving cell to stress conditions in degree, thus slows down aging and the survival rate under pressure stressed condition
There are much relations.This results show polypeptide LDENDIIL can significantly increase the oxidative stress ability of nematode, improve line
The survival rate of worm illustrates that certain density polypeptide LDENDIIL has anti-aging function for nematode.
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 embodiment 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 the present invention's
Within protection domain.
Sequence table
<110>The Shanghai bio tech ltd Bo Hui；Zhejiang Hui Tai life and healths Science and Technology Ltd.
<120>A kind of biologically active polypeptide LDENDIIL and its preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Leu Asp Glu Asn Asp Ile Ile Leu
1 5
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gctcgacgaa aatgatatta tttt 24
<210> 3
<211> 268
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Met Ala Met Met Asp Phe Ser Leu Gln Asn Leu Asp Tyr Glu Val Arg
1 5 10 15
Phe Leu Ile Glu Thr Ala Tyr Ala Ser Ser Ile Gly Lys Val Arg Lys
20 25 30
Ser Asn Gln Asp Phe Val Gln Val Phe Glu Asn Arg Glu Gly Val Thr
35 40 45
Leu Ala Ile Val Cys Asp Gly Met Gly Gly His Gln Gly Gly Asp Val
50 55 60
Ala Ser Thr Met Ala Val Thr His Leu Gly His Asn Phe Gly Met Thr
65 70 75 80
Asp Phe Thr Asp Ser Asn Ile Ala Arg Lys Trp Leu Asp Val Gln Leu
85 90 95
Asn Ser Glu Asn Glu Ile Ile Leu Lys Thr Ala Asp Lys Phe Pro Asp
100 105 110
Leu Asn Gly Met Gly Thr Thr Ile Val Leu Ala Phe Ala Phe Lys Asn
115 120 125
Asn Ala Leu Ile Ala His Leu Gly Asp Ser Arg Ala Tyr Ser Tyr Ser
130 135 140
Glu Gly Lys Phe Val Gln Leu Thr Glu Asp His Ser Leu Val Asn Glu
145 150 155 160
Leu Val Lys Met Gly Gln Ile Thr Lys Glu Gln Ala Lys His His Pro
165 170 175
Gln Lys Asn Ile Ile Thr Gln Ala Leu Gly Val Ser Ser Thr Ile Asp
180 185 190
Pro Glu Ile Lys Glu Ile Lys Leu Asp Glu Asn Asp Ile Ile Leu Leu
195 200 205
Cys Thr Asp Gly Leu Thr Asn Ser Leu Ser Asp Pro Gln Ile Gln Gln
210 215 220
Ile Leu Ala Thr Lys Glu Leu Ser Leu Lys Glu Arg Cys Asn Lys Leu
225 230 235 240
Ile Ser Glu Ser Asn Arg Leu Gly Gly Gly Asp Asn Ile Thr Val Cys
245 250 255
Leu Thr Trp Asn Lys Glu Asp Glu Ser Ser Asp Arg
260 265
Claims (10)
1. a kind of biologically active polypeptide LDENDIIL, which is characterized in that its amino acid sequence is Leu-Asp-Glu-Asn-Asp-
Ile-Ile-Leu。
2. a kind of biologically active polypeptide LDENDIIL 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 LDENDIIL 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 LDENDIIL as described in claim 1, which is characterized in that pass through genetic engineering
Method is artificial synthesized or Lactobacillus helveticus thalline 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 LDENDIIL as described in claim 1, which is characterized in that the biologically active polypeptide
Applications of the LDENDIIL in preparing the food with anti-inflammatory properties, health products, drug or cosmetics.
6. the application of biologically active polypeptide LDENDIIL as described in claim 1, which is characterized in that the biologically active polypeptide
Applications of the LDENDIIL in preparing the food with anti-senescence function, health products or drug.
7. the application of biologically active polypeptide LDENDIIL as described in claim 1, which is characterized in that the biologically active polypeptide
Applications of the LDENDIIL in preparing the food with anti-inflammatory properties and anti-senescence function, health products or drug.
8. a kind of anti-inflammatory products, which is characterized in that including biologically active polypeptide LDENDIIL or described lifes as described in claim 1
The derivative of object active peptides LDENDIIL；The anti-inflammatory products include anti-inflammatory food, anti-inflammatory health product, anti-inflammatory drug or anti-
Scorching cosmetics；The derivative of the biologically active polypeptide LDENDIIL refers to the amino acid in biologically active polypeptide LDENDIIL
On side-chain radical, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification or
Polypeptide derivative that is glycosylation modified, obtaining.
9. a kind of anti-aging product, which is characterized in that including biologically active polypeptide LDENDIIL or described as described in claim 1
The derivative of biologically active polypeptide LDENDIIL；The anti-aging product includes antisenility cistanche food, antisenescence health product or anti-
Senescence drug；The derivative of the biologically active polypeptide LDENDIIL refers to the amino acid in biologically active polypeptide LDENDIIL
On side-chain radical, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification or
Polypeptide derivative that is glycosylation modified, obtaining.
10. a kind of product with anti-inflammatory properties and anti-senescence function, which is characterized in that including biology as described in claim 1
The derivative of active peptides LDENDIIL or described biologically active polypeptides LDENDIIL；With anti-inflammatory properties and anti-senescence function
Product includes food, health products or drug；The derivative of the biologically active polypeptide LDENDIIL, refers in biologically active polypeptide
On the amino acid side groups of LDENDIIL, aminoterminal or c-terminus progress hydroxylating, carboxylated, it is carbonylated, methylates, acetyl
Change, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
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Citations (2)
| 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 |
| CN105254750A (en) * | 2015-10-16 | 2016-01-20 | 上海交通大学 | Bioactive polypeptide FGYSGAFKCL as well as preparation and application thereof |
-
2018
- 2018-06-29 CN CN201810701237.0A patent/CN108794586B/en active Active
Patent Citations (2)
| 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 |
| CN105254750A (en) * | 2015-10-16 | 2016-01-20 | 上海交通大学 | Bioactive polypeptide FGYSGAFKCL as well as preparation and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| JIEHUI,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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