Antibacterial peptide FP-CATH41 and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an antibacterial peptide FP-CATH41 and application thereof. The antibacterial peptide FP-CATH41 is composed of 41 amino acids, the amino acid sequence of the antibacterial peptide FP-CATH41 is shown as SEQ ID NO.1, and all the amino acids of the antibacterial peptide FP-CATH41 are L-shaped. The antibacterial peptide FP-CATH41 has a broad-spectrum and efficient antibacterial effect, has the characteristics of small molecular weight, simplicity in synthesis and low hemolytic activity, and can be applied to the fields of medicines, cosmetics, food preservation and breeding industry.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to an antimicrobial peptide FP-CATH41 and its application. Background Art

[0002] Traditional antibiotics have achieved remarkable results in combating bacterial infections. However, due to their overuse in medicine and aquaculture in recent years, microorganisms have gradually developed tolerance to traditional antibiotics, which has exacerbated the emergence of highly resistant superbugs. Faced with this situation, the traditional solution is to find new or alternative antibacterial drugs that have not yet been used by resistant bacteria. Unfortunately, over time, even these newly developed antibiotics cannot avoid the fate of being overcome by microorganisms. Therefore, the continuous development of new antimicrobial agents has become the key to coping with this problem.

[0003] Antimicrobial peptides have a unique antibacterial mechanism. Unlike antibiotics, they mainly exert their antibacterial effects by interacting with bacterial cell membranes, making it difficult for bacteria to develop drug resistance. Therefore, the discovery of new antimicrobial peptides can provide new ideas and methods for solving the problem of antibiotic resistance and provide more effective antimicrobial drug options for clinical treatment. The discovery and development of narrow-spectrum antimicrobial agents (also known as precision antimicrobial agents) reflects the current accurate strategy to reduce or slow the emergence and spread of antimicrobial resistance and minimize adverse side effects on the normal microbiome. This approach aims to inhibit or even kill pathogenic microorganisms through a different mode of action from the past, thus providing new hope for solving the increasingly serious problem of drug resistance. Therefore, narrow-spectrum antimicrobial peptides have the potential to become a new and highly effective class of antimicrobial therapeutic agents, showing great development potential and application prospects. Summary of the invention The purpose of the present invention is to solve the shortcomings of conventional antibiotics and provide an antimicrobial peptide FP-CATH41 and its application, which specifically adopts the following technical solutions: In a first aspect, the present invention provides an antimicrobial peptide FP-CATH41, wherein the antimicrobial peptide FP-CATH41 consists of 41 amino acids, and its amino acid sequence is shown in SEQ ID NO.1, and all amino acids are L-type.

[0004] As a further preferred embodiment, the antimicrobial peptide FP-CATH41 has a molecular weight of 4531.5 Da and an isoelectric point of 13.45.

[0005] SEQ ID NO.1: KRNGKVRKFIRKVKKLLPGGGSIIARVKPVRTLHIASAHVT or Lys Arg AsnGly Lys Val Arg Lys Phe Ile Arg Lys Val Lys Lys Leu Leu Pro Gly Gly Gly SerIle Ile Ala Arg Val Lys Pro Val Arg Thr Leu His Ile Ala Ser Ala His Val Thr.

[0006] The above antimicrobial peptide FP-CATH41 comes from the agkistrodon acutus of the Squamata order of the reptiles. The amino acid sequence of the mature peptide of the natural antimicrobial peptide FP-CATH41 was obtained, and its complete amino acid sequence was synthesized using an automatic peptide synthesis instrument. Subsequently, it was desalted and purified by reverse phase column chromatography technology 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.

[0007] In a second aspect, the present invention provides the use of the antimicrobial peptide FP-CATH41 in the preparation of antimicrobial drugs.

[0008] As a further preferred embodiment, the bacteria include Gram-negative bacteria.

[0009] As a further preferred embodiment, the Gram-negative bacteria include at least one of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

[0010] Experimental data show that the antimicrobial peptide FP-CATH41 provided by the present invention is highly selective for Gram-negative bacteria and has weak antimicrobial activity against Gram-positive bacteria and fungi. In addition, the antimicrobial peptide has low hemolytic activity and can be used to prepare antibacterial and bacterial growth-inhibiting drugs.

[0011] In a third aspect, the present invention provides a drug for inhibiting bacterial growth, comprising the above-mentioned antimicrobial peptide FP-CATH41.

[0012] As a further preferred embodiment, the drug further comprises a pharmaceutically acceptable excipient.

[0013] In a fourth aspect, the present invention provides the use of the antimicrobial peptide FP-CATH41 in the preparation of a food preservative.

[0014] In a fifth aspect, the present invention provides the use of the antimicrobial peptide FP-CATH41 in the preparation of an animal feed additive.

[0015] In a sixth aspect, the present invention provides the use of the antimicrobial peptide FP-CATH41 in the preparation of cosmetic additives.

[0016] The beneficial effects of the present invention are: The present invention provides an antimicrobial peptide FP-CATH41, which has a broad-spectrum and highly effective antimicrobial 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

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 Shown is the gene sequence of the antimicrobial peptide FP-CATH41 from Agkistrodon acutus; Figure 2 The results show the effect of the antimicrobial peptide FP-CATH41 from Agkistrodon acutus on the removal of bacteria that have formed biofilms; Figure 3 The graph shows the hemolytic activity of the antimicrobial peptide FP-CATH41 from Agkistrodon acutus on erythrocytes; Figure 4 The figure shows the cytotoxicity of the antimicrobial peptide FP-CATH41 from Agkistrodon acutus to Raw264.7 cells; Figure 5 Shown is a map of the cleavage site (-R130-V131-) of the mature peptide of FP-CATH41; Figure 6 Shown is the mass spectrum identification diagram of FP-CATH41. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0020] Example 1 Acquisition of antimicrobial peptide FP-CATH41 Step 1: Extraction of total RNA from liver tissue of Agkistrodon acutus All instruments used in the experiment must be treated with 0.1% DEPC water and sterilized by high pressure to ensure that there is no ribonuclease contamination. The specific steps are as follows: (1) Take 100 mg of fresh liver of Agkistrodon acutus, add 1 mL of Transzol, and grind thoroughly; (2) When there are no tissue pieces left, transfer the sample to a centrifuge tube without nuclease and let it stand at room temperature for 5 minutes. (3) Add 200 μL of chloroform, vortex for 15 seconds, and incubate at room temperature for 3 minutes; (4) Centrifuge at 12,000 rpm in a 4°C centrifuge for 15 minutes, carefully aspirate the colorless aqueous phase; transfer to a new centrifuge tube without nuclease, add appropriate amount of isopropanol according to the ratio of 1 ml of Transzol to 500 uL of isopropanol, mix well with a pipette tip, and let stand at room temperature for 10 minutes; (5) Centrifuge at 12,000 rpm in a 4°C centrifuge for 10 minutes, discard the supernatant, add 1 mL of pre-cooled 75% alcohol and wash repeatedly, and shake on a vortex shaker for 10 seconds; (6) Centrifuge at 10,000 rpm for 5 minutes at 4°C, discard the supernatant, and use a 10 μL pipette tip to remove as much liquid as possible from the tube wall. Incubate at room temperature for 5 minutes until the ethanol evaporates. (7) Dissolve the precipitate in 40 μL of nuclease-free water to obtain total RNA. Measure the RNA concentration using Nanodrop 2000 and store at -80°C.

[0021] Step 2: cDNA synthesis from liver tissue of Agkistrodon acutus According to the steps of TAKARA's reverse transcription kit, the reaction mixture was prepared according to Table 1 below, and the reaction conditions were set on the PCR instrument: 42°C for 2 minutes and stored at 4°C.

[0022] Table 1 The reverse transcription system was prepared according to Table 2 below. The reaction conditions were: first at 37°C for 15 minutes, then at 85°C for 5 seconds, and then stored in a -20°C refrigerator.

[0023] Table 2 Step 3: Cloning of the gene encoding the antimicrobial peptide from Agkistrodon acutus According to the signal peptide conserved region of the cDNA sequence of the snake cathelicidins family antimicrobial peptides submitted to the NCBI database, a forward primer (SEQ ID NO.2) was designed and synthesized: 5'-ATGCAGACCGGACGGGGCAGG-3', and the reverse primer sequence was (SEQ ID NO.3): 5'-AGTCCCACGTGCCACCTCGCACTCGCG-3'; the PCR reaction was 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, 30 cycles. After amplification, the target fragment was recovered using a gel recovery kit (Tiangen Biotechnology) and then nucleotide sequencing was performed.

[0024] The results of sequence alignment analysis of the gene sequence using blast X software on the NCBI website showed that the encoding product of the gene may be the precursor of the Cathelicidins family of antimicrobial peptides of Agkistrodon acutus. The polypeptide precursor sequence encoded by the gene was further aligned using protein blast software on the NCBI website, and compared with the sequences of the Cathelicidins family of antimicrobial peptides from other animal sources previously discovered, and the restriction site -R130-V131- (such as Figure 5 As shown), the mature peptide sequence of the antimicrobial peptide KRNGKVRKFIRKVKKLLPGGGSIIARVKPVRTLHIASAHVT (SEQ ID NO.1, amino acid single-letter abbreviation sequence) was obtained, named FP-CATH41. The amino acid sequence of FP-CATH41 is significantly different from that of all currently known antimicrobial peptides (compared with the database, FP-CATH41 has a maximum similarity of only 60% with other reported snake cathelicidin families, so FP-CATH41 is a new snake cathelicidin superfamily member).

[0025] The precursor encoding FP-CATH41 consists of 172 amino acid residues and the gene sequence is 519 bases long (e.g. Figure 1 as shown).

[0026] Example 2 The chemical synthesis method of the antimicrobial peptide FP-CATH41 is as follows: (1) The full sequence of FP-CATH41 was synthesized on solid phase using an automatic peptide synthesizer (433A, Applied Biosystems) and purified by desalting on a HPLC-C18 reverse phase column; (2) The molecular weight was determined by conventional matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) (e.g. Figure 6 shown); (3) The purity of the purified FP-CATH41 was determined to be >95% by HPLC, the isoelectric point was determined to be 10.16 by isoelectric focusing, and the amino acid sequence structure was determined by an automatic amino acid sequencer.

[0027] Example 3 Detection of antibacterial activity of Agkistrodon acutus antimicrobial peptide FP-CATH41 (1) Select the test strain stored at -80℃ and evenly spread it on the MH solid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) plate for activation. Select the monoclonal strain, adjust the bacterial solution concentration to 0.5 McFarland units with physiological saline, place a sterilized 6 mm diameter filter paper on the surface of the culture medium, add 10 μL of 2 mg / mL FP-CATH41 sample solution dissolved in sterile deionized water, invert and culture at 37℃ for 18-20 hours, and observe whether an inhibition zone is formed. If the sample has antibacterial activity, a clear and transparent inhibition zone will be formed around the filter paper. The larger the inhibition zone, the stronger the antibacterial activity of the sample.

[0028] (2) Determine the minimum inhibitory concentration using the 2-fold dilution method: Select the strain with an inhibition zone in the above experimental step (1) for the MIC determination experiment. Inoculate the test strain into MH liquid culture medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.), then culture it in an incubator at 37°C with shaking until the logarithmic growth phase, and then dilute the culture medium of the strain cultured to the logarithmic growth phase with fresh MH liquid culture medium to 2×105 cfu / mL for later use.

[0029] (3) Add 100 μL of MH liquid medium to each well of a sterile 96-well plate in advance, then add 100 μL of FP-CATH41 sample solution diluted to a certain concentration with MH liquid medium and filtered through a 0.22 μm pore filter membrane to the first well, mix well, take 100 μL and add it to the second well, dilute it in turn, aspirate 100 μL from the 11th well and discard it, and the 12th well is the control tube. Add 100 μL of the diluted strain culture solution to each well, then place the 96-well plate in an incubator at 37°C for 12 hours, and measure the light absorption at a wavelength of 600 nm. The minimum inhibitory concentration is the lowest sample concentration at which bacterial growth is not visible. The results are shown in Table 3.

[0030] Table 3 Antimicrobial activity of the antimicrobial peptide FP-CATH41 from Agkistrodon acutus As shown in Table 3, the antimicrobial peptide FP-CATH41 exhibits extremely strong antibacterial activity against Gram-negative bacteria, including clinically isolated drug-resistant bacteria, with MIC values ​​ranging from 3.12 μg / mL to 50 μg / mL. It has certain antifungal activity, but its activity against Gram-positive bacteria is weak.

[0031] Example 4 Determination of biofilm-clearing activity of the antimicrobial peptide FP-CATH41 from Agkistrodon acutus Pseudomonas aeruginosa ATCC 27853 was inoculated into MH liquid medium (Qingdao Haibo Biotechnology Co., Ltd.) and cultured at 37°C with shaking until the logarithmic growth phase. The bacterial solution was diluted to 1×10 7 CFU / mL for later use. The 96-well plate was sealed with sterile PBS, and 100 μL of diluted bacterial solution was added to each well. Incubate in a 37°C incubator for 48 h. Carefully remove the supernatant from each well and wash three times with sterile PBS. Add diluted FP-CATH41 (100 μL) to each well, and set up three 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 a 37°C incubator for 24 h. Discard the supernatant, wash three times with PBS, fix with 50 μL of methanol for 15 minutes, and blow dry on the edge of the clean bench. Add 100 μL of crystal violet stain to each well and let stand for 30 minutes. Discard the crystal violet stain, wash three times with PBS, and dry. Add 200 μL of anhydrous ethanol to dissolve the crystal violet for 30 minutes, and measure the absorbance at 600 nm.

[0032] The results are as follows Figure 2 As shown by Figure 2 It can be seen that the antimicrobial peptides from Agkistrodon acutus have a certain clearing effect on the biofilm formed by the Gram-negative bacteria Pseudomonas aeruginosa, which is concentration-dependent.

[0033] Example 5 Cytotoxicity test of the antimicrobial peptide FP-CATH41 from Agkistrodon acutus (1) Determination of hemolytic activity Take standard human blood cell reagent (Changchun Boxun Biotechnology Co., Ltd.), wash it with physiological saline three times, and prepare 10 7 cells / mL red blood cell suspension. The red blood cell suspension was mixed with different concentrations of antimicrobial peptide FP-CATH41 dissolved in physiological saline, incubated at 37°C for 1 hour, and then centrifuged at 1000 rpm and 4°C for 10 minutes; 100 μL of the supernatant was aspirated and OD 540 The absorbance value was measured under 40 °C. Physiological saline was used as a negative control. 阴性对照 Indicates that 1% TritonX-100 was used as a positive control. 阳性对照 Indicates that the peptide is added with A 样品 indicates; Figure 3 It can be seen that the antimicrobial peptide FP-CATH41 of the present invention has low hemolytic activity and good biocompatibility.

[0034] The hemolysis rate percentage is calculated according to the following formula: Hemolysis rate % = A 样品 - A 阴性对照 / A 阳性对照 ×100%.

[0035] (2) Determination of cytotoxicity The cytotoxicity of the antimicrobial peptide FP-CATH41 on mouse Raw264.7 cells was determined by CCK8 assay. The antimicrobial peptide FP-CATH41 was dissolved in serum-free RPMI 1640 medium, diluted to different concentration gradients, and then added to 96-well plates containing mouse Raw264.7 cells (2×10 4 Each peptide concentration was set up in 3 replicate wells, and serum-free RPMI 1640 culture medium without peptide was used as blank control. After incubation at 37°C for 24 hours, 10 μL CCK8 reagent was added to each well. After incubation for 4 hours, the expression of CCK8 in OD 450 The results of three independent experiments were averaged, and the cell viability was calculated as % = [A administration - blank] / [A0 administration - blank] × 100%. Among them, blank refers to the absorbance of the well containing culture medium and CCK8 solution without cells; A administration refers to the absorbance of the well containing CCK8 solution with peptide, culture medium and cells; A0 administration refers to the absorbance of the well containing CCK8 solution without peptide, culture medium and cells; the results are shown in Figure 4 shown.

[0036] Figure 3 The graph shows the hemolytic activity of the antimicrobial peptide FP-CATH41 on erythrocytes; Figure 3 It can be seen that the antimicrobial peptide FP-CATH41 of the present invention has a lower hemolytic activity, indicating that the antimicrobial peptide has low hemolytic toxicity.

[0037] Figure 4 The figure shows the cytotoxicity of the antimicrobial peptide FP-CATH41 to Raw264.7 cells; Figure 4 It can be seen that the antimicrobial peptide FP-CATH41 has lower cytotoxicity.

[0038] It can be seen from the above examples that the Agkistrodon acutus antimicrobial peptide FP-CATH41 in the present invention can be obtained by chemical synthesis, has high selectivity for Gram-negative bacteria, has weak antibacterial activity against Gram-positive bacteria and fungi, but has extremely strong antibacterial activity against Gram-negative bacteria including some clinically isolated pathogenic bacteria. Secondly, the 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.

[0039] The antimicrobial peptide FP-CATH41 from Agkistrodon acutus is combined with conventional auxiliary materials in conventional dosages and is prepared into corresponding medicines, health products, feed additives, cosmetic additives and other products using existing mature technologies.

[0040] The embodiments of the present application are described above in conjunction with the accompanying drawings. Specific examples are used herein to illustrate 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, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the inspiration of the present application, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present application and the claims, all of which are within the protection of the present application.

Claims

1. An antimicrobial 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 its amino acids are L-type.

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

45.

3. Use of the antimicrobial peptide FP-CATH41 according to any one of claims 1 to 2 in the preparation of antimicrobial drugs.

4. The use according to claim 3, characterized in that: The bacteria include Gram-negative bacteria or fungi.

5. The use according to claim 4, characterized in that: The Gram-negative bacteria or fungi include Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Candida albicans.

6. A drug for inhibiting bacterial growth, characterized in that: It comprises the antimicrobial peptide FP-CATH41 as claimed in claim 1.

7. The drug according to claim 6, characterized in that The medicament further comprises a pharmaceutically acceptable excipient.

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

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

10. Use of the antimicrobial peptide FP-CATH41 according to any one of claims 1 to 2 in the preparation of cosmetic additives.

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