CN112592396B

CN112592396B - Bioactive peptide VDPFSKKDW as well as preparation method and application thereof

Info

Publication number: CN112592396B
Application number: CN202011467505.0A
Authority: CN
Inventors: 张少辉; 周洲; 李政; 郭婷婷; 谢雨佳; 栾媛媛; 王娟; 张伯宇
Original assignee: Zhejiang Huitai Life Health Technology Co ltd; Shanghai Jiaotong University
Current assignee: Zhejiang Huitai Life Health Technology Co ltd; Shanghai Jiaotong University
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2022-10-21
Anticipated expiration: 2040-12-14
Also published as: CN112592396A

Abstract

The invention relates to the field of proteins, and particularly relates to a bioactive peptide VDPFSKKDW, a preparation method and application thereof. In-vitro immune regulation function experiments prove that the bioactive peptide VDPFSKKDW has good immune regulation function. The bioactive peptide VDPFSKKDW can promote macrophages to release cytokines, effectively inhibit inflammation caused by oxidation of an organism, has a certain immunoregulation effect, has a good protection effect on inflammatory animals, improves the capability of the organism in resisting external pathogen infection, reduces the morbidity of the organism, improves the quality of life, and has very important significance for developing foods, health-care products and medicines with immunoregulation function.

Description

Bioactive peptide VDPFSKKDW and preparation method and application thereof

Technical Field

The invention relates to the field of proteins, in particular to a bioactive peptide VDPFSKKDW, and a preparation method and application thereof.

Background

In recent years, bioactive peptides have become a word of great energy in the ear. Because of its many potential biological functions, it attracts more and more attention and becomes one of the hot spots of scientific research. The beneficial effects of many bioactive peptides, such as anti-cancer, blood pressure lowering, antibacterial, cholesterol lowering, anti-diabetic, etc., are well documented. Currently more than 3000 different bioactive peptides have been reported in the most authoritative bioactive peptide database BIOPEP-UMW.

Currently, studies on bioactive peptides are mostly focused on food-derived polypeptides, and studies and reports on non-food-derived polypeptides are few. And it has been confirmed from the research results that non-food-derived bioactive peptides have higher affinity and can effectively exert their bioactive functions, compared to food-derived bioactive peptides. Lymphocytes are central regulatory cells of the immune system, most of whose function is mediated by a group of small molecule polypeptides called lymphokines. Expression and secretion of these small molecule polypeptides are induced by antigen-stimulated cellular activation. Lymphocytes are therefore the primary source of immunoregulatory peptides produced in the animal body.

Immunomodulatory peptides are a class of bioactive peptides that were first obtained from milk following opioid peptide discovery and demonstrated their physiological activity. Jolles et al found for the first time in 1981 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 mouse bone marrow macrophages and a source peptide (PGPIPN), found that phagocytosis of rat peritoneal macrophages and red blood cell-related anti-inflammatory function were significantly enhanced. Bowdis et al, in studying the immune function of the 13 amino acid peptide indolicidin derived from bovine neutrophils, found that the polypeptide indolicidin inhibits LPS-induced TNF- α production in a macrophage-like cell line.

Research shows that the immunoregulation peptide can enhance the immunity of the organism, stimulate the proliferation of lymphocytes of the organism, enhance the phagocytic function of macrophages, promote the release of cytokines, 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 and cannot cause the immune rejection reaction of the organism.

Immunomodulatory peptides generally refer to small, relatively small molecular weight peptides with immunomodulatory activity. The immunomodulatory peptides presently disclosed are generally small peptides with specific immunomodulatory activity, isolated enzymatically from proteins or synthesized chemically. However, when these small peptides are not enzymatically separated from the protein, the protein itself is often not immunomodulatory. It is one of the directions in the field of protein research to find bioactive peptides with specific functions from a wide variety of proteins whose amino acid sequences are known, and to study the functions of these polypeptides.

The amino acid sequence of the 40S ribosomal protein S3a protein is shown in SEQ ID NO:2, respectively. At present, no research on related functions of the 40S ribosomal protein S3a protein polypeptide fragment exists in the prior art.

Disclosure of Invention

The invention aims to provide a bioactive peptide VDPFSKKDW, a preparation method and application thereof.

The purpose of the invention can be realized by the following technical scheme:

in the first aspect of the invention, a bioactive peptide VDPFSKKDW is provided, the amino acid sequence of which is Val-Asp-Pro-Phe-Ser-Lys-Lys-Asp-Trp, and is shown as SEQ ID NO:1 is shown.

Preferably, the bioactive peptide is mouse spleen derived lymphocyte peptide. Specifically, the protein is derived from 40S ribosomal protein S3a, and is the 22-30 amino acid residue of the 40S ribosomal protein S3a protein. The amino acid sequence of the 40S ribosomal protein S3a protein is shown in SEQ ID NO:2, respectively.

The amino acid sequence and the corresponding nucleotide sequence of the 40S ribosomal protein S3a protein are the prior art, and the nucleotide fragment for coding the 22 nd to 30 th amino acid residues of the 40S ribosomal protein S3a protein can code mature biological active peptide VDPFSKKDW.

Preferably, the bioactive peptide has an anti-inflammatory function and an immune-modulating function.

The invention also provides a polynucleotide for coding the bioactive peptide VDPFSKKDW.

In the second aspect of the invention, the preparation method of the bioactive peptide VDPFSKKDW is provided, which can be artificially synthesized by a genetic engineering method, can be directly obtained from cells by a separation and purification method, and can be directly prepared by chemical synthesis.

The artificial synthesis of the bioactive peptide VDPFSKKDW by genetic engineering is a technical scheme which can be realized by a person skilled in the art, and for example, the sequence synthesis of the polypeptide can be controlled by a suitable DNA template based on DNA recombination technology.

The method for directly obtaining the cell by the separation and purification method can be as follows: based on the amino acid sequence of the given bioactive peptide VDPFSKKDW, the bioactive peptide VDPFSKKDW is obtained from mouse spleen source lymphocytes by adopting a conventional enzymolysis and purification method in the biological technology.

The third aspect of the invention provides application of the bioactive peptide VDPFSKKDW in preparation of drugs or cosmetics with anti-inflammatory functions.

Further, the application of the bioactive peptide VDPFSKKDW in preparing a medicament for inhibiting inflammation caused by oxidation.

In the fourth aspect of the invention, the invention provides the application of the bioactive peptide VDPFSKKDW in the preparation of food or medicines with the immunoregulation function.

Further, the biological active peptide VDPFSKKDW is applied to the preparation of foods or medicines for promoting macrophages to secrete cytokines.

In a fifth aspect of the present invention, an anti-inflammatory product is provided, which comprises the bioactive peptide VDPFSKKDW or a derivative of the bioactive peptide VDPFSKKDW; the anti-inflammatory product comprises an anti-inflammatory drug or an anti-inflammatory cosmetic.

In a sixth aspect of the present invention, a product with immunoregulatory function is provided, which comprises the bioactive peptide VDPFSKKDW or a derivative of the bioactive peptide VDPFSKKDW; the product with immunoregulatory function comprises food with immunoregulatory function or medicine with immunoregulatory function.

The derivative of the bioactive peptide VDPFSKKDW refers to the derivative which has the same activity or better activity with the bioactive peptide VDPFSKKDW.

The derivative of the bioactive peptide VDPFSKKDW refers to a bioactive peptide derivative obtained by carrying out hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation and other modifications on an amino acid side chain group, an amino terminal or a carboxyl terminal of the bioactive peptide VDPFSKKDW.

The beneficial effects of the bioactive peptide VDPFSKKDW are as follows: the bioactive peptide VDPFSKKDW has better anti-inflammatory activity; the biological active peptide VDPFSKKDW can promote macrophages to release cytokines, effectively inhibit inflammation caused by oxidation of organisms, has a certain immunoregulation function, has a good protection effect on inflammatory animals, improves the ability of the organisms to resist infection of external pathogens, reduces the morbidity of the organisms, improves the quality of life, and has very important significance in developing foods, health care products and medicines with immunoregulation function.

Drawings

FIG. 1: a first order mass spectrum of a fragment with a mass to charge ratio of 374.5259 (m/z = 374.5259);

FIG. 2: a secondary mass spectrum of a fragment with a mass-to-charge ratio of 374.5259 and the breaking conditions of the bioactive peptides az and by;

Detailed Description

Before the present embodiments are further described, it is to be understood that the scope of the invention is not to be limited to the specific 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 are described in particular in Sambrook et al, molecular CLONING: 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 biologically active peptide VDPFSKKDW

1. Synthesis of bioactive peptides

Artificially synthesizing the bioactive peptide VDPFSKKDW.

2. Identification 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 phase, electrospray, quadrupole and 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)

Time(min) %A %B

0 95.0 5.0

1.50 80.0 20.0

3.50 60.0 40.0

5.00 40.0 60.0

7.00 15.0 85.0

8.00 0.0 100.0

11.00 0.0 100.0

11.50 95.0 5.0

13.00 95.0 5.0

2) Mass spectrometric analysis

The mass spectrometry conditions were as follows:

ion mode: ES + C

Mass range (m/z): 100. 1000A

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 biological active peptide VDPFSKKDW is subjected to chromatographic analysis and mass spectrometry by using ultra-high performance liquid phase, electrospray, a quadrupole and a time-of-flight mass spectrum. The primary mass spectrum of the bioactive peptide VDPFSKKDW is shown in figure 1, the secondary mass spectrum of the extracted peak and the az and by fracture conditions are shown in figure 2, the mass-to-charge ratio of the bioactive peptide of the peak is 374.5259, and the retention time is 27.5 min.

3) As a result, the

As can be seen from fig. 2, the fragment sequence with the mass-to-charge ratio 374.5259, which is Val, asp, pro, phe, ser, lys, asp, trp (VDPFSKKDW) according to the cases of az and by fragmentation, is analyzed and calculated by Mascot software, and is marked as SEQ ID NO:1. the fragment corresponds to the residue sequence of positions 22-30 of the 40S ribosomal protein S3a protein, the GenBank number of the amino acid sequence of the 40S ribosomal protein S3a protein is AAH81451.1, and the sequence is shown in SEQ ID NO:2.

example 2 immunological Activity assay of bioactive peptides

1. Experiment of biological active peptide VDPFSKKDW on effect of immune cell factor in serum

1. Experimental reagents and instruments:

reagent: experimental animal ICR mouse (male 5 weeks old), experimental animal center in shanghai city; d-gal, national pharmaceutical group chemical reagents, inc.; paraformaldehyde, chemical reagents of the national drug group, ltd; sodium chloride, national chemical group chemical agents ltd; the mouse spleen lymphocyte-derived bioactive peptide VDPFSKKDW obtained in example 1; BCA protein kit, nanjing Kaikyi Biotech Co., ltd; ELISA cytokine Rapid kits (TNF-. Alpha.and IL-6), wuhan Dr bioengineering, inc.

An instrument device: model CM-230 Moleji super purified Water, shanghai Mohler scientific instruments, inc.; millipore Milllex GP0.22 μm membrane filter, millipore USA; GL-22M high-speed refrigerated centrifuge, shanghai Luxiang apparatus centrifuge Instrument Co.

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 at a dose of 500mg/kg daily and the biologically active peptide VDPFSKKDW was intragastric at a dose of 0.5 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 1 mg/mouse was gavage with biologically active peptide VDPFSKKDW at a daily dose; 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 the D-gal and the gavage period of the bioactive peptide are both 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 assist 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 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.3pg/mL, 15.6 pg/mL and the like. Each concentration gradient solution was pipetted at 100. Mu.L in an antibody-coated microplate. And (4) 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 serum sample can be diluted properly and then needs to be converted proportionally when being detected and calculated). Cover the plate and incubate it at 37 ℃ for 90min. After the reaction is finished, carefully throwing off the liquid in the ELISA plate, placing the ELISA plate on absorbent paper, carefully beating, and removing the excess liquid. Adding preheated biotin 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 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 1 Change in cytokines in serum of mice in each group

	TNF-α(pg/mL)	IL-6 (pg/mL)
			Group 1	2.27±0.30**	74.29±10.38**
Group 2	2.53±0.39**	95.34±17.40**
			Group 3	2.12±0.42**	68.32±14.39**
Group 4	5.39±0.53	184.24±18.92

From Table 1, it can be found that the IL-6 and TNF-alpha contents in the mice of the model group in the experiment are 184.24 + -18.92 pg/mL and 5.39 + -0.53 pg/mL respectively, and show very significant increase (P < 0.01) compared with the normal group, so that the mice of the model group are considered to have symptoms of aging inflammation at the cytokine level due to continuous injection of the aging-causing factor, and the IL-6 and TNF-alpha contents in the serum of the mice of the bioactive peptide gavage group are effectively controlled. According to the experimental result of the cell factors, the secretion levels of serum inflammatory cell factors IL-6 and TNF-alpha of the mice in the bioactive peptide gavage group are lower than those of the mice in the animal model group, and the oxidation damage of the mice caused by free radical attack and peroxidation product accumulation can be inhibited to a certain degree from the perspective of the oxidation damage; from the viewpoint of inflammation, the inflammation of the mice caused by oxidation is effectively inhibited; from the aging point of view, a series of senile diseases of mice caused by aging caused by long-term injection of D-gal are likely to be controlled. Therefore, the VDPFSKKDW can be determined to effectively inhibit inflammation caused by oxidation of mice, has a certain immunoregulation effect, and can be used for research and development of health care products.

2. Experiment (ELISA method) for promoting macrophage to secrete cytokine of bioactive peptide VDPFSKKDW

1. Experimental reagents and instruments:

reagent: experimental animals balb/c mice (male 6, 8 week old), shanghai slek experimental animals limited; mouse lymphocyte extract, shanghai solibao biotechnology limited; RPMI1640 medium, GIBCO; bovine Serum Albumin (BSA), genebase; the mouse spleen lymphocyte-derived bioactive peptide VDPFSKKDW obtained in example 1; ELISA cytokine Rapid kits (TNF-. Alpha., IL-1. Beta. And IL-6), wuhan Dr bioengineering, inc.

The instrument equipment comprises: LRH, 250F biochemical incubator shanghai constant technology ltd; GL, 22M high speed refrigerated centrifuge Shanghai Luxiang apparatus centrifuge instruments ltd; hera cell 150 CO ₂ Incubator Heraeus; dragon Wellscan MK3 microplate reader Labsystems Inc.

2. The experimental method comprises the following steps:

the number of the added cells was 2X 10 ⁶ 100. Mu.l/well of cell suspension/ml, 200. Mu.l/well of peptide-containing RPMI1640 complete medium (10. Mu.g FBS) after adherent purification, LPS at 24 hours to a final concentration of 10. Mu.g/ml for the inflammatory group, continuous culture for 48 hours, 24 hours before termination of culture for the inflammatory groupLPS was added to a final concentration of 100ng/ml. After the termination of the culture, the cell culture supernatant was collected by centrifugation. Mu.l of supernatant was added to an enzyme-labeled plate coated with cytokine antibody, reacted at 37 ℃ for 90 minutes, then added with biotin-labeled antibody, reacted at 37 ℃ for 60 minutes, washed with PBS, and then added with avidin-peroxidase complex, reacted for 30 minutes. After washing with PBS, a developing solution was added thereto, and the reaction was carried out for 20 minutes. After addition of the chromogenic stop solution, the absorbance value (OD 450) was measured at a wavelength of 450nm using a microplate reader.

3. Experimental results and analysis:

TABLE 2 determination of the Effect of the biologically active peptide VDPFSKKDW on the level of macrophage cytokines

Experiment grouping	TNF-α	IL-1β	IL-6
				Cell blank	0.116±0.021	0.405±0.020	1.219±0.032
Bioactive peptide samples (0.1 mg/ml)	0.374±0.301**	0.572±0.102**	1.228±0.285
				Bioactive peptide samples (0.5 mg/ml)	0.472±0.138**	0.642±0.068**	1.482±0.482**

Note: * Compared with a negative control, the difference is significant (P is less than 0.05); * The difference between the positive control group and the negative control group is very significant (P < 0.01)

As can be seen from Table 2, in the experimental results of three cytokines of TNF-alpha, IL-1 beta and IL-6, the significant difference (P < 0.01) appears at 0.1mg/ml and above for TNF-alpha and IL-1 beta, and the significant difference (P < 0.01) appears at 0.5mg/ml for IL-6, thus proving that VDPFSKKDW under a certain concentration can promote the activation of mouse abdominal macrophages and release three cytokines of TNF-alpha, IL-1 beta, IL-6, TNF-alpha, IL-1 beta and IL-6 which are pro-inflammatory factors, can induce the differentiation of B cells and the generation of antibodies, and induce the activation, proliferation and differentiation of T cells, and participate in the immune response of the organism. Therefore, the VDPFSKKDW under a certain concentration can improve the effect of the cytokines under the resting state of normal macrophages, thereby regulating the immunity of the organism.

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 river peptide Life health science and technology Limited

<120> biological active peptide VDPFSKKDW and preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Val Asp Pro Phe Ser Lys Lys Asp Trp

1 5

<210> 2

<211> 264

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ala Val Gly Lys Asn Lys Arg Leu Thr Lys Gly Gly Lys Lys Gly

1 5 10 15

Ala Lys Lys Lys Val Val Asp Pro Phe Ser Lys Lys Asp Trp Tyr Asp

20 25 30

Val Lys Ala Pro Ala Met Phe Asn Ile Arg Asn Ile Gly Lys Thr Leu

35 40 45

Val Thr Arg Thr Gln Gly Thr Lys Ile Ala Ser Asp Gly Leu Lys Gly

50 55 60

Arg Val Phe Glu Val Ser Leu Ala Asp Leu Gln Asn Asp Glu Val Ala

65 70 75 80

Phe Arg Lys Phe Lys Leu Ile Thr Glu Asp Val Gln Gly Lys Asn Cys

85 90 95

Leu Thr Asn Phe His Gly Met Asp Leu Thr Arg Asp Lys Met Cys Ser

100 105 110

Met Val Lys Lys Trp Gln Thr Met Ile Glu Ala His Val Asp Val Lys

115 120 125

Thr Thr Asp Gly Tyr Leu Leu Arg Leu Phe Cys Val Gly Phe Thr Lys

130 135 140

Lys Arg Asn Asn Gln Ile Arg Lys Thr Ser Tyr Ala Gln His Gln Gln

145 150 155 160

Val Arg Gln Ile Arg Lys Lys Met Met Glu Ile Met Thr Arg Glu Val

165 170 175

Gln Thr Asn Asp Leu Lys Glu Val Val Asn Lys Leu Ile Pro Asp Ser

180 185 190

Ile Gly Lys Asp Ile Glu Lys Ala Cys Gln Ser Ile Tyr Pro Leu His

195 200 205

Asp Val Phe Val Arg Lys Val Lys Met Leu Lys Lys Pro Lys Phe Glu

210 215 220

Leu Gly Lys Leu Met Glu Leu His Gly Glu Gly Gly Ser Ser Gly Lys

225 230 235 240

Ala Ala Gly Asp Glu Thr Gly Ala Lys Val Glu Arg Ala Asp Gly Tyr

245 250 255

Glu Pro Pro Val Gln Glu Ser Val

260

Claims

1. The application of the bioactive peptide VDPFSKKDW in preparing the medicine for reducing the secretion level of immune cell factors IL-6 and TNF-alpha in serum under the aging state is characterized in that the amino acid sequence of the bioactive peptide VDPFSKKDW is Val-Asp-Pro-Phe-Ser-Lys-Lys-Asp-Trp.