CN112646023B

CN112646023B - Bioactive peptide with amino acid structure VNVVPTFGKKKGP, and preparation method and application thereof

Info

Publication number: CN112646023B
Application number: CN202110083619.3A
Authority: CN
Inventors: 张少辉; 张伯宇; 占文静
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: 2022-05-31
Anticipated expiration: 2041-01-21
Also published as: CN112646023A

Abstract

The invention belongs to the field of protein, and relates to a bioactive peptide with an amino acid structure VNVVPTFGKKKGP, and a preparation method and application thereof, wherein the bioactive peptide mainly comprises bioactive peptides RFVNVVPTFGKKKGP, RFVNVVPTFGKKKGPNA and VNVVPTFGKKKGP. In vitro immunoregulation function experiments show that the bioactive peptides RFVNVVPTFGKKKGP, RFVNVVPTFGKKKGPNA and VNVVPTFGKKKGP have good immunoregulation function. The RFVNVVPTFGKKKGP, RFVNVVPTFGKKKGPNA and VNVVPTFGKKKGP of the invention have obvious promotion effect on the ability of phagocytizing neutral red by macrophages in vitro under the condition of inflammation, can improve the immunity of organisms, reduce the morbidity of the organisms, promote the proliferation of the macrophages, improve the quality of life, and have very important significance for developing foods, health care products and medicines with immunoregulation function.

Description

Bioactive peptide with amino acid structure VNVVPTFGKKKGP, 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 VNVVPTFGKKKGP, a preparation method and application thereof.

Background

Bioactive peptides have attracted more and more attention because of their potential biological functions, and are one of the hot spots in 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, but since the number of live peptides is really too large, there are still a very large number of polypeptides to be investigated for their relevant properties.

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, using synthetic mouse bone marrow macrophages to feed rats with a peptide of origin (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.

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 40S ribosomal protein S30 protein is shown as SEQ ID NO: 4, respectively. At present, the related functions of the 40S ribosomal protein S30 protein polypeptide fragment are not studied in the prior art.

Disclosure of Invention

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

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

in a first aspect of the present invention, there is provided a biologically active peptide having amino acid structure VNVVPTFGKKKGP, selected from one or more of biologically active peptide RFVNVVPTFGKKKGP, biologically active peptide RFVNVVPTFGKKKGPNA, or biologically active peptide VNVVPTFGKKKGP:

the amino acid sequence of the bioactive peptide RFVNVVPTFGKKKGP is Arg-Phe-Val-Asn-Val-Val-Pro-Thr-Phe-Gly-Lys-Lys-Lys-Gly-Pro; as shown in SEQ ID NO: 1. as shown.

The amino acid sequence of the bioactive peptide RFVNVVPTFGKKKGPNA is Arg-Phe-Val-Asn-Val-Val-Pro-Thr-Phe-Gly-Lys-Lys-Lys-Gly-Pro-Asn-Ala; as shown in SEQ ID NO: 2, respectively.

The amino acid sequence of bioactive peptide VNVVPTFGKKKGP is Val-Asn-Val-Val-Pro-Thr-Phe-Gly-Lys-Lys-Lys-Gly-Pro as shown in SEQ ID NO: 3, respectively.

Preferably, the bioactive peptide RFVNVVPTFGKKKGP, bioactive peptide RFVNVVPTFGKKKGPNA or bioactive peptide VNVVPTFGKKKGP is mouse spleen-derived lymphocyte peptide. The protein is specifically derived from 40S ribosomal protein S30, and is respectively the amino acid residues at the 41 th to 55 th, 41 th to 57 th and 43 th to 55 th positions of the 40S ribosomal protein S30 protein.

The amino acid sequence of the 40S ribosomal protein S30 protein is shown as SEQ ID NO: 4, respectively. The amino acid sequence and the corresponding nucleotide sequence of the 40S ribosomal protein S30 protein are the existing technology, and the nucleotide fragments of the 41 th to 55 th, 41 th to 57 th and 43 th to 55 th amino acid residues of the 40S ribosomal protein S30 protein can code mature bioactive peptide RFVNVVPTFGKKKGP, bioactive peptide RFVNVVPTFGKKKGPNA or bioactive peptide VNVVPTFGKKKGP.

Preferably, the bioactive peptide RFVNVVPTFGKKKGP, bioactive peptide RFVNVVPTFGKKKGPNA or bioactive peptide VNVVPTFGKKKGP has anti-inflammatory and immunomodulatory effects.

The invention also provides polynucleotides encoding the biologically active peptides RFVNVVPTFGKKKGP, RFVNVVPTFGKKKGPNA, or VNVVPTFGKKKGP.

In the second aspect of the present invention, there is provided a method for producing the bioactive peptide RFVNVVPTFGKKKGP, the bioactive peptide RFVNVVPTFGKKKGPNA or the bioactive peptide VNVVPTFGKKKGP, which can be artificially synthesized by a genetic engineering method, can be directly obtained from cells by a method of separation and purification, or can be directly produced by chemical synthesis.

The artificial synthesis of the bioactive peptide RFVNVVPTFGKKKGP, the bioactive peptide RFVNVVPTFGKKKGPNA or the bioactive peptide VNVVPTFGKKKGP 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 a given bioactive peptide RFVNVVPTFGKKKGP, bioactive peptide RFVNVVPTFGKKKGPNA or bioactive peptide VNVVPTFGKKKGP, the bioactive peptide RFVNVVPTFGKKKGP, bioactive peptide RFVNVVPTFGKKKGPNA or bioactive peptide VNVVPTFGKKKGP 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 a use of one or more of the bioactive peptides RFVNVVPTFGKKKGP, RFVNVVPTFGKKKGPNA or VNVVPTFGKKKGP in the preparation of a medicament or cosmetic with anti-inflammatory activity.

In particular, one or more of bioactive peptide RFVNVVPTFGKKKGP, bioactive peptide RFVNVVPTFGKKKGPNA, or bioactive peptide VNVVPTFGKKKGP in combination according to the present invention can be used to prepare anti-inflammatory drugs.

Specifically, the combination of one or more of bioactive peptide RFVNVVPTFGKKKGP, bioactive peptide RFVNVVPTFGKKKGPNA or bioactive peptide VNVVPTFGKKKGP 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 a use of one or more of the bioactive peptides RFVNVVPTFGKKKGP, RFVNVVPTFGKKKGPNA or VNVVPTFGKKKGP in the preparation of a food or a medicament with immunoregulatory activity.

Further, the use of a combination of one or more of biologically active peptide RFVNVVPTFGKKKGP, biologically active peptide RFVNVVPTFGKKKGPNA, or biologically active peptide VNVVPTFGKKKGP in the preparation of a food or medicament for promoting macrophage proliferation in vitro.

In a fifth aspect of the present invention, there is provided an anti-inflammatory product comprising a combination of one or more of said bioactive peptides RFVNVVPTFGKKKGP, bioactive peptides RFVNVVPTFGKKKGPNA or bioactive peptides VNVVPTFGKKKGP or a combination of one or more of said bioactive peptides RFVNVVPTFGKKKGP, bioactive peptides RFVNVVPTFGKKKGPNA or bioactive peptides VNVVPTFGKKKGP derivatives; 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 more of the bioactive peptides RFVNVVPTFGKKKGP, RFVNVVPTFGKKKGPNA, VNVVPTFGKKKGP, RFVNVVPTFGKKKGP, RFVNVVPTFGKKKGPNA, VNVVPTFGKKKGP derivatives; the product with immunoregulatory function comprises food with immunoregulatory function or medicine with immunoregulatory function.

The derivatives of bioactive peptides RFVNVVPTFGKKKGP, bioactive peptides RFVNVVPTFGKKKGPNA, or bioactive peptides VNVVPTFGKKKGP are those having the same or better activity as bioactive peptides RFVNVVPTFGKKKGP, bioactive peptides RFVNVVPTFGKKKGPNA, or bioactive peptides VNVVPTFGKKKGP.

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

The biological active peptide RFVNVVPTFGKKKGP, the biological active peptide RFVNVVPTFGKKKGPNA or the biological active peptide VNVVPTFGKKKGP have the following beneficial effects: the bioactive peptide RFVNVVPTFGKKKGP, the bioactive peptide RFVNVVPTFGKKKGPNA or the bioactive peptide VNVVPTFGKKKGP have better anti-inflammatory activity; the biological active peptide RFVNVVPTFGKKKGP, the biological active peptide RFVNVVPTFGKKKGPNA or the biological active peptide VNVVPTFGKKKGP has a remarkable promoting effect on the ability of phagocytizing neutral red by macrophages in vitro under the condition of inflammation, can improve the immunity of the organism, reduce the morbidity of the organism, promote the proliferation of the macrophages, improve the quality of life and have a very important significance for developing foods, health-care products and medicines with immune regulation functions.

Drawings

FIG. 1: a first order mass spectrum of a fragment with a mass to charge ratio of 419.2504 (m/z 419.2504);

FIG. 2: a secondary mass spectrum of a segment with the mass-to-charge ratio of 419.2504 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 465.5225 (m/z 465.5225);

FIG. 4: a secondary mass spectrum of a segment with the mass-to-charge ratio of 465.5225 and the breaking conditions of the bioactive peptides az and by;

FIG. 5 is a schematic view of: a first order mass spectrum of a fragment with a mass to charge ratio of 457.6105 (m/z 457.6105);

FIG. 6: a secondary mass spectrum of a segment with the mass-to-charge ratio of 457.6105 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 are described in particular in Sambrook et al, Molecular CLONING: 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 RFVNVVPTFGKKKGP, RFVNVVPTFGKKKGPNA and VNVVPTFGKKKGP

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 decolorizing 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 Arg and a proper amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, adding 20ml of DMF to dissolve the amino acid Arg 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 proper amount of the second amino acid and a proper amount of HOBT in a 50ml centrifuge tube, adding 25ml of DMF to dissolve the second 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 shaker 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 Arg, Phe, Val, Asn, Val, Pro, Thr, Phe, Gly, Lys, Gly, and Pro are sequentially ligated according to steps 9 to 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 RFVNVVPTFGKKKGP was synthesized.

The synthesis of the bioactive peptides RFVNVVPTFGKKKGPNA and VNVVPTFGKKKGP can be performed by selecting the first amino acid corresponding to the specific bioactive peptide at step 5 and linking the first amino acid corresponding to the specific bioactive peptide at step 12, as described above.

Second, 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)

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 +

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 RFVNVVPTFGKKKGP, RFVNVVPTFGKKKGPNA and VNVVPTFGKKKGP were performed using ultra high performance liquid, electrospray, quadrupole, time-of-flight mass spectrometry. The primary mass spectrum of the bioactive peptide RFVNVVPTFGKKKGP 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 419.2504, and the retention time is 22.64 min. The mass chromatogram extraction diagram of the bioactive peptide RFVNVVPTFGKKKGPNA 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 465.5225, and the retention time is 21.83 min. The mass chromatogram extraction diagram of the bioactive peptide VNVVPTFGKKKGP is shown in FIG. 5, the secondary mass spectrum diagram of the extraction peak and the az and by fracture conditions are shown in FIG. 6, the mass-to-charge ratio of the bioactive peptide of the peak is 457.6105, and the retention time is 17.06 min.

3) Results

As can be seen from fig. 2, the fragment sequence having the mass-to-charge ratio 419.2504 obtained from az and by cleavage was Arg, Phe, Val, Asn, Val, Pro, Thr, Phe, Gly, Lys, Gly, and Pro (RFVNVVPTFGKKKGP), and is represented by SEQ ID NO: 1. the fragment corresponds to the residue sequence of 41-55 th site of 40S ribosomal protein S30 protein, the GenBank number of the amino acid sequence of 40S ribosomal protein S30 protein is AAF80246.1, and the sequence is shown in SEQ ID NO: 4.

as can be seen from fig. 4, the fragment sequence of mass-to-charge ratio 465.5225, which is calculated by Mascot software analysis based on az and by cleavage, is Arg, Phe, Val, Asn, Val, Pro, Thr, Phe, Gly, Lys, Gly, Pro, Asn, Ala (RFVNVVPTFGKKKGPNA), and is denoted as SEQ ID NO: 2. the fragment corresponds to residue sequences of 41-57 th sites of 40S ribosomal protein S30 protein, the GenBank number of the amino acid sequence of the 40S ribosomal protein S30 protein is AAF80246.1, and the sequence is shown in SEQ ID NO: 4.

as can be seen from fig. 6, the fragment sequences of mass-to-charge ratio 457.6105, which were analyzed and calculated by Mascot software according to the cases of az and by cleavage, were Val, Asn, Val, Pro, Thr, Phe, Gly, Lys, Gly, Pro (VNVVPTFGKKKGP), and are represented as SEQ ID NO: 3. the fragment corresponds to the residue sequence of 43-55 th site of 40S ribosomal protein S30 protein, the GenBank number of the amino acid sequence of 40S ribosomal protein S30 protein is AAF80246.1, and the sequence is shown in SEQ ID NO: 4.

example 2 immunomodulatory Activity assays of bioactive peptides

First, MTT method for testing in vitro macrophage proliferation ability experiment of biological active peptide RFVNVVPTFGKKKGP

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

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 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 the abdominal skin, sucking 4 ℃ Phosphate Buffer Solution (PBS) by using a syringe to repeatedly wash the abdominal cavity, centrifuging the washed solution by using a centrifuge tube for 10 minutes after collecting the washed solution, discarding the supernatant after centrifuging the washed solution (1000rpm and 4 ℃), washing the washed solution twice by using 4 ℃ RPMI1640 complete culture solution (containing 10% FBS), staining the washed solution by using 0.2% trypan blue solution to detect the vitality of the cells, and confirming 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.

The cell suspension that had been blown to complete suspension was added to a 96-well cell culture plate at 37 ℃ with 5% CO in an appropriate volume₂After culturing for 4 hours under the environment, removing liquid in the holes, carefully cleaning the bottom of the holes of the cell culture plate by using a complete culture solution RPMI1640 at 37 ℃, and washing the cells and cell fragments which are not attached to the walls to obtain the purified attached 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, adding 200 mul/hole RPMI1640 complete culture solution (10% FBS) dissolved with bioactive peptide (0.5mg/ml) into each hole of the experimental group, and continuously culturing for 48 h; negative control group added BSA (500. mu.g/mL) dissolved in RPMI1640 complete medium (10% FBS) 200. mu.l/well; the blank group was continuously cultured for 48 hours with the addition of 200. mu.l/well of RPMI1640 complete medium (10% FBS). 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 100 ng/ml; LPS is not added in a normal group; and the normal group and the inflammatory group were added with 5% MTT 20. mu.l/well at 44 h; 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 (OD570) 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 solution OD value)/(blank group OD value-blank culture solution 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 bioactive peptide RFVNVVPTFGKKKGP on macrophage proliferation in vitro

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

The results are shown in Table 1, and it is understood from Table 1 that macrophages were proliferated in both the normal group and the inflammatory group in the case of adding 0.5mg/ml bioactive peptide. Compared with the negative control group, the normal group has significant difference ((P < 0.05) and the inflammatory group has very significant difference (P < 0.01), which indicates that the bioactive peptide RFVNVVPTFGKKKGP has significant proliferation promoting effect on in vitro macrophages.

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

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 RFVNVVPTFGKKKGP 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 (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 2 determination of the ability of the bioactive peptide RFVNVVPTFGKKKGP to promote phagocytosis of neutral Red by macrophages

Experimental groups	Absorbance value (OD540)
		Blank group	0.1084±0.0323
Experimental group	0.1379±0.0152*

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 2, compared with the blank cell group, the macrophage phagocytosis ability of the inflammatory group added with 0.5mg/ml bioactive peptide is obviously increased, and compared with the blank cell group, the macrophage phagocytosis ability of the inflammatory group added with bioactive peptide is obviously different (P is less than 0.05). The biological active peptide RFVNVVPTFGKKKGP is proved to have obvious promotion effect on the ability of phagocytizing neutral red by macrophages in vitro under the condition of inflammation.

Third, MTT method measures the in vitro macrophage proliferation ability experiment of bioactive peptide RFVNVVPTFGKKKGPNA

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 RFVNVVPTFGKKKGPNA 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 solutions, aqueous solutions 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 apparatus centrifuge Instrument Co., Ltd; hera cell 150CO₂Incubator Heraeus; dragon Wellscan MK3 microplate reader Labsystems.

2. The test method comprises the following steps:

The cell suspension that had been blown to complete suspension was added to a 96-well cell culture plate at 37 ℃ with 5% CO in an appropriate volume₂After culturing for 4 hours under the environment, sucking away liquid in the holes, carefully cleaning the bottom of the holes of the cell culture plate by using a complete culture solution RPMI1640 at 37 ℃, and washing away nonadherent cells and cell fragments to obtain purified adherent abdomenLuminal 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 purified adherent abdominal cavity macrophages are obtained, 200 mul/hole of RPMI1640 complete culture solution (10% FBS) dissolved with bioactive peptide (0.5mg/ml) is added into each hole of the experimental group for continuous culture for 48 h; negative control group added BSA (500. mu.g/mL) dissolved in RPMI1640 complete medium (10% FBS) 200. mu.l/well; the blank group was continuously cultured for 48 hours with the addition of 200. mu.l/well of RPMI1640 complete medium (10% FBS). 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 100 ng/ml; LPS is not added in a normal group; and the normal group and the inflammatory group were added with 5% MTT 20. mu.l/well at 44 h; 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 (OD570) of each well was measured by a microplate reader at a wavelength of 570nm, and the Growth index (Growth Indices) was calculated as follows:

3. Results and analysis of the experiments

TABLE 3 Effect of bioactive peptide RFVNVVPTFGKKKGPNA on macrophage proliferation in vitro

Experiment grouping	Normal group GI	GI inflammation group
			Negative control group	1	1
Bioactive peptide (0.5mg/ml)	1.0365±0.0264*	1.123±0.0243**

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

The results are shown in Table 3, and it can be seen from Table 3 that macrophages were proliferated in both the normal group and the inflammatory group when 0.5mg/ml bioactive peptide was added. Compared with the negative control group, the normal group has significant difference ((P < 0.05) and the inflammatory group has very significant difference (P < 0.01), which indicates that the bioactive peptide RFVNVVPTFGKKKGPNA has significant proliferation effect on macrophages in vitro.

Fourth, experiment of macrophage phagocytosis neutral red promoting ability of bioactive peptide RFVNVVPTFGKKKGPNA

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 RFVNVVPTFGKKKGPNA 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 10⁶The cell suspension per ml is 100 μ l/well, and after adherent purification, the cell suspension is added with RPMI1640 complete culture solution (10% FBS) containing bioactive peptide (0.5mg/ml) 200 μ l/well as RPMI1640 complete culture solution (10% FBS) containing no bioactive peptide) The cells cultured at 200. mu.l/well were set as a 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 4 determination of the ability of the bioactive peptide RFVNVVPTFGKKKGPNA to promote phagocytosis of neutral Red by macrophages

Experiment grouping	Absorbance value (OD540)
		Blank group	0.1043±0.0289
Experimental group	0.2188±0.0132**

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

Fifth, MTT method measures the in vitro macrophage proliferation ability experiment of bioactive peptide VNVVPTFGKKKGP

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

2. The test method comprises the following steps:

3. Results and analysis of the experiments

TABLE 5 Effect of bioactive peptide VNVVPTFGKKKGP on macrophage proliferation in vitro

Experiment grouping	Normal group GI	GI inflammation group
			Negative control group	1	1
Bioactive peptide (0.5mg/ml)	1.1129±0.0483**	1.374±0.0375**

The results are shown in Table 5, and it is understood from Table 5 that macrophages were proliferated in both the normal group and the inflammatory group when 0.5mg/ml bioactive peptide was added. And compared with a negative control group, the difference is very significant (P < 0.01). It is shown that the bioactive peptide VNVVPTFGKKKGP has a significant proliferation effect on macrophages in vitro.

Sixthly, experiment of macrophage phagocytosis neutral red promoting capability of bioactive peptide VNVVPTFGKKKGP

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 VNVVPTFGKKKGP obtained in example 1; LPS, purchased from Sigma; neutral red staining solution, produced by Biyuntian biotechnology 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 (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. Washing the bottom of the well with PBS, adding 80 μ l/well of neutral red dye solution at 37 deg.C, sucking off the dye solution after 10 min, and washing the well with PBSAfter this time, 150 μ 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 6 determination of the ability of the bioactive peptide VNVVPTFGKKKGP to promote phagocytosis of neutral Red by macrophages

Experiment grouping	Absorbance value (OD540)
		Blank group	0.1056±0.0301
Experimental group	0.1869±0.0121**

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 6, compared with the blank cell group, the macrophage phagocytosis ability of the inflammatory group added with 0.5mg/ml bioactive peptide is obviously increased, and compared with the blank cell group, the macrophage phagocytosis ability of the inflammatory group added with bioactive peptide is very significant (P is less than 0.01). The result shows that the bioactive peptide VNVVPTFGKKKGP has a remarkable promoting effect on the ability of macrophages in vitro to phagocytose neutral red 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 ghrelin Life health science and technology Limited

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

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Arg Phe Val Asn Val Val Pro Thr Phe Gly Lys Lys Lys Gly Pro

1 5 10 15

<210> 2

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Arg Phe Val Asn Val Val Pro Thr Phe Gly Lys Lys Lys Gly Pro Asn

1 5 10 15

Ala

<210> 3

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Val Asn Val Val Pro Thr Phe Gly Lys Lys Lys Gly Pro

1 5 10

<210> 4

<211> 59

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Lys Val His Gly Ser Leu Ala Arg Ala Gly Lys Val Arg Gly Gln Thr

1 5 10 15

Pro Lys Val Ala Lys Gln Glu Lys Lys Lys Lys Lys Thr Gly Arg Ala

20 25 30

Lys Arg Arg Met Gln Tyr Asn Arg Arg Phe Val Asn Val Val Pro Thr

35 40 45

Phe Gly Lys Lys Lys Gly Pro Asn Ala Asn Ser

50 55

Claims

1. A biologically active peptide having amino acid structure VNVVPTFGKKKGP, wherein one or more of biologically active peptide RFVNVVPTFGKKKGP, biologically active peptide RFVNVVPTFGKKKGPNA, or biologically active peptide VNVVPTFGKKKGP:

the amino acid sequence of the bioactive peptide RFVNVVPTFGKKKGP is Arg-Phe-Val-Asn-Val-Val-Pro-Thr-Phe-Gly-Lys-Lys-Lys-Gly-Pro;

the amino acid sequence of the bioactive peptide RFVNVVPTFGKKKGPNA is Arg-Phe-Val-Asn-Val-Val-Pro-Thr-Phe-Gly-Lys-Lys-Lys-Gly-Pro-Asn-Ala;

the amino acid sequence of the bioactive peptide VNVVPTFGKKKGP is Val-Asn-Val-Val-Pro-Thr-Phe-Gly-Lys-Lys-Lys-Gly-Pro.

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

3. The method of claim 1, wherein the bioactive peptide having the amino acid structure VNVVPTFGKKKGP is produced directly by chemical synthesis.

4. Use of a biologically active peptide having amino acid structure VNVVPTFGKKKGP of claim 1, wherein said biologically active peptide RFVNVVPTFGKKKGP, biologically active peptide RFVNVVPTFGKKKGPNA, or a combination of one or more of biologically active peptides VNVVPTFGKKKGP 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 VNVVPTFGKKKGP of claim 1, wherein said biologically active peptide RFVNVVPTFGKKKGP, biologically active peptide RFVNVVPTFGKKKGPNA, or a combination of one or more of biologically active peptides VNVVPTFGKKKGP is used in the manufacture of a medicament for promoting macrophage proliferation.