KR20180053867A - Antimicrobial Peptides derived from abalone bactericidal??permeability-increasing protein and uses thereof - Google Patents

Abstract

The present invention provides peptides derived from abalone having excellent anti-microbial activity and anti-insect activity. According to another aspect of the present invention, provided is a polynucleotide encoding the anti-microbial peptide.

Description

Antimicrobial Peptides Derived from Increasing Sterile Permeability of Abalones and Uses Thereof < RTI ID = 0.0 > Antimicrobial Peptides < / RTI > derived from abalone bactericidal permeability-

The present invention relates to an antimicrobial peptide, and more particularly, to an antimicrobial peptide derived from a bactericidal permeability increasing protein of an abalone and its use.

An antimicrobial peptide (AMP) is the foremost biological defense factor of the innate immune system that exists in all types of living organisms, from prokaryotes to mammals. Antimicrobial peptides, unlike postmortem immune responses to specific microorganisms or antigens, act largely independent of microbial species and response times are very rapid within a few hours. It is a natural product that is naturally produced to protect the host from microorganisms in a wide range of biological systems without the problem of tolerance or immune problem or rejection problem, which is different from existing antibiotics which always contain the problem of resistant bacteria. (Hancock et al., Curr Drug Targets Infect Disord. Mar ; 2 (1): 79-83, 2002). Recently, a novel AMP has been designed based on the amino acid sequence of a protein having antibacterial or bactericidal activity in genomic / transcriptome database information (He et al., Insect Biochem Mol Biol. (2): 135-46. 2007). In particular, Bactericidal permeability-increasing protein (BPI) is highly homologous to the protein sequence of LBP (LPS-binding protein) known as other LPS binding proteins, but the biological roles of the two proteins are known to be different. LBP BPI is found in the lysosomal granules of polymorphonuclear neutrophils, while it is an inflammatory plasma protein that responds to LPS, has bactericidal properties, neutralizes the toxic effects of LPS . The functional and functional aspects of LBP and BPI are well studied in mammals, and the functional properties of these proteins in non-mammals, including invertebrates, are less well known. In this regard, Korean Patent No. 1384578 discloses a novel antimicrobial peptide derived from abalone and its use.

However, in the case of the prior art described above, it is difficult to expect a uniform effect due to factors such as potential toxicity without showing a sufficient antibacterial activity because the yield of peptides is not high.

It is an object of the present invention to provide an antibacterial peptide derived from abalone having excellent antimicrobial activity and antifungal activity for solving various problems including the above problems. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided an antimicrobial peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

According to another aspect of the present invention, there is provided a polynucleotide encoding said antimicrobial peptide.

According to another aspect of the present invention, there is provided a recombinant vector into which the polynucleotide of the above-mentioned antimicrobial peptide is inserted.

According to another aspect of the present invention, there is provided a transformed host cell obtained by transforming a host cell with the recombinant vector.

According to another aspect of the present invention, there is provided an antimicrobial composition comprising the antibacterial peptide or the transfected host cell as an active ingredient.

According to another aspect of the present invention, there is provided an antimicrobial cosmetic composition comprising the antibacterial peptide or the transfected host cell as an active ingredient.

According to another aspect of the present invention, there is provided an antimicrobial food additive comprising the antibacterial peptide or the transformed host cell as an active ingredient.

According to another aspect of the present invention, there is provided an antibacterial feed additive comprising the antibacterial peptide or the transformed host cell as an active ingredient.

According to one embodiment of the present invention as described above, it is possible to realize an antimicrobial activity against various strains and an antimicrobial peptide-producing effect from abalone showing an antitumor activity against a scuticacil. Of course, the scope of the present invention is not limited by these effects.

Figure 1 is a prediction result of a secondary structure for a part of the HDH-BPI protein sequence according to an embodiment of the present invention.
FIG. 2 is a schematic diagram showing that the N-terminus of a parent peptide and an analogue of HDH-BPI takes an α-helix structure according to an embodiment of the present invention.
FIG. 3 is a photograph showing antimicrobial activity of an HDH-BPI parent peptide and an analogue according to an embodiment of the present invention.
FIG. 4 is a photograph showing the thermal stability of an HDH-BPI parent peptide and an analogue according to an embodiment of the present invention.
FIG. 5 is a photograph showing the salt sensitivity of an HDH-BPI parent peptide and an analogue according to an embodiment of the present invention. FIG.
FIG. 6 is a photograph showing the ability of the HDH-BPI parent peptide and its analogues to inhibit proliferation of scuticacid according to an embodiment of the present invention.
FIG. 7 is a graph showing the inhibition of scuticacid activity by addition of an HDH-BPI parent peptide and an analogue according to an embodiment of the present invention.
FIG. 8 is a photograph showing the DNA-binding ability measurement results of an HDH-BPI parent peptide and an analogue according to an embodiment of the present invention.
FIG. 9 is a photograph showing the binding ability of an HDH-BPI parent peptide and an analogue to a DNA polymerase according to an embodiment of the present invention.
10 is a photograph showing the antimicrobial activity of the HDH-BPI maternal peptide against ampicillin-resistant antibiotics according to an embodiment of the present invention.

Definition of Terms:

As used herein, the term " Bactericidal permeability-increasing protein (BPI) "is a type of protein present in human neutrophils, which is a growth inhibitory effect against gram-negative bacteria and a neutralizing effect on exotoxins produced by Gram- Effect.

As used herein, the term "abalone" refers to a large, elliptical mollusk gastropod with a shell length of at least 10 cm. It is inhabited by an apparent island of 5 to 50 m in depth.

DETAILED DESCRIPTION OF THE INVENTION [

According to one aspect of the present invention, there is provided an antimicrobial peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

In the antimicrobial peptide, the tenth amino acid is a basic amino acid selected from the group consisting of lysine (K), arginine (R) and histidine (H), serine (S), threonine (T), asparagine Glutamine (Q), or a polar amino acid selected from the group consisting of glutamine (Q).

In the antimicrobial peptide, it may be composed of the amino acid sequence represented by SEQ ID NO: 2 or 3, and the C-terminal thereof may be amidated.

According to another aspect of the present invention, there is provided a polynucleotide encoding said antimicrobial peptide.

According to another aspect of the present invention, there is provided a recombinant vector into which the polynucleotide of the above-mentioned antimicrobial peptide is inserted.

In the recombinant vector, the polynucleotide may be an expression vector operably linked to a promoter.

According to another aspect of the present invention, there is provided a transformed host cell obtained by transforming a host cell with the recombinant vector.

In the transfected host cell, the host cell may be a probiotic strain.

According to another aspect of the present invention, there is provided an antimicrobial composition comprising the antibacterial peptide or the transfected host cell as an active ingredient.

According to another aspect of the present invention, there is provided a cosmetic composition comprising the antibacterial peptide or the transformed host cell as an active ingredient.

According to another aspect of the present invention, there is provided an antimicrobial food additive comprising the antibacterial peptide or the transformed host cell as an active ingredient.

According to another aspect of the present invention, there is provided an antibacterial feed additive comprising the antibacterial peptide or the transformed host cell as an active ingredient.

Functional and functional aspects of LPS-binding protein (LBP) and bactericidal permeability enhancing protein (BPI), which generally have antimicrobial activity, are well studied in mammals, such as those found in non-mammals, including lower vertebrates and invertebrates The functional properties are not well known. LBP and BPI are composed of N- and C-terminal domains and can be cleaved into nearly two identical fragments with approximately 200 amino acids, but each sequence is slightly different. The N-terminal domain in LBP binds to LPS, whereas the C-terminal domain is required to carry LPS to CD14, an important component of LPS-signaling (Fenton and Golenbock, 1998). The antibacterial and LPS neutralizing activity of BPI is completely expressed by the N-terminal domain. The recombinant 23-kDa N-terminal fragment of BPI has equal or greater activity than the holoprotein in the bactericidal and LPS binding assays. Additionally, three cleavage fragments and synthetic peptides designed to overlap the peptide 23-kDa N-terminal fragment of BPI were each assayed for biological activity and only one synthetic peptide (amino acids 85-99) had bactericidal activity Respectively. However, the function of the C-terminal portion has not yet been clarified (Iovine et al., J Cell Biol. May 19; 137 (4): 797-811. Accordingly, the present inventors have designed a series of peptides (HDH-BPI peptide analogs) based on the amino acid sequence of the C-terminal domain of the bacterial permeability increasing protein of Haliotis discus hannai to develop a novel antimicrobial peptide, By confirming that the peptide has antimicrobial activity against gram-positive and gram-negative bacteria, the antimicrobial peptide of the present invention, which can be used as a natural antibiotic, has been completed.

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

Example 1: Reagents and Materials

Tryptic soy broth (TSB) and agarose type I (Low EEO Agar) for measurement of antimicrobial activity used in the present invention were purchased from Merck (Merk, Darmstadt, Germany) and Sigma (St. Louis, USA), and all the reagents except the reagents were used for the Exp.

Example 2: Design of antimicrobial peptide

In order to develop a new antimicrobial peptide according to an embodiment of the present invention, a bactericidal penetration enhancing protein (BPI) which is an antibacterial protein of Haliotis discus hannai (HDH) was cloned and a bactericidal permeability increasing protein (BPI) sequence Based on the amino acid sequence of the α-helix structural fragment located at the C-terminal of HDH-BPI, the secondary structure prediction program of the HDH-BPI analog was predicted based on (HDSC002806) (FIG. 1) . Based on the predicted secondary structure, the most ideal site for analogue design was selected and the structural characteristics of the portion selected as the parent peptide (HDH-BPI-2N, SEQ ID NO: 2) were confirmed and the amidation of the C- And an amino acid substitution method (HDH-BPI-2A, SEQ ID NO: 3) (FIG. 2). Information on the designed peptides is shown in Table 1 below.

Peptides
designation Amino acid sequence Length
(mer) MW
(Da) pI Majesty Boman Index
(kcal / mol) SEQ ID NO: HDH-BPI-2N MALQLVMNGALKL-NH ₂ 13 mer 1401.8 9.9 +1 -1.17 2 HDH-BPI-2A MALQLVMNKALKL-NH ₂ 13 mer 1472.9 10.55 +2 -0.67 3

Example 3: Peptide synthesis and purification

According to one embodiment of the present invention, the peptide designed in Example 2 was designed with Peptron Inc. (Daejeon, Korea) at a purity of 95% and synthesized commercially. Specifically, the peptides were synthesized using the FmoC solid phase peptide synthesis (SPPS) method of ASP48S (Peptron Inc. Taejon, Korea) and analyzed using a Vydac Everest C18 column (250 mm × 22 mm, 10 μm; Grace, Deerfield, IL , USA) using reverse phase high performance liquid chromatography (HPLC). This was then eluted with a water-acetonitrile linear gradient (acetonitrile 3% -40% (v / v)) containing 0.1% (v / v) trifluoroacetic acid. The molecular weight of the purified peptide was confirmed using a liquid chromatography / mass spectrometer (HP100 series; Agilent, Santa Clara, Calif., USA), and all of the synthesized peptides were dissolved in 0.01% mL of a storage solution.

Example 4: Ultrasensitive Radial Diffusion Assay

Antibacterial activities of the purified HDH-BPI-2N and HDH-BPI-2A peptides were measured according to one embodiment of the present invention.

Specifically, a highly sensitive spin diffusion analysis for the measurement of antimicrobial activity was carried out according to a conventional method (Seo et al ., Biochem. Biophys. Res. Commun., 338: 1998-2004, 2005). Bacillus cereus , Staphylococcus aureus RM4220 , Streptococcus iniae Gram-positive bacteria including FP5229 and S. mutans ; Gram-negative bacteria including Pseudomonas aeruginosa KCTC204 , Vibrio anguillarum , Vibrio harveyi ; And yeast Candida albicans KCTC7965 . The strains were cultured in brain heart-leavening medium (BHI, BD Bioscience, USA) at a suitable temperature ( P. aeruginosa and S. iniae at 25 ° C and the rest of the strain at 37 ° C), and the yeast strain, C. albicans KCTC7965 And cultured in yeast medium (YM) at 25 ° C. The suspensions of the strains and yeast were then stored at ~ 10 ⁶ CFU / mL, McFarland turbidity standard of 0.5, Vitek Colorimeter # 52-1210, Hach, Loveland, CO, USA corresponding to C. albicans ) And 1/2 mL of the diluted germ or C. albicans suspension was resuspended in 10 mM phosphate buffer (PB; pH 6.6; pH 7.0) containing 0.03% TSB or 0.03% SDB and 1% Type I (low EEO) agarose Was added to 9.5 mL of underlay gel containing 5 x 10 < ⁶ > CFU / mL or 5 x 10 < ⁴ > The purified peptides were then serially diluted in duplicate in 5 [mu] l of acidified (0.01% HAc) and each dilution was added to a 2.5-mm diameter well made in 1-mm thick underlay gel. After incubation for 3 hours at 25 ° C. and 37 ° C. for 3 hours, P. aeruginosa , S. iniae , and C. albicans were incubated with 10 mM of phosphate buffered saline (PBS, pH 6.6) Or with 10 ml of double-strength overlay gel containing 6% YM in a 1% agarose suspension or yeast suspension. Plates were then added to incubate for a further 18-24 hours and the diameter of the clearing zone was measured and the diameter of the clear zone was expressed as units (0.1 mm = 1 U) after excluding the diameter of the wells . In addition, the minimum effective concentration of HDH-BPI-derived mutants was calculated as the minimal effective concentration (MEC, 占 퐂 / ml) of the synthetic peptide as the x-section of the unit plot against the log10 value of the peptide concentration, The results were averaged.

As a result, as shown in FIG. 3, HDH-BPI-2N and HDH-BPI-2A peptides showed strong antimicrobial activity against gram positive bacteria, gram negative bacteria and yeast.

Example 5: Effect of temperature and salt on antibacterial activity

In order to observe the antimicrobial activity of HDH-BPI-2N and HDH-BPI-2A peptides according to temperature and salt effects according to one embodiment of the present invention, strains B. cereus , E. coli , C. albicans was subjected to the same high-sensitivity radiation diffusion analysis (URDA) as in Example 4. [ In order to observe the thermal stability of HDH-BPI-2N and HDH-BPI-2A peptides, HDH-BPI-2N and HDH-BPI-2A were heat treated at 100 ℃ for 10 min. The peptides were treated with different concentrations of salts (0.5, 1 and 2%) and incubated for 30 min. The treated peptides were used for post-cooling analysis.

As shown in FIG. 4, the HDH-BPI-2N and HDH-BPI-2A peptides had thermal stability. However, as shown in FIG. 5, when the salt concentration was increased, The antimicrobial activity against some strains (such as C. albicans and S. mutans ) was confirmed to be lowered, and the salt tolerance was found to be somewhat lower.

Example 6: Confirming the activity of Hansucutika

In accordance with one embodiment of the present invention, the antimicrobial activity of the HDH-LGBP mutant was confirmed for the Philatelic acid dicentrarchi .

Specifically, for the in vitro antimicrobial activity of the anthelmintic insecticidal strain, Sukutika sacrificed from the flounder infected with the Sukutika infestation was used and the embryo cell line of the flounder (HINAE cell line ) Was used. HINAE cells are treated with 10% FBS and 1% penicillin / streptomycin (penicillin / streptomycin) in L15 culture medium was cultured at 20 ℃ the Surgical urticae charge for Surgical urticae charge proliferation experiments in 96-well plate 1 × 10 ⁴ / mL, and HDH-LGBP mutants were treated at 1, 5, 10, 25, and 50 ug / mL for 4 hours, respectively. Cells were observed under an optical microscope. In order to measure the activity of live Sutuca pupa, 10ul of WST-1 assay reagent was added at 24 hours after HDH-LGBP mutant treatment, and absorbance was measured at 450nm after 1 hour.

As a result, as shown in Figs. 6 and 7, the HDH-BPI-2A peptide showed an antinociceptive effect inhibiting the proliferation of the scutica.

Example 7: DNA-binding assay

DNA binding assays were conducted to investigate the interaction of HDH-BPI-2N and HDH-BPI-2A peptides prepared according to an example of the present invention with the intracellular molecules of microorganisms.

Specifically, DNA binding analysis was performed by measuring the inhibition of the rate of migration of the DNA band passing through the agarose gel by partially modifying the conventional method (Nam et al ., Marine Drugs, 5240-5257, 2014) (50 ng; Roche, Basel, Switzerland) was mixed with various concentrations of peptides (0, 0.157, 0.313, 0.625, 1.25, and 2.5 μg) dissolved in 0.01% acetic acid, After incubation at room temperature for 5 minutes, the cells were electrophoresed on a 1.0% agarose gel containing 0.5 μg / ml of ethidium bromide (EtBr).

As a result, HDH-BPI-2N and HDH-BPI-2A showed DNA binding ability as shown in Fig.

Example 8: DNA polymerase inhibition assay

PCR amplification was performed in order to evaluate inhibition of DNA polymerase activity of HDH-BPI-2N and HDH-BPI-2A peptides prepared according to one embodiment of the present invention.

Specifically, after adding the HDH-BPI analogs of various concentrations (2.5, 1.25, 0.625, 0.313 ㎍ / ㎖) to each reaction mixture E. coli gen mix DNA forward primer for the (genomic DNA, 100ng) (SEQ ID NO: 4) and a reverse primer (SEQ ID NO: 5). The PCR conditions consisted of 30 cycles of 30 sec at 90 ° C, 30 sec at 55 ° C, and 1 min at 72 ° C. After PCR amplification, all amplicons were electrophoresed on 1.5% agarose gel containing EtBr. The information on the primers used for the PCR amplification is shown in Table 2 below.

As a result, as shown in Fig. 9, HDH-BPI-2N and HDH-BPI-2A showed DNA polymerase inhibitory effect.

primer The nucleic acid sequence (5 '-> 3') SEQ ID NO: 16S-F1 CTCCTACGGGAGGCAGCAG 4 16S-R3 CCAGGGTATCTAATCCTG 5

Example 9: Identification of antimicrobial activity against antibiotic resistant bacteria

To confirm the antimicrobial activity against the antibiotic-resistant bacteria according to one embodiment of the present invention, five antibiotic-resistant strains ( Escherichia coli CCARM0238, Enterobacter cloacae CCARM 0252, Pseudomonas aeruginosa CCARM 0225, Klebsiella oxytoca CCARM 0248, Staphylococcus aureus CCARM 0203) were subjected to high sensitivity radiometric diffusion analysis. The antimicrobial activity of 5 ㎍ of HDH-BPI-2N was compared with 0.01% acetic acid as a negative control and 0.5 ㎍ and 5 ㎍ of ampicillin antibiotics as a positive control.

As a result, as shown in Fig. 10, in the wells to which 0.5 μg and 5 μg of ampicillin had been added, strains having resistance to ampicillin were proliferated. However, the growth of all test strains was inhibited around the wells to which HDH-BPI-2N peptide was added .

In conclusion, the peptides (HDH-BPI peptide analogs) derived from the Haliotis discus hannai sterilization permeability increasing protein of the present invention have strong leaking ability and membrane disturbance ability, are thermostable and exhibit binding ability to DNA or DNA polymerase Gram-positive, Gram-negative bacteria, fish pathogens and yeast. Therefore, it can be used as a candidate substance for the development of alternative antibiotics substitutes for existing antibiotics or development of anti-inflammatory drugs.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> Republic of Korea represented by National Fisheries Research & Development Institute <120> Antimicrobial peptides derived from abalone          bactericidal? permeability-increasing protein and uses thereof <130> PD16-5434 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial Peptide <400> 1 Met Ala Leu Gln Leu Val Met Asn Xaa Ala Leu Lys Leu   1 5 10 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> HDH-BPI-2N <400> 2 Met Ala Leu Gln Leu Val Met Asn Gly Ala Leu Lys Leu   1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> HDH-BPI-2A <400> 3 Met Ala Leu Gln Leu Val Met Asn Lys Ala Leu Lys Leu   1 5 10 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 16S-F1 <400> 4 ctcctacggg aggcagcag 19 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 16S-R3 <400> 5 ccagggtatc taatcctg 18

Claims (14)

An antimicrobial peptide consisting of the amino acid sequence represented by SEQ ID NO: 1. The method according to claim 1,
The tenth amino acid is composed of a basic amino acid selected from the group consisting of lysine (K), arginine (R) and histidine (H), serine (S), threonine (T), asparagine (N) and glutamine Or a polar amino acid or glycine (G) selected from the group consisting of the antimicrobial peptide. The method according to claim 1,
An antimicrobial peptide consisting of an amino acid sequence represented by SEQ ID NO: 2 or 3; The method according to claim 1,
An antimicrobial peptide wherein the C-terminus is amidated. A polynucleotide encoding the antimicrobial peptide of claim 1. A recombinant vector into which the polynucleotide of claim 5 is inserted. The method according to claim 6,
Wherein the polynucleotide is an expression vector operably linked to a promoter. A transformed host cell transformed with the recombinant vector of claim 6. A transformed host cell obtained by transforming a host cell with the recombinant vector of claim 7. 10. The method of claim 9,
Wherein the host cell is a probiotic strain. An antimicrobial composition comprising the antimicrobial peptide of any one of claims 1 to 4 or the transformed host cell of claim 10 as an active ingredient. An antimicrobial cosmetic composition comprising the antimicrobial peptide of any one of claims 1 to 4 or the transformed host cell of claim 10 as an active ingredient. An antimicrobial food additive comprising the antimicrobial peptide of any one of claims 1 to 4 or the transformed host cell of claim 10 as an active ingredient. An antibacterial feed additive comprising the antibacterial peptide of any one of claims 1 to 4 or the transformed host cell of claim 10 as an active ingredient.

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