CN112500468B

CN112500468B - Bioactive peptide RLAFIAHPKLG, and preparation method and application thereof

Info

Publication number: CN112500468B
Application number: CN202011467494.6A
Authority: CN
Inventors: 张少辉; 李政; 郭婷婷; 栾媛媛; 占文静; 张伯宇; 谢雨佳; 王娟
Original assignee: Zhejiang Huitai Life Health Technology Co ltd; Shanghai Jiaotong University
Current assignee: Zhejiang Huitai Life Health Technology Co ltd; Shanghai Jiaotong University
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2022-07-15
Anticipated expiration: 2040-12-14
Also published as: CN112500468A

Abstract

The invention relates to the field of proteins, and in particular relates to a bioactive peptide RLAFIAHPKLG, and a preparation method and application thereof, wherein the amino acid sequence of the bioactive peptide RLAFIAHPKLG is Arg-Leu-Ala-Phe-Ile-Ala-His-Pro-Lys-Leu-Gly. In vitro immune regulation function experiments show that the bioactive peptide RLAFIAHPKLG has better immune regulation function. The bioactive peptide RLAFIAHPKLG has the advantages of enhancing the in-vitro proliferation capacity of lymphocytes, promoting macrophages to secrete cell factors, improving the capability of an organism to resist infection of external pathogens, reducing the morbidity of the organism, improving the quality of life and having very important significance in developing foods, health-care products and medicines with immune regulation functions.

Description

Bioactive peptide RLAFIAHPKLG, and preparation method and application thereof

Technical Field

The invention relates to the field of protein, and in particular relates to a bioactive peptide RLAFIAHPKLG, and a preparation method and application thereof.

Background

In recent years, bioactive peptides have become a word of the human 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, over 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 few. 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 polypeptides are both 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 bioactive peptides that were first obtained from milk following opioid peptide discovery and demonstrated 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 not only enhance the immunity of organisms, stimulate the proliferation of lymphocytes of the organisms, enhance the phagocytic function of macrophages, promote the release of cell factors, promote the increase of the nitric oxide induction quantity of the macrophages, improve the capability of the organisms for resisting the infection of external pathogens, reduce the morbidity of the organisms, but also can not cause the immunological rejection reaction of the organisms.

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 is often not immunomodulatory. 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 60S ribosomal protein L29 protein is shown in SEQ ID NO: 2, respectively. At present, no research on the related functions of the polypeptide fragment of the 60S ribosomal protein L29 protein exists in the prior art.

Disclosure of Invention

The invention aims to provide a bioactive peptide RLAFIAHPKLG, 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 present invention, a bioactive peptide RLAFIAHPKLG is provided, which has an amino acid sequence of Arg-Leu-Ala-Phe-Ile-Ala-His-Pro-Lys-Leu-Gly, as shown in SEQ ID NO: 1 is shown.

Preferably, the bioactive peptide is mouse spleen derived lymphocyte peptide. Specifically, the protein is derived from 60S ribosomal protein L29 protein and is the amino acid residues from 93 th to 103 th positions of 60S ribosomal protein L29 protein. The amino acid sequence of the 60S ribosomal protein L29 is shown in SEQ ID NO: 2, respectively.

The amino acid sequence and the corresponding nucleotide sequence of the 60S ribosomal protein L29 protein are the existing technology, and the nucleotide fragment coding the 93 th to 103 th amino acid residues of the 60S ribosomal protein L29 protein can code mature bioactive peptide RLAFIAHPKLG.

Preferably, the bioactive peptide has anti-inflammatory and immunoregulatory functions.

The invention also provides polynucleotides encoding said biologically active peptides RLAFIAHPKLG.

In the second aspect of the present invention, there is provided a method for preparing the bioactive peptide RLAFIAHPKLG, which can be artificially synthesized by genetic engineering methods, can be directly obtained from cells by separation and purification methods, and can be directly prepared by chemical synthesis.

The artificial synthesis of the bioactive peptide RLAFIAHPKLG 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 RLAFIAHPKLG, the bioactive peptide RLAFIAHPKLG is obtained from mouse spleen-derived lymphocytes by a conventional enzymolysis and purification method in biological technology.

In a third aspect of the present invention, there is provided a use of the bioactive peptide RLAFIAHPKLG in the preparation of a medicament or a cosmetic having an anti-inflammatory effect.

In a fourth aspect, the present invention provides the use of the bioactive peptide RLAFIAHPKLG in the preparation of food or medicament with immunoregulatory function.

Further, the use of the bioactive peptide RLAFIAHPKLG in the preparation of a food or medicament for enhancing the in vitro proliferation capacity of lymphocytes.

Further, the use of the bioactive peptide RLAFIAHPKLG in the preparation of a food or a medicament for secretion of cytokines by macrophages.

In a fifth aspect of the present invention, there is provided a product with immunoregulatory function, comprising said biologically active peptide RLAFIAHPKLG or a derivative of said biologically active peptide RLAFIAHPKLG; the product with the immunoregulation function comprises food with the immunoregulation function or a medicine with the immunoregulation function; derivatives of said biologically active peptide RLAFIAHPKLG are meant to have the same activity or better activity than said biologically active peptide RLAFIAHPKLG. The derivative of the bioactive peptide RLAFIAHPKLG refers to a bioactive peptide derivative obtained by modifying the amino acid side chain group, amino terminal or carboxyl terminal of the bioactive peptide RLAFIAHPKLG by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation. In a sixth aspect of the invention, there is provided an anti-inflammatory product comprising said biologically active peptide RLAFIAHPKLG or a derivative of said biologically active peptide RLAFIAHPKLG; the anti-inflammatory product comprises anti-inflammatory food, anti-inflammatory health care product, anti-inflammatory drug or anti-inflammatory cosmetic; derivatives of said biologically active peptide RLAFIAHPKLG are meant to have the same activity or better activity than said biologically active peptide RLAFIAHPKLG; the derivative of the bioactive peptide RLAFIAHPKLG refers to a bioactive peptide derivative obtained by modifying the amino acid side chain group, amino terminal or carboxyl terminal of the bioactive peptide RLAFIAHPKLG by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation.

The bioactive peptide RLAFIAHPKLG has the following beneficial effects: the bioactive peptide RLAFIAHPKLG has better anti-inflammatory activity; the bioactive peptide RLAFIAHPKLG has the advantages of enhancing the in-vitro proliferation capacity of lymphocytes, promoting macrophages to secrete cytokines, improving the resistance of an organism to external pathogen infection, reducing the morbidity of the organism, improving the quality of life and having 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 408.2534 (m/z = 408.2534);

FIG. 2: a secondary mass spectrum of a segment with the mass-to-charge ratio of 408.2534 and the breaking conditions of the bioactive peptides az and by;

Detailed Description

Before the present embodiments are further described, it is to be understood that the scope of the invention is not to be limited to the specific 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, the invention may be practiced using any method, device, and material that is similar or equivalent to the methods, devices, and materials described in examples herein, in addition to those described in prior art practice and the description herein.

Unless otherwise indicated, the methods of testing, methods of preparation, and methods of preparation disclosed herein 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; METHOD 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 Synthesis of bioactive peptide RLAFIAHPKLG

Synthesis of bioactive peptide

Biologically active peptide RLAFIAHPKLG was synthesized.

Second, confirmation of biologically active peptides

1) UPLC analysis

The UPLC conditions were as follows:

the instrument comprises: 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 spectrometry

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 RLAFIAHPKLG 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 RLAFIAHPKLG is shown in figure 1, the secondary mass spectrum of the extracted peak and the az and by fracture conditions are shown in figure 2, the mass-to-charge ratio of the bioactive peptide of the peak is 408.2534, and the retention time is 25.52 min.

3) Results

As can be seen from fig. 2, the fragment sequences of mass-to-charge ratio 408.2534 obtained from az and by fragmentation were Arg, Leu, Ala, Phe, Ile, Ala, His, Pro, Lys, Leu, Gly (RLAFIAHPKLG) and are denoted as SEQ ID NO: 1. the fragment corresponds to the residue sequence of 93-103 of the 60S ribosomal protein L29 protein, the GenBank number of the amino acid sequence of the 60S ribosomal protein L29 protein is AAH82292.1, 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 peptide RLAFIAHPKLG

1. Experimental materials and instruments:

reagents and materials: experimental animals balb/c mice (male 6-8 weeks old, animal experimental center of college of agriculture and biology of Shanghai university of traffic); the mouse spleen lymphocyte-derived bioactive peptide RLAFIAHPKLG obtained in example 1; mouse lymphocyte extract (ex solibao); RPMI1640 medium (purchased from GIBCO); 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide (MTT, available from Amresco); concanavalin (ConA, available from Sigma); bovine serum albumin (BSA, available from Genebase); pepsin (purchased from Sigma); pancreatin (Corolase PP, from AB).

An instrument device: 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: mu.L mouse lymphocyte suspension, 100. mu.L RPMI1640 complete medium, 20. mu.L concanavalin, 100. mu.L bioactive peptide sample (0.5mg/ml, 1 mg/ml). In addition, a blank control group (pH7.2-7.4, 3mol/L PBS) 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 group of 3 parallel experimentsAnd (5) sampling. At 5% CO₂Culturing at 37 deg.C for 68 hr, adding 20 μ L MTT into each well under aseptic condition, culturing for 4 hr, carefully removing supernatant, adding 100 μ L dimethyl sulfoxide into each well, incubating at 37 deg.C for 10min, shaking, and measuring light absorption at 570nm with enzyme labeling instrument.

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 bioactive peptide RLAFIAHPKLG on lymphocyte proliferation in vitro

Experimental groups	Stimulation index SI
		BSA
	1
		Bioactive peptide samples (0.5mg/ml)	1.147±0.011*
Bioactive peptide samples (1 mg/ml)	1.032±0.010

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

The results are shown in Table 1. As shown in Table 1, under the condition that the mass concentration of the bioactive peptide RLAFIAHPKLG is 0.5mg/mL, the stimulation index of the bioactive peptide RLAFIAHPKLG is greater than that of BSA, which indicates that RLAFIAHPKLG can stimulate the proliferation of mouse lymphocytes in vitro to a certain extent. And RLAFIAHPKLG reached a stimulation index of 1.147, which was significantly different from the negative control group (P < 0.05). When the mass concentration reaches 1mg/ml, the proliferation effect on lymphocytes is not obvious, so that the active bioactive peptide RLAFIAHPKLG can be determined to have the capacity of remarkably promoting mouse lymphocyte proliferation in a certain concentration range, can be added into a health-care product as a substance with immunoregulation activity, and can improve the immunity of a human body.

Second, experiment (ELISA method) of promoting macrophage secretion cell factor of bioactive peptide RLAFIAHPKLG

1. Experimental reagents and instruments:

reagent: experimental animals balb/c mice (male 6, 8 week old), shanghai slek experimental animals limited; mouse lymphocyte extract, shanghai solibao biotechnology limited; RPMI1640 medium, GIBCO; bovine Serum Albumin (BSA), Genebase; the mouse spleen lymphocyte-derived bioactive peptide RLAFIAHPKLG obtained in example 1; ELISA cytokine Rapid kits (TNF-. alpha.and IL-1. beta.), Roche Biotechnology Ltd.

The instrument equipment comprises: LRH, 250F biochemical incubator shanghai constant technology ltd; GL, 22M high speed refrigerated centrifuge Shanghai Luxiang apparatus centrifuge instruments ltd; hera cell 150 CO₂Incubator Heraeus; dragon Wellscan MK3 microplate reader Labsystems Inc.

2. The experimental method comprises the following steps:

the number of the added cells was 2X 10⁶100 mul/well of cell suspension per ml, adding 200 mul/well of RPMI1640 complete culture medium (10% FBS) containing peptide after adherent purification, adding LPS to the final concentration of 10 mug/ml in the inflammation group at 24 hours, continuously culturing for 48 hours, culturing the inflammation group in cultureLPS was added 24 hours before termination to a final concentration of 100 ng/ml. After the termination of the culture, the cell culture supernatant was collected by centrifugation. Mu.l of supernatant was added to an enzyme-labeled plate coated with cytokine antibody, reacted at 37 ℃ for 90 minutes, then added with biotin-labeled antibody, reacted at 37 ℃ for 60 minutes, washed with PBS, and then added with avidin-peroxidase complex, reacted for 30 minutes. After washing with PBS, a developing solution was added thereto, and the reaction was carried out for 20 minutes. After addition of the chromogenic stop solution, the absorbance value (OD 450) was measured at a wavelength of 450nm using a microplate reader.

3. Experimental results and analysis:

TABLE 2 determination of the Effect of bioactive peptide RLAFIAHPKLG on macrophage cytokine levels

Experiment grouping	TNF-α	IL-1β
			Cell blank	0.119±0.015	0.417±0.043
Bioactive peptide samples (0.2mg/ml)	0.482±0.223**	0.792±0.034**
			Bioactive peptide samples (0.5mg/ml)	0.249±0.173**	0.567±0.025**

Note: significant difference compared to negative control (P < 0.05); the difference between the positive control group and the negative control group is very significant (P < 0.01)

As can be seen from Table 2, in the experimental results of two cytokines TNF-alpha and IL-1 beta, the significant difference (P < 0.01) between TNF-alpha and IL-1 beta at 0.2mg/ml and above appears, and it is proved that RLAFIAHPKLG at a certain concentration can promote the activation of mouse abdominal cavity macrophages and release TNF-alpha and IL-1 beta. TNF-alpha and IL-1 beta cytokines are proinflammatory factors, can induce B cells to differentiate and generate antibodies, and induce T cells to activate, proliferate and differentiate and participate in immune response of organisms. Therefore, RLAFIAHPKLG at a certain concentration can improve the action of these cytokines in the resting state of normal macrophages, thereby regulating the immunity of the organism.

The embodiments described above are intended to facilitate a person of ordinary skill in the art in understanding and using the invention. 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 modifications and alterations without departing from the scope of the present invention.

Sequence listing

<110> Shanghai university of transportation, Zhejiang river peptide Life health science and technology Limited

<120> bioactive peptide RLAFIAHPKLG, and preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Arg Leu Ala Phe Ile Ala His Pro Lys Leu Gly

1 5 10

<210> 2

<211> 160

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ala Lys Ser Lys Asn His Thr Thr His Asn Gln Ser Arg Lys Trp

1 5 10 15

His Arg Asn Gly Ile Lys Lys Pro Arg Ser Gln Arg Tyr Glu Ser Leu

20 25 30

Lys Gly Val Asp Pro Lys Phe Leu Arg Asn Met Arg Phe Ala Lys Lys

35 40 45

His Asn Lys Lys Gly Leu Lys Lys Met Gln Ala Asn Asn Ala Lys Ala

50 55 60

Val Ser Ala Arg Ala Glu Ala Ile Lys Ala Leu Val Lys Pro Gln Ala

65 70 75 80

Ile Lys Pro Lys Met Pro Lys Gly Pro Lys Leu Lys Arg Leu Ala Phe

85 90 95

Ile Ala His Pro Lys Leu Gly Lys Arg Ile Arg Ser Tyr Met Ala Lys

100 105 110

Gly Gln Arg Leu Cys Gln Pro Lys Pro Lys Val Gln Thr Lys Ala Gly

115 120 125

Ala Lys Ala Pro Ala Lys Ala Gln Ala Ser Ala Pro Ala Gln Ala Pro

130 135 140

Lys Gly Ala Gln Ala Pro Lys Gly Ala Gln Ala Pro Val Lys Ala Pro

145 150 155 160

Claims

1. The bioactive peptide RLAFIAHPKLG is characterized in that the amino acid sequence is Arg-Leu-Ala-Phe-Ile-Ala-His-Pro-Lys-Leu-Gly.

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

3. The process for preparing bioactive peptide RLAFIAHPKLG of claim 1, directly prepared by chemical synthesis.