CN108341855A - A kind of biologically active polypeptide ADVKIGNDTVIEGN and its preparation method and application - Google Patents
A kind of biologically active polypeptide ADVKIGNDTVIEGN and its preparation method and application Download PDFInfo
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- CN108341855A CN108341855A CN201810439290.8A CN201810439290A CN108341855A CN 108341855 A CN108341855 A CN 108341855A CN 201810439290 A CN201810439290 A CN 201810439290A CN 108341855 A CN108341855 A CN 108341855A
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- advkigndtviegn
- biologically active
- active polypeptide
- polypeptide
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Classifications
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Landscapes
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Abstract
The present invention relates to albumen fields, more particularly to a kind of biologically active polypeptide ADVKIGNDTVIEGN and its preparation method and application, its amino acid sequence of biologically active polypeptide ADVKIGNDTVIEGN is Ala Asp Val Lys Ile Gly Asn Asp Thr Val Ile Glu Gly Asn.By the experiment of ion vitro immunization function point analysis, internal Antisenility Experiment, demonstrating polypeptide A DVKIGNDTVIEGN has preferable immunoloregulation function and activity of fighting against senium, on the one hand, the biologically active polypeptide ADVKIGNDTVIEGN of the present invention can enhance the in-vitro multiplication ability of lymphocyte and macrophage, 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 immunoloregulation function, 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 ADVKIGNDTVIEGN and its preparation side
Method and application.
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.Newborn source immunomodulatory peptides (PGPIPN) the feeding rat of Li Su duckweeds 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 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 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 ADVKIGNDTVIEGN 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 ADVKIGNDTVIEGN, amino acid sequence Ala-
Asp-Val-Lys-Ile-Gly-Asn-Asp-Thr-Val-Ile-Glu-Gly-Asn, such as SEQ ID NO：Shown in 1.
Preferably, the biologically active polypeptide derives from Lactobacillus helveticus mycoprotein.It is derive specifically from LBH_0185 |
m.172LBH_0185|g.172ORF LBH_0185|g.172LBH_0185|m.172type:complete len:462(+)
LBH_0185:1-1386 (+) albumen, and the amino acid residue of the 264th~277, albumen thus.LBH_0185|
m.172LBH_0185|g.172ORF LBH_0185|g.172LBH_0185|m.172type:complete len:462(+)
LBH_0185:1-1386 (+) protein amino acid sequence such as SEQ ID NO：Shown in 3.
LBH_0185|m.172LBH_0185|g.172ORF LBH_0185|g.172LBH_0185|m.172type:
complete len:462(+)LBH_0185:The amino acid sequence and corresponding nucleotides sequence of 1-1386 (+) albumen are classified as both
There is technology, encodes the biologically active polypeptide of the nucleotide fragments energy encoding mature of this 264th~277 amino acids residue of albumen
ADVKIGNDTVIEGN。
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 ADVKIGNDTVIEGN,
Its sequence is：5 '-cag atg taa aaa ttg gca acg aca ctg tta ttg aag gca atg-3 ', such as SEQ
ID NO：Shown in 2.
Third aspect present invention provides the preparation method of the biologically active polypeptide ADVKIGNDTVIEGN, Ke Yitong
The method for crossing genetic engineering is artificial synthesized, can directly be obtained from Lactobacillus helveticus thalline by the method that clasmatosis isolates and purifies
, it can directly be prepared by chemical synthesis.
Fourth aspect present invention provides the biologically active polypeptide ADVKIGNDTVIEGN and is preparing with immunological regulation
Application in the food of function, health products, drug or cosmetics.
Fifth aspect present invention provides the biologically active polypeptide ADVKIGNDTVIEGN and is preparing with anti-aging work(
Can food, the application in health products or drug.
Sixth aspect present invention provides the biologically active polypeptide ADVKIGNDTVIEGN and is preparing while having immune
Application in the food of regulatory function and anti-senescence function, health products or drug.
Specifically, the biologically active polypeptide ADVKIGNDTVIEGN of the present invention, which can be used for preparing, reduces free radical to skin
The cosmetics of skin injury prepare the drug with immunological regulation and/or anti-aging；And due to the biologically active polypeptide of the present invention
Product after ADVKIGNDTVIEGN is degraded by gastrointestinal tract still has bioactivity, therefore can be also used for preparing Yoghourt etc.
Food, the health products for adjusting 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
The derivative of ADVKIGNDTVIEGN or described biologically active polypeptides ADVKIGNDTVIEGN；The immunological regulation product includes
Immunological regulation food, immunological regulation health products, immunoregulation medicament or immunological regulation cosmetics；The biologically active polypeptide
The derivative of ADVKIGNDTVIEGN refers on the amino acid side groups of biologically active polypeptide ADVKIGNDTVIEGN, ammonia
Cardinal extremity or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, the modifications such as esterification or glycosylation,
Obtained polypeptide derivative.
Eighth aspect present invention provides a kind of anti-aging product, including the biologically active polypeptide
The derivative of ADVKIGNDTVIEGN or described biologically active polypeptides ADVKIGNDTVIEGN；The anti-aging product includes anti-
Aging food, antisenescence health product or antiaging agent；The derivative of the biologically active polypeptide ADVKIGNDTVIEGN refers to
On the amino acid side groups of biologically active polypeptide ADVKIGNDTVIEGN, aminoterminal or c-terminus progress hydroxylating, carboxyl
Change, be carbonylated, methylating, acetylation, phosphorylation, the modifications such as esterification or glycosylation, 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 ADVKIGNDTVIEGN or described biologically active polypeptides ADVKIGNDTVIEGN；With exempting from
The product of epidemic disease regulatory function and anti-senescence function includes food, health products or drug；The biologically active polypeptide
The derivative of ADVKIGNDTVIEGN refers on the amino acid side groups of biologically active polypeptide ADVKIGNDTVIEGN, ammonia
Cardinal extremity or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, the modifications such as esterification or glycosylation,
Obtained polypeptide derivative.
Biologically active polypeptide ADVKIGNDTVIEGN's of the present invention has the beneficial effect that：The biologically active polypeptide of the present invention
ADVKIGNDTVIEGN has preferable adjusting immunity of organisms activity and activity of fighting against senium；On the one hand, biology of the invention is living
Property polypeptide A DVKIGNDTVIEGN can enhance the in-vitro multiplication ability of lymphocyte and macrophage, improve body and resist the external world
The ability of pathogenic infection reduces body incidence；On the other hand, the vigor of internal anti-peroxidation enzyme system can be improved, is enhanced
Body resists the function of external source sexual stimulus, to reduce organism aging process, aging and sick probability, has immunological regulation to exploitation
The dairy products and health products of function and anti-senescence function have a very important significance.
Description of the drawings
Fig. 1：Mass chromatography extraction figure (m/z=722.8600)；
Fig. 2：The second order ms figure for the segment that mass-to-charge ratio is 722.8600；
Fig. 3：Polypeptide az, by crack conditions that mass-to-charge ratio is 722.8600；
Fig. 4：The macrophages in vitro proliferative capacity of biologically active polypeptide ADVKIGNDTVIEGN is tested；
Fig. 5：Influence situations of the biologically active polypeptide ADVKIGNDTVIEGN to female Drosophila survival rate.
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 ADVKIGNDTVIEGN'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 Ala 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. according to step 9-11 connect successively amino acid Asp, Val, Lys, Ile, Gly, Asn, Asp, Thr, Val, Ile,
Glu, Gly and Asn.
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 ADVKIGNDTVIEGN.
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 ADVKIGNDTVIEGN carries out chromatography and mass spectral analysis, and mass chromatography extraction figure is as shown in Figure 1, extract the two of this peak
Grade mass spectrogram 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 722.8600Da, retention time
It is 36.9min.
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
722.8600Da fragment sequence be Ala-Asp-Val-Lys-Ile-Gly-Asn-Asp-Thr-Val-Ile-Glu-Gly-Asn
(ADVKIGNDTVIEGN), SEQ ID NO are denoted as：1.The segment and LBH_0185 | m.172LBH_0185 | g.172ORF LBH_
0185|g.172LBH_0185|m.172type:complete len:462(+)LBH_0185:1-1386 (+) albumen the 264th~
277 residue sequences are 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, mtt assay measures the macrophages in vitro proliferative capacity experiment of biologically active polypeptide ADVKIGNDTVIEGN
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 ADVKIGNDTVIEGN that embodiment 1 obtains；3- (4,5- dimethylthiazole -2) -2,5- hexichol
Base tetrazole bromide (MTT) Amresco companies；LPS (lipopolysaccharides) Sigma companies；Bovine serum albumin(BSA) (Bovine Serum
Albumin, BSA) Genebase companies；Three lysates, the water containing 10%SDS, 5% isobutanol and 0.012mol/L HCl
Solution.
Instrument and equipment：LRH-250F biochemical cultivation cases Shanghai perseverance Science and Technology Ltd.；On GL-22M high speed freezing centrifuges
Hai Luxiang instrument centrifuges Instrument Ltd.；Hera cell 150CO2Incubator Heraeus companies；Dragon Wellscan
MK3 microplate reader Labsystems companies.
2. experimental method：
Balb/c mouse peritoneals inject 2% (w/w) the sterilizing starch solutions 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, 4 DEG C of phosphate buffers (PBS) is drawn with syringe and rinses abdominal cavity repeatedly, from
After heart pipe collects flushing liquor, supernatant is abandoned in centrifugation (1000rpm, 4 DEG C) after ten minutes, (is contained with 4 DEG C of RPMI1640 complete culture solutions
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, liquid in hole is abandoned in suction, and cell culture plate well is carefully cleaned with 37 DEG C of RPMI1640 complete culture solutions
Bottom washes away not adherent cell and cell fragment, obtains adherent peritoneal macrophage after purification.0.2ml is added per hole
RPMI1640 complete mediums, experiment small peptide sample and LPS are added after being dissolved in culture medium in advance, start cell culture.
It is 2 × 10 that number of cells, which is added,5100 holes μ l/ of cell suspension of/ml, adherent addition after purification are more containing bioactivity
200 holes μ l/ of RPMI1640 complete culture solutions (10%FBS) of peptide (100,500,1000 μ g/mL), continuous culture 48 hours are scorching
LPS to final concentration 100ng/ml was added at 24 hours for disease group.20 holes μ l/ 5%MTT are added at 44 hours, after reaching 48 hours
Three lysates in 100 holes μ l/ are added to terminate culture, after dissolving overnight, survey the suction in each hole with microplate reader at wavelength 570nm
The calculation formula of shading value (OD570), growth index (Growth Indices) is as follows：
Wherein, blank group is not apply small peptide and the cell processing group of BSA, and BSA groups are negative control.
3. experimental result and analysis：
Experimental result is shown in Fig. 4, and the addition concentration of biologically active polypeptide (ADVKIGNDTVIEGN) is respectively in experimental group
1000,500,100 μ g/mL, blank group are added the PBS of corresponding amount as blank control, indicate in the case where no LPS is stimulated
The proliferative conditions of macrophage.Compared with blank control group, add the polypeptide A DVKIGNDTVIEGN experimental groups of various concentration with
The increase of experimental concentration, the proliferative capacity of macrophage is gradually increasing, and in a concentration of 1000,500 μ g/mL, is had notable
Sex differernce (P<0.05).Illustrate that biologically active polypeptide ADVKIGNDTVIEGN has the ability for promoting macrophage proliferation.
Two, the rush macrophage of biologically active polypeptide ADVKIGNDTVIEGN swallows dimethyl diaminophenazine chloride capacity experimental
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 ADVKIGNDTVIEGN that embodiment 1 obtains；LPS is purchased from Sigma companies；Neutral red staining
Liquid, green skies biotechnology research institute production.
Instrument and equipment：LRH-250F biochemical cultivation cases Shanghai perseverance Science and Technology Ltd.；On GL-22M high speed freezing centrifuges
Hai Luxiang instrument centrifuges Instrument Ltd.；Hera cell 150CO2 incubator Heraeus companies；Dragon Wellscan
MK3 microplate reader Labsystems companies.
2. experimental method：
It is 2 × 10 that number of cells, which is added,6100 holes μ l/ of cell suspension of/ml, adherent be added after purification contain active peptide
200 holes μ l/ of RPMI1640 complete culture solutions (10%FBS) of ADVKIGNDTVIEGN (1mg/ml) are experimental group, and addition is free of
What 200 holes μ l/ of RPMI1640 complete culture solutions (10%FBS) of active peptide were cultivated is set as blank group；And experimental group and
LPS to 10 μ g/ml of final concentration is added when culture is arrived for 24 hours for blank group；After continuing culture to 48h, cell culture fluid is abandoned in suction.PBS
37 DEG C of 80 holes μ l/ of dimethyl diaminophenazine chloride dye liquor are added after cleaning bottom hole, inhales abandon dye liquor after ten minutes, after being cleaned twice with PBS, add per hole
Enter 150 μ l cell pyrolysis liquid (glacial acetic acid：Absolute ethyl alcohol=1:1, v/v).After 4 DEG C of dissolvings overnight, measures and inhale at wavelength 540nm
Shading value (OD540).
3. experimental result and analysis：
1 biologically active polypeptide ADVKIGNDTVIEGN of table promotees the measurement of macrophage phagocytosis dimethyl diaminophenazine chloride ability
| Experiment packet | Inflammation group absorbance value (OD540) |
| Blank group | 0.1031±0.0846 |
| Experimental group | 0.1425±0.0462** |
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, compared with cell blank, addition 1mg/ml biologically active polypeptides ADVKIGNDTVIEGN's
Inflammation group macrophage phagocytosis dimethyl diaminophenazine chloride ability obviously increases, and compared with cell blank group, has significant difference (P ＜
0.01).In illustrating that biologically active polypeptide ADVKIGNDTVIEGN swallows macrophages in vitro in the case where there is inflammation generation
Property red ability there is significant facilitation.
The activity of fighting against senium of 3 biologically active peptide of embodiment is tested
One, biologically active polypeptide ADVKIGNDTVIEGN improves the experiment of drosophila survival ability
1. experiment reagent and instrument：
Reagent：Oregon K wild type Drosophila melanogasters, agricultural college of Shanghai Communications University genetics experiments room；Agar powder, state
Chemical reagent Co., Ltd of medicine group；The biologically active polypeptide ADVKIGNDTVIEGN that embodiment 1 obtains.
Instrument and equipment：The ultra-clean water of CM-230 types mole, Shanghai Moller scientific instrument Co., Ltd；G136T type Zealway intelligence
Energy high-temperature sterilization pot, Xiamen Zhi Wei instruments Science and Technology Ltd.；BJ-CD SERIES bio-incubators, Shanghai is rich to prove to be true after interrogation industry public affairs
Department；GRX-9073 type hot air sterilizers, the permanent Science and Technology Ltd. in Shanghai one.
2. experimental method：
Using drosophila as experimental model：The drosophila adult newly to sprout wings in 8 hours is collected, divides male and female random transferring to respectively after anesthesia
In experimental group, every group of each gender 100, every group of setting 3 is parallel, and control group gives conventional corn powder culture medium, experimental group
ADVKIGNDTVIEGN biologically active peptides-corn culture medium respectively containing 0.05mg/ml, 0.5mg/ml, 1mg/ml.Every 2
Its replacement fresh culture is primary, observes and records the death toll of different sexes drosophila daily, until drosophila is all dead.
Drosophila survival curve is drawn, and calculates the average life span of different sexes drosophila and maximum life span (takes 5 drosophilas of last death
It is counted).
3. experimental result and analysis：
This experiment is as follows to the result of study of the life span of drosophila melanogaster of feeding various concentration biologically active peptide：It can be sent out from Fig. 5
It is existing, relative to blank control group female Drosophila, when a concentration of 0.5mg/ml and 1mg/ml of feeding, in same time point, female fruit
The survival rate of fly increases.
Influence situations of the table 2-1 ADVKIGNDTVIEGN to the Male Drosophila service life
Note：* mark is compared with blank control group, significant difference (P<0.05)；Similarly hereinafter.
Influence situations of the table 2-2 ADVKIGNDTVIEGN to the female Drosophila service life
From table 2-1 it is found that relative to blank control group, low dose group Male Drosophila average life span does not have significant change,
But middle dose group and advanced amount group Male Drosophila average life span are improved, and respectively 16.21% and 8.57%, but only middle dosage
Group produces significant difference (p<0.05), illustrate that the average life span conspicuousness of middle dose group Male Drosophila improves.Meanwhile middle dose
The half death time of amount group and high dose group drosophila is improved, but does not have notable difference in terms of MaLS.By table
2-2 is it is found that female Drosophila low dose group, middle dose group and high dose group increase in terms of average life span, but do not produce
Raw significant difference.But the MaLS of middle dose group and high dose group increases, and extends 7 days respectively compared with blank control group
With 6 days, and produce significant difference (P<0.05).
This is the experiment results show that biologically active polypeptide ADVKIGNDTVIEGN can improve the flat of drosophila under a certain concentration
Equal service life and MaLS, but it is related with concentration and gender.This phenomenon related to tested material concentration, strain may be because
ADVKIGNDTVIEGN participates in the part biological metabolism of drosophila, or is reached by improving the antioxidant system of drosophila tissue
Extend the effect of life span of drosophila melanogaster.Since the metabolism of different lines drosophila can have any different, to cause the difference of result.And gender
Difference, it may be possible to since female Drosophila is inherently with certain conservative and to the resistance of external environment, so
ADVKIGNDTVIEGN, which extends the female Drosophila service life, to be not obvious.
Two, biologically active polypeptide ADVKIGNDTVIEGN improves the experiment of drosophila fertility
1. experiment reagent and instrument：
Reagent：Oregon K wild type Drosophila melanogasters, agricultural college of Shanghai Communications University genetics experiments room；Agar powder, state
Chemical reagent Co., Ltd of medicine group；The biologically active polypeptide ADVKIGNDTVIEGN that embodiment 1 obtains.
Instrument and equipment：The ultra-clean water of CM-230 types mole, Shanghai Moller scientific instrument Co., Ltd；G136T type Zealway intelligence
Energy high-temperature sterilization pot, Xiamen Zhi Wei instruments Science and Technology Ltd.；BJ-CD SERIES bio-incubators, Shanghai is rich to prove to be true after interrogation industry public affairs
Department；GRX-9073 type hot air sterilizers, the permanent Science and Technology Ltd. in Shanghai one.
2. experimental method：
The drosophila adult newly to sprout wings in 8 hours is collected, its male and female is separately fed, is separately added into culture medium a concentration of
The ADVKIGNDTVIEGN solution of 0mg/ml, 0.05mg/ml, 0.5mg/ml, 1mg/ml, continuous culture 12 days.It collects within 13rd day
The adult drosophila cultivated under same concentrations is simultaneously transferred in new Nostoc commune Vanch bottle, and each culture bottle ensures 1 female and 2 heros
Property (every group 5 bottles), gives accurate 24 hours for every bottle and lays eggs.Parent drosophila is transferred to new Nostoc commune Vanch bottle after oviposition
In, old culture bottle continues breeding culture, counts progeny size, METHOD FOR CONTINUOUS DETERMINATION 7 days after larva sprouts wings, and be repeated 3 times.
3. experimental result and analysis：
3 reproductive capacity measurement result of table
From table 3 it can be seen that low concentration experimental group reproductive capacity does not generate conspicuousness variation, but middle dosage compared with the control group
Experimental group and the reproductive capacity of high dose experimental group drosophila are significantly increased (P compared with blank control group<0.05).Illustrate certain dense
The ADVKIGNDTVIEGN of degree can promote the reproductive capacity of drosophila.Originally the experimental results showed that, the extension of life span of drosophila melanogaster is
It is that ADVKIGNDTVIEGN is directly acted on as a result, rather than ADVKIGNDTVIEGN pass through reduce reproductive capacity caused by two level physiology
Effect.Also illustrate that ADVKIGNDTVIEGN is safe to drosophila simultaneously, without toxic hazard.
Three, the experiment that biologically active polypeptide ADVKIGNDTVIEGN influences drosophila SOD and MAD content
1. experiment reagent and instrument：
Reagent：Oregon K wild type Drosophila melanogasters, agricultural college of Shanghai Communications University genetics experiments room；Agar powder, state
Chemical reagent Co., Ltd of medicine group；MDA lipid peroxide kits, Science and Technology Ltd. of Nanjing Keygen Biotech；SOD super oxygens
Compound is disproportionated enzyme reagent kit, and bio tech ltd is built up in Nanjing；The biologically active polypeptide that embodiment 1 obtains
ADVKIGNDTVIEGN。
Instrument and equipment：The ultra-clean water of CM-230 types mole, Shanghai Moller scientific instrument Co., Ltd；Organize homogenizer, Shanghai
Member is as bio tech ltd；G136T type Zealway intelligence high-temperature sterilization pots, Xiamen Zhi Wei instruments Science and Technology Ltd.；
BJ-CD SERIES bio-incubators, Shanghai Bo Xun industrial corporations；GRX-9073 type hot air sterilizers, the permanent science and technology in Shanghai one have
Limit company；Infinite type microplate reader, Austrian Di Ken Co., Ltds.
2. experimental method：
The drosophila adult newly to sprout wings in 8 hours is collected, is divided after anesthesia in male and female random transferring to each experimental group, every group each
Gender 100, every group of setting 3 is parallel, and control group gives conventional corn powder culture medium, and experimental group is respectively to contain 0.05mg/
ADVKIGNDTVIEGN biologically active peptides-corn culture medium of ml, 0.5mg/ml, 1mg/ml.Replace fresh culture one within every 2 days
Secondary, after raising 30 days, every group weighs drosophila 40mg, adds 0.5ml physiological saline, grinds and is homogenized in ice bath, interval 10s seconds, instead
It carries out 3 times again, homogenate is made, illustrate to measure every group of drosophila SOD activity and MDA levels according to kit.It is detected using MDA
Kit detects the levels of the lipid peroxidation product MDA in drosophila body, and the wavelength of spectrophotometer is 532nm.
3. experimental result and analysis：
Influences of 4 ADVKIGNDTVIEGN of table to drosophila SOD, MDA
As can be known from Table 4, relative to blank control group, the SOD contents in the female male drosophila body of polypeptide processing group are improved,
And for Male Drosophila group, when peptide concentration reaches 1mg/ml, there is significant difference in the SOD contents in drosophila body, and
Then there is significant difference when peptide concentration is 0.5mg/ml and 1mg/ml in female Drosophila group.Illustrate by taking in certain polypeptide,
Internal SOD contents can be improved, and body protective itself is helped to prevent oxidative damage.MDA contents can see from table 4,
MDA contents in experimental group Male Drosophila and female Drosophila body have reduction.Relative to male blank control group MDA contents 1.37
There is the reduction of conspicuousness in ± 0.21 μm of ol/L, the MDA contents of a concentration of 0.5mg/ml and 1mg/ml drosophilas group, and female Drosophila
In group, when 1mg/ml peptides are handled, there is the reduction of conspicuousness in the MDA contents in drosophila body.Since MDA is body lipid peroxide
Change and generate, the reduction of content illustrates that the Antioxidant Enzymes vigor of drosophila is improved indirectly, to protect body
Histoorgan not will produce a large amount of lipid peroxide.
From experimental result as can be seen that the experimental result of SOD and MDA is mutually proved, it may be said that gelatine/biological activity polypeptide
ADVKIGNDTVIEGN helps to improve the vigor of the Antioxidant Enzymes in body body, so as to effectively improve the anti-oxidant of body
Ability, reduce body is stimulated by the bad factor, to reduce organism aging process, aging and sick probability, all in all, for hero
The effect of property drosophila is better than female Drosophila.
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>Zhejiang panda dairy industry Group Plc；Zhang Shaohui
<120>A kind of biologically active polypeptide ADVKIGNDTVIEGN and its preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
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<211> 14
<212> PRT
<213>Artificial sequence (Artificial Sequence)
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Ala Asp Val Lys Ile Gly Asn Asp Thr Val Ile Glu Gly Asn
1 5 10
<210> 2
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cagatgtaaa aattggcaac gacactgtta ttgaaggcaa tg 42
<210> 3
<211> 461
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Met Glu Lys Tyr Val Val Val Leu Ala Ala Gly Lys Gly Thr Arg Met
1 5 10 15
Lys Ser Lys Leu Tyr Lys Val Leu His Lys Val Cys Gly Lys Thr Met
20 25 30
Val Glu His Val Val Asp Ala Ala Ser Gly Ile Asn Pro Thr Lys Ile
35 40 45
Val Thr Val Val Gly Thr Gly Ala Gly Glu Val Glu Lys Val Leu Ala
50 55 60
Asn Lys Ser Asp Phe Ala Phe Gln Glu Lys Gln Val Gly Thr Gly Asp
65 70 75 80
Ala Val Met Thr Ala Lys Glu Ala Leu Gly Asp Lys Glu Gly Ala Thr
85 90 95
Leu Val Val Thr Gly Asp Thr Pro Leu Phe Thr Thr Asp Thr Phe Asn
100 105 110
Glu Leu Phe Lys Tyr His Ala Glu Lys Gly Asn Ala Ala Thr Val Leu
115 120 125
Thr Ala Glu Ala Pro Asn Pro Phe Gly Tyr Gly Arg Ile Ile Arg Asp
130 135 140
Asn Gln Gly Asn Val Leu Arg Ile Val Glu Gln Lys Asp Gly Asn Pro
145 150 155 160
Asp Glu Leu Lys Val Lys Glu Ile Asn Thr Gly Val Phe Cys Phe Asp
165 170 175
Asn Gln Lys Leu Phe Glu Ala Leu Lys His Val Asp Asn Asp Asn Ala
180 185 190
Gln Gly Glu Tyr Tyr Leu Thr Asp Val Leu Glu Ile Leu Arg Asn Asn
195 200 205
Gly Glu Arg Val Gly Ala Tyr Lys Met Pro Asp Phe Ser Glu Ser Leu
210 215 220
Gly Val Asn Asp Arg Val Ala Leu Ala Gln Ala Thr Lys Thr Met Gln
225 230 235 240
Arg Arg Ile Asn Glu Ala His Met Arg Asp Gly Val Ser Phe Ile Asp
245 250 255
Pro Asp Thr Ala Tyr Ile Asp Ala Asp Val Lys Ile Gly Asn Asp Thr
260 265 270
Val Ile Glu Gly Asn Val Val Ile Lys Gly Asn Thr Glu Ile Gly Ser
275 280 285
Asp Cys Tyr Ile Thr Asn Gly Ser Arg Ile Val Asp Ser Lys Ile Gly
290 295 300
Asn Gly Val Thr Ile Thr Ser Ser Thr Ile Glu Glu Ala Glu Met Asp
305 310 315 320
Asp Asn Thr Asp Ile Gly Pro Asn Ser His Leu Arg Pro Lys Ala Ile
325 330 335
Ile Arg Lys Gly Ala His Ile Gly Asn Phe Val Glu Ile Lys Lys Ala
340 345 350
Glu Ile Gly Glu Asn Thr Lys Val Gly His Leu Thr Tyr Val Gly Asp
355 360 365
Ala Thr Leu Gly Lys Asp Ile Asn Ile Gly Cys Gly Thr Ile Phe Ser
370 375 380
Asn Tyr Asp Gly Val Lys Lys Phe His Thr Asn Val Gly Asp His Ser
385 390 395 400
Phe Ile Gly Ala Gly Ser Thr Leu Ile Ala Pro Ile Asn Val Ala Asp
405 410 415
His Ala Phe Ile Ala Ala Asp Ser Thr Ile Thr Lys Asp Val Gly Lys
420 425 430
Tyr Asp Met Ala Ile Ala Arg Gly Arg Gln Thr Asn Lys Glu Asp Tyr
435 440 445
Trp His Lys Leu Pro Leu Ser Lys Asp Lys Asp Trp Glu
450 455 460
Claims (10)
1. a kind of biologically active polypeptide ADVKIGNDTVIEGN, which is characterized in that its amino acid sequence is Ala-Asp-Val-
Lys-Ile-Gly-Asn-Asp-Thr-Val-Ile-Glu-Gly-Asn。
2. a kind of biologically active polypeptide ADVKIGNDTVIEGN according to claim 1, which is characterized in that the biology is living
Property polypeptide derive from Lactobacillus helveticus mycoprotein.
3. encoding the nucleotide fragments of biologically active polypeptide ADVKIGNDTVIEGN described in claim 1, which is characterized in that described
The sequence of nucleotide fragments such as SEQ ID NO：Shown in 2.
4. the preparation method of biologically active polypeptide ADVKIGNDTVIEGN as described in claim 1, which is characterized in that pass through gene
The method of engineering is artificial synthesized or Lactobacillus helveticus thalline is directly obtained by the method that clasmatosis isolates and purifies, or directly
It is prepared by chemical synthesis.
5. the application of biologically active polypeptide ADVKIGNDTVIEGN as described in claim 1, which is characterized in that the bioactivity
Applications of the polypeptide A DVKIGNDTVIEGN in preparing the food with immunoloregulation function, health products, drug or cosmetics.
6. the application of biologically active polypeptide ADVKIGNDTVIEGN as described in claim 1, which is characterized in that the bioactivity
Applications of the polypeptide A DVKIGNDTVIEGN in preparing the food with anti-senescence function, health products or drug.
7. the application of biologically active polypeptide ADVKIGNDTVIEGN as described in claim 1, which is characterized in that the bioactivity
Polypeptide A DVKIGNDTVIEGN answering in preparing the food with immunoloregulation function and anti-senescence function, health products or drug
With.
8. a kind of immunological regulation product, which is characterized in that including biologically active polypeptide as described in claim 1
The derivative of ADVKIGNDTVIEGN or described biologically active polypeptides ADVKIGNDTVIEGN；The immunological regulation product includes
Immunological regulation food, immunological regulation health products, immunoregulation medicament or immunological regulation cosmetics；The biologically active polypeptide
The derivative of ADVKIGNDTVIEGN refers on the amino acid side groups of biologically active polypeptide ADVKIGNDTVIEGN, ammonia
Cardinal extremity or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification or glycosylation modified, obtain
The polypeptide derivative arrived.
9. a kind of anti-aging product, which is characterized in that including biologically active polypeptide ADVKIGNDTVIEGN as described in claim 1
Or the derivative of the biologically active polypeptide ADVKIGNDTVIEGN；The anti-aging product includes antisenility cistanche food, anti-ageing
Old health products or antiaging agent；The derivative of the biologically active polypeptide ADVKIGNDTVIEGN refers to more in bioactivity
On the amino acid side groups of peptide ADVKIGNDTVIEGN, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, first
Base, acetylation, phosphorylation, esterification or glycosylation modified, 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 ADVKIGNDTVIEGN or described biologically active polypeptides ADVKIGNDTVIEGN；With immune tune
The product for saving function and anti-senescence function includes food, health products or drug；The biologically active polypeptide ADVKIGNDTVIEGN
Derivative, refer on the amino acid side groups of biologically active polypeptide ADVKIGNDTVIEGN, aminoterminal or c-terminus into
Row hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derives
Object.
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| CN112724237A (en) * | 2021-01-19 | 2021-04-30 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide GGSDGYGSGRGF, and preparation method and application thereof |
| CN112745379A (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 RDNKKTRIIPR, and preparation method and application thereof |
| CN112759636A (en) * | 2021-01-21 | 2021-05-07 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide with amino acid structure ESLKGVDPKFLR, and preparation method and application thereof |
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| CN107759681A (en) * | 2017-12-01 | 2018-03-06 | 浙江熊猫乳业集团股份有限公司 | A kind of biologically active polypeptide INNQFLPYPYYAKPA and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107759681A (en) * | 2017-12-01 | 2018-03-06 | 浙江熊猫乳业集团股份有限公司 | A kind of biologically active polypeptide INNQFLPYPYYAKPA and its preparation method and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112724237A (en) * | 2021-01-19 | 2021-04-30 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide GGSDGYGSGRGF, and preparation method and application thereof |
| CN112759636A (en) * | 2021-01-21 | 2021-05-07 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide with amino acid structure ESLKGVDPKFLR, and preparation method and application thereof |
| CN112745379A (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 RDNKKTRIIPR, and preparation method and application thereof |
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| CB02 | Change of applicant information |
Address after: 325800 LITE-ON East Road, Ling Xi Town, Cangnan County, Zhejiang 650-668 Applicant after: Panda Dairy Group Limited by Share Ltd Applicant after: Zhang Shaohui Address before: 325800 LITE-ON East Road, Ling Xi Town, Cangnan County, Wenzhou, Zhejiang 650-668 Applicant before: ZHEJIANG PANDA DAIRY CORPORATION Applicant before: Zhang Shaohui |
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Application publication date: 20180731 |