KR102394213B1 - Antimicrobial peptide derived from from the marine bivalve, Mytilus coruscus and uses thereof - Google Patents

Abstract

The present invention relates to an antibacterial peptide derived from M. coruscus and its use, and more particularly, it provides an antibacterial peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 7.

Description

Antimicrobial peptide derived from the marine bivalve, Mytilus coruscus and uses thereof

The present invention relates to an antibacterial peptide and its use, and more particularly, to an antibacterial peptide derived from bluefin mussel and its use.

Mollusks, such as mussels, clams, oysters and scallops, are the main phylum of marine invertebrates and obtain their nutrients from seawater through filter feeding. The marine molluscs, mussels, use two exhalants and inhalants to water because they are immobile because they are attached to each other or to rocks by proteinous byssus in the middle of their feet. , feed on plankton and microbes. Nutrient organic matter entering the water outlet is trapped in the gills and is aggregated by mucus and carried to the mouth. For this reason, molluscs are exposed to a microbial environment and an effective immune system is required to prevent microbial infection (JA Tincu, et al., Agents Chemother . 48. 3645-3654. 2004). As previously reported, the acquired immune system of invertebrates, such as mollusks, is underdeveloped, whereas the innate immune system is well-developed, and in order to defend itself from pathogenic microorganisms, It is mainly dependent on the innate immune system (C. Zannella, et al., Mar. Drugs. 15. 182. 2017). Innate immunity is the first line of defense of the nonspecific host defense system and immediately inhibits the growth of various invading microbial body fluids before phagocytosis or the adaptive immune system immediately after infection (A. Charles, et al., Immunobiology, fifth ed, Garland publishing, New York, 2001). Among these defense systems, antibacterial peptide (AMP) is known as a major factor in the innate immune system of mollusks (D. Destoumieux, et al., J. Biol. Chem. 272, 28398-28406. 1997).

Antibacterial peptides are host defense peptides with an alpha-helical or beta-sheet folding structure, and are short oligopeptides (less than about 100 amino acids) encoded by genes. Being amphipathic molecules with hydrophobic and cationic amino acids, they exhibit broad antibacterial activity against bacteria, fungi, parasites and even certain enveloped viruses (M. Zasloff. et al., Nature. 415. 389). -395. 2002). The action of cationic antimicrobial peptides depends on their secondary structure, overall net charge, amphiphilicity, hydrophobicity, size, and balance between hydrophobic and polar regions (KR Reddy, et al., Int). J. Antimicrob. Agents 24. 536-547. 2004). And antimicrobial peptides are classified into non-membrane-targeting peptides and membrane-targeting peptides according to the region of the targeted molecule such as cell wall, membrane or cytoplasm (CB Park et al., Biochem. Biophys. Res. Commun. 6;244 (1). ) 253-257. 1998). Although more than 1,000 antimicrobial peptides have been reported to have antimicrobial activity against several pathogens, studies on antimicrobial peptides derived from marine mussels have only been studied in some species.

The present invention is to solve various problems including the above problems, and unlike conventional antibacterial peptides, haemolytic activity is reduced and antibacterial activity is increased, showing excellent antibacterial and anti-parasitic activity against various indicator strains. An object of the present invention is to provide an antibacterial peptide derived therefrom and uses thereof. However, these problems are exemplary, and the scope of the present invention is not limited thereto.

According to one aspect of the present invention, consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 7, there is provided an antibacterial peptide.

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

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

According to another aspect of the present invention, containing a peptide as an active ingredient, an anti-Scouchica ciliate controlling composition is provided.

According to one embodiment of the present invention made as described above, the antibacterial peptide derived from mussels not only exhibits excellent antibacterial activity against gram-negative and gram-positive bacteria, but also exhibits anti-parasitic activity, so it can be effectively used as a substitute for antibiotics. can Of course, the scope of the present invention is not limited by these effects.

1a is a graph analyzing the purification and antibacterial activity of a purified peptide derived from M. coruscus mantle extract, and analyzing the antibacterial activity of the purified peak (30-32 minutes) against Bacillus subtilis . am. The acidified extract was injected onto a CapCell-Pak C18 reversed phase column with a gradient of 5-65% ACN in 0.1% TFA over 60 minutes at a flow rate of 1.0 mL/min.
Figure 1b is an analysis of the purification and antibacterial activity of purified peptides derived from M. coruscus mantle extract, and a graph analyzing the antibacterial activity of the purified peak (31-33 minutes) against Bacillus subtilis . am. The active fraction was applied to a TSK-gel ODS-80TM C18 reversed phase column and eluted with a linear gradient of 5% to 65% ACN (pH 2.2) in 0.1% TFA for 60% at a flow rate of 0.2 mL/min, elution was 220 was monitored in nm.
Figure 1c is the analysis of the purification and antibacterial activity of purified peptides derived from M. coruscus mantle extract, and 11,182.003 Da measured by Edman digestion and mass spectrometry of a single mass having an N-terminal sequence (23 amino acids) It is a graph.
2 is a diagram showing the cDNA sequence and the derived amino acid sequence of recombinant myticusin-beta of the present invention. The reduced amino acid sequence of the ORF is shown below the nucleotide sequence, the asterisk indicates the stop codon and the outline indicates the signal peptide of myticusin-beta.
3 is a diagram showing the multiple sequence alignment of recombinant myticusin-beta and isoform protein of the present invention. myticusin-beta represents MC1 and two isoforms, MC2 and MC3.
4 is a gel photograph showing overexpression and purification of recombinant myticusin-beta of the present invention. The TrxA fusion recombinant protein was purified using Ni-NTA affinity chromatography. lane M, molecular markers; lane 1, uninduced protein; lane 2, inducing protein; lane 3, soluble protein; lane 4, inclusion body; Lane 5, purified myticusin-beta protein (indicated by red arrow), lane 6, results of western blot using purified myticusin-beta protein.
5 is a graph showing the analysis of the hemolytic activity of phycidin 1 of recombinant myticusin-beta of the present invention on red blood cells of halibut, and analyzing the concentration of each sample at 100 μg/mL (A). It is a graph analyzing the effect (100, 50, 25, 12.5, 6.25 μg/mL) (B).
6 is an analysis of the anti-Scuchica ciliate activity of recombinant myticusin-beta using WST-1 reagent, and the aggregation of parasites treated with different concentrations of recombinant myticusin-beta (0-200 μg/mL) was observed. It is a photomicrograph (A), and is a graph analyzing the survival rate (%) of the parasite (Ciliary Scotia) by the concentration of recombinant myticusin-beta (0-200 μg/mL) (B). Error bars represent the mean standard deviation of three replicates.
7 is a diagram analyzing the antibacterial activity and spectrum of purified recombinant myticusin-beta of the present invention. The value of the antimicrobial activity against the strain was expressed as the diameter of the clear zone (diameter of the well; 2.2 mm).
8 is a graph showing the results of quantitative analysis of myticusin-beta expression in mussel tissues. Error bars represent the mean standard deviation of three replicates. ADD; adductor muscle, FOO;foot, GIL;gill, HEM; blood cells, HEP; Hepatopancreas, MAN: mantle, SIO; outlet pipe.
9 is a diagram showing a helical wheel depiction of a recombinant myticusin-beta-like peptide of the present invention. Arrows indicate substituted amino acid residues in the peptide. The amino acid residues were substituted with increased net charge and hydrophobicity. Hydrophobic residues are blue, acidic residues are red, basic residues are orange, and uncharged residues are purple.

Definition of Terms:

As used herein, the term "sea mackerel ( Mytilus coruscus )" is distributed all over the coast of Korea, and is a representative floating material filtration plant. The size is 150mm in length and 50mm in height, and the shell is thicker than the Mediterranean mussel and is purple in color. The growth surface is rough, and a lot of seaweed and barnacles attach to it. The apex is curved downward from the left end, and the back side is close to a straight line, and the front side is also straight.

As used herein, the term "Scuticociliate" is a single-celled protist that infects and parasitizes marine organisms such as halibut, seahorse, and salmon. Primary infection occurs in the epidermal layer of the host, and when it penetrates the epidermis and penetrates into the tissue, it engulfs various tissues of the host, including immune cells, brain and kidney, causing damage and death. Flounder is a major aquaculture source raised in Korea, Japan, and China, and when the fry are infected with ciliate scutica, they cause mass death, causing enormous industrial damage.

DETAILED DESCRIPTION OF THE INVENTION:

According to one aspect of the present invention, consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 7, there is provided an antibacterial peptide.

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

In the antimicrobial composition, the Gram-negative bacteria may be Vibrio alginolyticus , Vibrio parahaemolyticus , Escherichia coli , Enterobacter cloacae , Klebsiella pneumoniae , Edwardsiella tarda , Proteus mirabilis , Providencia stuartii or Pseudomonas aeruginosa , and the Gram-positive bacteria may be B. Enterococcus faecalis , Streptococcus mutan s , Streptococcus iniae , Staphylococcus aureous , Streptococcus vestibularis , Streptococcus parauberis or Lactococcus garvieae , and the fungus may be Candida albicans .

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

According to another aspect of the present invention, containing a peptide as an active ingredient, an anti-Scouchica ciliate controlling composition is provided.

Antibacterial peptides (AMPs) are host defense peptides with alpha-helical or beta-sheet folding structures and short oligopeptides (less than about 100 amino acids) encoded in genes. Antibacterial peptides, amphipathic molecules with hydrophobic and cationic amino acids, exhibit broad antibacterial activity against bacteria, fungi, parasites and even certain enveloped viruses. Although more than 1000 antimicrobial peptides have been reported to have antimicrobial activity against various pathogens, studies on antimicrobial peptides derived from marine mussels have been studied only in some species. Antibacterial peptides identified in mussels are classified into eight groups, including defensin, mytilin, myticin, mytimycin, mytimacin, big defencin, mytichitin-CBD, and myticusin. Several antimicrobial peptides have been purified and identified, focusing on marine mussels, particularly Mytilus edulis and Mytilus galloprovinvialis , but few studies have been conducted on antimicrobial peptides from Mytilus coruscus (JA Tincu, et al., Antimicrob. Agents Chemother. 48. 3645). -3654. 2004).

On the other hand, M. coruscus (also called "hardshell mussel") belongs to the bivalve mollusk of the Mytilidae family, and has long been cultivated as a traditional health food in East Asia, and is also treated as an economically important seafood in Korea. M. coruscus -derived antibacterial peptides mytichitin-CBD and myticusin were identified in hemolymph and foot muscle extracts of M. coruscus (CL Qin, et al., Fish shellfish Immunol. 41. 362-370. 2014). The peptide exhibits excellent antibacterial activity against gram-positive bacteria and constitutes an important component of innate immune defense.

The present inventors paid attention to the fact that some studies were conducted on antibacterial peptides only in some species derived from mussel, and isolated and purified the antibacterial peptide from the mantle of mussel. The peptide was purified by C18 reverse-phase high-performance liquid chromatography (HPLC), which exhibited antibacterial activity against Bacillus subtilis . The total molecular mass by matrix-assisted laser desorption ionization time-flight mass spectrometry (MALDI-TOF/MS) was 11,182 Da, and the 23 N-terminal amino acid sequences of the purified peak were obtained from Edman digestion, and myticusin from mussels -1 and 82% homology were shown. In addition, the complete sequence of the target peptide was determined by cDNA cloning and rapid amplification of cDNA ends (RACE). A full-sequence peptide consisting of 574 bp with an open reading frame (ORF) of 387 bp encoding 24 amino acids of the signal peptide and 104 amino acids of the mature peptide was named myticusin-beta, and two additional novel isoforms of myticusin-beta Proteins (isoforms) were found. The myticusin-beta-derived short antibacterial peptide analog was designed and synthesized based on the sequence of myticusin-beta, and as a result of measuring the antimicrobial activity of the analogs, Gram-positive bacteria ( Bacillus cereus, B. subtilis, Clostridium perfringens, Staphylococcus aureous) , Streptococcus iniae, Streptococcus mutan s) and Gram-negative bacteria ( Escherichia coli, Pseudomonas aeruginosa, Vibrio alginolyticus, Klebsiella pneumoniae ) and showed anti-parasitic activity at different concentrations. The expression level of myticusin-beta prepared according to an embodiment of the present invention was detected at the highest level in the mantle membrane and then in hemocytes. In conclusion, the present inventors isolated, purified, and characterized the myticusin-beta antibacterial peptide from the mantle of M. coruscus , and the results suggest that myticusin-beta can be utilized as a substitute for antibiotics and immune system-related peptides.

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

Example 1: Tissue Extraction

M. coruscus used in the present invention was purchased from the Busan Fish Market (Korea), washed gently with fresh water, and artificially infected by injecting Vibrio parahaemolyticus into the adductor muscle with a syringe. The mantle was then removed under aseptic conditions and heated with 4 volumes of preheated 1% acetic acid (HAc) for 5 minutes. The heated tissue was centrifuged (4000 rpm, 30 min, 4°C), and the supernatant was filtered through a syringe filter with a pore size of 0.45 μm and stored at -70°C until use.

Example 2: Ultrasensitive Radial Diffusion Analysis (URDA)

The present inventors performed antibacterial analysis by ultra-sensitive radial diffusion analysis (URDA). Specifically, antibacterial activity was confirmed using gram-positive, negative bacteria and fungi as reference toxic microorganisms (RI Lehrer, et al., J. Immunol. Meth. 137. 167-173. 1991). First, 19 bacterial strains were incubated in appropriate media and temperatures (25°C, 30°C and 37°C), respectively, and reference bacteria and fungi were cultured with McFarland turbidity standards (corresponding to ~10 ⁸ CFU/mL or ~10 ⁶ CFU/mL). turbidity standard) was diluted with 0.1. 0.5 mL of the diluted microbial culture medium was added to 9.5 mL of underlay gel containing 1.2% type I agarose, and poured into 1 mm thick underlay gel with 2.2 mm diameter wells. Samples were then added to wells and bacterial or fungal suspensions were overlaid with 10 mL of overlay gel containing 1.2% type I agarose and 10 mM phosphate buffer, pH 6.5, and incubated at 25, 30 and 37° C. for 18 hours. After that, the clear zone was measured. Information on the pathogenic strains used in the URDA analysis is summarized in Table 1 below.

Pathogenic strains used for URDA analysis strain reference badge Temperature (℃) Gram-positive bacteria Bacillus cereus KCTC 1012 NB 30 Bacillus subtilis KCTC 1021 TSB 37 Clostridium perfringens ECa TSB 37 Enterococcus faecalis KCTC 3206 BHI 37 Staphylococcus aureus KCCM 11335 TSB 37 Streptococcus iniae KCTC 3657 BHI+1% Nacl 37 Streptococcus mutans KCTC 3065 BHI 37 Streptococcus vestibularis KCTC 3650 TSB+0.3%YE 37 Streptococcus parauberis KCTC 3651 TSB+0.3%YE 37 Lactococcus garviae KCTC 3772 MRS 30 Gram-negative bacteria Escherichia coli KCCM 40271 NB 37 Enterobacter cloacae KCTC 2361 NB 37 Klebsiella pneumoniae KCTC 12385 NB 37 Proteus mirabilis KCTC 2510 NB 37 Providencia stuartii KCTC 2569 NB 37 Pseudomonas aeruginosa KCTC 1636 NB 37 Vibrio alginolyticus KCTC 2472 BHI 37 Vibrio harveyi KCCM 40866 BHI+1% Nacl 25 Vibrio parahaemolyticus KCCM 41664 BHI+1% Nacl 37 fungus Candida albicans KCCM 11282 YM 30

Example 3: HPLC purification of peptides

The present inventors isolated the mantle tissue of tuna tuna, homogenized, purified and acidified it. The acidified mantle extract was applied to URDA and showed antibacterial activity against Gram-negative bacteria including E. coli and Psuodomonas aeruginosa and Gram-positive bacteria including Bacillus cereus , B. subtilis and Streptococcus mutans . Based on the initial screening results, Bacillus subtilis showing high sensitivity to the extract was selected as a reference strain for antibacterial analysis and subjected to RP-HPLC for separation and purification of the acidified mantle extract.

Specifically, the purification process of the present invention comprises two types of reversed-phase columns, CapCell-Pak C18 reversed-phase column (5 μm, 120A, 4.6 x 250 mm) and TSK-Gel ODS-80™ C18 reversed-phase column (4.6 x 150 mm; Japan). TOSOH) with a reversed-phase HPLC system (DIONEX UltiMate 3000 HPLC system, Thermo Fisher Scientific, USA). In a first step, the acidified extract was applied to a CapCell-Pak C18 reversed-phase column with a gradient of 5-65% acetonitrile (ACN) in 0.1% trifluoroacetic acid (TFA) over 60 minutes at a flow rate of 1.0 mL/min. injected. In a second step, the collected fractions were applied to a TSK-gel ODS-80TM C18 reversed phase column with a gradient of 5-65% ACN in 0.1% TFA over 1 hour at a flow rate of 0.2 mL/min. Then, the eluted fractions were monitored at 220 nm and vacuum dried for further characterization, and each eluted peak was evaluated for Bacillus subtilis (KCTC 1021) by applying the URDA method to confirm the antimicrobial activity.

As a result, the fraction with the strongest activity was eluted at 30-32 minutes by the CapCell-Pak C18 reversed-phase column (FIG. 1a). In addition, the fractions were collected, concentrated, and applied to a TSK-gel ODS-80TM C18 reversed-phase column for further purification. Finally, the purified active fraction was eluted at 31-33 minutes, and trypsin was used to identify proteinaceous substances. (trypsin). After the reaction, it was confirmed that the antibacterial activity against Bacillus subtilis was completely disappeared (FIG. 1b). These results suggest that the purified peak is a proteinaceous component with antibacterial activity.

In previous studies, most of the antimicrobial peptides found in mussel species were isolated from blood cells or blood lymph, defensin from M. edulis and M. galloprovincialis , mytilin isoforms A and B from M. edulis , mytilin isoforms C, D, and G1 from M. galloprovincialis , myticins A, B, mytimycin from M. galloprovincialis , mytichitin-CBD from M. coruscus , and myticusin-1 from M. coruscus were isolated and reported (M. Gerdol, et al., Dev. Comp. Immunol. 36: 390-399, 2012). However, the antibacterial peptide of the present invention was uniquely purified from the mantle of mussel, and the eluted fraction showed antibacterial activity against the Gram-positive Bacillus subtilis, unlike the previously known myticusin-1 of M. coruscus (Z. Liao). et al., Fish shellfish Immunol. 34: 610-616, 2013).

Example 4: Digestion of purified peptides with proteases

In order to finally identify the proteinaceous compound, the purified single peak was reacted with 2 μL (1,000 μg/mL) of trypsin at 37° C. for 1 hour. The trypsin hydrolyzes peptide bonds and digested proteins into peptides by breaking the carboxyl terminus of arginine and lysine residues. Thereafter, the sample was incubated at 4° C. for inactivation of trypsin, and the single peak purified after protease reaction was subjected to URDA test, and the antibacterial activity was compared with that of the trypsin-free peptide.

Example 5: Sequence identification of purified peptides

To measure the molecular weight of the purified peptide, an Ultraflex III matrix-assisted laser desorption ionization time-flight mass spectrometer (MALDI-TOF/MS, Bruker Daltonik GmbH, Germany) was used. Specifically, the purified peptide was dissolved in α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution (10 mg/mL, 1:1,v/v in 0.1% TFA/50% ACN) and MALDI plate loaded on In addition, the N-terminal amino acid sequence was obtained by Edman digestion method using a pulse liquid automatic sequencer (Model 473A; Applied Biosystems, USA).

As a result, the molecular weight of the purified peptide was confirmed to be 11,182.003 Da through MALDI-TOF/MS analysis, and 23 amino acid residues of the purified peptide were obtained by Edman digestion: SDHQMAQSACMGLAQDAAYAS-AI (Fig. 1c, SEQ ID NO: 5). The identified 23 residues of the N-terminal partial sequence were 91% identical to the hypothetical protein portion (OPL33655) of M. galloprovincialis and 82% identical to the myticusin alpha precursor (AFJ04410) of M. coruscus (M. Murgarella, et al. , PLoS One. 11(3): e0151561, 2016). For this reason, the purified peptide was named myticusin-beta.

Among the antibacterial peptides found in various organisms, amino-terminal copper and nickel (ATCUN)-containing peptides (ATCUN-AMPs) having a tripeptide known as an amino acid sequence of Xaa-Xaa-His (where Xaa is amino) at the amino group terminus (ATCUN-AMPs) have been reported (MDJ Libardo, et al., Biochimie. 113: 143-155, 2015). An ATCUN-AMPs peptide having a common amino-terminal copper and Nicket (ATCUN) site, an antibacterial peptide from mussels, is also known. Antibacterial peptides isolated from mussels were found to be antibacterial peptides having the ends of threonine-aspartic acid-histidine (TDH), hisditine-proline-histidine (HPH) and histidine-serine-histidine (HSH). For example, the ATCUN motif region of myticusin-1 from M. coruscus is TDH (Thr-Asp-His), Myticin B from M. galloprovincialis is His-Pro-His (HPH), and myticin A from M. galloprovincialis is His- Ser-His (HSH). Myticusin-beta of the present invention has serine-aspartic acid-histidine (SDH) at the N-terminus of the peptide. Like other reported antibacterial peptides, myticusin-beta antibacterial peptide shows the characteristics of ATCUN-AMP with 3 amino acid residues containing histidine at the N-terminus, and the hypothetical protein portion of M. galloprovincialis (OPL33655) of the peptide Contains serine-aspartic acid-histidine (SDH) at the N-terminus. The spectrum of the antimicrobial activity of these antimicrobial peptides can be determined by ATCUN.

Example 6: RNA extraction, cDNA preparation and nucleotide sequencing

Accordingly, the present inventors cloned a gene encoding myticusin-beta identified in Example 5 above. Specifically, RNA was first extracted and purified from the mussel mantle using TRI reagent (Sigma-Aldrich, USA), and the purity of RNA was measured with a spectrophotometer (Novue plus spectrophotometer, USA). Then, in order to determine the complete DNA sequence, cDNA of myticusin-beta derived from M. coruscus was synthesized using the ^SMARTer RACE5'/3' kit (Clontech, USA). Then, first strand cDNA was synthesized with poly(A) polymerase (Takara, Japan). The 5'-RACE-Ready cDNA synthesis reaction was performed in a final volume of 11 µL containing 1.0 µL of RNA and 1.0 µL of 5'-DNS primer A. In addition, 3'-RACE-Ready cDNA synthesis reaction was performed in a final volume of 12 µL containing 1.0 µL of RNA and 1.0 µL of 3'-DNS primer A for the preparation of 3'-RACE-Ready cDNA. The tube was then incubated at 72° C. for 3 min and cooled to 42° C. for 2 min. Then, 1.0 μL of oligonucleotide was added to the 5'-RACE cDNA synthesis reaction, and the master mix was added to the denatured RNA from the 5'- and 3'-RACE-Ready cDNA synthesis reaction, followed by 90 minutes at 42°C. Then incubated for 10 minutes at 70 ℃. The 3'- and 5'-RACE-Ready cDNA samples were stored at -20°C for use and two gene-specific oligonucleotide primer pairs (Myticusin-3RACE: SEQ ID NO: 1 and Myticusin-5RACE: SEQ ID NO: 2) were used. prepared. In addition, ^Myicusin -beta was cloned into pGEM and T-easy vector system (Promega, USA), and E. coli XL-1 blue host cells were transformed with the cloned vector. Thereafter, the plasmid was isolated and purified using a DNA-spin plasmid purification kit (Intron, Korea), and sequence analysis was performed using a 3130XL gene analyzer (Applied Biosystems, USA). After that, the amino acid sequence was searched using BLAST (Basic Local Alignment Search Tool) of the National Center for Biotechnology Information (NCBI, blast.ncbi.nlm.nih.gov/Blast), and BioEdit version 7.2.5 and GENETYX version 4.0 program was used to align the sequences for comparison and analysis. In addition, the theoretical molecular weight and pI value of the purified peptide were evaluated using the Expasy-online tool (www.expasy.org/rools/protparam.html). Information on the prepared primers and the primers used in the present invention is summarized in Table 2 below.

Base sequence information of primers designation base sequence (5'-->3') SEQ ID NO: Myticusin-3RACE GATTACGCCAAGCTT GAC CAC CAG ATG GCA CAG TCG GCC TGC One Myticusin-5RACE GATTACGCCAAGCTT TAT TGC GGA TGC ATA TGC IGC GTCTG 2 BamHI-linker F GGATCC ATG TCA GAC CAT CAG ATG GCA 3 XhoI-linker R CTCGAG TTA AAC GAT ACA ACA GCA GAA GTT 4

Example 7: Construction of Recombinant Plasmids

The present inventors screened cDNA clones to identify the sequence of myticusin-beta. Specific primers were designed to amplify the cDNA of myticusin-beta from M. coruscus and amplify the complete cDNA. Specifically, for the construction of the expression plasmid, the target gene was amplified by polymerase chain reaction (PCR) using a sense Bam HI-linker primer (SEQ ID NO: 3) and an antisense Xho I-linker primer (SEQ ID NO: 4). Specifically, PCR was performed in 50 μL of a final volume containing 1.0 μl of DNA template, 1X PCR buffer containing MgCl ₂ , 0.2 mmol/L of dNTPs, 0.6 U of Taq polymerase and 1.0 pmol of each primer. Amplification conditions were 25 cycles of 1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C using a PCR thermal cycler TP600 (Takara, Japan), followed by final extension at 72°C for 7 minutes. extension). After that, the amplification product was confirmed by 1.5% agarose gel electrophoresis and purified using QIAquick ^ㄾ PCR purification kit (QIAGEN, Germany), and the final product was Bam H I, Xho I sites and N-terminal 6X His-tagged (CH Kim, et al., FEBS. 3332-3338. 2017) were cloned into the pET-28a(+)-thioredoxin A (TrxA) fusion vector. Finally, a recombinant plasmid encoding the myicusin-beta gene was isolated and transformed into E. coli DH5α and BL21 (DE3) host cells (competent cells).

As a result, it was confirmed that the complete sequence of myticusin-beta was 574 bp and consisted of an ORF sequence of 387 bp (FIG. 2). A purified peptide encoding 128 amino acids discloses a signal peptide of 24 amino acids identified using the SIGNALP 4.0 online tool (http://www.cbs.dtu.dk/services/SignalP). The active site peptide contained 10 basic residues; The total hydrophobic ratio of 4 Lys, 4 Arg and 2 His was 39%. The cleavage site for secretion of the signal peptide is between glycine ²⁴ and serine ²⁵ . The active peptide consisting of 104 amino acids has 87% homology with myticusin-1 of M. coruscus and 88% homology with the hypothetical protein of M. galloprovincialis . In addition, two or more isoforms of myticusin-beta were identified in the mantle of M. coruscus , and as a result of comparing amino acid sequences, myticusin-beta (MC1) and two isoforms myticusin-beta 2 (MC2) and By comparing the 128 amino acid sequence of myticusin-beta 3 (MC3) with myticusin-1, 74.2% homology of amino acids was confirmed (FIG. 3). Myticusin-beta showed very close amino acid sequence similarity to the two isoform proteins, and the compared homology was 98.4% and 99.2% in MC2 and MC3, respectively. According to the 128 amino acid sequence comparison of myticusin-beta and myticusin-1 with isoforms, threonine ⁷⁴ in myticusin-1 was replaced with alanine ⁷⁴ and glycine ⁷⁴ , and histidine ¹¹⁸ was replaced with serine ¹¹⁸ and asparagine ¹¹⁸ . Consequently, two types of isoforms were performed with myticusin-beta.

Since differences in amino acid sequence can lead to various properties of protein function, further analysis of antimicrobial activity and structural properties of synthetic peptides is required. A calculated molecular weight of 11,082.53 Da was obtained for 104 amino acids of the active peptide, and the detected molecular weight was found to be 11,182.003 Da by mass spectrometry ( FIG. 1C ). The molecular weight results are nearly identical and the 0.527 Da difference can be attributed to various post-transformation. The difference is allysine (lose 1.03 Da), amidation (lose 0.98 Da), 2,3-didehydroalanine (lose 18.02 Da), 2,3-didehydrobutyrine (lose 18.02 Da), 3-oxoalanine cysteine (lose 18.08 Da), 3 -oxoalanine serine (lose 2.02 Da), 2-oxobutanoic acid (lose 17.03 Da), pyrrolidone carboxylic acid (lose 18.02 Da), pyrrolidone carboxylic acid (lose 17.03 Da) and pyruvic acid (lose 33.10) known to reduce molecular mass Da), such as post-translational modifications (C. Zannella, et al., immunology and antimicrobial defense, Mar. Drugs. 15. 182. 2017).

Example 8: Expression and Purification of Recombinant Proteins

The present inventors used 300 mL of Luria-Bertain (LB) liquid medium containing 50 μg/mL of kanamycin at 37° C. for expression and purification of the recombinant protein, until the optical density reached a maximum of 0.4 at 600 nm. Cells were cultured. Induced expression was performed with isopropyl-D-1 thiogalactopyranoside (IPTG) at 37°C for 4 hours, the cells were centrifuged at 4,000 rpm for 30 minutes, and the pellet was washed with 40 mL of 50 mM Tris-HCl buffer. (pH 8.0). The suspended cells were crushed by ultrasonication in an ice-water bath and centrifuged at 12,000 rpm at 4°C for 10 minutes. Thereafter, the supernatant was subjected to Ni-NTA His Bind® Purification was performed using Superflow™ (Novagen, USA), and after purification, the eluted fraction was dialyzed against 20 mM Tris-HCl buffer (pH 8.0) and analyzed using an EzWay-PAG gel (KOMABIOTECH, Korea) composed of 10% aspartate. For quantitative analysis of the purified target protein, Bradford assay was performed, and antibacterial activity was measured by the URDA method using the purified recombinant protein of the present invention for the above-mentioned strain.

The present inventors amplified the myticusin-beta gene based on cDNA sequencing using the RACE method and cloned it into a TrxA fusion pET-28a(+) vector (CH Kim, et al., FEBS. 3332-3338. 2017). The pET-28a(+)-TrxA fusion vector containing an N-terminal 6X his-tag fused to an E. coli -derived TrxA protein was used as an expression vector useful for the production of high levels of soluble protein in the E. coli cytoplasm. E. coli BL21 was transformed for expression of the constructed plasmid, and the induced recombinant protein expression was analyzed by EzWay-PAG gel, and protein expression was confirmed in the supernatant and pellet. Thereafter, the target protein having 12 kDa thioredoxin was purified, and the results were consistent with the calculated molecular weights of thioredoxin and the target protein. Then, Western blot analysis was performed to confirm the expression of the recombinant protein, and a clear single band corresponding to the expected molecular weight of the protein was observed (FIG. 4). Finally, the purified recombinant peptide was desalted and lyophilized to evaluate antibacterial activity.

The protein expression system using E. coli as a host has several problems in the expression of small cationic antibacterial peptides. The target recombinant protein gene was recognized as an unnecessary protein introduced from the outside and expressed in the form of insoluble aggregation. And insoluble aggregates must be refolded to the correct form through a refolding process for activity (Y Shan, et al., J. Korean Soc. Appl. Biol. Chem. 58(6) (2015) 807-812. 2015) ). Various methods have been tried to overcome the shortcomings of the E. coli expression method, and among them, it was reported that the technology of fusion expression with a protein that helps the expression and folding process is the most suitable for high-yield expression of the target protein. For this reason, we ligated myticusin-beta into the pET-28a(+)-TrxA fusion vector, which was suitable for cationic peptide expression (KL Piers, et al., Gene. 134(1). 7-13 (1993). Expression methods through various fusion proteins include glutathione S-transferase (GST), N-utilization substance (NusA), maltose binding protein (MBP) and Trx (S. Costa, et al., the novel Fh8 system). 5 (63) . Among the fusion partners, GST (20 kDa), NusA (55 kDa) and MBP (43 kDa) were proteins that were too large in size than the antibacterial peptide, so their co-expression was reserved. However, the TrxA protein has a small size of 12 kDa and has an effect of inducing production in its expression and active form, making it suitable for this study (S. Costa, et al., the novel Fh8 system. 5(63). 1-20. 2014). In the above study, myticusin-beta was expressed in a water-soluble form, and the thioredoxin gene (trxA) fusion expression system was useful for the expression of antibacterial peptides by avoiding the formation of inclusion bodies in the E. coli cytoplasm. can

Example 9: Western blotting

We heated the samples at 100° C. for 10 minutes and separated them on a 10% aspartate gel for western blot. Then, bands were obtained using the ^{WesternBreeze®} chromogenic Western Blot Immunodetection kit (Invitrogen, USA) according to the manufacturer's instructions, and the gel was washed and then trans-blotted on a nitrocellulose (NC) membrane at 15V for 1 hour ( trans-blotted) was performed. The NC membrane was incubated with an anti-his antibody (1:5000, Invitrogen, USA) and incubated with an alkaline phosphatase-conjugated secondary antibody for 1 hour. The band was confirmed.

Example 10: Quantitative analysis of myticusin-beta expression

The mRNA expression level of myticusin-beta of the present invention was analyzed by real-time qPCR. transcriptional level) was measured. The β-actin gene was used as an internal reference gene for expression level analysis, and the amplification specificity for myticusin-beta and β-actin was analyzed using a melting curve. Specifically, for quantitative analysis, the tissue-specific expression level of myticusin-beta was measured by quantitative real-time PCR (qRT-PC) after the mussel was infected with V. parahaemolyticus to activate the immune system. Specifically, total RNA was extracted from adductor muscle, paws, gills, blood cells, hepatopancreas, mantle and excurrent siphon using TRI reagent (Sigma-Aldrich, USA). cDNA was synthesized as the first strand of the transcript using a cDNA Synthesis Kit (Roche, Germany) and used as a template. Amplification was performed in a final volume of 20 µL containing 1.0 µL of diluted cDNA, 10 µL of 2X Fast SYBR Green Master Mix (ABI, USA), 0.5 µL (10 pmol/µL) of each primer, and 8 µL of DW. As: 1 cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec, 55°C for 1 min and 72°C for 30 sec on an ABI 7500 real-time PCR system (Applied Biosystems, USA). Then, to demonstrate the correct specific amplification, a melting curve was performed and analyzed and the expression level was compared with the 18S rRNA expression level using the 2 ^-ΔΔCt method and presented as relative expression (mean ± standard deviation). (KK Livak, et al., Methods. 25. 402-408. 2001).

As a result, gene expression of myticusin-beta was expressed in all tissues including mantle, blood cells, adductor muscle, paw, hepatopancreas, gills and foramina (FIG. 8). The mRNA of myticusin-beta was highest expressed in the mantle, and then the expression was measured in blood cells. The expression of myticucin-beta showed moderate activity in adductor muscle and gills, and was low in paws, hepatopancreas, and draining ducts. Mollusks, mussels, have an open circulatory system, so when antibacterial peptides are produced, they are transported to all tissues through blood lymph. Therefore, myticusin-1 derived from M. coruscus , which is known to be expressed in blood cells, was isolated from blood cells (Z. Liao, et al., Fish shellfish Immunol. 34. 610-616. 2013). However, the highest expression level of mycusin-beta mRNA was detected in mantle tissue after bacterial infection ( FIG. 8 ). Therefore, these results suggest that myticusin-beta is produced in a tissue-specific manner and is associated with the immune system of M. coruscus .

Experimental Example 1: Anti-Scuticociliate activity

Scuticociliates are parasites considered to be the causative agents of scuticociliatosis disease, causing mass mortality and enormous economic losses in the aquaculture industry (MC Piazzon, et al., Dev. Comp. immunol. 41. 248-256. 2013 ). The incidence of scuticociliatosis disease in farmed fish is increasing worldwide, and it is necessary to develop anti-scuchica-related substances to solve the problem. Some marine antibacterial peptides are known to inhibit the growth of protozoa. For example, Pc-Pis, a cationic peptide derived from Psuedosciaena corcea , has antibacterial activity against bacteria, fungi and parasites and is a piscidin family ) and (SF Niu, et al., Fish Shellfish Immunol. 35 (2) 513-524. 2010). Therefore, the present inventors investigated the anti-Scuchica ciliate activity of the purified recombinant peptide against ciliate scotch. number was evaluated.

As a result, it was found that about 50% of parasites were reduced at 200 μg/mL, the highest concentration of recombinant protein after 24 hours. It was observed under a microscope that the shape of the spindle of the ciliated Scotica swelled or ruptured (FIG. 6A), and it was confirmed that the movement of the fast moving ciliates Scotica was slowed or stopped. In addition, the ciliate Scotica treated with the recombinant protein of the present invention showed a lower viability than the untreated group, and it was observed that the viability of ciliate Scotica decreased due to the concentration of the recombinant protein through the WST-1 cell proliferation assay (FIG. 6B). ). These results suggest that recombinant myticusin-beta exhibits excellent anti-parasitic activity against Scutica ciliates.

Experimental Example 2: antimicrobial activities

The present inventors observed the antibacterial activity of myticusin-beta of the present invention using the URDA method using various indicator strains in Table 1 above. As a result, recombinant myticusin-beta exhibited antibacterial activity against both Gram-positive and Gram-negative bacteria (FIG. 7). Specifically, Myticusin-beta showed strong activity against Gram-positive bacteria including B. subtils, Clostridium perfringens, Staphylococcus aureus and S. mutans and Gram-negative bacteria including P. aeuruginosa and Vibro alginolyticus, and moderately against B. cereus and Klebsiella pneumoniae . It exhibited moderate activity, but rather weak activity against Streptococcus iniae and E. coli . Specifically, Gram-positive bacteria showed higher sensitivity to myticusin-beta than Gram-negative bacteria.

Antibacterial peptides derived from mussels such as denfensins, mytilins, mytichitin-CBD, myticusin-1 and myticin have been reported to have antibacterial activity against bacteria and fungi, and mytimycin has only known antibacterial activity against fungi (G. Mitta, et al. , Dev. Comp. Immunol. 24. 381-393. 2000). Among the previously reported antimicrobial peptides, myticin, mytichitin-CBD and myticusin-1 showed very low activity against Gram-negative bacteria and fungi (Z. Liao, et al., Fish shellfish Immunol. 34. 610-616. 2013). . In addition, the present inventors confirmed that myticusin-beta had antibacterial activity against gram-positive and gram-negative bacteria, but had no activity against fungi. . The difference in the activity of the antimicrobial peptide against these strains occurs depending on the components of the cell membrane and cell wall of the strains. Gram-positive bacteria have several layers of peptidoglycan in the cell wall, and the membrane of gram-negative bacteria has a monolayer of peptidoglycan surrounded by an outer membrane containing LPS that protects the bacteria as a permeability barrier (V. Teixeira) , et al., Prog. Lipid. Res. 51(2).149-177. 2012). Antimicrobial peptides interact with or pass through cell envelopes to reach intracellular targets. The fungal cell wall contains complex polysaccharides such as chitin and glucan, and is tightly composed even to the cytoplasmic membrane. As such, the active range of antibacterial peptides is broad or narrow, and strong or weak, so it is necessary to consider whether to use them alone or in combination with several peptides. And in this way, harmful strains can be effectively controlled, and for this, it is necessary to develop and study antibacterial peptides having various activities.

Experimental Example 3: Antibacterial activity of myticusin-beta peptide analogs

The present inventors used myticusin-beta to design analogues as short-length antibacterial compounds with a low synthesis cost and improved activity and diffusion. A short antibacterial peptide analogue was designed based on the unique sequence of myticusin-beta including the signal peptide, and the analogue was prepared by substituting some amino acids. In order to analyze the properties of the analog, Schiffer-Edmundson helical wheel projection was performed with EMBOSS Pepwheel (European Bioinformatics Institute, UK). To increase the antibacterial activity and cation of the peptide, arginine (Arg) and leucine (Leu) amino acids were substituted. Information on net charge, molecular weight, Boman index (potential protein interaction index), pI values and hydrophobicity is available in the Antimicrobial Peptides Database v2.34 (APD2; http://aps.unmc.edu/AP/main.php). ) was calculated using (G, Wang, et al., Nucleic Acids Res. 37. D933-D937. 2009). For the development of a short antibacterial peptide as a substitute for antibiotics, the peptide fragment VDAFHIYSRR (SEQ ID NO: 6) was selected and synthesized from the active peptide of myticusin-beta in consideration of the peptide length, charge, hydrophobicity, Boman index, and amino acid residue structure. (See Table 3). The physicochemical properties of the peptide variants of the present invention are summarized in Table 3 below.

Physicochemical properties of peptide analogs natural analogues order VDAFHIYSRR VRAFHILLRL Length 10 10 majesty +1 +2 Molecular Weight (Da) 1,263.42 1,237.56 Borman index (kcal/mol) 3.3 0.59 pI 8.72 11.8 Hydrophobicity (%) 40 70 Hydrophobicity (kcal/mol) 1.01 -1.09 area 84-93 transform D→R, Y→L
S→L, R→L SEQ ID NO: 6 7

In addition, in order to predict the hydrophobic and hydrophilic regions in the secondary structure of the synthetic peptide, Schiffer-Edmundson helical wheel projection was performed (FIG. 9). Hydrophobic and hydrophilic regions were identified in the secondary structures of native and analog peptides using the EMOSS pepwheel program, which can predict the interactions and effects between adjacent amino acids and residues. The present inventors designed a cationic antibacterial peptide in consideration of the amino acids above and below the secondary structure of the alpha helix as well as the adjacent amino acids before and after in the primary structure to improve the antibacterial activity. The arginine (Arg), tyrosine (Tyr), aspartic acid (Asp) and serine (Ser) residues in the native form of myticusin-beta were modified to Arg or Leu in their analogs. In the reconstituted analog peptide, the hydrophobic and hydrophilic regions are located at opposite positions, and this amphiphilicity contributes to the antimicrobial activity. The cationic antibacterial peptide is an amphiphilic peptide with a total positive charge that interacts with negatively charged bacterial membranes and forms transmembrane pores (BH Nam, et al., Drugs. 12. 5240-5257. 2014). Increased net charge (+1 to +4), baseline pI values (>10), decreased Boman index values (≤1), and converted structures recorded antimicrobial activity against a broader range of strains.

As a result of measuring the antimicrobial activity of the peptide analogs of the present invention, it showed improved antibacterial activity against all indicator strains (9 Gram-positive bacteria, 9 Gram-negative bacteria and the fungus Candida albicans ), whereas the antibacterial peptide in its natural form before modification was B. subtilis, S Aureus, and showed antibacterial activity only against C. albicans (see Table 4). The synthetic analog peptide induced enhanced activity against Gram-positive bacteria, negative bacteria and fungi due to the low Boman index and increased net charge. Therefore, studies on the total hydrophobic ratio of analogs and synthetic analog peptides suggest that antibacterial peptides can be activated and can greatly contribute to the study of novel antibiotic substitutes. The antimicrobial activity of the native peptides and their analogs is summarized in Table 4 below.

Antimicrobial activity of peptide variants natural analogues Suppression Diameter Area (mm) Gram-positive bacteria B. cereus - 4.9 B. subtilis 4.5 7.9 E. faecalis - 5.1 S. mutans - 8.0 S. iniae - 5.1 S. aureus 2.5 7.2 S. vestibularis - 2.5 S. parauberis - 13.1 L. garvieae - 7.9 Gram-negative bacteria V. alginolyticus - 6.6 V. parahaemolyticus - 7.6 E. coli - 11.7 E. cloacae - 8.6 K. pneumoniae - 9.9 E. tarda - 4.3 P. mirabilis - 6.6 P. stuartii - 9.1 P. aeruginosa - 9.2 fungus C. albicans 2.5 9.3

*Diameter of well: 2.2 mm

Experimental Example 4: Hemolytic activity

In order to determine the toxicity of the recombinant peptide of the present invention, the present inventors measured the hemolytic activity of erythrocytes of flounder ( Paralichthys olivaceus ). For haemolytic activity analysis, halibut blood cells were collected, diluted with sodium phosphate buffer (150 mM NaCl, 50 mM sodium phosphate pH 7.4), and centrifuged at 5,000 rpm at 4°C for 1 minute. After that, the blood cells were washed to collect red blood cells except for buffy coat and plasma, and 90 μL of 3% red blood cells in phosphate buffered saline (PBS) were mixed with 10 μL of sample and incubated at 37° C. for 1 hour. . Then, 100 μL of 3% red blood cells and samples in PBS were centrifuged at 5,000 rpm, 4° C. for 1 minute, and 70 μL of the supernatant was injected into a 96-well plate. Then, hemolysis was monitored by measuring absorbance at 405 nm with a VICTOR3 1420 plate reader (Perkin Elmer, USA), 1% Triton X-100 and picidine 1 (1 mg/mL) were used as positive controls, 0.01% HAc and PBS were used as negative controls. Percent hemolytic activity was calculated as follows: hemolytic activity (%) = [(Abs _{405 nm} test-Abs 405 _nm buffer)/(Abs _{405 nm} 1% Triton X-100-Abs _{405 nm} buffer)] x 100.

As a result, the positive controls Piscidin 1 and Triton X-100 showed strong hemolytic activity, but the recombinant myticusin-beta peptide of the present invention did not show hemolytic activity (FIG. 5A). In addition, the Piscidin 1 showed very high hemolytic activity at a high concentration and sharply decreased at a low concentration, whereas the myticusin-beta peptide did not show a concentration-dependent hemolytic activity (FIG. 5B). The above results demonstrate that the recombinant peptide of the present invention does not exhibit hemolytic activity.

Some antibacterial peptides have disadvantages such as cytotoxicity, such as hemolytic activity to host cells. Antibacterial peptides indirectly bind to bacterial surfaces through electrostatic interactions or interact directly with host cells and increase cell membrane permeability to disrupt membranes ( membrane disruption) (G. Diamond, et al., Curr. Pharm. Des. 15. 2377-2392. 2009). Therefore, non-hemolytic antibacterial peptides are required to use antibacterial peptides as alternative antibiotics, so the above results suggest that recombinant protein myticusin-beta exhibits low hemolytic activity and applicability as a substitute for antibiotics.

Although the present invention has been described with reference to the above-described examples and experimental examples, these are merely exemplary, and those of ordinary skill in the art can make various modifications and equivalent other examples and experiments therefrom. will understand Therefore, the true technical protection scope of the present invention is defined by the technical spirit of the appended claims.

will have to be determined

<110> Republic of Korea represented by National Fisheries Research & Development Institute <120> Antimicrobial peptide derived from the marine bivalve, Mytilus coruscus and uses thereof <130> PD19-5841 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Myticusin-3RACE <400> 1 gattacgcca agcttgacca ccagatggca cagtcggcct gc 42 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Myticusin-5RACE <400> 2 gattacgcca agctttattg cggatgcata tgcgcgtctg 40 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> BamHI-linker F <400> 3 ggatccatgt cagaccatca gatggca 27 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> XhoI-linker R <400> 4 ctcgagttaa acgatacaac agcagaagtt 30 <210> 5 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> myticusin-beta <400> 5 Ser Asp His Gln Met Ala Gln Ser Ala Cys Met Gly Leu Ala Gln Asp 1 5 10 15 Ala Ala Tyr Ala Ser Ala Ile 20 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> myticusin-beta peptiede fragment <400> 6 Val Asp Ala Phe His Ile Tyr Ser Arg Arg 1 5 10 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> myticusin-beta peptiede analogs <400> 7 Val Arg Ala Phe His Ile Leu Leu Arg Leu 1 5 10

Claims (8)

An antibacterial peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 7. An antibacterial composition containing the peptide of claim 1 as an active ingredient. 3. The method of claim 2,
An antibacterial composition having antibacterial activity against Gram-negative bacteria, Gram-positive bacteria or fungi. 4. The method of claim 3,
The Gram-negative bacteria are Vibrio alginolyticus , Vibrio parahaemolyticus , Escherichia coli , Enterobacter cloacae , Klebsiella pneumoniae , Edwardsiella tarda , Proteus mirabilis , Providencia stuartii or Pseudomonas aeruginosa , antibacterial composition. 4. The method of claim 3,
The gram-positive bacteria are Bacillus cereus , B. subtilis , Enterococcus faecalis , Streptococcus mutan s, Streptococcus iniae , Staphylococcus aureous , Streptococcus vestibularis , Streptococcus parauberis or Lactococcus garvieae . 4. The method of claim 3,
wherein the fungus is Candida albicans . A feed additive containing the peptide of claim 1 as an active ingredient. Claim 1 containing the peptide as an active ingredient, anti-Scuchika ciliate controlling composition.

Images