CN112500469B

CN112500469B - Bioactive polypeptide AAPAAPAAAPPAE, and preparation method and application thereof

Info

Publication number: CN112500469B
Application number: CN202011485260.4A
Authority: CN
Inventors: 张少辉; 李锡安; 王娟; 占文静; 陈承余; 肖珊珊; 石毅; 洪志骏; 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: 2022-03-29
Anticipated expiration: 2040-12-16
Also published as: CN112500469A

Abstract

The invention relates to the field of proteins, in particular to a bioactive polypeptide AAPAAPAAAPPAE, and a preparation method and application thereof, wherein the amino acid sequence of the bioactive polypeptide AAPAAPAAAPPAE is Ala-Ala-Pro-Ala-Ala-Pro-Ala-Ala-Ala-Pro-Pro-Ala-Glu. In vitro immune function regulating experiment proves that the polypeptide AAPAAPAAAPPAE has better immune regulating function. The bioactive peptide AAPAAPAAAPPAE has obvious promotion effect on the ability of phagocytizing neutral red by macrophages in vitro under the condition of inflammation occurrence, has good protection effect on inflammatory animals, reduces the morbidity of organisms, improves the quality of life, promotes the proliferation of lymphocytes, improves the immunity of the organisms, and has very important significance for developing foods, health-care products and medicines with immunoregulation function.

Description

Bioactive polypeptide AAPAAPAAAPPAE, and preparation method and application thereof

Technical Field

The invention relates to the field of proteins, in particular to a bioactive polypeptide AAPAAPAAAPPAE, 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. Lymphocytes are central regulatory cells of the immune system, most of whose function is mediated by a group of small molecule polypeptides called lymphokines. Expression and secretion of these small molecule polypeptides are induced by antigen-stimulated cellular activation. Lymphocytes are therefore the primary source of immunoregulatory peptides produced in the animal body.

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.

Immunomodulatory peptides generally refer to small, relatively small molecular weight peptides with immunomodulatory activity. 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 Histone H1.2 protein is shown as SEQ ID NO: 2, respectively. At present, the related functions of the polypeptide fragments of Histone H1.2 protein are not researched in the prior art.

Disclosure of Invention

The invention aims to provide a bioactive polypeptide AAPAAPAAAPPAE, 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, there is provided a biologically active polypeptide AAPAAPAAAPPAE having the amino acid sequence Ala-Ala-Pro-Ala-Ala-Pro-Ala-Ala-Ala-Pro-Pro-Ala-Glu as set forth in SEQ ID NO: 1 is shown.

Preferably, the bioactive polypeptide is mouse spleen-derived lymphocyte peptide. Specifically, the protein is derived from Histone H1.2 protein and is the amino acid residue at the 4 th to 16 th positions of the Histone H1.2 protein. The amino acid sequence of Histone H1.2 protein is shown as SEQ ID NO: 2, respectively.

The amino acid sequence and the corresponding nucleotide sequence of the Histone H1.2 protein are the prior art, and the nucleotide fragment for coding the 4 th to 16 th amino acid residues of the Histone H1.2 protein can code mature bioactive polypeptide AAPAAPAAAPPAE.

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

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

In the second aspect of the present invention, a method for preparing the bioactive polypeptide AAPAAPAAAPPAE 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 AAPAAPAAAPPAE 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 AAPAAPAAAPPAE, the bioactive peptide AAPAAPAAAPPAE 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 AAPAAPAAAPPAE in preparing medicines or cosmetics with anti-inflammatory function is provided.

Further, the application of the bioactive peptide AAPAAPAAAPPAE in preparing a medicament for promoting the ability of macrophages in vitro to phagocytose neutral red under the condition of inflammation occurrence.

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

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

In a fifth aspect of the invention, there is provided an anti-inflammatory product comprising said biologically active polypeptide AAPAAPAAAPPAE or a derivative of said biologically active polypeptide AAPAAPAAAPPAE; the anti-inflammatory product comprises an anti-inflammatory drug or an 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 AAPAAPAAAPPAE or a derivative of said biologically active peptide AAPAAPAAAPPAE; the product with immunoregulatory function comprises food with immunoregulatory function or medicine with immunoregulatory function.

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

The derivative of the biologically active polypeptide AAPAAPAAAPPAE 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 AAPAAPAAAPPAE.

The bioactive polypeptide AAPAAPAAAPPAE has the following beneficial effects: the bioactive polypeptide AAPAAPAAAPPAE has good anti-inflammatory activity; the bioactive peptide AAPAAPAAAPPAE has obvious promotion effect on the ability of phagocytizing neutral red by macrophages in vitro under the condition of inflammation occurrence, has good protection effect on inflammatory animals, reduces the morbidity of organisms, improves the quality of life, promotes the proliferation of lymphocytes, improves the immunity of the organisms, and has very important significance for developing foods, health-care products and medicines with immunoregulation function.

Drawings

FIG. 1: a primary mass spectrum of a fragment with a mass to charge ratio of 552.7877 (m/z = 552.7877);

FIG. 2: a secondary mass spectrum of a fragment with a mass-to-charge ratio of 552.7877 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 AAPAAPAAAPPAE

Synthesis of bioactive peptide

Biologically active peptide AAPAAPAAAPPAE 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 AAPAAPAAAPPAE 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 AAPAAPAAAPPAE 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 552.7877, and the retention time is 18.61 min.

3) Results

As can be seen from fig. 2, the fragment sequence of mass-to-charge ratio 552.7877 was calculated by Mascot software analysis based on az and by cleavage, and was designated as Ala, Pro, Ala, Pro, Ala, Glu (AAPAAPAAAPPAE), and as SEQ ID NO: 1. the fragment corresponds to residue sequences of 4 th to 16 th sites of Histone H1.2 protein, the GenBank number of the amino acid sequence of the Histone H1.2 protein is AAO06216.1, 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 AAPAAPAAAPPAE

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 AAPAAPAAAPPAE 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 the spleen of a mouse under the aseptic condition, extracting the lymphocyte of the mouse by using the lymphocyte extracting solution, and carrying out 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 AAPAAPAAAPPAE on in vitro lymphocyte proliferation

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

The results are shown in Table 2. As shown in Table 2, under the condition that the mass concentration of the bioactive peptide AAPAAPAAAPPAE is 200. mu.g/mL, the stimulation index of the bioactive peptide AAPAAPAAAPPAE is greater than that of BSA, which indicates that AAPAAPAAAPPAE can stimulate the proliferation of mouse lymphocytes in vitro to a certain extent. And the stimulation index of AAPAAPAAAPPAE reached 1.1372, which was significantly different from that of the negative control group (P < 0.05). Therefore, the active polypeptide AAPAAPAAAPPAE 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, experiment of macrophage phagocytosis neutral red ability of bioactive polypeptide AAPAAPAAAPPAE

1. Experimental reagents and instruments:

reagent: experimental animals balb/c mice (male 6-8 weeks old) were collected at the animal Experimental center of the college of agriculture and biology of Shanghai university of transportation; the mouse spleen lymphocyte-derived bioactive polypeptide AAPAAPAAAPPAE obtained in example 1; LPS, purchased from Sigma; neutral red staining solution, produced by Biyuntian biotechnological research institute.

The instrument equipment comprises: shanghai Hengheng LRH-250F biochemical incubatorScience and technology limited; GL-22M high speed refrigerated centrifuge Shanghai Luxiang apparatus centrifuge Instrument Co., Ltd; hera cell 150 CO₂Incubator 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, after adherent purification, 200 μ l/well of RPMI1640 complete medium (10% FBS) containing active peptide AAPAAPAAAPPAE (0.5 mg/ml) was added as an experimental group, and 200 μ l/well of RPMI1640 complete medium (10% FBS) containing no active peptide was added as a blank group; and LPS is added into the experimental group and the blank group when the culture time reaches 24h to reach the final concentration of 10 mug/ml; after further culturing for 48h, the cell culture solution was aspirated. After washing the bottom of the well with PBS, 80. mu.l/well of neutral red dye solution at 37 ℃ was added, and after 10 minutes the dye solution was aspirated and washed twice with PBS, 150. mu.l of cell lysate (glacial acetic acid: absolute ethanol =1:1, v/v) was added to each well. After overnight dissolution at 4 ℃ the absorbance value (OD 540) was determined at a wavelength of 540 nm.

3. Experimental results and analysis:

TABLE 2 determination of the ability of the biologically active polypeptide AAPAAPAAAPPAE to promote phagocytosis of neutral Red by macrophages

Experiment grouping	Absorbance value (OD 540)
		Blank group	0.1134±0.0493
Experimental group	0.2842±0.0382**

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

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

The experimental results are shown in Table 2, compared with the blank cell group, the macrophage phagocytosis ability of the inflammatory group macrophage added with 0.5mg/ml bioactive polypeptide AAPAAPAAAPPAE is obviously increased, and compared with the blank cell group, the macrophage phagocytosis ability has very significant difference (P is less than 0.01). The result shows that the bioactive polypeptide AAPAAPAAAPPAE has obvious promotion effect on the ability of phagocytizing neutral red by macrophages in vitro under the condition of inflammation.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Sequence listing

<110> panda milk group GmbH, Zhejiang ghui peptide Life health science & technology, Inc

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

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ala Ala Pro Ala Ala Pro Ala Ala Ala Pro Pro Ala Glu

1 5 10

<210> 2

<211> 212

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ser Glu Ala Ala Pro Ala Ala Pro Ala Ala Ala Pro Pro Ala Glu

1 5 10 15

Lys Ala Pro Ala Lys Lys Lys Ala Ala Lys Lys Pro Ala Gly Val Arg

20 25 30

Arg Lys Ala Ser Gly Pro Pro Val Ser Glu Leu Ile Thr Lys Ala Val

35 40 45

Ala Ala Ser Lys Glu Arg Ser Gly Val Ser Leu Ala Ala Leu Lys Lys

50 55 60

Ala Leu Ala Ala Ala Gly Tyr Asp Val Glu Lys Asn Asn Ser Arg Ile

65 70 75 80

Lys Leu Gly Leu Lys Ser Leu Val Ser Lys Gly Ile Leu Val Gln Thr

85 90 95

Lys Gly Thr Gly Ala Ser Gly Ser Phe Lys Leu Asn Lys Lys Ala Ala

100 105 110

Ser Gly Glu Ala Lys Pro Gln Ala Lys Lys Ala Gly Ala Ala Lys Ala

115 120 125

Lys Lys Pro Ala Gly Ala Ala Lys Lys Pro Lys Lys Ala Thr Gly Ala

130 135 140

Ala Thr Pro Lys Lys Ala Ala Lys Lys Thr Pro Lys Lys Ala Lys Lys

145 150 155 160

Pro Ala Ala Ala Ala Val Thr Lys Lys Val Ala Lys Ser Pro Lys Lys

165 170 175

Ala Lys Val Thr Lys Pro Lys Lys Val Lys Ser Ala Ser Lys Ala Val

180 185 190

Lys Pro Lys Ala Ala Lys Pro Lys Val Ala Lys Ala Lys Lys Val Ala

195 200 205

Ala Lys Lys Lys

210

Claims

1. A biologically active polypeptide AAPAAPAAAPPAE, having an amino acid sequence of Ala-Ala-Pro-Ala-Ala-Pro-Ala-Ala-Pro-Pro-Ala-Glu.

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

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