CN107814840B - Bioactive polypeptide PKYPVEPF as well as preparation method and application thereof - Google Patents
Bioactive polypeptide PKYPVEPF as well as preparation method and application thereof Download PDFInfo
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- CN107814840B CN107814840B CN201711317979.5A CN201711317979A CN107814840B CN 107814840 B CN107814840 B CN 107814840B CN 201711317979 A CN201711317979 A CN 201711317979A CN 107814840 B CN107814840 B CN 107814840B
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- 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
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
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- 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
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
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Abstract
The invention relates to the field of protein, and in particular relates to a bioactive polypeptide PKYPVEPF, a preparation method and application thereof. In-vitro anti-inflammatory activity experiments and in-vivo anti-aging experiments prove that the polypeptide PKYPVEPF has better anti-inflammatory activity and anti-aging activity, and on one hand, the bioactive polypeptide PKYPVEPF can promote macrophages to secrete cytokines, promote the increase of the induced amount of nitric oxide of the macrophages, improve the capability of resisting the infection of external pathogens of an organism and reduce the morbidity of the organism; on the other hand, the activity of an anti-peroxidase system in vivo can be improved, and the function of resisting exogenous stimulation of the organism is enhanced, so that the probability of aging, aging and illness of the organism is reduced, and the method has very important significance for developing foods, health-care products and medicines with anti-inflammatory and anti-aging functions.
Description
Technical Field
The invention relates to the field of protein, in particular to a bioactive polypeptide PKYPVEPF as well as a preparation method and application thereof.
Background
In the process of fermenting the cow milk by the lactic acid bacteria, a part of protein in the cow milk is metabolized and utilized by the lactic acid bacteria, and a series of physiological and biochemical reactions occur, so that the protein is changed into polypeptide or free amino acid which is digested and absorbed by a human body or directly enters the blood circulation of the human body through the absorption and transportation of small intestinal epithelial cells. Among these polypeptides, some have a specific physiological function and are called "bioactive peptides".
It is particularly important to find safe bioactive peptides in natural food sources. 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. 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.
Immunoactive peptides are a class of bioactive polypeptides that are first obtained from milk following opioid peptide discovery and demonstrate their physiological activity. Jolles et al found in 1981 for the first time that a hexapeptide with an amino acid sequence Val-Glu-Pro-Ile-Pro-Tyr can be obtained by hydrolyzing human milk protein with trypsin, and in vitro experiments prove that the hexapeptide can enhance the phagocytosis of mouse abdominal cavity macrophages to sheep erythrocytes. Migliore-Samour et al found that the casein-derived hexapeptide Thr-Thr-Met-Pro-Leu-Trp was able to stimulate phagocytosis of murine peritoneal macrophages by sheep red blood cells and to enhance resistance to Klebsiella pneumoniae, with anti-inflammatory properties. Lemna hexandra et al, fed rats with synthetic milk-derived peptide (PGPIPN), found that phagocytosis of macrophages in the abdominal cavity of rats and the anti-inflammatory function associated with erythrocytes were significantly enhanced.
Researches show that the immune active peptide can not only enhance the immunity of the organism, stimulate the proliferation of lymphocytes of the organism, enhance the phagocytic function of macrophages, promote the release of cell factors, promote the increase of the induced amount of nitric oxide of the macrophages, improve the capability of the organism for resisting the infection of external pathogens, reduce the morbidity of the organism, but also can not cause the immune rejection reaction of the organism.
Aging is a natural phenomenon, and the process is often accompanied by the changes of antioxidant level, organ tissues and immune factors, wherein the cytokines are changed in a complex way, such as proinflammatory cytokines IL-6, IL-4, TNF- α and the like show a growing trend, and IL-6 and TNF-a are considered to play important roles in the process of the senile diseases.
The anti-aging peptide has the advantages that the anti-aging peptide is a novel anti-aging agent, has incomparable advantages with amino acid in the aspect of physiological function, can promote or inhibit enzymes in organisms, improve the absorption and utilization of minerals and other nutrient elements, clear away free radicals in the bodies, enhance the self anti-oxidation capability of the organisms and delay aging. Therefore, the nutrition and health care effects of bioactive peptides have become the focus of research on the subjects of scholars at home and abroad. Experiments and researches by meaningful people find that the milk-derived bioactive small peptide can effectively prolong the life of the drosophila and delay the aging of the drosophila, and has better antioxidation effect, and presumably is rich in thiopeptides. The results of Zhou Zhi Hui et al show that the bovine colostrum extract can obviously improve the SOD activity in serum of the elderly, reduce lipid peroxides of the SOD, enhance the oxidation resistance of organisms and have certain anti-aging function.
At present, there are many researches on bioactive polypeptides, for example, chinese patent CN105254738A discloses a milk-derived bioactive polypeptide DELQDKIH derived from β -casein, chinese patent CN105254739A discloses a milk-derived bioactive polypeptide GTQYTD derived from α s 1-casein, and chinese patent CN105254740A discloses a milk-derived bioactive polypeptide NQFYQKF derived from α s 2-casein.
Disclosure of Invention
The invention aims to provide a bioactive polypeptide PKYPVEPF as well as 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, a biologically active polypeptide, PKYPVEPF, is provided, the amino acid sequence of which is Pro-Lys-Tyr-Pro-Val-Glu-Pro-Phe, as shown in SEQ ID NO: 1 is shown.
Preferably, the bioactive polypeptide is milk-derived, is specifically derived from β -casein, and is β -amino acid residues at positions 112-119 of the casein variant B, and the amino acid sequence of β -casein variant B is shown in SEQ ID NO. 3.
β -casein and the corresponding nucleotide sequence are the existing technology, the nucleotide fragment coding the 112 th to 119 th amino acid residues of β -casein variant B can code the mature bioactive polypeptide PKYPVEPF.
Preferably, the bioactive polypeptide has an anti-inflammatory function and an anti-aging function.
In a second aspect of the invention, there is provided a nucleotide fragment encoding the biologically active polypeptide PKYPVEPF, having the sequence: 5'-cct aaa tat cca gtt gag ccc ttt-3', as shown in SEQ ID NO: 2, respectively.
In the third aspect of the invention, the preparation method of the bioactive polypeptide PKYPVEPF is provided, can be artificially synthesized by a genetic engineering method, can be directly obtained from dairy products by a separation and purification method, and can be directly prepared by chemical synthesis.
In the fourth aspect of the invention, the application of the bioactive polypeptide PKYPVEPF in preparing foods, health-care products, medicines or cosmetics with anti-inflammatory functions is provided.
In the fifth aspect of the invention, the application of the bioactive polypeptide PKYPVEPF in preparing foods, health-care products or medicines with an anti-aging function is provided.
The sixth aspect of the invention provides application of the bioactive polypeptide PKYPVEPF in preparing foods, health-care products or medicines with anti-inflammatory and anti-aging functions.
In particular, the bioactive polypeptide PKYPVEPF can be used for preparing cosmetics for reducing free radical damage to skin, medicaments for resisting inflammation and/or aging; and because the product of the biological active polypeptide PKYPVEPF degraded by gastrointestinal tract still has biological activity, the biological active polypeptide PKYPVEPF can be used for preparing foods such as yoghourt and the like, health-care products for regulating immunity and oral medicines for resisting inflammation and/or resisting aging.
In a seventh aspect of the invention, there is provided an anti-inflammatory product comprising the biologically active polypeptide PKYPVEPF or a derivative of the biologically active polypeptide PKYPVEPF; the anti-inflammatory product comprises anti-inflammatory food, anti-inflammatory health product, anti-inflammatory drug or anti-inflammatory cosmetic; the derivative of the bioactive polypeptide PKYPVEPF refers to a polypeptide derivative obtained by carrying out modification 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 bioactive polypeptide PKYPVEPF.
In an eighth aspect of the invention, there is provided an anti-ageing product comprising the biologically active polypeptide PKYPVEPF or a derivative of the biologically active polypeptide PKYPVEPF; the anti-aging product comprises anti-aging food, anti-aging health care product or anti-aging drug; the derivative of the bioactive polypeptide PKYPVEPF refers to a polypeptide derivative obtained by carrying out modification 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 bioactive polypeptide PKYPVEPF.
In a ninth aspect of the invention, there is provided a product having both anti-inflammatory and anti-aging functions, comprising the biologically active polypeptide PKYPVEPF or a derivative of the biologically active polypeptide PKYPVEPF; products with anti-inflammatory and anti-aging effects include foods, health products or drugs; the derivative of the bioactive polypeptide PKYPVEPF refers to a polypeptide derivative obtained by carrying out modification 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 bioactive polypeptide PKYPVEPF.
The biological active polypeptide PKYPVEPF has the beneficial effects that: the milk-derived bioactive polypeptide PKYPVEPF has good anti-inflammatory activity and anti-aging activity; on one hand, the bioactive polypeptide PKYPVEPF can promote macrophages to secrete cytokines, promote the increase of the induction quantity of nitric oxide of the macrophages, improve the capability of resisting the infection of external pathogens of organisms and reduce the morbidity of the organisms; on the other hand, the activity of an anti-peroxidase system in vivo can be improved, and the function of resisting exogenous stimulation of the organism is enhanced, so that the probability of aging, aging and illness of the organism is reduced, and the method has very important significance for developing foods, health-care products and medicines with anti-inflammatory and anti-aging functions.
Drawings
FIG. 1: mass chromatogram extraction (m/z 488.7625);
FIG. 2: a secondary mass spectrum of a fragment with a mass to charge ratio of 488.7625;
FIG. 3: fragmentation of polypeptide az and by with mass-to-charge ratio of 488.7625;
FIG. 4: an IL-4 standard curve;
FIG. 5: the effect of the biologically active polypeptide PKYPVEPF on the amount of cytokine IL-4 secretion;
FIG. 6: spleen change of each group of experimental animal mice;
(a) spleen tissue maps of mice in the low-dose gavage group; (b) spleen tissue maps of mice in the high-dose gavage group; (c) spleen tissues of blank mice; (d) spleen tissue map of mice in animal model group
FIG. 7: serum IL-6 profiles for each group of mice;
FIG. 8 is a table showing the changes in serum TNF- α in each group of mice.
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, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS Inmolecular BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATINSTRUCUTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) Methods Inenzymolygy, 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 PKYPVEPF
Synthesis of bioactive peptide
1.3 g 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 a proper amount of amino acid Pro and a proper amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, adding 20ml of DMF to dissolve the amino acid Pro and the 1-hydroxy-benzotriazole (HOBT), then adding 3ml of N, N diisopropyl carbodiimide (DIC) to shake and shake for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor into a30 ℃ 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 Lys, Tyr, Pro, Val, Glu, Pro and Phe are attached 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, the biologically active peptide PKYPVEPF was artificially 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, 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 PKYPVEPF, 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 488.7625Da, and the retention time is 38.0 min.
3) Results
As can be seen from FIG. 3, according to the cases of az and by fragmentation, the sequence of the fragment with the mass-to-charge ratio of 488.7625Da is Pro-Lys-Tyr-Pro-Val-Glu-Pro-Phe (PKYPVEPF) obtained by analysis and calculation of Mascot software, and is marked as SEQ ID NO: 1, the fragment corresponds to the residue sequence of 112-119 th sites of β -casein variant B, the GenBank number of the β -casein amino acid sequence is AAA30431.1, and the sequence is shown as SEQ ID NO: 3.
Example 2 anti-inflammatory Activity assay of bioactive peptides
Experiment (ELISA method) for promoting macrophage to secrete cytokine by bioactive polypeptide PKYPVEPF
1. Experimental reagents and instruments:
reagent: experimental animals balb/c mice (male 6-8 weeks old), Shanghai Slek Experimental animals, Inc.; mouse lymphocyte extract, shanghai solibao biotechnology limited; RPMI1640 medium, GIBCO; bovine Serum Albumin (BSA), Genebase; the milk-derived bioactive polypeptide PKYPVEPF obtained in example 1; ELISA cytokine Rapid kit (IL-4), Wuhan doctor De bioengineering, Inc.
The instrument equipment comprises: model CM-230 Mohr super Water, Shanghai Mole scientific instruments, Inc.; LRH-250F Biochemical incubator, Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge, shanghai luxiang instrument centrifuge instruments ltd; heracell 150CO2Incubator, Heraeus corporation; dragon Wellscan MK3 microplate reader, Labsystems Inc.
2. The experimental method comprises the following steps:
(1) preparation of the Standard Curve
Making an IL-4 standard curve: IL-4 standard substances with the concentrations of 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL, 15.6pg/mL and 7.8pg/mL are respectively and sequentially added into the holes of the ELISA plate, then biotin-labeled anti-mouse IL-4 antibody (ELISA cytokine rapid kit) is added, the ELISA plate is covered, and the reaction is carried out for 90min at 37 ℃. And (3) throwing off liquid in the ELISA plate, and sequentially adding 0.1mL of avidin-peroxidase complex (ELISA cytokine rapid kit) into each hole. The reaction was carried out at 37 ℃ for 60 min. 0.01M PBS was washed 3 times, and 0.1mL of ABC working solution was added to each well and reacted at 37 ℃ for 30 min. Washing with 0.01M PBS for 5 times, adding 90ul of TMB color development solution into each well, and reacting at 37 deg.C in dark for 25 min. 0.1mL of TMB stop solution was added to each well, and the absorbance was measured at 450nm using a microplate reader. The standard curve for IL-4 detection was prepared as shown in FIG. 4. The IL-4 standard curve was fitted by first regression using the concentration as abscissa (unit pg/mL) and the absorbance at 450nm as ordinate to obtain a standard curve Y of 0.0038X +0.1224, R20.9979. Wherein X represents the IL-4 concentration in pg/mL and Y represents the absorbance at OD 450.
(2) Detection of macrophage secretion promoting cytokine of polypeptide PKYPVEPF
Taking mouse spleen lymphocytes under aseptic condition, adjusting cell concentration to 5 × 105The concentration of the biological active polypeptide PKYPVEPF is adjusted to 100, 50 and 10 mu g/mL respectively, and the cytokine IL-4 is measured after the biological active polypeptide PKYPVEPF and the lymphocytes are cultured for 36 hours. Blank groupThe bioactive polypeptide PKYPVEPF is added and cultured for 36h as a control.
3. Experimental results and analysis:
the experimental results are shown in FIG. 5, and compared with the blank control group, the secretion amount of IL-4 is gradually increased along with the increase of the polypeptide concentration; when the polypeptide addition concentration reaches 50 and 100 mu g/mL, the IL-4 secretion amount is obviously greater than that of a blank group; the bioactive polypeptide PKYPVEPF has the function of promoting lymphocyte proliferation, and has the function of regulating the humoral immunity of the organism through regulating the secretion of IL-4 cytokines.
Second, determination of macrophage-promoting nitric oxide induction quantity of biological active polypeptide PKYPVEPF (Griess method)
1. Experimental reagents and instruments:
reagent: experimental animals balb/c mice (male 6-8 weeks old) were collected at the animal Experimental center of the college of agriculture and biology of Shanghai university of transportation; the milk-derived bioactive polypeptide PKYPVEPF obtained in example 1; LPS, purchased from Sigma; neutral red staining solution, produced by Biyuntian biotechnological research institute.
The instrument equipment comprises: LRH-250F Biochemical incubator Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge Shanghai Luxiang apparatus centrifuge Instrument Co., Ltd; hera cell 150CO2 incubator Heraeus; dragon WellscanMK3 microplate reader Labsystems.
2. The test method comprises the following steps:
the number of the added cells was 2X 106100 μ l/well of a cell suspension per ml, 200 μ l/well of a complete peptide-containing RPMI1640 culture medium (10% FBS) was added after adherent purification, LPS was added to a final concentration of 10 μ g/ml at 24 hours in an inflammation group, 50 μ l/well of a culture supernatant was collected after continuous culture for 48 hours, 50 μ l/well of Griess reagent 1 and Griess reagent 2 were sequentially added to the culture supernatant, and after reaction at room temperature for 10 minutes, an absorbance value (OD540) was measured at a wavelength of 540 nm.
3. Experimental results and analysis:
TABLE 1 determination of the amount of nitric oxide-inducing PKYPVEPF-promoted macrophages
| Experiment grouping | Normal group | Inflammation group |
| Cell blank | 0.0592±0.00525 | 0.3241±0.0381 |
| PKYPVEPF 1mg/ml | 0.1392±0.0225** | 0.4972±0.0268** |
| PKYPVEPF 0.5mg/ml | 0.1257±0.0361** | 0.3983±0.0373** |
| PKYPVEPF 0.1mg/ml | 0.2685±0.0271** |
Note: significant difference compared to negative control (P < 0.05);
significant difference compared with negative control group (P <0.01)
The results are shown in Table 1, and it is understood from Table 1 that addition of the bioactive polypeptide PKYPVEPF to the experimental groups at concentrations of 1mg/mL and 0.5mg/mL, respectively, promotes the amount of NO induced in macrophages, both when grown under normal conditions and when grown under conditions of inflammation induced by LPS. There was a significant difference (P <0.05) compared to the cell blank. When the addition concentration of the bioactive polypeptide PKYPVEPF is 0.1mg/mL, the increase of the nitric oxide induction quantity of macrophages can be promoted compared with that in the situation of inflammation caused by LPS, and the obvious difference is achieved (P < 0.05). But there were no significant differences compared to the cell blank grown under normal conditions. The biological active polypeptide PKYPVEPF has the capacity of promoting the increase of the nitric oxide induction quantity of macrophage under the condition of certain concentration.
Example 3 anti-aging Activity assay of bioactive peptides
Experiment of effect of bioactive polypeptide PKYPVEPF on in-vivo spleen tissue structure
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 milk-derived bioactive polypeptide PKYPVEPF obtained in example 1.
The instrument equipment comprises: model CM-230 Mohr super Water, Shanghai Mole scientific instruments, Inc.; milliporem Milllex GP0.22 μm filter membrane, 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 gavage group, mice were injected subcutaneously in the neck and back at a dose of 500mg/kg daily and gavage the bioactive polypeptide PKYPVEPF at a dose of 1 mg/day; group 2 was a high-dose gavage group, mice were injected subcutaneously in the neck and back at a dose of 500mg/kg daily and the bioactive polypeptide PKYPVEPF was gavage at a daily dose of 3 mg/mouse; 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
Preparation of tissue sections: mouse spleen samples were fixed in 4% paraformaldehyde solution for at least 24 hours. The preparation of wax block, slicing and HE staining of spleen tissue were completed by Shanghai Weiao Biotech Co., Ltd.
3. Experimental results and analysis:
in this experiment, there were 4 groups of mice, of which the blank group did not undergo any external stimulation for normal growth, and the remaining 3 groups received long-term injections of D-gal. By observing spleen sections of different groups of mice by using an optical microscope, as can be seen from fig. 6, compared with spleen sections of each group of mice, compared with blank groups of mice, spleen red marrow and white marrow of animal model mice have fuzzy boundaries and atrophy of the white marrow, which indicates that long-term D-gal injection causes sugar metabolism pathways of the mice to be disordered, so that the antioxidant enzyme activity is reduced, peroxide is accumulated, and further spleen aging and atrophy are possibly caused. Compared with the mice of the animal model group, the spleen tissues of the mice of the gavage polypeptide group have lighter atrophy degree of the white marrow and have better boundary between the red marrow and the white marrow. This result suggests that the experimental animals are continuously stimulated by the senescence-causing factor throughout the injection cycle of D-gal, resulting in senescence and atrophy of the spleen. Therefore, from the condition of tissue structure change, the bioactive polypeptide PKYPVEPF disclosed in the experiment has a certain protective effect on spleen aging and atrophy caused by stimulation of adverse factors.
Second, experiment of the effect of the bioactive polypeptide PKYPVEPF on the antioxidant level of the 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 milk-derived bioactive polypeptide PKYPVEPF 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, established Biotech limited.
The instrument equipment comprises: model CM-230 Mohr super Water, Shanghai Mole scientific instruments, Inc.; milliporem Milllex GP0.22 μm filter membrane, 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 gavage group, mice were injected subcutaneously in the neck and back at a dose of 500mg/kg daily and gavage the bioactive polypeptide PKYPVEPF at a dose of 1 mg/day; group 2 was a high-dose gavage group, mice were injected subcutaneously in the neck and back at a dose of 500mg/kg daily and the bioactive polypeptide PKYPVEPF was gavage at a daily dose of 3 mg/mouse; 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 and taking supernate, removing precipitates, and operating according to a kit instruction or placing the mixture in a refrigerator at minus 80 ℃ for detection.
3. Experimental results and analysis:
TABLE 2 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, 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 in the injection period, the experimental animals are continuously stimulated by the aging-causing factors, so that the SOD content in different organs is reduced, and meanwhile, a certain amount of polypeptide PKYPVEPF is taken to have a certain protection effect on oxidative damage in the mice.
TABLE 3 variation of MDA content in different organs of various groups of experimental animal mice
As can be seen from Table 3, the liver MDA content of the mice in the animal model group is 26.86 +/-7.04 nmol/L, and compared with the animal model group, the MDA content of the livers of the two groups of mice with the polypeptide gavage shows a significant difference (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 reduce the activity of antioxidant 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 PKYPVEPF can effectively protect important tissues and organs from being stimulated by adverse factors to generate a large amount of lipid peroxides.
Experiment of bioactive polypeptide PKYPVEPF on immune cell factor in serum
1. Experimental reagents and instruments:
reagents including an experimental animal ICR mouse (male 5 weeks old), Shanghai's center for experimental animals, D-gal, national drug group chemical reagent Co., Ltd, paraformaldehyde, national drug group chemical reagent Co., Ltd, sodium chloride, national drug group chemical reagent Co., Ltd, the milk-derived bioactive polypeptide PKYPVEPF obtained in example 1, a BCA protein kit, Nanjing Kayji Biotech Co., Ltd, ELISA cytokine rapid kits (TNF- α and IL-6), Wuhan doctor Germany bioengineering Co., Ltd.
The instrument equipment comprises: model CM-230 Mohr super Water, Shanghai Mole scientific instruments, Inc.; milliporem Milllex GP0.22 μm filter membrane, 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 gavage group, mice were injected subcutaneously in the neck and back at a dose of 500mg/kg daily and gavage the bioactive polypeptide PKYPVEPF at a dose of 1 mg/day; group 2 was a high-dose gavage group, mice were injected subcutaneously in the neck and back at a dose of 500mg/kg daily and the bioactive polypeptide PKYPVEPF was gavage at a daily dose of 3 mg/mouse; 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 and serum
After the experiment period is finished, blood of the mouse is obtained by an eyeball-picking blood-taking method, the mouse is killed by breaking the neck after the blood is obtained, then the body of the mouse is placed on a low-temperature ice box, the blood of the mouse is stood for 1 hour at room temperature, and then is centrifuged for 15min at 3000g, and serum is separated. The serum was stored in a freezer at-80 ℃ for testing. 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
According to the instruction of the kit, firstly, a standard curve is drawn, standard powder is prepared into a solution of 1000pg/mL by using a standard diluent, and then the solution is continuously diluted into different concentrations of 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL, 15.6pg/mL and the like. Each concentration gradient solution was pipetted at 100. mu.L in an antibody-coated microplate. And (3) sucking 100 mu L of mouse serum sample, and adding the mouse serum sample into the same enzyme label plate (if the serum sample is insufficient, the mouse serum sample can be diluted properly and then needs to be converted proportionally when being detected and calculated). The plate was covered and incubated at 37 ℃ for 90 min. After the reaction is finished, carefully throwing off the liquid in the ELISA plate, placing the ELISA plate on absorbent paper, carefully beating the absorbent paper, and removing the redundant liquid. Adding preheated biotin anti-antibody working solution into each hole of the ELISA plate according to 100 mu L of each hole, and reacting for 60min at 37 ℃. After the reaction was completed, the reaction solution was washed 3 times with 0.01M PBS, 100. mu.L of PBS was added to each well, and the solution was removed after soaking for 1min, and the reaction was repeated 3 times. The preheated ABC working solution is added into each hole according to the volume of 0.1ml in turn, and the reaction is carried out for 30min at the temperature of 37 ℃. After the reaction, the reaction mixture was washed with 0.01M PBS for 5 times, and soaked for about 1min each time. Adding TMB color development solution which is balanced at 37 ℃ for 30min in turn according to 90 mu L per hole, and reacting for 8-12min at 37 ℃ in a dark place. TMB stop solution was added in an amount of 0.1ml per well in this order, and the color blue was immediately changed to yellow, and the OD value was measured at 450nm using a microplate reader. The standard protein of the cell factor is serially diluted in known concentration, an OD value is measured, a standard curve is drawn, and the content of the cell factor in the specimen can be calculated according to the standard curve.
3. Experimental results and analysis:
TABLE 4 Change in cytokines in serum of mice in each group
It can be found from table 4, fig. 7and fig. 8 that the contents of IL-6 and TNF- α in the mice of the model group in the experiment were 168.01 ± 26.38pg/mL and 4.34 ± 0.76pg/mL, respectively, and showed significant increase (P <0.01) compared to the normal group, so that it is considered that the mice of the model group showed symptoms of aging inflammation at the cytokine level due to continuous injection of senescence factors, while the contents of IL-6 and TNF- α in the serum of the mice of the polypeptide gavage group were effectively controlled, and according to the results of the cytokine experiment, the secretion levels of IL-6 and TNF- α in the serum of the inflammatory cytokines of the mice of the polypeptide gavage group were lower than those of the animal model group, and from the viewpoint of oxidation injury, the mice might be inhibited to a certain extent by free radical attack and accumulation of peroxide products, and from the viewpoint of aging, the mice might be inhibited by oxidation caused by a series of D-gal injection.
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> Zhejiang ghui peptide Life health science and technology Limited; shanghai platinum Biotech Ltd
<120> bioactive polypeptide PKYPVEPF, preparation method and application thereof
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>8
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Pro Lys Tyr Pro Val Glu Pro Phe
1 5
<210>2
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
cctaaatatc cagttgagcc cttt 24
<210>3
<211>209
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
Arg Glu Leu Glu Glu Leu Asn Val Pro Gly Glu Ile Val Glu Ser Leu
1 5 10 15
Ser Ser Ser Glu Glu Ser Ile Thr Arg Ile Asn Lys Lys Ile Glu Lys
20 25 30
Phe Gln Ser Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln Asp Lys
35 40 45
Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly
50 55 60
Pro Ile Pro Asn Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr
65 70 75 80
Pro Val Val Val Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser
85 90 95
Lys Val Lys Glu Ala Met Ala Pro Lys His Lys Glu Met Pro Phe Pro
100 105 110
Lys Tyr Pro Val Glu Pro Phe Thr Glu Arg Gln Ser Leu Thr Leu Thr
115120 125
Asp Val Glu Asn Leu His Leu Pro Leu Pro Leu Leu Gln Ser Trp Met
130 135 140
His Gln Pro His Gln Pro Leu Pro Pro Thr Val Met Phe Pro Pro Gln
145 150 155 160
Ser Val Leu Ser Leu Ser Gln Ser Lys Val Leu Pro Val Pro Gln Lys
165 170 175
Ala Val Pro Tyr Pro Gln Arg Asp Met Pro Ile Gln Ala Phe Leu Leu
180 185 190
Tyr Gln Glu Pro Val Leu Gly Pro Val Arg Gly Pro Phe Pro Ile Ile
195 200 205
Val
Claims (9)
1. A bioactive polypeptide PKYPVEPF, characterized in that its amino acid sequence is Pro-Lys-Tyr-Pro-Val-Glu-Pro-Phe.
2. A nucleotide fragment encoding the biologically active polypeptide PKYPVEPF of claim 1, wherein the nucleotide fragment has the sequence of SEQ ID NO: 2, respectively.
3. The method for producing PKYPVEPF of claim 1, wherein the biologically active polypeptide is artificially synthesized by genetic engineering or is directly produced by chemical synthesis.
4. The use of the biologically active polypeptide PKYPVEPF of claim 1, in the preparation of a food, a health product, a pharmaceutical or a cosmetic product with anti-inflammatory properties.
5. The use of the biologically active polypeptide PKYPVEPF of claim 1 in the preparation of a food, health product or pharmaceutical with anti-aging effect.
6. The use of the biologically active polypeptide PKYPVEPF of claim 1, in the preparation of a food, health care product or medicament having an anti-inflammatory and anti-aging function.
7. An anti-inflammatory product comprising the biologically active polypeptide PKYPVEPF of claim 1; the anti-inflammatory product comprises anti-inflammatory food, anti-inflammatory health product, anti-inflammatory drug or anti-inflammatory cosmetic.
8. An anti-aging product comprising the biologically active polypeptide PKYPVEPF of claim 1; the anti-aging product comprises anti-aging food, anti-aging health care products or anti-aging drugs.
9. A product having anti-inflammatory and anti-aging functions, comprising the biologically active polypeptide PKYPVEPF of claim 1; the product with anti-inflammatory and anti-aging effects comprises food, health product or medicine.
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| KR20080003637A (en) * | 2006-07-03 | 2008-01-08 | 한상기 | New functional fermented milk (yogurt) for use in dieting |
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