CN110938128B - Bioactive polypeptide PKCPKCDKEVYFAERVTSL, and preparation method and application thereof - Google Patents
Bioactive polypeptide PKCPKCDKEVYFAERVTSL, and preparation method and application thereof Download PDFInfo
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- CN110938128B CN110938128B CN201911086868.7A CN201911086868A CN110938128B CN 110938128 B CN110938128 B CN 110938128B CN 201911086868 A CN201911086868 A CN 201911086868A CN 110938128 B CN110938128 B CN 110938128B
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- pkcpkcdkevyfaervtsl
- polypeptide
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- aging
- inflammatory
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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
-
- 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
-
- 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]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
-
- 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
- A61Q19/08—Anti-ageing preparations
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to the field of protein, and in particular relates to a bioactive polypeptide PKCPKCDKEVYFAERVTSL, a preparation method and application thereof, wherein the amino acid sequence of the bioactive polypeptide PKCPKCDKEVYFAERVTSL is Pro-Lys-Cys-Pro-Lys-Cys-Asp-Lys-Glu-Val-Tyr-Phe-Ala-Glu-Arg-Val-Thr-Ser-Leu. In vitro anti-inflammatory activity experiments and in vivo anti-aging experiments prove that the polypeptide PKCPKCDKEVYFAERVTSL has good anti-inflammatory function and anti-aging activity, and on one hand, the bioactive polypeptide PKCPKCDKEVYFAERVTSL disclosed by the invention can promote macrophages to secrete cytokines, improve the capability of an organism in resisting external pathogen infection and reduce the morbidity of the organism; on the other hand, the compound nematode feed has better anti-aging activity, can improve the reproductive capacity of nematodes and the quality of life, and has very important significance for developing foods, health-care products and medicines with anti-inflammatory and anti-aging functions.
Description
Technical Field
The invention relates to the field of proteins, in particular to a bioactive polypeptide PKCPKCDKEVYFAERVTSL, and a preparation method and application thereof.
Background
With the improvement of living standard, the requirement of people on diet changes from 'pursuit amount' to 'quality'. Therefore, research on bioactive peptides having specific functions has been hot. In recent years, some food-derived polypeptides, such as short peptides of corn, soybean peptides, milk polypeptides, etc., have been found to have good biological activity. And experiments prove that the health-care tea has the functions of resisting aging, bacteria and cancers, resisting inflammation, reducing blood pressure and the like.
The polypeptides can be obtained through various ways such as microbial fermentation, digestion and enzymolysis and the like, and most of the polypeptides with biological activity consist of 2-20 amino acid residues, have the molecular weight of less than 6000Da and contain a certain amount of hydrophobic amino acids and aromatic amino acids.
Macrophages are the second defense line of the body against the invasion of external harmful substances, are widely present in various tissues of the body, are main immune response cells, participate in biological functions such as immune response, anti-inflammation and the like through phagocytosis and secretion of cytokines, and play an important role in an immune system. The function of the polypeptide present in macrophages was investigated.
Currently, there are some researches on anti-aging bioactive peptides in the prior art, but new bioactive polypeptides having anti-oxidation or anti-aging functions different from the existing polypeptides are still the current direction of further research needed to further expand the diversity of bioactive polypeptides.
Disclosure of Invention
The invention aims to provide a bioactive polypeptide PKCPKCDKEVYFAERVTSL, 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 invention, a biologically active polypeptide PKCPKCDKEVYFAERVTSL is provided, having an amino acid sequence Pro-Lys-Cys-Pro-Lys-Cys-Asp-Lys-Glu-Val-Tyr-Phe-Ala-Glu-Arg-Val-Thr-Ser-Leu as shown in SEQ ID NO: 1 is shown.
Preferably, the bioactive polypeptide is mouse bone marrow-derived macrophage peptide. Specifically, the protein is derived from Cysteine-rich protein and is the amino acid residue at the 2 nd to 20 th positions of the Cysteine-rich protein. The amino acid sequence of Cysteine-rich protein is shown as SEQ ID NO: 2, respectively.
The amino acid sequence and the corresponding nucleotide sequence of Cysteine-rich protein are the existing technology, and the nucleotide fragment for coding the 2 nd to 20 th amino acid residues of the Cysteine-rich protein can code mature bioactive polypeptide PKCPKCDKEVYFAERVTSL.
Preferably, the bioactive polypeptide has anti-inflammatory and anti-aging functions.
In the second aspect of the present invention, a method for preparing the bioactive polypeptide PKCPKCDKEVYFAERVTSL is provided, which can be artificially synthesized by genetic engineering methods, can be directly obtained from cells by a separation and purification method, and can be directly prepared by chemical synthesis.
In a third aspect of the invention, an application of the bioactive polypeptide PKCPKCDKEVYFAERVTSL in preparing foods, health products, medicines or cosmetics with anti-inflammatory functions is provided.
In the fourth aspect of the invention, the application of the bioactive polypeptide PKCPKCDKEVYFAERVTSL in preparing food, health-care products or medicines with the anti-aging function is provided.
In the fifth aspect of the invention, the application of the bioactive polypeptide PKCPKCDKEVYFAERVTSL in preparing food, health-care products or medicines with anti-inflammatory and anti-aging functions is provided.
In particular, the biologically active polypeptide PKCPKCDKEVYFAERVTSL of the present invention can be used for preparing cosmetics for reducing free radical damage to skin, and medicines for anti-inflammation and/or anti-aging.
In a sixth aspect of the invention, there is provided an anti-inflammatory product comprising said biologically active polypeptide PKCPKCDKEVYFAERVTSL or a derivative of said biologically active polypeptide PKCPKCDKEVYFAERVTSL; the anti-inflammatory product comprises anti-inflammatory food, anti-inflammatory health product, anti-inflammatory drug or anti-inflammatory cosmetic; the derivative of the biologically active polypeptide PKCPKCDKEVYFAERVTSL refers to a polypeptide derivative obtained by performing modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on an amino acid side chain group, an amino terminal or a carboxyl terminal of the biologically active polypeptide PKCPKCDKEVYFAERVTSL.
In a seventh aspect of the invention, there is provided an anti-aging product comprising the biologically active polypeptide PKCPKCDKEVYFAERVTSL or a derivative of the biologically active polypeptide PKCPKCDKEVYFAERVTSL; the anti-aging product comprises anti-aging food, anti-aging health care product or anti-aging drug; the derivative of the biologically active polypeptide PKCPKCDKEVYFAERVTSL refers to a polypeptide derivative obtained by performing modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on an amino acid side chain group, an amino terminal or a carboxyl terminal of the biologically active polypeptide PKCPKCDKEVYFAERVTSL.
In the eighth aspect of the present invention, there is provided a product having both anti-inflammatory and anti-aging functions, comprising the biologically active polypeptide PKCPKCDKEVYFAERVTSL or a derivative of the biologically active polypeptide PKCPKCDKEVYFAERVTSL; products with anti-inflammatory and anti-aging effects include foods, health products or drugs; the derivative of the biologically active polypeptide PKCPKCDKEVYFAERVTSL refers to a polypeptide derivative obtained by performing modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on an amino acid side chain group, an amino terminal or a carboxyl terminal of the biologically active polypeptide PKCPKCDKEVYFAERVTSL.
The bioactive polypeptide PKCPKCDKEVYFAERVTSL has the following beneficial effects: the mouse bone marrow-derived macrophage bioactive polypeptide PKCPKCDKEVYFAERVTSL has good anti-inflammatory activity and anti-aging activity; on one hand, the bioactive polypeptide PKCPKCDKEVYFAERVTSL can promote macrophage to secrete cytokine, improve the capability of an organism to resist infection of external pathogens and reduce the morbidity of the organism; on the other hand, the compound nematode feed has better anti-aging activity, can improve the reproductive capacity of nematodes and the quality of life, and has very important significance for developing foods, health-care products and medicines with anti-inflammatory and anti-aging functions.
Drawings
FIG. 1: mass chromatogram extract (m/z = 776.3888);
FIG. 2: a secondary mass spectrum of a fragment with a mass to charge ratio of 776.3888;
FIG. 3: fragmentation of polypeptide az and by with mass-to-charge ratio of 776.3888;
FIG. 4: experimental results for the effect of biologically active polypeptide PKCPKCDKEVYFAERVTSL on the reproductive capacity of caenorhabditis elegans.
Detailed Description
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989 and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1 Artificial Synthesis of active peptide PKCPKCDKEVYFAERVTSL
Synthesis of bioactive peptide
Biologically active peptide PKCPKCDKEVYFAERVTSL was synthesized.
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-electrospray-quadrupole-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): 100-1000
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, the ultra-high performance liquid chromatography-electrospray-quadrupole-time-of-flight mass spectrometry is used for carrying out chromatographic analysis and mass spectrometric analysis on the bioactive peptide PKCPKCDKEVYFAERVTSL, the mass chromatogram extraction diagram is shown in figure 1, the secondary mass spectrogram of the peak and the az and by fracture conditions are shown in figures 2 and 3, the polypeptide mass-to-charge ratio of the peak is 776.3888Da, and the retention time is 37.4 min.
3) Results
As can be seen from FIG. 3, the fragment sequence with the mass-to-charge ratio of 776.3888Da obtained from the cases of az and by cleavage was Pro-Lys-Cys-Pro-Lys-Cys-Asp-Lys-Glu-Val-Tyr-Phe-
Ala-Glu-Arg-Val-Thr-Ser-Leu (PKCPKCDKEVYFARVTSL), SEQ ID NO: 1. the fragment corresponds to the 2 nd to 20 th residue sequences of Cysteine-rich protein, the GenBank number of the amino acid sequence of the Cysteine-rich protein is AAA37458.1, and the sequence is shown in SEQ ID NO: 2.
example 2 anti-inflammatory Activity assay of bioactive peptides
Experiment (ELISA method) for promoting macrophage to secrete cytokine by bioactive polypeptide PKCPKCDKEVYFAERVTSL
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 bone marrow macrophage-derived bioactive polypeptide PKCPKCDKEVYFAERVTSL obtained in example 1; ELISA cytokine Rapid kits (TNF-. alpha., IL-1. beta. and IL-6), Wuhan Dr bioengineering, Inc.
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 150 CO2 incubator Heraeus; dragon Wellscan MK3 microplate reader Labsystems.
2. The experimental method comprises the following steps:
the number of the added cells was 2X 106100 μ l/well of cell suspension/ml, 200 μ l/well of peptide-containing RPMI1640 complete medium (10% FBS) after adherent purification, LPS to a final concentration of 10 μ g/ml at 24 hours in the inflammation group, continuous culture for 48 hours, and LPS to a final concentration of 100ng/ml at 24 hours before termination of the 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 enzyme-linked plate coated with cytokine antibody, reacting at 37 deg.C for 90 min, adding biotin-labeled antibody, reacting at 37 deg.C for 60 min, washing with PBS, and washing with waterThe avidin-peroxidase complex was added and reacted 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 1 determination of the Effect of bioactive peptide PKCPKCDKEVYFAERVTSL on macrophage cytokine levels
| Experiment grouping | TNF-α | IL-1β | IL-6 |
| Cell blank | 0.140±0.021 | 0.450±0.010 | 1.309±0.036 |
| PKCPKCDKEVYFAERVTSL0.5mg/ml | 0.865±0.205** | 0.558±0.026* | 2.596±0.143** |
| PKCPKCDKEVYFAERVTSL0.2mg/ml | 0.292±0.242** | 0.595±0.058** | 1.622±0.013 |
Note: significant difference compared to negative control (P < 0.05); the difference in the negative control group was very significant (P < 0.01)
As can be seen from Table 1, in the experimental results of three cytokines, namely TNF-alpha, IL-1 beta and IL-6, significant differences (P < 0.01) appear between TNF-alpha and IL-1 beta at 0.2mg/ml and above, and significant differences (P < 0.01) appear between IL-6 at 0.5mg/ml, which proves that PKCPKCDKEVYFAERVTSL at a certain concentration can promote the activation of macrophages in abdominal cavity of mice and release TNF-alpha, IL-1 beta and IL-6, and the bottom values of the cytokines in the resting state of normal macrophages are improved, so that the immunity of the body is adjusted.
Example 3 anti-aging Activity assay of bioactive peptides
Experiment on influence of bioactive polypeptide PKCPKCDKEVYFAERVTSL on reproductive capacity of caenorhabditis elegans
1. Experimental reagents and instruments:
reagent: caenorhabditis elegans, subsidiary of the institute for combined Chinese and Western medicine, university of Compound Dane; coli OP50, subsidiary of the university of fudan; agar powder, national drug group chemical reagents limited; yeast powder, national drug group chemical reagents limited; the mouse bone marrow macrophage-derived bioactive peptide PKCPKCDKEVYFAERVTSL obtained in example 1.
The instrument equipment comprises: likang RO15 pure water system, Likang biomedical science and technology, Inc.; model G136T Zealway intelligent high temperature sterilization pot, xiamen micro instrument science and technology ltd; THZ-32 type desk type constant temperature oscillator, shanghai smart dense testing equipment ltd; TDL-40B centrifuge, Shanghai' an pavilion scientific instrument factory; luxiang apparatus GL-22M high speed refrigerated centrifuge, Shanghai Luxiang apparatus instruments Ltd; boxun BJ-CD SERIES biosafety cabinet, Shanghai Boxun industries, Inc.; nikko inverted Electron microscope, Nikon corporation.
2. The experimental method comprises the following steps:
(1) preparation of NGM plate
Taking an escherichia coli strain to streak on an LB plate, picking a single colony in 10ml of an LB liquid culture medium, performing shaking culture at 37 ℃ and 200rpm for 24h until OD600=0.4 is used for inoculating NGM plates to feed nematodes. 100 mu L of bacterial liquid is applied to a 60mm NGM plate, and the distance between the edge of the bacterial liquid and the edge of the plate is about 0.5 cm. The coated NGM plates were ready for use overnight at room temperature (21-25 ℃).
(2) Nematode culture
The nematodes used in the experiment are hermaphrodite and grow under standard culture conditions (temperature 20 ℃, humidity 40-60%).
(3) Synchronization treatment of nematodes
1) Bleaching with sodium perchlorate
Preparing a pregnant insect growth plate (more than 80% of insects in the plate are in a reproductive period) 2-3 plates, washing 5ml of M9 buffer solution for 2 times, sucking the buffer solution into a 15ml centrifuge tube, centrifuging at 1000 r/min for 3min, and discarding the supernatant. 5ml of fresh contemporaneous bleaching solution was added and shaken vigorously at room temperature for 2.5min to erode the adult worms. Centrifuged and the supernatant discarded. Ensuring that the total treatment time cannot exceed 5min and preventing insect eggs from being damaged. And adding M9 buffer solution to resuspend the precipitate, mixing uniformly, centrifuging, discarding supernatant, and repeating the process for 3 times.
2) Time-limited spawning method
Selecting a plurality of nematodes in the egg laying period in the same plate, wherein the specific quantity is based on the number of the nematodes needing to be synchronized. Under general conditions, one nematode can lay eggs for about 6 within 1 h. After 0.5h incubation in the plates, the nematodes were picked out of the plates and the eggs in the plates were in the same growth phase.
(4) Index measurement
In the experiment, caenorhabditis elegans is used as an animal model, and L4 stage nematodes after the synchronization treatment are picked into NGM plates with corresponding concentrations. Each concentration of at least 8 nematodes, one for each NGM plate, and recorded as day 0, after which the plate is moved to a new plate every day until the nematodes basically no longer lay eggs, and the total number of eggs laid by the nematodes is counted before they enter the egg laying period.
3. Experimental results and analysis:
the results are shown in FIG. 4, which shows that the average egg production is increased to different extents in the groups fed with different concentrations of polypeptide PKCPKCDKEVYFAERVTSL compared with the blank group not fed with polypeptide PKCPKCDKEVYFAERVTSL. The average number of eggs laid by the nematodes is very significantly different (P < 0.01) when the fed polypeptide PKCPKCDKEVYFAERVTSL is 300mg/L compared with the blank group, and only significantly different (P < 0.05) when the fed polypeptide PKCPKCDKEVYFAERVTSL is 400mg/L and 500mg/L compared with the blank group, which further proves that 300mg/L is the optimal concentration of the mixed peptide polypeptide PKCPKCDKEVYFAERVTSL, and the effect of the mixed peptide polypeptide is reduced without inhibiting the reproduction of the nematodes as the concentration of the peptide is increased. In conclusion, the polypeptide PKCPKCDKEVYFAERVTSL can obviously improve the reproductive capacity of the nematode under a certain concentration. Meanwhile, the experimental result shows that the polypeptide PKCPKCDKEVYFAERVTSL 300mg/L is the optimal concentration. However, with increasing concentration, the reproductive capacity of the nematodes is no longer significantly improved.
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> Shanghai university of transportation; zhejiang ghui peptide Life health science and technology Limited
<120> a bioactive polypeptide PKCPKCDKEVYFAERVTSL, and its preparation method and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Pro Lys Cys Pro Lys Cys Asp Lys Glu Val Tyr Phe Ala Glu Arg Val
1 5 10 15
Thr Ser Leu
<210> 2
<211> 77
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Pro Lys Cys Pro Lys Cys Asp Lys Glu Val Tyr Phe Ala Glu Arg
1 5 10 15
Val Thr Ser Leu Gly Lys Asp Trp His Arg Pro Cys Leu Lys Cys Glu
20 25 30
Lys Cys Gly Lys Thr Leu Thr Ser Gly Gly His Ala Glu His Glu Gly
35 40 45
Lys Pro Tyr Cys Asn His Pro Cys Tyr Ser Ala Met Phe Gly Pro Lys
50 55 60
Gly Phe Gly Arg Gly Gly Ala Glu Ser His Thr Phe Lys
65 70 75
Claims (3)
1. A bioactive polypeptide PKCPKCDKEVYFAERVTSL, characterized in that its amino acid sequence is Pro-Lys-Cys-Pro-Lys-Cys-Asp-Lys-Glu-Val-Tyr-Phe-Ala-Glu-Arg-Val-Thr-Ser-Leu.
2. A polynucleotide encoding the biologically active polypeptide PKCPKCDKEVYFAERVTSL of claim 1.
3. The method of claim 1, wherein the biologically active polypeptide PKCPKCDKEVYFAERVTSL is produced directly by chemical synthesis.
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