CN110922466B - Bioactive polypeptide KSWNETFHARLA, and preparation method and application thereof - Google Patents
Bioactive polypeptide KSWNETFHARLA, and preparation method and application thereof Download PDFInfo
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- CN110922466B CN110922466B CN201911086895.4A CN201911086895A CN110922466B CN 110922466 B CN110922466 B CN 110922466B CN 201911086895 A CN201911086895 A CN 201911086895A CN 110922466 B CN110922466 B CN 110922466B
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- China
- Prior art keywords
- kswnetfharla
- aging
- ala
- polypeptide
- bioactive polypeptide
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to the field of protein, and in particular relates to a bioactive polypeptide KSWNETFHARLA, a preparation method and application thereof, wherein the amino acid sequence of the bioactive polypeptide KSWNETFHARLA is Lys-Ser-Trp-Asn-Glu-Thr-Phe-His-Ala-Arg-Leu-Ala. Through in vitro immunoregulation activity experiments and in vivo anti-aging experiments, the polypeptide KSWNETFHARLA is verified to have better immunoregulation function and aging activity; on one hand, the bioactive polypeptide KSWNETFHARLA can enhance the in vitro proliferation capacity of lymphocytes, improve the capacity of an organism for resisting infection of external pathogens and reduce the morbidity of the organism; on the other hand, the compound has better anti-aging activity, can remove free radicals in the organism, improves the quality of life, and has very important significance for developing foods, health-care products and medicines with immunoregulation function and anti-aging function.
Description
Technical Field
The invention relates to the field of proteins, in particular to a bioactive polypeptide KSWNETFHARLA, and a preparation method and application thereof.
Background
With the improvement of living standard, the requirement of people on diet changes from 'pursuit amount' to 'quality'. Therefore, research on bioactive peptides having specific functions has been hot. In recent years, some food-derived polypeptides, such as short peptides of corn, soybean peptides, milk polypeptides, etc., have been found to have good biological activity. And experiments prove that the health-care tea has the functions of resisting aging, resisting bacteria and cancers, regulating immunity, reducing blood pressure and the like.
The polypeptides can be obtained through various ways such as microbial fermentation, digestion and enzymolysis and the like, and most of the polypeptides with biological activity consist of 2-20 amino acid residues, have the molecular weight of less than 6000Da and contain a certain amount of hydrophobic amino acids and aromatic amino acids.
Macrophages are the second defense line of the body against the invasion of external harmful substances, are widely present in various tissues of the body, are main immune response cells, participate in biological functions such as immune response, immune regulation and the like through phagocytosis and secretion of cytokines, and play an important role in an immune system. The function of the polypeptide present in macrophages was investigated.
Currently, there are some researches on anti-aging bioactive peptides in the prior art, but new bioactive polypeptides having anti-oxidation or anti-aging functions different from the existing polypeptides are still the current direction of further research needed to further expand the diversity of bioactive polypeptides.
Disclosure of Invention
The invention aims to provide a bioactive polypeptide KSWNETFHARLA, and a preparation method and application thereof.
The purpose of the invention can be realized by the following technical scheme:
in a first aspect of the invention, there is provided a biologically active polypeptide KSWNETFHARLA having the amino acid sequence Lys-Ser-Trp-Asn-Glu-Thr-Phe-His-Ala-Arg-Leu-Ala as set forth in SEQ ID NO: 1 is shown.
Preferably, the bioactive polypeptide is mouse bone marrow-derived macrophage peptide. Specifically, the protein is derived from ATP-synthase subunit D protein and is the 32 th to 43 th amino acid residues of the ATP-synthase subunit D protein. The amino acid sequence of the ATP-synthase outburnit D protein is shown as SEQ ID NO: 2, respectively.
The amino acid sequence and the corresponding nucleotide sequence of the ATP-synthase outbunint D protein are the prior art, and the nucleotide fragment which codes the 32 th to 43 th amino acid residues of the ATP-synthase outbunint D protein can code mature bioactive polypeptide KSWNETFHARLA.
Preferably, the bioactive polypeptide has the functions of immunoregulation and anti-aging.
In the second aspect of the present invention, a method for preparing the bioactive polypeptide KSWNETFHARLA is provided, which can be artificially synthesized by genetic engineering methods, can be directly obtained from cells by a separation and purification method, and can be directly prepared by chemical synthesis.
In a third aspect of the invention, an application of the bioactive polypeptide KSWNETFHARLA in preparing food, health products, medicines or cosmetics with immunoregulation function is provided.
In the fourth aspect of the invention, the application of the bioactive polypeptide KSWNETFHARLA in preparing food, health-care products or medicines with the anti-aging function is provided.
In the fifth aspect of the invention, the application of the bioactive polypeptide KSWNETFHARLA in preparing food, health-care products or medicines with immune regulation and anti-aging functions is provided.
In particular, the biologically active polypeptide KSWNETFHARLA of the present invention can be used for preparing cosmetics for reducing free radical damage to skin, and medicines for regulating immunity and/or resisting aging.
In a sixth aspect of the invention, there is provided an immunomodulatory product comprising said biologically active polypeptide KSWNETFHARLA or a derivative of said biologically active polypeptide VASNLNLKPGECLKVRGEV A; the immunoregulation product comprises immunoregulation food, immunoregulation health-care products, immunoregulation medicaments or immunoregulation cosmetics; the derivative of the biologically active polypeptide KSWNETFHARLA refers to a polypeptide derivative obtained by performing modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on an amino acid side chain group, an amino terminal or a carboxyl terminal of the biologically active polypeptide KSWNETFHARLA.
In a seventh aspect of the invention, there is provided an anti-aging product comprising the biologically active polypeptide KSWNETFHARLA or a derivative of the biologically active polypeptide VASNLNLKPGECLKVRGEV A; the anti-aging product comprises anti-aging food, anti-aging health care product or anti-aging drug; the derivative of the biologically active polypeptide KSWNETFHARLA refers to a polypeptide derivative obtained by performing modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on an amino acid side chain group, an amino terminal or a carboxyl terminal of the biologically active polypeptide KSWNETFHARLA.
In the eighth aspect of the present invention, a product having both immunoregulatory function and anti-aging function is provided, which comprises the bioactive polypeptide KSWNETFHARLA or a derivative of the bioactive polypeptide KSWNETFHARLA; products with immunoregulatory and anti-aging functions include foods, health products or drugs; the derivative of the biologically active polypeptide KSWNETFHARLA refers to a polypeptide derivative obtained by performing modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on an amino acid side chain group, an amino terminal or a carboxyl terminal of the biologically active polypeptide KSWNETFHARLA.
The bioactive polypeptide KSWNETFHARLA has the following beneficial effects: the mouse bone marrow-derived macrophage bioactive polypeptide KSWNETFHARLA has good immunoregulation activity and anti-aging activity; on one hand, the bioactive polypeptide KSWNETFHARLA can enhance the in vitro proliferation capacity of lymphocytes, improve the capacity of an organism for resisting infection of external pathogens and reduce the morbidity of the organism; on the other hand, the compound has better anti-aging activity, can remove free radicals in the organism, improves the quality of life, and has very important significance for developing foods, health-care products and medicines with immunoregulation function and anti-aging function.
Drawings
FIG. 1: mass chromatogram extraction (m/z 487.2544);
FIG. 2: a secondary mass spectrum of a fragment with a mass to charge ratio of 487.2544;
FIG. 3: fragmentation of polypeptide az and by with mass-to-charge ratio of 487.2544;
Detailed Description
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989 and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1 Artificial Synthesis of active peptide KSWNETFHARLA
Synthesis of bioactive peptide
1. 3g of RINK resin (degree of substitution 0.3mmol/g) was weighed into a 150ml reactor and soaked with 50ml of Dichloromethane (DCM).
After 2.2 hours, the resin was washed with 3 resin volumes of N-Dimethylformamide (DMF) and then drained, and this was repeated four times and the resin was drained until use.
3. The Fmoc protecting group on the resin was removed by adding a quantity of 20% piperidine (piperidine/DMF: 1:4, v: v) to the reactor and shaking on a decolourising shaker for 20 min. After deprotection, the resin was washed four times with 3 resin volumes of DMF and then drained.
4. And (3) detecting a small amount of resin by a ninhydrin (ninhydrin) method (detecting A and B, respectively, and reacting at 100 ℃ for 1min), wherein the resin is colored, which indicates that the deprotection is successful.
5. Weighing an appropriate amount of amino acid Lys and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, adding 20ml of DMF to dissolve the amino acid Lys and the 1-hydroxy-benzotriazole (HOBT), then adding 3ml of N, N Diisopropylcarbodiimide (DIC) to shake and shake for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor into a 30 ℃ shaking table to react.
After 6.2 hours, the column was capped with a suitable amount of acetic anhydride (acetic anhydride: DIEA: DCM ═ 1:1:2, v: v: v) for half an hour, then washed four times with 3 resin volumes of DMF and drained until needed.
7. The Fmoc protecting group on the resin was removed by adding a quantity of 20% piperidine (piperidine/DMF: 1:4, v: v) to the reactor and shaking on a decolourising shaker for 20 min. After deprotection was washed four times with DMF and then drained.
8. And (3) detecting a small amount of resin by a ninhydrin (ninhydrin) method (detecting A and B, respectively, and reacting at 100 ℃ for 1min), wherein the resin is colored, which indicates that the deprotection is successful.
9. Weighing a second proper amount of amino acid and a proper amount of HOBT in a 50ml centrifuge tube, adding 25ml of DMF to dissolve the amino acid and the HOBT, adding 2.5ml of DIC to shake and shake for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor in a shaking table at 30 ℃ to react.
After 10.1 hours, a small amount of resin is taken for detection, and the detection is carried out by an indanthrone method (two drops are respectively detected A and B, and the reaction is carried out for 1min at 100 ℃), if the resin is colorless, the reaction is complete; if the resin is colored, the condensation is not complete and the reaction is continued.
11. After the reaction was completed, the resin was washed four times with DMF and then drained, and a certain amount of 20% piperidine (piperidine/DMF ═ 1:4, v: v) was added to the reactor, and the mixture was shaken on a decolorizing shaker for 20min to remove the Fmoc-protecting group from the resin. After the protection is removed, washing with DMF for four times, and then draining to detect whether the protection is removed.
12. The amino acids Ser, Trp, Asn, Glu, Thr, Phe, His, Ala, Arg, Leu and Ala are grafted in sequence according to steps 9-11.
13. After the last amino acid had been grafted, the protection was removed, washed four times with DMF and the resin was drained with methanol. The polypeptide was then cleaved from the resin with 95 cleavage medium (trifluoroacetic acid: 1,2 ethanedithiol: 3, isopropylsilane: water: 95:2:2:1, v: v: v) (10 ml of cleavage medium per gram of resin) and centrifuged four times with glacial ethyl ether (cleavage medium: ethyl ether: 1:9, v: v).
To this end, bioactive peptide KSWNETFHARLA was synthesized.
Confirmation of biologically active peptides
1) UPLC analysis
UPLC conditions were as follows:
the instrument comprises the following steps: waters ACQUITY UPLC ultra-high performance liquid-electrospray-quadrupole-time-of-flight mass spectrometer
Specification of chromatographic column: BEH C18 chromatographic column
Flow rate: 0.4mL/min
Temperature: 50 deg.C
Ultraviolet detection wavelength: 210nm
Sample introduction amount: 2 μ L
Gradient conditions: solution A: water containing 0.1% formic acid (v/v), liquid B: acetonitrile containing 0.1% formic acid (v/v)
2) Mass spectrometric analysis
The mass spectrometry conditions were as follows:
ion mode: ES +
Mass range (m/z): 100-1000
Capillary voltage (Capillary) (kV): 3.0
Sampling cone (V): 35.0
Ion source temperature (. degree. C.): 115
Desolvation temperature (. degree. C.): 350
Desolventizing gas stream (L/hr): 700.0
Collision energy (eV): 4.0
Scan time (sec): 0.25
Inner scan time (sec): 0.02
According to the analysis method, the ultra-high performance liquid chromatography-electrospray-quadrupole-time-of-flight mass spectrometry is used for carrying out chromatographic analysis and mass spectrometric analysis on the bioactive peptide KSWNETFHARLA, the mass chromatogram extraction diagram is shown in figure 1, the secondary mass spectrogram of the peak and the az and by fracture conditions are shown in figures 2 and 3, the polypeptide mass-to-charge ratio of the peak is 487.2544Da, and the retention time is 27.6 min.
3) Results
As can be seen from FIG. 3, according to the cases of az and by fragmentation, the fragment sequence with mass-to-charge ratio of 487.2544Da obtained by analysis and calculation of Mascot software is Lys-Ser-Trp-Asn-Glu-Thr-Phe-His-Ala-Arg-Leu-Ala (KSWNETFHARA), and is marked as SEQ ID NO: 1. the fragment corresponds to the residue sequence of 32-43 th sites of ATP-synthase subbunit D protein, the GenBank number of the amino acid sequence of the ATP-synthase subbunit D protein is AAL83962.1, and the sequence is shown in SEQ ID NO: 2.
example 2 immunomodulatory Activity assays of bioactive peptides
First, MTT method for testing in vitro lymphocyte proliferation capacity experiment of bioactive polypeptide KSWNETFHARLA
1. Experimental materials and instruments:
reagents and materials: experimental animals balb/c mice (male 6-8 weeks old, animal experiment center of Shanghai university of transportation, college of agriculture and biology); the mouse bone marrow macrophage-derived bioactive peptide KSWNETFHARLA obtained in example 1; mouse lymphocyte extract (ex solibao); RPMI1640 medium (purchased from GIBCO); 3- (4, 5-Dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide salt (MTT, available from Amresco, Inc.); concanavalin (ConA, available from Sigma); bovine serum albumin (BSA, available from Genebase); pepsin (available from Sigma); pancreatin (Corolase PP, from AB).
The instrument equipment comprises: LRH-250F Biochemical incubator, Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge, shanghai luxiang instrument centrifuge instruments ltd; hera cell 150 CO2 incubator, Heraeus; dragon Wellscan MK3 microplate reader, Labsystems Inc.; ALPHA 1-2-LD vacuum freeze drier, Christ company; ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometer, waters corporation.
2. The experimental method comprises the following steps:
taking mouse spleen under aseptic condition, extracting mouse lymphocyte with lymphocyte extract, and performing primary culture. The cell density was adjusted to 2.5X 10 with complete RPMI1640 medium6one/mL. To a 96-well cell culture plate were added in sequence: 100 μ L mouse lymphocyte suspension, 100 μ L RPMI1640 complete medium, 20 μ L concanavalin, 100 μ L sample. In addition, a blank control group (PBS with pH7.2-7.4 and 3 mol/L) and a negative control group (500 mu g/mL BSA) are arranged, and the research shows that the blank control group has no influence on the in vitro lymphocyte proliferation. Each set of 3 replicates. At 5% CO2After incubation at 37 ℃ for 68h, 20. mu.L MTT was added to each well under aseptic conditions and incubation was continued for 4h, taking careDiscarding supernatant, adding 100 μ L dimethyl sulfoxide into each well, incubating at 37 deg.C for 10min, shaking, and measuring absorbance at 570nm with microplate reader.
The in vitro lymphocyte proliferation capacity is expressed by a stimulation index and is calculated as follows:
in the formula: a. the1Absorbance at 570nm for the blank; a. the2Absorbance at 570nm for the negative control, A3The absorbance at 570nm for the experimental group.
3. Experimental results and analysis:
TABLE 1 Effect of biologically active polypeptide KSWNETFHARLA on in vitro lymphocyte proliferation
| Experiment grouping | Stimulation index SI |
| Negative control group | 1 |
| KSWNETFHARLA | 1.178±0.041* |
Note: the number marked as significant difference (P <0.05) compared to the negative control.
The results are shown in Table 1. As can be seen from Table 1, under the condition that the mass concentration of the bioactive peptide KSWNETFHARLA is 100 mug/mL, the stimulation index of the milk-derived bioactive peptide KSWNETFHARLA is greater than that of BSA, which indicates that KSWNETFHARLA can stimulate the proliferation of mouse lymphocytes in vitro to a certain extent. And KSWNETFHARLA reached a stimulation index of 1.169 with a significant difference from the negative control group (P < 0.05). Therefore, the active polypeptide KSWNETFHARLA can be considered to have the capacity of remarkably promoting the mouse lymphocyte proliferation, can be eaten as a health product or an additive, and can improve the immunity of animals and human bodies.
Example 3 anti-aging Activity assay of bioactive peptides
Experiment of the effect of bioactive polypeptide KSWNETFHARLA on the immunoregulation level of organs in vivo
1. Experimental reagents and instruments:
reagent: experimental animal ICR mouse (male 5 weeks old), shanghai city experimental animal center; d-gal, national pharmaceutical group chemical reagents, Inc.; paraformaldehyde, chemical reagents of the national drug group, ltd; sodium chloride, national pharmaceutical group chemical reagents ltd; the mouse bone marrow macrophage-derived bioactive peptide KSWNETFHARLA obtained in example 1; BCA protein kit, Nanjing Kaikyi Biotech Co., Ltd; MDA lipid peroxide kit, south kyo kaiky biotechnology limited; SOD superoxide dismutase kit, Nanjing, biological technology Limited; T-AOC general immunoregulation kit, Nanjing, established Biotechnology Limited.
The instrument equipment comprises: model CM-230 Mohr super Water, Shanghai Mole scientific instruments, Inc.; millipore Milllex GP0.22 μm membrane filter, Millipore USA; GL-22M high-speed refrigerated centrifuge, Shanghai Luxiang apparatus centrifuge instruments Inc.
2. The experimental method comprises the following steps:
(1) model for animal aging
After one week of adaptive ICR mouse feeding, 4 groups of 6 mice were divided. Group 1 was a low dose intragastric group, mice were injected subcutaneously in the neck and back with D-gal at a dose of 500mg/kg daily and intragastric bioactive polypeptide KSWNETFHARLA at a dose of 1 mg/day; group 2 was a high dose intragastric group, mice were injected subcutaneously in the neck and back with D-gal at a dose of 500mg/kg daily, and 3 mg/mouse a day dose of intragastric bioactive polypeptide KSWNETFHARLA; group 3 was blank, mice grew normally; group 4 was an animal model group, and mice were injected subcutaneously into the neck and back with D-gal at a dose of 500mg/kg daily, and gavage with 0.9% normal saline; the injection period of D-gal and the gavage period of polypeptide were 42 days. The bedding is replaced every 3 days and the feed and distilled water supply is ensured. The weight of the mice was weighed once every five days, D-gal injection was prepared according to the weight of the mice, and the D-gal injection was filtered through a 0.22 μm syringe filter to ensure sterility.
(2) Obtaining animal viscera
After the experiment period is finished, blood of a mouse is obtained by an eyeball-picking blood-taking method, the mouse is killed by breaking the neck after the blood is obtained, then a body of the mouse is placed on a low-temperature ice box, the brain, the spleen, the liver and the kidney of the mouse are quickly picked, the obtained viscera are placed in a pre-sterilized 1.5mL centrifuge tube, and all organ samples are stored in a refrigerator at the temperature of-80 ℃ for inspection. All procedures in the procedure of treating the experimental animals followed the guidance comments on the animals being treated in good care published by the department of scientific technology in 2006. The spleen of the mouse is directly soaked in a prepared 4% paraformaldehyde solution to fix the shape. The paraformaldehyde powder is relatively insoluble, and a trace amount of sodium bicarbonate can be added to adjust the pH value to be alkaline so as to aid dissolution. The preparation of the paraformaldehyde solution needs to be completed in a fume hood.
(3) Sample detection
Grinding all organs to be detected in a low-temperature environment, diluting the ground organs into 10% tissue homogenate by using a 4 ℃ sterile PBS solution, centrifuging 4000g at 4 ℃, sucking a supernatant, removing a precipitate, and operating according to a kit instruction or placing the mixture in a refrigerator at minus 80 ℃ for detection.
3. Experimental results and analysis:
as can be seen from Table 2-1, the SOD content in the liver and kidney of the mice in the polypeptide gavage group showed significant increase (P <0.01) compared to the mice in the animal model group. The fact that the mice in the polypeptide gavage group are stimulated by large dose of D-gal for a long time and the SOD enzyme system in the mice is not completely destroyed even if the D-gal is excessively injected indicates that the experimental animals are continuously stimulated by the aging-causing factors in the injection period to reduce the SOD content in different organs, but simultaneously ingest a certain amount of polypeptide KSWNETFHARLA to have certain protection effect on oxidative damage in the mice.
TABLE 2-1 variation of SOD content in different organs of each group of experimental animal mice
Note: significant differences (P <0.05) in plots compared to model group controls; the plot showed significant differences (P <0.01) compared to the model group control, as follows.
As can be seen from Table 2-2, the liver MDA content of the mice in the animal model group is 26.80 + -7.03 nmol/L, and compared with the animal model group, the MDA content of the liver of the two groups of mice with the polypeptide gavage is significantly different (P < 0.01). As MDA can be used for estimating the accumulation condition of lipid peroxides in animals, it can be known that in the process of forming an aging model, sugar metabolism pathways of mice in an animal model group are disordered due to long-term injection of excessive D-gal, a large amount of free radicals are generated to cause oxidative damage, a large amount of lipid peroxides are generated in liver tissues of the mice, and MDA is used as the lipid peroxides, so that the increase of the content of the lipid peroxides in the animals can be laterally reflected to the reduction of the activity of the immunomodulatory enzyme systems in the mice. The obvious reduction of the MDA content in the liver of the mice in the polypeptide gavage group shows that the ingestion of the polypeptide KSWNETFHARLA can effectively protect important tissues and organs from being stimulated by adverse factors to generate a large amount of lipid peroxides.
TABLE 2-2 changes in MDA content in different organs of various groups of experimental animal mice
As can be seen from tables 2-3, the liver T-AOC value of the mice in the animal model group is 0.70 +/-0.23U/mgprot, and compared with the mice in the model group, the mice in the high-dose and low-dose gavage groups of the polypeptide show significant difference (P < 0.05); the content of the kidney T-AOC in the mice of the animal model group is 0.60 +/-0.19U/mgprot, and compared with the mice of the animal model group, the mice of the low-dose gavage group have significant difference (P <0.05) and the mice of the high-dose gavage group also have significant difference (P < 0.01). The results show that in the whole experimental period, because the experimental animals are continuously stimulated by the senescence-causing factors, the liver and kidney tissues of the mice in the animal model group are damaged, so that the total immunoregulatory capacity of the mice is reduced. Compared with animal model group and blank group, the total immunoregulation capability of main organs of mice in polypeptide gavage group is always maintained at a higher level in the process of being stimulated by aging-causing factors, which indicates that the animal body and the main organs thereof have higher self-protection function by taking the bioactive polypeptide KSWNETFHARLA.
TABLE 2-3 Change in T-AOC in groups of Experimental animals mice
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Sequence listing
<110> Shanghai university of transportation; zhejiang ghui peptide Life health science and technology Limited
<120> a bioactive polypeptide KSWNETFHARLA, and its preparation method and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Lys Ser Trp Asn Glu Thr Phe His Ala Arg Leu Ala
1 5 10
<210> 2
<211> 161
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Ala Gly Arg Lys Leu Ala Leu Lys Thr Ile Asp Trp Val Ser Phe
1 5 10 15
Val Glu Val Met Pro Gln Asn Gln Lys Ala Ile Gly Asn Ala Leu Lys
20 25 30
Ser Trp Asn Glu Thr Phe His Ala Arg Leu Ala Ser Leu Ser Glu Lys
35 40 45
Pro Pro Ala Ile Asp Trp Ala Tyr Tyr Arg Ala Asn Val Ala Lys Pro
50 55 60
Gly Leu Val Asp Asp Phe Glu Lys Lys Tyr Asn Ala Leu Lys Ile Pro
65 70 75 80
Val Pro Glu Asp Lys Tyr Thr Ala Leu Val Asp Gln Glu Glu Lys Glu
85 90 95
Asp Val Lys Ser Cys Ala Glu Phe Val Ser Gly Ser Gln Leu Arg Ile
100 105 110
Gln Glu Tyr Glu Lys Gln Leu Glu Lys Met Arg Asn Ile Ile Pro Phe
115 120 125
Asp Gln Met Thr Ile Asp Asp Leu Asn Glu Ile Phe Pro Glu Thr Lys
130 135 140
Leu Asp Lys Lys Lys Tyr Pro Tyr Trp Pro His Gln Pro Ile Glu Asn
145 150 155 160
Leu
Claims (6)
1. The application of the bioactive polypeptide KSWNETFHARLA is characterized in that the bioactive polypeptide KSWNETFHARLA is applied to the preparation of food, health products, medicines or cosmetics with immunoregulation function; the amino acid sequence of the bioactive polypeptide KSWNETFHARLA is Lys-Ser-Trp-Asn-Glu-Thr-Phe-His-Ala-Arg-Leu-Ala.
2. The application of the bioactive polypeptide KSWNETFHARLA is characterized in that the bioactive polypeptide KSWNETFHARLA is applied to the preparation of foods, health-care products or medicines with the anti-aging function; the amino acid sequence of the bioactive polypeptide KSWNETFHARLA is Lys-Ser-Trp-Asn-Glu-Thr-Phe-His-Ala-Arg-Leu-Ala.
3. The application of the bioactive polypeptide KSWNETFHARLA is characterized in that the bioactive polypeptide KSWNETFHARLA is applied to the preparation of food, health products or medicines with immunoregulation and anti-aging functions; the amino acid sequence of the bioactive polypeptide KSWNETFHARLA is Lys-Ser-Trp-Asn-Glu-Thr-Phe-His-Ala-Arg-Leu-Ala.
4. An immunomodulatory product comprising a biologically active polypeptide KSWNETFHARLA; the immunoregulation product comprises immunoregulation food, immunoregulation health-care products, immunoregulation medicaments or immunoregulation cosmetics; the amino acid sequence of the bioactive polypeptide KSWNETFHARLA is Lys-Ser-Trp-Asn-Glu-Thr-polypeptide KSWNETFHARLA, and the technical scheme is Phe-His-Ala-Arg-Leu-Ala with better immunoregulation function and aging activity.
5. An anti-aging product comprising a biologically active polypeptide KSWNETFHARLA; the anti-aging product comprises anti-aging food, anti-aging health care product, anti-aging drug or anti-aging cosmetic; the amino acid sequence of the bioactive polypeptide KSWNETFHARLA is Lys-Ser-Trp-Asn-Glu-Thr-Phe-His-Ala-Arg-Leu-Ala.
6. A product having immunomodulating and anti-aging properties, comprising a biologically active polypeptide KSWNETFHARLA; products with immunoregulatory and anti-aging functions include foods, health products or drugs; the amino acid sequence of the bioactive polypeptide KSWNETFHARLA is Lys-Ser-Trp-Asn-Glu-Thr-Phe-His-Ala-Arg-Leu-Ala.
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