Polypeptide with bacterial infection resistance and immunomodulatory effect and application thereof

Abstract

The invention discloses a polypeptide with bacterial infection resistance and immunoregulation effects and application thereof, and is characterized in that the polypeptide comprises the following polypeptide A1 or A2: A1 is a polypeptide with an amino acid sequence of SEQ ID NO.1, A2 is a polypeptide with an amino acid sequence of SEQ ID NO.2, and A3 is a polypeptide with an amino acid sequence of SEQ ID NO.3; a2 is a polypeptide which is obtained by substituting and / or deleting and / or adding a plurality of amino acid residues to the amino acid sequence with the amino acid sequence shown as SEQ ID NO.1, has 90% or more identity with the SEQ ID NO.1 and has the same function as A1. The polypeptide disclosed by the invention is convenient to recombine and express and has better antibacterial and immunomodulatory effects, and the antibacterial infection and immunomodulatory drugs prepared from the polypeptide FY36 have better clinical application prospects.

Description

Technical Field

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

[0002] Bacterial resistance is one of the most serious public health threats currently, and it is expected to cause 10 million deaths each year by 2050. Antibiotics are the most widely used drugs for treating a variety of infectious diseases worldwide. Due to the misuse and overuse of antibiotics, resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), have emerged and spread rapidly, leading to a serious crisis of antimicrobial resistance. Therefore, there is an urgent need to develop new technologies and methods to identify broad-spectrum and potent antibiotics, especially antimicrobial peptides (AMPs), to address the crisis of antimicrobial resistance and effectively treat infectious diseases. It is urgent to develop new potent and broad-spectrum antimicrobial peptides (AMPs) to help overcome the crisis of antimicrobial resistance. Defensins are a class of disulfide-rich cationic polypeptides that are widely distributed in fungi, plants and animals and are important regulatory molecules in the biological immune system. In addition, defensins have direct bactericidal functions and are an important class of antimicrobial peptides. The discovery of new defensins provides new ideas for the development of new antimicrobial drugs. Summary of the invention

[0003] In view of the above problems, the object of the present invention is to provide a polypeptide having anti-bacterial infection and immunomodulatory effects and its application. The polypeptide of the present invention (named as polypeptide FY36 in the present invention) is easy to recombinantly express, has good antibacterial and immunomodulatory effects, and the anti-bacterial infection and immunomodulatory drugs prepared using polypeptide FY36 have good clinical application prospects.

[0004] To achieve the above object, the present invention adopts the following technical solution: The first object of the present invention is to provide a polypeptide with anti-bacterial infection and immunomodulatory effects, wherein the polypeptide comprises the following polypeptide A1 or A2: A1 is a polypeptide with an amino acid sequence of SEQ ID NO.1; A2 is a polypeptide with 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.

[0005] 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.

[0006] Specifically, the products include pharmaceutical compositions, daily chemical products and health products.

[0007] 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.

[0008] 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.

[0009] Specifically, the pharmaceutical excipients can be excipients widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical excipients can be inert fillers, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of 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 delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0010] Specifically, the pharmaceutical composition of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes, etc. The effective dose of the active substance can range widely, and is usually administered in a pharmaceutically effective amount. However, it is understood that the amount of compound actually administered is usually determined by the physician based on relevant circumstances, including the condition being treated, the route of administration selected, the actual compound administered; the age, weight and response of the individual patient; the severity of the patient's symptoms, etc.

[0011] Specifically, the pharmaceutical composition of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or delayed release dosage form (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.

[0012] The fourth object of the present invention is to provide a daily chemical product, including auxiliary materials allowed to be added to daily chemical products, characterized in that it also includes the above-mentioned polypeptide or a pharmaceutically acceptable salt thereof.

[0013] Furthermore, the daily chemical products include shampoo, anti-dandruff lotion / cream, skin care ointment / cream, skin care mask, and skin care gel dressing.

[0014] 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.

[0015] The sixth object of the present invention is to provide a biomaterial having antibacterial infection and immunomodulatory effects, characterized in that it is any one of the following B1 to B6: B1: a nucleic acid molecule encoding the polypeptide having anti-bacterial infection and immunomodulatory effects as claimed in 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.

[0016] Preferably, the nucleic acid molecule is any one of the following: (1) The coding sequence is the DNA molecule of SEQ ID NO.2 in the sequence listing; (2) The DNA molecule shown as SEQ ID NO.2 in the sequence listing.

[0017] The seventh object of the present invention is to provide the use of the above-mentioned biomaterial in the preparation of products having anti-bacterial infection and / or immunomodulatory effects.

[0018] 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 a biological cell to obtain a protein having anti-bacterial infection and immunomodulatory effects; the biological cell is a microbial cell.

[0019] 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-leaf spider ( Agelena labyrinthica ) is a new type of defensin with antibacterial and immunomodulatory effects that was first identified in the transcriptome of the human genome. The artificial recombinant expression and fermentation of polypeptide FY36 is convenient, green and energy-saving, and easy to prepare for industrialization. Polypeptide FY36 has multiple pairs of disulfide bonds, stable structure, strong anti-degradation ability, good antibacterial and immunomodulatory effects, and anti-bacterial infection and immunomodulatory drugs prepared using polypeptide FY36 have good clinical application prospects. The present invention constructs a recombinant plasmid and recombinant cell expression to efficiently prepare a polypeptide (FSNGCPSSSACSSKCKLNGFKWGMCTGPDKGTCRCY) with an amino acid sequence of SEQ ID NO.1 with antibacterial and immunomodulatory effects, with a molecular weight of 3812.37 Da, composed of 36 amino acid residues, and having 3 pairs of disulfide bonds. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the gel electrophoresis identification diagram of the recombinant expression of polypeptide FY36 in Example 1; Figure 2 This is a diagram showing the effect of peptide FY36 on the microstructure of Staphylococcus aureus; 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

[0021] In order to make the purpose, technical scheme and advantages of the present invention clearer, the specific embodiments of the present invention are further described in detail below in conjunction with specific examples and drawings. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0022] The polypeptide having antibacterial and immunomodulatory effects in the present invention is equivalent to polypeptide FY36. Example 1

[0023] Preparation of polypeptide FY36.

[0024] Step S1, synthesizing the nucleotide sequence of the gene encoding FY36. The FY36 gene was synthesized by chemical methods and provided by Shanghai Bioengineering Company. 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; SEQ ID NO: 1: FSNGCPSSACSSKCKLNGFKWGMCTGPDKGTCRCY.

[0025] SEQ ID NO.2:tttagcaacggctgcccgagcagcagcgcgtgcagcagcaaatgcaaactgaacggcttt Aaatggggcatgtgcaccggcccggataaaggcacctgccgctgctat.

[0026] Step S2, cloning the coding gene of FY36 obtained in step S1 into the plasmid vector pET-32a to obtain the recombinant plasmid pET-32a-FY36, optimizing the expressed target gene sequence according to the codon usage preference of Escherichia coli, and introducing the specific recognition coding sequence ENLYFQG at the N-terminus of FY36 so that the TEV enzyme can effectively cut the recombinant protein to remove the fusion tag.

[0027] The coding gene of FY36 and the plasmid vector pET-32a were double-digested with NcoI and XhoI, the target fragment was recovered, the gene fragment of FY36 and the pET-32a vector fragment were connected with T4 ligase, and the obtained connection was a recombinant plasmid, and the recombinant plasmid was subjected to DNA sequencing; Step S3, introducing the recombinant plasmid pET-32aFY36 obtained in step S2 into the host bacteria, transferring 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.), incubating overnight at 37°C, picking a single colony the next day and culturing it in 1 mL of culture medium containing corresponding resistance to obtain genetically engineered bacteria; Step S4, inoculate the host bacteria obtained in step S3 into LB medium containing ampicillin and shake culture, and efficiently express and produce soluble recombinant protein Trx-FY36 after IPTG induction; shake culture the genetically engineered bacteria obtained in step S3 on a large scale, and when the OD600 value reaches about 0.8, add IPTG with a final concentration of 1 mg / mL, and shake culture at 28°C and 120rpm for 16 hours. After the induction, enrich the bacteria in a refrigerated centrifuge, resuspend the bacteria, and break the bacteria with a high-pressure homogenizer to obtain soluble recombinant protein. After high-speed centrifugation, use nickel ion resin to separate the soluble recombinant protein Trx-FY36 with a His tag from the supernatant; 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 the impurities with 10mM and 30mM imidazole, wash the target protein on the column with 250mM imidazole, add an appropriate amount of Tev enzyme to the target protein, the enzyme cleavage temperature is 16°C, the enzyme cleavage time is 12 hours, dialyze to remove impurities such as imidazole, perform chromatographic purification and separation, collect the target liquid, and freeze-dry to obtain the target polypeptide FY36.

[0028] 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

[0029] Evaluation of the antibacterial activity of peptide FY36 against bacteria / fungi.

[0030] 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, Malassezia).

[0031] The cultured bacteria were measured by UV spectrophotometer at OD600, according to 1 OD600 = 1 × 10 9 CFU / mL method, the bacterial solution was diluted to 1×10 6 CFU / mL. Add 100 μl of diluted bacterial solution to a sterile 96-well plate, then add 100 μl of gradient diluted drugs, pipette and mix well, use untreated culture medium as negative control, and clindamycin as positive control. After mixing, culture at 30℃ for 1 day. Use an enzyme marker to measure the absorbance of the bacterial solution at 630 nm. The results are shown in Table 1. FY36 has a good bactericidal effect on Staphylococcus aureus, Propionibacterium acnes and Escherichia coli, with MIC values ​​of 8, 32 and 16 μg / mL, respectively.

[0032] Table 1 MIC values ​​of FY36 against different strains Example 3

[0033] Effects of FY36 on the microstructure of Staphylococcus aureus and Propionibacterium acnes.

[0034] Scanning electron microscopy was used to observe the microscopic morphological changes of bacteria after FY36 treatment. The specific operation steps were as follows: the bacteria in the exponential growth phase were centrifuged at 5000g for 5 minutes and the precipitate was collected, washed three times with sterile PBS solution, and the bacterial precipitate was collected and resuspended to OD600 = 0.2; 10 mL of the above bacterial solution was added to the FY36 solution with a final concentration of 1×MIC, and cultured at 37°C for 30 minutes. The treatment group without peptide was used as a negative control; 4°C, 5000g centrifuged for 5 minutes, the precipitate was collected, 0.6 mL (pH = 7.4) of 2.5% glutaraldehyde was added, and fixed at 4°C overnight; after washing with sterile PBS three times, the samples were dehydrated with 50%, 70%, 90% and 100% ethanol gradients for 10 minutes respectively. Then, the samples were treated with pure ethanol, a 1:1 mixture of pure ethanol and tert-butanol, and pure tert-butanol for 15 minutes respectively. 0.6 mL of pure tert-butanol was added to dry and plated. Finally, a scanning electron microscope (TECNAIG2, BIOTWIN) was used to observe the surface morphology of the bacterial membrane. The experimental results are shown in Figure 2 shown.

[0035] 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

[0036] Effect of peptide FY36 on cytokine production by mouse splenocytes induced by LPS.

[0037] First, Kunming mice were killed by cervical dislocation, the spleen was removed and the adipose tissue was peeled off, rinsed with RPMI1640 medium and cut into pieces, and then the spleen was ground into single cells in a 200-mesh copper screen using a 5mL syringe piston. The cell suspension was centrifuged at 1000rpm for 10min, and the supernatant was discarded. Red blood cell lysis buffer was added, gently blown to mix, lysed on ice for 3-5min, centrifuged at 1000rpm for 10min, and the supernatant was discarded. The cells were then resuspended in RPMI 1640 medium containing 5% fetal bovine serum, gently blown to mix, centrifuged at 1000rpm for 10min, and the supernatant was discarded. The cells were resuspended again and centrifuged to wash away the red blood cell lysis buffer. Resuspend the cells and count them with a hemocytometer to make the cell concentration reach 1×106 / mL, add them to a 96-well plate for culture, 180μ1 per well, and culture in a 37℃, 5% CO2 incubator for 4h, then add LPS (2μg / mL) for stimulation, and add 20μ1 of sample at the same time (the sample is dissolved in RPMI1640 culture medium and filtered with a 0.22μm filter to a final concentration of 5μg / ml, 10μg / ml, 20μg / ml, and 40μg / ml).

[0038] After 48 hours of continuous culture at 37°C and 5% CO2, the cell supernatant was collected and centrifuged at 2000 rpm for 15 minutes. The cytokine content in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA). In this experiment, wells without LPS and LPS stimulation without sample were set as controls, and 3 replicates were set for each group. The operation method was carried out according to the instructions. The results are shown in Figure 3 .

[0039] Depend on Figure 3 It can be seen that compared with the LPS stimulation group, different concentrations (10, 20, 30 μg / ml) of FY36 can reduce the production of TNF-a by 13.9%, 29.5%, 66.6%, 77% and the production of IL-6 by 35.2%, 77.9% and 97.4% respectively. FY36 can significantly inhibit the production of cytokines TNF-a and IL-6 by mouse spleen cells induced by LPS in a concentration-dependent manner, and has a good immunomodulatory effect.

Claims

1. A polypeptide having anti-bacterial infection and immunomodulatory effects, characterized in that: A polypeptide comprising the following 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 90% or more identity with SEQ ID NO.1 and has the same function as A1.

2. Use of the polypeptide as claimed in claim 1 in the preparation of products having anti-bacterial infection and / or immunomodulatory effects.

3. The use according to claim 2, characterized in that The products include pharmaceutical compositions, daily chemical products and health care products.

4. A pharmaceutical composition comprising a pharmaceutical excipient, characterized in that: It also includes the polypeptide with anti-bacterial infection and immunomodulatory effects as described in claim 1 or a pharmaceutically acceptable salt thereof.

5. A daily chemical product, comprising auxiliary materials allowed to be added to daily chemical products, characterized in that: It also includes the polypeptide with anti-bacterial infection and immunomodulatory effects as described in claim 1 or a pharmaceutically acceptable salt thereof.

6. A health product, comprising an auxiliary material allowed to be added to the health product, characterized in that: It also includes the polypeptide with anti-bacterial infection and immunomodulatory effects as described in claim 1 or a pharmaceutically acceptable salt thereof.

7. A biomaterial having anti-bacterial infection and immunomodulatory effects, characterized in that: Any one of B1 to B6 below: B1: a nucleic acid molecule encoding the polypeptide having anti-bacterial infection and immunomodulatory effects as claimed in 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.

8. The biomaterial having anti-bacterial infection and immunomodulatory effects as claimed in claim 7, characterized in that: The nucleic acid molecule is any of the following: (1) The coding sequence is the DNA molecule of SEQ ID NO.2 in the sequence listing; (2) The DNA molecule shown as SEQ ID NO.2 in the sequence listing.

9. Use of the biomaterial according to any one of claims 7 to 8 in the preparation of products having anti-bacterial infection and / or immunomodulatory effects.

10. A method for preparing a polypeptide having anti-bacterial infection and immunomodulatory effects, 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 anti-bacterial infection and immunomodulatory effects; the biological cells are microbial cells.

Images