CN110938113A

CN110938113A - Application of immunoregulation peptide in preparing immunopotentiation medicine

Info

Publication number: CN110938113A
Application number: CN201911307096.5A
Authority: CN
Inventors: 曹彦; 季晨博; 崔县伟
Original assignee: Nanjing Maternity and Child Healthcare Hospital
Current assignee: Nanjing Maternity and Child Healthcare Hospital
Priority date: 2019-12-17
Filing date: 2019-12-17
Publication date: 2020-03-31

Abstract

The invention discloses an application of immunoregulation peptide in preparing immune enhancement drugs, wherein the polypeptide is found in human breast milk β -casein, has no more than 21 amino acids, can enter cells and is distributed in cytoplasm and nucleus, and the polypeptide has no obvious influence on the cell proliferation activity and apoptosis of human mononuclear cells, and in vitro experiments prove that the polypeptide can obviously promote the migration of the human mononuclear cells, thereby improving the immune response mechanism of an organism.

Description

Application of immunoregulation peptide in preparing immunopotentiation medicine

Technical Field

The invention relates to a new application of biological source polypeptide, in particular to an application of immunoregulation peptide in preparing immunity-enhancing medicines.

Background

The discovery of antibiotics is one of the greatest achievements of modern medicine, but with the widespread use of antibiotics, many microorganisms have resistance to common antibiotics, and infection caused by multidrug-resistant bacteria has gradually become a long-standing challenge in the medical field, so that the development of novel antibacterial preparations is urgent. In recent years, polypeptide therapy has been increasingly applied to clinical applications, and antibacterial peptides having various biological functions such as antimicrobial activity have been developed, but there is still a risk of drug resistance to drugs that kill pathogenic bacteria. The innate immune response is the first line of defense against pathogenic microorganisms. Therefore, new approaches to anti-infective therapy, which modulate the innate immune system function, are receiving increasing attention. The function of the immune system is adjusted, so that the immune system has the effect similar to that of antibiotic treatment, has wider potential application value, and can effectively reduce or even avoid the problem of drug resistance.

The successful use of many known polypeptides with immunopotentiating activity as viable adjuvants for established therapeutic modalities provides a novel avenue for the treatment of infections and other malignant diseases in the next decades. Since 1974, a minimal bacterial peptidoglycan fragment, Muramyl Dipeptide (MDP), was found to have immunostimulatory activity, researchers have struggled to develop immunopotentiators of chemically defined, low molecular weight substances. Thus, a number of powerful compounds are gradually being discovered, some of which are now entering the clinical trial phase. Novel, chemically defined, clinically useful immunomodulators, such as many glycopeptides and lipopeptides structurally similar to MDP, have been designed and synthesized. However, most of them contain components of microbial origin and thus have some toxic side effects.

The human breast milk is used as a natural nutrient source of newborns, and a large amount of endogenous polypeptides are generated under the hydrolysis action of in vivo protease, the polypeptides not only provide amino acid sources for the newborns, but also play various biological functions and protect the newborns from infection.

Disclosure of Invention

The invention aims to provide a novel application of polypeptide found in human breast milk β -casein, which can be used for preparing immunopotentiating or immunoregulatory medicines, and a composition containing the polypeptide.

The technical scheme is as follows: the general formula of the immunoregulatory peptide is as follows:

R₁-A-P-B-R₂

wherein, P is an amino acid sequence shown in SEQ ID NO. 1;

R₁any one selected from amino and acetyl;

R₂any one selected from hydroxyl and amino;

a is a peptide chain consisting of 0-5 amino acids;

b is a peptide chain consisting of 0-5 amino acids.

The immunoregulatory peptide provided by the invention comprises but is not limited to a sequence shown in SEQ ID NO.1, the sequence is taken as a common continuous polypeptide chain, and polypeptides with homology of more than 66.67 percent have certain immune enhancement or immunoregulation application, such as the following amino acid sequences:

SEQ ID NO.2：VLKSPTIPFFDPQIPK

SEQ ID NO.3：MPVLKSPTIPFFDPQIP

SEQ ID NO.4：MPVLKSPTIPFFDPQIPK

SEQ ID NO.5：GRVMPVLKSPTIPFFDPQIP

the polypeptides are found in amino acid 112-132 of human breast milk β -casein, can be prepared by chemical synthesis (liquid phase method and solid phase method), biological preparation (prokaryotic expression system and eukaryotic expression system) and other methods, and can be used for enhancing the stability by acetylating the nitrogen end of a peptide chain and amidating the carbon end of the peptide chain.

Wherein, the immunoregulation peptide shown in SEQ ID NO.5 has better activity compared with other polypeptides. The polypeptide has a molecular weight (Mw) of 2238.2, an isoelectric point (pI) of 8.8, a fat index of 87.5, a hydrophilicity of 0.17, 90% of the amino acids in random coil form, and 10% of the amino acids in secondary structure of extended chain.

The immunoregulatory peptide is added into a human monocyte THP-1 to be processed for 3 hours, then the cells are flaked by a flaker, fixed by 4 percent paraformaldehyde, dripped with an anti-quenching agent containing DAPI dye, sealed and photographed by a confocal microscope. The results show that the polypeptide is able to enter the cell and distribute in the cytoplasm and nucleus.

Through the determination of the proliferation activity of in vitro cells, the immunoregulation peptide has no obvious influence on the proliferation activity of human monocytes, and the flow detection result shows that the polypeptide has no obvious influence on the apoptosis of the human monocytes.

Transwell in vitro cell migration experiments show that the immunomodulatory peptide can significantly promote migration of human monocytes.

Real-time fluorescent quantitative PCR results show that the immune regulatory peptide can remarkably promote the expression of human monocyte chemotactic factors MCP-1, MCP-3 and a cytokine TNF α, and it is well known that the effective control and elimination of pathogen infection depends on the migration of monocytes to the infected part, and the migration depends on the environment of the chemotactic factors, including monocyte chemotactic proteins 1 and 3(MCP-1 and MCP-3), and active monocyte-produced cytokines such as TNF α can generate effective preventive inflammatory response to various pathogens.

The invention also discovers that the immunomodulatory peptide can significantly increase the expression of phosphorylated I kappa B α in human monocytes through a protein immunoblotting test, and indicates that the immunomodulatory peptide can activate an NF-kappa B signal pathway.

The innate immunity is the first line of defense of the immune system, monocytes are key effector cells of innate immunity response, and are more important links between innate immunity and adaptive immunity, so that the enhancement of the function of the innate immunity monocytes will help to eliminate pathogens.

The polypeptide of the invention can smoothly enter human monocytes, has no obvious influence on the proliferation activity and apoptosis of the human monocytes, can obviously enhance the migration capacity of the monocytes, promotes the expression of monocyte chemotactic factors MCP-1, MCP-3 and cytokine TNF α, and can activate NF kappa B signal channels.

The five immunoregulatory peptides provided by the invention can be optionally prepared into a composition by using any two or more different immunoregulatory peptides, and can be used for preparing an immunopotentiating medicament or an immunoregulatory medicament.

Drawings

FIG. 1 is a schematic diagram of the spatial structure of a polypeptide of the present invention predicted based on the I-TASSER database;

FIG. 2 is a photograph taken by a fluorescence microscope of the polypeptides of test example 1;

FIG. 3 is a control graph of Experimental example 2 to determine the effect of polypeptides on human monocyte proliferation activity;

FIG. 4 is a control flow scattergram of test example 3 for determining the effect of a polypeptide on human monocyte apoptosis;

FIG. 5 is a control bar graph of the microscopic staining contrast and counting statistics for the polypeptides promoting migration of human monocytes in test example 4;

FIG. 6 is a bar graph showing that the polypeptides of test example 5 promote the expression of human monocyte MCP-1, MCP-3 and TNF- α;

FIG. 7 is a control bar graph of five polypeptides promoting the expression of human peripheral blood mononuclear cell MCP-1 in test example 6;

FIG. 8 is a control diagram of the Western blotting experiment for polypeptide-activated human monocyte NF к B signaling pathway in test example 7;

Detailed Description

The invention is further described below with reference to the accompanying drawings.

Example 1

The polypeptide SEQ ID NO.1 has the amino acid sequence of VLKSPTIPFFDPQIP, the molecular weight (Mw) of 1699.02, the isoelectric point (pI) of 5.81, the fat index of 97.33 and the hydrophilicity of 0.253.

Example 2

Polypeptide SEQ ID NO.2, amino acid sequence VLKSPTIPFFDPQIPK, molecular weight (Mw) 1827.2, isoelectric point (pI) 8.56, fat index 91.25, hydrophilicity-0.006.

Example 3

The polypeptide SEQ ID NO.3 has the amino acid sequence of MPVLKSPTIPFFDPQIP, the molecular weight (Mw) of 1927.33, the isoelectric point (pI) of 5.59, the fat index of 85.88 and the hydrophilicity of 0.241.

Example 4

The polypeptide SEQ ID NO.4 has the amino acid sequence of MPVLKSPTIPFFDPQIPK, the molecular weight (Mw) of 2055.51, the isoelectric point (pI) of 8.35, the fat index of 81.11 and the hydrophilicity of 0.011.

Example 5

Polypeptide SEQ ID No.5, amino acid sequence GRVMPVLKSPTIPFFDPQIP, molecular weight (Mw) 2238.2, isoelectric point (pI) 8.8, fat index 87.5, hydrophilicity 0.17, 90% of the amino acids in random coil, 10% of the amino acids in extended chain secondary structure. The predicted spatial structure of the polypeptide in the I-TASSER database is shown in FIG. 1. As can be seen from the spatial structure diagram of the polypeptide in FIG. 1, the light color part of the polypeptide is random coil, and the dark color part is the extended chain structure.

The polypeptides corresponding to the above five examples were obtained by the method of Shanghai peptide Biotech Co., LtdObtained by phase chemical synthesis, and acetylating the nitrogen end of the peptide chain and amidating the carbon end of the peptide chain (Ac-GRVMPVLKSPTIPFFDPQIP-NH)₂). The end blocking makes it closer to the parent protein to enhance its stability and ability to enter cells to enhance the biological activity of the polypeptide.

Test example 1 cellular Performance of the polypeptide

After the polypeptides described in examples 1 to 5 were added to the human monocyte THP-1, respectively, and treated for 3 hours, the cells were flaked by a flaker (Thermo, USA), fixed with 4% paraformaldehyde (Biosharp, China) for 15 minutes, and an anti-quencher (Life P36966, USA) containing DAPI dye was added dropwise, and the cells were sealed, and photographed by a confocal microscope, and it was observed that the polypeptides were able to enter the cells and distribute to cytoplasm and nucleus, indicating that the polypeptides were able to enter the cells and function. FIG. 2 is a photograph taken with a fluorescence microscope of the polypeptide shown in example 5.

Test example 2 Effect of the Polypeptides on the proliferation Activity of human monocytes

The test adopts a method (Dojindo CK04, Japan) of a CCK8 kit to measure the cell proliferation activity, and comprises the following specific steps:

(1) THP-1 cells were cultured in RPMI1640 medium (Gibco, USA) containing 10% FBS (Gibco, USA). Preparation of 5X 10 in 96-well plates³Cell suspension at 100. mu.l/well, 3 replicates per group. Plates were incubated at 37 ℃ with 5% CO₂Pre-culturing for 24 hours in the incubator.

(2) To the plates were added 10. mu.l of complete medium containing the above-described examples, at a working concentration of 100. mu.M of polypeptide. Control wells (medium containing cells, CCK-8, without the polypeptide) and blank wells (medium without cells and the polypeptide, CCK-8) were set.

(3) Plates were incubated at 37 ℃ with 5% CO₂For 6 hours, 24 hours or 48 hours.

(4) 100 μ l/well of fresh medium was replaced and 10 μ l CCK-8 solution was added to each well.

(5) The plates were incubated in an incubator for 1 hour.

(6) Absorbance at 450nm (OD450nm) was measured with a microplate reader.

(7) The effect of the polypeptide on cell proliferation viability was determined based on the absorbance at 450nm and the corresponding formula (cell survival ═ As-Ab ]/(Ac-Ab) ] × 100%).

Test results show that the polypeptides have no significant influence on the proliferation activity of human monocyte cells. A control graph of the effect of the polypeptide on human monocyte proliferation activity based on the polypeptide assay of example 5 is shown in figure 3.

Experimental example 3 Effect of the polypeptide on apoptosis of human monocytes

The method adopts Annexin V-FITC/PI apoptosis detection kit (BD 556547, USA), and comprises the following steps:

(1) 100 μ M of THP-1 cells and control cells treated with the polypeptide as described in the above example for 24 hours were collected, and centrifuged at 300g and 4 ℃ for 5 min.

(2) The cells were washed 2 times with pre-chilled PBS, 300g each time, and centrifuged at 4 ℃ for 5 min.

(3) PBS was aspirated off and 100. mu.11 XBinding Buffer was added to resuspend the cells.

(4) Add 5. mu.l Annexin V-FITC and 10. mu.l PI stabilizing Solution and mix gently.

(5) And (4) keeping out of the light and reacting at room temperature for 10-15 min.

(6) Add 400. mu.l of 1 XBinding Buffer, mix well and place on ice, and the sample is detected by flow cytometry within 1 hour.

The test result shows that the polypeptide of the invention has no obvious influence on the human monocyte apoptosis. A control graph of the effect of polypeptides on human monocyte apoptosis based on the polypeptide assay of example 5 is shown in figure 4.

The results of the test example 2 and the test example 3 show that the polypeptide has no obvious influence on the cell proliferation activity and the apoptosis of the human mononuclear cells and no toxic and side effects of the cells.

Test example 4 Effect of the polypeptide on migration of human monocytes

The detection is carried out by adopting a Transwell migration chamber experiment, and the specific steps are as follows:

human monocytes treated with 100. mu.M of the polypeptides of the above examplesThe culture supernatant is collected 24h after the THP-1 cells are cultured, and the supernatant is taken for immediate experiment after 15min of centrifugation at 4 ℃ and 1000 g. Mu.l of culture supernatant 24 hours after the polypeptide-treated THP-1 cells described in example 1 was added to the lower chamber of the migration chamber, and the THP-1 cells cultured normally were treated at 5X 10⁴Carefully add to the upper chamber suspended in 100. mu.l of serum-free RPMI1640 medium containing 0.5% BSA (Solarbio, A8020-5g, USA) to avoid air bubbles. 600. mu.l of supernatant obtained after 24h of culture of THP-1 cells without polypeptide treatment was added to the lower chamber of the control group. After 24h of culture, the medium was discarded, rinsed with 1 XPBS (Gibco, USA), fixed for 20min by adding 4% paraformaldehyde, rinsed with 1 XPBS, stained with 1% crystal violet (Solambio, G1062-10ml, USA) for 20min, rinsed with clear water to remove excess color, the cell on the upper surface of the chamber was wiped with a cotton swab, naturally dried, observed under a microscope, and photographed.

Test results show that the supernatant cultured by the polypeptide treatment group can obviously promote the migration of human monocytes. Fig. 5 shows the results of cell migration based on the polypeptide Transwell migration experiment of example 5, stained under the microscope, and the histogram is significant for the expression polypeptide group.

Test example 5 Effect of the polypeptide on the expression of human monocyte chemotactic factor and cytokine

Human monocyte THP-1 cells were treated with 100. mu.M of the polypeptide of the above example, centrifuged at 1000rpm for 5min after 6h, the supernatant was discarded, washed once with 1 XPBS, the cells were collected, and total RNA was extracted using an RNA extraction kit (OMEGA R6834-01, USA). The method comprises the following steps:

1. adding β -mercaptoethanol 20 μ l per 1ml of TRK lysis buffer solution before use, collecting cells (no more than 1, 500rpmor 400 g) for 5min, discarding supernatant, adding TRK) lysis solution, mixing by vortex oscillation, lysing cells with TRK lysis buffer solution in a centrifuge tube to loosen cell mass, spinning by flicking EP tube or blowing with a gun head, and mixing by vortex oscillation.

2. Adding equal volume of 70% ethanol into the cracking product, and mixing.

3. The HiBind RNA Mini column was placed in a 2ml collection tube, and the whole sample was applied to the HiBind RNA Mini column and centrifuged at 10000g for 1min at room temperature. The effluent was discarded.

4. The column was placed in a new 2ml collection tube, 300. mu.l of RNA wash buffer I was added and centrifuged at 10000g for 1min at room temperature. The effluent was discarded.

5. Add 500. mu.l RNA wash buffer II and centrifuge at 10000g for 1min at room temperature. The effluent was discarded.

6. Repeating the fifth step once.

7.20000g was centrifuged for 2min to dry the Hibind RNA Mini column.

8. The RNA was eluted. The HiBind RNA Mini column was transferred to a new enzyme-free 1.5ml EP tube, the column was eluted with 30. mu.l DEPC water and centrifuged at 10000g for 1min at room temperature. The effluent was collected as total RNA.

And 9, measuring the RNA concentration by the Nano Drop for later use or freezing and storing by a refrigerator at minus 80 ℃.

The reverse transcription kit (TAKARA RR047A, Japan) was used to reverse mRNA to cDNA as follows:

42 degrees, 2min, 4 degrees.

37 degrees 15min, 85 degrees 5s, 4 degrees.

The expression of human monocyte chemotactic protein 1(MCP-1), monocyte chemotactic protein 3(MCP-3) and tumor necrosis factor α (TNF α) mRNA was detected using Power SYBR Green Master Mix (ABI 4367659, USA) and fluorescent quantitative PCR instrument (ABIViA 7, USA).

The primer sequences are as follows:

MCP-1 upstream primer: 5'-TCATAGCAGCCACCTTCATTC-3'

MCP-1 downstream primer: 5'-TAGCGCAGATTCTTGGGTTG-3'

MCP-3 upstream primer: 5'-AAGCAGAGGCTGGAGAGCTACA-3'

MCP-3 downstream primer: 5'-GGGTCAGCACAGATCTCCTTG-3'

TNF α upstream primer 5'-GACCTCTCTCTAATCAGCCCTCTG-3'

TNF α downstream primer 5'-CAGCCTTGGCCCTTGAAGAGGAC-3'

GAPDH upstream primer: 5'-GTCGCTGTTGAAGTCAGAGG-3'

GAPDH downstream primer: 5'-GAAACTGTGGCGTGATGG-3' are provided.

FIG. 6 shows that the polypeptide treatment of example 5 can significantly promote the expression of human monocyte chemotactic factors MCP-1, MCP-3 and cytokine TNF α, thereby enhancing monocyte migration and pathogen defense.

Test example 6 Effect of the polypeptide on the expression of human peripheral blood mononuclear cell chemokine

Using EasySep^TMNormal Human peripheral blood (EDTA anticoagulated) monocytes were isolated using Direct Human monoclonal Isolation Kit immunomagnetic bead sorting Kit according to the instructions. That is, 50. mu.L of Isolation Cocktail/mL of blood sample and 50. mu.L of RapidSpheres are added^TMAnd (3) standing the blood sample at room temperature for 5min, adding 4 times of volume of the complete culture medium in the experimental example 2, uniformly blowing, putting into a magnetic frame, standing at room temperature for 3min, and repeating the steps to obtain the human peripheral blood mononuclear cells.

The expression of human peripheral blood monocyte chemotactic factor MCP-1 was treated with 100. mu.M of each of the polypeptides of the above examples in the same manner as in test example 5.

FIG. 7 shows that five polypeptides can promote the expression of human peripheral blood monocyte chemotactic factor MCP-1, wherein the polypeptide of example 5 has the best effect of promoting the expression of the peripheral blood monocyte MCP-1. It is further demonstrated that the above polypeptides have similar immunopotentiation effect on human monocytes of different sources.

Experimental example 7 Effect of the polypeptide on the NF-. kappa.B Signal pathway in human monocytes

The method comprises the steps of treating human monocyte THP-1 cells with 100 mu M of the polypeptide as described in example 5, centrifuging at 1000rpm for 5min after 1h, discarding the supernatant, washing once with 1 XPBS, collecting the cells, adding RIPA protein lysate (Beyotime P0013B, China) into the cell sediment to lyse the cells, placing the cells on ice for 30min, centrifuging at 4 degrees and 12000g for 20min, and taking the supernatant as the total protein, adding a BCA protein quantification kit (Thermo 23225, USA) according to the instructions, detecting the absorbance of the sample at 562nm by using a plate reader, quantifying the total protein, adding a BCA protein supernatant buffer solution at 30 mu g of protein per sample to prepare the sample, performing 5min of 95 ℃ denaturation by using a protein blot assay (Western blot), performing loading, electrophoresis, transferring, blocking with 5% skim milk, performing primary anti-kappa 4 ℃ and 1 XPTBST three times, incubating with 10min, incubating with Millibn-TBST, performing primary anti kappa 4 ℃ TBST, washing with 10 x protein wash, performing primary antibody wash with CST wash, PCR wash, and detecting with a key rabbit monoclonal antibody (SAP) as anti-goat anti-rabbit monoclonal antibody, such as anti kappa wash, and anti kappa wash antibody, and anti-rabbit protein wash antibody, and anti-protein wash antibody, wherein the steps are shown as anti-wash, and anti-protein wash antibody, and anti-protein wash.

The results are shown in fig. 8, and western blot experiments show that polypeptide treatment can obviously increase the expression of phosphorylated h κ B α in human monocytes, indicating that NF- κ B signaling pathway can be activated.

Sequence listing

<110> Nanjing City health care hospital for women and children

Application of immunoregulation peptide in preparation of immunopotentiation medicines

<141>2019-12-17

<160>5

<170>SIPOSequenceListing 1.0

<210>1

<211>15

<212>PRT

<213>Homo sapiens β-casein

<400>1

Val Leu Lys Ser Pro Thr Ile Pro Phe Phe Asp Pro Gln Ile Pro

1 5 10 15

<210>2

<211>16

<212>PRT

<213>Homo sapiens β-casein

<400>2

Val Leu Lys Ser Pro Thr Ile Pro Phe Phe Asp Pro Gln Ile Pro Lys

1 5 10 15

<210>3

<211>17

<212>PRT

<213>Homo sapiens β-casein

<400>3

Met Pro Val Leu Lys Ser Pro Thr Ile Pro Phe Phe Asp Pro Gln Ile

1 5 10 15

Pro

<210>4

<211>18

<212>PRT

<213>Homo sapiens β-casein

<400>4

Met Pro Val Leu Lys Ser Pro Thr Ile Pro Phe Phe Asp Pro Gln Ile

1 5 10 15

Pro Lys

<210>5

<211>20

<212>PRT

<213>Homo sapiens β-casein

<400>5

Gly Arg Val Met Pro Val Leu Lys Ser Pro Thr Ile Pro Phe Phe Asp

1 5 10 15

Pro Gln Ile Pro

20

Claims

1. Application of immunoregulation peptide in preparing immunity enhancing medicine is provided.

2. Application of immunoregulation peptide in preparing immunoregulation medicine is provided.

3. Use according to claim 1 or 2, characterized in that: the general formula of the immunoregulation peptide is as follows:

R₁-A-P-B-R₂

wherein, P is an amino acid sequence shown in SEQ ID NO. 1;

R₁any one selected from amino and acetyl;

R₂any one selected from hydroxyl and amino;

a is a peptide chain consisting of 0-5 amino acids;

b is a peptide chain consisting of 0-5 amino acids.

4. Use according to claim 3, characterized in that: the immunoregulation peptide is an amino acid sequence shown by any one of SEQ ID NO. 1-SEQ ID NO. 5.

5. A polypeptide composition characterized by: comprises two or more different polypeptides selected from SEQ ID NO. 1-SEQ ID number 5.