CN108558991A - A kind of biologically active polypeptide GIQDPKEP and its preparation method and application - Google Patents

A kind of biologically active polypeptide GIQDPKEP and its preparation method and application Download PDF

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CN108558991A
CN108558991A CN201810712843.2A CN201810712843A CN108558991A CN 108558991 A CN108558991 A CN 108558991A CN 201810712843 A CN201810712843 A CN 201810712843A CN 108558991 A CN108558991 A CN 108558991A
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giqdpkep
biologically active
active polypeptide
polypeptide
inflammatory
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CN108558991B (en
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张少辉
占文静
张伯宇
陈静
李婉茹
汪超
李阜烁
范梦珠
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Zhejiang Peptide Life Health Science And Technology Co Ltd
Shanghai Platinum Hui Biological Technology Co Ltd
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Zhejiang Peptide Life Health Science And Technology Co Ltd
Shanghai Platinum Hui Biological Technology Co Ltd
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, 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
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17Amino acids, peptides or proteins
    • A23L33/18Peptides; Protein hydrolysates
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64Proteins; Peptides; Derivatives or degradation products thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00General protective or antinoxious agents
    • A61P39/06Free radical scavengers or antioxidants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • A61Q19/08Anti-ageing preparations
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

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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 GIQDPKEP and its preparation method and application, biologically active polypeptide GIQDPKEP is Gly Ile Gln Asp Pro Lys Glu Pro.By extracorporeal anti-inflammatory activity experiment, internal Antisenility Experiment, demonstrating polypeptide GIQDPKEP has preferable anti-inflammatory activity and activity of fighting against senium, on the one hand, the biologically active polypeptide GIQDPKEP 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

A kind of biologically active polypeptide GIQDPKEP and its preparation method and application
Technical field
The present invention relates to albumen fields, more particularly, to a kind of biologically active polypeptide GIQDPKEP 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 GIQDPKEP 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 GIQDPKEP, amino acid sequence Gly-Ile- Gln-Asp-Pro-Lys-Glu-Pro, such as SEQ ID NO:Shown in 1.
Preferably, the biologically active polypeptide derives from Lactobacillus helveticus mycoprotein.It is derive specifically from LBH_1578 | m.1470LBH_1578|g.1470ORF LBH_1578|g.1470LBH_1578|m.1470type:complete len:310 (+)LBH_1578:1-930 (+) albumen, and the amino acid residue of the 199th~206, albumen thus.LBH_1578| m.1470LBH_1578|g.1470ORF LBH_1578|g.1470LBH_1578|m.1470type:complete len:310 (+)LBH_1578:1-930 (+) protein amino acid sequence such as SEQ ID NO:Shown in 3.
LBH_1578|m.1470LBH_1578|g.1470ORF LBH_1578|g.1470LBH_1578|m.1470type: complete len:310(+)LBH_1578:The amino acid sequence and corresponding nucleotides sequence of 1-930 (+) albumen are classified as both There is technology, encodes the biologically active polypeptide of the nucleotide fragments energy encoding mature of this 199th~206 amino acids residue of albumen GIQDPKEP。
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 GIQDPKEP, sequence For:5 '-ttc cct ggg ccc atc cct aac agc-3 ', such as SEQ ID NO:Shown in 2.
Third aspect present invention provides the preparation method of the biologically active polypeptide GIQDPKEP, 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 GIQDPKEP 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 GIQDPKEP 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 GIQDPKEP 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 GIQDPKEP 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 GIQDPKEP 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 GIQDPKEP or described The derivative of biologically active polypeptide GIQDPKEP;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 GIQDPKEP refers to the amino in biologically active polypeptide GIQDPKEP 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 GIQDPKEP or institute State the derivative of biologically active polypeptide GIQDPKEP;The anti-aging product include antisenility cistanche food, antisenescence health product or Antiaging agent;The derivative of the biologically active polypeptide GIQDPKEP refers to the amino in biologically active polypeptide GIQDPKEP 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 GIQDPKEP or described biologically active polypeptides GIQDPKEP;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 GIQDPKEP, refers in bioactivity On the amino acid side groups of polypeptide GIQDPKEP, 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 GIQDPKEP's of the present invention has the beneficial effect that:The biologically active polypeptide GIQDPKEP tools of the present invention There are preferable anti-inflammatory activity and activity of fighting against senium;On the one hand, biologically active polypeptide GIQDPKEP 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=884.4524);
Fig. 2:The second order ms figure for the segment that mass-to-charge ratio is 884.4524;
Fig. 3:Polypeptide az, by crack conditions that mass-to-charge ratio is 884.4524;
Fig. 4:Influence situations of the biologically active polypeptide GIQDPKEP to drosophila survival rate;
Fig. 5:Hydrogen peroxide (H2O2) acute experiment.
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:ALABORATORY 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 GIQDPKEP'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 Gly 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 Ile, Gln, Asp, Pro, Lys, Glu and Pro 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 GIQDPKEP.
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 GIQDPKEP 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 884.4524Da, and retention time is 61.0min。
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 884.4524Da is Gly-Ile-Gln-Asp-Pro-Lys-Glu-Pro (GIQDPKEP), is denoted as SEQ ID NO:1.The segment and LBH_1578 | m.1470LBH_1578 | g.1470ORF LBH_1578 | g.1470LBH_1578 | m.1470type:complete len:310(+)LBH_1578:The residue sequence of 199-206,1-930 (+) 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 GIQDPKEP
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 GIQDPKEP that embodiment 1 obtains; ELISA cell factors Quick kit (TNF-α, IL-1 β and IL-6), Wuhan Boster Biological Technology Co., Ltd..
Instrument and equipment:LRH-250F biochemical cultivation cases Shanghai perseverance Science and Technology Ltd.;On GL-22M high speed freezing centrifuges Hai Luxiang instrument centrifuges Instrument Ltd.;Hera cell 150CO2 incubator Heraeus companies;Dragon Wellscan MK3 microplate reader Labsystems companies.
2. experimental method:
It is 2 × 10 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 to 10 μ g/ml of final concentration was added at 24 hours for 200 holes μ l/ of RPMI1640 complete culture solutions (10%FBS), inflammation group, even Continuous culture 48 hours, inflammation group terminate first 24 hours in culture and LPS to final concentration 100ng/ml are added.After culture terminates, centrifugation Collect cell culture supernatant liquid.100 μ l supernatants, 37 DEG C of reactions 90 are added in the ELISA Plate of coated cell factor antibody After minute, biotin labelled antibodies are added, 37 DEG C are reacted 60 minutes, and after PBS washings, it is compound that Avidin-peroxidase is added Object reacts 30 minutes.Developing solution is added after PBS washings, reacts 20 minutes.After colour developing terminate liquid is added, using microplate reader in wave Absorbance value (OD450) is measured under long 450nm.
3. experimental result and analysis:
The measurement that 1 biologically active peptide GIQDPKEP of table influences Macrophage Cell factor level
Note:*, compared with negative control, significant difference (P < 0.05);* has significantly compared with negative control group Sex differernce (P < 0.01)
As can be known from Table 1, in TNF-α, the experimental result of IL-1 β and IL-6 these three cell factors, TNF-α, IL-1 β In 0.2mg/ml and significant difference (P < 0.01) is appeared above, IL-6 significant difference (P < occurs in 0.5mg/ml 0.01), it was demonstrated that the GIQDPKEP under a certain concentration can promote the Turnover of Mouse Peritoneal Macrophages to activate and discharge TNF-α, IL-1 β, IL-6 improves floors of these cell factors under normal macrophages quiescent condition, to adjust the immunity of body.
Two, the measurement (Griess methods) of the rush macrophage nitric oxide amount of inducing of biologically active polypeptide GIQDPKEP
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 GIQDPKEP 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.;Hera cell 150CO2 incubator Heraeus companies;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:
2 biologically active polypeptide GIQDPKEP of table promotees the measurement of the macrophage nitric oxide amount of inducing
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 2, as shown in Table 2, biologically active polypeptide GIQDPKEP 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 GIQDPKEP, 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 GIQDPKEP 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, biologically active polypeptide GIQDPKEP 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 GIQDPKEP 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 GIQDPKEP biologically active peptides-corn culture medium respectively containing 0.05mg/ml, 0.5mg/ml, 1mg/ml.It replaces within every 2 days Fresh culture is primary, observes and records the death toll of different sexes drosophila daily, until drosophila is all dead.Draw fruit Fly survivorship curve, and calculate the average life span of different sexes drosophila and maximum life span (takes 5 drosophilas of last death to unite Meter).
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 with from Fig. 4 (A) It was found that for blank control group Male Drosophila, the GIQDPKEP of a concentration of 0.05mg/ml of feeding is not significantly changed The survival rate of Male Drosophila, and when peptide concentration reaches 0.5mg/ml and 1mg/ml, same time point, the survival rate of Male Drosophila It is significantly improved.From Fig. 4 (B), relative to blank control group female Drosophila, feeding a concentration of 0.5mg/ml and 1mg/ml When, in same time point, the survival rate of female Drosophila increases, but result difference unobvious.
Influence situations of the table 3-1GIQDPKEP 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 3-2GIQDPKEP to the female Drosophila service life
From table 3-1 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 16.17% and 9.21%, 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 3-2 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 GIQDPKEP can improve the average life span of drosophila under a certain concentration And MaLS, but it is related with concentration and gender.This phenomenon related to tested material concentration, strain may be because GIQDPKEP participates in the part biological metabolism of drosophila, or the antioxidant system by improving drosophila tissue extends fruit to reach The effect in fly service life.Since the metabolism of different lines drosophila can have any different, to cause the difference of result.And the difference of gender, May be since female Drosophila is inherently with certain conservative and to the resistance of external environment, so GIQDPKEP is to female Property life span of drosophila melanogaster extension be not obvious.
Two, biologically active polypeptide GIQDPKEP 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 GIQDPKEP 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 GIQDPKEP solution of 0mg/ml, 0.05mg/ml, 0.5mg/ml, 1mg/ml, continuous culture 12 days.It collects within 13rd day identical dense The lower adult drosophila cultivated of degree is simultaneously transferred in new Nostoc commune Vanch bottle, and each culture bottle ensures that 1 female and 2 males are (every 5 bottles of group), it gives accurate 24 hours for every bottle and lays eggs.Parent drosophila is transferred in new Nostoc commune Vanch bottle after oviposition, it is 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:
4 reproductive capacity measurement result of table
From table 4, 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 GIQDPKEP of degree can promote the reproductive capacity of drosophila.Originally the experimental results showed that, the extension of life span of drosophila melanogaster is GIQDPKEP direct Effect as a result, rather than GIQDPKEP pass through reduce reproductive capacity caused by two level physiological effect.Also illustrate GIQDPKEP simultaneously It is safe to drosophila, without toxic hazard.
Three, biologically active polypeptide GIQDPKEP hydrogen peroxide Acute oxidative is tested
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;Hydrogen peroxide, Shanghai Ling Feng chemical reagent Co., Ltd;The biology that embodiment 1 obtains is living Property polypeptide GIQDPKEP.
Instrument and equipment:The ultra-clean water of CM-230 types mole, Shanghai Moller scientific instrument Co., Ltd;Mi Libo Millipore MILLEX GP0.22 μm filter membranes, Millipore Corp. of the U.S.;GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument centrifuge instruments Co., Ltd.
2. experimental method:
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, takes the service life real The preferable peptide concentration culture medium of middle result is tested, blank control group is set and experimental group, control group give conventional corn powder culture medium. Every group of male and female gender drosophila is 50, is cultivated three weeks drosophila.Then 5 males and 5 female Drosophilas is taken to be transferred to every time In one new container, new container is included there are one papery disk, disk contain the sucrose solution of 300 μ L a concentration of 5% with And a concentration of 30% hydrogen peroxide 1ml, blank group and experimental group are exposed to the toxicity peroxide that this hydrogen peroxide generates In environment, 10 Duplicate Samples of every group of setting observe its oxidation resistance.Every 4 hour record drosophila The dead quantity and gender, until Drosophila is all dead.
3. experimental result and analysis:
From Fig. 5 (A) as can be seen that for Male Drosophila, after GIQDPKEP feedings, in Each point in time, male The survival rate of drosophila is above the drosophila without GIQDPKEP feedings, and the time-to-live increases compared with blank control group, explanation After feeding GIQDPKEP, Male Drosophila oxidation resistance increases.In Fig. 5 (B), the female Drosophila of feeding GIQDPKEP, The apparent high and control group of survival rate in 15h, illustrates the anti-oxidant energy of female Drosophila this period in the hydrogen peroxide environment of high concentration Power increases.But later experiments group and control group survival curve essentially coincide, and illustrate the female Drosophila of feeding GIQDPKEP Oxidation resistance gradually weakens, by not having difference with control group after a certain period of time.This experimental results showed that, GIQDPKEP can be with Improve the oxidation resistance of drosophila.According to H2O2Acute toxicity testing is as a result, can speculate that GIQDPKEP may be by adjusting peroxide Change hydrogen enzyme CAT activity to improve drosophila to H2O2The resistivity of damage.
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 GIQDPKEP 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
Gly Ile Gln Asp Pro Lys Glu Pro
1 5
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cggtatccaa gatccaaaag agcc 24
<210> 3
<211> 309
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Met Ile Lys Asp Leu Ala Leu Glu Gln Val Val Gly His His His Ala
1 5 10 15
Leu Gly Thr Ser Ile Thr Leu Gln Ile Phe Gly Thr Gln Asp Arg Ser
20 25 30
Leu Leu Lys Lys Ser Phe Asp Leu Ile Asp His Tyr Glu Asp Ile Phe
35 40 45
Thr Val Asn Arg Asp Gln Ser Glu Val Met Asp Ile Asn His Ala Ser
50 55 60
Gly Asp His Pro Val Gln Val Ser Ser Ala Val Tyr Ser Leu Val Lys
65 70 75 80
Leu Ala Val Glu Lys Ser Arg Glu Asn Phe Gly Phe Asn Ala Leu Ile
85 90 95
Gly Pro Val Val Lys Leu Trp His Ile Gly Phe Lys Gly Ala His Val
100 105 110
Pro Lys Asp Glu Glu Ile Lys Asp Lys Met Leu Met Thr Asp Pro Phe
115 120 125
Lys Val Glu Leu Asn Asp Gln Asp Gln Thr Val Phe Leu Gln Met Lys
130 135 140
Gly Met Glu Leu Asp Leu Gly Gly Ile Ala Lys Gly Trp Ile Ala Asp
145 150 155 160
Arg Ile Arg Asp Leu Trp Leu Ala Tyr Gly Val Glu Ala Gly Ile Ile
165 170 175
Asn Leu Gly Gly Asn Ile Leu Leu Val Gly Asp Ser Pro Lys Arg Asp
180 185 190
Asn Gly Lys Trp Val Ile Gly Ile Gln Asp Pro Lys Glu Pro Arg Gly
195 200 205
Asp Asn Ile Thr Ser Val Met Val Gly Glu Cys Ser Ala Val Thr Ser
210 215 220
Gly Thr Tyr Glu Arg Tyr Leu Val Val Asp Gly His Lys Tyr His His
225 230 235 240
Leu Ile Asp Pro Arg Thr Gly Tyr Pro Val Lys Thr Asn Leu Ala Gly
245 250 255
Val Thr Thr Phe Thr Lys Thr Ser Val Glu Ala Glu Ile Glu Cys Lys
260 265 270
Arg Leu Phe Phe Val Gly His Pro Leu Glu Asn Trp His Asp Asn Pro
275 280 285
Asp Arg Ile Gly Ala Ile Phe Val Tyr Asn Asp Glu His Val Glu Tyr
290 295 300
Asp Asn Leu Asn Lys
305

Claims (10)

1. a kind of biologically active polypeptide GIQDPKEP, which is characterized in that its amino acid sequence is Gly-Ile-Gln-Asp-Pro- Lys-Glu-Pro。
2. a kind of biologically active polypeptide GIQDPKEP 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 GIQDPKEP 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 GIQDPKEP 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 GIQDPKEP as described in claim 1, which is characterized in that the biologically active polypeptide Applications of the GIQDPKEP in preparing the food with anti-inflammatory properties, health products, drug or cosmetics.
6. the application of biologically active polypeptide GIQDPKEP as described in claim 1, which is characterized in that the biologically active polypeptide Applications of the GIQDPKEP in preparing the food with anti-senescence function, health products or drug.
7. the application of biologically active polypeptide GIQDPKEP as described in claim 1, which is characterized in that the biologically active polypeptide Applications of the GIQDPKEP 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 GIQDPKEP or described lifes as described in claim 1 The derivative of object active peptides GIQDPKEP;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 GIQDPKEP refers to the amino acid in biologically active polypeptide GIQDPKEP 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 GIQDPKEP or described as described in claim 1 The derivative of biologically active polypeptide GIQDPKEP;The anti-aging product includes antisenility cistanche food, antisenescence health product or anti- Senescence drug;The derivative of the biologically active polypeptide GIQDPKEP refers to the amino acid in biologically active polypeptide GIQDPKEP 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 GIQDPKEP or described biologically active polypeptides GIQDPKEP;With anti-inflammatory properties and anti-senescence function Product includes food, health products or drug;The derivative of the biologically active polypeptide GIQDPKEP, refers in biologically active polypeptide On the amino acid side groups of GIQDPKEP, 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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