Polypeptides and uses thereof

Abstract

The application discloses a polypeptide and application thereof, and belongs to the technical field of peptide inhibitors. The polypeptide has an amino acid sequence as shown in SEQ ID NO:1; the polypeptide has a bacteriostatic and / or bactericidal function. The polypeptide can inhibit the growth of Vibrio parahaemolyticus at 128 muM, can inhibit the growth of Vibrio harveyi, Vibrio alginolyticus and Vibrio anguillarum at 256 muM, and can inhibit the growth of Aeromonas hydrophila at 512 muM, and can be used for preparing products with bacteriostatic and / or bactericidal properties, such as food or aquaculture products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of peptide inhibitors, in particular, to polypeptides and applications thereof. More particularly, to polypeptides, methods for inhibiting and / or killing bacteria, use of polypeptides in the preparation of products with antibacterial and / or bactericidal properties, food preservatives containing polypeptides, fresh food preservatives, aquaculture feed additives, aquaculture facility treatment agents, disease control preparations, pharmaceutical compositions and bacteriostatic additives. BACKGROUND

[0002] Microbial infection is one of the serious problems in aquaculture. For a long time, antibiotics have been widely used in aquaculture to prevent and control microbial infection diseases, but this has led to an increase in bacterial drug resistance, seriously affecting the safety of aquatic products and the sustainable development of aquaculture. Vibrio parahaemolyticus ( Vibrio parahaemolyticus ), Vibrio harveyi ( Vibrio harveyi ), Vibrio alginolyticus ( Vibrio alginolyticus ), Vibrio anguillarum ( Vibrio anguillarum ) and Aeromonas hydrophila ( Aeromonas hydrophila ) are common pathogenic bacteria in aquaculture, which show intestinal inflammation, septicemia, skin ulceration, liver lesions and other symptoms on aquatic animals such as fish and shellfish, and can cause mass mortality in severe cases. In addition, Vibrio parahaemolyticus and Aeromonas hydrophila can also be transmitted to humans through aquatic products, causing foodborne diseases and wound infections, etc.

[0003] Antibacterial peptides are a class of small peptides that can resist a variety of microorganisms, including bacteria, fungi, viruses and parasites. These peptides mainly bind to the cell membrane or cell wall of microorganisms, causing intracellular substances to leak, and ultimately leading to the death of microorganisms. In addition, antibacterial peptides have the advantages of not being prone to drug resistance, no residue, etc., and will not pollute the water body and aquatic products after use, ensuring the safety of aquatic products.

[0004] However, there are still limited antibacterial peptides suitable for aquatic products at present, so it is necessary to continuously explore new antibacterial peptide resources. SUMMARY

[0005] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, one object of the present application is to provide polypeptides and applications thereof, which have broad-spectrum antibacterial activity against Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum and Aeromonas hydrophila. The polypeptides are not prone to drug resistance, have low mammalian cell toxicity, low hemolytic toxicity and high cell selectivity, and can be used to prepare antibacterial compositions for treating Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum and Aeromonas hydrophila, and also can be applied to the fields of aquaculture and food as peptide inhibitors.

[0006] Specifically, the technical solution of the present application is as follows:

[0007] In a first aspect, the present application provides a polypeptide. According to embodiments of the present application, the polypeptide has an amino acid sequence as set forth in SEQ ID NO: 1; the polypeptide has a bacteriostatic and / or bactericidal function.

[0008] FPSGLDRVDRLVDLVHKLVRG (SEQ ID NO: 1).

[0009] In a second aspect, the present application provides a method for inhibiting bacteria and / or killing bacteria. According to embodiments of the present application, the method comprises contacting a sample to be treated with the polypeptide of the first aspect of the present application.

[0010] According to embodiments of the present application, the bacteria include at least one of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum, and Aeromonas hydrophila.

[0011] According to embodiments of the present application, the bacteria include Vibrio parahaemolyticus, and the polypeptide has a minimum concentration of 128 μM in the contacting system.

[0012] According to embodiments of the present application, the bacteria include at least one of Vibrio harveyi, Vibrio alginolyticus, and Vibrio anguillarum, and the polypeptide has a minimum concentration of 256 μM in the contacting system.

[0013] According to embodiments of the present application, the bacteria include Aeromonas hydrophila, and the polypeptide has a minimum concentration of 512 μM in the contacting system.

[0014] In a third aspect, the present application provides use of the polypeptide of the first aspect in the preparation of a product having a bacteriostatic and / or bactericidal function.

[0015] It can be understood that the aforementioned product includes but is not limited to bacteriostatic agents, preservatives, feed, preservatives, etc.

[0016] According to embodiments of the present application, the bacteria include at least one of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum, and Aeromonas hydrophila.

[0017] According to embodiments of the present application, the polypeptide has a minimum bacteriostatic concentration of 128 μM against Vibrio parahaemolyticus.

[0018] According to embodiments of the present application, the polypeptide has a minimum bacteriostatic concentration of 256 μM against Vibrio harveyi, Vibrio alginolyticus, or Vibrio anguillarum.

[0019] According to embodiments of the present application, the polypeptide has a minimum bacteriostatic concentration of 512 μM against Aeromonas hydrophila.

[0020] In a fourth aspect, the present application provides a food preservative or fresh food preservative. According to an embodiment of the present application, the food preservative or fresh food preservative comprises the polypeptide of the first aspect of the present application. The food preservative or fresh food preservative is not related to disease prevention or treatment function. It can be understood that the aforementioned polypeptide is an active ingredient of the food preservative or fresh food preservative.

[0021] According to an embodiment of the present application, the fresh food preservative is selected from at least one of fruit preservative and vegetable preservative.

[0022] In a fifth aspect, the present application provides an aquaculture feed additive, aquaculture facility treatment agent or disease control preparation. According to an embodiment of the present application, the aquaculture feed additive, aquaculture facility treatment agent or disease control preparation comprises the polypeptide of the first aspect of the present application. It can be understood that the aforementioned polypeptide is an active ingredient of the aquaculture feed additive, aquaculture facility treatment agent or disease control preparation.

[0023] In a sixth aspect, the present application provides a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition comprises the polypeptide of the first aspect of the present application. It can be understood that the aforementioned polypeptide is an active ingredient of the pharmaceutical composition.

[0024] According to an embodiment of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, such as thickening agent, dispersing agent, emulsifying agent, preservative, etc.

[0025] In a seventh aspect, the present application provides a bacteriostatic additive, which comprises the polypeptide of the first aspect of the present application. It can be understood that the aforementioned polypeptide is an active ingredient of the bacteriostatic additive.

[0026] The polypeptide of the present application has the following beneficial technical effects:

[0027] 1) It has been verified that the polypeptide of the present application has broad-spectrum bacteriostatic activity on Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum or Aeromonas hydrophila;

[0028] 2) It has been verified that the polypeptide of the present application is not prone to drug resistance, has low mammalian cell toxicity, low hemolytic toxicity and high cell selectivity, and can be used for preparing an antibacterial composition for Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum or Aeromonas hydrophila infection; in addition, the polypeptide can be used as a peptide inhibitor in the fields of food preservation, aquaculture and medicine.

[0029] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0030] The above and / or additional aspects and advantages of the present application will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings in which:

[0031] Figure 1 is a schematic diagram of HPLC detection results of the antibacterial peptide cAMP093 provided in an embodiment of the present application;

[0032] Figure 2 is a schematic diagram of LC-MS detection results of the antibacterial peptide cAMP093 provided in an embodiment of the present application;

[0033] Figure 3 is a schematic diagram of minimum inhibitory concentration determination of the antibacterial peptide cAMP093 provided in an embodiment of the present application; wherein (a) is Vibrio parahaemolyticus V. parahaemolyticus ; (b) is Vibrio harveyi V. harveyi ; (c) is Vibrio alginolyticus V. alginolyticus ; (d) is Vibrio anguillarum V. anguillarum ; (e) is Aeromonas hydrophila A. hydrophila ;

[0034] Figure 4 is a schematic diagram of mammalian cell toxicity determination results of the antibacterial peptide cAMP093 provided in an embodiment of the present application;

[0035] Figure 5 is a schematic diagram of hemolytic toxicity determination results of the antibacterial peptide cAMP093 provided in an embodiment of the present application. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0037] It has to be noted that, as used herein, the terms "first", "second", etc. merely serve for differentiating between two entities or steps whose names might otherwise be identical, but do not necessarily indicate a specific order or chronology. It is to be understood that data thus designated can be interchanged, where appropriate, so that embodiments of the application described herein can be implemented in other than the order illustrated or described herein. Moreover, the terms "comprising", "having", "including" and "containing" are to be construed open- ended, i.e., to mean "including, but not limited to", unless otherwise noted. Thus, these terms are to be interpreted to allow for the possibility that additional steps, elements, etc. can be added, or that some steps, elements, etc. can be omitted, without departing from the scope of the application.

[0038] In the present context, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any one of the methods well known in the pharmaceutical art. All methods include the step of bringing into association active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active antibody or antigen-binding fragment with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0039] In the present context, the term "pharmaceutically acceptable excipient" can include any solvent, solid or liquid excipient, diluent, or other liquid vehicle, etc., as appropriate for the particular dosage form contemplated. The use of any of these excipients is contemplated herein, except to the extent that any conventional excipient is incompatible with the antibody or antigen-binding fragment according to the application, for example, for producing any adverse biological effect or interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition.

[0040] The present application is based on the global extreme environment microbiome database (The Extreme Environment Microbiome Catalogue, EEMC) constructed by Sanya Huada Life Science Research Institute to mine and screen new antibacterial peptides. First, the antiSMASH (v7.0) (Kai Blin, Simon Shaw, Hannah E Augustijn, et al., antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualisation, Nucleic Acids Research, Volume 51, Issue W1, 5 July 2023, Pages W46-W50) tool is used to identify biosynthesis gene clusters (BGCs) in extreme environment microbial genomes with the parameter set to --minlength 5000, and a deep learning model (RNN, LSTM) is used to predict the core peptide sequence in the RiPPs type BGCs after ribosome synthesis. Further, three pre-trained protein language models (ESM2-3B, ESM3, PTRANS) are used to predict low-toxicity antibacterial peptides for the obtained core peptide sequence, and the polypeptide sequences with the top 50% of each model prediction score are screened as candidate low-toxicity antibacterial peptides. The candidate antibacterial peptides are aligned with the peptides in the known antibacterial peptide database, and the polypeptide sequences identical to the known database are removed to obtain new candidate low-toxicity antibacterial peptide sequences, which are verified by experiments. Through the above sequence mining and experimental verification steps, an antibacterial peptide is finally obtained, which is named cAMP093 and derived from a deep-sea microbial genome. The following is the process of verifying the antibacterial function and toxicity of the antibacterial peptide through wet experiments.

[0041] The technology of the present application is described below through specific examples. If the specific technology or condition is not specified in the examples, it is performed according to the technology or condition described in the literature in the art or according to the product manual. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0042] In the following examples, V. parahaemolyticus Vibrio parahaemolyticus (North American, ATCC 17802), V. harveyi Vibrio harveyi (North American, ATCC 14126), V. alginolyticus Vibrio alginolyticus (North American, ATCC 17749),V. anguillarum Vibrio anguillarum (North American isolate, ATCC 43307), A. hydrophila Aeromonas hydrophila (North American isolate, ATCC 7966).

[0043] Example 1: Synthesis and purification of antibacterial peptide cAMP093

[0044] The antibacterial peptide cAMP093 was synthesized by solid-phase peptide synthesis method by Shanghai Generay Biotech Co., Ltd. The purity of the antibacterial peptide was determined by high performance liquid chromatography (HPLC), and the accurate molecular weight of the antibacterial peptide was detected by liquid chromatography-mass spectrometry (LC-MS).

[0045] HPLC determination: The HPLC analysis column used was Shimadzu Shim-pack GIST C18 column (4.6 mm x 250 mm, 5 μm), mobile phase A was deionized water containing 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% trifluoroacetic acid. 0.1 mg of antibacterial peptide was dissolved in a mixture of formic acid: acetonitrile: water at a volume ratio of 10:30:60, and the final volume was 0.5 mL. The detection sample was injected 30 μL, the total flow rate of the mobile phase was set to 1 mL / min, the detection wavelength was 214 nm, and the linear gradient elution program was used, B phase from 20% to 80% in 0-20 min, A phase from 80% to 20% accordingly. The chromatogram was recorded and the retention time and peak shape characteristics were analyzed, and the results are shown in Figure 1 .

[0046] LC-MS detection: The liquid chromatography-mass spectrometer used was Shimadzu LCMS-2020. The mobile phase was a mixture of methanol and water at a ratio of 1:1 (v / v), and the isocratic elution was carried out at a flow rate of 0.2 mL / min. The sample preparation method was as follows: 0.1 mg of antibacterial peptide was dissolved in a mixture of 50% acetonitrile-50% water (v / v), and the final volume was 0.5 mL. The detection conditions were set as follows: atomization gas flow rate 1.50 L / min, CDL temperature 250℃, CDL voltage 0 v, module temperature 200℃, pre-rod deviation +4.5 kv, detector -0.2 kv, T.Flow 0.2 mL / min. After sample detection, the mass spectrum signal was recorded, including mass-to-charge ratio (m / z) and ion intensity, to confirm the molecular weight information of the antibacterial peptide. The detection results are shown in Figure 2 . Figure 1 The results shown in

[0047] According to the amino acid sequence, the theoretical molecular weight of the antibacterial peptide cAMP093 is 2391.79, Figure 2 The results shown indicate that the molecular weight of the synthetic antibacterial peptide cAMP093 is consistent with the theoretical molecular weight.

[0048] Example 2: Activity analysis of antibacterial peptide

[0049] (1) Minimum inhibitory concentration (MIC) determination

[0050] Five strains of aquatic pathogenic bacteria, including Vibrio parahaemolyticus V. parahaemolyticus , Vibrio harveyi V. harveyi , Vibrio alginolyticus V. alginolyticus , Vibrio anguillarum V. anguillarum , and Aeromonas hydrophila A. hydrophila were streaked on Columbia blood agar medium and incubated at 37°C overnight.

[0051] Individual colonies of the above five strains of aquatic pathogenic bacteria were picked into Mueller-Hinton Broth (MHB) liquid medium and incubated at 37°C with 120 rpm shaking overnight. Fresh MHB was used to dilute the culture by 1:100, followed by incubation to the exponential phase (OD 600 0.4-0.6) and adjustment of the cell concentration to 1×10 6 cfu / mL. The antibacterial peptide cAMP093 was dissolved in MHB to prepare a stock solution with an initial concentration of 1024 μM, and serial dilutions were performed in a two-fold decreasing manner (1:1) to obtain working solutions with concentrations ranging from 1024 μM, 512 μM, 256 μM, 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, 2 μM. Then 100 μL of antibacterial peptide solution at each concentration was added to a 96-well plate, followed by the addition of 100 μL of bacterial solution, resulting in a final antibacterial peptide concentration in the wells ranging from 512 μM to 1 μM. The negative control group was 100 μL of MHB liquid medium added with 100 μL of bacterial solution, and the blank group was 200 μL of MHB medium. Incubation was carried out at 37°C for 16-18 hours. Considering that some antibacterial peptides have poor solubility in the culture medium, which may cause slight turbidity and interfere with visual judgment, spectrophotometry was used to determine the absorbance (OD 600 ) of each well at 600 nm wavelength as an indirect quantitative indicator of bacterial growth. OD 600 is a parameter widely used in microbiology to reflect the cell density in bacterial suspension, with good linear correlation. OD 600Values close to the negative control group usually reflect an increase in bacterial concentration, i.e. good bacterial growth; significantly lower than the negative control group indicates that the bacteria are inhibited or not grown. The blank group is used to correct the OD 600 values of the negative control group and the polypeptide group. The concentration group with a significant decrease in OD 600 value is defined as the MIC value of the antibacterial peptide, which is the lowest concentration of the antibacterial peptide at which no bacterial growth is observed. All experiments were repeated 3 times, and the results are shown in Figure 3 .

[0052] As shown in the experimental results Figure 3 , the MIC of antibacterial peptide cAMP093 against V. parahaemolyticus is 128 μM, the MIC against V. harveyi , V. alginolyticus or V. anguillarum is 256 μM, and the MIC against A. hydrophila is 512 μM.

[0053] (2) Mammalian cell toxicity assay

[0054] To evaluate the toxicity of antibacterial peptide cAMP093 to mammalian cells, four experimental conditions were set: blank group (Blank), negative control group (Control), positive control group (cisplatin treatment group) and antibacterial peptide treatment group (cAMP093). In the 96-well plate, the blank group was only added with 10% fetal bovine serum (04-001-1acs, Biological industries, Israel) MHB culture solution per well. L-02 human liver cells (Beina Biological Technology) and 293T human embryonic kidney cells (Pulunosi) were prepared into single cell suspensions with MHB culture solution containing 10% fetal bovine serum, and 90 μL of 5×10 4 / mL of adherent cells and incubated in a 37°C constant temperature incubator with 5% CO2 for 24 hours. After 24 hours, 10 μL of different treatment solutions were added to each group: PBS buffer was added to the negative control group; a cisplatin solution prepared with PBS (600 μM, D8810, Solarbio) was added to the positive control group, so that the final cisplatin concentration in the well was 60 μM; an antibacterial peptide cAMP093 solution prepared with PBS (600 μM) was added to the antibacterial peptide group, so that the final antibacterial peptide concentration in the well was 60 μM. Each group was further incubated for 48 hours. After the incubation was completed, the culture medium and drug solution in the well were discarded, and 100 μL of CCK-8 working solution (BS350A, Baishabiological) diluted ten times with serum-free MHB medium to a final concentration of 10% (v / v) was added to each well of the 96-well plate. The plate was further incubated in the dark at 37°C with 5% CO2 for 1 hour. The absorbance at 450 nm was measured using a microplate reader, and the raw data results were recorded. The mammalian cell inhibition rate was calculated according to the formula: (OD Control -OD cAMP093 / 顺铂 ) / (OD Control - OD Blank ) x 100%. All experiments were repeated three times, and the results are shown in Figure 4 .

[0055] Figure 4 The results show that, compared with the positive control group (cisplatin treatment group), the antibacterial peptide cAMP093 group has a lower inhibition rate and has significantly lower mammalian cell toxicity. Calculation shows that the inhibition rate of antibacterial peptide cAMP093 on L-02 human liver cells is 0.45%, and the inhibition rate on 293T human embryonic kidney cells is 5.90%.

[0056] (3) Hemolytic toxicity determination

[0057] To evaluate the hemolytic toxicity of the antimicrobial peptide cAMP093, blank group (Blank), positive control group (Control, Triton X-100) and antimicrobial peptide group (cAMP093) were set. Fresh defibrinated sheep blood (Bikeman Biological, China) was taken and centrifuged at 1500 rpm for 10 minutes to separate the red blood cells, and the plasma was discarded. Then the red blood cells were washed repeatedly 3-4 times with PBS buffer until the supernatant was clear and colorless. The washed red blood cells were resuspended with PBS and prepared into a red blood cell suspension with a volume fraction of 4% (v / v). 100 μL of red blood cell suspension was distributed into each well of a 96-well plate. In the positive control group, Triton X-100 (final concentration 1%) was added to each well to induce complete hemolysis. In the antimicrobial peptide group, cAMP093 solution prepared with PBS was added to each well to achieve a final concentration of 60 μM. The blank group only contained red blood cell suspension and PBS buffer, without adding any hemolytic agent or antimicrobial peptide. The 96-well plate was placed in a 37°C incubator and incubated for 1 hour. After incubation, centrifugation was performed at 4°C and 1500 rpm for 10 minutes, and the supernatant of each well was collected into a new 96-well plate. The absorbance at 570 nm was measured by a microplate reader, and the raw data results were recorded. The hemolysis rate calculation formula is (OD cAMP093 - OD Blank ) / (OD Control - OD Blank ) x 100%. All experiments were repeated 4 times, and the results are shown in Figure 5 .

[0058] Figure 5 The results shown in

[0059] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the different embodiments or examples described in the present specification and the features of the different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0060] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and that changes, modifications, substitutions and variations can be made by those skilled in the art without departing from the scope of the present application.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1; The polypeptide has a bacteriostatic and / or bactericidal function.

2. A method for bacteriostatic and / or bactericidal purposes other than therapeutic purposes, characterized in that, The polypeptide of claim 1 is contacted with a sample to be treated; the bacteria include at least one of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum and Aeromonas hydrophila.

3. The method of claim 2, wherein, The bacteria include Vibrio parahaemolyticus, and the minimum concentration of the polypeptide in the contact system is 128 μM.

4. The method of claim 2, wherein, The bacteria include at least one of Vibrio harveyi, Vibrio alginolyticus and Vibrio anguillarum, and the minimum concentration of the polypeptide in the contact system is 256 μM.

5. The method of claim 2, wherein, The bacteria include Aeromonas hydrophila, and the minimum concentration of the polypeptide in the contact system is 512 μM.

6. Use of the polypeptide of claim 1 in the preparation of a product having bacteriostatic and / or bactericidal properties. The bacteria include at least one of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum and Aeromonas hydrophila.

7. A food preservative or fresh food preservative, characterized by, The polypeptide of claim 1 is included; The food preservative or fresh food preservative does not involve disease prevention and treatment function.

8. An aquaculture feed additive, aquaculture facility treatment, or disease control formulation, characterized in that, The polypeptide of claim 1 is included.

9. A pharmaceutical composition, characterized by, Included are: The polypeptide of claim 1 is included.

10. A bacteriostatic additive characterized in that, Included are: The polypeptide of claim 1 is included.

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