CN112724237B

CN112724237B - Bioactive peptide GGSDGYGSGRGF, and preparation method and application thereof

Info

Publication number: CN112724237B
Application number: CN202110071799.3A
Authority: CN
Inventors: 张少辉; 占文静; 张伯宇
Original assignee: Zhejiang Huitai Life Health Technology Co ltd
Current assignee: Zhejiang Huitai Life Health Technology Co ltd
Priority date: 2021-01-19
Filing date: 2021-01-19
Publication date: 2022-05-31
Anticipated expiration: 2041-01-19
Also published as: CN112724237A

Abstract

The invention relates to the field of protein, and in particular relates to a bioactive peptide GGSDGYGSGRGF, a preparation method and application thereof, wherein the amino acid sequence of the bioactive peptide GGSDGYGSGRGF is Gly-Gly-Ser-Asp-Gly-Tyr-Gly-Ser-Gly-Arg-Gly-Phe. In-vitro immune function verification experiments prove that the bioactive peptide GGSDGYGSGRGF has a good immune regulation function according to the experimental results. The bioactive peptide GGSDGYGSGRGF can improve the phagocytic capacity of macrophage to neutral red, improve the immunity of organism to external stimulation, reduce the disease incidence of organism, promote the induction quantity of macrophage nitric oxide, improve the quality of life, and has very important significance for developing food, health care products and medicines with immunoregulation function.

Description

Bioactive peptide GGSDGYGSGRGF, and preparation method and application thereof

Technical Field

The invention relates to the field of protein, in particular to a bioactive peptide GGSDGYGSGRGF, 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, over 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.

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.

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 heterologous nucleic acids A2/B1 protein is shown in SEQ ID NO: 2, respectively. At present, the related functions of the polypeptide fragments of hetereogenous nucleosclerotins A2/B1 protein are not researched in the prior art.

Disclosure of Invention

The invention aims to provide a bioactive peptide GGSDGYGSGRGF, and 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 present invention, a bioactive peptide GGSDGYGSGRGF is provided, wherein the amino acid sequence thereof is Gly-Ser-Asp-Gly-Tyr-Gly-Ser-Gly-Arg-Gly-Phe, as shown in SEQ ID NO: 1 is shown.

Preferably, the bioactive peptide is mouse spleen derived lymphocyte peptide. Specifically, the amino acid residues are derived from heterologous ribonucleoproteins A2/B1 protein and are the 229-240 amino acid residues of heterologous ribonucleoproteins A2/B1 protein. The amino acid sequence of the heterologous nucleic acids A2/B1 protein is shown as SEQ ID NO: 2, respectively.

The amino acid sequence and the corresponding nucleotide sequence of the heterologous nucleic acid proteins A2/B1 are the prior art, and the nucleotide fragment of the 229 to 240 amino acid residues of the heterologous nucleic acid proteins A2/B1 protein can encode the mature bioactive peptide GGSDGYGSGRGF.

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

The present invention also provides polynucleotides encoding the biologically active peptide GGSDGYGSGRGF.

In the second aspect of the present invention, there is provided a method for preparing the bioactive peptide GGSDGYGSGRGF, 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 GGSDGYGSGRGF by genetic engineering is a technical solution that can be realized by those skilled in the art, and for example, the synthesis of the sequence 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 GGSDGYGSGRGF, the bioactive peptide GGSDGYGSGRGF is obtained from mouse spleen-derived lymphocytes by a conventional enzymolysis and purification method in biological technology.

In a third aspect of the present invention, there is provided a use of the bioactive peptide GGSDGYGSGRGF in the preparation of a medicament or a cosmetic having an anti-inflammatory function.

In particular, the bioactive peptide GGSDGYGSGRGF of the present invention may be used in the preparation of medicaments with anti-inflammatory and/or anti-oxidant properties.

In a fourth aspect of the present invention, there is provided a use of the bioactive peptide GGSDGYGSGRGF in the preparation of food or medicine with immunoregulatory function.

Further, the use of the biologically active peptide GGSDGYGSGRGF in the manufacture of a food or medicament for promoting an increase in nitric oxide-inducing amount of macrophages.

Further, the use of the bioactive peptide GGSDGYGSGRGF in the preparation of a food or a medicament for promoting the ability of macrophages to phagocytose neutral red.

In a fifth aspect of the present invention, there is provided a product having an immunoregulatory function, comprising said biologically active peptide GGSDGYGSGRGF or a derivative of said biologically active peptide GGSDGYGSGRGF; the product with immunoregulatory function comprises food with immunoregulatory function or medicine with immunoregulatory function.

In a sixth aspect of the invention, there is provided an anti-inflammatory product comprising said biologically active peptide GGSDGYGSGRGF or a derivative of said biologically active peptide GGSDGYGSGRGF; the anti-inflammatory product comprises anti-inflammatory food, anti-inflammatory health product, anti-inflammatory drug or anti-inflammatory cosmetic.

Derivatives of the bioactive peptides YFGSGFAAPFFIVRHQLLKK are meant to have the same activity or better activity than the bioactive peptides YFGSGFAAPFFIVRHQLLKK.

The derivative of the bioactive peptide GGSDGYGSGRGF refers to a polypeptide derivative obtained by modifying the amino acid side chain group, amino terminal or carboxyl terminal of the bioactive peptide GGSDGYGSGRGF by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation.

The bioactive peptide GGSDGYGSGRGF has the beneficial effects that: the bioactive peptide GGSDGYGSGRGF has good anti-inflammatory activity; the bioactive peptide GGSDGYGSGRGF of the invention can increase the ability of phagocytosis of neutral red by macrophages, improve the immunity of organisms to external stimulation, reduce the morbidity of the organisms, promote the induction of nitric oxide in the macrophages, improve the quality of life and have very important significance for 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 558.7396 (m/z 558.7396);

FIG. 2: a secondary mass spectrum of a fragment with the mass-to-charge ratio of 558.7396 and the cleavage conditions of the polypeptides 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, 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 Synthesis of active peptide GGSDGYGSGRGF

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 a proper amount of amino acid Gly and a proper amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, adding 20ml of DMF to dissolve the amino acid Gly and the 1-hydroxy-benzotriazole (HOBT), then adding 3ml of N, N Diisopropylcarbodiimide (DIC) to shake and shake for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor into a 30 ℃ shaking table to react.

After 6.2 hours, the column was capped with a suitable amount of acetic anhydride (acetic anhydride: DIEA: DCM ═ 1:1:2, v: v: v) for half an hour, then washed four times with 3 resin volumes of DMF and drained until needed.

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

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

9. Weighing a second proper amount of amino acid and a proper amount of HOBT in a 50ml centrifuge tube, adding 25ml of DMF to dissolve the amino acid and the HOBT, adding 2.5ml of DIC to shake and shake for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor in a shaking table at 30 ℃ to react.

After 10.1 hours, a small amount of resin is taken for detection, and the detection is carried out by an indanthrone method (two drops are respectively detected A and B, and the reaction is carried out for 1min at 100 ℃), if the resin is colorless, the reaction is complete; if the resin is colored, the condensation is not complete and the reaction is continued.

11. After the reaction was completed, the resin was washed four times with DMF and then drained, and a certain amount of 20% piperidine (piperidine/DMF ═ 1:4, v: v) was added to the reactor, and the mixture was shaken on a decolorizing shaker for 20min to remove the Fmoc-protecting group from the resin. After the protection is removed, washing with DMF for four times, and then draining to detect whether the protection is removed.

12. And sequentially grafting amino acids Gly, Ser, Asp, Gly, Tyr, Gly, Ser, Gly, Arg, Gly and Phe according to the 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 GGSDGYGSGRGF was synthesized.

Confirmation of biologically active peptides

1) UPLC analysis

UPLC conditions were as follows:

the instrument comprises: 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 method, the bioactive peptide GGSDGYGSGRGF was subjected to chromatographic analysis and mass spectrometric analysis using ultra high performance liquid, electrospray, quadrupole, time-of-flight mass spectrometry. The primary mass spectrum of the bioactive peptide GGSDGYGSGRGF 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 558.7396, and the retention time is 12.94 min.

3) Results

As can be seen from fig. 2, the fragments with mass-to-charge ratios 558.7396 obtained from az and by fragmentation were analyzed and calculated by Mascot software, and have the sequence Gly, Ser, Asp, Gly, Tyr, Gly, Ser, Gly, Arg, Gly, Phe (GGSDGYGSGRGF), and are represented as SEQ ID NO: 1. the fragment corresponds to the 229-240 residue sequence of heterologous nucleic acid nucleoproteins A2/B1 protein, the GenBank accession number of the amino acid sequence of heterologous nucleic acid nucleoproteins A2/B1 protein is AAC26867.1, and the sequence is shown in SEQ ID NO: 2.

example 2 immunological Activity assay of bioactive peptides

Measurement of macrophage-promoting nitric oxide-inducing amount of bioactive peptide GGSDGYGSGRGF (Griess method)

1. Experimental reagents and instruments:

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

The instrument equipment comprises: LRH-250F Biochemical incubator Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge Shanghai Luxiang apparatus centrifuge Instrument Co., Ltd; hera cell 150CO2 incubator Heraeus; dragon Wellscan MK3 microplate reader Labsystems.

2. The test method comprises the following steps:

the number of the added cells was 2X 10⁶100 μ l/well of a cell suspension per ml, 200 μ l/well of a complete peptide-containing RPMI1640 culture medium (10% FBS) was added after adherent purification, LPS was added to a final concentration of 10 μ g/ml at 24 hours in an inflammation group, 50 μ l/well of a culture supernatant was collected after continuous culture for 48 hours, 50 μ l/well of Griess reagent 1 and Griess reagent 2 were sequentially added to the culture supernatant, and after reaction at room temperature for 10 minutes, an absorbance value (OD540) was measured at a wavelength of 540 nm.

3. Experimental results and analysis:

TABLE 1 determination of macrophage-promoting nitric oxide-inducing amount of bioactive peptide GGSDGYGSGRGF

Experiment grouping	Normal group	Inflammation group
			Cell blank	0.0593±0.0517	0.3238±0.0382
GGSDGYGSGRGF 1mg/ml	0.1286±0.0255**	0.4890±0.0243**
			GGSDGYGSGRGF 0.5mg/ml	0.1229±0.0133**	0.3359±0.0306**
GGSDGYGSGRGF 0.1mg/ml	0.0596±0.0037	0.3253±0.0189*

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

there was a very significant difference (P <0.01) compared to the negative control group.

The results are shown in Table 1, and it is understood from Table 1 that the addition of the bioactive peptide GGSDGYGSGRGF at concentrations of 1mg/mL and 0.5mg/mL to the test group promotes the nitric oxide-inducing amount of macrophages that grow under normal conditions and under conditions of inflammation caused by LPS. Compared with the cell blank group, the cell blank group has a very significant difference (P < 0.01). When the addition concentration of the bioactive peptide GGSDGYGSGRGF is 0.1mg/mL, the increase of the macrophage nitric oxide induction amount can be promoted compared with that in the case of LPS inflammation, and the obvious difference is achieved (P is less than 0.05). But there were no significant differences compared to the cell blank grown under normal conditions. The biological active peptide GGSDGYGSGRGF is shown to have the ability to promote the increase of the nitric oxide induction amount of macrophage under certain concentration condition.

Second, the experiment of macrophage phagocytosis neutral red promoting ability of bioactive peptide GGSDGYGSGRGF

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; milk-derived bioactive peptide GGSDGYGSGRGF 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/well of cell suspension per ml, adding 200 mul/well of RPMI1640 complete culture solution (10% FBS) containing active peptide GGSDGYGSGRGF (1mg/ml) after adherent purification as experimental group, adding 200 mul/well of RPMI1640 complete culture solution (10% FBS) containing no active 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 2 determination of the ability of the bioactive peptide GGSDGYGSGRGF to promote phagocytosis of neutral Red by macrophages

Experiment grouping	Absorbance values for inflammatory group (OD540)
		Blank group	0.1079±0.0314
Experimental group	0.1351±0.0114**

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

Significant difference compared with negative control group (P <0.01)

The experimental results are shown in table 2, compared with the blank cell group, the macrophage phagocytosis ability of the inflammatory group added with 1mg/ml bioactive peptide GGSDGYGSGRGF is obviously increased, and compared with the blank cell group, the macrophage phagocytosis ability of the inflammatory group added with 1mg/ml bioactive peptide GGSDGYGSGRGF has significant difference (P is less than 0.01). The biological active peptide GGSDGYGSGRGF is proved to have obvious promotion effect on the ability of phagocytizing neutral red by macrophages in vitro under the condition of inflammation.

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 GGSDGYGSGRGF, and preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gly Gly Ser Asp Gly Tyr Gly Ser Gly Arg Gly Phe

1 5 10

<210> 2

<211> 353

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Glu Lys Thr Leu Glu Thr Val Pro Leu Glu Arg Lys Lys Arg Glu

1 5 10 15

Lys Glu Gln Phe Arg Lys Leu Phe Ile Gly Gly Leu Ser Phe Glu Thr

20 25 30

Thr Glu Glu Ser Leu Arg Asn Tyr Tyr Glu Gln Trp Gly Lys Leu Thr

35 40 45

Asp Cys Val Val Met Arg Asp Pro Ala Ser Lys Arg Ser Arg Gly Phe

50 55 60

Gly Phe Val Thr Phe Ser Ser Met Ala Glu Val Asp Ala Ala Met Ala

65 70 75 80

Ala Arg Pro His Ser Ile Asp Gly Arg Val Val Glu Pro Lys Arg Ala

85 90 95

Val Ala Arg Glu Glu Ser Gly Lys Pro Gly Ala His Val Thr Val Lys

100 105 110

Lys Leu Phe Val Gly Gly Ile Lys Glu Asp Thr Glu Glu His His Leu

115 120 125

Arg Asp Tyr Phe Glu Glu Tyr Gly Lys Ile Asp Thr Ile Glu Ile Ile

130 135 140

Thr Asp Arg Gln Ser Gly Lys Lys Arg Gly Phe Gly Phe Val Thr Phe

145 150 155 160

Asp Asp His Asp Pro Val Asp Lys Ile Val Leu Gln Lys Tyr His Thr

165 170 175

Ile Asn Gly His Asn Ala Glu Val Arg Lys Ala Leu Ser Arg Gln Glu

180 185 190

Met Gln Glu Val Gln Ser Ser Arg Ser Gly Arg Gly Gly Asn Phe Gly

195 200 205

Phe Gly Asp Ser Arg Gly Gly Gly Gly Asn Phe Gly Pro Gly Pro Gly

210 215 220

Ser Asn Phe Arg Gly Gly Ser Asp Gly Tyr Gly Ser Gly Arg Gly Phe

225 230 235 240

Gly Asp Gly Tyr Asn Gly Tyr Gly Gly Gly Pro Gly Gly Gly Asn Phe

245 250 255

Gly Gly Ser Pro Gly Tyr Gly Gly Gly Arg Gly Gly Tyr Gly Gly Gly

260 265 270

Gly Pro Gly Tyr Gly Asn Gln Gly Gly Gly Tyr Gly Gly Gly Tyr Asp

275 280 285

Asn Tyr Gly Gly Gly Asn Tyr Gly Ser Gly Ser Tyr Asn Asp Phe Gly

290 295 300

Asn Tyr Asn Gln Gln Pro Ser Asn Tyr Gly Pro Met Lys Ser Gly Asn

305 310 315 320

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

325 330 335

Gly Pro Gly Gly Ser Gly Gly Ser Gly Gly Tyr Gly Gly Arg Ser Arg

340 345 350

Tyr

Claims

1. A bioactive peptide GGSDGYGSGRGF is characterized in that the amino acid sequence is Gly-Gly-Ser-Asp-Gly-Tyr-Gly-Ser-Gly-Arg-Gly-Phe.

2. A polynucleotide encoding the biologically active peptide GGSDGYGSGRGF of claim 1.

3. The process for preparing bioactive peptide GGSDGYGSGRGF of claim 1, directly prepared by chemical synthesis.

4. Use of the bioactive peptide GGSDGYGSGRGF of claim 1 in the manufacture of a medicament for promoting increased nitric oxide-induced production of macrophages.

5. Use of the bioactive peptide GGSDGYGSGRGF of claim 1 in the manufacture of a medicament for promoting phagocytosis of neutral red blood by macrophages.