CN112574291B

CN112574291B - Bioactive peptide SHRKFSAPRHGLGFLPR and preparation method and application thereof

Info

Publication number: CN112574291B
Application number: CN202011468805.0A
Authority: CN
Inventors: 张少辉; 彭小杰; 谢雨佳; 栾媛媛; 王娟; 郭婷婷; 占文静; 李政
Original assignee: Zhejiang Huitai Life Health Technology Co ltd; Shanghai Jiaotong University
Current assignee: Zhejiang Huitai Life Health Technology Co ltd; Shanghai Jiaotong University
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2022-10-21
Anticipated expiration: 2040-12-14
Also published as: CN112574291A

Abstract

The invention relates to the field of protein, in particular to a bioactive peptide SHRKFSAPRHGSLFLPR and a preparation method and application thereof. In-vitro immune regulation function experiments show that the bioactive peptide SHRKFSAPRHGSLFLPR has a better immune regulation function. The bioactive peptide SHRKFSAPRHGLGFLPR has obvious promotion effect on in-vitro macrophage proliferation, can improve the immunity of an organism, promote the macrophage to be activated and release cell factors, reduce the morbidity of the organism and has very important significance on the development of foods, health care products and medicines with immune regulation functions.

Description

Bioactive peptide SHRKFSAPRHGLGFLPR and preparation method and application thereof

Technical Field

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

Background

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

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

Immunomodulatory peptides are a class of bioactive peptides that were first obtained from milk following opioid peptide discovery and demonstrated their physiological activity. Jolles et al found 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.

Research shows that the immunoregulation peptide can not only enhance the immunity of organisms, stimulate the proliferation of lymphocytes of the organisms, enhance the phagocytic function of macrophages, promote the release of cell factors, promote the increase of the nitric oxide induction quantity of the macrophages, improve the capability of the organisms for resisting the infection of external pathogens, reduce the morbidity of the organisms, but also can not cause the immunological rejection reaction of the organisms.

Immunomodulatory peptides generally refer to small, relatively small molecular weight peptides with immunomodulatory activity. The presently disclosed immunomodulatory peptides are generally small peptides having specific immunomodulatory activity, either enzymatically isolated from proteins or chemically synthesized. 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 60S ribosol protein L3 protein is shown as SEQ ID NO:2, respectively. At present, no research on the related functions of the polypeptide fragment of the 60S ribosomal protein L3 protein exists in the prior art.

Disclosure of Invention

The invention aims to provide a bioactive peptide SHRKFSAPRHGLGFLPR as well as a preparation method and application thereof.

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

in the first aspect of the invention, the amino acid sequence of the bioactive peptide SHRKFSAPRHGSLFLPR is Ser-His-Arg-Lys-Phe-Ser-Ala-Pro-Arg-His-Gly-Ser-Leu-Gly-Phe-Leu-Pro-Arg, and is shown in SEQ ID NO:1 is shown.

Preferably, the bioactive peptide is mouse spleen derived lymphocyte peptide. Particularly derived from the 60S ribosol protein L3 protein and is the amino acid residue at the 2 nd to 19 th positions of the 60S ribosol protein L3 protein. The amino acid sequence of the 60S ribosol protein L3 protein is shown as SEQ ID NO:2, respectively.

The amino acid sequence and the corresponding nucleotide sequence of the 60S ribosol protein L3 protein are the prior art, and the nucleotide fragment for coding the 2 nd to 19 th amino acid residues of the 60S ribosol protein L3 protein can code mature bioactive peptide SHRKFSAPRHGSGFLPR.

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

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

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

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

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

In a third aspect of the invention, the application of the bioactive peptide SHRKFSAPRHGSLFLPR in the preparation of medicines or cosmetics with anti-inflammatory function is provided.

In the fourth aspect of the invention, the application of the bioactive peptide SHRKFSAPRHGSLFLPR in preparing food or medicines with immunoregulation function is provided.

Further, the application of the bioactive peptide SHRKFSAPRHGSLFLPR in preparing food or medicine for promoting macrophage proliferation in vitro.

Further, the application of the bioactive peptide SHRKFSAPRHGLGFLPR in the preparation of foods or medicines for promoting macrophage activation and releasing cytokines is provided. In a fifth aspect of the invention, there is provided an anti-inflammatory product comprising the biologically active peptide shrksfplg flpr or a derivative of the biologically active peptide shrksaprhgslgflpr; the anti-inflammatory product comprises anti-inflammatory food, anti-inflammatory health product, anti-inflammatory drug or anti-inflammatory cosmetic; the derivative of the bioactive peptide SHRKFSAPRHGSLFLPR is the same or better activity as the bioactive peptide SHRKFSAPRHGSLFLPR.

In a sixth aspect of the invention, there is provided a product with immunomodulatory function, comprising the bioactive peptide shrksaprhgslgflpr or a derivative of the bioactive peptide shrksaprhgslgflpr; the product with immunoregulation function comprises food with immunoregulation function or medicine with immunoregulation function; the derivatives of the biologically active peptide SHRKFSAPRHGLGFLPR are meant to have the same or better activity as the biologically active peptide SHRKFSAPRHGLGFLPR.

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

The biological active peptide SHRKFSAPRHGLGFLPR has the beneficial effects that: the bioactive peptide SHRKFSAPRHGSLFLPR has good anti-inflammatory activity; the bioactive peptide SHRKFSAPRHGSLFLPR has obvious promotion effect on in-vitro macrophage proliferation, can improve the immunity of the organism, promote the macrophage to activate and release cell factors, reduce the morbidity of the organism and has very important significance for developing foods, health-care products and medicines with the immunoregulation function.

Drawings

FIG. 1: a first order mass spectrum of a fragment with a mass to charge ratio of 410.8319 (m/z = 410.8319);

FIG. 2: a secondary mass spectrogram of a segment with a mass-to-charge ratio of 410.8319 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 number between the two endpoints are optional unless otherwise specified in the invention. 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, the invention may be practiced using any method, device, and material that is similar or equivalent to the methods, devices, and materials described in examples herein, in addition to those described in prior art practice and the description herein.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature and are described in particular in Sambrook et al, molecular CLONING: a LABORATORY MANUAL, second edition, cold Spring Harbor LABORATORY Press,1989 and Third edition, 2001; ausubel et al, current PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987 and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego, 1998; METHOD 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 the biologically active peptide SHRKFSAPRHGLGFLPR

1. Synthesis of bioactive peptides

Artificially synthesizing the bioactive peptide SHRKFSAPRHGLGFLPR.

2. Identification of biologically active peptides

1) UPLC analysis

The UPLC conditions were as follows:

the instrument comprises the following steps: waters ACQUITY UPLC ultra-high performance liquid phase, electrospray, quadrupole and time-of-flight mass spectrometer

Specification of chromatographic column: BEH C18 chromatographic column

Flow rate: 0.4mL/min

Temperature: 50 deg.C

Ultraviolet detection wavelength: 210nm

Sample introduction amount: 2 μ L

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

Time(min) %A %B

0 95.0 5.0

1.50 80.0 20.0

3.50 60.0 40.0

5.00 40.0 60.0

7.00 15.0 85.0

8.00 0.0 100.0

11.00 0.0 100.0

11.50 95.0 5.0

13.00 95.0 5.0

2) Mass spectrometric analysis

The mass spectrometry conditions were as follows:

ion mode: ES + C

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

Capillary voltage (Capillary) (kV): 3.0

Sampling cone (V): 35.0

Ion source temperature (. Degree. C.): 115

Desolvation temperature (. Degree. C.): 350

Desolventizing gas stream (L/hr): 700.0

Collision energy (eV): 4.0

Scan time (sec): 0.25

Inner scan time (sec): 0.02

According to the analysis method, chromatographic analysis and mass spectrometry are carried out on the bioactive peptide SHRKFSAPRHGSLFLPR by using ultra-high performance liquid phase, electrospray, a four-stage rod and time-of-flight mass spectrometry. The primary mass spectrum of the bioactive peptide SHRKFSAPRHGSLFLPR is shown in figure 1, the secondary mass spectrum of the extracted peak and the az and by fracture conditions are shown in figure 2, the mass-to-charge ratio of the bioactive peptide of the peak is 410.8319, and the retention time is 16.65 min.

3) Results

As can be seen from fig. 2, according to the cases of az and by fragmentation, the fragment sequence with the mass-to-charge ratio of 410.8319 is analyzed and calculated by Mascot software, and is Ser, his, arg, lys, phe, ser, ala, pro, arg, his, gly, ser, leu, gly, phe, leu, pro, arg (shrksfsaprhglgflpr), and is marked as SEQ ID NO:1. the fragment corresponds to residue sequences of 2 nd to 19 th positions of a 60S ribosol protein L3 protein, the GenBank number of the amino acid sequence of the 60S ribosol protein L3 protein is BAE25922.1, and the sequence is shown in SEQ ID NO:2.

example 2 immunological Activity assay of bioactive peptides

1. In vitro macrophage proliferation capacity experiment for measuring bioactive peptide SHRKFSAPRHGSGFLPR by MTT method

1. Experimental reagent and instrument

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 SHRKFSAPRHGLGFLPR obtained in example 1; 3- (4, 5-Dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide (MTT) Amresco; LPS (lipopolysaccharide) Sigma company; bovine Serum Albumin (BSA) Genebase; triple lysate, aqueous solution containing 10% SDS, 5% isobutanol and 0.012mol/L HCl.

An instrument device: LRH-250F Biochemical incubator Shanghai Hengshi Co., ltd; GL-22M high speed refrigerated centrifuge shanghai luxiang instrument centrifuge instruments ltd; hera cell 150 CO ₂ Incubator Heraeus; dragon Wellscan MK3 microplate reader Labsystems.

2. The test method comprises the following steps:

balb/c mice were injected intraperitoneally with 2ml of 2% (w/w) sterile starch solution for three consecutive days, and sacrificed by cervical dislocation 24 hours after the last injection. Peeling off abdominal skin, repeatedly washing abdominal cavity with 4 deg.C Phosphate Buffer Solution (PBS) by syringe, centrifuging (1000rpm, 4 deg.C) for 10 min after collecting washing liquid by centrifuge tube, discarding supernatant, washing twice with 4 deg.C RPMI1640 complete culture solution (containing 10% FBS), staining with 0.2% trypan blue solution for cell viability detection, and determining that the collected viable macrophages account for more than 95%. After reading the cell counting plate, the cell concentration was adjusted to the appropriate concentration.

Adding the cell suspension blown to complete suspension to a 96 well cell culture plate in an appropriate volume, 37 ℃, 5% CO ₂ After culturing for 4 hours under the environment, sucking liquid in the holes, carefully cleaning the bottom of the holes of the cell culture plate by using 37 ℃ RPMI1640 complete culture solution, and washing away nonadherent cells and cell fragments to obtain purified adherent abdominal cavity macrophages. 0.2ml of RPMI1640 complete medium was added to each well, and the small peptide sample for experiment and LPS were dissolved in the medium in advance and then added to start cell culture.

After obtaining purified adherent abdominal cavity macrophages, each well of the experimental group was added with 200. Mu.l/well of RPMI1640 complete culture medium (10% FBS) dissolved with bioactive peptide SHRKFSAPRHGLGFLPR (1 mg/ml), and continuously cultured for 48h; negative control group adding BSA (500. Mu.g/mL) dissolved in RPMI1640 complete medium (10% FBS) 200. Mu.l/well per well; the blank group was incubated for 48 hours with RPMI1640 complete medium (10% FBS) at 200. Mu.l/well. In addition, the experimental group, the negative control group and the blank group are respectively provided with a normal group and an inflammation group; LPS is added into the inflammation group when the inflammation group is cultured for 24 hours until the final concentration is 100ng/ml; LPS is not added in a normal group; and 5% MTT 20. Mu.l/well was added at 44h in the normal group and the inflammatory group; after the cell culture reached 48h, 100. Mu.l/well of the triple lysis buffer was added to terminate the culture, and after overnight lysis, the absorbance value (OD 570) of each well was measured by a microplate reader at a wavelength of 570nm, and the Growth index (Growth Indices) was calculated as follows:

growth index GI = (small peptide group OD value-blank culture OD value)/(blank group OD value-blank culture OD value)

Wherein the blank culture solution is RPMI1640 complete culture solution containing 10% FBS.

3. Results and analysis of the experiments

TABLE 1 Effect of the biologically active peptide SHRKFSAPRHGLGFLPR on macrophage proliferation in vitro

Experiment grouping	Normal group GI	GI inflammation group
			Negative control group	1	1
SHRKFSAPRHGSLGFLPR（1mg/ml）	1.1538±0.0482**	1.2382±0.0293**

Note: * Indicating significant difference (P < 0.05) compared with negative control; * Indicates that there is a highly significant difference (P < 0.01) compared to the negative control group

The results are shown in Table 1, and it can be seen from Table 1 that macrophages of both the normal group and the inflammatory group proliferated under the condition of adding 1mg/ml of the bioactive peptide SHRKFSAPRHGLGFLPR. Compared with a negative control group, the two groups have significant difference (P is less than 0.01). The result shows that the bioactive peptide SHRKFSAPRHGLGFLPR has obvious proliferation effect on in-vitro macrophages.

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

1. Experimental reagents and instruments:

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

An instrument device: LRH, 250F biochemical incubator shanghai constant technology ltd; GL, 22M high speed refrigerated centrifuge shanghai luxiang instrument centrifuge instruments ltd; hera cell 150 CO ₂ Incubator Heraeus; dragon Wellscan MK3 microplate reader Labsystems.

2. The experimental method comprises the following steps:

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

3. Experimental results and analysis:

TABLE 2 determination of the Effect of the biologically active peptide SHRKFSAPRHGLGFLPR on macrophage cytokine levels

Experimental groups	TNF-α	IL-1β	IL-6
				Cell blank	0.122±0.029	0.449±0.038	1.211±0.021
Bioactive peptide samples (0.1 mg/ml)	0.374±0.038**	0.633±0.020**	1.482±0.186**
				Bioactive peptide samples (0.5 mg/ml)	0.396±0.072**	0.683±0.042**	1.830±0.185**

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

As can be seen from Table 2, in the experimental results of three cytokines of TNF-alpha, IL-1 beta and IL-6, the differences (P < 0.01) are very significant at 0.1mg/ml and above, and the fact that SHRKFSAPRHGSLFLPR at a certain concentration can promote the activation of mouse abdominal cavity macrophages and release three cytokines of TNF-alpha, IL-1 beta and IL-6, TNF-alpha, IL-1 beta and IL-6 which are pro-inflammatory factors can induce the differentiation and the production of B cells and the activation, the proliferation and the differentiation of T cells and participate in the immune response of organisms is proved. Therefore, SHRKFSAPRHGLGFLPR at a certain concentration can play a role in regulating the immunity of the body.

The embodiments described above are intended to facilitate a person of ordinary skill in the art in understanding and using the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Sequence listing

<110> Shanghai university of transportation, zhejiang river peptide Life health science and technology Limited

<120> bioactive peptide SHRKFSAPRHGLGFLPR as well as preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ser His Arg Lys Phe Ser Ala Pro Arg His Gly Ser Leu Gly Phe Leu

1 5 10 15

Pro Arg

<210> 2

<211> 403

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ser His Arg Lys Phe Ser Ala Pro Arg His Gly Ser Leu Gly Phe

1 5 10 15

Leu Pro Arg Lys Arg Ser Ser Arg His Arg Gly Lys Val Lys Ser Phe

20 25 30

Pro Lys Asp Asp Ala Ser Lys Pro Val His Leu Thr Ala Phe Leu Gly

35 40 45

Tyr Lys Ala Gly Met Thr His Ile Val Arg Glu Val Asp Arg Pro Gly

50 55 60

Ser Lys Val Asn Lys Lys Glu Val Val Glu Ala Val Thr Ile Val Glu

65 70 75 80

Thr Pro Pro Met Val Val Val Gly Ile Val Gly Tyr Val Glu Thr Pro

85 90 95

Arg Gly Leu Arg Thr Phe Lys Thr Val Phe Ala Glu His Ile Ser Asp

100 105 110

Glu Cys Lys Arg Arg Phe Tyr Lys Asn Trp His Lys Ser Lys Lys Lys

115 120 125

Ala Phe Thr Lys Tyr Cys Lys Lys Trp Gln Asp Asp Thr Gly Lys Lys

130 135 140

Gln Leu Glu Lys Asp Phe Asn Ser Met Lys Lys Tyr Cys Gln Val Ile

145 150 155 160

Arg Ile Ile Ala His Thr Gln Met Arg Leu Leu Pro Leu Arg Gln Lys

165 170 175

Lys Ala His Leu Met Glu Ile Gln Val Asn Gly Gly Thr Val Ala Glu

180 185 190

Lys Leu Asp Trp Ala Arg Glu Arg Leu Glu Gln Gln Val Pro Val Asn

195 200 205

Gln Val Phe Gly Gln Asp Glu Met Ile Asp Val Ile Gly Val Thr Lys

210 215 220

Gly Lys Gly Tyr Lys Gly Val Thr Ser Arg Trp His Thr Lys Lys Leu

225 230 235 240

Pro Arg Lys Thr His Arg Gly Leu Arg Lys Val Ala Cys Ile Gly Ala

245 250 255

Trp His Pro Ala Arg Val Ala Phe Ser Val Ala Arg Ala Gly Gln Lys

260 265 270

Gly Tyr His His Arg Thr Glu Ile Asn Lys Lys Ile Tyr Lys Ile Gly

275 280 285

Gln Gly Tyr Leu Ile Lys Asp Gly Lys Leu Ile Lys Asn Asn Ala Ser

290 295 300

Thr Asp Tyr Asp Leu Ser Asp Lys Ser Ile Asn Pro Leu Gly Gly Phe

305 310 315 320

Val His Tyr Gly Glu Val Thr Asn Asp Phe Ile Met Leu Lys Gly Cys

325 330 335

Val Val Gly Thr Lys Lys Arg Val Leu Thr Leu Arg Lys Ser Leu Leu

340 345 350

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

355 360 365

Asp Thr Thr Ser Lys Phe Gly His Gly Arg Phe Gln Thr Met Glu Glu

370 375 380

Lys Lys Ala Phe Met Gly Pro Leu Lys Lys Asp Arg Ile Ala Lys Glu

385 390 395 400

Glu Gly Ala

Claims

1. The application of the bioactive peptide SHRKFSAPRHGSLFLPR in the preparation of the medicine for promoting macrophage proliferation is characterized in that the amino acid sequence of the bioactive peptide SHRKFSAPRHGSLFLPR is Ser-His-Arg-Lys-Phe-Ser-Ala-Pro-Arg-His-Gly-Ser-Leu-Gly-Phe-Leu-Pro-Arg.