CN112812171B - Bioactive peptide with amino acid structure VVRKPLNKEGKKP, and preparation method and application thereof - Google Patents
Bioactive peptide with amino acid structure VVRKPLNKEGKKP, and preparation method and application thereof Download PDFInfo
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- CN112812171B CN112812171B CN202110090097.XA CN202110090097A CN112812171B CN 112812171 B CN112812171 B CN 112812171B CN 202110090097 A CN202110090097 A CN 202110090097A CN 112812171 B CN112812171 B CN 112812171B
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- vvrkplnkegkkp
- amino acid
- bioactive peptide
- vvrkplnkegkkpr
- peptide
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Images
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention relates to the field of protein, in particular to a bioactive peptide with an amino acid structure of VVRKPLNKEGKKP, a preparation method and application thereof, wherein the amino acid sequence of the bioactive peptide VVRKPLNKEGKKP is shown as SEQ ID NO: 1, the amino acid sequence of the bioactive peptide VVRKPLNKEGKKPR is shown as SEQ ID NO: 2, respectively. In vitro immunoregulation function experiments prove that the bioactive peptides VVRKPLNKEGKKP and VVRKPLNKEGKKPR have good immunoregulation function. The VVRKPLNKEGKKP and VVRKPLNKEGKKPR of the invention have the capability of promoting the increase of the induced amount of the nitric oxide of the macrophage under the condition of certain concentration, thereby improving the capability of the body for resisting the infection of external pathogens, reducing the morbidity of the body, simultaneously promoting the proliferation of the macrophage in vitro, exerting the immunologic function of the body and having very important significance for developing foods, health care products and medicines with immunoregulation function.
Description
Technical Field
The invention relates to the field of protein, in particular to a bioactive peptide with an amino acid structure VVRKPLNKEGKKP, 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.
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.
Often, these proteins themselves are not immunomodulatory when the polypeptide is not enzymatically separated from the protein. 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 ribosol protein S6 is shown in SEQ ID NO: 3, respectively. At present, the related functions of the 40S ribosomal protein S6 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 VVRKPLNKEGKKP, and a preparation method and application thereof. In particular to two biological active peptides VVRKPLNKEGKKP and VVRKPLNKEGKKPR with highly similar structures, 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 invention, there is provided a biologically active peptide having amino acid structure VVRKPLNKEGKKP, selected from the group consisting of biologically active peptide VVRKPLNKEGKKP or biologically active peptide VVRKPLNKEGKKPR or a combination of both, the amino acid sequence of biologically active peptide VVRKPLNKEGKKP being Val-Arg-Lys-Pro-Leu-Asn-Lys-Glu-Gly-Lys-Pro, as set forth in SEQ ID NO: 1, the amino acid sequence of the bioactive peptide VVRKPLNKEGKKPR is Val-Val-Arg-Lys-Pro-Leu-Asn-Lys-Glu-Gly-Lys-Lys-Pro-Arg, as shown in SEQ ID NO: 2, respectively.
Preferably, the bioactive peptide VVRKPLNKEGKKP or bioactive peptide VVRKPLNKEGKKPR is mouse spleen derived lymphocyte peptide. Specifically, the amino acid residues are derived from 40S ribosol protein S6 protein and are the 157 th to 169 th amino acid residues and 157 th to 170 th amino acid residues of 40S ribosol protein S6 protein. The amino acid sequence of the 40S ribosol protein S6 is shown in SEQ ID NO: 3, respectively.
The amino acid sequence and the corresponding nucleotide sequence of the 40S ribosomal protein S6 protein are the prior art, and the nucleotide fragment which codes the 157 th to 169 th and 157 th to 170 th amino acid residues of the 40S ribosomal protein S6 protein can code mature bioactive peptides VVRKPLNKEGKKP and VVRKPLNKEGKKPR.
Preferably, the bioactive peptide VVRKPLNKEGKKP or bioactive peptide VVRKPLNKEGKKPR has anti-inflammatory and immunomodulatory effects.
The invention also provides polynucleotides encoding the biologically active peptide VVRKPLNKEGKKP or biologically active peptide VVRKPLNKEGKKPR.
In the second aspect of the present invention, there is provided a method for preparing the bioactive peptide VVRKPLNKEGKKP or the bioactive peptide VVRKPLNKEGKKPR, 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 VVRKPLNKEGKKP or the bioactive peptide VVRKPLNKEGKKPR by genetic engineering methods is a technical solution that can be realized by those skilled in the art, and for example, the sequence synthesis of the polypeptide can be controlled by a suitable DNA template based on DNA recombination technology.
The method for directly obtaining the cell by the separation and purification method can be as follows: based on the given amino acid sequence of the bioactive peptide VVRKPLNKEGKKP or the bioactive peptide VVRKPLNKEGKKPR, the bioactive peptide VVRKPLNKEGKKP or the bioactive peptide VVRKPLNKEGKKPR is obtained from mouse spleen-derived lymphocytes by a conventional enzymolysis and purification method in a biological technology.
In a third aspect of the present invention, there is provided the use of a biologically active peptide having amino acid structure VVRKPLNKEGKKP in the manufacture of a medicament or cosmetic product having anti-inflammatory properties, wherein the biologically active peptide having amino acid structure VVRKPLNKEGKKP is selected from the group consisting of biologically active peptide VVRKPLNKEGKKP or biologically active peptide VVRKPLNKEGKKPR or a combination thereof.
Specifically, the bioactive peptide VVRKPLNKEGKKP or the bioactive peptide VVRKPLNKEGKKPR can be used for preparing medicaments with anti-inflammatory effects.
In a fourth aspect of the present invention, there is provided the use of a biologically active peptide having amino acid structure VVRKPLNKEGKKP in the preparation of a food or a medicament having an immunomodulatory effect, wherein the biologically active peptide having amino acid structure VVRKPLNKEGKKP is selected from the group consisting of biologically active peptide VVRKPLNKEGKKP and biologically active peptide VVRKPLNKEGKKPR, or a combination thereof.
Specifically, the invention relates to the application of the bioactive peptide VVRKPLNKEGKKP or the bioactive peptide VVRKPLNKEGKKPR or the composition of the two in preparing food or medicine with macrophage-promoting nitric oxide inducing amount.
Specifically, the invention relates to application of the bioactive peptide VVRKPLNKEGKKP or the bioactive peptide VVRKPLNKEGKKPR or the composition of the two in preparation of food or medicines for promoting in vitro macrophage proliferation.
In a fifth aspect of the present invention, an anti-inflammatory product is provided, comprising one or a combination of two of said bioactive peptide VVRKPLNKEGKKP or bioactive peptide VVRKPLNKEGKKPR or one or a combination of several of said bioactive peptide VVRKPLNKEGKKP or a derivative of bioactive peptide VVRKPLNKEGKKPR; the anti-inflammatory product comprises an anti-inflammatory drug or an anti-inflammatory cosmetic.
In a sixth aspect of the present invention, a product with immunoregulatory function is provided, which comprises one or two of the bioactive peptides VVRKPLNKEGKKP or bioactive peptides VVRKPLNKEGKKPR or one or more of the bioactive peptides VVRKPLNKEGKKP or derivatives of bioactive peptides VVRKPLNKEGKKPR; the product with immunoregulatory function comprises food with immunoregulatory function or medicine with immunoregulatory function.
In the present invention, the derivative of the bioactive peptide VVRKPLNKEGKKP or the bioactive peptide VVRKPLNKEGKKPR means the same activity or better activity as the bioactive peptide VVRKPLNKEGKKP or the bioactive peptide VVRKPLNKEGKKPR.
In the present invention, the derivative of the bioactive peptide VVRKPLNKEGKKP or the bioactive peptide VVRKPLNKEGKKPR refers to a bioactive peptide derivative obtained by modifying the amino acid side chain group, the amino terminus or the carboxyl terminus of the bioactive peptide VVRKPLNKEGKKP or the bioactive peptide VVRKPLNKEGKKPR with a modification such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification, or glycosylation.
The bioactive peptide with the amino acid structure of VVRKPLNKEGKKP has the following beneficial effects: the bioactive peptide with the amino acid structure of VVRKPLNKEGKKP has better anti-inflammatory activity; the bioactive peptide with the amino acid structure of VVRKPLNKEGKKP has the capacity of promoting the increase of the induced amount of the nitric oxide of macrophages under the condition of certain concentration, thereby improving the capacity of resisting the infection of external pathogens of organisms, reducing the morbidity of the organisms, promoting the proliferation of the macrophages in vitro, exerting the immune function of the organisms and having very important significance for developing foods, health care products and medicines with the immune regulation function.
Drawings
FIG. 1: a first order mass spectrum of a fragment with a mass to charge ratio of 498.3153 (m/z 498.3153);
FIG. 2: a secondary mass spectrum of a segment with the mass-to-charge ratio of 498.3153 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 413.0139 (m/z 413.0139);
FIG. 4: a secondary mass spectrum of a segment with the mass-to-charge ratio of 413.0139 and the breaking conditions of the bioactive peptides az and by;
Detailed Description
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1 Artificial Synthesis of active peptides VVRKPLNKEGKKP and VVRKPLNKEGKKPR
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 Val and a proper amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, adding 20ml of DMF to dissolve the amino acid Val 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. The amino acids Val, Arg, Lys, Pro, Leu, Asn, Lys, Glu, Gly, Lys, Pro are sequentially linked according to the 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 VVRKPLNKEGKKP was synthesized.
Method for the synthesis of bioactive peptide VVRKPLNKEGKKPR referring to the above method, only the amino acids corresponding to the specific bioactive peptide need to be linked in 12 steps.
Confirmation of biologically active peptides
1) UPLC analysis
UPLC conditions were as follows:
the instrument comprises the following steps: waters ACQUITY UPLC ultra-high performance liquid phase, electrospray, quadrupole and time-of-flight mass spectrometer
Specification of chromatographic column: BEH C18 chromatographic column
Flow rate: 0.4mL/min
Temperature: 50 deg.C
Ultraviolet detection wavelength: 210nm
Sample introduction amount: 2 μ L
Gradient conditions: solution A: water containing 0.1% formic acid (v/v), liquid B: acetonitrile containing 0.1% formic acid (v/v)
2) Mass spectrometric analysis
The mass spectrometry conditions were as follows:
ion mode: ES +
Mass range (m/z): 100. 1000A
Capillary voltage (Capillary) (kV): 3.0
Sampling cone (V): 35.0
Ion source temperature (. degree. C.): 115
Desolvation temperature (. degree. C.): 350
Desolventizing gas stream (L/hr): 700.0
Collision energy (eV): 4.0
Scan time (sec): 0.25
Inner scan time (sec): 0.02
According to the above analysis methods, chromatographic analysis and mass spectrometric analysis of bioactive peptides VVRKPLNKEGKKP and VVRKPLNKEGKKPR were performed using ultra high performance liquid, electrospray, quadrupole, time-of-flight mass spectrometry. The primary mass spectrum of the bioactive peptide VVRKPLNKEGKKP 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 498.3153, and the retention time is 6.23 min. The mass chromatogram extraction diagram of the bioactive peptide VVRKPLNKEGKKPR is shown in FIG. 3, the secondary mass spectrum of the extracted peak and the az and by fracture conditions are shown in FIG. 4, the mass-to-charge ratio of the bioactive peptide of the peak is 413.0139, and the retention time is 4.46 min.
3) Results
As can be seen from fig. 2, when az and by are cleaved, the fragment sequence having the mass-to-charge ratio 498.3153 obtained by Mascot software analysis and calculation is Val, Arg, Lys, Pro, Leu, Asn, Lys, Glu, Gly, Lys, Pro (VVRKPLNKEGKKP), and is represented as SEQ ID NO: 1. the fragment corresponds to residue sequences of 157 th to 169 th positions of 40S ribosomal protein S6 protein, the GenBank number of the amino acid sequence of the 40S ribosomal protein S6 protein is BAB28498.1, and the sequence is shown in SEQ ID NO: 3.
as can be seen from fig. 4, the fragment sequence having the mass-to-charge ratio 413.0139 obtained from az and by cleavage was Val, Arg, Lys, Pro, Leu, Asn, Lys, Glu, Gly, Lys, Pro, Arg (VVRKPLNKEGKKPR), and represented by SEQ ID NO: 2. the fragment corresponds to residue sequences of 157 th to 170 th positions of 40S ribosomal protein S6 protein, the GenBank number of the amino acid sequence of the 40S ribosomal protein S6 protein is BAB28498.1, and the sequence is shown in SEQ ID NO: 3.
example 2 immunomodulatory Activity assays of bioactive peptides
Measurement of macrophage-promoting nitric oxide-inducing amount of bioactive peptide VVRKPLNKEGKKP (Griess method)
1. Experimental reagents and instruments:
reagent: experimental animal balb/c mouse (male 6-8 weeks old) spleen lymphocyte source bioactive peptide VVRKPLNKEGKKP; LPS, purchased from Sigma; neutral red staining solution, produced by Biyuntian biotechnological research institute.
The instrument equipment comprises: LRH-250F Biochemical incubator Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge Shanghai Luxiang apparatus centrifuge Instrument Co., Ltd; hera cell 150CO2Incubator Heraeus; dragon Wellscan MK3 microplate reader Labsystems.
2. The 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 VVRKPLNKEGKKP
Note: significant difference compared to negative control (P < 0.05);
significant difference compared with negative control group (P <0.01)
The results are shown in table 1, and it is seen from table 1 that when bioactive peptide VVRKPLNKEGKKP was added to the test groups at concentrations of 1mg/mL and 0.5mg/mL, respectively, the peptides both promoted nitric oxide-induced macrophage growth under normal growth and under LPS-induced inflammation, and were significantly different from the cell blank group (P < 0.01). When the bioactive peptide VVRKPLNKEGKKP was added at a concentration of 0.1mg/mL, there was no significant difference between the normal and LPS-induced inflammation compared to the cell blank. Indicating that the bioactive peptide VVRKPLNKEGKKP has the ability to promote an increase in the nitric oxide-inducing amount of macrophages over a range of concentrations.
Second, MTT method for testing in vitro macrophage proliferation ability experiment of bioactive peptide VVRKPLNKEGKKP
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 VVRKPLNKEGKKP 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.
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 VVRKPLNKEGKKP (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 2 Effect of bioactive peptide VVRKPLNKEGKKP on macrophage proliferation in vitro
| Experiment grouping | Normal group GI | GI inflammation group |
| |
1 | 1 |
| Bioactive peptide (0.5mg/ml) | 1.0794±0.0142** | 1.1563±0.0349** |
Note: indicates a significant difference (P < 0.05) compared to the 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 2, and it is understood from Table 2 that macrophages were proliferated in both the normal group and the inflammatory group when 0.5mg/ml of bioactive peptide VVRKPLNKEGKKP was added. And compared with a negative control group, the difference is highly significant (P is less than 0.01). It is shown that the bioactive peptide VVRKPLNKEGKKP has a significant proliferation effect on macrophages in vitro.
Third, determination of macrophage-promoting nitric oxide-inducing amount of bioactive peptide VVRKPLNKEGKKPR (Griess method)
1. Experimental reagents and instruments:
reagent: experimental animal balb/c mouse (male 6-8 weeks old) spleen lymphocyte source bioactive peptide VVRKPLNKEGKKPR; LPS, purchased from Sigma; neutral red staining solution, produced by Biyuntian biotechnological research institute.
The instrument equipment comprises: LRH-250F Biochemical incubator Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge Shanghai Luxiang apparatus centrifuge Instrument Co., Ltd; hera cell 150CO2Incubator Heraeus; dragon Wellscan MK3 microplate reader Labsystems.
2. The 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 3 determination of macrophage-promoting nitric oxide-inducing amount of bioactive peptide VVRKPLNKEGKKPR
| Experiment grouping | Normal group | Inflammation group |
| Cell blank | 0.0453±0.0185 | 0.3375±0.0392 |
| Bioactive peptide (1mg/ml) | 0.1085±0.0116** | 0.4793±0.0369** |
| Bioactive peptide (0.5mg/ml) | 0.1299±0.0223** | 0.5154±0.0363** |
Note: significant difference compared to negative control (P < 0.05);
significant difference compared with negative control group (P <0.01)
The results are shown in table 3, and it is seen from table 3 that when bioactive peptide VVRKPLNKEGKKPR was added to the test groups at concentrations of 1mg/mL and 0.5mg/mL, respectively, the peptides both promoted nitric oxide-induced macrophage growth under normal growth and under LPS-induced inflammation, and were significantly different from the cell blank group (P < 0.01). Indicating that the bioactive peptide VVRKPLNKEGKKPR has the ability to promote an increase in the nitric oxide-inducing amount of macrophages over a range of concentrations.
Fourth, MTT method measures the in vitro macrophage proliferation ability experiment of bioactive peptide VVRKPLNKEGKKPR
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 VVRKPLNKEGKKPR 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.
The instrument equipment comprises: LRH-250F Biochemical incubator Shanghai Hengshi Co., Ltd; shanghai Luxiang instrument separation of GL-22M high-speed refrigerated centrifugeCardio-mechanical instruments 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 VVRKPLNKEGKKPR (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 4 Effect of bioactive peptide VVRKPLNKEGKKPR on macrophage proliferation in vitro
| Experiment grouping | Normal group GI | GI inflammation group |
| |
1 | 1 |
| Bioactive peptide (0.5mg/ml) | 1.0829±0.0112** | 1.1377±0.0285** |
Note: indicates a significant difference (P < 0.05) compared to the negative control; indicates that there is a highly significant difference (P <0.01) compared to the negative control group
The results of the experiment are shown in Table 4, and it is understood from Table 4 that macrophages were proliferated in both the normal group and the inflammatory group when 0.5mg/ml of bioactive peptide VVRKPLNKEGKKPR was added. And compared with a negative control group, the difference is highly significant (P is less than 0.01). It is shown that the bioactive peptide VVRKPLNKEGKKPR has a significant proliferation effect on macrophages in vitro.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Sequence listing
<110> Zhejiang ghui peptide Life health science and technology Limited
<120> bioactive peptide with amino acid structure VVRKPLNKEGKKP, and preparation method and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Val Val Arg Lys Pro Leu Asn Lys Glu Gly Lys Lys Pro
1 5 10
<210> 2
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Val Val Arg Lys Pro Leu Asn Lys Glu Gly Lys Lys Pro Arg
1 5 10
<210> 3
<211> 249
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Lys Leu Asn Ile Ser Phe Pro Ala Thr Gly Cys Gln Lys Leu Ile
1 5 10 15
Glu Val Asp Asp Glu Arg Lys Leu Arg Thr Phe Tyr Glu Lys Arg Met
20 25 30
Ala Thr Glu Val Ala Ala Asp Ala Leu Gly Glu Glu Trp Lys Gly Tyr
35 40 45
Val Val Arg Ile Ser Gly Gly Asn Asp Lys Gln Gly Phe Pro Met Lys
50 55 60
Gln Gly Val Leu Thr His Gly Arg Val Arg Leu Leu Leu Ser Lys Gly
65 70 75 80
His Ser Cys Tyr Arg Pro Arg Arg Thr Gly Glu Arg Lys Arg Lys Ser
85 90 95
Val Arg Gly Cys Ile Val Asp Ala Asn Leu Ser Val Leu Asn Leu Val
100 105 110
Ile Val Lys Lys Gly Glu Lys Asp Ile Pro Gly Leu Thr Asp Thr Thr
115 120 125
Val Pro Arg Arg Leu Gly Pro Lys Arg Ala Ser Arg Ile Arg Lys Leu
130 135 140
Phe Asn Leu Ser Lys Glu Asp Asp Val Arg Gln Tyr Val Val Arg Lys
145 150 155 160
Pro Leu Asn Lys Glu Gly Lys Lys Pro Arg Thr Lys Ala Pro Lys Ile
165 170 175
Gln Arg Leu Val Thr Pro Arg Val Leu Gln His Lys Arg Arg Arg Ile
180 185 190
Ala Leu Lys Lys Gln Arg Thr Lys Lys Asn Lys Glu Glu Ala Ala Glu
195 200 205
Tyr Ala Lys Leu Leu Ala Lys Arg Met Lys Glu Ala Lys Glu Lys Arg
210 215 220
Gln Glu Gln Ile Ala Lys Arg Arg Arg Leu Ser Ser Leu Arg Ala Ser
225 230 235 240
Thr Ser Lys Ser Glu Ser Ser Gln Lys
245
Claims (5)
1. A biologically active peptide having amino acid structure VVRKPLNKEGKKP, selected from the group consisting of biologically active peptide VVRKPLNKEGKKP, biologically active peptide VVRKPLNKEGKKPR, and combinations thereof, wherein the amino acid sequence of biologically active peptide VVRKPLNKEGKKP is as set forth in SEQ ID NO: 1, the amino acid sequence of bioactive peptide VVRKPLNKEGKKPR is shown as SEQ ID NO: 2, respectively.
2. A polynucleotide encoding the biologically active peptide of claim 1 having the amino acid structure VVRKPLNKEGKKP.
3. The method of claim 1, wherein the bioactive peptide having the amino acid structure VVRKPLNKEGKKP is produced directly by chemical synthesis.
4. Use of a biologically active peptide having amino acid structure VVRKPLNKEGKKP of claim 1, wherein said biologically active peptide VVRKPLNKEGKKP or biologically active peptide VVRKPLNKEGKKPR or a combination of both is used in the preparation of a macrophage nitric oxide-inducing amount of a medicament.
5. Use of a biologically active peptide having amino acid structure VVRKPLNKEGKKP of claim 1, wherein said biologically active peptide VVRKPLNKEGKKP or biologically active peptide VVRKPLNKEGKKPR or a combination of both is used in the manufacture of a medicament for promoting macrophage proliferation.
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| EP2565202A1 (en) * | 2010-04-30 | 2013-03-06 | Sanwa Kagaku Kenkyusho Co., Ltd | Peptide for improving in vivo stability of physiologically active substance or the like and physiologically active substance with improved in vivo stability |
| CN109608550A (en) * | 2012-12-05 | 2019-04-12 | 生控基因疫苗股份有限公司 | Fusion protein as the immunogenicity reinforcing agent for inducing T cells with antigenic specificity reaction |
| CN107163136A (en) * | 2017-07-06 | 2017-09-15 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide WNIPMGLIVNQ and its preparation method and application |
| CN108017702A (en) * | 2017-11-14 | 2018-05-11 | 上海交通大学 | A kind of biologically active polypeptide FPPQSVLS and its preparation method and application |
| CN107746428A (en) * | 2017-12-01 | 2018-03-02 | 浙江熊猫乳业集团股份有限公司 | A kind of biologically active polypeptide DERFFSDKIA and its preparation method and application |
| CN107814836A (en) * | 2017-12-01 | 2018-03-20 | 浙江熊猫乳业集团股份有限公司 | A kind of biologically active polypeptide FPGPIPNS and its preparation method and application |
| CN108017707A (en) * | 2017-12-12 | 2018-05-11 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide QPVLGPVRGP and its preparation method and application |
| CN110938129A (en) * | 2019-11-08 | 2020-03-31 | 上海交通大学 | Bioactive polypeptide SKLVPVGYGIRKL, and preparation method and application thereof |
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