An antibacterial peptide FP-CATH41 and its applications

Abstract

This application belongs to the field of biotechnology, and specifically relates to an antibacterial peptide FP-CATH41 and its applications. This antibacterial peptide FP-CATH41 consists of 41 amino acids, and its amino acid sequence is shown as SEQ ID NO.1, and all of its amino acids are of the L type. This antibacterial peptide FP-CATH41 has a broad-spectrum and highly efficient antibacterial effect, and has the characteristics of small molecular weight, simple synthesis, and low hemolytic activity, and can be applied to the fields of medicine, cosmetics, food preservation, and aquaculture.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, and particularly relates to an antibacterial peptide FP-CATH41 and its applications. Background Art

[0002] Traditional antibiotics have achieved remarkable results in combating bacterial infections. However, in recent years, due to their overuse in medicine and aquaculture, microorganisms have gradually developed tolerance to traditional antibiotics, which has exacerbated the emergence of super bacteria with strong drug resistance. In the face of this situation, the traditional solution strategy is to search for new or alternative antibacterial drugs that have not been utilized by drug-resistant bacteria. Unfortunately, over time, even these newly developed antibiotics cannot avoid the fate of ultimately being overcome by microorganisms. Therefore, continuously researching and developing new antibacterial agents has become the key to solving this problem.

[0003] Antibacterial peptides have a unique antibacterial mechanism. Different from antibiotics, they mainly exert their antibacterial effects by interacting with the bacterial cell membrane, making it difficult for bacteria to develop drug resistance. Therefore, the discovery of new antibacterial peptides can provide new ideas and methods for solving the problem of antibiotic resistance and offer more effective antibacterial drug options for clinical treatment. The discovery and development of narrow-spectrum antibacterial agents (also known as precision antibacterial agents) represent the current accurate strategy for reducing or slowing down the emergence and spread of antimicrobial resistance and minimizing the adverse side effects on the normal microbiome. This method aims to inhibit or even kill pathogenic microorganisms through a mode of action different from the past, thus providing new hope for solving the increasingly serious drug resistance problem. Therefore, narrow-spectrum antibacterial peptides have the potential to become a new type of highly efficient antibacterial therapeutic agent, showing great development potential and application prospects. Summary of the Invention

[0004] The object of the present invention is to solve the deficiencies of existing traditional antibiotics and provide an antibacterial peptide FP-CATH41 and its applications, specifically adopting the following technical solutions:

[0005] In a first aspect, the present invention provides an antibacterial peptide FP-CATH41, which is composed of 41 amino acids, and its amino acid sequence is as shown in SEQ ID NO.1, and all of its amino acids are of the L type.

[0006] As a further preferred embodiment, the molecular weight of the antibacterial peptide FP-CATH41 is 4531.5 Da, and the isoelectric point is 13.45.

[0007] SEQ ID NO.1: KRNGKVRKFIRKVKKLLPGGGSIIARVKPVRTLHIASAHVT or Lys Arg Asn Gly Lys Val Arg Lys Phe Ile Arg Lys Val Lys Lys Leu Leu Pro Gly Gly Gly Ser Ile Ile Ala Arg Val Lys Pro Val Arg Thr Leu His Ile Ala Ser Ala His Val Thr.

[0008] The above-mentioned antimicrobial peptide FP-CATH41 is derived from Deinagkistrodon acutus, a snake in the suborder Serpentes of the class Reptilia. Based on the amino acid sequence of the mature peptide of the natural antimicrobial peptide FP-CATH41 obtained, its complete amino acid sequence was synthesized using an automatic polypeptide synthesizer. Subsequently, desalting and purification were performed by reverse-phase column chromatography of high-performance liquid chromatography (HPLC). High-performance liquid chromatography (HPLC) was used to identify the purity of the synthetic peptide, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) was used to determine the molecular weight.

[0009] In a second aspect, the present invention provides the use of the above-mentioned antimicrobial peptide FP-CATH41 in the preparation of antibacterial drugs.

[0010] As a further preferred embodiment, the bacteria include Gram-negative bacteria.

[0011] As a further preferred embodiment, the Gram-negative bacteria include at least one of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

[0012] Experimental data show that the antimicrobial peptide FP-CATH41 provided by the present invention has a high selectivity for Gram-negative bacteria, has weak antibacterial activity against Gram-positive bacteria and fungi, and in addition, the antimicrobial peptide has low hemolytic activity and can be used in the preparation of drugs for antibacterial and inhibiting bacterial growth.

[0013] In a third aspect, the present invention provides a drug for inhibiting bacterial growth, comprising the above-mentioned antimicrobial peptide FP-CATH41.

[0014] As a further preferred embodiment, the drug further comprises a pharmaceutically acceptable excipient.

[0015] In a fourth aspect, the present invention provides the use of the above-mentioned antimicrobial peptide FP-CATH41 in the preparation of food preservatives.

[0016] In a fifth aspect, the present invention provides the use of the above-mentioned antimicrobial peptide FP-CATH41 in the preparation of animal feed additives.

[0017] In a sixth aspect, the present invention provides the use of the above-mentioned antibacterial peptide FP-CATH41 in the preparation of a cosmetic additive.

[0018] The beneficial effects of the present invention are as follows:

[0019] The present invention provides an antibacterial peptide FP-CATH41, which has a broad-spectrum and highly efficient antibacterial effect, and has the characteristics of small molecular weight, simple synthesis, and low hemolytic activity, and can be applied to the fields of medicine, cosmetics, food preservation, and aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 Shown is the gene sequence of the antibacterial peptide FP-CATH41 from Deinagkistrodon acutus;

[0022] Figure 2 Shown are the results of the clearance effect of the antibacterial peptide FP-CATH41 from Deinagkistrodon acutus on the bacterial biofilm that has formed;

[0023] Figure 3 Shown is the hemolytic activity graph of the antibacterial peptide FP-CATH41 from Deinagkistrodon acutus against red blood cells;

[0024] Figure 4 Shown is the cytotoxicity graph of the antibacterial peptide FP-CATH41 from Deinagkistrodon acutus against Raw264.7 cells;

[0025] Figure 5 Shown is the map of the cleavage site (-R130-V131-) of the mature peptide of FP-CATH41;

[0026] Figure 6 Shown is the mass spectrometry identification graph of FP-CATH41. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some, but not all, of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts fall within the scope of protection of the present application.

[0028] Example 1

[0029] Obtaining of Antibacterial Peptide FP-CATH41

[0030] Step 1: Extraction of Total RNA from the Liver Tissue of Deinagkistrodon acutus

[0031] All instruments used in the experiment should be treated with 0.1% DEPC water and autoclaved to ensure no ribonuclease contamination. The specific steps are as follows:

[0032] (1) Take 100 mg of the liver of fresh Deinagkistrodon acutus and add 1 mL of Transzol, then grind thoroughly;

[0033] (2) When there are no tissue lumps after grinding, transfer it to a nuclease-free centrifuge tube and let it stand at room temperature for 5 minutes;

[0034] (3) Add 200 μL of chloroform, vortex for 15 seconds, and incubate at room temperature for 3 minutes;

[0035] (4) Centrifuge at 12000 rpm in a 4℃ centrifuge for 15 minutes, carefully aspirate the colorless aqueous phase; transfer it to a new nuclease-free centrifuge tube, add appropriate isopropanol according to the ratio of 500 uL of isopropanol per 1 mL of Transzol, pipette and mix well, and let it stand at room temperature for 10 minutes;

[0036] (5) Centrifuge at 12000 rpm in a 4℃ centrifuge for 10 minutes, pour off the supernatant, add 1 mL of pre-cooled 75% alcohol, pipette and wash repeatedly, and vortex on the vortex shaker for 10 seconds;

[0037] (6) Centrifuge at 10000 rpm in a 4℃ centrifuge for 5 minutes, pour off the supernatant, use a 10 μL pipette tip to aspirate and discard as much of the liquid remaining on the tube wall as possible, and let the ethanol evaporate completely at room temperature for 5 minutes

[0038] (7) Dissolve the precipitate with 40 μL of nuclease-free water to obtain the total RNA, measure the RNA concentration with Nanodrop 2000, and store it at -80℃.

[0039] Step 2: Synthesis of cDNA from the Liver Tissue of Deinagkistrodon acutus

[0040] Operate according to the steps of the reverse transcription kit of TAKARA. The reaction mixture is prepared as shown in Table 1 below, and set the reaction conditions on the PCR instrument: 42℃ for 2 minutes, and store at 4℃.

[0041] Table 1

[0042]

[0043] Prepare the reverse transcription system according to Table 2 below. Reaction conditions: First, carry out at 37°C for 15 minutes, then at 85°C for 5 s, and after completion, store in a -20°C refrigerator.

[0044] Table 2

[0045]

[0046] Step 3: Cloning of the coding gene for the antibacterial peptide from Deinagkistrodon acutus

[0047] Design and synthesize the forward primer (SEQ ID NO.2): 5’-ATGCAGACCGGACGGGGCAGG-3’ according to the signal peptide conserved region of the antibacterial peptide cDNA sequence of the snake Cathelicidins family submitted in the NCBI database, and the reverse primer sequence is (SEQ ID NO.3): 5’ -AGTCCCACGTGCCACCTCGCACTCGCG-3’; The PCR reaction is carried out under the following conditions: 95°C for 4 minutes, 95°C for 30 sec, 58°C for 30 sec, and 72°C for 1 minute, for 30 cycles. After amplification, use a gel recovery kit (Tiangen Biotech) to recover the target fragment, and then carry out nucleotide sequencing.

[0048] Sequence alignment analysis of this gene sequence using the blast X software on the NCBI website showed that the coding product of this gene might be the precursor of the antibacterial peptide of the Deinagkistrodon acutus Cathelicidins family. Further, sequence alignment of the polypeptide precursor sequence encoded by this gene was carried out using the protein blast software on the NCBI website, and comparison and analysis were carried out with the antibacterial peptide sequences of the Cathelicidins family from other animal sources found previously to determine the cleavage site -R130-V131- during the maturation of this antibacterial peptide (as Figure 5 shown), thereby obtaining the mature peptide sequence of this antibacterial peptide KRNGKVRKFIRKVKKLLPGGGSIIARVKPVRTLHIASAHVT (SEQ ID NO.1, amino acid single-letter abbreviation sequence), named FP-CATH41. FP-CATH41 has obvious differences from the amino acid sequences of all known antibacterial peptides currently (compared with the database, FP-CATH41 has a similarity of only 60% at most with other reported snake cathelicidin families, so FP-CATH41 is a new member of the snake cathelicidin superfamily).

[0049] The precursor encoding FP-CATH41 consists of 172 amino acid residues, and the gene sequence length is 519 bases (as Figure 1 shown).

[0050] Example 2

[0051] Chemical synthesis method of antibacterial peptide FP-CATH41, the process is as follows:

[0052] (1)Solid-phase synthesize the full sequence of FP-CATH41 using an automatic peptide synthesizer (433A, Applied Biosystems), and desalt and purify it by HPLC-C18 reverse-phase column chromatography;

[0053] (2)Determine the molecular weight by the conventional matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) method (as Figure 6 shown);

[0054] (3)Identify the purity of the purified FP-CATH41 by high-performance liquid chromatography HPLC>95%, determine the isoelectric point to be 10.16 by isoelectric focusing electrophoresis, and determine its amino acid sequence structure with an automatic amino acid sequencer.

[0055] Example 3

[0056] Antibacterial activity detection of agkistrodon acutus antibacterial peptide FP-CATH41

[0057] (1)Pick the test strains stored at -80°C and evenly coat them on the MH solid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) plate for activation. Pick single clone strains, adjust the bacterial liquid concentration to 0.5 McFarland units with normal saline, prepare to place the sterilized filter paper with a diameter of 6 mm on the medium surface, drop 10 μL of 2 mg / mL FP-CATH41 sample solution dissolved in sterilized deionized water, and incubate it upside down at 37°C for 18-20 hours, and observe whether an antibacterial circle is formed. If the sample has antibacterial activity, a clear and transparent antibacterial circle will be formed around the filter paper, and the larger the antibacterial circle, the stronger the antibacterial activity of the sample.

[0058] (2)Determine the minimum inhibitory concentration by the two-fold dilution method: Select the strains with antibacterial circles in the above experimental step (1) for MIC determination experiment. Inoculate the test strains into the MH liquid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.), then shake and culture them in an incubator at 37°C until the logarithmic growth phase, and then dilute the bacterial liquid culture of the strains cultured to the logarithmic growth phase to 2×105 cfu / mL with fresh MH liquid medium for use.

[0059] (3) Add 100 μL of MH liquid medium to each well of a sterile 96-well plate in advance. Then add 100 μL of the FP-CATH41 sample solution filtered through a 0.22 μm pore filter membrane and diluted to a certain concentration with MH liquid medium to the first well. After mixing, take 100 μL and add it to the second well, and perform serial dilution in turn. Aspirate 100 μL from the 11th well and discard it. The 12th well is the control tube. Add 100 μL of the diluted bacterial strain culture solution to each well, and then place the 96-well plate in an incubator and incubate at 37 °C for 12 hours. Measure the light absorption at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which no bacterial growth is visible. The results are shown in Table 3.

[0060] Table 3 Antibacterial activity of the antivenom peptide FP-CATH41 from Deinagkistrodon acutus

[0061]

[0062] As can be seen from Table 3, the antimicrobial peptide FP-CATH41 exhibits extremely strong antibacterial activity against Gram-negative bacteria, including clinically isolated drug-resistant bacteria. Its MIC value ranges from 3.12 μg / mL to 50 μg / mL, and it has certain antifungal activity, but its activity against Gram-positive bacteria is weak.

[0063] Example 4

[0064] Determination of the biofilm removal activity of the antivenom peptide FP-CATH41 from Deinagkistrodon acutus

[0065] Pseudomonas aeruginosa ATCC 27853 was inoculated into MH liquid medium (Qingdao Haibo Biotechnology Co., Ltd.) and cultured with shaking at 37 °C until the logarithmic growth phase. The bacterial solution was diluted to 1×10 7 CFU / mL for standby. The 96-well plate was blocked with sterile PBS, and 100 μL of the diluted bacterial solution was added to each well. Incubate in an incubator at 37 °C for 48 h. Carefully remove the supernatant from each well and wash 3 times with sterile PBS. Add the diluted FP-CATH41 (100 μL) to each well, and set 3 parallels for each well according to the concentrations of 0.5×MIC, 1×MIC, 2×MIC, 4×MIC, and 8×MIC. Add 100 μL of normal saline to the blank group. Incubate in an incubator at 37 °C for 24 h. Discard the supernatant, wash 3 times with PBS, fix with 50 μL of methanol for 15 minutes, and dry at the edge of the laminar flow hood. Add 100 μL of crystal violet staining solution to each well and let it stand for 30 minutes. Discard the crystal violet staining solution, wash 3 times with PBS, and blot dry. Add 200 μL of absolute ethanol to dissolve the crystal violet for 30 minutes, and measure the absorbance at 600 nm.

[0066] The results are as Figure 2 shown, and from Figure 2It can be seen that the agkistrodon acutus antibacterial peptide has a certain scavenging effect on the biofilms formed by the Gram-negative bacterium Pseudomonas aeruginosa, showing a concentration-dependent manner.

[0067] Example 5

[0068] Cytotoxicity detection experiment of agkistrodon acutus antibacterial peptide FP-CATH41

[0069] (1) Determination of hemolytic activity

[0070] Take the standard human blood cell reagent (Changchun Boxun Biotechnology Co., Ltd.), wash it 3 times with physiological saline, and prepare a 10 7 cells / mL red blood cell suspension. Mix the red blood cell suspension with different concentrations of antibacterial peptide FP-CATH41 dissolved in physiological saline, incubate at 37 °C for 1 hour, and then centrifuge at 1000 rpm and 4 °C for 10 minutes; aspirate 100 μL of the supernatant and measure the absorbance value at OD 540 . Use physiological saline as the negative control, denoted as A 阴性对照 ; use 1% TritonX-100 as the positive control, denoted as A 阳性对照 ; denote the addition of the polypeptide as A 样品 ; It can be seen from Figure 3 that the antibacterial peptide FP-CATH41 of the present invention has low hemolytic activity and good biocompatibility.

[0071] The percentage of hemolysis rate is calculated according to the following formula: Hemolysis rate % = A 样品 - A 阴性对照 / A 阳性对照 × 100%.

[0072] (2) Determination of cytotoxicity

[0073] Use the CCK8 method to determine the cytotoxicity of antibacterial peptide FP-CATH41 against mouse Raw264.7 cells. Dissolve antibacterial peptide FP-CATH41 in serum-free RPMI 1640 medium, dilute and configure it into different concentration gradients, and then add them to 96-well plates containing mouse Raw264.7 cells (2 × 10 4 cells / well) respectively. Set 3 replicates for each concentration of the peptide, and use serum-free RPMI 1640 culture medium without peptide as the blank control; after incubating at a constant temperature of 37 °C for 24 hours, add 10 μL of CCK8 reagent to each well; after incubating for 4 hours, measure at OD 450Absorbance at this position; The results of three independent experiments were averaged, and cell viability was calculated as % = [A drug - blank] / [A0 drug - blank] × 100%. Among them, blank represents the absorbance of the well containing the culture medium and CCK8 solution without cells; A drug represents the absorbance of the well containing the peptide, culture medium and cells in the CCK8 solution; A0 drug represents the absorbance of the well containing the culture medium and cells in the CCK8 solution without the peptide; The results are as Figure 4 shown.

[0074] Figure 3 Shown is the hemolytic activity diagram of the antimicrobial peptide FP-CATH41 against red blood cells; It can be seen from Figure 3 that the antimicrobial peptide FP-CATH41 of the present invention has low hemolytic activity, indicating that the antimicrobial peptide has low hemolytic toxicity.

[0075] Figure 4 Shown is the cytotoxicity diagram of the antimicrobial peptide FP-CATH41 against Raw264.7 cells; It can be seen from Figure 4 that the antimicrobial peptide FP-CATH41 has low cytotoxicity.

[0076] As can be seen from the above examples, the agkistrodon acutus antimicrobial peptide FP-CATH41 in the present invention can be obtained by chemical synthesis. It has a high selectivity for Gram-negative bacteria, has weak antibacterial activity against Gram-positive bacteria and fungi, but has extremely strong antibacterial activity against some clinically isolated pathogenic Gram-negative bacteria. Secondly, this antimicrobial peptide has the characteristics of small molecular weight, simple synthesis and low hemolytic activity, and can be applied to the fields of medicine, cosmetics, food preservation and aquaculture.

[0077] The agkistrodon acutus antimicrobial peptide FP-CATH41 is combined with conventional dosages and conventional excipients, and corresponding products such as drugs, health products, feed additives, and cosmetic additives are prepared by using existing mature technologies.

[0078] The embodiments of the present application have been described above in conjunction with the accompanying drawings. Specific examples have been used in this article to elaborate on the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the core idea of the present application. However, the present application is not limited to the above specific implementation methods. The above specific implementation methods are only illustrative and not restrictive. Under the inspiration of the present application, those of ordinary skill in the art can also make many forms without departing from the purpose of the present application and the scope protected by the claims, and all belong to the protection scope of the present application.

Claims

1. An antibacterial peptide FP-CATH41, characterized in that, The antimicrobial peptide FP-CATH41 consists of 41 amino acids, and its amino acid sequence is shown in SEQ ID NO.1, and all of its amino acids are of the L type.

2. The antimicrobial peptide FP-CATH41 according to claim 1, wherein The molecular weight of the antimicrobial peptide FP-CATH41 is 4531.5 Da, and its isoelectric point is 13.

45.

3. Use of the antimicrobial peptide FP-CATH41 according to any one of claims 1-2 in the preparation of an antimicrobial drug, characterized in that, The bacteria include Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Candida albicans.

4. A drug for inhibiting the growth of bacteria, characterized in that, It includes the antimicrobial peptide FP-CATH41 described in claim 1; the bacteria include Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

5. The drug according to claim 4, characterized in that, The drug also includes a pharmaceutically acceptable excipient.

6. Use of the antimicrobial peptide FP-CATH41 according to any one of claims 1-2 in the preparation of a food preservative.

7. Use of the antimicrobial peptide FP-CATH41 according to any one of claims 1-2 in the preparation of an animal feed additive.

8. Use of the antimicrobial peptide FP-CATH41 according to any one of claims 1-2 in the preparation of a cosmetic additive.

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