CN110563814B

CN110563814B - Polypeptide with immunoregulation function and application thereof

Info

Publication number: CN110563814B
Application number: CN201911020432.8A
Authority: CN
Inventors: 容明强; 廖琼
Original assignee: Hunan Normal University
Current assignee: Hunan Normal University
Priority date: 2019-10-24
Filing date: 2019-10-24
Publication date: 2022-11-22
Anticipated expiration: 2039-10-24
Also published as: CN110563814A

Abstract

The invention discloses a polypeptide with antibacterial infection and immunoregulation functions and application thereof, belonging to the technical field of biomedicine. The polypeptide of the invention is convenient for artificial synthesis, has better bacteriostatic action and immunoregulation action, and the antibacterial infection resisting medicine and the immunoregulation medicine prepared by the polypeptide of the invention have better clinical application prospect.

Description

Polypeptide with immunoregulation function and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a polypeptide with antibacterial infection and immunoregulation functions and application thereof.

Background

The wide application of antibiotics, the increasing number of drug-resistant strains and multi-drug-resistant strains, and the clinical anti-infective treatment face serious challenges, and the search for broad-spectrum and efficient antibacterial and antiviral drugs is still a very difficult task. The antibacterial peptide is an extremely important innate immune substance in an animal body, and is a bioactive factor with high-efficiency antibacterial, antiviral and immune functions. The antibacterial peptide is a peptide active substance which is generated by a host non-specific immune defense system and is used for resisting exogenous pathogens, and is an effector molecule of natural immunity. Research shows that the antibacterial peptide has completely different action mechanism from the traditional antibiotics and is not easy to induce the generation of drug-resistant strains; it has broad antibacterial spectrum, and can be used for treating bacteria, fungi, viruses, protozoa, and cancer cells. On poultry, 3 families of antimicrobial peptides have been found to include cathelicidin, lever-expressed antimicrobial peptide (LEAP), and β -defensin. These antimicrobial peptides are of vital importance for poultry against bacterial and viral diseases. Mutations or deletions of these related genes will have a significant effect on the ability of poultry to resist microbial infections. The antibacterial peptides not only have broad-spectrum antibacterial activity, but also have high-efficiency antifungal, antiviral, antiprotozoal and/or antitumor activity, for example, bat5 and IMc7 can kill leptospira, and have good killing effect on candida albicans, cryptococcus, enveloped virus and parasites; some antibiotic peptides have obvious killing power to herpes virus, influenza virus, AIDS virus and other enveloped viruses. In addition, some antibacterial peptides also have various other regulation functions, for example, cathelicidin also has the functions of promoting wound healing, repairing tissue injury, chemochemotaxis, promoting angiogenesis, resisting parasites and the like, and has important biological activity on regulating the immunity of an animal body.

Disclosure of Invention

Aiming at the problems, the invention aims to provide the polypeptide with the functions of resisting bacterial infection and regulating immunity and the application thereof, the polypeptide (named as polypeptide GC35 in the invention) is convenient to artificially synthesize and has better antibacterial and regulating immunity, and the antibacterial and regulating immunity medicine prepared by adopting the polypeptide GC35 has better clinical application prospect.

The invention is realized by the following technical scheme.

The polypeptide with the functions of resisting bacterial infection and regulating immunity is characterized in that the amino acid sequence of the polypeptide is shown in SEQ ID NO. 1; the polypeptide GC35 used in the invention can be obtained by adopting a conventional chemical synthesis method, and the amino acid sequence of the polypeptide GC35 is GCPLDQMQCHNHCQSVRYRGGYCTNFLKMTCKCYG.

The application of the polypeptide in preparing medicines for resisting bacterial infection and regulating immunity.

As a preferable technical scheme, the preparation type of the antibacterial infection medicament is a solution type, a colloidal solution type, an emulsion type or a suspension type.

As a preferred technical scheme, the anti-bacterial infection medicine comprises a medicine for treating escherichia coli, staphylococcus aureus, bacillus subtilis or candida albicans infection.

As a preferable technical scheme, the administration dosage form of the immunoregulation medicament is an oral instant medicinal membrane, oral liquid, a capsule, an injection or a transdermal absorption preparation.

The invention has the beneficial effects that: the polypeptide GC35 of the invention is convenient to artificially synthesize, has better antibacterial and immunoregulation functions, and the antibacterial infection and immunoregulation medicine prepared by the polypeptide GC35 has better clinical application prospect.

Drawings

FIG. 1 is a graph showing the inhibitory effects of the polypeptide GC35 of the present invention on Escherichia coli, staphylococcus aureus, bacillus subtilis and Candida albicans.

FIG. 2 shows the effect of GC35 polypeptide on LPS-induced cytokine production of IFN-. Gamma.TNF-. Alpha.and IL-6 by mouse splenocytes.

FIG. 3 shows the effect of GC35 according to the invention on the proliferation of mouse splenocytes.

FIG. 4 shows the effect of GC35 according to the invention on the viability of mouse splenocytes.

Detailed Description

The present invention will be further described with reference to the following detailed description, which should be construed as illustrative only, and not limiting the scope of the invention, which is to be given the full breadth of the appended claims, and all changes that can be made by those skilled in the art and which are, therefore, intended to be embraced therein.

Example 1

The polypeptide GC35 is prepared by adopting an Fmoc solid phase synthesis method, and the specific steps are as follows.

Weighing 0.2g of resin, placing the resin in a dry and clean reaction tube, adding a proper amount of N, N-dimethyl amide (DMF), activating for 30 minutes, adding 1mmol of the first amino acid residue and 150mg of DMAP into the reaction tube, and reacting the DMF as a solvent for 3 hours; washing the mixture for 3 to 6 times by using DMF after the reaction is finished, adding proper pyridine and acetic anhydride according to the volume ratio of 1:1, and reacting for 30min; after the reaction is finished, washing with DMF for 3-6 times; then, the protecting group Fmoc of the amino acid is eluted with piperidine twice, each time for 15min, and then washed with DMF for 4 times and methanol for 2 times.

Weighing a second amino acid 3mmol and HBTU 3mmol in a reaction tube, adding 0.5ml of DIEA, reacting for 40 minutes, washing with DMF for 3-6 times, adding piperidine solution for twice eluting the protecting group Fmoc of the amino acid, each time for 10 minutes, washing with DMF for 4 times, and washing with methanol for 2 times.

The second step is repeated until the last amino acid residue.

And after the last amino acid is reacted, cutting the reaction product by trifluoroacetic acid for 2 hours, performing reaction and suction filtration to obtain a trifluoroacetic acid solution of the polypeptide, precipitating by using ether, centrifuging, washing by using ether for 3-5 times to obtain a white solid, and desalting and freeze-drying by HPLC to obtain a polypeptide GC35 sample. The polypeptide GC35 has the molecular mass of 4046.70Da and the isoelectric point of 8.65.

Example 2

The polypeptide GC35 has the bacteriostatic strength on escherichia coli, staphylococcus aureus, bacillus subtilis and candida albicans.

The paper method is adopted to determine the bacteriostatic strength of the polypeptide GC35 on escherichia coli, staphylococcus aureus, bacillus subtilis and candida albicans. Heating and melting the prepared high-column broth agar culture medium, cooling to about 50 ℃, adding 1mL broth culture medium bacterial liquid (bacterial liquid cultured for 18 hours at constant temperature of 37 ℃) into the broth culture medium, slightly shaking the broth culture medium bacterial liquid to be uniform, pouring the broth culture medium bacterial liquid into a sterile plate, slightly shaking the plate to enable the culture medium to be uniformly and flatly paved on the plate, after cooling and solidifying, putting polypeptide GC35 dry paper sheets into plain blood in order by using sterilized forceps, covering the plain blood with Tao Wagai, marking the mark, culturing the culture medium in a constant-temperature incubator at 37 ℃ for 24 hours, taking out the culture medium after 24 hours, measuring the size of a circle by using a caliper, recording the size of the circle, and comparing the bacteriostatic strength of the polypeptide GC35 on drug-resistant Candida albicans with the dosages of 50 mu g/mL, 50 mu g/mL escherichia coli, 50 mu g/mL staphylococcus, 50 mu g/mL bacillus subtilis, 50 mu g/mL Candida albicans and 6 strains, wherein the bacteriostatic strength is shown in figure 1.

In the graph of FIG. 1, the large intestine shows the inhibitory effect of the polypeptide GC35 of the present invention on Escherichia coli at 50. Mu.g/ml; the aureococcus is a graph of the inhibitory effect of the polypeptide GC35 of the invention on 50 mu g/ml of aureostaphylococcus; the Candida albicans is a graph of the inhibitory effect of the polypeptide GC35 of the invention on Candida albicans of 50 mug/ml; the hay is a figure of the inhibition effect of the polypeptide GC35 on 50 mu g/ml of the hay bacillus; candida 08022710, candida 08032821, candida 08030102, candida 08030401, candida 08030809, candida 08032815 are graphs of the inhibitory effect of the polypeptide GC35 of the present invention against 6 strains of drug-resistant Candida albicans. Wherein 6 drug-resistant Candida albicans strains are drug-resistant strains provided by clinical screening in hospitals.

As can be seen from FIG. 1, the polypeptide GC35 has strong inhibitory effects on Escherichia coli at 50. Mu.g/ml, staphylococcus aureus at 50. Mu.g/ml, bacillus subtilis at 50. Mu.g/ml and Candida albicans at 50. Mu.g/ml, wherein the polypeptide GC35 also has significant inhibitory effects on clinically drug-resistant Candida albicans.

Example 3

Minimum inhibitory concentrations of polypeptide GC35 against different bacteria.

The minimal inhibitory concentrations of polypeptide GC35 against different bacteria were determined by the macrobroth dilution method and the results are shown in table 1.

TABLE 1 minimal inhibitory concentrations of polypeptide GC35 against different bacteria

Species of	Minimum inhibitory concentration
		Escherichia coli	17.5μg/ml
Staphylococcus aureus	10.0μg/ml
		Bacillus subtilis	5.0μg/ml
Candida albicans	10.0μg/ml

As can be seen from Table 1, the minimum inhibitory concentrations of the polypeptide GC35 against Escherichia coli, staphylococcus aureus, bacillus subtilis and Candida albicans were: 17.5ug/ml, 10.0ug/ml, 5.0ug/ml, 10.0ug/ml.

Example 4

Effect of polypeptide GC35 on LPS-induced cytokine production by mouse splenocytes.

First, kunming mice were sacrificed by cervical dislocation, spleens were removed and adipose tissues were dissected off, and RPMI 1640 medium was usedAfter washing, the cells were minced, the spleen was ground and dispersed into single cells by using a 5mL syringe piston in a 200 mesh copper mesh, the cell suspension was centrifuged at 1000rpm for 10min, and the supernatant was discarded. Adding erythrocyte lysate, gently blowing and mixing, cracking on ice for 3-5min, centrifuging at 1000rpm for 10min, and discarding the supernatant. Then, the cells were resuspended in RPMI 1640 medium containing 5% fetal bovine serum, and the mixture was gently pipetted and centrifuged at 1000rpm for 10min, and the supernatant was discarded. The cells were resuspended and centrifuged again to wash the red blood cell lysate. Resuspend the cells and count with a hemocytometer to a cell concentration of 1X 10 ⁶ Perml, added to a 96-well plate for culturing at 180. Mu.l per well at 37 ℃ in 5% CO ₂ The cells were incubated in an incubator for 4h and stimulated with LPS (2. Mu.g/mL) while adding 20. Mu.l of the sample (dissolved in RPMI 1640 medium and filtered through a 0.22 μm filter to a final concentration of 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL).

At 37 ℃ C, 5% CO ₂ And (3) continuously culturing in the incubator for 48h, collecting cell supernatant, centrifuging at 2000rpm for 15min, and detecting the content of the cell factor in the supernatant by adopting an enzyme-linked immunosorbent assay (ELISA). In this experiment, wells with no LPS added, LPS stimulated but no sample added were set simultaneously as controls, and 3 replicates were set for each group. The procedure was carried out as described and the results are shown in FIG. 2.

As can be seen from FIG. 2, GC35 was able to significantly inhibit LPS-induced cytokine production IFN-. Gamma.TNF-. Alpha.and IL-6 by mouse splenocytes in a concentration-dependent manner. Compared with the LPS stimulation group, the GC35 with different concentrations (5, 10, 20, 40 mu g/ml) can reduce IFN-gamma produced by mouse spleen cells by 6.3 percent, 20 percent, 48 percent and 64 percent respectively, reduce TNF-alpha produced by 4.9 percent, 24 percent, 68 percent and 77 percent respectively, and reduce IL-6 produced by 50 percent, 53 percent, 93 percent and 94 percent respectively.

Example 5

The MTT method detects the influence of the polypeptide GC35 on the mouse splenocyte proliferation.

Mouse splenocytes were aseptically isolated and suspended in RPMI 1640 medium containing 5% fetal bovine serum and 4. Mu.g/mL concanavalin A (ConA) to a cell concentration of 1X 10 ⁶ /mL, addition to 96-well plates, 180. Mu.l per well, 5% CO at 37% ₂ In the incubatorAfter 4h incubation, the samples (dissolved in RPMI 1640 medium and filtered through a 0.22 μm filter) were added to final concentrations of 5, 10, 20, 40 and 80 μ g/mL and incubation continued for 44h. 20 μ l of MTT (methanol/methanol) 5mg/mL is added into each well, the mixture is incubated for 4h, 150 μ l of dimethyl sulfoxide (DMSO) is added into each well, and the mixture is uniformly blown to fully dissolve purple crystals. The enzyme-linked detector measures the absorption value at 570nm, and the reference wavelength is 630nm. Blank control wells without ConA and sample were also set up with 6 replicates per experimental group.

In this experiment, different concentrations of GC35 were used to stimulate splenocytes from mice with ConA, and the absorbance at 570nm was measured and the cell viability was calculated, as shown in FIG. 3. As can be seen from FIG. 3, the cell viability was not greatly different at GC35 concentrations of 5, 10, 20, 40 and 80. Mu.g/mL compared to the control group stimulated with ConA alone, indicating that GC35 did not inhibit the proliferation of mouse splenocytes.

Example 6

The MTT method detects the influence of the polypeptide GC35 on the activity of mouse spleen cells.

Aseptically separating mouse splenocytes, diluting to 5 × 10 ⁴ Cell suspension per mL. Adding to a 96-well plate, adding 180. Mu.l of cell suspension per well, at 37 ℃,5% ₂ Culturing in an incubator for 5-6h. After the cells are attached, 20. Mu.l of samples diluted with RPMI 1640 medium to different concentrations are added to each well, each concentration is repeated for 6 times, and an equal volume of RPMI 1640 medium is added to the blank control for 24h of culture. Then 20. Mu.l of MTT at 5mg/mL was added to each well and incubated for 4h, and bluish purple crystals were observed. The medium was aspirated, 150. Mu.l DMSO was added to each well, and the mixture was shaken and mixed to dissolve the crystals sufficiently. The light absorption at 570nm was measured on an enzyme-linked detector with a reference wavelength of 630nm. Each experimental group was set up for 6 replicates.

The cell viability calculation formula is as follows:

cell viability (%) = Ax/A0 × 100 (A0 is blank light absorption, ax is sample light absorption)

The spleen cells of mice were cultured for 24h with various concentrations of GC35, and the light absorption at 570nm was measured and the cell viability was calculated, and the results are shown in FIG. 4. Experimental results figure 4 shows that GC35 had substantially no effect on mouse splenocytes at concentrations of 10, 20, 40, and 80 μ g/mL. Cell proliferation was slightly promoted at 160. Mu.g/mL. Therefore, GC35 had substantially no effect on mouse splenocyte viability.

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Sequence listing

<110> university of Master in Hunan

<120> polypeptide having immunoregulation action and use thereof

<130> 2019

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 35

<212> PRT

<213> Artificial sequence ()

<400> 1

Gly Cys Pro Leu Asp Gln Met Gln Cys His Asn His Cys Gln Ser Val

1 5 10 15

Arg Tyr Arg Gly Gly Tyr Cys Thr Asn Phe Leu Lys Met Thr Cys Lys

20 25 30

Cys Tyr Gly

35

Claims

1. The polypeptide with the functions of resisting bacterial infection and regulating immunity is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

2. Use of the polypeptide of claim 1 for the manufacture of a medicament for the treatment of a bacterial infection.

3. Use of the polypeptide of claim 1 for the preparation of a medicament against Candida albicans infection.

4. The use according to claim 2, wherein the antibacterial agent is formulated in the form of a solution, a colloidal solution, an emulsion or a suspension.

5. The use according to claim 2, wherein the anti-bacterial infection medicament comprises a medicament for the treatment of an infection with escherichia coli, staphylococcus aureus or bacillus subtilis.