CN112501140B

CN112501140B - Bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and preparation method and application thereof

Info

Publication number: CN112501140B
Application number: CN202011485267.6A
Authority: CN
Inventors: 张少辉; 郭婷婷; 栾媛媛; 李锡安; 占文静; 陈承余; 肖珊珊; 石毅; 梁世排; 梁世赞
Original assignee: Zhejiang Huitai Life Health Technology Co ltd; Panda Dairy Group Co ltd
Current assignee: Zhejiang Huitai Life Health Technology Co ltd; Panda Dairy Group Co ltd
Priority date: 2020-12-16
Filing date: 2020-12-16
Publication date: 2022-03-18
Anticipated expiration: 2040-12-16
Also published as: CN112501140A

Abstract

The invention relates to the field of protein, and in particular relates to a bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, a preparation method and application thereof, wherein the amino acid sequence of the bioactive polypeptide YFGSGFAAPFFIVRHQLLKK is Tyr-Phe-Gly-Ser-Gly-Phe-Ala-Ala-Pro-Phe-Phe-Ile-Val-Arg-His-Gln-Leu-Leu-Lys-Lys. In vitro immune function regulating experiment proves that the polypeptide YFGSGFAAPFFIVRHQLLKK has better immune regulating function. The bioactive peptide YFGSGFAAPFFIVRHQLLKK can promote the increase of the induction quantity of macrophage nitric oxide, improve the capability of an organism for resisting the infection of external pathogens, reduce the morbidity of the organism, obviously promote the proliferation of mouse lymphocytes, improve the quality of life and have very important significance for developing foods, health-care products and medicines with the immunoregulation function.

Description

Bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and preparation method and application thereof

Technical Field

The invention relates to the field of proteins, in particular to a bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and a preparation method and application thereof.

Background

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

Currently, studies on bioactive peptides are mostly focused on food-derived polypeptides, and studies and reports on non-food-derived polypeptides are 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.

Immunomodulatory peptides are a class of biologically active 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, with anti-inflammatory properties. Lemna hexandra et al, fed rats with synthetic mouse bone marrow macrophages and a source peptide (PGPIPN), found that phagocytosis of rat peritoneal macrophages and red blood cell-related anti-inflammatory function were significantly enhanced. Bowdis et al, in studying the immune function of the 13 amino acid peptide indolicidin derived from bovine neutrophils, found that the polypeptide indolicidin inhibits LPS-induced TNF- α production in a macrophage-like cell line.

Research shows that the immunoregulation peptide can enhance the immunity of the organism, stimulate the proliferation of lymphocytes of the organism, enhance the phagocytic function of macrophages, promote the release of cytokines, promote the increase of the induced amount of nitric oxide of the macrophages, improve the capability of the organism for resisting the infection of external pathogens, reduce the morbidity of the organism and cannot cause the immune rejection reaction of the organism.

The immunomodulatory peptides presently disclosed are generally small peptides with specific immunomodulatory activity, isolated enzymatically from proteins or synthesized chemically. However, when these small peptides are not enzymatically separated from the protein, the protein itself often has no immunomodulatory activity. It is one of the directions in the field of protein research to find bioactive peptides with specific functions from a wide variety of proteins whose amino acid sequences are known, and to study the functions of these polypeptides.

The amino acid sequence of the Cytochrome C oxidase subunit 7C and mitondrial protein is shown as SEQ ID NO: 2, respectively. At present, no research on related functions of the polypeptide fragment of the Cytochrome C oxidase subunit 7C, mitochondrial protein exists in the prior art.

Disclosure of Invention

The invention aims to provide a bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, 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 YFGSGFAAPFFIVRHQLLKK is provided, having an amino acid sequence of Tyr-Phe-Gly-Ser-Gly-Phe-Ala-Ala-Pro-Phe-Phe-Ile-Val-Arg-His-Gln-Leu-Leu-Lys-Lys, as set forth in SEQ ID NO: 1 is shown.

Preferably, the bioactive polypeptide is mouse spleen-derived lymphocyte peptide. The protein is specifically derived from Cytochrome C oxidase subunit 7C and mitochondrial protein, and is an amino acid residue at the 44 th-63 th position of the Cytochrome C oxidase subunit 7C and mitochondrial protein. The amino acid sequence of the Cytochrome C oxidase subunit 7C and mitondrial protein is shown as SEQ ID NO: 2, respectively.

The amino acid sequences and the corresponding nucleotide sequences of the Cytochrome C oxidase subunit 7C and the mitochondrial protein are the prior art, and the nucleotide fragment which codes the Cytochrome C oxidase subunit 7C and the 44 th to 63 th amino acid residues of the mitochondrial protein can code mature bioactive polypeptide YFGSGFAAPFFIVRHQLLKK.

Preferably, the bioactive polypeptide has an anti-inflammatory function and an immunoregulatory function.

The present invention also provides polynucleotides encoding the biologically active peptide YFGSGFAAPFFIVRHQLLKK.

In the second aspect of the present invention, a method for preparing the bioactive polypeptide YFGSGFAAPFFIVRHQLLKK 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.

The artificial synthesis of the bioactive peptide YFGSGFAAPFFIVRHQLLKK by genetic engineering is a technical solution that can be realized by those skilled in the art, and for example, the synthesis of the sequence of the polypeptide can be controlled by a suitable DNA template based on DNA recombination technology.

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

In a third aspect of the invention, the application of the bioactive polypeptide YFGSGFAAPFFIVRHQLLKK in preparing medicines or cosmetics with anti-inflammatory function is provided.

In a fourth aspect of the present invention, there is provided a use of the bioactive peptide YFGSGFAAPFFIVRHQLLKK in the preparation of food or medicine with immunoregulatory function.

Further, the use of the biologically active peptide YFGSGFAAPFFIVRHQLLKK in the manufacture of a food or medicament for promoting an increase in nitric oxide-inducing amount of macrophages.

Further, the use of the bioactive peptide YFGSGFAAPFFIVRHQLLKK in the preparation of a food or medicament for promoting lymphocyte proliferation.

In a fifth aspect of the invention, there is provided an anti-inflammatory product comprising said biologically active polypeptide YFGSGFAAPFFIVRHQLLKK or a derivative of said biologically active polypeptide YFGSGFAAPFFIVRHQLLKK; the anti-inflammatory product comprises anti-inflammatory food, anti-inflammatory health product, anti-inflammatory drug or anti-inflammatory cosmetic.

In a sixth aspect of the present invention, there is provided a product having an immunoregulatory function, comprising said biologically active peptide YFGSGFAAPFFIVRHQLLKK or a derivative of said biologically active peptide YFGSGFAAPFFIVRHQLLKK; the product with immunoregulatory function comprises food with immunoregulatory function or medicine with immunoregulatory function.

Derivatives of the bioactive peptides YFGSGFAAPFFIVRHQLLKK are meant to have the same activity or better activity than the bioactive peptides YFGSGFAAPFFIVRHQLLKK.

The derivative of the biologically active polypeptide YFGSGFAAPFFIVRHQLLKK 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 YFGSGFAAPFFIVRHQLLKK.

The bioactive polypeptide YFGSGFAAPFFIVRHQLLKK has the following beneficial effects: the bioactive polypeptide YFGSGFAAPFFIVRHQLLKK has good anti-inflammatory activity; the bioactive peptide YFGSGFAAPFFIVRHQLLKK can promote the increase of the induction quantity of macrophage nitric oxide, improve the capability of an organism for resisting the infection of external pathogens, reduce the morbidity of the organism, obviously promote the proliferation of mouse lymphocytes, improve the quality of life and have very important significance for developing foods, health-care products and medicines with the immunoregulation function.

Drawings

FIG. 1: a primary mass spectrum of a fragment with a mass to charge ratio of 582.3296 (m/z = 582.3296);

FIG. 2: a secondary mass spectrum of a fragment with a mass-to-charge ratio of 582.3296 and the breakage conditions of the polypeptides 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, 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 YFGSGFAAPFFIVRHQLLKK

Synthesis of bioactive peptide

Biologically active peptide YFGSGFAAPFFIVRHQLLKK 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 phase, electrospray, quadrupole and time-of-flight mass spectrometer

Specification of chromatographic column: BEH C18 chromatographic column

Flow rate: 0.4mL/min

Temperature: 50 deg.C

Ultraviolet detection wavelength: 210nm

Sample introduction amount: 2 μ L

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

Time(min) %A %B

0 95.0 5.0

1.50 80.0 20.0

3.50 60.0 40.0

5.00 40.0 60.0

7.00 15.0 85.0

8.00 0.0 100.0

11.00 0.0 100.0

11.50 95.0 5.0

13.00 95.0 5.0

2) Mass spectrometric analysis

The mass spectrometry conditions were as follows:

ion mode: ES +

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

Capillary voltage (Capillary) (kV): 3.0

Sampling cone (V): 35.0

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

Desolvation temperature (. degree. C.): 350

Desolventizing gas stream (L/hr): 700.0

Collision energy (eV): 4.0

Scan time (sec): 0.25

Inner scan time (sec): 0.02

According to the above analysis method, the bioactive peptide YFGSGFAAPFFIVRHQLLKK was subjected to chromatographic analysis and mass spectrometric analysis using ultra high performance liquid, electrospray, quadrupole, time-of-flight mass spectrometry. The primary mass spectrum of the bioactive peptide YFGSGFAAPFFIVRHQLLKK 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 polypeptide mass-to-charge ratio of the peak is 582.3296, and the retention time is 51.58 min.

3) Results

As can be seen from fig. 2, the fragment sequence of mass-to-charge ratio 582.3296 was determined by Mascot software analysis and calculation based on az and by cleavage, to be Tyr, Phe, Gly, Ser, Gly, Phe, Ala, Pro, Phe, Ile, Val, Arg, His, Gln, Leu, Lys (YFGSGFAAPFFIVRHQLLKK), and was identified as SEQ ID NO: 1. the fragment corresponds to a residue sequence of 44-63 sites of Cytochrome C oxidase subunit 7C and mitochondrial protein, the GenBank number of the amino acid sequence of Cytochrome C oxidase subunit 7C and mitochondrial protein is BAE20843.1, and the sequence is shown in SEQ ID NO: 2.

example 2 immunological Activity assay of bioactive peptides

In vitro lymphocyte proliferation capacity experiment (MTT method) of bioactive polypeptide YFGSGFAAPFFIVRHQLLKK

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 mouse spleen lymphocyte-derived bioactive peptide YFGSGFAAPFFIVRHQLLKK 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 150 CO₂Incubator, Heraeus corporation; dragon Wellscan MK3 microplate reader, Labsystems Inc.; ALPHA 1-2-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 concanavalin, 100 μ L polypeptide 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 2 Effect of the bioactive polypeptide YFGSGFAAPFFIVRHQLLKK on in vitro lymphocyte proliferation

Note: the number marked as significant difference (P < 0.05) compared to the negative control.

The difference in the negative control group was very significant (P < 0.01)

The results are shown in Table 2. As shown in Table 2, under the condition that the mass concentration of the bioactive peptide YFGSGFAAPFFIVRHQLLKK is 200. mu.g/mL, the stimulation index of the bioactive peptide YFGSGFAAPFFIVRHQLLKK is greater than that of BSA, which indicates that YFGSGFAAPFFIVRHQLLKK can stimulate the proliferation of mouse lymphocytes in vitro to a certain extent. And YFGSGFAAPFFIVRHQLLKK reached a stimulation index of 1.482, which was very significantly different from the negative control group (P < 0.01). Therefore, the active polypeptide YFGSGFAAPFFIVRHQLLKK can be considered to have the capacity of remarkably promoting the mouse lymphocyte proliferation, can be used as a substance with immunoregulation activity to be added into health products, and can improve the immunity of human bodies.

Second, determination of macrophage-promoting nitric oxide-inducing amount of bioactive polypeptide YFGSGFAAPFFIVRHQLLKK (Griess method)

1. Experimental reagents and instruments:

reagent: experimental animal balb/c mouse (male 6-8 weeks old) spleen lymphocyte source bioactive peptide YFGSGFAAPFFIVRHQLLKK; 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 150 CO₂Incubator Heraeus corporation; 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 (OD 540) was measured at a wavelength of 540 nm.

3. Experimental results and analysis:

TABLE 1 determination of macrophage nitric oxide-inducing amount of biologically active polypeptide YFGSGFAAPFFIVRHQLLKK

Note: significant difference compared to negative control (P < 0.05);

the difference in the negative control group was very significant (P < 0.01)

The results are shown in table 1, and it is understood from table 1 that the addition of biologically active polypeptide YFGSGFAAPFFIVRHQLLKK at a concentration of 0.5mg/mL to the experimental group promoted the nitric oxide-induced amount of macrophages both under normal conditions and under conditions of LPS-induced inflammation, and showed a significant difference (P < 0.01) compared to the cell blank group. When the added concentration of the bioactive polypeptide YFGSGFAAPFFIVRHQLLKK is 1 mg/mL, the increase of the nitric oxide induction amount of macrophages can be promoted under the condition of inflammation caused by LPS, and the difference is very significant (P < 0.01). But there were no significant differences compared to the cell blank grown under normal conditions. The biologically active polypeptide YFGSGFAAPFFIVRHQLLKK is shown to have the ability to promote an increase in nitric oxide-induced levels in macrophages at certain concentrations.

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> panda milk group GmbH, Zhejiang ghui peptide Life health science & technology, Inc

<120> a bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and its preparation method and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Tyr Phe Gly Ser Gly Phe Ala Ala Pro Phe Phe Ile Val Arg His Gln

1 5 10 15

Leu Leu Lys Lys

20

<210> 2

<211> 63

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Leu Gly Gln Ser Ile Arg Arg Phe Thr Thr Ser Val Val Arg Arg

1 5 10 15

Ser His Tyr Glu Glu Gly Pro Gly Lys Asn Leu Pro Phe Ser Val Glu

20 25 30

Asn Lys Trp Arg Leu Leu Ala Met Met Thr Val Tyr Phe Gly Ser Gly

35 40 45

Phe Ala Ala Pro Phe Phe Ile Val Arg His Gln Leu Leu Lys Lys

50 55 60

Claims

1. A bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, wherein the amino acid is Tyr-Phe-Gly-Ser-Gly-Phe-Ala-Ala-Pro-Phe-Phe-Ile-Val-Arg-His-Gln-Leu-Leu-Lys-Lys.

2. A polynucleotide encoding the biologically active peptide YFGSGFAAPFFIVRHQLLKK of claim 1.

3. The method of claim 1, wherein the biologically active polypeptide YFGSGFAAPFFIVRHQLLKK is produced directly by chemical synthesis.