KR102160484B1 - Antimicrobial peptide derived from the phospholipase of rock bream and uses thereof - Google Patents

Abstract

The present invention relates to an antimicrobial peptide derived from Dole Dome phospholipidase (CDP), and more specifically, the amino acid sequence described in SEQ ID NO: 1 that exhibits high antibacterial activity against various labeled strains and has excellent salt and thermal stability (VKLX ₁ VKLVX ₂ X ₃ LIK) containing, to provide an antibacterial peptide derived from dolphin phospholipidase (CDP).

Description

Antimicrobial peptide derived from the phospholipase of rock bream and uses thereof

The present invention relates to an antimicrobial peptide derived from doldom phospholipidase, and more particularly, to a phospholipidase antibacterial peptide derived from doldom, having excellent antibacterial activity against various strains, and its use.

Antibiotics have been used very usefully for humans for the past hundred years, as they have helped humans escape from bacterial diseases and increase the productivity of livestock and aquatic products. However, the use of antibiotics is very limited due to the recent occurrence and accumulation of antibiotics in the body and the emergence of antibiotic-resistant strains through them, and antibiotics used as feed additives for livestock and aquatic products are banned altogether. Therefore, there is a demand for the development of a material that can replace these antibiotics, and various alternative materials research for this is being conducted. Among various alternative materials, antibacterial peptides are natural antibiotics produced by living organisms and do not have the risk of resistant strains, have broad antibacterial activity against various microorganisms, and are easy to manufacture through solid phase synthesis or microbial cultivation processes, which attracts the most attention. to be.

Antimicrobial peptides (AMPs) are short-length peptides of about 100 or less with antimicrobial activity first discovered in the epidermal secretion of frogs in the 1960s, and are positively charged such as lysine, arginine and histidine It contains a large number of amino acids. Therefore, it has antibacterial activity by attaching to the microbial cell membrane having negative charges and destroying the microbial cell membrane. It is known to be produced by almost all living organisms, from microorganisms to plants, invertebrates, vertebrates and humans, and exhibits unique antibacterial properties against a wide range of microorganisms such as bacteria, fungi, viruses and parasites. Antibacterial peptides have been found to be the most important defenses in the innate immune system because they are in charge of a defense system against various external invading microorganisms in a short time.In addition to these antibacterial activities, antioxidant, anti-inflammatory, anti-insect and wound healing activities, etc. It has been reported to have the versatility characteristics of. In addition, there is an advantage that the possibility of the appearance of resistant strains according to the physical antimicrobial mechanism of action is low and the size is small, so that the synthesis is easy. Therefore, the antimicrobial peptide, which is a natural bioprotective material, is a material that is expected to be widely used in recent years, and is particularly attracting attention in the field of antibiotic substitutes, various additives, and drug development. In this regard, Korean Patent No. 1660258 discloses a novel antimicrobial peptide derived from dolphin and its use.

However, in the case of the prior art, since the yield is not high when the peptide is isolated, it is difficult to expect a uniform effect due to factors such as potential toxicity and not exhibiting sufficient antibacterial activity.

The present invention is to solve a number of problems including the above problems. Unlike conventional antibacterial peptides, the hemolytic activity is decreased and the antibacterial activity is increased, thereby decomposing doldom phospholipids showing excellent antibacterial activity and multifunctional effect against various indicator strains. It is an object to provide an enzyme-derived antibacterial peptide. However, these problems are exemplary, and the scope of the present invention is not limited thereby.

According to one aspect of the present invention, there is provided a peptide comprising the amino acid sequence described in SEQ ID NO: 1 (VKLX ₁ VKLVX ₂ X ₃ LIK) and having antibacterial activity.

(In the above amino acid sequence, X ₁ is tyrosine, tryptophan or phenylalanine, X ₂ is threonine, serine, arginine, histidine or lysine, and X ₃ is proline, leucine, methionine, valine, or isoleucine.)

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

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

According to another aspect of the present invention, there is provided a composition for controlling anti-scotch worms containing the peptide as an active ingredient.

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

According to an embodiment of the present invention made as described above, not only exhibits high antibacterial activity against various indicator strains, but is stable against heat and salt, exhibits multifunctional effects such as antiparasitic activity, and toxicity such as hemolytic activity is reduced. It can realize the production effect of antibacterial peptide derived from degrading enzyme. Of course, the scope of the present invention is not limited by these effects.

1 is a schematic diagram showing the RB-CDP-IN amino acid sequence, secondary structure, and amphiphilic properties for the preparation of an RB-CDP-IN antibacterial peptide derived from CDP (catalytic domain of phospholipase) obtained through the analysis of the dolmens genome of the present invention. .
2 is a photograph of an analysis of the antimicrobial activity of the natural form of RB-CDP-IN antibacterial peptide against various indicator strains.
3 is an analysis result of the secondary structure, substituted residues, and amphiphilic properties of RB-CDP-I-A1 and RB-CDP-I-A2 for increasing the activity of RB-CDP-IN antimicrobial peptide.
Figure 4 is a photograph of analyzing the antibacterial activity of the antimicrobial peptides ① RB-CDP-IN, ②RB-CDP-I-A1 and ③ RB-CDP-I-A2 for various indicator strains using the URDA method. Indicator strains used in the evaluation of antimicrobial activity were (A) B. cereus , (B) B. subtilis , (C) E. coli , (D) S. mutans , (E) C. albicans (F) P. aeruginosa , and (G) V. alginolyticus , (H) V. parahaemolyticus (I) S. iniae , (J) S. aureus , (K) L. garvieae , (L) E. cloacae , (M) E. faecalis , (N) ) K. pneumoniae , (O) S. parauberis , (P) E. tarda , (Q) P. mirabilis , (R) P. stuartii were used.
5 is a photograph showing the stability of the representative indicator strains ① RB-CDP-IN, ② RB-CDP-I-A1 and ③ RB-CDP-I-A2 against heat and salt using the URDA method. Each sample was evaluated for the stability of antimicrobial activity after (A) 1,000 ㎍/ml, (B) heated at 100° C. for 10 minutes, (C) 0.5% NaCl treatment, (D) 1% NaCl treatment, and (E) 2% NaCl treatment. I did.
6 is a result of comparing and analyzing the hemolytic activity of RB-CDP-IN, RB-CDP-I-A1 and RB-CDP-I-A2 using red blood cell components of flounder.
7 is a result of analyzing the amino acid sequence, secondary structure and amphiphilicity of RB-CDP-I-A2 derivatives designed to reduce hemolytic activity while maintaining functions such as antimicrobial activity and antiparasitic activity.
Figure 8a is a photograph of analyzing the antimicrobial activity of RB-CDP-I-A2 and hemolytic activity-reducing antibacterial peptide derivatives against various indicator strains using the URDA method:
A2: RB-CDP-I-A2;
1: RB-CDP-I-A2-R1;
2: RB-CDP-I-A2-R2;
3: RB-CDP-I-A2-R3;
4: RB-CDP-I-A2-R4; And
5: RB-CDP-I-A2-R5.
8B is a photograph of an analysis of the antibacterial activity of RB-CDP-I-A2 and hemolytic activity-reducing antimicrobial peptide derivatives against various indicator strains using the URDA method.
8C is a photograph of an analysis of the antibacterial activity of RB-CDP-I-A2 and hemolytic activity-reducing antimicrobial peptide derivatives against various indicator strains using the URDA method.
9 is a photograph of an analysis of the antimicrobial activity mechanism of RB-CDP-I-A2 and synthesized RB-CDP-I-A2 hemolytic activity-reducing antibacterial peptide derivatives through analysis of DNA synthesis inhibitory ability of Taq DNA polymerase in PCR process:
M: DNA ladder;
(+): positive control;
A2: RB-CDP-I-A2;
R1: RB-CDP-I-A2-R1;
R2: RB-CDP-I-A2-R2;
R3: RB-CDP-I-A2-R3;
R4: RB-CDP-I-A2-R4;
R5: RB-CDP-I-A2-R5;
1: 2.5 μg,
2: 1.25 μg,
3: 0.62 μg,
4: 0.31 μg, and
5: 0.15 μg.
FIG. 10 is a photograph of an analysis of the antimicrobial activity mechanism of RB-CDP-I-A2 and hemolytic activity-reducing antimicrobial peptide derivatives as a result of evaluating DNA-binding activity using agarose-gel electrophoresis:
M: DNA ladder;
(+): positive control,
A2: RB-CDP-I-A2;
R1: RB-CDP-I-A2-R1;
R2: RB-CDP-I-A2-R2;
R3: RB-CDP-I-A2-R3;
R4: RB-CDP-I-A2-R4;
R5: RB-CDP-I-A2-R5;
1: 2.5 μg,
2: 1.25 μg,
3: 0.62 μg, and
4: 0.31 μg.
11 is a photograph comparing and analyzing the hemolytic activity of RB-CDP-I-A2 and RB-CDP-I-A2 hemolytic activity-reducing antibacterial peptide derivatives synthesized using red blood cells of flounder.
12 is a graph analyzing the antiparasitic activity of RB-CDP-I-A2 and RB-CDP-I-A2 hemolytic activity-reducing antibacterial peptide derivatives synthesized according to the present invention.
13 is a graph analyzing the anti-cancer activity of RB-CDP-I-A2 and RB-CDP-I-A2 hemolytic activity-reducing antimicrobial peptide derivatives synthesized of the present invention against uterine cancer cells.
14 is a graph analyzing the anti-cancer activity of RB-CDP-I-A2 and RB-CDP-I-A2 hemolytic activity-reducing antibacterial peptide derivatives synthesized according to the present invention against lung cancer cells.

Definition of Terms:

The term "AMPs (Antimicrobial peptides)" used in this document is an antimicrobial peptide, and the following properties are known. Numerous organisms on Earth have an immune system that can protect themselves from the environment in which they are located, and various bioactive substances as a primary defense means that have antimicrobial activity that is typically produced to protect themselves against invading foreign substances. As a natural antibiotic, it has been reported to exist in almost all living organisms on the planet from mammals to insects and plants (Zasloff, Nature 415:389-395, 2002). Antimicrobial peptides were first discovered in epidermal secretions of frogs ( Bombina variegate ) in 1962 (Kiss and Michl, Toxicon 1:33-39,1962; Monatshefte fㆌr Chemie-Chemical Monthly, 101:182-189.1970), 1971. Various antimicrobial peptides have begun to be identified from various organisms such as bee venom, and since then, many studies have been conducted on their properties and functions. Among the hypotheses of the mechanism of action of antimicrobial peptides so far, the most validly suggested is the Shai-Matsuzaki-Huang (SMH) model. The peptide synthesized in the body has a suitable structure with hydrophobic and hydrophilic parts, and a positively charged hydrophilic part is It binds to the cell membrane of negatively charged bacteria (Matsuzaki, Biochimica et Biophysica Acta 1462:1-10. 1999; Shai, Biochimica et Biophysica Acta, 1462:55-70. 1999; Huang, Biochemistry 39:8347-8352. 2000) ; Yang et al., Biophysical Journal, 79:2002-2009.2000). At this time, it is known that the binding force between the peptide and the cell membrane is about 2-3 times stronger than that of general Ca ²⁺ and Mg ²⁺ . After that, the hydrophobic part of the peptide bound to the cell membrane interacts with the hydrophobic part of the cell membrane phospholipid to form a hole, thereby changing the permeability of the cell membrane and eventually destroying the cell (Christensen et al., Proceeding of the National Academy of Sciences). , 85:5072-5076.1988).

The term "phospholipase" used in this document is a lipid metabolizing enzyme that degrades phospholipids present in cell membranes, and is a protein involved in remodeling lipids, synthesis of lipid molecules other than phospholipids, and various signaling in cells. . Lipid-derived products produced by phospholipidases act as secondary signaling substances in cells to regulate various physiological phenomena in cells.

Detailed description of the invention:

According to one aspect of the present invention, there is provided a peptide comprising the amino acid sequence described in SEQ ID NO: 1 (VKLX ₁ VKLVX ₂ X ₃ LIK) and having antibacterial activity.

(In the above amino acid sequence, X ₁ is tyrosine, tryptophan or phenylalanine, X ₂ is threonine, serine, arginine, histidine or lysine, and X ₃ is proline, leucine, methionine, valine, or isoleucine.)

In the antimicrobial peptide, the C-terminus may be amidated.

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

In the antibacterial composition, it may have antimicrobial activity against gram-negative bacteria, gram-positive bacteria, or fungi.

In the antimicrobial composition, the gram-positive bacteria may be B. cereus, B. subtilis, S. mutans, S. iniae, S. aureus, L. garvieae, S. vestibularis , E. faecalis, or S. parauberis , and the S. aureus may be S. aureus 0203 , S. aureus 0204 , S. pyogenes 0206 , or S. aureus 3795.

In the antimicrobial composition, the gram-negative bacteria are E. coli, E. cloacae, P. aeruginosa, V. alginoalyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, V. harveyi, K. pneumoniae, E. tarda, and P. mirabilis, P. stuartii, S. typhimurium, K. oxytoca 0248 , K. aerogenes 0249 , or E. cloacae 0252, and the E. coli is E. coli ML35p, E. coli 0238 or E. coli 1A814 days The P. aeruginosa may be P. aeruginosa 0225, the S. thypimurium may be S. typhimurium 0240, and the fungus may be C. albicans .

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

According to another aspect of the present invention, there is provided a composition for controlling anti-scotch worms containing a peptide as an active ingredient.

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

In the anticancer composition, the cancer may be uterine cancer or lung cancer.

The effective amount of the antimicrobial peptide of the present invention may vary depending on the type of the affected part of the patient, the application site, the number of times of treatment, the treatment time, the formulation, the condition of the patient, the type of adjuvant, and the like. The amount used is not particularly limited, but may be 0.01 μg/kg/day to 10 mg/kg/day. The daily dose may be administered once a day, divided into 2-3 times a day at appropriate intervals, or intermittently administered at intervals of several days.

The antibacterial peptide of the present invention may be contained in an amount of 0.1-100% by weight based on the total weight of the composition, and may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. In addition, solid or liquid additives for preparation may be used in the preparation of pharmaceutical compositions. The additive for formulation may be either organic or inorganic. Examples of excipients include lactose, sucrose, sucrose, glucose, cornstarch, starch, talc, sorbit, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silicon dioxide. As a binder, for example, polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, And dextrin and pectin. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any colorant that is permitted to be added to pharmaceuticals can be used. These tablets and granules can be appropriately coated with a sugar coat, gelatin coating, or other necessary. In addition, preservatives, antioxidants, and the like may be added as necessary.

The antimicrobial peptide of the present invention may be prepared in any formulation conventionally prepared in the art (for example, Remington's Pharmaceutical Science, the latest edition; Mack Publishing Company, Easton PA), and the form of the formulation is not particularly limited. These formulations are described in Remington's Pharmaceutical Science, 15 ^th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour), a formula generally known for all pharmaceutical chemistry.

The antimicrobial peptide of the present invention can be administered orally or parenterally, preferably parenteral administration by intravenous injection, subcutaneous injection, intracerebroventricular injection, intracerebrospinal fluid injection, intramuscular injection And intraperitoneal injection.

The inventors of the present inventors believe that research on bio-specific antimicrobial peptides based on antimicrobial activity requires various analysis processes such as pure separation of the target substance in each process, confirmation of the antibacterial activity of the separated fractions, post-translational modification process and terminal sequence analysis, and pure separation. As a result of noticing the existence of difficulties in sequencing and sequencing, and as a result of polite efforts to solve the existing analysis method, a variety of antimicrobial peptide candidates were secured by conducting research on antibacterial peptides based on the sequence of the dolmens phospholipidase catalytic domain using genomic analysis information. . By analyzing the excellent antimicrobial activity and properties of the antibacterial peptide derived from dolphin showing excellent antimicrobial activity among the candidate groups, unlike the conventional peptide, the hemolytic activity was reduced, but the antibacterial, antiparasitic, and anticancer activity was maintained or improved.

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 may be implemented in various different forms, and the following embodiments make the disclosure of the present invention complete, and the scope of the invention to those of ordinary skill in the art. It is provided to fully inform you.

Example 1: genomic analysis

According to an embodiment of the present invention, various antimicrobial peptide candidates were secured by conducting a sequence-based antibacterial peptide study using genome analysis information. Using the result of genome analysis of Oplegnathus fasciatus , based on the protein sequence after translation, helix secondary structure, which is a characteristic of antibacterial peptide, short primary sequence of 2 to 50 or less, contains a large number of positively charged amino acids, has both hydrophobicity and hydrophilicity. As a result of analyzing a short protein sequence that satisfies the amphiphilicity, low Boman index, etc. through the program, some short peptide sequences were obtained as antibacterial peptide candidates. Among the nucleotide sequence-based amino acid sequences in the obtained genomic data, 20 or more various antibacterial peptide candidate sequences were obtained. Among them, the catalytic domain of the doldom phospholipidase (phospholipase) phospholipase D superfamily sequence As a result of analyzing the sequence that satisfies the number of amino acids between 2 to 50, helix structure, charge value, and boman index, and is most suitable for antibacterial peptide properties, VKLWVKLVTPLIK among the 18 amino acid sequences of RHVKLWVKLVTPLIKNFF among the peptide candidates It was confirmed that the 13 amino acid sequence portion of phosphorus (SEQ ID NO: 2) has a value that best matches the properties of the antibacterial peptide (Table 1 and Fig. 1). The information on the antibacterial peptide candidate derived from the dolphin CDP gene is shown in Table 1 below.

Information on Antimicrobial Peptide Candidate Derived from the CDP Gene order
number Peptide name order M.W. p.I. Net charge Hydrophobicity (kcal/mol) Boman index (kcal/mol) 2 RB-CDP-I-N VKLWVKLVTPLIK-NH ₂ 1536.9 10.3 +3 -0.07 -1.14 3 RB-CDP-I-A1 VKLWVKLVKPLIK-NH ₂ 1564 10.48 +4 0.78 -0.91 4 RB-CDP-I-A2 VKLWVKLVKLLIK-NH ₂ 1580.1 10.48 +4 -0.23 -1.29

Example 2: Preparation of antibacterial peptide

In order to prepare an antimicrobial peptide having improved antibacterial activity, the present inventors analyzed the helix wheel, which is a protein secondary structure, using the sequence of Table 1, and then substituted some amino acid residues. Peptides (SEQ ID NO: 3) were prepared from 13 amino acid sequences in Doldom CDP protein, which were substituted according to the amphiphilic properties of the antimicrobial peptide, and were named RB-CDP-I-A1 (Table 1, FIG. 3). The designed peptide was obtained by requesting solid-phase synthesis from EnsolBioscience, Inc., a company specializing in peptide synthesis. In addition, in order to prepare an antimicrobial peptide with increased antibacterial activity by analyzing the secondary structure of 13 amino acid sequences, the sequence of consecutive front and rear amino acids in the helix structure, the structure of the upper and lower amino acids, and the characteristics of the residues were analyzed. To evaluate amphiphilicity, some amino acids were substituted with other amino acid residues that match the properties of the antimicrobial peptide. Specifically, a peptide obtained by substituting leucine for the tenth amino acid (proline) of the RB-CDP-I-A1 peptide was prepared and named as RB-CDP-I-A2 (SEQ ID NO: 4) (Table 1). In addition, in order to increase the structural stability and antimicrobial activity of the peptides, a peptide was designed with amidation of the carboxyl terminal (C-term) with NH ₂ , and solid-phase synthesis was performed by EnsolBioscience, Inc., a specialized peptide synthesis company. It was requested and secured.

Example 3: Antimicrobial activity measurement assay (URDA)

Dissolve RB-CDP-IN and RB-CDP-I-A2 antibacterial peptides obtained according to an embodiment of the present invention in 0.01% acetic acid solution at a concentration of 1,000 to 31.3 μg/ml, and one fungus, Gram-positive bacteria Antimicrobial activity measurement analysis was performed against a total of 35 different pathogenic microorganisms, including 9 species, 16 Gram-negative bacteria, and antibiotic-resistant strains.

First, using the optimal growth medium of each indicator strain, after incubation for 18 hours at an appropriate culture temperature, OD ₆₀₀ = 0.1 for bacteria 10 ⁸ CFU / ml or yeast 10 ⁶ CFU / ml was diluted with the same medium. Then, 0.5 ml of the diluted strain culture solution was added to 9.5 ml of an under-lay gel containing 0.01% of culture medium, 1% agarose, and 10 mM phosphate buffer (pH 6.5), and mixed, and then plated ( plate) to solidify. After drilling a 2.2 mm diameter hole in the hardened under-lay gel, the peptides dissolved in 10 µl of 0.01% acetic acid were injected, respectively. In addition, 10 ml of an over-lay gel containing 2% culture medium, 1% agarose, and 10 mM phosphate buffer (pH 6.5) was dispensed onto the under-lay gel to solidify it, and 18 hours at the optimum culture temperature of the indicator strains. During incubation. After that, the size of the transparent ring formed around each sample was measured to confirm the antibacterial activity.

As a result, the RB-CDP-I-A2 antibacterial peptide, which increased antibacterial activity by substituting some amino acids of RB-CDP-IN, showed antibacterial activity against various indicator strains, but in the case of RB-CDP-IN antibacterial peptide, Vibrio alginolyticus, V. parahaemolyticus, Streptococcus iniae, Enterobacter cloacae, Proteus mirabilis, Providencia stuartii strains were very weak in activity (Fig. 2). In addition, the RB-CDP-I-A2 antibacterial peptide was observed for antimicrobial activity against a total of 34 strains excluding Staphylococcus haemolyticus strain in various concentration ranges (FIG. 4).

In the case of some antibacterial peptides, it is often reported that only gram-positive or negative bacteria are active, or fungi have no antimicrobial activity at all, but RB-CDP-I-A2 antibacterial peptides exhibit antimicrobial activity not only against fungi, but also against both Gram-positive and negative strains. In addition, some antibiotic-resistant strains also showed high antibacterial activity, and the activity showed a slight significant difference in activity depending on the species. In addition, the minimum effective concentration (MEC) for each strain of the RB-CDP-I-A2 antimicrobial peptide was analyzed through the above results. The list of indicator strains used in the antibacterial activity assay and the minimum effect concentration (㎍/ml) are shown in Table 2 below.

List of indicator strains and minimum effective concentration (㎍/ml) Type of microorganism MIC
(㎍/ml) Type of microorganism MIC
(㎍/ml) leaven Candida albicans KCCM 11282 3.3 Gram-positive bacteria Bacillus cereus KCTC 1012 5.3 Bacillus subtilis KCTC 1021 1.4 Enterococcus faecalis KCTC 3206 4.9 Lactococcus garvieae KCTC 3772 2.3 Staphylocuccus aureus KCCM 11335 3.5 Streptococcus mutans KCTC 3065 5.0 Streptococcus parauberis KCTC 3651 0.8 Streptococcus iniae KCTC 3657 9.5 Gram-negative bacteria Enterobacter cloacae CCARM 0252 1.7 Enterobacter cloacae KCTC 2361 1.7 Escherichia coli ACTT 8739 1.6 Klebsiella pneumonia KCTC 12385 0.5 Providencia stuartii KCTC 2569 16 Proteus mirabilis KCTC 2510 2.3 Pseudomonas aeruginosa KCTC 1636 3.5 Vibrio alginolyticus KCTC 2472 9.3 Vibrio parahaemolyticus KCCM 41664 7.2

Example 4: Evaluation of stability against heat and salt

Stability against heat and salt was evaluated using the RB-CDP-I-A2 antibacterial peptide prepared according to an embodiment of the present invention. In the case of some antibacterial peptides, the antimicrobial activity disappears after heating at 100℃, or in the case of the antibacterial peptide isolated from terrestrial organisms, the activity is inhibited and disappeared by NaCl present in the marine environment. In order to evaluate the salt stability against NaCl, RB-CDP-I-A2 antibacterial peptide was heated at 100°C for 10 minutes and the antimicrobial activity was evaluated under 0.5~2% NaCl conditions.

As a result, the RB-CDP-I-A2 antibacterial peptide of the present invention exhibited stable antimicrobial activity without being affected by heat and NaCl (FIG. 5).

Example 5: Evaluation of hemolytic activity on flounder red blood cells

The present inventors analyzed the hemolytic activity of the antimicrobial peptides RB-CDP-I-N, RB-CDP-I-A1 and RB-CDP-I-A2 of the present invention. Specifically, 50 mM sodium phosphate buffer (PBS, pH 7.4) containing the same amount of 150 mM NaCl was added to the blood isolated from the flounder, mixed, and then centrifuged for 1 minute at 5,000 rpm at 4°C to obtain a precipitate. . The red blood cells obtained by repeating the same washing process 3 times were used after adding PBS to a 3% mixed solution. Thereafter, 90 μl of a 3% red blood cell mixture and 10 μl of antimicrobial peptides for each concentration were mixed and reacted at 37° C. for 1 hour. After centrifuging the reaction solution, the degree of red blood cell outflow of the supernatant was measured with a spectrophotometer (405 nm), 1% Triton X-100 was used as a positive control, and 0.01% acetic acid and PBS were used as a negative control. Then, the hemolytic activity was evaluated by the following formula.

% Hemolytic activity=[(ABS _405nm peptide absorbance-ABS _405nm buffer absorbance)/(ABS _405nm 1% Triton X-100 absorbance-ABS _405nm buffer absorbance)] × 100

As a result, RB-CDP-I-N and RB-CDP-I-A1 had very low hemolytic activity, but RB-CDP-I-A2 showed high hemolytic activity (FIG. 6 ).

Example 6: Hemolytic activity-reducing derivative antibacterial peptide preparation and activity evaluation

The present inventors prepared an antimicrobial peptide derivative with reduced hemolytic activity while maintaining high antimicrobial activity of the antibacterial peptide RB-CDP-I-A2, and analyzed its functionality. Specifically, the tenth leucine (Leucine, Leu, L) residue, which is a residue that induces hemolytic activity, is referred to as methionine (Methioninie, Met, M), lysine (Lysine, Lys, K), valine (Valine, Val, V), and isoleucine ( Isoleucine, Ile, I), and derivatives in which only the fourth residue, Tryptophan, Trp, W, was substituted with phenylalanine (Phe, F) were prepared (Table 3. FIG. 7). The information of the prepared derivatives is summarized in Table 3 below.

Sequence information of RB-CDP-I-A2 antibacterial peptide derivative Sequence number Peptide name Peptide sequence 5 RB-CDP-I-A2-R1 VKLWVKLVKMLIK-NH ₂ 6 RB-CDP-I-A2-R2 VKLWVKLVKKLIK-NH ₂ 7 RB-CDP-I-A2-R3 VKLWVKLVKVLIK-NH ₂ 8 RB-CDP-I-A2-R4 VKLWVKLVKILIK-NH ₂ 9 RB-CDP-I-A2-R5 VKLFVKLVKLLIK-NH ₂

As a result, the antimicrobial activity of the hemolytic activity-reducing derivatives prepared to decrease hemolytic activity was similar to that of RB-CDP-I-A2 (RB-CDP-I-A2-R3, or -R4) or further increased the activity ( RB-CDP-I-A2-R1, or -R2), especially RB-CDP-I-A2-R5, showed a reduced activity against S. iniae strains (FIGS. 8A to 8C ).

Example 7: Evaluation of antibacterial activity of antibacterial peptide derivatives 1 (polymerase inhibitory reaction)

The antimicrobial activity of the antibacterial peptide RB-CDP-I-A2 and the hemolytic activity-reducing derivatives of the present invention was analyzed by evaluating the inhibitory ability of the gene amplification activity of the polymerase (Taq polymerase). Specifically, the antimicrobial activity by inhibiting the DNA amplification reaction was evaluated by adding each of the prepared antimicrobial peptides during the gene amplification reaction. As a result, all of the derivatives showed an enzyme inhibition reaction similar to that of the RB-CDP-I-A2 antibacterial peptide (FIG. 9).

Example 8: Evaluation of antibacterial activity of antibacterial peptide derivatives 2 (Evaluation of DNA adhesion ability)

The antimicrobial activity of the antimicrobial peptide RB-CDP-I-A2 and the hemolytic activity-reducing derivatives of the present invention through DNA attachment was analyzed. Specifically, in the process of separating and deploying DNA markers through agarose electrophoresis, each antimicrobial peptide was added and then attached to DNA during electrophoresis to measure the activity to interfere.

As a result, RB-CDP-I-A2-R3 and -R4 exhibited similar DNA adhesion activity to RB-CDP-I-A2 except for the result showing decreased adhesion at 0.31 μg (Fig. 10). .

Example 9: Evaluation of hemolytic activity of antibacterial peptide derivatives

The hemolytic activity of the antimicrobial peptide RB-CDP-I-A2 and the hemolytic activity-reducing derivatives of the present invention on red blood cells of flounder was evaluated. As for the evaluation method, the degree of hemolytic activity by concentration was evaluated in the same manner as in Example 5.

As a result, the high concentration of 100 μg/ml showed similar hemolytic activity, but RB-CDP-I-A2-R2 decreased hemolytic activity by more than 50% from the concentration of 50 μg/ml, and the hemolytic activity was remarkably reduced below 20 μg/ml. It was confirmed that the hemolytic activity was significantly decreased compared to RB-CDP-I-A2 except for RB-CDP-I-A2-R1 and -R4 by concentration, and RB-CDP-I-A2-R1 and R -4 decreased hemolytic activity by 50% or more from a concentration of 12.5 μg/ml compared to RB-CDP-I-A2 (FIG. 11).

Example 10: Evaluation of Sukuchika parasite killing activity

The antimicrobial peptide RB-CDP-I-A2 of the present invention and Antiparasitic activity was analyzed to evaluate the versatility of derivatives with reduced hemolytic activity. Specifically, after diluting in a medium so that the ratio of scutica insects was 1x10 ⁶ cells/100 µl, 100 µl was dispensed into a 96-well plate (about 50% per well), and the sample was mixed with the medium. After making each 100 µl concentration, it was dispensed into wells containing 100 µl of parasites (100 µl of sample + 100 µl of parasites = 200 µl in total). Thereafter, the culture was cultured for 24 hours under the conditions of 20°C, and the change in morphology and motility of the Sukuchika insect was photographed under a microscope. After the reaction was over, 20 µl of Premix WST-1 was added per well, and absorbance (450 nm) was repeatedly measured using an ELISA reader at various reaction times (0.5, 1, 2, 3, 4 and 5 hours). Was determined. When 0.01% acetic acid was treated, the survival rate of the Scotch worms was measured according to the concentration of the antimicrobial peptide of 25 to 200 μg/ml based on 100%. The formula for the experimental conditions and survival rate is summarized as follows:

*Blank = medium (L-15) + 0.01% acetic acid

Control = Scuchika + 0.01% acetic acid

Sample treatment group = Sukuchika + peptide (1, 5, 10, 15 ㎍/ml)

(sample-blank / control-blank) x 100 = survival rate.

As a result, it was confirmed that RB-CDP-I-A2 and RB-CDP-I-A2-R1 exhibited antiparasitic activity against Sukuchika insects up to a concentration of 10 μg/ml in addition to antibacterial activity. However, at concentrations of 10 µg/ml or more, about 80% or more of killing activity was shown against Sukuchika insects, but at concentrations of 5 µg/ml or less, about 20% of mortality was recorded. In addition, the derivatives showed lower antiparasitic activity than RB-CDP-I-A2 except for RB-CDP-I-A2-R1, but in the case of RB-CDP-I-A2-R4 and -R5, the concentration of more than 15 μg/ml At least 70% of antiparasitic activity was recorded (Fig. 12).

Example 11: Anti-cancer activity assay

Anticancer activity was analyzed to evaluate the versatility of the RB-CDP-I-A2 antimicrobial peptide and hemolytic activity-reducing derivatives of the present invention. In order to observe the anticancer effect among the multifunctional effects, RB-CDP-I-A2 antimicrobial peptides were added to uterine cancer cells (HeLa), lung cancer cells (A549), and colon cancer cells (HCT116) by concentration in the same manner as in Example 10 ( 0-50 μg/ml) and the survival rate was analyzed.

As a result, it showed low anticancer activity in colon cancer cells (HCT116), but in uterine cancer cells (HeLa) and lung cancer cells (A549), RB-CDP-I-A2 and derivatives RB-CDP-I-A2-R1, -R2 It was confirmed that it exhibits excellent anticancer activity (Figs. 13 and 14).

In conclusion, the antimicrobial peptides of the present invention prepared based on the post-translational phospholipidase catalytic domain sequence using the results of the dolmens genome analysis showed not only high antibacterial activity against various marker strains, but also showed antiparasitic activity The stability is also proven after overheating, so it can be used as an antibiotic substitute for the control of various pathogenic and harmful bacteria.

The present invention has been described with reference to the above-described embodiments, but these are merely exemplary, and those of ordinary skill in the art will appreciate that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

<110> Republic of Korea represented by National Fisheries Research & Development Institute <120> Antimicrobial peptide derived from the phospholipase of rock bream and uses thereof <130> PD18-5663 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> antimicrobial peptide <220> <221> VARIANT <222> (4) <223> Xaa is Tyr, Trp or Phe. <220> <221> VARIANT <222> (9) <223> Xaa is Thr, Ser, Arg, His or Lys. <220> <221> VARIANT <222> (10) <223> Xaa is Pro, Leu, Met or Ile. <400> 1 Val Lys Leu Xaa Val Lys Leu Val Xaa Xaa Leu Ile Lys 1 5 10 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> RB-CDP-I-N <400> 2 Val Lys Leu Trp Val Lys Leu Val Thr Pro Leu Ile Lys 1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> RB-CDP-I-A1 <400> 3 Val Lys Leu Trp Val Lys Leu Val Lys Pro Leu Ile Lys 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> RB-CDP-I-A2 <400> 4 Val Lys Leu Trp Val Lys Leu Val Lys Leu Leu Ile Lys 1 5 10 <210> 5 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> RB-CDP-I-A2-R1 <400> 5 Val Lys Leu Trp Val Lys Leu Val Lys Met Leu Ile Lys 1 5 10 <210> 6 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> RB-CDP-I-A2-R2 <400> 6 Val Lys Leu Trp Val Lys Leu Val Lys Lys Leu Ile Lys 1 5 10 <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> RB-CDP-I-A2-R3 <400> 7 Val Lys Leu Trp Val Lys Leu Val Lys Val Leu Ile Lys 1 5 10 <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> RB-CDP-I-A2-R4 <400> 8 Val Lys Leu Trp Val Lys Leu Val Lys Ile Leu Ile Lys 1 5 10 <210> 9 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> RB-CDP-I-A2-R5 <400> 9 Val Lys Leu Phe Val Lys Leu Val Lys Leu Leu Ile Lys 1 5 10

Claims (15)

A peptide having antimicrobial activity consisting of the amino acid sequence shown in SEQ ID NO: 1 (VKLX ₁ VKLVX ₂ X ₃ LIK).
(In the above amino acid sequence, X ₁ is tryptophan, X ₂ is lysine, and X ₃ is leucine, methionine, or lysine.) The method of claim 1,
C-terminal amidated, peptide. An antimicrobial composition containing the peptide of claim 1 or 2 as an active ingredient. The method of claim 3,
Having antibacterial activity against gram-negative bacteria, gram-positive bacteria, or fungi. The method of claim 4,
The gram-positive bacteria are B. cereus, B. subtilis, S. mutans, S. iniae, S. aureus, L. garvieae, S. vestibularis , E. faecalis, or S. parauberis , antibacterial composition. The method of claim 5,
The S. aureus is S. aureus 0203 , S. aureus 0204 , S. pyogenes 0206 , or S. aureus 3795, antibacterial composition. The method of claim 4,
The gram-negative bacteria are E. coli, E. cloacae, P. aeruginosa, V. alginoalyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, V. harveyi, K. pneumoniae, E. tarda, P. mirabilis, P. stuartii, S. typhimurium, K. oxytoca 0248 , K. aerogenes 0249 , or E. cloacae 0252, antibacterial composition. The method of claim 7,
The E. coli is E. coli ML35p, E. coli 0238 or E. coli 1A814, antibacterial composition. The method of claim 7,
The P. aeruginosa is P. aeruginosa 0225, antibacterial composition. The method of claim 7,
The S. thypimurium is S. typhimurium 0240, antibacterial composition. The method of claim 4,
The fungus is C. albicans , antibacterial composition. A feed additive containing the peptide of claim 1 or 2 as an active ingredient. Claims 1 or 2, containing the peptide of claim 2 as an active ingredient, anti-Skuchika insect control composition. An anticancer composition containing the peptide of claim 1 or 2 as an active ingredient. The method of claim 14,
The cancer is uterine cancer or lung cancer, anticancer composition.

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