KR101720539B1 - Antimicrobial peptide analogues Ov1-1, Ov1-2 derived from Octopus variabilis and antimicrobial composition containing the same - Google Patents

Abstract

The present invention relates to an octopus-derived antimicrobial peptide analogue Ov1-1, Ov1-2 and an antimicrobial composition containing the same.
The antimicrobial peptide analogue according to the present invention is newly designed to be commercialized with peptides having excellent antimicrobial activity derived from octopus. Namely, the number of amino acids is designed so as to enable commercialization, and the antimicrobial activity is excellently achieved both in vivo and in vitro, thereby solving the problem that the parent peptide significantly reduces the antimicrobial activity in vitro of the octopus. In addition, despite the commercialization, the problem of reducing the antimicrobial activity due to the structural change caused by the synthesis was solved.

Description

[Antimicrobial peptide analogues Ov1-1, Ov1-2 derived from Octopus variabilis and antimicrobial composition containing the same}

The present invention relates to an antimicrobial peptide analog derived from octopus and an antimicrobial composition comprising the same.

The mechanism by which antimicrobial peptides act on pathogens is very different from that of existing antibiotics. They mainly bind to the cell surface of microorganisms and form pores in the cell membrane, thereby disturbing the normal permeability of the cell membrane and kill pathogens at a rapid rate (Pharmacol. Rev. 55: 27-55, 2003). In addition, they are known to prevent sepsis caused by pathogen endotoxic factors (Biochim. Biophys. Acta. 1562: 32-36, 2002), and are said to act as signaling molecules mediating acquired immune responses in higher animals. Reported (J. Leukoc. Biol. 60: 415-422, 1996). Due to these characteristics, antibacterial peptides are attracting attention that they can be used as novel antibiotics or new bioimmune activating factors that can control antibiotic-resistant bacteria. Thus, in order to find an antibiotic that can replace existing antibiotics, peptide-type antibiotics have been isolated and tested from various organisms. However, the actual commercialization and use cases are extremely rare, because the stability of the peptide antibiotic when injected into the body or the safety of the human body has been raised.

In order to solve these difficulties, it is necessary to compare and analyze the amino acid sequence and structure of peptides having antibacterial or antifungal activity to find a structure capable of efficiently killing microorganisms without harming the human body. To this end, information on the amino acid sequence and structure of various antimicrobial active peptides beyond those reported so far is required.

In addition to these problems, in order to commercialize the found antibacterial peptide, the structure of the peptide is not complicated and the amino acid sequence does not exceed 12-15, so that it can be easily synthesized and has excellent antibacterial activity. The effort to do is lacking a lot.

On the other hand, both vertebrates and invertebrates have an immune system to defend themselves. In particular, marine invertebrates are always exposed to infectious agents existing in the sea unlike the terrestrial environment, so the immune system by their own defense system Have. Therefore, despite the necessity of efforts to discover antibacterial peptides by utilizing the defense system of these marine invertebrates, related studies are lacking. In particular, in marine invertebrates such as octopus, although various antibacterial peptides may exist as a defense mechanism against external invading substances through gills, efforts to discover antibacterial peptides by paying attention to this fact have been insufficient. In addition, even if antimicrobial peptides are found from them, a separate process of artificially synthesizing these peptides as a parent is required in order to commercialize them, but the antimicrobial activity in vivo and in vitro is significantly different. In addition, there is a concern that the antimicrobial activity may decrease due to structural changes according to the synthesis, and thus there are many problems in commercializing such antimicrobial peptides.

As a prior art document related to the present invention, Korean Patent Registration No. 10-1374267 (Patent Document 1) is disclosed, and Patent Document 1 relates to an antibiotic peptide having a novel amino acid sequence, and more specifically, from abalone As for the isolated antibiotic peptide, it relates to a peptide having excellent antibacterial activity against Escherichia coli or Staphylococcus aureus. In Patent Document 1, only the contents of the antimicrobial peptide isolated from abalone are disclosed, and Octopus variabilis ), there is no disclosure or even suggestion of antimicrobial peptides.

Patent Document 1. Korean Patent Registration No. 10-1374267

The present invention was conceived to solve the above-described problems, and an object of the present invention is an octopus ( Octopus), which is a marine invertebrate. variabilis ), and has found a peptide having excellent antimicrobial activity, designed appropriately the number of amino acids so that it can be commercialized with it, and designed and provided an antibacterial peptide analogue having excellent antibacterial activity. In particular, it is an object to provide an antimicrobial peptide analogue having excellent antimicrobial activity both in vivo or in vitro unlike the parental peptide, while the antimicrobial activity is not lowered even with structural changes due to synthesis.

The present invention for solving the above problems provides an antibacterial peptide derived from octopus having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

In addition, the present invention provides an antimicrobial composition comprising an antibacterial peptide derived from octopus having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

The antimicrobial peptide analogue according to the present invention is derived from octopus and is newly designed to be commercially available with a peptide having weak antibacterial activity. That is, the number of amino acids was properly designed to be commercially available, and the antibacterial activity was excellently achieved both in vivo or in vitro, thereby solving the problem that the parental peptide significantly reduced the antimicrobial activity of octopus in vitro. In addition, in spite of the synthesis process according to commercialization, the problem of reduction of antimicrobial activity due to structural changes due to synthesis was solved.

1 is a measurement result of antimicrobial activity of an octopus gill extract before and after trypsin treatment.
2 is a diagram showing the antimicrobial activity and activity distribution of Prep-cell pooled # against E. coli D31.
Figure 3 is an AU-PAGE of active prep-cell # of octopus gill extracts.
4 is a reverse phase HPLC result for purifying an antimicrobial peptide from prep-cell #53, an active fraction of an octopus gill extract.
5 is a molecular weight measurement and edman degradation results of the octopus gill extract peptides.
6 is a secondary structure prediction of Ov 1 (A) and Ov 2 (B) derived from octopus gill extract.
7 is a sequence alignment of Ov 1 (A) and Ov 2 (B) derived from octopus gill extract.
8 is a result of measuring the antimicrobial activity of Ov 1 and Ov 2.
9 is a result of measuring the antimicrobial activity of Ov 1 analogs (Ov1-1, Ov1-2).
10 is a result of measuring the hemolytic activity of Ov 1 and Ov 2 analogs and piscidin 1 on human red blood cells.
11 is a result of measuring the intima permeability of E. coli ML35p of Ov 1 and Ov 2 analogs and piscidin 1. FIG.
12 is a result of measuring the DNA binding ability of Ov 1 and Ov 2 analogs.

Accordingly, the inventors of the present invention are octopus ( Octopus variabilis ) from the antimicrobial peptide derived from the antibacterial activity, and as a result of intensive research efforts to develop a commercially available peptide analogue, the present invention was completed by discovering the antibacterial peptide analogue according to the present invention and an antimicrobial composition comprising the same. .

Specifically, the antimicrobial peptide according to the present invention is characterized in that the present invention is an antimicrobial peptide analog derived from octopus having an amino acid sequence represented by SEQ ID NO: 1 or 2.

The antimicrobial peptide including the antimicrobial peptide analogue of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 may be an analogue of the peptide of the amino acid sequence of SEQ ID NO: 4.

These antimicrobial peptides having the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5 are derived from the gills of octopus, and the present inventors confirmed the antibacterial activity of these peptides. However, after artificially synthesizing these antimicrobial peptides, it was confirmed that the antimicrobial activity was significantly lowered. This may be due to a structural difference accompanying the synthesis process, or may be due to a significant decrease in antimicrobial activity in vitro due to an environmental difference due to synthesis. Thus, while properly designing the number of amino acids to enable commercialization, it maintains excellent antibacterial activity, and is an antimicrobial peptide having excellent antibacterial activity as in vivo even when it is changed to an in vitro environment by synthesis: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO. An analogue of an antimicrobial peptide having an amino acid sequence selected from the group represented by 3 was designed. Specifically, as described above, the antibacterial peptide of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 may be an analog of the peptide of the amino acid sequence of SEQ ID NO: 4, and the antibacterial of the amino acid sequence represented by SEQ ID NO: 3 The peptide may be an analog of the peptide of the amino acid sequence of SEQ ID NO: 5.

In addition, the peptide of the amino acid sequence represented by SEQ ID NO: 4 may be the parent peptide of the antimicrobial peptide of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and its experimental molecular weight is not particularly limited, but may be 1211.59 Da. , But is not limited thereto. In addition, the peptide of the amino acid sequence represented by SEQ ID NO: 5 may be the parent peptide of the antibacterial peptide of the amino acid sequence represented by SEQ ID NO: 3, and the experimental molecular weight thereof is not particularly limited, but may be 2502.24 Da, and is limited thereto. It is not.

In addition, the peptide of the amino acid sequence represented by SEQ ID NO: 4 can form a mixed structure in which the β-sheet structure and the turn structure are mixed, and the peptide of the amino acid sequence represented by SEQ ID NO: 5 is α-helix (α -helix) as the main structure may be a secondary structure in which a β-sheet structure and a turn structure are slightly mixed.

In addition, the antimicrobial peptide of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 can be designed by replacing the Gly residue or Gln residue in SEQ ID NO: 4 with another amino acid, and amidating the C-terminus. In addition, as a result of predicting the secondary structure of SEQ ID NO: 5, the antimicrobial peptide of the amino acid sequence represented by SEQ ID NO: 3 can design analogues through the amino acid substitution method and amidation of the C-terminus by preferentially selecting the part that takes the α-helix structure. have.

The antimicrobial peptide according to the present invention designed in this way has excellent antibacterial activity in both in vivo or in vitro, and can be commercialized. In addition, the antibacterial peptide according to the present invention may be an antibacterial peptide without a problem of cytotoxicity because it does not show hemolytic activity. Meanwhile, among the antibacterial peptides according to the present invention, the antimicrobial peptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 has weak inner membrane permeability, but the antibacterial peptide having the amino acid sequence of SEQ ID NO: 3 has strong inner membrane permeability. In addition, all of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 have strong binding power to DNA. Therefore, the antimicrobial peptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 may achieve antimicrobial activity indirectly by targeting the intracellular target site of DNA rather than targeting the membrane directly. That is, the antimicrobial peptide comprising the amino acid of SEQ ID NO: 1 or SEQ ID NO: 2 may exhibit antimicrobial activity by binding to a target DNA and interfering with transcription and translation. In addition, the antimicrobial peptide having the amino acid sequence of SEQ ID NO: 3 may directly attack cell membranes to achieve antibacterial activity.

In addition, the antimicrobial peptide may have an amino group (-NH ₂ ) bonded to the C-terminus. In this way, when an amino group (-NH ₂ ) is bound to the C-terminus of the antimicrobial peptide, the effect of improving resistance to protease and improving net-charge may be further improved.

In addition, the antimicrobial peptide is B. subtilis , S. epidermidis , S. mutans , A. hydrophila. E. coli , S. enterica , S. flexni , S. sonnei , P. aeruginosa, and C. albicans may have antibacterial activity against any one or more selected from the group consisting of.

In addition, although there is no particular limitation, as a preferred embodiment of the present invention, if an amino group (-NH ₂ ) is bonded to the C-terminus of the amino acid represented by SEQ ID NO: 1 or 2, the antimicrobial activity It is excellent, does not exhibit hemolytic activity, does not have high membrane permeability, and has high binding ability with DNA.In this case, it is bound to intracellular substances (e.g., DNA) after penetration into the cell rather than directly attacking the bacterial membrane. It could mean that antimicrobial action is achieved by doing so or by another indirect method (e.g. by binding to DNA and interfering with transcription and translation).

In addition, although there is no particular limitation, as corresponding to a preferred embodiment of the present invention, looking at the effect of the case _{where the amino group (-NH 2) is bonded to the C-terminus of the amino acid sequence represented by SEQ ID NO: 3} Hemolytic activity did not appear and the DNA binding power was also high, but unlike the other analogues, the membrane permeability was high, and the antibacterial activity could be achieved by directly attacking the membrane.

The antimicrobial composition according to another aspect of the present invention includes an antimicrobial peptide analog derived from octopus represented by SEQ ID NO: 1 or SEQ ID NO: 2.

The antimicrobial pharmaceutical composition according to the present invention contains another antimicrobial peptide of the present invention as an active ingredient, thereby achieving a pharmacological effect of antibacterial activity.

The antibacterial peptide is derived from a peptide found in the baby rice of octopus, and the antibacterial peptide may have an amino group (-NH ₂ ) bound to the C-terminus.

In addition, the antimicrobial peptide is B. subtilis , S. epidermidis , S. mutans , A. hydrophila. E. coli , S. enterica , S. flexni , S. sonnei , P. aeruginosa, and C. albicans may have antibacterial activity against any one or more selected from the group consisting of.

On the other hand, the method of administering the antimicrobial composition is not particularly limited, but is preferably injected into an artery or vein, or subcutaneously, rectal, nasal, or any other parenteral administration, and more preferably It is preferred to be injected into an artery or vein, orally or directly into muscle cells.

In addition, the dosage level selected from the composition will depend on the activity of the compound, the route of administration, the severity of the condition being treated and the condition and previous medical history of the patient being treated. However, it is within the knowledge of the art to start with a dose of the compound at a lower level than required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved, and the preferred dosage is age, sex. , It can be determined according to body type and weight. The composition may be further processed prior to being formulated into a pharmaceutically acceptable pharmaceutical formulation, preferably pulverized or ground into smaller particles. Also, the composition will depend on the condition and the patient being treated, but this can be determined non-ingeniously.

In addition, the antimicrobial peptide has antimicrobial activity in B. subtilis , S. epidermidis , S. mutans, A. hydrophila . E. coli , S. enterica , S. flexni , S. sonnei , P. aeruginosa, or C. albicans , as it is possible to prevent or treat diseases such as food poisoning, candism, typhoid fever, cholera, etc. By containing such an antimicrobial peptide as an active ingredient, it can be used as a pharmaceutical composition for preventing or treating the above diseases.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments so that those of ordinary skill in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Example

<Materials and methods>

Experimental animals

The octopus used in the experiment (Mt. Samcheonpo) was purchased alive at Haemang-dong, Gunsan-si, Jeollabuk-do, and was immediately transferred to the laboratory for tissue collection and extraction, and each of the obtained extracts was stored at -70°C until use. .

Reagents and materials

Tryptic soy broth (TSB) and agarose type I (Low EEO Agar) for measuring antibacterial activity were obtained from Merck (Merck, Darmstadt, Germany) and Sigma (St. Louis, MO, USA), respectively. It was purchased and used. _{HPLC water and acetonitrile (CH 3} CN) used in the purification process of natural products and synthetics were purchased from Tedia (Ohio, USA), and all other reagents were used with special grade.

Marine invertebrates Tissue extraction

After each tissue was collected, the extraction process using weak acid was immediately performed. The collected tissue was boiled for 5 minutes by adding 1% acetic acid (v/v) corresponding to 4 times the tissue volume, heated, and then completely cooled while being stored on ice. The cooled tissue was completely crushed using a homogenizer and then centrifuged. The supernatant was taken and stored at -70°C until use for antibacterial activity detection and peptide purification.

Antibacterial activity measurement method and Strain used

E. coli D31 was used in the experiment for purification of the antibacterial peptide, and 3 gram-positive bacteria (B. subtilis, S. epidermidis, S. mutans) and 4 gram-negative bacteria (E. coli D31) were used to measure the antimicrobial activity of analogs. , S. enterica, Shigella sonnei, P aeruginosa) and one yeast (Candida albicans) were used. As a method of measuring antimicrobial activity, an ultrasensitive radial diffusion assay (URDA) method using two layers of medium containing different concentrations was used. The strain used for URDA was first pre-cultured for 18 hours at each medium and culture temperature, and then the bacterial concentration was 84%T (≒ 1 x 10) using a colorimeter (ProductNo.52-1210, BioMerieux, Inc., USA). ⁸ CFU/mL). Then, 0.5 mL of the bacterial solution diluted at each concentration was added to an underlay gel containing 9.5 mL of 0.03% TSB, 1% Type I agarose, and 10 mM phosphate buffer (PB) (pH 6.5), and mixed well, and then plated. Pour flat on and harden. After piercing a well with a diameter of 2.5 mm using a punch in the hardened plate, 5 μL of the purification solution or 5 μL of the peptide solution (1000 ug/mL in 0.01% HAc) was introduced. All samples were confirmed to have no influence by the solvent using 5 μl of 0.01% acetic acid. After the peptide has soaked into the medium, incubate the first for 3 hours, then pour the overlay gel containing 10 mL of 6% TSB, 1% Type I agarose, and 10 mM phosphate buffer (pH 6.5) on top of it, and the same temperature after hardening. Incubated for 18 hours in the secondary. The next day, the size of the clearing zone generated around the well was measured to confirm the antibacterial activity. As a control, piscidin 1 was used as an antibacterial peptide derived from mast cells of a hybrid perch from the United States, which has excellent antibacterial activity but strong hemolytic activity.

Hemolytic activity ( Hemolysis ) Measure

In order to measure the hemolytic activity of human red blood cells, the same amount of phosphate buffered saline (PBS, 50 mM sodium phosphate, 150 mM NaCl, pH 7.4) was added to human blood (blood type B), mixed, and then 8000 xg, The supernatant was removed by centrifugation at 4° C. for 1 minute. After repeating this washing process 3 times, PBS was added to the obtained red blood cells to become 3% hematocrit. In order to measure the hemolytic activity, 90 μL of 3% hematocrit was introduced into an e-tube, and 10 μL of a peptide solution of each concentration was added. Each e-tube was reacted at 37° C. for 1 hour, followed by centrifugation at 13,000 x g at 4° C. for 10 minutes. 70 μL of each of the obtained supernatant was taken, transferred to a 96-well microtiter plate, and the degree of hemoglobin outflow was measured at 542 nm. Triton X-100 (0.1%) was used as an experimental control for hemolysis of 100% red blood cells, and the degree of hemolytic activity was calculated using the following equation.

% Hemolysis = [(Abs 542nm in the peptide solution-Abs 542nm in buffer) / (Abs 542nm in 0.1% Triton X-100-Abs 542nm in buffer)] x 100

Acid - urea PAGE ( AU - PAGE ) And bug-blot ( bug - blot )

Acid-urea PAGE was performed to confirm the distribution and activity of the peptidic material. 20 uL of each extract was mixed with 20 uL of 2x sample buffer in a gel containing acetic acid and urea, and then introduced into the gel, followed by electrophoresis at 150 V for 50 minutes. After electrophoresis, the gel was stained with coomassie brilliant blue (CBB) R-250 dye, and the results were confirmed. In addition, in order to perform bug-blot, after electrophoresis, rinse the gel without performing the staining process, and blot it on an LB plate containing E. coli D31 and blot at 37°C for 3 hours. After the primary culture, the gel was removed, and the resulting clearing zone after secondary culture at 37° C. for 16-18 hours was compared with the results of the stained gel.

Prep - cell Run

Using Bio-rad Prep cell 491 (Bio-rad), a crude purification process of the octopus gill extract was performed by preparative continuous acid-urea PAGE (CAU-PAGE). For the prep-cell run, a gel with a height of 6.8 cm and 5% HAc were used as a buffer, and pre-run was first performed at 40 mA for 90 minutes. After the pre-run process, 10 mL of gill extract was mixed with 2x sample buffer, introduced into the gel, and separated by 30 mA for 7 hours to obtain fractions.

Isolation and purification of antibacterial active substances

The octopus gill extract was separated and purified from the active material using HPLC (YL9100 HPLC system, Youngrin, Korea). That is, the sample was placed on a Capcell-Pak C18 column (4.6 mm×250 mm, Shiseido, Japan) equilibrated with 95% A solvent (0.1% TFA in HPLC grade water) and 5% B solvent (0.1% TFA in HPLC CH3CN). After injection, it was eluted with a concentration gradient of solvent B (5-65%) for 60 minutes. At this time, the flow rate was maintained at 1.0 mL/min, and each separated fraction was measured for antimicrobial activity against E. coil D31 by the URDA method. The active peaks were purified as a single peak after performing the final step under the same conditions.

Trypsin ( Trypsin ) Confirmation of proteinaceous active substances by treatment

In order to confirm whether the active substance present in the octopus gill extract is a proteinaceous substance, the change in antimicrobial activity before and after trypsin treatment was confirmed using the URDA method. 1 μL of trypsin (1 mg/mL) was added to the octopus extract and reacted at 37° C. for 1 hour, and then the activity change was measured by the URDA method, and 1 μL of 0.01% HAc was added to 4 μL as a control. If the activity is lost or decreased due to the decomposition of the active substance after sufficiently reacting by treatment with trypsin, it was determined that the substance involved in the activity would be a protein.

Molecular weight measurement

The molecular weight of the peptide was measured using the MALDI-TOF/TOF 5800 system (AB SCIEX). The matrix used was α-Cyano-4-hydroxycinnamic acid (CHCA) dissolved in a concentration of 5 mg/mL (0.1% TFA/50% acetonitrile). Each peptide was dissolved in 10 uL of 0.1% TFA solvent, mixed with the matrix in 1:1 (v/v) and introduced into the device, and the molecular weight was measured in MS Reflector (Positive) operation mode, and Calmix 2 was used as the standard. I did.

Amino acid sequencing

In order to determine the amino acid sequence of the purified peptides, it was analyzed by Edman digestion method using Procise 491 HT protein sequencer (Applied Biosystems, USA) of Korea Basic Science Institute.

Molecular Weight, pI , homology search And sequence alignment

The molecular weight and pI of the analyzed primary structure were calculated using the Expasy tool (http://expasy.org/tools/). And homology search was confirmed using NCBI BlastP (http://www.ncbi..nlm.gov/BLAST). Alignment of primary sequences with similar substances was performed using the ClustalX program.

Analog design

For analog design, the secondary structure of the purified peptide was predicted using a secondary structure prediction program, and the most ideal site for analog design was selected based on the predicted secondary structure. The structural properties of the portion selected as the parent peptide were confirmed, and analogs were designed by C-terminal amidation and amino acid substitution.

Antibacterial Peptide Synthesis and purification

The designed peptide was synthesized and purified with a purity of 95% or higher from Peptron Co., Ltd. (Daejeon) and supplied. The analogs were synthesized by the F-moc solid phase conjugation method using the peptide synthesizer ASP48S (Peptron, Daejeon Korea), and purified by RP-HPLC using a Shiseido Capcell-Pak C18 column (250 mm × 4.6 mm, 10 μm). _{Purification conditions were carried out with a solvent system of H 2} O-CH ₃ CN containing 0.1% (v/v) trifluoroacetic acid at a flow rate of 1 mL/min at 220 nm under a concentration gradient of 3-40%. Molecular weight measurement of the purified peptide was performed using liquid chromatography/mass spectrometry (LC/MS; HP1100 series; Agilent, Santa Clara, CA, USA). The purified peptides were dissolved in 0.01% acetic acid at a concentration of 1000 ug/mL and used in subsequent experiments.

Bacterial permeability to the inner membrane ( Cytoplasmic membrane permeabilization ) Measure

Bacterial permeability measurement was performed by measuring the activity of β-galactosidase in the cytoplasm of E. coli ML35p using O-nitrophenyl-β-D-galactopyranoside (ONPG), a nonmembrane permeative chromogenic substrate. It was done by doing. The cultured Mid-logarithmic phase of E. coli ML35p was washed with 10 mM sodium phosphate buffer (pH 7.4), and then dissolved in the same buffer containing 1.5 mM ONPG. Thereafter, after adding the peptide to be measured, the degree of hydrolysis of ONPG to O-nitrophenol by β-galactosidase of E. coli ML35p was measured at 405 nm. Piscidin1 and 0.01% acetic acid were used as controls, respectively.

DNA - Binding assay

The DNA-binding assay of the peptide was confirmed as the degree of inhibition of DNA migration by agarose-gel electrophoresis after culturing the DNA and the peptide. To this end, 1 Kb DNA marker, λ-Hind III-digested DNA (50 ng) (Roche, Basel, Switzerland) and each peptide (1 ug) were mixed and reacted at 37° C. for 30 minutes, followed by 0.5 μg/mL ethidium bromide ( EtBr) was confirmed by performing electrophoresis on a 1.0% agarose gel.

Experimental example

< Experimental example 1: antibacterial Peptide Purification and Characterization>

1) Antibacterial activity of gill extract

To confirm the antimicrobial activity of the octopus gill extract and the presence of proteinaceous antibacterial substances, the antibacterial activity of the gill extracts before and after trypsin treatment was measured for E. coil D31 and B. subtilis KCTC1021 using the URDA method (Fig. One). In FIG. 1, Before is the antibacterial activity of the extract before trypsin treatment, and after is the antibacterial activity of the extracts after trypsin treatment. As a result, the gill extract showed strong antimicrobial activity against the two strains, and it was found that this antimicrobial activity was almost lost after treatment with trypsin. These results indicate that the octopus gill extract contains proteinaceous antibacterial substances. Therefore, an experiment for purification of the antibacterial peptide present in the gill extract was performed.

2) Continuous AU-PAGE

The gill extract was introduced into Bio-rad Prep-cell 491 to carry out the conditioning agent process. As a result, the fractionated fraction #37 started to exhibit antimicrobial activity and exhibited activity over a wide range from fraction #103, and particularly, fractions #43 to #63 showed strong activity (Fig. 2A). These results mean that antimicrobial substances having various molecular weights and net-charges exist in the octopus gill extract (FIG. 2B). Fig. 2 is a diagram showing the antimicrobial activity and activity distribution of this Prep-cell pooled # against E. coli D31. 2A shows that each prep-cell# was concentrated and then dissolved in 0.01% HAc, and the antimicrobial activity was measured against E. coli D31 using the URDA method. In addition, Figure 2B is a graph showing the clearing zone diameter obtained as a result of the URDA of each fraction. Fractions introduced into reverse phase HPLC for purification of antimicrobial substances are indicated by red bars.

In addition, for the separation of the antimicrobial active peptide, the active fractions were introduced into acid-urea PAGE to select a fraction containing a low molecular weight substance and to perform a smooth purification process, thereby confirming the molecular weight of each fraction (FIG. 3). As a result, prep-cell fractions #43 to 53 contained a band corresponding to a molecular weight of less than about 6.5 kDa, and those from #57 to 63 were found to have a molecular weight of ~11 kDa or more. 3 is an AU-PAGE of active prep-cell #s of octopus gill extracts, and electrophoresis after mixing 20 uL of prep-cell #33-65(A) and prep-cell #69-97(B) with 2x sample buffer Yeongdong was performed. Human histone H1 (~21 kDa) 1 ug, lysozyme (~11 kDa, 2 ug), aprotinin (~6.5 kDa, 3 ug) and synthetic peptide Cg3 (~1.4 kDa, 1 ug) were used as markers.

3) HPLC purification

#53, the prep-cell active fraction of the octopus gill extract, was introduced into reverse-phase HPLC to perform the purification process of the antimicrobial active peptide (FIG. 4). As a result, it was confirmed that prep-cell #53 contained two active peaks with retention times similar to those of the two peaks whose activity was confirmed in reverse phase HPLC of the gill extract (32% and 34% acetonitrile concentration). , The final purification process of each of the active peaks was performed using the same purification conditions (FIGS. 4B and 4C). As a result, two antimicrobial active substances (designated as Ov1 1 and 2) were finally purified. The two purified antimicrobial substances were subjected to primary structure analysis through molecular weight measurement and edman degradation method.

4) Peptide identification

The molecular weight of the two antimicrobial peptides finally purified by reverse-phase HPLC of #53, the prep-cell active fraction of the octopus gill extract, was measured using the MALDI-TOF/TOF™™ 5800 system (AB SCIEX). As a result of molecular weight measurement, it was confirmed that the molecular weights of the purified peptides 1 and 2 were 1211.29 Da and 2502.24 Da, respectively (FIGS. 5A and B).

Peptide sequencing of the two antimicrobial peptides finally purified from the octopus gill extract was performed using a PROCISE 491 HT protein sequencer (Applied Biosystems, USA). As a result, peptides 1 and 2 were found to be peptides composed of 11 and 24 amino acids, respectively (Fig. 5C, D). In order to confirm the accuracy of the primary structure analysis data, the theoretical molecular weights (1211 Da and 1211 Da) calculated based on the experimental molecular weights (1211.59 Da and 2502.24 Da) of each peptide obtained by measuring the actual molecular weight and the amino acid constituents of each peptide identified by edman degradation. As a result of comparing 2502 Da), it was confirmed that the two molecular weights were exactly the same.

5) Prediction of secondary structure of purified peptides

The secondary structure prediction of the purified peptide was performed using the garnier method of EMBOSS GUI. As a result, Ov 1 was predicted to have a mixed structure in which a β-sheet structure and a turn structure are mixed (FIG. 6A). On the other hand, Ov 2 was found to have a secondary structure in which a β-sheet structure and a turn structure are slightly mixed with α-helix as the main structure (FIG. 6B).

6) Sequence alignment of purified peptides

The homology confirmation of the purified peptides was performed using Genome Net's BLASTP 2.2.10 and TBLASTN 2.2.10. As a result, Ov 1 (SEQ ID NO: 4) showed complete homology with the C-terminal portion of histone H4 common to various organisms, and Ov 2 (SEQ ID NO: 5) was also N- It showed complete homology with the terminal portion (Fig. 7A, B). These results indicate that purified peptides are predicted as fragments derived from histone proteins present in octopus, and that histone proteins are also involved in innate immunity.

< Experimental example 2: Marine invertebrates Octopus-derived antibacterial Peptide Analogue development>

1) Antibacterial activity of purified peptides (Ov 1 & Ov 2)

In order to measure the antibacterial activity of the purified peptides, peptides were synthesized by solid-phase synthesis according to the identified amino acid sequence, and the antimicrobial activity was measured using the synthesized peptide (FIG. 8). As a result, the synthesized peptides showed weak antibacterial activity. This result is probably due to the structural difference between the natural and synthesized forms of the peptides, or the difference between the in vivo and in vitro environments in which they exist. Therefore, in order to improve the activity and increase stability of Ov 1 (SEQ ID NO: 4) and Ov 2 (SEQ ID NO: 5), the primary structure of these peptides is used as a template to select the leading peptide with increased activity with fragmented length. Was designed.

2) Design of analogs derived from Ov 1, 2

Sequence information for analogs designed based on the primary structures of Ov 1 and Ov 2 are shown in Table 1. Analogs of Ov 1 consist of 11 amino acids, and the analogs were designed by substituting other amino acids for Gly or Gln residues in the molecule and amide at the C-terminus. The analogue of Ov 2 is also designed through the amino acid substitution method and C-terminal amideization by preferentially selecting the part that takes the α-helix structure from the secondary structure prediction result of Ov 2, which is the parent. I did. It was also designed for the purpose of fragmenting the length of Ov 2 through the selection of the N-terminal part.

< Experimental example 3: Ov Lesson 1 Ov Antimicrobial activity of analogs of 2>

Analogs of Ov 1 (SEQ ID NO: 4) and Ov 2 (SEQ ID NO: 5) and piscidin 1 (antibacterial peptide derived from hybrid perch from the United States) as a control were used for 7 bacteria and 1 yeast (C. albicans). Antimicrobial activity was measured using the URDA method (Fig. 9, Table 2). As a result of measuring antimicrobial activity, three analogs showed strong antimicrobial activity. In particular, Ov1-1 (SEQ ID NO: 1) and Ov1-2 (SEQ ID NO: 2) showed similar or strong antimicrobial activity to the activity of piscidin 1 for all strains used for the measurement. Interesting results are that piscidin 1 used as a control showed weak activity against C. albicans, but Ov1-1 and Ov1-2 showed strong activity against C. albicans. These results indicate that the weak activity of Ov 1 and Ov 2 can be used as a parent for the development of leading materials showing improved results of fragmentation of length and increase of activity through analog design, and development of new antimicrobial peptides is also possible. It means that.

< Experimental example 4: Ov Lesson 1 Ov Of analogues of 2 Hemolytic activity Measurement>

In order to confirm the toxicity of the analogs, the hemolytic activity of piscidin 1, an antimicrobial peptide used as analogs and a control, against human red blood cells (blood type B) was measured (FIG. 10). As a result, Ov 1-2 showed a little hemolytic activity, but the rest of the analogs hardly showed hemolytic activity against human red blood cells, but piscidin 1 used as a control began to show strong hemolytic activity at a concentration of 12.5 μg/mL. . These results show that, in contrast to piscidin 1, which has strong hemolytic activity, Ov 1-1 (SEQ ID NO: 1), Ov 1-2 (SEQ ID NO: 2) and Ov 2-2 (SEQ ID NO: 3) analogs exhibit significant antimicrobial activity. It indicates that there is no toxicity to human red blood cells at a concentration much higher than the indicated concentration. Therefore, it means that the above three analogs exhibiting strong antimicrobial activity can be used in the development of new antimicrobial peptides.

< Experimental example 5: Cytoplasmic membrane permeabilization Measurement>

In order to confirm the permeability of the Ov 1 and Ov 2 analogs and the antimicrobial peptide piscidin 1 used as a control to the bacteria membrane, the inner membrane permeability to E. coli ML35p was confirmed by measuring the absorbance at 405 nm. As a result of measuring membrane permeability, piscidn 1, an antimicrobial peptide used as a control, showed strong bacterial permeability. Among Ov 1 and Ov 2 analogs, only Ov 2-2 showed strong membrane permeability, and other analogs showed strong membrane permeability. Almost not shown (Fig. 11). These results indicate that Ov 2-2 is a method of directly attacking the bacterial membrane, indicating the possibility of exhibiting antimicrobial activity, and Ov 1-1 and Ov 1-2 exhibit antimicrobial activity through indirect action rather than directly attacking the membrane. It means that there is a possibility to show.

< Experimental example 6: DNA - binding assay >

The binding force of Ov 1 and Ov 2 analogs to DNA was measured using an electrophoretic mobility shift assay (EMSA) (FIG. 12). As a result, piscidin 1 used as a control completely inhibited the electrical migration of DNA, and among analogs, Ov 1-1, Ov 1-2, and Ov 2-2 completely inhibited the electrical migration of DNA. On the other hand, other analogs did not affect the electrical movement of DNA. When comparing the results of this DNA-binding assay with the membrane permeability, it is judged that Ov 1-1 and Ov 1-2 among analogs exhibiting antimicrobial activity show antimicrobial activity by targeting the intracellular target site of bacteria. It is judged that 2 has a mechanism of action that directly attacks the cell membrane. The data on the expected mechanisms of action of these analogs are thought to be useful as important basic data for the development of new peptide lead substances.

<Result>

The present inventors purified two antimicrobial peptides (Ov1, Ov2) from the gills of octopus, and as a result of clarifying the primary structure, the two peptides were the C-terminal portion of histone H4 and the N-terminal portion of histone H2B. It showed complete homology with the distal portion. Both peptides had no hemolytic activity and showed antimicrobial activity against Gram-(+) and (-) bacteria. In addition, six analogues were designed and synthesized by solid-phase synthesis in order to select antimicrobial peptides with improved short-length activity using the two peptides as parent. Analogs were designed by amino acid substitution and amideization of the C-terminus based on the primary structures of Ov1 and Ov2. Various activity measurements were performed to select effective analogues, and as a result, three effective analogues (Ov1-1, Ov1-2, Ov2-2) were selected. Ov 2-2 is predicted to exhibit antimicrobial activity by directly attacking the bacterial membrane, and Ov 1-1 and Ov 1-2 are indirect mechanisms that target the intracellular target site rather than directly attack the membrane. It was predicted to exhibit antibacterial activity.

Finally, through the present invention, two antimicrobial peptides (Ov1 and Ov2) were purified from octopus gills, and analogs of fragments with improved activity were designed and selected using these as parent peptides.

In the above, preferred embodiments of the present invention have been described, but the present invention is not limited thereto, and it is possible to implement various modifications within the scope of the technical spirit of the present invention, and this also belongs to the appended claims. It is natural.

<110> Industry-Academic Cooperation Foundation, Kunsan National University <120> Antimicrobial peptide analogues Ov1-1, Ov1-2 derived from Octopus variabilis and antimicrobial composition containing the same <130> HPC6216-Ov1-1 <160> 5 <170> KopatentIn 2.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptide analogue Ov1-1 <400> 1 Arg Gln Leu Arg Thr Leu Tyr Arg Phe Leu Arg 1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptide analogue Ov1-2 <400> 2 Arg Leu Leu Arg Thr Leu Tyr Arg Phe Leu Arg 1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptide analogue Ov2-2 <400> 3 Leu Ala Lys His Ala Val Lys Lys Ala Leu Lys Ala Val 1 5 10 <210> 4 <211> 11 <212> PRT <213> Octopus variabilis <400> 4 Arg Gln Gly Arg Thr Leu Tyr Gly Phe Gly Gly 1 5 10 <210> 5 <211> 24 <212> PRT <213> Octopus variabilis <400> 5 Leu Pro Gly Glu Leu Ala Lys His Ala Val Ser Glu Gly Thr Lys Ala 1 5 10 15 Val Thr Lys Tyr Thr Ser Ser Lys 20

Claims (10)

An antimicrobial peptide derived from octopus of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.
The method according to claim 1,
Wherein the antimicrobial peptide has an amino group (-NH ₂ ) bonded to the C-terminal thereof.
The method according to claim 1,
The antimicrobial peptide may be selected from the group consisting of Bacillus subtilis , Staphylococcus epidermidis , Streptococcus mutans , Aeromonas hydrophila , E. coli , Is characterized by having an antimicrobial activity against any one or more selected from the group consisting of Salmonella enterica , Shigella sonnei , Pseudomonas aeruginosa , and Candida albicans Octopus-derived antimicrobial peptides.
An antimicrobial peptide derived from an octopus of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.
5. The method of claim 4,
Wherein the antimicrobial peptide has an amino group (-NH ₂ ) bonded at its C-terminal.
5. The method of claim 4,
The antimicrobial peptide may be selected from the group consisting of Bacillus subtilis , Staphylococcus epidermidis , Streptococcus mutans , Aeromonas hydrophila , E. coli , Is characterized by having an antimicrobial activity against any one or more selected from the group consisting of Salmonella enterica , Shigella sonnei , Pseudomonas aeruginosa , and Candida albicans Lt; / RTI &gt;
A pharmaceutical composition for preventing or treating food poisoning, comprising an octopus-derived antimicrobial peptide having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.
A pharmaceutical composition for preventing or treating candidiasis comprising an octopus-derived antimicrobial peptide having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.
A pharmaceutical composition for preventing or treating typhoid fever comprising an octopus-derived antimicrobial peptide having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.
A pharmaceutical composition for preventing or treating cholera comprising an octopus-derived antimicrobial peptide having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

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