Polypeptide with antibacterial infection and immunomodulatory effects and its application

Abstract

The present invention discloses a polypeptide with antibacterial and immunomodulatory effects and its application, characterized in that the polypeptide comprises the following polypeptides: A1 is a polypeptide having an amino acid sequence of SEQ ID NO. 1; A2 is a polypeptide having an amino acid sequence of SEQ ID NO. 1 obtained by substitution and / or deletion and / or addition of certain amino acid residues, which is 90% or greater identical to SEQ ID NO. 1 and has the same function as A1. The polypeptide of the present invention is easily recombinantly expressed and exhibits strong antibacterial and immunomodulatory effects. Antibacterial and immunomodulatory drugs prepared using the polypeptide FY36 have promising clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a polypeptide with anti-bacterial infection and immunomodulatory effects and applications thereof. Background Art

[0002] Bacterial resistance is one of the most serious public health threats currently facing the public, projected to cause 10 million deaths annually by 2050. Antibiotics are the most widely used drugs worldwide for treating numerous infectious diseases. Due to the misuse and overuse of antibiotics, drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), have emerged and rapidly spread, leading to a serious antimicrobial resistance crisis. Therefore, there is an urgent need to develop new technologies and methods to identify broad-spectrum, potent antibiotics, particularly antimicrobial peptides (AMPs), to address the antimicrobial resistance crisis and effectively treat infectious diseases. The development of novel, potent, broad-spectrum AMPs to help overcome the antimicrobial resistance crisis is urgent. Defensins are a class of disulfide-rich cationic peptides widely distributed in fungi, plants, and animals. They are important regulatory molecules in the immune system and, moreover, possess direct bactericidal activity, making them an important class of antimicrobial peptides. The discovery of novel defensins provides new avenues for the development of novel antimicrobial drugs. Summary of the Invention

[0003] To address the above-mentioned issues, the present invention aims to provide a polypeptide having antibacterial and immunomodulatory effects and its use. The polypeptide of the present invention (designated herein as polypeptide FY36) is easily recombinantly expressed and exhibits excellent antibacterial and immunomodulatory effects. Antibacterial and immunomodulatory drugs prepared using polypeptide FY36 have promising clinical application prospects.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] The first object of the present invention is to provide a polypeptide having anti-bacterial infection and immunomodulatory effects, wherein the polypeptide comprises the following polypeptide A1 or A2: A1 is a polypeptide having an amino acid sequence of SEQ ID NO.1; A2 is a polypeptide having an amino acid sequence of SEQ ID NO.1 obtained by substitution and / or deletion and / or addition of several amino acid residues, which has an identity of 90% or more with SEQ ID NO.1 and has the same function as A1.

[0006] The second object of the present invention is to provide the use of the above polypeptide in the preparation of products having anti-bacterial infection and / or immunomodulatory effects.

[0007] Specifically, the products include pharmaceutical compositions, daily chemical products and health products.

[0008] The third object of the present invention is to provide a pharmaceutical composition comprising pharmaceutical excipients, characterized in that it also comprises the above-mentioned polypeptide or a pharmaceutically acceptable salt thereof.

[0009] Preferably, in the pharmaceutical composition, the amount of the polypeptide having anti-bacterial infection and immunomodulatory effects or a pharmaceutically acceptable salt thereof can be a therapeutically effective amount.

[0010] Specifically, the pharmaceutical excipients can be those widely used in the field of pharmaceutical production. Excipients are primarily used to provide a safe, stable, and functional pharmaceutical composition. They can also provide methods to dissolve the active ingredient at a desired rate after administration to a subject, or to promote effective absorption of the active ingredient after administration of the composition to a subject. The pharmaceutical excipients can be inert fillers or provide a certain function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient in the composition. The pharmaceutical excipients can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0011] Specifically, the pharmaceutical compositions of the present invention can be prepared using any method known to those skilled in the art in accordance with the disclosure. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding, or lyophilizing processes can be used. The effective dosage of the active substance can range widely, and is generally administered in a pharmaceutically effective amount. However, it will be understood that the actual amount of compound administered is generally determined by the physician based on the relevant circumstances, including the condition being treated, the selected route of administration, the actual compound administered; the age, weight, and response of the individual patient; and the severity of the patient's symptoms.

[0012] Specifically, the pharmaceutical compositions of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ophthalmic, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical compositions of the present invention can also be controlled-release or delayed-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and lozenges.

[0013] The fourth object of the present invention is to provide a daily chemical product, comprising excipients permitted to be added to daily chemical products, characterized in that it further comprises the above-mentioned polypeptide or a pharmaceutically acceptable salt thereof.

[0014] Furthermore, the daily chemical products include shampoo, anti-dandruff lotion / cream, skin care ointment / cream, skin care mask, and skin care gel dressing.

[0015] The fifth object of the present invention is to provide a health product, including auxiliary materials allowed to be added to health products, characterized in that it also includes the above-mentioned polypeptide or a pharmaceutically acceptable salt thereof.

[0016] A sixth object of the present invention is to provide a biomaterial having antibacterial and immunomodulatory effects, characterized in that it is any one of the following B1 to B6:

[0017] B1: a nucleic acid molecule encoding the polypeptide having anti-bacterial infection and immunomodulatory effects;

[0018] B2: an expression cassette containing the nucleic acid molecule described in B1;

[0019] B3: a recombinant vector containing the nucleic acid molecule described in B1;

[0020] B4: a recombinant microorganism containing the nucleic acid molecule described in B1;

[0021] B5: a recombinant microorganism containing the expression cassette described in B2;

[0022] B6: A recombinant microorganism containing the recombinant vector described in B3.

[0023] Preferably, the nucleic acid molecule is any one of the following:

[0024] (1) The coding sequence is the DNA molecule of SEQ ID NO.2 in the sequence listing;

[0025] (2) The DNA molecule shown as SEQ ID NO. 2 in the sequence listing.

[0026] The seventh object of the present invention is to provide the use of the above-mentioned biomaterial in the preparation of products with anti-bacterial infection and / or immunomodulatory effects.

[0027] The eighth object of the present invention is to provide a method for preparing a polypeptide having anti-bacterial infection and immunomodulatory effects, characterized in that it includes the steps of expressing the coding gene of the above-mentioned polypeptide having anti-bacterial infection and immunomodulatory effects in biological cells to obtain a protein having anti-bacterial infection and immunomodulatory effects; the biological cells are microbial cells.

[0028] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects: the polypeptide FY36 of the present invention is isolated from the labyrinthine funnel-shaped spider ( Agelena labyrinthica ) transcriptome, a novel defensin with antimicrobial and immunomodulatory properties. The polypeptide FY36 is easily expressed and fermented through recombinant expression, is energy-efficient, and readily commercially viable. The polypeptide FY36 possesses multiple disulfide bonds, a stable structure, and strong resistance to degradation, demonstrating promising antimicrobial and immunomodulatory properties. Antibacterial and immunomodulatory drugs prepared using the polypeptide FY36 have promising clinical application prospects. This invention efficiently produces the antimicrobial and immunomodulatory polypeptide (FSNGCPSSSACSSKCKLNGFKWGMCTGPDKGTCRCY) with the amino acid sequence SEQ ID NO. 1 (FSNGCPSSSACSSKCKLNGFKWGMCTGPDKGTCRCY) by constructing a recombinant plasmid and expressing it in recombinant cells. The polypeptide has a molecular weight of 3812.37 Da, is composed of 36 amino acid residues, and has three disulfide bonds. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the gel electrophoresis identification image of the recombinant expression of polypeptide FY36 in Example 1;

[0030] Figure 2 This is a diagram showing the effect of peptide FY36 on the microstructure of Staphylococcus aureus;

[0031] Figure 3 This is the effect of polypeptide FY36 on LPS-induced production of cytokines TNF-a and IL-6 by mouse spleen cells. DETAILED DESCRIPTION

[0032] To make the purpose, technical solutions and advantages of the present invention clearer, the specific embodiments of the present invention are described in further detail below in conjunction with specific examples and accompanying drawings. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0033] The polypeptide having antibacterial and immunomodulatory effects in the present invention is equivalent to polypeptide FY36. Example 1

[0034] Preparation of polypeptide FY36.

[0035] Step S1, synthesizing the nucleotide sequence of the gene encoding FY36. The FY36 gene was synthesized by chemical methods and provided by Shanghai Bioengineering Co., Ltd. The nucleotide sequence encoding FY36 is shown in SEQ ID NO: 2, and the amino acid sequence of FY36 is shown in SEQ ID NO: 1;

[0036] SEQ ID NO: 1: FSNGCPSSACSSKCKLNGFKWGMCTGPDKGTCRCY.

[0037] SEQ ID NO.2:tttagcaacggctgcccgagcagcagcgcgtgcagcagcaaatgcaaactgaacggcttt

[0038] Aaatggggcatgtgcaccggcccggataaaggcacctgccgctgctat.

[0039] In step S2, the coding gene of FY36 obtained in step S1 is cloned into the plasmid vector pET-32a to obtain the recombinant plasmid pET-32a-FY36. The expressed target gene sequence is optimized according to the codon usage preference of Escherichia coli. In order to enable the TEV enzyme to effectively cut the recombinant protein to remove the fusion tag, the specific recognition coding sequence ENLYFQG is introduced at the N-terminus of FY36.

[0040] The FY36 coding gene and plasmid vector pET-32a were double-digested with NcoI and XhoI to recover the target fragment. The FY36 gene fragment and the pET-32a vector fragment were ligated with T4 ligase to obtain a recombinant plasmid, which was then subjected to DNA sequencing.

[0041] Step S3, introducing the recombinant plasmid pET-32aFY36 obtained in step S2 into the host bacteria, and transforming the pET-32a-FY36 expression plasmid successfully constructed in step S2 into competent cells of Escherichia coli BL21 (DE3) strain (purchased from Beijing Qingke Biotechnology Co., Ltd.), culturing them inverted at 37°C overnight, and picking a single colony the next day to culture in 1 mL of culture medium containing the corresponding resistance to obtain genetically engineered bacteria;

[0042] Step S4: The host bacteria obtained in step S3 are inoculated into LB medium containing ampicillin and shake-cultured. After IPTG induction, the soluble recombinant protein Trx-FY36 is efficiently expressed. The genetically engineered bacteria obtained in step S3 are shake-cultured on a large scale. When the OD600 value reaches approximately 0.8, IPTG is added at a final concentration of 1 mg / mL and shake-cultured at 28°C and 120 rpm for 16 hours. After induction, the cells are enriched in a refrigerated centrifuge, resuspended, and then disrupted using a high-pressure homogenizer to obtain the soluble recombinant protein. After high-speed centrifugation, the His-tagged soluble recombinant protein Trx-FY36 is separated from the supernatant using nickel ion resin.

[0043] Step S5: extract and purify the soluble recombinant protein Trx-FY36 obtained in step S4, and cut off Trx with TEV enzyme to obtain FY36; elute impurities with 10 mM and 30 mM imidazole, wash the target protein on the column with 250 mM imidazole, add an appropriate amount of Tev enzyme to the target protein, and cut at a temperature of 16°C for 12 hours. After dialysis to remove impurities such as imidazole, perform chromatographic purification and separation, collect the target solution, and freeze-dry to obtain the target polypeptide FY36.

[0044] The results of gel electrophoresis identification of recombinant expression of polypeptide FY36 are as follows Figure 1 As shown, the recombinant protein can be observed to be cleaved before and after enzyme digestion. Example 2

[0045] Evaluation of the antibacterial activity of peptide FY36 against bacteria / fungi.

[0046] Antimicrobial spectrum experiment: Detect the inhibitory ability of peptide FY36 against other bacteria / fungi (such as Staphylococcus aureus, Escherichia coli, Candida albicans, Streptococcus mutans, Bacillus subtilis, Propionibacterium acnes, and Malassezia).

[0047] The cultured bacteria were measured under UV spectrophotometer for OD600, according to the formula 1 OD600 = 1 × 10 9 CFU / mL method, the bacterial solution was diluted to 1×10 6CFU / mL. Add 100 μl of diluted bacterial culture to a sterile 96-well plate, followed by 100 μl of serially diluted drug. Mix thoroughly using a pipette. Use untreated culture medium as a negative control, and clindamycin as a positive control. Incubate at 30°C for 1 day. Measure the absorbance of the bacterial culture at 630 nm using a microplate reader. As shown in Table 1, FY36 exhibits potent bactericidal activity against Staphylococcus aureus, Propionibacterium acnes, and Escherichia coli, with MICs of 8, 32, and 16 μg / mL, respectively.

[0048] Table 1 MIC values ​​of FY36 against different strains

[0049] Example 3

[0050] Effects of FY36 on the microstructure of Staphylococcus aureus and Propionibacterium acnes.

[0051] Scanning electron microscopy was used to observe the microscopic morphological changes of bacteria after FY36 treatment. The specific steps were as follows: Bacteria in the exponential growth phase were centrifuged at 5000g for 5 minutes, and the pellet was collected. The cells were rinsed three times with sterile PBS, and the pellet was resuspended to an OD600 of 0.2. To 10 mL of the bacterial suspension, FY36 solution at a final concentration of 1× the MIC was added and incubated at 37°C for 30 minutes. A negative control group was treated without peptide. The cells were centrifuged at 5000g for 5 minutes at 4°C, and the pellet was collected and fixed with 0.6 mL of 2.5% glutaraldehyde (pH 7.4) at 4°C overnight. The cells were washed three times with sterile PBS and dehydrated with a gradient of 50%, 70%, 90%, and 100% ethanol for 10 minutes, respectively. The samples were then treated with pure ethanol, a 1:1 mixture of pure ethanol and tert-butanol, and pure tert-butanol for 15 minutes, respectively. The cells were dried with 0.6 mL of pure tert-butanol, and then plated. Finally, the surface morphology of the bacterial membrane was observed using a scanning electron microscope (TECNAIG2, BIOTWIN). Figure 2 shown.

[0052] Depend on Figure 2 It can be seen that after Staphylococcus aureus and Propionibacterium acnes were treated with FY36 at a concentration of 1×MIC for 60 minutes, Staphylococcus aureus and Propionibacterium acnes showed phenomena such as increased cell volume, destruction of cell wall integrity, and rupture or thickening of the cytoplasmic membrane. Example 4

[0053] Effect of polypeptide FY36 on cytokine production by mouse splenocytes induced by LPS.

[0054] First, Kunming mice were killed by cervical dislocation. The spleens were removed and adipose tissue was removed. The spleens were rinsed with RPMI 1640 medium and minced. The spleens were then ground into single cells using a 5 mL syringe plunger on a 200-mesh copper screen. The cell suspension was centrifuged at 1000 rpm for 10 minutes, and the supernatant discarded. Red blood cell lysis buffer was added, gently pipetted to mix, and the suspension was lysed on ice for 3-5 minutes. The suspension was centrifuged at 1000 rpm for 10 minutes, and the supernatant discarded. The cells were then resuspended in RPMI 1640 medium supplemented with 5% fetal bovine serum, gently pipetted to mix, and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded. The cells were resuspended and centrifuged again to wash away the red blood cell lysis buffer. Resuspend the cells and count them with a hemocytometer to a cell concentration of 1×106 / mL. Add them to a 96-well plate for culture, 180 μl per well, and culture in a 37°C, 5% CO2 incubator for 4 h. Then add LPS (2 μg / mL) for stimulation and add 20 μl of sample (the sample was dissolved in RPMI1640 medium and filtered with a 0.22 μm filter to a final concentration of 5 μg / ml, 10 μg / ml, 20 μg / ml, and 40 μg / ml).

[0055] After 48 hours of incubation at 37°C, 5% CO₂, the cell supernatant was collected and centrifuged at 2000 rpm for 15 minutes. Cytokine levels in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA). In this experiment, wells without LPS and LPS-stimulated wells without sample were also set up as controls. Three replicates were set up for each group. The procedure was performed according to the manufacturer's instructions. Results are shown in Table 1. Figure 3 .

[0056] Depend on Figure 3 Compared with the LPS-only stimulation group, FY36 at different concentrations (10, 20, and 30 μg / ml) reduced TNF-α production by mouse splenocytes by 13.9%, 29.5%, 66.6%, and 77%, respectively, and IL-6 production by 35.2%, 77.9%, and 97.4%, respectively. FY36 significantly inhibited LPS-induced production of TNF-α and IL-6 by mouse splenocytes in a concentration-dependent manner, demonstrating a potent immunomodulatory effect.

Claims

1. A polypeptide having an antibacterial and / or antifungal infection effect, characterized in that: The polypeptide of A1 is as follows: A1 is a polypeptide having an amino acid sequence of SEQ ID NO.

1.

2. Use of the polypeptide according to claim 1 in the preparation of a product having an antibacterial and / or antifungal effect, wherein the bacteria are Staphylococcus aureus, Escherichia coli, Streptococcus mutans, Bacillus subtilis and Propionibacterium acnes, and the fungi are Candida albicans and Malassezia.

3. The use according to claim 2, characterized in that The products include pharmaceutical compositions.

4. A pharmaceutical composition comprising pharmaceutical excipients, characterized in that: It also includes the polypeptide having anti-bacterial and / or fungal infection effect according to claim 1 or a pharmaceutically acceptable salt thereof.

5. A biomaterial having an antibacterial and / or antifungal effect, characterized in that: Any one of the following B1 to B6: B1: a nucleic acid molecule encoding the polypeptide having antibacterial and / or fungal infection effect according to claim 1; B2: an expression cassette containing the nucleic acid molecule described in B1; B3: a recombinant vector containing the nucleic acid molecule described in B1; B4: a recombinant microorganism containing the nucleic acid molecule described in B1; B5: a recombinant microorganism containing the expression cassette described in B2; B6: A recombinant microorganism containing the recombinant vector described in B3.

6. The biomaterial having antibacterial and / or antifungal infection effect according to claim 5, characterized in that: The nucleic acid molecule is: a DNA molecule of SEQ ID NO.1 in the coding sequence table.

7. The biomaterial having antibacterial and / or antifungal infection effect according to claim 6, characterized in that: The nucleic acid molecule is: a DNA molecule shown in SEQ ID NO.2 in the sequence table.

8. Use of the biomaterial according to any one of claims 5 to 7 in the preparation of a product for resisting bacterial and / or fungal infections, wherein the bacteria are Staphylococcus aureus, Escherichia coli, Streptococcus mutans, Bacillus subtilis and Propionibacterium acnes, and the fungi are Candida albicans and Malassezia.

9. A method for preparing a polypeptide having an antibacterial and / or fungal infection effect, characterized in that: The method comprises the steps of expressing the coding gene of the polypeptide according to claim 1 in biological cells to obtain a protein having bacterial and / or fungal infection effects; the biological cells are microbial cells.

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