CN107903316B - Bioactive polypeptide LPYPYYA and preparation method and application thereof - Google Patents
Bioactive polypeptide LPYPYYA and preparation method and application thereof Download PDFInfo
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- CN107903316B CN107903316B CN201711320807.3A CN201711320807A CN107903316B CN 107903316 B CN107903316 B CN 107903316B CN 201711320807 A CN201711320807 A CN 201711320807A CN 107903316 B CN107903316 B CN 107903316B
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- lpypyya
- aging
- polypeptide
- bioactive polypeptide
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Abstract
The invention relates to the field of protein, and in particular relates to a bioactive polypeptide LPYPYYA and a preparation method and application thereof. Through in vitro immune function regulation experiments and in vivo anti-aging experiments, the polypeptide LPYPYYA is verified to have better immune regulation function and anti-aging activity, on one hand, the bioactive polypeptide LPYPYYA can enhance the in vitro proliferation capacity of lymphocytes and macrophages, improve the capacity of an organism for resisting external pathogen infection, and reduce the morbidity of the organism; on the other hand, the activity of an anti-peroxidase system in vivo can be improved, and the function of resisting exogenous stimulation of an organism is enhanced, so that the probability of aging, aging and illness of the organism is reduced, and the method has very important significance for developing foods, health-care products and medicines with immunoregulation function and anti-aging function.
Description
Technical Field
The invention relates to the field of protein, in particular to a bioactive polypeptide LPYPYYA and a preparation method and application thereof.
Background
In the process of fermenting the cow milk by the lactic acid bacteria, a part of protein in the cow milk is metabolized and utilized by the lactic acid bacteria, and a series of physiological and biochemical reactions occur, so that the protein is changed into polypeptide or free amino acid which is digested and absorbed by a human body or directly enters the blood circulation of the human body through the absorption and transportation of small intestinal epithelial cells. Among these polypeptides, some have a specific physiological function and are called "bioactive peptides".
It is particularly important to find safe bioactive peptides in natural food sources. 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. 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.
Immunoactive peptides are a class of bioactive polypeptides that are first obtained from milk following opioid peptide discovery and demonstrate 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. Lemna hexandra et al, fed rats with synthetic milk-derived immunoregulatory peptide (PGPIPN), found that phagocytosis of macrophages in the abdominal cavity of rats and immunoregulatory function related to erythrocytes were significantly enhanced.
Researches show that the immune active peptide can not only enhance the immunity of the organism, stimulate the proliferation of lymphocytes of the organism, enhance the phagocytic function of macrophages, promote the release of cell factors, improve the capability of the organism for resisting the infection of external pathogens, reduce the morbidity of the organism, but also can not cause the immune rejection reaction of the organism.
Aging is a natural phenomenon, and the process is often accompanied by the changes of antioxidant level, organ tissues and immune factors, wherein the cytokines are changed in a complex way, such as proinflammatory cytokines IL-6, IL-4, TNF- α and the like show a growing trend, and IL-6 and TNF-a are considered to play important roles in the process of the senile diseases.
The anti-aging peptide has the advantages that the anti-aging peptide is a novel anti-aging agent, has incomparable advantages with amino acid in the aspect of physiological function, can promote or inhibit enzymes in organisms, improve the absorption and utilization of minerals and other nutrient elements, clear away free radicals in the bodies, enhance the self anti-oxidation capability of the organisms and delay aging. Therefore, the nutrition and health care effects of bioactive peptides have become the focus of research on the subjects of scholars at home and abroad. Experiments and researches by meaningful people find that the milk-derived bioactive small peptide can effectively prolong the life of the drosophila and delay the aging of the drosophila, and has better antioxidation effect, and presumably is rich in thiopeptides. The results of Zhou Zhi Hui et al show that the bovine colostrum extract can obviously improve the SOD activity in serum of the elderly, reduce lipid peroxides of the SOD, enhance the oxidation resistance of organisms and have certain anti-aging function.
At present, there are many researches on bioactive polypeptides, for example, chinese patent CN105254738A discloses a milk-derived bioactive polypeptide DELQDKIH derived from β -casein, chinese patent CN105254739A discloses a milk-derived bioactive polypeptide GTQYTD derived from α s 1-casein, and chinese patent CN105254740A discloses a milk-derived bioactive polypeptide NQFYQKF derived from α s 2-casein.
Chinese patent CN1566152A discloses an immunoactive peptide with antibacterial activity, which is named as "tyrosine-rich polypeptide (Trpi). The amino acid sequence is as follows: Cys-Glu-Lys-Asp-Glu-Arg-Phe-Phe-Ser-Asp-Lys-Ile-Ala-Lys-Tyr-Ile-Pro-Ile-Gln-Tyr-V al-Leu-Ser-Arg-Tyr-Pro-Ser-Tyr-Gly-Leu-Asn-Tyr-Gln-Gln-Lys-Pro-Val-Ala-Leu-Ile-Asn-Asn-Gln-Phe-Leu-Pro-Tyr-Pro-Tyr-Tyr-Ala-Lys. The polypeptide of this patent is part of bovine casein. This patent discloses a macromolecular substance which is poorly digestible and poorly absorbable, while the macromolecular biological activity disclosed therein is immunologically active and does not disclose whether it has other properties.
Disclosure of Invention
The invention aims to provide a bioactive polypeptide LPYPYYA 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 bioactive polypeptide LPYPYYA is provided, the amino acid sequence of which is Leu-Pro-Tyr-Pro-Tyr-Tyr-Ala, as shown in SEQ ID NO: 1 is shown.
Preferably, the biologically active polypeptide is milk-derived. Particularly derived from kappa-casein, and is the 77 th to 83 th amino acid residues of the kappa-casein variant A. The amino acid sequence of the kappa-casein variant A is shown as SEQ ID NO: 3, respectively.
The amino acid sequence and the corresponding nucleotide sequence of the kappa-casein are the prior art, and the nucleotide fragment for coding the 77 th-83 th amino acid residues of the kappa-casein variant A can code a mature bioactive polypeptide LPYPYYA.
Preferably, the bioactive polypeptide has an immunoregulatory function and an anti-aging function.
In a second aspect of the present invention, there is provided a nucleotide fragment encoding said biologically active polypeptide, LPYPYYA, having the sequence: 5'-ctg cca tac cca tat tat gca-3', as shown in SEQ ID NO: 2, respectively.
In the third aspect of the invention, the preparation method of the bioactive polypeptide LPYPYYA is provided, which can be artificially synthesized by a genetic engineering method, can be directly obtained from a dairy product by a separation and purification method, and can be directly prepared by chemical synthesis.
In the fourth aspect of the invention, the application of the bioactive polypeptide LPYPYYA in preparing food, health-care products, medicines or cosmetics with immunoregulation function is provided.
In the fifth aspect of the invention, the application of the bioactive polypeptide LPYPYYA in preparing food, health-care products or medicines with anti-aging function is provided.
The sixth aspect of the invention provides the application of the bioactive polypeptide LPYPYYA in preparing food, health-care products or medicines with immune regulation function and anti-aging function.
Specifically, the bioactive polypeptide LPYPYYA can be used for preparing cosmetics for reducing free radical damage to skin, medicines for regulating immunity and/or resisting aging; and because the product of the bioactive polypeptide LPYPYYA of the invention after being degraded by gastrointestinal tract still has bioactivity, the bioactive polypeptide LPYPYYA can also be used for preparing foods such as yoghourt and health products for regulating immunity, and can be used for preparing medicines with immunoregulation and/or anti-aging by oral administration.
In a seventh aspect of the invention, there is provided an immunomodulatory product comprising the biologically active polypeptide LPYPYYA, or a derivative of the biologically active polypeptide LPYPYYA; the immunoregulation product comprises immunoregulation food, immunoregulation health-care products, immunoregulation medicaments or immunoregulation cosmetics; the derivative of the bioactive polypeptide LPYPYYA refers to a polypeptide derivative obtained by carrying out modification 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 bioactive polypeptide LPYPYYA.
In an eighth aspect of the invention, there is provided an anti-ageing product comprising the biologically active polypeptide LPYPYYA or a derivative of the biologically active polypeptide LPYPYYA; the anti-aging product comprises anti-aging food, anti-aging health care product or anti-aging drug; the derivative of the bioactive polypeptide LPYPYYA refers to a polypeptide derivative obtained by carrying out modification 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 bioactive polypeptide LPYPYYA.
In the ninth aspect of the invention, a product having both immunoregulatory and anti-aging functions is provided, comprising the bioactive polypeptide lpyya or a derivative of the bioactive polypeptide lpyya; products with immunoregulatory and anti-aging functions include foods, health products or drugs; the derivative of the bioactive polypeptide LPYPYYA refers to a polypeptide derivative obtained by carrying out modification 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 bioactive polypeptide LPYPYYA.
The bioactive polypeptide LPYPYYA has the beneficial effects that: the milk-derived bioactive polypeptide LPYPYYA has good activity of regulating the immunity of organisms and anti-aging activity; on one hand, the bioactive polypeptide LPYPYYA can enhance the in-vitro proliferation capacity of lymphocytes and macrophages, improve the capability of an organism for resisting infection of external pathogens and reduce the morbidity of the organism; on the other hand, the activity of an anti-peroxidase system in vivo can be improved, and the function of resisting exogenous stimulation of an organism is enhanced, so that the probability of aging, aging and illness of the organism is reduced, and the method has very important significance for developing dairy products and health care products with immunoregulation function and anti-aging function.
The polypeptide of the present application is essentially different from the prior art (e.g., rich in the tyrosine polypeptide Trpi in the background art) in structure and function: the bioactive polypeptide LPYPYYA is a small molecular bioactive fragment, and belongs to a core fragment; the bioactive polypeptide LPYPYYA of the invention has more digestibility and absorbability. Meanwhile, the bioactive polypeptide LPYPYYA has the functions of immunoregulation and anti-aging, and has obvious difference in functional activity from the polypeptide disclosed in the prior art.
Drawings
FIG. 1: mass chromatogram extraction (m/z 886.4363);
FIG. 2: a secondary mass spectrum of a fragment with a mass to charge ratio of 886.4363;
FIG. 3: fragmentation of polypeptide az and by with mass-to-charge ratio of 886.4363;
FIG. 4: spleen change of each group of experimental animal mice;
(a) spleen tissue maps of mice in the low-dose gavage group; (b) spleen tissue maps of mice in the high-dose gavage group; (c) spleen tissues of blank mice; (d) is a spleen tissue map of mice in an animal model group;
FIG. 5: serum IL-6 profiles for each group of mice;
FIG. 6 is a table of changes in serum TNF- α for each group of mice;
FIG. 7: serum IL-2 changes in each group of mice.
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: ALABORATORY MANUAL, Second edition, Cold Spring harbor laboratory Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS Inmolecular BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATINSTRUCUTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) Methods Inenzymolygy, 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 an active peptide, LPYPYYA
Synthesis of bioactive peptide
1.3 g 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 Leu and a proper amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, adding 20ml of DMF to dissolve the amino acid Leu and the 1-hydroxy-benzotriazole (HOBT), then adding 3ml of N, N diisopropyl carbodiimide (DIC) to shake and shake for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor into a30 ℃ shaking table to react.
After 6.2 hours, the column was capped with a suitable amount of acetic anhydride (acetic anhydride: DIEA: DCM ═ 1:1:2, v: v: v) for half an hour, then washed four times with 3 resin volumes of DMF and drained until needed.
7. The Fmoc protecting group on the resin was removed by adding a quantity of 20% piperidine (piperidine/DMF: 1:4, v: v) to the reactor and shaking on a decolourising shaker for 20 min. After deprotection was washed four times with DMF and then drained.
8. And (3) detecting a small amount of resin by a ninhydrin (ninhydrin) method (detecting A and B, respectively, and reacting at 100 ℃ for 1min), wherein the resin is colored, which indicates that the deprotection is successful.
9. Weighing a second proper amount of amino acid and a proper amount of HOBT in a 50ml centrifuge tube, adding 25ml of DMF to dissolve the amino acid and the HOBT, adding 2.5ml of DIC to shake and shake for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor in a shaking table at 30 ℃ to react.
After 10.1 hours, a small amount of resin is taken for detection, and the detection is carried out by an indanthrone method (two drops are respectively detected A and B, and the reaction is carried out for 1min at 100 ℃), if the resin is colorless, the reaction is complete; if the resin is colored, the condensation is not complete and the reaction is continued.
11. After the reaction was completed, the resin was washed four times with DMF and then drained, and a certain amount of 20% piperidine (piperidine/DMF ═ 1:4, v: v) was added to the reactor, and the mixture was shaken on a decolorizing shaker for 20min to remove the Fmoc-protecting group from the resin. After the protection is removed, washing with DMF for four times, and then draining to detect whether the protection is removed.
12. And sequentially grafting amino acids Pro, Tyr and Ala according to the steps 9-11.
13. After the last amino acid had been grafted, the protection was removed, washed four times with DMF and the resin was drained with methanol. The polypeptide was then cleaved from the resin with 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).
Thus, the bioactive peptide LPYPYYA is artificially 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)
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, 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 LPYPYYA, 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 886.4363Da, and the retention time is 55.0 min.
3) Results
As can be seen from FIG. 3, according to the cases of az and by breakage, the fragment sequence with the mass-to-charge ratio of 886.4363Da, which is Leu-Pro-Tyr-Pro-Tyr-Ala (LPYPYYA), is obtained by analysis and calculation of Mascot software and is marked as SEQ ID NO: 1. the fragment corresponds to residue sequences of 77 th to 83 th sites of a kappa-casein variant A, the GenBank number of a kappa-casein amino acid sequence is AAA30433.1, and the sequence is shown in SEQ ID NO: 3.
example 2 experiment of the Activity of bioactive peptides in regulating the Immunity of the organism
First, MTT method for testing in vitro lymphocyte proliferation capacity experiment of bioactive polypeptide LPYPYYA
1. Experimental materials and instruments:
reagents and materials: experimental animals balb/c mice (male 6-8 weeks old, animal experiment center of Shanghai university of transportation, college of agriculture and biology); the milk-derived bioactive polypeptide LPYPYYA obtained in example 1; mouse lymphocyte extract (ex solibao); RPMI1640 medium (purchased from GIBCO); 3- (4, 5-Dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide salt (MTT, available from Amresco, Inc.); concanavalin (ConA, available from Sigma); bovine serum albumin (BSA, available from Genebase); pepsin (available from Sigma); pancreatin (Corolase PP, from AB).
The instrument equipment comprises: LRH-250F Biochemical incubator, Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge, shanghai luxiang instrument centrifuge instruments ltd; hera cell 150CO2 incubator, Heraeus; DragonWellscan MK3 microplate reader, Labsystems corporation; ALPHA1-2-LD vacuum freeze drier, Christ company; ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometer, waters corporation.
2. The experimental method comprises the following steps:
taking mouse spleen under aseptic condition, extracting mouse lymphocyte with lymphocyte extract, and performing primary culture. The cell density was adjusted to 2.5X 10 with complete RPMI1640 medium6one/mL. To a 96-well cell culture plate were added in sequence: 100 μ L mouse lymphocyte suspension, 100 μ L RPMI1640 complete medium, 20 μ L concanavalis canadensisProtein, 100 μ L sample. In addition, a blank control group (PBS with pH7.2-7.4 and 3 mol/L) and a negative control group (500 mu g/mL BSA) are arranged, and the research shows that the blank control group has no influence on the in vitro lymphocyte proliferation. Each set of 3 replicates. At 5% CO2Culturing at 37 deg.C for 68h, adding 20 μ L MTT into each well under aseptic condition, culturing for 4h, carefully removing supernatant, adding 100 μ L dimethyl sulfoxide into each well, incubating at 37 deg.C for 10min, shaking, and measuring absorbance at 570nm with microplate reader.
The in vitro lymphocyte proliferation capacity is expressed by a stimulation index and is calculated as follows:
in the formula: a. the1Absorbance at 570nm for the blank; a. the2Absorbance at 570nm for the negative control, A3The absorbance at 570nm for the experimental group.
3. Experimental results and analysis:
TABLE 1 Effect of the bioactive polypeptide LPYPYYA on lymphocyte proliferation in vitro
| Experiment grouping | Stimulation index SI |
| |
1 |
| LPYPYYA | 1.173±0.053* |
Note: the number marked as significant difference (P <0.05) compared to the negative control.
The results are shown in Table 1. As can be seen from Table 1, the stimulation index of the milk-derived bioactive peptide LPYPYYA was greater than that of BSA at a mass concentration of 100. mu.g/mL of the bioactive peptide LPYPYYA, indicating that LPYPYYA can stimulate the proliferation of lymphocytes in vitro mice to some extent. And the stimulation index of LPYPYYA reaches 1.173, which is obviously different from that of a negative control group (P < 0.05). Therefore, the active polypeptide LPYPYYA can be considered to have the capacity of remarkably promoting the mouse lymphocyte proliferation, can be eaten as a health-care product or an additive, and can improve the immunity of animals and human bodies.
Second, MTT method for testing in vitro macrophage proliferation capacity of bioactive polypeptide LPYPYYA
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 milk-derived bioactive polypeptide LPYPYYA 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 WellscanMK3 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 polypeptide LPLP (1mg/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:
wherein the blank culture solution is RPMI1640 complete culture solution containing 10% FBS.
3) Results and analysis of the experiments
TABLE 2 Effect of the biologically active polypeptide LPYPYYA on macrophage proliferation in vitro
| Experiment grouping | Normal group GI | GI inflammation group |
| |
1 | 1 |
| LPYPYYA(1mg/ml) | 1.0842±0.0562** | 1.1632±0.0764** |
Note: indicates a significant difference (P <0.05) compared to the negative control; indicates that there is a significant difference (P <0.01) compared with the negative control group
The results are shown in Table 2, and it is clear from Table 2 that macrophages were proliferated in both the normal group and the inflammatory group in the presence of the bioactive polypeptide LPYPYYA at a concentration of 1 mg/ml. And compared with a negative control group, the two groups have significant difference (P is less than 0.01). The biological active polypeptide LPYPYYA has obvious proliferation effect on in vitro macrophage.
Example 3 anti-aging Activity assay of bioactive peptides
Experiment of effect of bioactive polypeptide LPYPYYA on in-vivo spleen tissue structure
1. Experimental reagents and instruments:
reagent: experimental animal ICR mouse (male 5 weeks old), shanghai city experimental animal center; d-gal, national pharmaceutical group chemical reagents, Inc.; paraformaldehyde, chemical reagents of the national drug group, ltd; sodium chloride, national pharmaceutical group chemical reagents ltd; the milk-derived bioactive polypeptide LPYPYYA obtained in example 1.
The instrument equipment comprises: model CM-230 Mohr super Water, Shanghai Mole scientific instruments, Inc.; milliporem Milllex GP0.22 μm filter membrane, Millipore, USA; GL-22M high-speed refrigerated centrifuge, Shanghai Luxiang apparatus centrifuge instruments Inc.
2. The experimental method comprises the following steps:
(1) model for animal aging
After one week of adaptive ICR mouse feeding, 4 groups of 6 mice were divided. Group 1 is a low-dose intragastric group, and the mice are injected with D-gal subcutaneously in the neck and back at a dose of 500mg/kg every day, and the bioactive polypeptide LPYPYYA is intragastric at a dose of 1 mg/day; group 2 was a high-dose gavage group, mice were injected subcutaneously in the neck and back at a dose of 500mg/kg daily and the bioactive polypeptide LPYPYYA was gavage at a daily dose of 3 mg/mouse; group 3 was blank, mice grew normally; group 4 was an animal model group, and mice were injected subcutaneously into the neck and back with D-gal at a dose of 500mg/kg daily, and gavage with 0.9% normal saline; the injection period of D-gal and the gavage period of polypeptide were 42 days. The bedding is replaced every 3 days and the feed and distilled water supply is ensured. The weight of the mice was weighed once every five days, D-gal injection was prepared according to the weight of the mice, and the D-gal injection was filtered through a 0.22 μm syringe filter to ensure sterility.
(2) Obtaining animal viscera
After the experiment period is finished, blood of a mouse is obtained by an eyeball-picking blood-taking method, the mouse is killed by breaking the neck after the blood is obtained, then a body of the mouse is placed on a low-temperature ice box, the brain, the spleen, the liver and the kidney of the mouse are quickly picked, the obtained viscera are placed in a pre-sterilized 1.5mL centrifuge tube, and all organ samples are stored in a refrigerator at the temperature of-80 ℃ for inspection. All procedures in the procedure of treating the experimental animals followed the guidance comments on the animals being treated in good care published by the department of scientific technology in 2006. The spleen of the mouse is directly soaked in a prepared 4% paraformaldehyde solution to fix the shape. The paraformaldehyde powder is relatively insoluble, and a trace amount of sodium bicarbonate can be added to adjust the pH value to be alkaline so as to aid dissolution. The preparation of the paraformaldehyde solution needs to be completed in a fume hood.
(3) Sample detection
Preparation of tissue sections: mouse spleen samples were fixed in 4% paraformaldehyde solution for at least 24 hours. The preparation of wax block, slicing and HE staining of spleen tissue were completed by Shanghai Weiao Biotech Co., Ltd.
3. Experimental results and analysis:
in this experiment, there were 4 groups of mice, of which the blank group did not undergo any external stimulation for normal growth, and the remaining 3 groups received long-term injections of D-gal. By observing spleen sections of different groups of mice by using an optical microscope, as can be seen from fig. 4, compared with spleen sections of each group of mice, compared with blank groups of mice, spleen red marrow and white marrow of animal model mice have fuzzy boundaries and atrophy of the white marrow, which indicates that long-term D-gal injection causes sugar metabolism pathways of the mice to be disordered, so that the antioxidant enzyme activity is reduced, peroxide is accumulated, and further spleen aging and atrophy are possibly caused. Compared with the mice of the animal model group, the spleen tissues of the mice of the gavage polypeptide group have lighter atrophy degree of the white marrow and have better boundary between the red marrow and the white marrow. This result suggests that the experimental animals are continuously stimulated by the senescence-causing factor throughout the injection cycle of D-gal, resulting in senescence and atrophy of the spleen. Therefore, from the aspect of tissue structure change, the bioactive polypeptide LPYPYYA invented in the experiment has certain protection effect on spleen aging and atrophy caused by stimulation of adverse factors.
Second, experiment of bioactive polypeptide LPYPYYA on antioxidant level of in vivo organ
1. Experimental reagents and instruments:
reagent: experimental animal ICR mouse (male 5 weeks old), shanghai city experimental animal center; d-gal, national pharmaceutical group chemical reagents, Inc.; paraformaldehyde, chemical reagents of the national drug group, ltd; sodium chloride, national pharmaceutical group chemical reagents ltd; the milk-derived bioactive polypeptide LPYPYYA obtained in example 1; BCA protein kit, Nanjing Kaikyi Biotech Co., Ltd; SOD superoxide dismutase kit, Nanjing, biological technology Limited; T-AOC total antioxidant activity kit, Nanjing, established Biotechnology Limited.
The instrument equipment comprises: model CM-230 Mohr super Water, Shanghai Mole scientific instruments, Inc.; milliporem Milllex GP0.22 μm filter membrane, Millipore, USA; GL-22M high-speed refrigerated centrifuge, Shanghai Luxiang apparatus centrifuge instruments Inc.
2. The experimental method comprises the following steps:
(1) model for animal aging
After one week of adaptive ICR mouse feeding, 4 groups of 6 mice were divided. Group 1 is a low-dose intragastric group, and the mice are injected with D-gal subcutaneously in the neck and back at a dose of 500mg/kg every day, and the bioactive polypeptide LPYPYYA is intragastric at a dose of 1 mg/day; group 2 was a high-dose gavage group, mice were injected subcutaneously in the neck and back at a dose of 500mg/kg daily and the bioactive polypeptide LPYPYYA was gavage at a daily dose of 3 mg/mouse; group 3 was blank, mice grew normally; group 4 was an animal model group, and mice were injected subcutaneously into the neck and back with D-gal at a dose of 500mg/kg daily, and gavage with 0.9% normal saline; the injection period of D-gal and the gavage period of polypeptide were 42 days. The bedding is replaced every 3 days and the feed and distilled water supply is ensured. The weight of the mice was weighed once every five days, D-gal injection was prepared according to the weight of the mice, and the D-gal injection was filtered through a 0.22 μm syringe filter to ensure sterility.
(2) Obtaining animal viscera
After the experiment period is finished, blood of a mouse is obtained by an eyeball-picking blood-taking method, the mouse is killed by breaking the neck after the blood is obtained, then a body of the mouse is placed on a low-temperature ice box, the brain, the spleen, the liver and the kidney of the mouse are quickly picked, the obtained viscera are placed in a pre-sterilized 1.5mL centrifuge tube, and all organ samples are stored in a refrigerator at the temperature of-80 ℃ for inspection. All procedures in the procedure of treating the experimental animals followed the guidance comments on the animals being treated in good care published by the department of scientific technology in 2006. The spleen of the mouse is directly soaked in a prepared 4% paraformaldehyde solution to fix the shape. The paraformaldehyde powder is relatively insoluble, and a trace amount of sodium bicarbonate can be added to adjust the pH value to be alkaline so as to aid dissolution. The preparation of the paraformaldehyde solution needs to be completed in a fume hood.
(3) Sample detection
Grinding all organs to be detected in a low-temperature environment, diluting the ground organs into 10% tissue homogenate by using a 4 ℃ sterile PBS solution, centrifuging 4000g at 4 ℃, sucking and taking supernate, removing precipitates, and operating according to a kit instruction or placing the mixture in a refrigerator at minus 80 ℃ for detection.
3. Experimental results and analysis:
TABLE 3 variation of SOD content in different organs of each group of experimental animal mice
Note: significant differences (P <0.05) in plots compared to model group controls; the plot showed significant differences (P <0.01) compared to the model group control, as follows.
As can be seen from table 3, the SOD content in the liver and kidney of the mice in the peptide gavage group showed significant increase (P <0.01) compared to the mice in the animal model group. The fact that the mice in the polypeptide gavage group are stimulated by large dose of D-gal for a long time and the SOD enzyme system in the mice is not completely destroyed even if the D-gal is excessively injected indicates that the experimental animals are continuously stimulated by the aging-causing factors in the injection period to reduce the SOD content in different organs, but the mice are protected from oxidative damage by taking a certain amount of the polypeptide LPYPYYA.
TABLE 4 Change in T-AOC in groups of Experimental animals mice
As can be seen from Table 4, the liver T-AOC value of the mice in the animal model group is 0.68 +/-0.21U/mgprot, and compared with the mice in the model group, the mice in the high-dose and low-dose gavage groups of the polypeptide show significant difference (P < 0.05); the kidney T-AOC content of the blank group of mice is 0.61 +/-0.25U/mgprot, and compared with the mice in the animal model group, the mice in the low-dose intragastric group show significant difference (P <0.05), and the mice in the high-dose intragastric group also show significant difference (P < 0.01). The results show that in the whole experimental period, because the experimental animals are continuously stimulated by the senescence-causing factors, the liver and kidney tissues of the mice in the animal model group are damaged, so that the total antioxidant capacity of the mice is reduced. Compared with animal model group and blank group, the total antioxidant capacity of main organs of mice in polypeptide gavage group is always maintained at a higher level in the process of being stimulated by aging-causing factors, which indicates that the animal body and the main organs thereof have higher self-protection function by taking the bioactive polypeptide LPYPYYA.
Experiment of bioactive polypeptide LPYPYYA on effect of immune cell factor in serum
1. Experimental reagents and instruments:
the reagent comprises an experimental animal ICR mouse (male 5 weeks old), Shanghai's center for experimental animals, D-gal, national drug group chemical reagent limited, paraformaldehyde, national drug group chemical reagent limited, sodium chloride, national drug group chemical reagent limited, the milk-derived bioactive polypeptide LPYPYYA obtained in example 1, a BCA protein kit, Nanjing Kayji biotechnology limited, an ELISA cytokine rapid kit (TNF- α, IL-2 and IL-6) and Wuhan doctor biological engineering limited.
The instrument equipment comprises: model CM-230 Mohr super Water, Shanghai Mole scientific instruments, Inc.; milliporem Milllex GP0.22 μm filter membrane, Millipore, USA; GL-22M high-speed refrigerated centrifuge, Shanghai Luxiang apparatus centrifuge instruments Inc.
2. The experimental method comprises the following steps:
(1) model for animal aging
After one week of adaptive ICR mouse feeding, 4 groups of 6 mice were divided. Group 1 is a low-dose intragastric group, and the mice are injected with D-gal subcutaneously in the neck and back at a dose of 500mg/kg every day, and the bioactive polypeptide LPYPYYA is intragastric at a dose of 1 mg/day; group 2 was a high-dose gavage group, mice were injected subcutaneously in the neck and back at a dose of 500mg/kg daily and the bioactive polypeptide LPYPYYA was gavage at a daily dose of 3 mg/mouse; group 3 was blank, mice grew normally; group 4 was an animal model group, and mice were injected subcutaneously into the neck and back with D-gal at a dose of 500mg/kg daily, and gavage with 0.9% normal saline; the injection period of D-gal and the gavage period of polypeptide were 42 days. The bedding is replaced every 3 days and the feed and distilled water supply is ensured. The weight of the mice was weighed once every five days, D-gal injection was prepared according to the weight of the mice, and the D-gal injection was filtered through a 0.22 μm syringe filter to ensure sterility.
(2) Obtaining animal viscera and serum
After the experiment period is finished, blood of the mouse is obtained by an eyeball-picking blood-taking method, the mouse is killed by breaking the neck after the blood is obtained, then the body of the mouse is placed on a low-temperature ice box, the blood of the mouse is stood for 1 hour at room temperature, and then is centrifuged for 15min at 3000g, and serum is separated. The serum was stored in a freezer at-80 ℃ for testing. All procedures in the procedure of treating the experimental animals followed the guidance comments on the animals being treated in good care published by the department of scientific technology in 2006. The spleen of the mouse is directly soaked in a prepared 4% paraformaldehyde solution to fix the shape. The paraformaldehyde powder is relatively insoluble, and a trace amount of sodium bicarbonate can be added to adjust the pH value to be alkaline so as to aid dissolution. The preparation of the paraformaldehyde solution needs to be completed in a fume hood.
(3) Sample detection
According to the instruction of the kit, firstly, a standard curve is drawn, standard powder is prepared into a solution of 1000pg/mL by using a standard diluent, and then the solution is continuously diluted into different concentrations of 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL, 15.6pg/mL and the like. Each concentration gradient solution was pipetted at 100. mu.L in an antibody-coated microplate. And (3) sucking 100 mu L of mouse serum sample, and adding the mouse serum sample into the same enzyme label plate (if the serum sample is insufficient, the mouse serum sample can be diluted properly and then needs to be converted proportionally when being detected and calculated). The plate was covered and incubated at 37 ℃ for 90 min. After the reaction is finished, carefully throwing off the liquid in the ELISA plate, placing the ELISA plate on absorbent paper, carefully beating the absorbent paper, and removing the redundant liquid. Adding preheated biotin anti-antibody working solution into each hole of the ELISA plate according to 100 mu L of each hole, and reacting for 60min at 37 ℃. After the reaction was completed, the reaction solution was washed 3 times with 0.01M PBS, 100. mu.L of PBS was added to each well, and the solution was removed after soaking for 1min, and the reaction was repeated 3 times. The preheated ABC working solution is added into each hole according to the volume of 0.1ml in turn, and the reaction is carried out for 30min at the temperature of 37 ℃. After the reaction, the reaction mixture was washed with 0.01M PBS for 5 times, and soaked for about 1min each time. Adding TMB color development solution which is balanced at 37 ℃ for 30min in turn according to 90 mu L per hole, and reacting for 8-12min at 37 ℃ in a dark place. TMB stop solution was added in an amount of 0.1ml per well in this order, and the color blue was immediately changed to yellow, and the OD value was measured at 450nm using a microplate reader. The standard protein of the cell factor is serially diluted in known concentration, an OD value is measured, a standard curve is drawn, and the content of the cell factor in the specimen can be calculated according to the standard curve.
3. Experimental results and analysis:
TABLE 5 cytokine profile in serum of groups of mice
From Table 5, FIG. 5 and FIG. 6, it can be seen that the mice in the model group of the experiment had in vivo IL-6 and TNF- α contents of 168.01. + -. 26.38pg/mL and 4.34. + -. 0.76pg/mL, respectively, which showed significant increases (P <0.01) compared to the normal group, and thus it is considered that the mice in the animal model group had symptoms of aging inflammation at the cytokine level due to continuous injection of senescence factors, while the mice in the polypeptide gavage group had serum IL-6 and TNF- α contents effectively controlled, and according to the results of the cytokine experiments, the mice in the polypeptide gavage group had serum inflammatory cytokines IL-6 and TNF- α levels lower than those in the animal model group, and from the standpoint of oxidative damage, the mice might have a certain degree of inhibition due to free radical attack and accumulation of peroxide products, and from the standpoint of inflammation, the mice were found that the mice had no significant inhibition of inflammation due to oxidation, and the results of aging were found in the model group of aging, and the aging model group had no significant aging factors, and the results of the aging factor change was considered as a long-aging index in the model group, and the aging factor model group was not considered that the aging factor-induced by long-aging.
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; shanghai platinum Biotech Ltd
<120> a bioactive polypeptide LPYPYYA and a preparation method and application thereof
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>7
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Leu Pro Tyr Pro Tyr Tyr Ala
1 5
<210>2
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
ctgccatacc catattatgc a 21
<210>3
<211>190
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
Met Met Lys SerPhe Phe Leu Val Val Thr Ile Leu Ala Leu Thr Leu
1 5 10 15
Pro Phe Leu Gly Ala Gln Glu Gln Asn Gln Glu Gln Pro Ile Arg Cys
20 25 30
Glu Lys Asp Glu Arg Phe Phe Ser Asp Lys Ile Ala Lys Tyr Ile Pro
35 40 45
Ile Gln Tyr Val Leu Ser Arg Tyr Pro Ser Tyr Gly Leu Asn Tyr Tyr
50 55 60
Gln Gln Lys Pro Val Ala Leu Ile Asn Asn Gln Phe Leu Pro Tyr Pro
65 70 75 80
Tyr Tyr Ala Lys Pro Ala Ala Val Arg Ser Pro Ala Gln Ile Leu Gln
85 90 95
Trp Gln Val Leu Ser Asn Thr Val Pro Ala Lys Ser Cys Gln Ala Gln
100 105 110
Pro Thr Thr Met Ala Arg His Pro His Pro His Leu Ser Phe Met Ala
115 120 125
Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile Asn
130 135 140
Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val
145 150 155 160
Glu Ser Thr Val Ala Thr LeuGlu Asp Ser Pro Glu Val Ile Glu Ser
165 170 175
Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val
180 185 190
Claims (9)
1. A bioactive polypeptide LPYPYYA is characterized in that the amino acid sequence is Leu-Pro-Tyr-Pro-Tyr-Tyr-Ala.
2. A nucleotide fragment encoding the biologically active polypeptide LPYPYYA of claim 1, wherein the nucleotide fragment has the sequence of SEQ ID NO: 2, respectively.
3. The method for producing the bioactive polypeptide LPYPYYA according to claim 1, wherein the bioactive polypeptide LPYPYYA is artificially synthesized by genetic engineering methods or is produced directly by chemical synthesis.
4. The use of the bioactive polypeptide LPYPYYA of claim 1 in the preparation of a food, health product, pharmaceutical or cosmetic product with immunomodulatory activity.
5. The use of the bioactive polypeptide LPYPYYA of claim 1, wherein the bioactive polypeptide LPYPYYA is used in the preparation of food, health care products or drugs with anti-aging function.
6. The use of the bioactive polypeptide LPYPYYA of claim 1 in the preparation of a food, health product or medicament with immunomodulatory and anti-aging effects.
7. An immunomodulatory product comprising the biologically active polypeptide LPYPYYA of claim 1; the immunoregulation product comprises an immunoregulation food, an immunoregulation health product, an immunoregulation medicament or an immunoregulation cosmetic.
8. An anti-aging product comprising the biologically active polypeptide LPYPYYA of claim 1; the anti-aging product comprises anti-aging food, anti-aging health care products or anti-aging drugs.
9. A product having immunoregulatory and anti-aging functions comprising the biologically active polypeptide LPYPYYA of claim 1; the product with immunoregulation function and anti-aging function comprises food, health product or medicine.
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| CN112759635B (en) * | 2021-01-21 | 2022-05-31 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide with amino acid structure VAKVTGGAASKL, and preparation method and application thereof |
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| CN1313487C (en) * | 2003-06-10 | 2007-05-02 | 天津商学院 | Immunoreactive peptide Trpi possessing antibacterial action |
| JP5686680B2 (en) * | 2011-06-24 | 2015-03-18 | カルピス株式会社 | Method for producing casein-derived peptide by lactic acid bacteria fermentation |
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