KR102529289B1 - Antimicrobial peptide analogues derived from mud loach NK-lysin - Google Patents

Abstract

The present invention provides an antibacterial peptide derivative derived from loach NK-lysin with an antibacterial effect, which has no side effects or rejection reactions to acquired immunity, is stable against heat and salt, and exhibits excellent antibacterial activity against various indicator strains.

Description

Antimicrobial peptide analogues derived from mud loach NK-lysin having antibacterial effect {Antimicrobial peptide analogues derived from mud loach NK-lysin}

The present invention relates to antibacterial peptides, and more particularly, to an antibacterial peptide derivative derived from loach NK-lysin having an antibacterial effect.

Antimicrobial peptides (AMPs) are part of the innate immune system of all living organisms and have broad-spectrum antibacterial activity against gram-positive and gram-negative bacteria, fungi, viruses and parasites. AMP is an oligopeptide composed of a variable number of amino acids, generally consisting of 5-50 amino acid residues. Endogenous AMP is produced when infection occurs and is secreted from the tissues of all animals and plants as a rapid and efficient primary defense mechanism. The peptide has a broad spectrum of antibacterial activity and can induce repair and adaptive immune responses in concert against multidrug-resistant bacteria. Typical characteristics of AMPs are net-cationic charge, hydrophobicity and amphipathic nature. Various strategies have been applied to design new synthetic analogues of these AMPs. In order to develop new AMPs that increase antibacterial activity and reduce toxicity against eukaryotic cells, many studies have been conducted on peptide chain length, net charge, etc. (net-charge), hydrophobicity and structural modifications are adopted. Sequence-based approaches that link antibacterial activity to the presence of specific amino acids or fragments at specific positions are a direct and efficient method among various strategies when designing new analogues of AMP (Le et al., Sci. Rep . 5, 9761, 2015).

Meanwhile, NK-lysin is a cationic, amphipathic alpha-helical AMP produced by cytotoxic T-lymphocytes and natural killer (NK)-cells, and has antibacterial activity against various microorganisms. NK-lysin derived from pigs and cattle shows a wide range of antibacterial activities against various pathogens, including gram-positive and gram-negative bacteria, fungi, viruses, and parasites. In this regard, conventional disease control methods have been universal, but as concerns about the human body increase due to the emergence of antibiotic-resistant bacteria, the development of antibiotic substitutes using natural antibacterial substances such as NK-lysin is required.

In the case of the prior art, there is a problem of low antibacterial activity to be used as an antibiotic substitute for existing chemical preparations.

The present invention is intended to solve various problems, including the above problems, and has no side effects or rejection reactions for acquired immunity and has an antibacterial effect showing excellent antibacterial activity against various indicator strains. An antibacterial peptide derivative derived from loach NK-lysin It aims to provide However, these tasks are illustrative, and the scope of the present invention is not limited thereby.

According to one aspect of the present invention, an antimicrobial peptide composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 is provided.

According to another aspect of the present invention, an antimicrobial composition containing the antimicrobial peptide as an active ingredient is provided.

According to another aspect of the present invention, a feed additive containing the antimicrobial peptide as an active ingredient is provided.

According to another aspect of the present invention, there is provided a composition for preventing caries, comprising the antimicrobial peptide as an active ingredient.

The loach NK-lysin-derived antimicrobial peptide derivative having an antibacterial effect of the present invention made as described above is stable to heat and salt, has no side effects, and has a high antibacterial effect against various indicator strains by using loach NK-lysin protein sequence information, which is a natural antibacterial substance. Since it shows antibacterial activity, it can be used as an antibiotic substitute that can solve the problem of resistant bacteria of existing antibiotics. Of course, the scope of the present invention is not limited by these effects.

1 is a diagram showing the full-length amino acid sequence and cDNA sequence of NK-lysin protein (MM-NKL) derived from loach according to an embodiment of the present invention. Saposin B superfamily domains are boxed and marked in gray.
2 is a diagram showing the amino acid sequences of two parent peptides, MM-NKL-1 and MM-NKL-2, using Jpred 4, a protein secondary structure prediction program.
Figure 3 is a schematic diagram of the secondary structure and amino acids of MM-NKL-1 for preparing antimicrobial peptides MM-NKL-1 (CKWVMDKIKK), MM-NKL-1A (CKWVMKKIKK), and MM-NKL-2 (FLKKACNFFI) peptides of the present invention This is a drawing of the design of the derivative, MM-NKL-1A, through substitution.
Figure 4 shows the antibacterial synthetic peptides of the present invention MM-NKL-1, MM-NKL-1A and MM-NKL-2 as experimental strains E. coli, E. tarda, P. aeruginosa, V. anguillarum, V. harveyi, These are photos of B. cereus, S. aureus, S. mutans and C. albicans treated and antibacterial activity observed.
Figure 5a is a graph showing the results of analyzing the hemolytic activity of the antimicrobial peptides MM-NKL-1, MM-NKL-1A and MM-NKL-2 of the present invention.
5B is a graph showing the results of analyzing the membrane permeability of E. coli of the antimicrobial peptides MM-NKL-1, MM-NKL-1A and MM-NKL-2 of the present invention.
Figure 6a is a gel photograph showing the results of analyzing the DNA-binding ability of the antimicrobial synthetic peptides MM-NKL-1, MM-NKL-1A and MM-NKL-2 of the present invention.
Figure 6b is a gel photograph showing the results of analyzing whether the antibacterial synthetic peptides MM-NKL-1, MM-NKL-1A, and MM-NKL-2 of the present invention inhibit DNA polymerase activity.
Figure 7a is a photograph of the stability of MM-NKL-2, an antibacterial peptide of the present invention, against heat.
Figure 7b is a photograph of observing the stability of MM-NKL-2, an antimicrobial peptide of the present invention, against salt (salt, NaCl).

Definition of Terms:

As used herein, the term "antimicrobial peptide (AMP)" is a front-line biological defense factor of the innate immune system present in all types of living organisms from prokaryotes to mammals. Unlike acquired immunity, which reacts to specific microorganisms or antigens, antimicrobial peptides act regardless of the type of microorganisms, and the reaction time is very fast, either right away or within a few hours. It is expected to replace existing antibiotics with natural products that are naturally produced to protect the host from microorganisms in a wide range of biological systems without problems of resistance, immunity or rejection, different from existing antibiotics that always have the problem of resistant bacteria. It is a potential drug candidate (Hancock et al, , Curr. Drug Targets Infect Disord . Mar;2(1):79-83. 2002).

As used herein, the term "NK-lysin" is a cationic, amphiphilic alpha-helical AMP produced by cytotoxic T-lymphocytes and natural killer (NK)-cells, and is a peptide that exhibits antibacterial activity against various microorganisms. It was first isolated from the small intestine of pigs, and it is a saposin-like protein family and has homology to granulysin. It typically consists of 74 to 78 amino acid residues and possesses the Saposin B superfamily domain, which contains six well-conserved cysteine residues that form three disulfide bonds.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect of the present invention, an antimicrobial peptide composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 is provided.

In the antimicrobial peptide, the peptide may be derived from the NK-lysin protein of loach and may be a peptide having an amidated C-terminus.

According to another aspect of the present invention, an antimicrobial composition containing an antimicrobial peptide as an active ingredient is provided.

In the antimicrobial composition, it may have antibacterial activity against gram-negative bacteria, gram-positive bacteria or fungi, and the gram-negative bacteria may be Vibrio anguillarum EC, Vibrio harveyi KCCM 40866 , Escherichia coli KCTC1116, Edwardsiella tarda H-4, or Pseudomonas aeruginosa KCTC2004 The Gram-positive bacteria may be Bacillus cereus ATCC21772, Streptococcus mutans KCTC3065, Streptococcus iniae FP5229, or Staphylococcus aureous RM4220, and the fungus may be Candida albicans KCTC7965.

According to another aspect of the present invention, a feed additive containing the antimicrobial peptide as an active ingredient is provided.

According to another aspect of the present invention, there is provided a composition for preventing caries, comprising the antimicrobial peptide as an active ingredient.

As antibiotics were banned from being mixed in animal feed from July 1, 2011, there is a steady demand for the development of non-residual next-generation antibiotics to replace existing antibiotics. In particular, it is very urgent to take measures to solve the misuse and abuse of pesticides and antibiotics used in feed and livestock due to the recent pesticide detection egg scandal and to secure the safety of national food. For this reason, if natural antibiotics using antimicrobial peptides derived from aquatic organisms are developed and used in the aquaculture industry, it is expected that it will be possible to develop a safe food brand free from residual antibiotics. However, until now, synthetic new drugs account for an absolutely high share in the pharmaceutical market, and in the case of protein drugs such as antibacterial peptides, the annual growth rate is 18%, but the production cost is still high compared to the production cost of synthetic antibiotics.

In order to solve the above problems, the development of antibiotic substitutes using natural antibacterial substances such as NK-lysin is required. NK-lysin structurally contains the Sapoin B superfamily domain, which adopts an alpha-helical structure. The saposin B superfamily domain, which consists of about 80 amino acids, is named after saposin, a small lysosomal protein that acts as a sphingolipid hydrolase-activating protein in vertebrates. Like NK-lysin, granulysin is a protein with a similar structure that contains the domain of the saposin B superfamily. NK-lysin and granulysin are generally synthesized as precursor proteins of about 15 kDa in size, and both ends are cleaved to convert into about 9 kDa of active protein having antibacterial activity.

Therefore, the inventors of the present invention, without the problem of resistant bacteria, which is a problem of existing antibiotics, using loach NK-lysin (MM-NKL) protein sequence information, a natural antibacterial substance with low side effects or rejection for acquired immunity, short-length peptide variants The present invention was completed by observing that it has no side effects, is stable to heat and salt, and exhibits high antibacterial activity as a result of being prepared and applied to various strains.

Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms. It is provided to fully inform you.

Example 1: Antimicrobial Peptide Design and Synthesis

1-1: Design of MM-NKL peptide analogues

The novel antimicrobial peptide (AMP) design of the present invention is derived from the loach whole genome (NCBI Bioproject accession no. PRJNA751427) (Shin et al., Front. Mar. Sci ., 8:799148, 2022) to single-headed fish (Megalobrama amblycephala, Wuchang bream ) of NK-lysin (NCBI accession no. XP_048046947) and loach NK-lysin (MM-NKL) showing 50% homology were used (FIG. 1). To design a series of MM-NKL peptide analogs, the secondary structure of MM-NKL was predicted using Jpred 4 (compbio.dundee.ac.uk/jpred), a protein secondary structure prediction program (FIG. 2). ). Specifically, five helical regions were predicted, one from the MM-NKL N-terminus and four from the saposin B superfamily domain. For the region predicted as a helical structure in the secondary structure prediction program, it is cut into 10 amino acid residues from the N-terminus, and the physicochemical properties of the amino acid sequence, such as hydrophobicity, hydrophobic moment, and net-charge value and amphipathic structures were investigated (heliquest.ipmc.cnrs.fr).

As a result, from the full-length peptide sequence, amino acid residues 53-62 (MM-NKL-1) and amino acid residues 86-95 (MM-NKL-2) were predicted to match the physicochemical properties of the antimicrobial peptide. In addition, in order to increase the amphiphilicity of MM-NKL1, the 58th aspartic acid (D) was substituted with lysine (K) to design a derivative of MM-NKL-1 (MM-NKL-1A) (see Table 1 below). ). The amino acid sequences of the antimicrobial peptide analogs derived from loach NK-lysin (MM-NKL) are summarized in Table 1 below.

Antimicrobial peptide amino acid sequence peptide Length (a.a.) amino acid sequence sequence number MM-NKL-1 10 CKWVMDKIKK One MM-NKL-1A 10 CKWVMKKIKK 2 MM-NKL-2 10 FLKKACNFFI 3

1-2: MM-NKL peptide analog characterization

The present inventors determined the physicochemical characteristics of the antimicrobial peptide designed according to an embodiment of the present invention, such as pI value, net charge, hydrophobicity, and protein-binding potential (Boman Index). investigated (see Table 2 below). The physicochemical properties of the MM-NKL peptide analogues are summarized in Table 2 below.

Physicochemical properties of peptide analogues sequence number peptide M.W. (Da) pI net charge hydrophobicity Boman index One MM-NKL-1 1278.634 10.38 +3 2.63 1.6 2 MM-NKL-1A 1291.715 10.86 +5 2.39 1.28 3 MM-NKL-2 1230.534 9.7 +2 -1.93 -0.41

1-3: MM-NKL-derived peptide synthesis

The present inventors obtained MM-NKL-1, MM-NKL-1A and MM-NKL-2, which are peptide derivatives derived from loach NK-lysin, with a purity of >95% (Anygen Inc.) (Gwangju, Korea). ) was commercially synthesized by requesting solid phase synthesis. In addition, in order to increase the structural stability and antibacterial activity of the MM-NKL-1, MM-NKL-1A, and MM-NKL-2 peptides, the carboxyl group terminal (C-terminal) was amidated with NH ₂ (amidation).

Example 2: High Sensitivity Radiation Diffusion Analysis (URDA)

The present inventors performed an ultrasensitive radial diffusion assay (URDA) to measure the antimicrobial activity of MM-NKL-1, MM-NKL-1A, and MM-NKL-2 peptides obtained according to an embodiment of the present invention. ) was performed (Seo et al., Biochem. Biophys. Res. Commun. 338: 1998-2004, 2005).

Specifically, Bacillus cereus ATCC21772, Staphylococcus aureus RM4220, Streptococcu siniae FP5229, and S. mutans KCTC3065 were used as Gram-positive bacteria for antimicrobial activity analysis, and Escherichia coli KCTC1116, Edwasiella tarda H-4, and Pseudomonas aeruginosa KCTC20 as Gram-negative bacteria 04 , Vibrio anguillarum EC, V. harveyi KCCM 40866 were used, and Candida albicans KCTC7965 was used as a fungus yeast. For the measurement of antimicrobial activity, the bacterial strains were cultured in brain-heart infusion medium (BHI; BD Biosciences, San Jose, CA, USA) at appropriate temperature conditions (25°C for P. aeruginosa and S. iniae and 37°C for other strains). The yeast strain C. albicans was cultured in a yeast medium at 25°C. After approximately 16-18 hour incubation, the bacterial suspension was ∼10 ⁸ CFU/mL and McFarland Turbidity Standard 0.5 (Vitek Colorimeter #52-1210; Hach, Loveland, CO) corresponding to ∼10 ⁶ CFU/mL of C. albicans suspension. , USA). A 500-mL aliquot of the above diluted bacterial or C. albicans suspension was mixed with 0.03% Tryptic Soy Broth (TSB) or 0.03% Sabouraud Dextrose Broth (SDB) and 10% type I low-EEO agarose. 9.5 mL of an underlay gel containing 5×10 ⁶ CFU/mL or 5×10 ⁴ CFU/mL in mM PBS (pH 6.6) was added. Then, the purified peptide was serially diluted 2-fold in 5 μL of acidified water (0.01% HAc), and each dilution was added to a 2.5 mm diameter well prepared in a 1 mm thick underlay gel. Then, after incubation at 25 ° C ( P. aeruginosa, S. iniae and C. albicans ) or 37 ° C (other strains) for 3 hours, the bacterial or yeast suspension was mixed with 10 mL 10% PBS (pH 6.6) and 1% agar. Rose was used to overlay the underlay gel with a 10 mL double strength overlay gel containing 6% BHI or 6% YM. Then, after incubating the plate for an additional 18-24 hours, the diameter of the clearing zone was measured, the diameter of the well was subtracted, and the diameter of the clearing zone was expressed in units (0.1 mm = 1 U). The minimum effective concentration (MEC, μg/mL) of the synthetic peptide was calculated as the x-intercept of the plot of the unit described above for log ₁₀ of the peptide concentration, and the antibacterial assay was performed three times and the results were averaged.

As a result, MM-NKL-1, MM-NKL-1A, and MM-NKL-2 peptides all showed broad antibacterial activity, but in the case of MM-NKL-1 peptide, MM-NKL-1A and MM-NKL-2 It showed relatively low antibacterial activity compared to the peptide (FIG. 4). MM-NKL-1 exhibited antibacterial activity (MECs 13.695 μg/mL) at a minimum effective concentration at a peptide concentration of 5 μg/mL against Gram-negative bacteria, E. coli . MM-NKL-1A showed antibacterial activity (MECs 8.733-47.755 μg/mL) at the minimum effective concentration at a peptide concentration of 5 μg/mL against Gram-negative bacteria P. aeruginosa, E. coli, and V. anguillarum , In Gram-positive bacteria, only S. mutans showed a distinct antibacterial activity (MECs 11.267) at the minimum effective concentration at a peptide concentration of 5 μg/mL. In addition, MM-NKL-2 showed antibacterial activity (11.609-99.493 μg/mL) at a peptide concentration of 5 μg/mL against all tested microorganisms except Gram-negative bacteria V. harveyi and E. tarda . Table 3 summarizes the analysis results of the minimum effective concentrations (MECs) of the antimicrobial peptides synthesized in the present invention.

Antibacterial activity of MM-NKL peptide analogues microbe Minimum effective concentration (μg/mL) MM-NKL-1 MM-NKL-1A MM-NKL-2
Gram-positive bacteria B. cereus ATCC21772 - N.D. 99.493 S. aureus RM4220 N.D. N.D. 21.657 S. iniae FP5229 N.D. - 68.015 S. mutans KCTC3065 N.D. 11.267 11.609


Gram-negative bacteria P. aeruginosa KCTC2004 N.D. 45.755 16.517 E. coli KCTC1116 13.695 8.733 12.677 V. anguillarum EC N.D. 47.755 16.517 V. harveyi KCCM 40866 - - N.D. E. tarda H-4 - - N.D. leaven C. albicans KCTC7965 N.D. N.D. 31.775

※ N.D.: Not determined, (-) No activity.

Example 3: Hemolytic activity and membrane permeability assay

To investigate the cell toxicity of the MM-NKL-1, MM-NKL-1A, and MM-NKL-2 antimicrobial peptides of the present invention, the present inventors measured the hemolytic activity using human red blood cells. Specifically, the hemolytic activity of MM-NKL-1, MM-NKL-1A, and MM-NKL-2 peptides was determined using human red blood cells (blood type B). First, citric acid-treated blood was centrifuged at 3,000 g for 5 minutes to obtain red blood cells, and washed with 10 nM phosphate buffer (PB) (pH 7.4) containing 150 mM NaCl. Plasma and buffy coat were removed. Thereafter, the suspension at 3% hematocrit was incubated at 37°C for 60 minutes in a buffer with or without the MM-NKL-1, MM-NKL-1A, and MM-NKL-2 peptides of the present invention. After centrifugation at 3,000 g for 5 minutes, the hemolytic capacity was measured based on the hemoglobin content obtained from the absorbance of the supernatant at 405 nm. 100% hemolysis was determined by hemoglobin release after the addition of 0.1% Triton X-100, and the hemolysis percentage of the peptide was calculated using Equation 1 below, and hemolysis analysis was performed three times, and the results were averaged.

(Equation 1)

% hemolysis = [(Abs _{405 nm} in peptide solution - Abs _{405 nm} in buffer)/(Abs 405 _nm in 0.1% Triton X-100 - Abs _{405 nm} in buffer)] × 100

As a result, the MM-NKL-1 and MM-NKL-1A peptides showed almost no hemolytic activity up to 100 μg/mL concentration, but the MM-NKL-2 peptide showed more than 50% hemolytic activity at 100 μg/mL concentration ( Fig. 5a).

Example 4: Membrane Permeability Assay

o-nitrophenyl-β-D-galactoside (ONPG) and total nucleotide leakage (total nucleotide Leakage) was performed in E. coli . Specifically, it was determined by measuring the β-galactosidase activity of E. coli ML35p, a lactose permease deficient strain, using ONPG as a chromogenic substrate. In summary, an overnight culture of E. coli ML35p was inoculated with fresh Lauri-Bertanni (LB) containing 1 mM IPTG to induce the expression of β-galactosidase, and at each time point (0, 10, 20, 30, 40 , 50, and 60 min), the supernatant was collected and ONPG was added to a final concentration of 0.4 mM and incubated at 37° C. for 10 min. Thereafter, ONP generated upon cleavage of ONPG by β-galactosidase was detected as an A405 value with a spectrophotometer.

As a result, the MM-NKL-2 peptide, which had previously confirmed hemolytic activity, and the antibacterial peptide piscidin 1 used as a control showed strong bacterial inner membrane permeability, while the MM-NKL-1 and MM-NKL-1A peptides were membrane permeable. did not show much (Fig. 5b). The above results suggest that the MM-NKL-2 peptide of the present invention exhibits antimicrobial activity by directly attacking the bacterial membrane, and the MM-NKL-1 and MM-NKL-1A peptides indirectly attack the membrane rather than directly. This means that there is a possibility of exhibiting antibacterial action by action.

Example 5: DNA-binding assay and DNA polymerase inhibition assay

Electrophoretic migration to examine the interaction between MM-NKL peptide variants and intracellular DNA and DNA polymerase using the MM-NKL-1, MM-NKL-1A, and MM-NKL-2 peptides of the present invention Analysis (EMSA, electrophoretic mobility shift assay) and DNA polymerase inhibition assay (DNA polymerase inhibition assay) were performed. Specifically, in order to evaluate the peptide-DNA binding by examining the inhibition of the rate of migration of the DNA band passing through the agarose gel, a commercially available λ-HindIII DNA marker was mixed with various amounts (0.156, 0.313) in 0.01% acetic acid. , 0.625, 1.25 μg) of MM-NKL-1, MM-NKL-1A, and MM-NKL-2 peptides and incubated for 5 minutes at room temperature, followed by 0.5 μg/mL ethidium bromide (EtBr). Electrophoresis was performed on a 1.0% agarose gel.

As a result, the MM-NKL-1A and MM-NKL-2 peptides did not affect the electrical migration of DNA, but in the case of the MM-NKL-1 peptide, inhibition was observed from the highest concentration of 1.25 μg/mL among the peptide concentrations tested. confirmed (Fig. 6a). Subsequently, in order to evaluate the inhibition of DNA polymerase activity of MM-NKL-1, MM-NKL-1A, and MM-NKL-2 peptides, peptide variants at various concentrations (1.25, 0.625, 0.313 μg/mL) were respectively was added to the reaction mixture. Then, PCR amplification was performed using E. coli genomic DNA as a template. Primers for amplification were 16S-F1 (SEQ ID NO: 4) and 16S-R3 (SEQ ID NO: 5), and the predicted amplicon was ~1.5 kb in length (see Table 4). Each PCR consisted of 30 cycles of 90°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute each. After PCR, all amplification products were electrophoresed on a 1.5% agarose gel containing EtBr. As a result, among the synthesized peptides of the present invention, both MM-NKL-1A and MM-NKL-2 peptides showed strong DNA polymerase inhibitory activity, but in the case of MM-NKL-1, the highest peptide concentration was 1.25 μg / It was found to completely inhibit the activity of DNA polymerase only in mL (Fig. 6b). The base sequences of the primers used for the PCR amplification are summarized in Table 4 below.

Primer sequence information primer Nucleic acid sequence (5'->3') sequence number 16S-F1 CTCCTACGGGAGGCAGCAG 4 16S-R3 CCAGGGTATCTAATCCTG 5

Example 6: Evaluation of stability to heat and salt

The present inventors evaluated the stability against heat or salt using the MM-NKL-2 peptide, which has the most excellent antibacterial activity among the synthetic peptides of the present invention. Specifically, in order to evaluate heat and salt stability, the MM-NKL-2 peptide was heated at 100 ° C. for 10 minutes or left in 0.5-2% NaCl for 60 minutes, and then the antibacterial activity thereof was confirmed by the URDA method.

As a result, the MM-NKL-2 peptide exhibited stable antibacterial activity without being affected by heat or salt (FIGS. 7a and 7b). These results suggest that the MM-NKL-derived peptide mutant has high stability against heat and salt.

In conclusion, MM-NKL-1, MM-NKL-1A, and MM-NKL-2, which are antibacterial peptides derived from loach NK-lysine protein of the present invention, are stable to heat and salt, and are resistant to Gram-positive bacteria and Since it showed excellent antibacterial activity against gram-negative bacteria, it can be used as a candidate material for developing antibiotic substitutes that can replace existing antibiotics or antibiotics.

Although the present invention has been described with reference to the above-described embodiments, these are only examples, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical scope of protection of the present invention should be determined by the technical spirit of the appended claims.

Claims (10)

Consisting of the amino acid sequence represented by SEQ ID NO: 3, antibacterial peptide. According to claim 1,
The peptide is derived from the NK-lysin protein of loach, an antimicrobial peptide. According to claim 1,
Antibacterial peptide, amidated at the C-terminus. Gram selected from the group consisting of Vibrio anguillarum EC, Vibrio harveyi KCCM 40866, Escherichia coli KCTC1116, Edwardsiella tarda H-4, and Pseudomonas aeruginosa KCTC2004 containing the antimicrobial peptide of any one of claims 1 to 3 as an active ingredient Gram-positive bacteria selected from the group consisting of negative bacteria, Bacillus cereus ATCC21772, Streptococcus mutans KCTC3065, Streptococcus iniae FP5229, and Staphylococcus aureous RM4220 and Candida albicans KCTC7965 having antibacterial activity against fungi, antibacterial pharmaceutical composition. delete delete delete delete A feed additive comprising the antimicrobial peptide of any one of claims 1 to 3 as an active ingredient. A pharmaceutical composition for preventing tooth decay, comprising the antimicrobial peptide of any one of claims 1 to 3 as an active ingredient.

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