CN112592396B - A kind of biologically active peptide VDPFSKKDW and its preparation method and application - Google Patents

Abstract

The invention relates to the field of proteins, in particular to a biologically active peptide VDPFSKKDW and a preparation method and application thereof. The amino acid sequence of the biologically active peptide VDPFSKKDW is Val-Asp-Pro-Phe-Ser-Lys-Lys-Asp-Trp. After in vitro immune regulation function experiments, it is proved that the biologically active peptide VDPFSKKDW has a good immune regulation function. The bioactive peptide VDPFSKKDW of the present invention can promote the release of cytokines from macrophages, effectively inhibit the inflammation caused by oxidation in the body, has a certain immune regulation effect, has a good protective effect on inflammatory animals, and improves the body's ability to resist infection by external pathogens. It is of great significance for the development of foods, health products and drugs with immune regulation functions.

Description

A kind of biologically active peptide VDPFSKKDW and its preparation method and application

technical field

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

Background technique

In recent years, bioactive peptides have become a familiar term. Because of its many potential biological functions, it has attracted more and more attention and has become one of the hot spots of scientific research. At present, the beneficial effects of many bioactive peptides have also been well proven, such as anticancer, blood pressure lowering, antibacterial, cholesterol lowering, antidiabetic and other properties. More than 3000 different bioactive peptides have been reported in BIOPEP-UMW, the most authoritative bioactive peptide database.

At present, the research on bioactive peptides mostly focuses on food-derived peptides, and there are few studies and reports on non-food-derived peptides. And research results have confirmed that, compared with food-derived bioactive peptides, non-food-derived bioactive peptides have higher affinity and can effectively exert their bioactive functions. Lymphocytes are the central regulatory cells of the immune system, and most of their functions are mediated by a group of small-molecule polypeptides called lymphokines. Both the expression and secretion of these small molecule polypeptides are induced by antigen-stimulated cell activation. Therefore, lymphocytes are the main source of immunomodulatory peptides produced in animals.

Immunomodulatory peptides are a class of bioactive peptides that were obtained from milk for the first time after the discovery of opioid peptides and proved their physiological activities. In 1981, Jolles et al. first discovered that a hexapeptide with an amino acid sequence of Val-Glu-Pro-Ile-Pro-Tyr could be obtained by using trypsin to hydrolyze human milk protein. In vitro experiments proved that this peptide can enhance mouse peritoneal macrophages. Phagocytosis of sheep erythrocytes. Migliore-Samour et al. found that the casein-derived hexapeptide Thr-Thr-Met-Pro-Leu-Trp can stimulate the phagocytosis of sheep erythrocytes on mouse peritoneal macrophages and enhance the resistance to Klebsiella pneumoniae. Anti-inflammatory function. Li Suping et al. used synthetic mouse bone marrow macrophages to feed rats with the source peptide (PGPIPN) and found that the phagocytosis of rat peritoneal macrophages and erythrocyte-related anti-inflammatory functions were significantly enhanced. Bowdis et al. studied the immune function of the 13 amino acid peptide indolicidin derived from bovine neutrophils and found that the peptide indolicidin inhibited LPS-induced TNF-α production in a macrophage-like cell line.

Studies have shown that immunomodulatory peptides can not only enhance the body's immunity, stimulate the proliferation of lymphocytes, enhance the phagocytic function of macrophages, promote the release of cytokines, promote the increase in the amount of nitric oxide induced by macrophages, and improve the body's defense. The ability of external pathogens to infect, reduce the body's morbidity, and will not cause the body's immune rejection.

Immunomodulatory peptides generally refer to small peptides with relatively small molecular weights that have immunomodulatory activity. The immunomodulatory peptides disclosed now are generally small peptides with specific immunomodulatory activity obtained by enzymatic separation or chemical synthesis from proteins. But when these small peptides are not enzymatically separated from proteins, these proteins themselves often do not have immunomodulatory activity. How to find bioactive peptides with specific functions from a wide variety of proteins with known amino acid sequences and study the functions of these peptides is one of the research directions in the protein field.

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

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a biologically active peptide VDPFSKKDW and its preparation method and application.

The object of the present invention can be realized through the following technical solutions:

The first aspect of the present invention provides a biologically active peptide VDPFSKKDW, the amino acid sequence of which is Val-Asp-Pro-Phe-Ser-Lys-Lys-Asp-Trp, as shown in SEQ ID NO: 1.

Preferably, the biologically active peptide is a mouse spleen-derived lymphocyte peptide. Specifically derived from 40S ribosomal protein S3a protein, and is the amino acid residues at positions 22 to 30 of 40S ribosomal protein S3a protein. The amino acid sequence of 40Sribosomal protein S3a protein is shown in SEQ ID NO:2.

The amino acid sequence of the 40S ribosomal protein S3a protein and the corresponding nucleotide sequence are in the prior art, and the nucleotide fragment encoding the 22nd to 30th amino acid residues of the 40S ribosomal protein S3a protein can encode the mature biologically active peptide VDPFSKKDW.

Preferably, the bioactive peptide has anti-inflammatory and immunomodulatory functions.

The present invention also provides a polynucleotide encoding the biologically active peptide VDPFSKKDW.

The second aspect of the present invention provides a method for preparing the biologically active peptide VDPFSKKDW, which can be artificially synthesized by genetic engineering, can be directly obtained from cells by separation and purification, and can be directly prepared by chemical synthesis.

Artificial synthesis of the biologically active peptide VDPFSKKDW by genetic engineering is a technical solution that can be achieved by those skilled in the art. For example, it can be based on DNA recombination technology, and the sequence synthesis of the polypeptide can be controlled by a suitable DNA template.

The way to obtain directly from cells by separation and purification can be as follows: based on the amino acid sequence of a given biologically active peptide VDPFSKKDW, using conventional enzymatic hydrolysis and purification methods in biological technology to obtain the organism from mouse spleen-derived lymphocytes Active peptide VDPFSKKDW.

The third aspect of the present invention provides the application of the biologically active peptide VDPFSKKDW in the preparation of medicines or cosmetics with anti-inflammatory function.

Further, the application of the biologically active peptide VDPFSKKDW in the preparation of a medicine for inhibiting inflammation caused by oxidation.

The fourth aspect of the present invention provides the application of the biologically active peptide VDPFSKKDW in preparing food or medicine with immunomodulatory function.

Further, the application of the biologically active peptide VDPFSKKDW in the preparation of food or medicine for promoting the secretion of cytokines by macrophages.

A fifth aspect of the present invention provides an anti-inflammatory product comprising the bioactive peptide VDPFSKKDW or a derivative of the bioactive peptide VDPFSKKDW; the anti-inflammatory product includes anti-inflammatory drugs or anti-inflammatory cosmetics.

The sixth aspect of the present invention provides a product with immunomodulatory function, comprising the biologically active peptide VDPFSKKDW or a derivative of the biologically active peptide VDPFSKKDW; the product with immunomodulatory function includes food with immunomodulatory function Or drugs with immunomodulatory functions.

The derivative of the biologically active peptide VDPFSKKDW refers to having the same activity or superior activity as the biologically active peptide VDPFSKKDW.

The derivative of the biologically active peptide VDPFSKKDW refers to hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, amino-terminal or carboxyl-terminal of the amino acid side chain group of the biologically active peptide VDPFSKKDW. The bioactive peptide derivatives obtained by modification such as esterification or glycosylation.

The beneficial effects of the biologically active peptide VDPFSKKDW of the present invention are as follows: the biologically active peptide VDPFSKKDW of the present invention has better anti-inflammatory activity; the biologically active peptide VDPFSKKDW of the present invention can promote the release of cytokines from macrophages and effectively inhibit the body's inflammation caused by oxidation. Inflammation has a certain immunomodulatory effect, has a good protective effect on inflammatory animals, improves the body's ability to resist infection by external pathogens, reduces the body's morbidity, and improves the quality of life. Medicines are very important.

Description of drawings

Figure 1: First-order mass spectrum of a fragment with a mass-to-charge ratio of 374.5259 (m/z = 374.5259);

Figure 2: The MS spectrum of the fragment with a mass-to-charge ratio of 374.5259 and the fragmentation of bioactive peptides az and by;

Detailed ways

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, It is not intended to limit the protection scope of the present invention.

When numerical ranges are given in the examples, it is to be understood that, unless otherwise indicated herein, both endpoints of each numerical range and any number between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment and materials used in the embodiments, according to the mastery of the prior art by those skilled in the art and the description of the present invention, the methods, equipment and materials described in the embodiments of the present invention can also be used Any methods, devices and materials similar or equivalent to those of the prior art can be used to implement the present invention.

Unless otherwise specified, the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. conventional technology. These techniques have been well described in the existing literature. For details, see 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 INMOLECULAR BIOLOGY, John Wiley & Co. Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODSIN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), AcademicPress, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999, etc.

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

Example 1 Artificial synthesis of bioactive peptide VDPFSKKDW

1. Synthesis of Bioactive Peptides

Synthetic bioactive peptide VDPFSKKDW.

2. Confirmation of Bioactive Peptides

1) UPLC analysis

The UPLC conditions are as follows:

Instrument: Waters ACQUITY UPLC ultra-high performance liquid phase, electrospray, quadrupole, time-of-flight mass spectrometer

Column Specifications: BEH C18 Column

Flow rate: 0.4mL/min

Temperature: 50℃

UV detection wavelength: 210nm

Injection volume: 2μL

Gradient conditions: solution A: water containing 0.1% formic acid (v/v), solution 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 spectrometry

The mass spectrometry conditions are 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 (℃): 115

Desolvation temperature (℃): 350

Desolvation gas flow (L/hr): 700.0

Collision energy (eV): 4.0

Scan time (sec): 0.25

Inner scan time (sec): 0.02

According to the above analysis methods, using ultra-high performance liquid phase, electrospray, quadrupole, and time-of-flight mass spectrometry, the biological active peptide VDPFSKKDW was analyzed by chromatography and mass spectrometry. The primary mass spectrum of the bioactive peptide VDPFSKKDW is shown in Figure 1, and the secondary mass spectrum of the extracted peak and the az, by fragmentation are shown in Figure 2. The mass-to-charge ratio of the bioactive peptide of this peak is 374.5259, and the retention time is 27.5 min.

3) Results

It can be seen from Fig. 2 that according to the az and by breakage, after analysis and calculation by the Mascot software, the fragment sequence with a mass-to-charge ratio of 374.5259 is obtained as Val, Asp, Pro, Phe, Ser, Lys, Lys, Asp, Trp (VDPFSKKDW), denoted as VDPFSKKDW. is SEQ ID NO:1. This fragment corresponds to the residue sequence of the 22nd-30th position of 40S ribosomal protein S3a protein. The GenBank number of the amino acid sequence of 40Sribosomal protein S3a protein is AAH81451.1, and the sequence is shown in SEQ ID NO: 2.

Example 2 Immunoactivity test of biologically active peptides

1. Experiment on the effect of bioactive peptide VDPFSKKDW on immune cytokines in serum

1. Experimental reagents and instruments:

Reagents: ICR mice (male, 5 weeks old), Shanghai Experimental Animal Center; D-gal, Sinopharm Chemical Reagent Co., Ltd.; Paraformaldehyde, Sinopharm Chemical Reagent Co., Ltd.; Sodium Chloride, Sinopharm Chemical Reagent Co., Ltd. Co., Ltd.; the mouse spleen lymphocyte-derived bioactive peptide VDPFSKKDW obtained in Example 1; BCA protein kit, Nanjing KGI Biotechnology Co., Ltd.; ELISA Cytokine Rapid Kit (TNF-α and IL-6), Dr. Wuhan German Bioengineering Co., Ltd.

Equipment: CM-230 Mole ultra-clean water, Shanghai Moller Scientific Instrument Co., Ltd.; Millipore MILLEX GP 0.22 μm filter membrane, Millipore Corporation, USA; GL-22M high-speed refrigerated centrifuge, Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.

2. Experimental method:

(1) Animal aging model modeling

After one week of adaptive feeding, ICR mice were divided into 4 groups with 6 mice in each group. Group 1 was a low-dose gavage group, the mice were subcutaneously injected with D-gal on the back of the neck at a dose of 500 mg/kg every day, and the bioactive peptide VDPFSKKDW was administered by gavage at a dose of 0.5 mg/mice per day; group 2 was a high-dose gavage. In group 3, mice were injected with D-gal subcutaneously on the back of the neck at a dose of 500 mg/kg every day, and the bioactive peptide VDPFSKKDW was intragastrically administered at a dose of 1 mg/day; group 3 was a blank group, and the mice grew normally; group 4 was an animal model group, the mice were injected with D-gal subcutaneously on the back of the neck at a dose of 500 mg/kg every day, and the concentration of normal saline was 0.9% by gavage; the injection cycle of D-gal and the gavage cycle of bioactive peptides were both 42 days. Change the litter every 3 days, and ensure the supply of feed and distilled water. The weight of the mice was weighed every five days, and the 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 organs and serum

After the experimental period was completed, the blood of the mice was obtained by removing the eyeballs and blood. After obtaining the blood, the mice were killed by neck dislocation, and then the bodies of the mice were placed on a low-temperature ice box. The serum was separated by centrifugation at 3000g for 15min. Serum was stored in a -80°C refrigerator until testing. All operations in the process of handling laboratory animals followed the "Guiding Opinions on Treating Laboratory Animals Kindly" issued by the Ministry of Science and Technology in 2006. The extracted mouse spleen was directly immersed in a pre-prepared 4% paraformaldehyde solution to fix its shape. Paraformaldehyde powder is relatively insoluble, and a small amount of sodium bicarbonate can be added to adjust the pH value to alkaline to help dissolve. The preparation of the paraformaldehyde solution needs to be done in a fume hood.

(3) Sample detection

According to the instructions of the kit, first draw a standard curve, prepare a 1000pg/mL solution of the standard powder with the standard dilution, and then serially dilute to 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL. mL, 31.3pg/mL, 15.6pg/mL and other different concentrations. Pipette 100 μL of each concentration gradient solution into the antibody-coated ELISA plate. Aspirate 100 μL of mouse serum sample and add it to the same ELISA plate (if the serum sample is not enough, it can be appropriately diluted and then calculated according to the proportion). Cover the microtiter plate and incubate it at 37°C for 90min. After the reaction is completed, carefully shake off the liquid in the ELISA plate, place the ELISA plate on absorbent paper, and tap carefully to remove excess liquid. Add 100 μL of preheated biotin antibody working solution to each well of the ELISA plate in turn, and react at 37°C for 60 min. After the reaction, wash three times with 0.01M PBS solution, add 100 μL of PBS to each well each time, soak for 1 min and then pour off the solution, repeating three times. The preheated ABC working solution was added in sequence at 0.1 ml per well, and the reaction was carried out at 37 °C for 30 min. After the reaction was completed, wash with 0.01MPBS for 5 times, soaking for about 1min each time. 90 μL of each well was sequentially added with TMB chromogenic solution that had been equilibrated at 37°C for 30 minutes, and the reaction was performed at 37°C for 8-12 minutes in the dark. Add 0.1 ml of TMB stop solution to each well in turn, at this time, the blue turns to yellow, and the OD value is measured at 450 nm with a microplate reader. The standard protein of cytokines is used for serial dilution of known concentration, and the standard curve is drawn after measuring the OD value. According to the standard curve, the content of cytokines in the sample can be calculated.

3. Experimental results and analysis:

Table 1 Changes of 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

It can be found from Table 1 that the levels of IL-6 and TNF-α in the model group mice in this experiment were 184.24±18.92pg/mL and 5.39±0.53pg/mL, respectively, which were significantly higher than those in the normal group. increased (P<0.01), so it can be considered that due to the continuous injection of senescent factors, the mice in the animal model group had symptoms of senescent inflammation at the cytokine level, while the serum IL- 6 and TNF-α content were effectively controlled. According to the experimental results of cytokines, the secretion levels of serum inflammatory cytokines IL-6 and TNF-α in the mice in the bioactive peptide gavage group were lower than those in the animal model group. Oxidative damage caused by attack and accumulation of peroxidative products may be inhibited to a certain extent; from the perspective of inflammation, the inflammation caused by oxidation in mice is effectively inhibited; from the perspective of aging, mice treated by long-term injection A series of senile diseases caused by aging caused by D-gal may be controlled. Therefore, it can be determined that VDPFSKKDW can effectively inhibit the inflammation caused by oxidation in mice, has a certain immunomodulatory effect, and can be used in the research and development of health care products.

2. The experiment of promoting the secretion of cytokines by macrophages with the bioactive peptide VDPFSKKDW (ELISA method)

1. Experimental reagents and instruments:

Reagents: experimental animal balb/c mice (male, 6 and 8 weeks old), Shanghai Slack Laboratory Animal Co., Ltd.; mouse lymphocyte extract, Shanghai Soleibao Biotechnology Co., Ltd.; RPMI1640 medium, GIBCO company; cattle Serum albumin (bovine serum albumin, BSA), Genebase company; mouse spleen lymphocyte-derived bioactive peptide VDPFSKKDW obtained in Example 1; ELISA cytokine rapid kit (TNF-α, IL-1β and IL-6) , Wuhan Boster Biological Engineering Co., Ltd.

Instruments and equipment: LRH, 250F biochemical incubator, Shanghai Heng Technology Co., Ltd.; GL, 22M high-speed refrigerated centrifuge, Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.; Hera cell 150 CO ₂ incubator Heraeus Company; DragonWellscan MK3 Microplate Reader Labsystems Company.

2. Experimental method:

Add 100 μl/well of cell suspension with a cell number of 2×10 ⁶ /ml, and add 200 μl/well of peptide-containing RPMI1640 complete culture medium (10% FBS) after adherence purification. In the inflammation group, add LPS to the end at 24 hours. The concentration was 10 μg/ml, and the culture was continued for 48 hours. In the inflammation group, LPS was added to the final concentration of 100 ng/ml 24 hours before the culture was terminated. After the culture was terminated, the cell culture supernatant was collected by centrifugation. Add 100 μl of supernatant to the ELISA plate that has been coated with cytokine antibodies. After 90 minutes of reaction at 37°C, add biotin-labeled antibodies, and then react at 37°C for 60 minutes. After washing with PBS, add avidin and peroxidase. The complex was reacted for 30 minutes. After washing with PBS, a color developing solution was added, and the reaction was performed for 20 minutes. After adding the color stop solution, use a microplate reader to measure the absorbance value (OD450) at a wavelength of 450 nm.

3. Experimental results and analysis:

Table 2 Determination of the effect of bioactive peptide VDPFSKKDW on cytokine levels in macrophages

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

Note: *, compared with the negative control group, there is a significant difference (P <0.05); **, compared with the negative control group, there is a very significant difference (P <0.01)

It can be seen from Table 2 that in the experimental results of the three cytokines TNF-α, IL-1β and IL-6, TNF-α and IL-1β showed extremely significant differences at 0.1 mg/ml and above (P <0.01), IL-6 showed a very significant difference at 0.5mg/ml (P<0.01), which proved that VDPFSKKDW at a certain concentration can promote the activation of mouse peritoneal macrophages and release TNF-α, IL-1β, The three cytokines IL-6, TNF-α, IL-1β and IL-6 are pro-inflammatory factors, which can induce B cells to differentiate and produce antibodies, and induce T cells to activate, proliferate and differentiate, and participate in the body's immune response. Therefore, VDPFSKKDW at a certain concentration can enhance the effect of these cytokines in normal macrophages at rest, thereby regulating the body's immunity.

The foregoing description of the embodiments is provided to facilitate understanding and use of the invention by those of ordinary skill in the art. It will be apparent to those skilled in the art that various modifications to these embodiments can be readily made, and the generic principles described herein can be applied to other embodiments without inventive step. Therefore, the present invention is not limited to the above-mentioned embodiments, and improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should all fall within the protection scope of the present invention.

sequence listing

<110> Shanghai Jiaotong University, Zhejiang Huitide Life and Health Technology Co., Ltd.

<120> A kind of biologically active peptide VDPFSKKDW and its preparation method and application

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<170> SIPOSequenceListing 1.0

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<211> 9

<212> PRT

<213> Artificial Sequence

<400> 1

Val Asp Pro Phe Ser Lys Lys Asp Trp

1 5

<210> 2

<211> 264

<212> PRT

<213> 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)

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.

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