CN112759634B

CN112759634B - Bioactive peptide with amino acid structure FEYIEENKY, and preparation method and application thereof

Info

Publication number: CN112759634B
Application number: CN202110082302.8A
Authority: CN
Inventors: 张少辉; 亚辛·黑马尔; 张伯宇; 占文静; 石毅; 洪志骏
Original assignee: Zhejiang Huitai Life Health Technology Co ltd
Current assignee: Zhejiang Huitai Life Health Technology Co ltd
Priority date: 2021-01-21
Filing date: 2021-01-21
Publication date: 2022-04-08
Anticipated expiration: 2041-01-21
Also published as: CN112759634A

Abstract

The invention relates to the field of protein, in particular to a bioactive peptide with an amino acid structure FEYIEENKY, a preparation method and application thereof, and a preparation method and application thereof, wherein the amino acid sequence of the bioactive peptide FEYIEENKY is Phe-Glu-Tyr-Ile-Glu-Glu-Asn-Lys-Tyr, and the amino acid sequence of the bioactive peptide FEYIEENKYSR is Phe-Glu-Tyr-Ile-Glu-Glu-Asn-Lys-Tyr-Ser-Arg. In vitro immune function regulation experiments prove that the bioactive peptides FEYIEENKY and FEYIEENKYSR have better immune regulation function. The FEYIEENKY and FEYIEENKYSR of the invention have the capacity of promoting the increase of the induction quantity of nitric oxide of macrophages, improve the immunity of organisms to external stimulation, reduce the morbidity of the organisms, have obvious promotion effect on the ability of phagocytosing neutral red by macrophages in vitro under the condition of inflammation occurrence, have good protection effect on inflammatory animals, and have very important significance for developing foods, health care products and medicines with immunoregulation function.

Description

Bioactive peptide with amino acid structure FEYIEENKY, and preparation method and application thereof

Technical Field

The invention relates to the field of protein, in particular to a bioactive peptide with an amino acid structure FEYIEENKY, a preparation method and application thereof, 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 less. 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 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 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.

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 often has no immunomodulatory activity. 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 hetereogenous nucleoroperetin U protein is shown as SEQ ID NO: 3, respectively. At present, the related functions of the polypeptide fragments of heterologous nuclear ribonucleoprotein U protein are not studied in the prior art.

Disclosure of Invention

The invention aims to provide a bioactive peptide with an amino acid structure FEYIEENKY, and a preparation method and application thereof, and particularly mainly relates to a bioactive peptide FEYIEENKY and a bioactive peptide FEYIEENKYSR.

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

in a first aspect of the invention, a biologically active peptide having amino acid structure FEYIEENKY is provided, being biologically active peptide FEYIEENKY or biologically active peptide FEYIEENKYSR.

The amino acid sequence of the bioactive peptide FEYIEENKY is Phe-Glu-Tyr-Ile-Glu-Glu-Asn-Lys-Tyr as shown in SEQ ID NO: 1 is shown.

The amino acid sequence of bioactive peptide FEYIEENKYSR is: Phe-Glu-Tyr-Ile-Glu-Glu-Asn-Lys-Tyr-Ser-Arg as shown in SEQ ID NO: 2, respectively.

Preferably, the bioactive peptide FEYIEENKY or bioactive peptide FEYIEENKYSR is mouse spleen derived lymphocyte peptide. The amino acid residues are respectively 234-242 th and 234-244 th amino acid residues of the heterologous nucleoside subunit U protein. The amino acid sequence of the hetereogenous nucleoroperetin U protein is shown as SEQ ID NO: 3, respectively.

The amino acid sequence and the corresponding nucleotide sequence of the heterologous nuclear nucleoprotein U protein are the prior art, and the nucleotide fragments which code the 234 th to 242 th and 234 th to 244 th amino acid residues of the heterologous nuclear nucleoprotein U protein can code mature bioactive peptides FEYIEENKY and FEYIEENKYSR.

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

The invention also provides polynucleotides encoding the biologically active peptide FEYIEENKY or biologically active peptide FEYIEENKYSR.

In the second aspect of the present invention, there is provided a method for preparing the bioactive peptide FEYIEENKY or the bioactive peptide FEYIEENKYSR, which can be artificially synthesized by genetic engineering methods, can be directly obtained from cells by separation and purification methods, and can be directly prepared by chemical synthesis.

The artificial synthesis of the bioactive peptide FEYIEENKY or the bioactive peptide FEYIEENKYSR by genetic engineering methods is a technical solution that can be realized by those 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 a given bioactive peptide FEYIEENKY or bioactive peptide FEYIEENKYSR, the bioactive peptide FEYIEENKY or bioactive peptide FEYIEENKYSR is obtained from mouse spleen-derived lymphocytes by a conventional enzymatic hydrolysis and purification method in biological technology.

In a third aspect of the present invention, there is provided the use of one or a combination of two of bioactive peptide FEYIEENKY or bioactive peptide FEYIEENKYSR in the manufacture of a medicament or cosmetic product with anti-inflammatory properties.

Specifically, the bioactive peptide FEYIEENKY or bioactive peptide FEYIEENKYSR of the present invention can be used for preparing drugs with anti-inflammatory properties.

Specifically, the bioactive peptide FEYIEENKY or the bioactive peptide FEYIEENKYSR disclosed by the invention is used for preparing a medicament for promoting the ability of macrophages in vitro to phagocytose neutral red.

In a fourth aspect of the present invention, there is provided the use of a combination of one or both of bioactive peptide FEYIEENKY or bioactive peptide FEYIEENKYSR in the preparation of a food or medicament having immunomodulatory properties.

Further, the use of one or a combination of two of the bioactive peptides FEYIEENKY or FEYIEENKYSR in the preparation of a macrophage-promoting nitric oxide-inducing amount of a food or a medicament.

In a fifth aspect of the present invention, there is provided an anti-inflammatory product comprising one or a combination of two of bioactive peptide FEYIEENKY or bioactive peptide FEYIEENKYSR or one or a combination of several of bioactive peptide FEYIEENKY or a derivative of bioactive peptide FEYIEENKYSR; 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 one or two of bioactive peptide FEYIEENKY or bioactive peptide FEYIEENKYSR, or one or more of bioactive peptide FEYIEENKY or a derivative of bioactive peptide FEYIEENKYSR; the product with immunoregulatory function comprises food with immunoregulatory function or medicine with immunoregulatory function.

The derivative of bioactive peptide FEYIEENKY or bioactive peptide FEYIEENKYSR refers to the same activity or better activity as bioactive peptide FEYIEENKY or bioactive peptide FEYIEENKYSR.

The derivative of bioactive peptide FEYIEENKY or bioactive peptide FEYIEENKYSR refers to a polypeptide derivative obtained by modifying amino acid side chain groups, amino terminals or carboxyl terminals of bioactive peptide FEYIEENKY or bioactive peptide FEYIEENKYSR by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation.

The bioactive peptide with the amino acid structure of FEYIEENKY has the following beneficial effects: the bioactive peptide with the amino acid structure of FEYIEENKY has better anti-inflammatory activity; the biological active peptide FEYIEENKY or FEYIEENKYSR of the invention has the capacity of promoting the increase of the induction quantity of the nitric oxide of macrophages, improves the immunity of the organism to external stimulation, reduces the morbidity of the organism, has obvious promotion effect on the ability of phagocytosing neutral red by the macrophages in vitro under the condition of inflammation occurrence, has good protection effect on inflammatory animals, and has very important significance for developing foods, health care products and medicines with the function of immunoregulation.

Drawings

FIG. 1: a first order mass spectrum of a fragment with a mass to charge ratio of 617.7855 (m/z 617.7855);

FIG. 2: a secondary mass spectrum of a segment with the mass-to-charge ratio of 617.7855 and the breaking conditions of the bioactive peptides az and by;

FIG. 3: a first order mass spectrum of a fragment with a mass to charge ratio of 739.3525 (m/z 739.3525);

FIG. 4: a secondary mass spectrum of a segment with the mass-to-charge ratio of 739.3525 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 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 IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

The invention is described in detail below with reference to the figures and specific embodiments.

Example 1 Artificial Synthesis of active peptides FEYIEENKY and FEYIEENKYSR

Synthesis of bioactive peptide

1. 3g of RINK resin (degree of substitution 0.3mmol/g) was weighed into a 150ml reactor and soaked with 50ml of Dichloromethane (DCM).

After 2.2 hours, the resin was washed with 3 resin volumes of N-Dimethylformamide (DMF) and then drained, and this was repeated four times and the resin was drained until use.

3. The Fmoc protecting group on the resin was removed by adding a quantity of 20% piperidine (piperidine/DMF: 1:4, v: v) to the reactor and shaking on a decolourising shaker for 20 min. After deprotection, the resin was washed four times with 3 resin volumes of DMF and then drained.

4. And (3) detecting a small amount of resin by a ninhydrin (ninhydrin) method (detecting A and B, respectively, and reacting at 100 ℃ for 1min), wherein the resin is colored, which indicates that the deprotection is successful.

5. Weighing an appropriate amount of amino acid Phe and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, adding 20ml of DMF to dissolve the amino acid Phe and the 1-hydroxy-benzotriazole (HOBT), then adding 3ml of N, N Diisopropylcarbodiimide (DIC) to the centrifuge tube, shaking and shaking for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor into a shaker at 30 ℃ 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. Amino acids Phe, Glu, Tyr, Ile, Glu, Asn, Lys and Tyr are grafted in sequence according to steps 9-11.

13. After the last amino acid had been grafted, the protection was removed, washed four times with DMF and the resin was drained with methanol. The biologically active peptide was then cleaved from the resin using 95 cleavage medium (trifluoroacetic acid: 1,2 ethanedithiol: 3, isopropylsilane: water: 95:2:2:1, v: v: v) (10 ml of cleavage medium per gram of resin) and centrifuged four times with glacial ethyl ether (cleavage medium: ethyl ether: 1:9, v: v).

To this end, bioactive peptide FEYIEENKY was synthesized.

The method for synthesizing the bioactive peptide FEYIEENKYSR is based on the above method, and only needs to select the first amino acid corresponding to the specific bioactive peptide at the 5 th step and link the amino acid corresponding to the specific bioactive peptide at the 12 th step.

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 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)

2) Mass spectrometric analysis

The mass spectrometry conditions were as follows:

ion mode: ES +

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 above analysis methods, chromatographic analysis and mass spectrometric analysis of bioactive peptides FEYIEENKY and FEYIEENKYSR were performed using ultra high performance liquid, electrospray, quadrupole, time-of-flight mass spectrometry. The primary mass spectrum of the bioactive peptide FEYIEENKY is shown in figure 1, the secondary mass spectrum of the extracted peak and the az and by breaking conditions are shown in figure 2, the mass-to-charge ratio of the bioactive peptide of the peak is 617.7855, and the retention time is 29.04 min. The mass chromatogram extraction diagram of the bioactive peptide FEYIEENKYSR is shown in FIG. 3, the secondary mass spectrum and az and by fracture conditions of the extraction peak are shown in FIG. 4, the mass-to-charge ratio of the bioactive peptide of the peak is 739.3525, and the retention time is 21.12 min.

3) Results

As can be seen from fig. 2, the fragment sequence of mass-to-charge ratio 617.7855 obtained from az and by cleavage was Phe, Glu, Tyr, Ile, Glu, Asn, Lys, Tyr (FEYIEENKY), and was represented as SEQ ID NO: 1. the fragment corresponds to the residue sequence of 234-242 th positions of heterologous ribonucleoprotein U protein, the GenBank number of the amino acid sequence of the heterologous ribonucleoprotein U protein is EDL13177.1, and the sequence is shown in SEQ ID NO: 3.

as can be seen from fig. 4, the fragment sequence of mass-to-charge ratio 739.3525 obtained from az and by cleavage was Phe, Glu, Tyr, Ile, Glu, Asn, Lys, Tyr, Ser, Arg (FEYIEENKYSR), and is represented as SEQ ID NO: 2. the fragment corresponds to the residue sequence of 234-244 th positions of heterologous ribonucleoprotein U protein, the GenBank number of the amino acid sequence of the heterologous ribonucleoprotein U protein is EDL13177.1, and the sequence is shown in SEQ ID NO: 3.

example 2 immunomodulatory Activity assays of bioactive peptides

First, experiment of macrophage phagocytosis neutral red promoting ability of biological active peptide FEYIEENKY

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 mouse spleen lymphocyte-derived bioactive peptide FEYIEENKY 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 150CO₂Incubator Heraeus; dragon Wellscan MK3 microplate reader Labsystems.

2. The experimental method comprises the following steps:

the number of the added cells was 2X 10⁶100 mul/hole of cell suspension per ml, adding 200 mul/hole of RPMI1640 complete culture solution (10% FBS) containing bioactive peptide FEYIEENKY (0.5mg/ml) after adherent purification as experimental group, adding 200 mul/hole of RPMI1640 complete culture solution (10% FBS) containing no bioactive peptide for culture as blank group; and LPS is added into the experimental group and the blank group when the culture time reaches 24h to reach the final concentration of 10 mug/ml; after further culturing for 48h, the cell culture solution was aspirated. After washing the bottom of the well with PBS, 80. mu.l/well of neutral red dye solution at 37 ℃ was added, and after 10 minutes the dye solution was aspirated and washed twice with PBS, 150. mu.l of cell lysate (glacial acetic acid: absolute ethanol ═ 1:1, v/v) was added to each well. After overnight dissolution at 4 ℃ the absorbance value (OD540) was determined at a wavelength of 540 nm.

3. Experimental results and analysis:

TABLE 1 determination of the ability of the bioactive peptide FEYIEENKY to promote phagocytosis of neutral Red by macrophages

Experiment grouping	Absorbance value (OD540)
		Blank group	0.1239±0.0402
Experimental group	0.1738±0.0299**

Note: significant difference compared to negative control (P <0.05)

The difference in the negative control group was very significant (P <0.01)

The experimental results are shown in table 1, compared with the blank cell group, the macrophage phagocytosis ability of the inflammatory group added with 0.5mg/ml bioactive peptide FEYIEENKY is obviously increased, and compared with the blank cell group, the macrophage phagocytosis ability of the inflammatory group added with the bioactive peptide is very significant difference (P is less than 0.01). The biological active peptide FEYIEENKY is proved to have obvious promotion effect on the ability of phagocytizing neutral red by macrophages in vitro under the condition of inflammation.

Second, determination of macrophage-promoting nitric oxide-inducing amount of bioactive peptide FEYIEENKY (Griess method)

1. Experimental reagents and instruments:

reagent: experimental animal balb/c mouse (male 6-8 weeks old) spleen lymphocyte source bioactive peptide FEYIEENKY; LPS, purchased from Sigma; neutral red staining solution, produced by Biyuntian biotechnological research institute.

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 2 determination of macrophage-promoting nitric oxide-inducing amount of bioactive peptide FEYIEENKY

Experiment grouping	Normal group	Inflammation group
			Cell blank	0.0382±0.0694	0.3391±0.0437
Bioactive peptide (0.5mg/ml)	0.0892±0.0149**	0.728±0.0201**
			Bioactive peptide (0.2mg/ml)	0.0421±0.0024*	0.3401±0.0023*

Note: significant difference compared to negative control (P < 0.05);

the difference in the negative control group was very significant (P <0.01)

The results are shown in table 2, and it is seen from table 2 that when bioactive peptide FEYIEENKY was added to the test group at a concentration of 0.5mg/mL, the effect of promoting the nitric oxide-induced amount of macrophages, both when growing under normal conditions and when growing under conditions of LPS-induced inflammation, was very significant (P <0.01) compared to the cell blank group. When the addition concentration of the bioactive peptide FEYIEENKY is 0.2mg/mL, the biological active peptide also has a promoting effect on the nitric oxide induction quantity of macrophages under the growth condition and the growth condition under the inflammation condition caused by LPS, and has a significant difference (P <0.05) compared with a cell blank group. The biological active peptide FEYIEENKY is shown to have the ability to promote the increase of the nitric oxide induction amount of macrophage under certain concentration condition.

Third, experiment of macrophage phagocytosis neutral red promoting ability of bioactive peptide FEYIEENKYSR

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 mouse spleen lymphocyte-derived bioactive peptide FEYIEENKYSR obtained in example 1; LPS, purchased from Sigma; neutral red staining solution, produced by Biyuntian biotechnological research institute.

2. The experimental method comprises the following steps:

the number of the added cells was 2X 10⁶100 mul/hole of cell suspension per ml, adding 200 mul/hole of RPMI1640 complete culture solution (10% FBS) containing bioactive peptide FEYIEENKYSR (0.5mg/ml) after adherent purification as experimental group, adding 200 mul/hole of RPMI1640 complete culture solution (10% FBS) containing no bioactive peptide for culture as blank group; and LPS is added into the experimental group and the blank group when the culture time reaches 24h to reach the final concentration of 10 mug/ml; after further culturing for 48h, the cell culture solution was aspirated. After washing the bottom of the well with PBS, 80. mu.l/well of neutral red dye solution at 37 ℃ was added, and after 10 minutes the dye solution was aspirated and washed twice with PBS, 150. mu.l of cell lysate (glacial acetic acid: absolute ethanol ═ 1:1, v/v) was added to each well. After overnight dissolution at 4 ℃ the absorbance value (OD540) was determined at a wavelength of 540 nm.

3. Experimental results and analysis:

TABLE 3 determination of the ability of the bioactive peptide FEYIEENKYSR to promote phagocytosis of neutral Red by macrophages

Experiment grouping	Absorbance value (OD540)
		Blank group	0.1375±0.0389
Experimental group	0.2058±0.0185**

Note: significant difference compared to negative control (P <0.05)

The difference in the negative control group was very significant (P <0.01)

The experimental results are shown in table 3, compared with the blank cell group, the macrophage phagocytosis ability of the inflammatory group added with 0.5mg/ml bioactive peptide FEYIEENKYSR is obviously increased, and compared with the blank cell group, the macrophage phagocytosis ability of the inflammatory group added with the bioactive peptide is very significant difference (P is less than 0.01). The biological active peptide FEYIEENKYSR is proved to have obvious promotion effect on the ability of phagocytizing neutral red by macrophages in vitro under the condition of inflammation.

Fourthly, measurement of macrophage-promoting nitric oxide induction quantity of biological active peptide FEYIEENKYSR (Griess method)

1. Experimental reagents and instruments:

reagent: experimental animal balb/c mouse (male 6-8 weeks old) spleen lymphocyte source bioactive peptide FEYIEENKYSR; 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 150CO₂Incubator Heraeus Inc.; dragon Wellscan MK3 microplate reader Labsystems.

2. The test method comprises the following steps:

4. Experimental results and analysis:

TABLE 4 determination of macrophage-promoting nitric oxide-inducing amount of bioactive peptide FEYIEENKYSR

Experiment grouping	Normal group	Inflammation group
			Cell blank	0.0375±0.0637	0.3429±0.0421
Bioactive peptide (0.5mg/ml)	0.0958±0.0127**	0.875±0.0284**
			Bioactive peptide (0.2mg/ml)	0.0521±0.0021*	0.3410±0.0033*

Note: significant difference compared to negative control (P < 0.05);

the difference in the negative control group was very significant (P <0.01)

The results are shown in table 4, and it is seen from table 4 that when bioactive peptide FEYIEENKYSR was added to the test groups at concentrations of 0.5mg/mL, the concentrations of both peptides promoted the nitric oxide-induced amount of macrophages that were normally grown and that were grown under the conditions of inflammation induced by LPS, and the difference was very significant (P <0.01) compared to the cell blank group. When the addition concentration of the bioactive peptide FEYIEENKYSR is 0.2mg/mL, the biological active peptide also has a promoting effect on the nitric oxide induction quantity of macrophages under the growth condition and the growth condition under the inflammation condition caused by LPS, and has a significant difference (P <0.05) compared with a cell blank group. The biological active peptide FEYIEENKYSR is shown to have the ability to promote the increase of the nitric oxide induction amount of macrophage under certain concentration condition.

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

<120> bioactive peptide with amino acid structure FEYIEENKY, and preparation method and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Phe Glu Tyr Ile Glu Glu Asn Lys Tyr

1 5

<210> 2

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Phe Glu Tyr Ile Glu Glu Asn Lys Tyr Ser Arg

1 5 10

<210> 3

<211> 800

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ser Ser Ser Pro Val Asn Val Lys Lys Leu Lys Val Ser Glu Leu

1 5 10 15

Lys Glu Glu Leu Lys Lys Arg Arg Leu Ser Asp Lys Gly Leu Lys Ala

20 25 30

Asp Leu Met Asp Arg Leu Gln Ala Ala Leu Asp Asn Glu Ala Gly Gly

35 40 45

Arg Pro Ala Met Glu Pro Gly Asn Gly Ser Leu Asp Leu Gly Gly Asp

50 55 60

Ala Ala Gly Arg Ser Gly Ala Gly Leu Glu Gln Glu Ala Ala Ala Gly

65 70 75 80

Ala Glu Asp Asp Glu Glu Glu Glu Gly Ile Ala Ala Leu Asp Gly Asp

85 90 95

Gln Met Glu Leu Gly Glu Glu Asn Gly Ala Ala Gly Ala Ala Asp Ala

100 105 110

Gly Ala Met Glu Glu Glu Glu Ala Ala Ser Glu Asp Glu Asn Gly Asp

115 120 125

Asp Gln Gly Phe Gln Glu Gly Glu Asp Glu Leu Gly Asp Glu Glu Glu

130 135 140

Gly Ala Gly Asp Glu Asn Gly His Gly Glu Gln Gln Ser Gln Pro Pro

145 150 155 160

Ala Ala Ala Ala Gln Gln Gln Pro Ser Gln Gln Arg Gly Ala Gly Lys

165 170 175

Glu Ala Ala Gly Lys Ser Ser Gly Pro Thr Ser Leu Phe Ala Val Thr

180 185 190

Val Ala Pro Pro Gly Ala Arg Gln Gly Gln Gln Gln Ala Gly Gly Asp

195 200 205

Gly Lys Thr Glu Gln Lys Gly Gly Asp Lys Lys Arg Gly Val Lys Arg

210 215 220

Pro Arg Glu Asp His Gly Arg Gly Tyr Phe Glu Tyr Ile Glu Glu Asn

225 230 235 240

Lys Tyr Ser Arg Ala Lys Ser Pro Gln Pro Pro Val Glu Glu Glu Asp

245 250 255

Glu His Phe Asp Asp Thr Val Val Cys Leu Asp Thr Tyr Asn Cys Asp

260 265 270

Leu His Phe Lys Ile Ser Arg Asp Arg Leu Ser Ala Ser Ser Leu Thr

275 280 285

Met Glu Ser Phe Ala Phe Leu Trp Ala Gly Gly Arg Ala Ser Tyr Gly

290 295 300

Val Ser Lys Gly Lys Val Cys Phe Glu Met Lys Val Thr Glu Lys Ile

305 310 315 320

Pro Val Arg His Leu Tyr Thr Lys Asp Ile Asp Ile His Glu Val Arg

325 330 335

Ile Gly Trp Ser Leu Thr Thr Ser Gly Met Leu Leu Gly Glu Glu Glu

340 345 350

Phe Ser Tyr Gly Tyr Ser Leu Lys Gly Ile Lys Thr Cys Asn Cys Glu

355 360 365

Thr Glu Asp Tyr Gly Glu Lys Phe Asp Glu Asn Asp Val Ile Thr Cys

370 375 380

Phe Ala Asn Phe Glu Thr Asp Glu Val Glu Leu Ser Tyr Ala Lys Asn

385 390 395 400

Gly Gln Asp Leu Gly Val Ala Phe Lys Ile Ser Lys Glu Val Leu Ala

405 410 415

Asp Arg Pro Leu Phe Pro His Val Leu Cys His Asn Cys Ala Val Glu

420 425 430

Phe Asn Phe Gly Gln Lys Glu Lys Pro Tyr Phe Pro Ile Pro Glu Asp

435 440 445

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

450 455 460

Lys Gly Pro Glu Glu Lys Lys Asp Cys Glu Val Val Met Met Ile Gly

465 470 475 480

Leu Pro Gly Ala Gly Lys Thr Thr Trp Val Thr Lys His Ala Ala Glu

485 490 495

Asn Pro Gly Lys Tyr Asn Ile Leu Gly Thr Asn Thr Ile Met Asp Lys

500 505 510

Met Met Val Ala Gly Phe Lys Lys Gln Met Ala Asp Thr Gly Lys Leu

515 520 525

Asn Thr Leu Leu Gln Arg Ala Pro Gln Cys Leu Gly Lys Phe Ile Glu

530 535 540

Ile Ala Ala Arg Lys Lys Arg Asn Phe Ile Leu Asp Gln Thr Asn Val

545 550 555 560

Ser Ala Ala Ala Gln Arg Arg Lys Met Cys Leu Phe Ala Gly Phe Gln

565 570 575

Arg Lys Ala Val Val Val Cys Pro Lys Asp Glu Asp Tyr Lys Gln Arg

580 585 590

Thr Gln Lys Lys Ala Glu Val Glu Gly Lys Asp Leu Pro Glu His Ala

595 600 605

Val Leu Lys Met Lys Gly Asn Phe Thr Leu Pro Glu Val Ala Glu Cys

610 615 620

Phe Asp Glu Ile Thr Tyr Val Glu Leu Gln Lys Glu Glu Ala Gln Lys

625 630 635 640

Leu Leu Glu Gln Tyr Lys Glu Glu Ser Lys Lys Ala Leu Pro Pro Glu

645 650 655

Lys Lys Gln Asn Thr Gly Ser Lys Lys Ser Asn Lys Asn Lys Ser Gly

660 665 670

Lys Asn Gln Phe Asn Arg Gly Gly Gly His Arg Gly Arg Gly Gly Phe

675 680 685

Asn Met Arg Gly Gly Asn Phe Arg Gly Gly Ala Pro Gly Asn Arg Gly

690 695 700

Gly Tyr Asn Arg Arg Gly Asn Met Pro Gln Arg Gly Gly Gly Gly Gly

705 710 715 720

Ser Gly Gly Ile Gly Tyr Pro Tyr Pro Arg Gly Pro Val Phe Pro Gly

725 730 735

Arg Gly Gly Tyr Ser Asn Arg Gly Asn Tyr Asn Arg Gly Gly Met Pro

740 745 750

Asn Arg Gly Asn Tyr Asn Gln Asn Phe Arg Gly Arg Gly Asn Asn Arg

755 760 765

Gly Tyr Lys Asn Gln Ser Gln Gly Tyr Asn Gln Trp Gln Gln Gly Gln

770 775 780

Phe Trp Gly Gln Lys Pro Trp Ser Gln His Tyr His Gln Gly Tyr Tyr

785 790 795 800

Claims

1. A biologically active peptide having an amino acid structure FEYIEENKY, characterized in that, being biologically active peptide FEYIEENKY or biologically active peptide FEYIEENKYSR, the amino acid sequence of biologically active peptide FEYIEENKY is Phe-Glu-Tyr-Ile-Glu-Asn-Lys-Tyr, and the amino acid sequence of biologically active peptide FEYIEENKYSR is: Phe-Glu-Tyr-Ile-Glu-Glu-Asn-Lys-Tyr-Ser-Arg.

2. A polynucleotide encoding the biologically active peptide of claim 1 having the amino acid structure FEYIEENKY.

3. The method of claim 1, wherein said biologically active peptide FEYIEENKY or said biologically active peptide FEYIEENKYSR is produced directly by chemical synthesis.

4. Use of a biologically active peptide having amino acid structure FEYIEENKY of claim 1, wherein one or a combination of both of biologically active peptide FEYIEENKY or biologically active peptide FEYIEENKYSR is used in the manufacture of a medicament for promoting the ability of macrophages to phagocytose neutral red.

5. Use of a biologically active peptide having amino acid structure FEYIEENKY of claim 1, wherein one or a combination of both of biologically active peptide FEYIEENKY or biologically active peptide FEYIEENKYSR is used in the preparation of a macrophage nitric oxide-inducing amount of a medicament.