KR20170114319A - Novel antimicrobial peptide isolated from Python bivittatus and screening method thereof - Google Patents

Abstract

The present invention relates to a novel antimicrobial peptide isolated from python and a method for its discovery. The antimicrobial peptide or its analogue sequence is extracted from a python by comparing with an existing antimicrobial peptide sequence, And a novel antimicrobial peptide as a candidate for use as a new antibiotic substance.
According to the present invention, peptides having a high similarity to katelicidin in the genome of python are extracted, and then an amino acid containing a domain that can be judged to exhibit antibacterial activity is extracted and synthesized to obtain a novel It is effective to present an antimicrobial peptide.

Description

(Novel antimicrobial peptide isolated from Python bivittatus and screening method)

The present invention relates to a novel antimicrobial peptide isolated from python and a method for its discovery. More particularly, the present invention relates to a novel antimicrobial peptide selected based on comparison of similarities among protein sequences derived from python based on known protein sequence information of reptiles And the method of discovery thereof.

Antimicrobial peptides (AMPs) are known to be part of the innate immune system used by most living organisms to protect themselves from external hazards such as infections. These insects and some microorganisms, including plants and animals, are widely available throughout life. In the 1960s, their presence in frog skin mucus was first recognized. Today, thousands of antimicrobial peptides are found in various species, .

Since its first discovery, cationic antimicrobial peptides such as bombinin and melittin, which are composed of 24 amino acids, have been found in amphibians and bees, respectively, and their structure and mechanisms have been studied in earnest It began to progress. To date, more than 5,500 antimicrobial peptides are known to exist, including 3900 naturally occurring and 1600 synthesized peptides from various species including bacteria, invertebrates, vertebrates and plants. Among them, antimicrobial, antiviral, Antibacterial, antiparasitic, anti-cancer effects associated with each of 5300, 1100, 1500, 10, 100 peptides were found.

The antimicrobial peptide has a broad action range that is active in some cancer cells including microorganisms such as bacteria, fungi and viruses, and it is known that the antimicrobial effect is immediate due to the unique action mechanism. In addition to its excellent antibiotic action, it has been shown that it plays an important role in the functional regulation of the body, such as wound healing and angiogenesis, as well as the role of intermediates connecting innate immune and post-immune immunity, and its relationship with autoimmune diseases and inflammation is also studied have.

Biological, biochemical, and structural studies using key antimicrobial peptides have shown that bases and amino acids differ in sequence and length depending on species and peptide types, but they generally have a positive charge, . ≪ / RTI >

Antimicrobial peptides are classified into several types according to their structural characteristics. Among them, cathelicidin and defensin family are representative examples. The cathelicidin family refers to antimicrobial peptides with various characteristics sharing the cathelin domain, and its presence has been confirmed especially in various vertebrates. Diphenes is an important antimicrobial peptide that is found widely in plants and animals and has a long evolutionary origin. It is characterized by a bond between cysteines, which plays a role in maintaining the stability of the peptide.

Antimicrobial peptides usually consist of about 20 amino acids, many of which are composed of amino acids with positive charges such as lysine, arginine, and histidine. The cationicity of positively charged amino acids plays an important role in the interaction between the outer membrane of the bacteria and the cytoplasmic membrane. In addition, hydrophobicity is also important because the hydrophobic helix core of the peptide can be attached to the membrane lipid acyl chains by hydrophobic interaction to be.

In other words, amphipathicity, which simultaneously possesses the cationic and hydrophobic properties of the peptide, plays an important role in the initial interaction between the peptide and the bacterial cell membrane. In particular, Gram-negative bacteria have LPS (lipopolysaccharide) on the cell wall, so cationic peptides are stuck to increase the permeability of the membrane, which is known to have an antimicrobial action. In the case of a modified form in which the hydrophobic part of the peptide is removed, .

A typical hypothesis for the mechanism of antibacterial action is barrel-stave, toroidal pore, carpet model. The positively charged portion of the antimicrobial peptide binds to the cell membrane of a negative charge and the hydrophobic portion of the peptide interacts with the head group (hydrophobic portion) of the cell membrane phospholipid 2 It is known to break cells by entering into the middle membrane and forming pores, changing membrane permeability and membrane fluidity.

The specificity of this activity for prokaryotic cells, rather than eukaryotic cells, is due to differences in constituents of the cell membrane. The cell membrane of the bacteria is located on the surface of the lipid phospholipid, which is in contact with the external environment, while in the case of plant and animal cells, the head also contacts the external environment and cytoplasm. Because of this, the net charge on the surface of cell membranes of prokaryotic and eukaryotic cells is different, and the affinity for the antimicrobial peptide is different. In eukaryotic cells, cholesterol is present in the cell membrane to stabilize the phospholipid bilayer, And the activity thereof is inhibited.

Alternative approaches to overcome resistance to existing antibiotics through these specific mechanisms have been studied and in fact, it has been shown that five types of cathelicidin exhibit strong activity against antibiotic resistant strains isolated from patients infected with pathogenic microorganisms Respectively. In the case of indolicidin, CAMA [cecropin (1-7) -melittin A (2-9) amide] and nisin in combination with existing antibiotics in vitro, methicillin-resistant Staphylococcus aureus) has been reported to exhibit synergistic synergistic effects.

Conventional antibiotics chemically inhibit the synthesis of bacterial cell membrane components such as peptidoglycan precursor lipids I and II and the activity of cells. Bacteria, however, are either 1) making enzymes that degrade antibiotics, 2) altering the biochemical structure and composition of antibiotic-acting subjects, making them no longer active, or 3) releasing antibiotics And 4) to develop a mechanism of bypassing the existing metabolic pathway, which is affected by antibiotics, so that it can be tolerated against antibiotics. On the other hand, antimicrobial peptides have a big difference in that they have antimicrobial action by a physical method, not a chemical action, as described above.

Efforts to discover new antimicrobial peptides are still ongoing, and as information on the genomes of animals living in diverse environments is revealed, more information is being sought. In recent evolution, an analysis of the genomes of animals such as Burmese python (Python bivittatus), which have a wide range of external and physiological adaptations such as loss of limbs, small lungs, body and organ kidneys As a result, there is a growing opportunity to find information on antimicrobial peptides that have evolved in humans and in different environments. In order to utilize this, it is necessary to study antimicrobial peptides that have already been discovered and antimicrobial peptides using new species of dielectrics.

On the other hand, the use of genomic, proteomic, functional information databases, chemo-informatics, and bioinformatics tools in terms of methodology has led to the discovery of antimicrobial drugs research has become increasingly important. These computational approaches can be applied to molecular simulation, molecular docking, structural class prediction, quantitative structure-activity relationships, ) Can be utilized.

As a related related art, Korean Patent No. 10-1444281 (Apr. 18, 2014) discloses a new method for detecting a foreign gene using a SVM (Support Vector Machine) algorithm based on genetic information on Wangzine transcripts We have presented the scolopendrazine Ⅰ peptide.

An object of the present invention is to provide a novel antimicrobial peptide as a candidate for use as a new antibiotic substance by discovering an antimicrobial peptide-related or similar sequence in python through comparison with an existing antimicrobial peptide sequence, .

In order to achieve the above object, the present invention provides a novel antimicrobial peptide comprising the amino acid sequence of any one of SEQ ID NOS: 7 to 9, wherein the amino acid sequence may be isolated from pythons.

The present invention also provides a method for detecting an antimicrobial peptide comprising the steps of: a) selecting antimicrobial peptide sequence information from a database having protein sequence information of reptiles; b) selecting a python-derived protein sequence highly similar to the selected antimicrobial peptide in a); And c) selectively selecting an amino acid sequence comprising an antimicrobial activity domain in the protein sequence selected through comparison of similarity in b) above.

The protein sequence selected in b) above is selected from the group consisting of the cathelicidin family, the crotamine-myotoxin family, the flavin monoamine oxidizing family FIG1 subfamily, the glycosyl hydrolase family 22, the phospholipase A2 family- -49 sub-subfamily, the phospholipase A2 family-the group II subfamily-D49 sub-sub family, the phospholipase A2 family-the group II subfamily-K49 sub-subfamily, the snake- Or at least one protein family selected from the [M12B] family-P-III subfamily-P-IIIa sub-subfamily.

The protein sequence selected in b) above may have an e value of 0.001 or less as a result of BLAST of a reptile-derived antimicrobial peptide sequence.

The antimicrobial activity domain of c) is located at the C-terminal and can exhibit an a-helix structure upon formation of the secondary structure.

According to the present invention, an amino acid having a domain which can be judged to exhibit antimicrobial activity can be extracted and synthesized by extracting peptides highly similar to the known antimicrobial peptide katerlicidin in the genome of python, There is an effect of presenting a novel antimicrobial peptide exhibiting antibacterial activity.

FIG. 1 shows the result of analysis of the entire sequence of GBP01, a candidate antimicrobial peptide, using the AMPA database according to Example 3. The results are shown in (A) AI graph of the average value of each amino acid to the control sequence, (B) PV and the position where the antimicrobial activity domain is presumed to exist, and (C) the location of the estimated antimicrobial activity domain.
FIG. 2 is a result of a secondary structure analysis for selecting an amino acid sequence deduced as an antimicrobial activity domain from the peptide GBP01 according to Example 3. FIG.
Figure 3 shows the amino acid composition of a given peptide labeled with (A) the hydrophobic amino acid (red) and proline and glycine (blue), and (B) (Red, underline) on the surface.
FIG. 4 is a result of analyzing the similarity with antimicrobial peptides already known for the peptide GBP01 selected according to Example 3. FIG.
5 is a graph showing antimicrobial activity of antimicrobial peptides against (A) Escherichia coli and (B) P. aeruginosa.

Hereinafter, the present invention will be described in detail.

In order to achieve the above object, the present invention provides a novel antimicrobial peptide comprising the amino acid sequence as set forth in any one of SEQ ID NOS: 7 to 9, wherein the amino acid sequence may be isolated from python bivittatus. In addition, the amino acid sequence is located at the C-terminal and can exhibit an a-helix structure during the formation of the secondary structure.

The antimicrobial peptide may exhibit an antimicrobial activity against Gram-negative bacteria. The Gram-negative bacteria may be Escherichia coli (E. coli) ATCC 25922 or Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853.

The present invention also provides a method for detecting an antimicrobial peptide comprising the steps of: a) selecting antimicrobial peptide sequence information from a database having protein sequence information of reptiles; b) selecting a protein sequence derived from Python bivittatus which is highly similar to the antimicrobial peptide selected in a) above; And c) selectively selecting an amino acid sequence comprising an antimicrobial activity domain in the protein sequence selected through comparison of similarity in b) above.

The protein sequence selected in b) above is selected from the group consisting of the cathelicidin family, the crotamine-myotoxin family, the Flavin monoamine oxidase family-FIG 1 subfamily, , Glycosyl hydrolase 22 family, Phospholipase A2 Family-Group I subfamily-D49 sub-subfamily, Phospholipase A2 family-Group I subfamily-D49 sub-subfamily, Family II subfamily, Group II subfamily, Group II subfamily, Group II subfamily, Group II subfamily, Group II subfamily-K49 sub-subfamily (Phospholipase A2 family-Group II subfamily-K49 sub-subfamily, Snake waprin family, and poison metalloproteinase [M12B] family-P-III sub-subfamily (Venom metalloproteinase [M12B] family- P-III subfamily y-P-IIIa sub-subfamily).

The protein sequence selected in b) above is characterized in that the e value of the reptile-derived antimicrobial peptide sequence is 0.001 or less as a result of BLAST.

The antimicrobial activity domain of c) can be searched for its position, AI and sequence information using AMPA. The AMPA (an automated web server for prediction of protein antimicrobial regions, http://tcoffee.crg.cat/apps / ampa / do) is an antimicrobial peptide database based on a web server built in 2011. Based on the results of 50% half maximal inhibitory concentration (IC50) of variants in which all amino acids constituting the antimicrobial peptide bactenecin were released in 2005, The bactericidal propensity values (PV) for the effect on the activity were calculated. Therefore, the smaller the PV, the higher the antimicrobial activity, and the amino acids that the antimicrobial peptides have in common have a relatively smaller PV.

For example, amino acids such as positively charged amino acids arginine and lysine, hydrophobic tryptophan and tyrosine have small PVs, while negatively charged amino acids have large PVs. Using AMPA, the PV of the amino acids in a given protein sequence can be applied to search for an antimicrobial domain or a portion expected to have a pattern.

The antimicrobial activity domain of c) is located at the C-terminus and is characterized by exhibiting an a-helix structure during the formation of the secondary structure.

In the above or in one embodiment, the antimicrobial peptide of the present invention has amphipathicity and may exhibit positive charge.

In one or more embodiments, the antimicrobial peptide of the present invention may exhibit an antimicrobial activity against Gram-negative bacteria. The Gram-negative bacteria may be Escherichia coli (E. coli) ATCC 25922 or Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853.

In the above or in one embodiment, there are a liquid-phase method and a solid-phase method as chemical methods for synthesizing the antimicrobial peptide of the present invention. Initially, the liquid phase process has been used predominantly after the solid phase process has been introduced, except for the case where mass production is required for industrial purposes.

Solid phase peptide synthesis (SPPS) can initiate synthesis by attaching functional units called linkers to small porous beads to induce peptide chains to continue. Unlike the liquid phase method, the peptides covalently bind to the beads and prevent them from being removed by filtration until they are cleaved by certain reagents such as TFA (trifluoroacetic acid). The protection process in which the N-terminal amine of the peptide attached to the solid phase binds to the N-protected amino acid unit, the deprotection process, the re-exposed amine group and the new amine group Synthesis is carried out by repeating a cycle (deprotection-wash-coupling-wash) in which an amino acid binds.

The SPPS method can be performed using a microwave technique. Microwave techniques can reduce the time required for coupling and deprotection of each cycle by applying heat in the course of peptide synthesis. The thermal energy can prevent aggregation of the extended peptide chains by folding and aggregation and promote chemical bonding.

The synthesized peptides can be analyzed by mass spectrometry (MS), mass spectrometry (QC), quality control (QC) such as purity measurement by RP-HPLC, solubility, (GeneScript, Piscataway Township, NJ, USA) after final quality assurance (QA) procedures to perform additional tests.

In order to measure the antimicrobial activity of the synthesized antimicrobial peptide, the bacteria to be tested are preliminarily cultured on a culture dish, and a certain amount of antibiotic substance is dropped on a certain area. After culturing, the diameter of the plaque is measured to confirm the activity level A disc diffusion method in which a concentration of antibiotics at different concentrations is added to a certain cell concentration and cultured in a culture dish to determine the concentration at the time point when the number is first decreased by counting the number of colonies, Select one of the colorimetric methods that use a colorimetric indicator by generating an aqueous colorimetric indicator as the electron mediator in the colored reagent is oxidized by the agar dilution method and the metabolism of the living microorganism .

The disk diffusion method and the agar dilution method have disadvantages in that the incubation time takes more than one day, so that the colorimetric method can be preferably selected.

Hereinafter, the present invention will be described in more detail through examples (and test examples). It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1 Selection of Antibacterial Peptides Using a Peptide Database

Amino acids and nucleic acid sequences, previously known as antimicrobial peptides, were collected to identify new antimicrobial peptides in the python genome. Based on information from UniProt's UniProtKB / Swiss-Prot (manually annotated knowledgebase record) and a database of antimicrobial peptides (Table 1). The amino acid sequences of 94 antimicrobial peptides derived from 47 reptiles whose genetic distances are close to each other and which are presumed to have relatively high peptide similarity have been searched and stored in a local server. These proteins consisted of 10 protein families as shown below.

The beta-defensin family, the cathelicidin family, the crotamine-myotoxin family, the flavin monoamine oxidase family-FIG1 subfamily, FIG. 1 subfamily, Glycosyl hydrolase 22 family, Phospholipase A2 family-D49 sub-subfamily, Phospholipase A2 family-Group I subfamily-D49 sub-subfamily, The phospholipase A2 family, the Group II subfamily-D49 sub-subfamily, the phospholipase A2 family-group II subfamily-K49 sub-subfamily (Phospholipase A2 family -Group II subfamily-K49 sub-subfamily, the Snake waprin family and the poison metalloproteinase [M12B] family-P-III subfamily-Venom metalloproteinase [M12B ] family-P-III subf amily-P-IIIa sub-subfamily).

The keywords used in the search using UniProtKB / Swiss-Prot were "antimicrobial peptides" and a total of 2,488 results were searched. After the search, the AMP (antimicrobial peptide) amino acid sequence of the reptile was directly isolated based on the species. About 5,500 AMP amino acid sequences included in the AMP database of Table 1 were also isolated on the same basis. The separated AMP amino acid sequences were arranged in FASTA format (format) for easy use in experiments.

Database Website Contents UniProt http://www.uniprot.org/ genel peptide database APD3 http://aps.unmc.edu/AP/main.php nature AMPs CAMP _R3 http://www.camp.bicnirrh.res.in/index.php AMPs with family classification AMPER http://marray.cmdr.ubc.ca/cgi-bin/amp.pl clustered mature and pro-AMPs PhytAMP http://phytamp.pfba-lab-tun.org/main.php plant AMPs BACTIBASE http://bactibase.pfba-lab-tun.org/main.php bacterial AMPs LAMP http://biotechlab.fudan.edu.cn/database/lamp/ general AMPs DADP http://split4.pmfst.hr/dadp/ Anuran AMPs Defensins http://defensins.bii.a-i.star.edu.sg/ defensins Peptaibol http://peptaibol.cryst.bbk.ac.uk/home.shtml fungal AMPs

Example 2. Detection of AMP-Associated Peptides Using Python Genome Sequence Information

In order to find python-derived peptide sequences with amino acid sequence similar to a total of 94 AMPs, the basic local alignment search tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi) Redundant protein sequence database of "python bivittatus" (taxid: 176946) and 94 reptilian AMPs using the tblastn algorithm, which is used to search for gene sequences from protein sequences. Respectively. In the case of a similar sequence, the result of alignment between each query and python sequence is confirmed based on the similarity through blastp (Max score, Total score, Query cover, E-value, Identity) .

As a result, candidate proteins based on a total of 96 python genomes similar to 91 AMPs except for three queries (Defensin-like turtle egg white protein, Crotamine-IV-3 and L-amino acid oxidase) The 96 candidate proteins are those with e-value of 0.001 or less in the BLAST results compared to the existing 91 AMPs, and the e-value indicates the probability of coincidence of a specific sequence. . In addition, 96 sequences consisted of the following 9 protein families except Beta defensin family.

D4 sub-subfamily, Phospholipase A2 family-Group II subfamily-D49 sub-subfamily, Phospholipase A2 family, Phospholipase A2 family-Group I subfamily-D49 sub-subfamily, -Group II subfamily-K49 sub-subfamily, Snake waprin family and Venom metalloproteinase (M12B) family-P-III subfamily-P-IIIa sub-subfamily.

Regarding the Beta defensin family, we could not find a similar sequence from the BLAST analysis.

Example 3. Screening of Peptides for Antibacterial Effectiveness

In order to further increase the efficiency of the study among 96 antimicrobial peptides, six (GBP01 ~ GBP06) peptides similar to the one of the already tested and studied AMP family catecholizidine were characterized and stored in FASTA format Respectively. The amino acid sequences of the peptides GBP01 to GBP06 are shown in SEQ ID NOS: 1 to 6, respectively.

SEQ ID NO: 1 (N-terminal- > C-terminal):

> GBP1 | XP_007442672.1 | Cath-OH

MEIHPGRILLVLSLVVRGSVWAVEGEILSYDAALSLAVNLYNQESGWDVVFQLLEAKPQPEWDPSSKARQKLDFTLKETTCPTSQNLNLEVCDFKEQGVVVECSGSSLAQPGAPIIQFSCETATQGNHRVKRNGFRKFMRRLKKFFAGGGSSIAHIKLH

SEQ ID NO: 2 (N-terminal- > C-terminal):

> GBP2 | XP_007442673.1 | Cath-2

MGLILLGAAWVALGILGSAASPTAEAPWLVLPRDAARLAVEDYNHRSPTPPSVFRLFKLRSTHKTRLEWGIHFSLHFTIKETHCQKTAGYRIGDCRYKPNGLIQDCSAEVSFLNLMWDSPVTSMKCGQAKWKKTKPHASPPQAMGFPPQVNVEHYIPASYSVAALTVTEEE

SEQ ID NO: 3 (N-terminal- > C-terminal):

> GBP3 | XP_007443269.1 | Cath-3

MHSFWVLLLFISPATTNFLSLSLTYPEALEEAVRLYNEEEGVKFLYRLVRAEPRPDWDPEAEGVQSLKFSMKETVCSAIEGLDFSKCDFKDDGEVKVCSASYKYQKKPQMNHVDVLCYCRQFCLFLFRQKAHTRLPVFRKSPHRFEAQAGQRSEGETGIPRPAMFRRPREGSKRAGGGRAGGPARPALRCHLEARRGRADVSGEARGLRRARAAPQRRLRAMARLKKFAEAGGADPDSGGLRARFPER

SEQ ID NO: 4 (N-terminal- > C-terminal):

> GBP4 | XP_007443270.1 | Cath-OH

MMEGCFWRILLVAGALSASGAAALPHRPLTYEEAVAFGVELYNKKAGEDSRYRLLEAVPQPDWDPTSESIQELNFTIKETVCLVQEERAEDECDFKDDGLVKECSGYYFFDETPPVAVLTCETVGGNEEETEEEKEEEKQPKRVKRFKKFFRKIKKGFRKIFKKTKIFIGGTIPI

SEQ ID NO: 5 (N-terminal- > C-terminal):

> GBP5 | XP_007445036.1 | Cath-OH

MTGVWALLLLLGVAAAAPPAQVVTYDQAIASAVNLYNQQKTTPFAFRLLEAEPQPNWDPRGKTTQGLKFTIKETVCPSAQSQNLTQCNFKEDGVDQDCSGTYSTQQEPPNLTVQCENIDQELNRITRSRWRRFIRGAGRFARRYGWRIALGL

SEQ ID NO: 6 (N-terminal- > C-terminal):

> GBP6 | XP_007445262.1 | Cath-OH

MMEGCFWRILLVAGALSASGAAPPPHKPLIYEKAVALGMELYNEKAGEDSQYRLLEAVPQPDWDPTSESTQELNFTIKETVCLVQEERAKDECDFKDDGLVKECSGYYFFDETPPVAVLTCETVGGNEEETEEEK

For the above SEQ ID NOS: 1 to 6, a portion expected to have an antimicrobial activity domain or pattern was searched using the AMPA database (Fig. 1A). As a result of plotting the AI (antimicrobial index) obtained by dividing the amino acid by 7 into the input sequence and calculating the PV average value of each amino acid, the average PV (Mean) of the given whole sequence and the antimicrobial activity domain (SEQ ID NO: 2), and a region having a smaller value based on the threshold value of 0.225 was predicted to be an antimicrobial active domain (FIG. 1B). The predicted position of the antimicrobial active domain moiety within the entire sequence was also readily ascertained (FIG. 1C).

Antimicrobial peptides are also classified as structural features, the largest α-helical protein family (peptide family) being the most universal in nature and the most studied of the cationic peptides, with amphiphilic, positively charged . It is known that an aqueous solution does not form a secondary structure but is folded into an α-helix form, which is a functional structure attached to a bacterial membrane. Therefore, even if the antimicrobial activity domain was predicted, the secondary structure was analyzed using PSI-BLAST (Protein-specific iterated BLAST) Protein Sequence Analysis Workbench (PSI-BLAST) 2 and Table 2).

Antimicrobial peptide Position in the entire sequence Sequence information of the predicted part Number of amino acids Secondary structure GBP01 128-149 HRVKRNGFRKFMRRLKKFFAGG 22 alpha-helix GBP02 56 ~ 68 LFKLRSTHKTRLE 13 루프 영역 125-137 KCGQAKWKKTKPH 13 루프 영역 GBP03 99 ~ 111 SASYKYQKKPQMN 13 β-sheet,
루프 영역 118 ~ 139 YCRQFCLFLFRQKAHTRLPVFR 22 loop region,
alpha-helix 218 ~ 229 RLRAMARLKKFA 12 alpha-helix GBP04 142-172 KRVKRFKKFFRKIKKGFRKIFKKTKIFIGGT 31 alpha-helix GBP05 126-150 TRSRWRRFIRGAGRFARRYGWRIAL 25 alpha-helix GBP06 it does not exist ㅡ ㅡ ㅡ

As shown in Table 2, the analysis of the AI (amino acid number 7, average PV, antimicrobial index) of six full length sequences using the AMPA database revealed that the amino acids of GBP01 were 128 ~ 149, No. 56 to No. 68 of the GBP02, No. 125 to No. 137 of the GBP03, No. 99 to No. 111, No. 118 to No. 139, No. 218 to No. 229 of the GBP03, No. 142 to No. 172 of the GBP04, and No. 126 to No. 150 of the GBP05. . As an additional parameter in the analysis, the threshold value was set to 0.225.

Based on PSI-BLAST, local alignment of all sequences and query sequences of one or more protein databases improves the accuracy of the secondary structure prediction results and the sensitivity of PSI-BLAST using repeated analysis (profile) sensitivity.

Based on the results of the secondary structure analysis, it was judged that antimicrobial activity would not be obtained when the candidate having the feature of the antimicrobial peptide does not have a functional specific structure or the corresponding position is not the C-terminal. Therefore, it was confirmed that the candidate peptides of Table 2 have the α-helix structure of GBP02, GBP03 except 99-111, 118-139 of GBP01, and GBP03 of 218-229, GBP04 and GBP05. Of these, GBP03 was predicted to have an α-helix structure in the case of 218 to 229, but since only a part of the predicted α-helix structure was included, selective peptides GBP01, GBP04 and GBP05 were selected for further analysis using the APD3 database Respectively.

Depending on the peptide input, information on the number, composition and characteristics of amino acids can be obtained. Among the given sequences, the hydrophobic amino acids are red, while the glycine and proline, which play an important role in the protein structure, are blue (FIG. 3A). In addition, it can be determined whether several of the hydrophobic residues are located on the same surface (Fig. 3B).

As a result, they all have a common α-helix structure and their hydrophobic ratios are 36%, 39%, 37%, and net charges of +9, +14 and +9, And it was confirmed that the peptide had hydrophobicity and positive charge characteristic of the antimicrobial peptide. As a result of comparing sequences with antimicrobial peptides previously identified, it was confirmed that they have similarities of 38.46%, 50.00% and 39.28% for the most similar sequences (FIG. 4). And exhibited similarity of 50% or less with that of the existing antimicrobial peptide sequence, it was confirmed that these were novel peptides which were not previously known (Table 3).

Antimicrobial peptide Amino acid sequence Number of amino acids Molecular weight (Da) Hydrophobic Ratio (%) Net charge (+) Similarity with existing antimicrobial peptide sequences (%) GBP01 HRVKRNGFRKFMRRLKKFFAGG 22 2737 36 9 38.46 GBP04 RVKRFKKFFRKIKKGFRKIFKKTKIFIG 28 3575 39 14 50.00 GBP05 TRSRWRRFIRGAGRFARRYGWRIA 24 3053 37 9 39.28

Example 4. Synthesis of Selected Antimicrobial Peptides

A common solid-phase peptide synthesis that initiates synthesis by attaching functional units called linkers to small porous beads by chemical methods for protein synthesis to induce peptide chains to continue SPPS) method was used. The amino acid sequences of the synthesized antimicrobial peptides GBP01, GBP04 and GBP05 (SEQ ID NOS: 7 to 9, respectively) are as follows.

SEQ ID NO: 7 (N-terminal- > C-terminal):

HRVKRNGFRKFMRRLKKFFAGG

SEQ ID NO: 8 (N-terminal- > C-terminal):

RVKRFKKFFRKIKKGFRKIFKKTKIFIG

SEQ ID NO: 9 (N-terminal- > C-terminal):

TRSRWRRFIRGAGRFARRYGWRIA

The three selected peptides were synthesized by SPPS method and microwave technique. The synthesized GBP01, GBP04 and GBP05 peptides were prepared to a final concentration using respective 1X PBS (phosphate-buffered saline) with 1 ㎎ / ㎖ (* 1X PBS : 10 mM PO 4 3- + 137 mM NaCl + 2.7 mM KCl , pH 7.4).

Test Example 1. Measurement of minimum inhibitory concentration and confirmation of antimicrobial activity

In a culture plate in which Escherichia coli (E. coli) ATCC 25922, Staphylococcus aureus (S. aureus) ATCC 25213, Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853 were formed, a single colony ) Was removed and cultured overnight in a 14 ml culture tube containing 5 ml of LB (Luria-Bertani) medium. The cultured cells were centrifuged at 4000 rpm for 4 minutes, the supernatant was discarded, and the remaining pellet-shaped cells were suspended in 3 ml of LB medium. Then 100 μl of the solution was taken out and mixed with 900 μl of LB medium, diluted, and the OD 600 value was measured using a UV spectrophotometer.

This is a procedure for adjusting the number of cells in a minimal inhibitory concentration assay (MIC assay), and 1 ml of LB medium was set as a reference (* MIC value: inhibition of cell growth The lower the value, the higher the activity of the substance used.

MIC measurement was performed according to the manufacturer's protocol using Microbial Viability Assay Kit-WST (Dojindo, DC, USA). For each strain, the final OD600 value standard was set to 0.125. To 96 well-microplates containing three kinds of antimicrobial peptides each containing 1, 2, 3, 4, 7, 8, 16, 32 and 64 占 퐂 / ml (prepared by dissolving in 1X PBS), 180 占 퐇 of LB medium Respectively. The amount of cells used in the experiment was 10 ⁵ CFU / well (* CFU: colony-forming unit, unit for measuring the number of bacteria or fungi visible). 1X PBS without peptide was used as a negative control.

The prepared 96-well microplate was placed in a shaking incubator and cultured at 37 ° C and 220 rpm for 6 hours. A coloring reagent prepared by mixing 900 μl of WST solution (WST-8) and 100 μl of electron mediator was prepared and stored at 4 ° C. After incubation for 6 hours, 10 μl of each well was added to all wells Respectively. The total volume per well containing the antimicrobial peptide, cells, LB medium, and colored reagent was adjusted to 210 μl each. After incubation for 2 hours, absorbance was measured at 450 nm using a microplate reader.

The genomic information of E. coli was confirmed by NCBI (accession number: CP009072). E. aeruginosa strains were found to be effective against inflammation in plants as well as humans, And it is known that it can cause diseases accompanied by sepsis. These two strains are typical Gram-negative organisms and are a commonly used quality control strain after sensitivity testing for 34 antibiotics, including ampicillin, in 1979. Gram-positive bacteria, S. aureus, are also harmful to humans and often cause infection through the skin. aeruginosa, and the emergence and spread of S. aureus resistant to antibiotics has become a global problem.

MIC measurement was performed to confirm whether the three peptides synthesized against E. coli, S. aureus and P. aeruginosa had an actual antibacterial action, and interesting results were obtained. They showed antimicrobial activity against Gram-negative bacteria such as E. coli and P. aeruginosa, but did not show antimicrobial activity against Gram-positive S. aureus even when the concentration of the sample was increased to 64 ㎍ / ㎖. The MICs of the GBP01, GBP04 and GBP05 peptides were 3, 2 and 1 ㎍ / ㎖ for E. coli, and GBP05 peptide for P. aeruginosa at 8, 3 and 3 ㎍ / ㎖, respectively, in both strains And showed the best activity (Fig. 5, Table 4).

Antibacterial
Peptides The minimum inhibitory concentration ([mu] g / ml) E. coli
ATCC 25922 S. aureus
ATCC 25213 P. aeruginosa
ATCC 27853 GBP01 3 Inactive 8 GBP04 2 Inactive 3 GBP05 One Inactive 3

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation Corp <120> Novel antimicrobial peptide isolated from Python bivittatus and          screening method thereof <130> P160140 <160> 9 <170> KoPatentin 3.0 <210> 1 <211> 159 <212> PRT <213> Unknown <220> <223> Python bivittatus <400> 1 Met Glu Ile His Pro Gly Arg Ile Leu Leu Val Leu Ser Leu Val Val   1 5 10 15 Arg Gly Ser Val Trp Ala Val Glu Gly Glu Ile Leu Ser Tyr Asp Ala              20 25 30 Ala Leu Ser Leu Ala Val Asn Leu Tyr Asn Gln Glu Ser Gly Trp Asp          35 40 45 Val Val Phe Gln Leu Leu Glu Ala Lys Pro Gln Pro Glu Trp Asp Pro      50 55 60 Ser Ser Lys Ala Arg Gln Lys Leu Asp Phe Thr Leu Lys Glu Thr Thr  65 70 75 80 Cys Pro Thr Ser Gln Asn Leu Asn Leu Glu Val Cys Asp Phe Lys Glu                  85 90 95 Gln Gly Val Val Val Glu Cys Ser Gly Ser Ser Leu Ala Gln Pro Gly             100 105 110 Ala Pro Ile Ile Gln Phe Ser Cys Glu Thr Ala Thr Gln Gly Asn His         115 120 125 Arg Val Lys Arg Asn Gly Phe Arg Lys Phe Met Arg Arg Leu Lys Lys     130 135 140 Phe Phe Ala Gly Gly Gly Ser Ser Ile Ala His Ile Lys Leu His 145 150 155 <210> 2 <211> 171 <212> PRT <213> Unknown <220> <223> Python bivittatus <400> 2 Met Gly Leu Ile Leu Leu Gly Ala Ala Trp Val Ala Leu Gly Ile Leu   1 5 10 15 Gly Ser Ala Ala Ser Pro Thr Ala Glu Ala Pro Trp Leu Val Leu Pro              20 25 30 Arg Asp Ala Ala Arg Leu Ala Val Glu Asp Tyr Asn His Arg Ser Pro          35 40 45 Thr Pro Pro Ser Val Phe Arg Leu Phe Lys Leu Arg Ser Thr His Lys      50 55 60 Thr Arg Leu Glu Trp Gly Ile His Phe Ser Leu His Phe Thr Ile Lys  65 70 75 80 Glu Thr His Cys Gln Lys Thr Ala Gly Tyr Arg Ile Gly Asp Cys Arg                  85 90 95 Tyr Lys Pro Asn Gly Leu Ile Gln Asp Cys Ser Ala Glu Val Ser Phe             100 105 110 Leu Asn Leu Met Trp Asp Ser Pro Val Thr Ser Met Lys Cys Gly Gln         115 120 125 Ala Lys Trp Lys Lys Thr Lys Pro His Ala Ser Pro Pro Gln Ala Met     130 135 140 Gly Phe Pro Pro Gln Val Asn Val Glu His Tyr Ile Pro Ala Ser Tyr 145 150 155 160 Ser Val Ala Leu Thr Val Thr Glu Glu Glu                 165 170 <210> 3 <211> 248 <212> PRT <213> Unknown <220> <223> Python bivittatus <400> 3 Met His Ser Phe Trp Val Leu Leu Leu Phe Ile Ser Pro Ala Thr Thr   1 5 10 15 Asn Phe Leu Ser Leu Ser Leu Thr Tyr Pro Glu Ala Leu Glu Glu Ala              20 25 30 Val Arg Leu Tyr Asn Glu Glu Glu Gly Val Lys Phe Leu Tyr Arg Leu          35 40 45 Val Arg Ala Glu Pro Arg Pro Asp Trp Asp Pro Glu Ala Glu Gly Val      50 55 60 Gln Ser Leu Lys Phe Ser Met Lys Glu Thr Val Cys Ser Ala Ile Glu  65 70 75 80 Gly Leu Asp Phe Ser Lys Cys Asp Phe Lys Asp Asp Gly Glu Val Lys                  85 90 95 Val Cys Ser Ala Ser Tyr Lys Tyr Gln Lys Lys Pro Gln Met Asn His             100 105 110 Val Asp Val Leu Cys Tyr Cys Arg Gln Phe Cys Leu Phe Leu Phe Arg         115 120 125 Gln Lys Ala His Thr Arg Leu Pro Val Phe Arg Lys Ser Pro His Arg     130 135 140 Phe Glu Ala Gln Ala Gly Gln Arg Ser Glu Gly Glu Thr Gly Ile Pro 145 150 155 160 Arg Pro Ala Met Phe Arg Arg Pro Arg Glu Gly Ser Lys Arg Ala Gly                 165 170 175 Gly Gly Arg Ala Gly Gly Pro Ala Arg Pro Ala Leu Arg Cys His Leu             180 185 190 Glu Ala Arg Arg Gly Arg Ala Asp Val Ser Gly Glu Ala Arg Gly Leu         195 200 205 Arg Arg Ala Arg Ala Ala Pro Gln Arg Arg Leu Arg Ala Met Ala Arg     210 215 220 Leu Lys Lys Phe Ala Glu Ala Gly Gly Ala Asp Pro Asp Ser Gly Gly 225 230 235 240 Leu Arg Ala Arg Phe Pro Glu Arg                 245 <210> 4 <211> 175 <212> PRT <213> Unknown <220> <223> Python bivittatus <400> 4 Met Met Glu Gly Cys Phe Trp Arg Ile Leu Leu Val Ala Gly Ala Leu   1 5 10 15 Ser Ala Ser Gly Ala Ala Leu Pro His Arg Pro Leu Thr Tyr Glu              20 25 30 Glu Ala Val Ala Phe Gly Val Glu Leu Tyr Asn Lys Lys Ala Gly Glu          35 40 45 Asp Ser Arg Tyr Arg Leu Leu Glu Ala Val Pro Gln Pro Asp Trp Asp      50 55 60 Pro Thr Ser Glu Ser Ile Gln Glu Leu Asn Phe Thr Ile Lys Glu Thr  65 70 75 80 Val Cys Leu Val Gln Glu Glu Arg Ala Glu Asp Glu Cys Asp Phe Lys                  85 90 95 Asp Asp Gly Leu Val Lys Glu Cys Ser Gly Tyr Tyr Phe Phe Asp Glu             100 105 110 Thr Pro Pro Val Ala Val Leu Thr Cys Glu Thr Val Gly Gly Asn Glu         115 120 125 Glu Glu Thr Glu Glu Glu Lys Glu Glu Glu Lys Gln Pro Lys Arg Val     130 135 140 Lys Arg Phe Lys Lys Phe Phe Arg Lys Ile Lys Lys Gly Phe Arg Lys 145 150 155 160 Ile Phe Lys Lys Thr Lys Ile Phe Ile Gly Gly Thr Ile Pro Ile                 165 170 175 <210> 5 <211> 152 <212> PRT <213> Unknown <220> <223> Python bivittatus <400> 5 Met Thr Gly Val Trp Ala Leu Leu Leu Leu   1 5 10 15 Ala Pro Pro Ala Gln Val Val Thr Tyr Asp Gln Ala Ile Ala Ser Ala              20 25 30 Val Asn Leu Tyr Asn Gln Gln Lys Thr Thr Pro Phe Ala Phe Arg Leu          35 40 45 Leu Glu Ala Glu Pro Gln Pro Asn Trp Asp Pro Arg Gly Lys Thr Thr      50 55 60 Gln Gly Leu Lys Phe Thr Ile Lys Glu Thr Val Cys Pro Ser Ala Gln  65 70 75 80 Ser Gln Asn Leu Thr Gln Cys Asn Phe Lys Glu Asp Gly Val Asp Gln                  85 90 95 Asp Cys Ser Gly Thr Tyr Ser Thr Gln Gln Glu Pro Pro Asn Leu Thr             100 105 110 Val Gln Cys Glu Asn Ile Asp Gln Glu Leu Asn Arg Ile Thr Arg Ser         115 120 125 Arg Trp Arg Arg Phe Ile Arg Gly Ala Gly Arg Phe Ala Arg Arg Tyr     130 135 140 Gly Trp Arg Ile Ala Leu Gly Leu 145 150 <210> 6 <211> 135 <212> PRT <213> Unknown <220> <223> Python bivittatus <400> 6 Met Met Glu Gly Cys Phe Trp Arg Ile Leu Leu Val Ala Gly Ala Leu   1 5 10 15 Ser Ala Ser Gly Ala Ala Pro Pro His Lys Pro Leu Ile Tyr Glu              20 25 30 Lys Ala Val Ala Leu Gly Met Glu Leu Tyr Asn Glu Lys Ala Gly Glu          35 40 45 Asp Ser Gln Tyr Arg Leu Leu Glu Ala Val Pro Gln Pro Asp Trp Asp      50 55 60 Pro Thr Ser Glu Ser Thr Gln Glu Leu Asn Phe Thr Ile Lys Glu Thr  65 70 75 80 Val Cys Leu Val Gln Glu Glu Arg Ala Lys Asp Glu Cys Asp Phe Lys                  85 90 95 Asp Asp Gly Leu Val Lys Glu Cys Ser Gly Tyr Tyr Phe Phe Asp Glu             100 105 110 Thr Pro Pro Val Ala Val Leu Thr Cys Glu Thr Val Gly Gly Asn Glu         115 120 125 Glu Glu Thr Glu Glu Glu Glu Lys     130 135 <210> 7 <211> 22 <212> PRT <213> Unknown <220> <223> Python bivittatus <400> 7 His Arg Val Lys Arg Asn Gly Phe Arg Lys Phe Met Arg Arg Leu Lys   1 5 10 15 Lys Phe Phe Ala Gly Gly              20 <210> 8 <211> 28 <212> PRT <213> Unknown <220> <223> Python bivittatus <400> 8 Arg Val Lys Arg Phe Lys Lys Phe Phe Arg Lys Ile Lys Lys Gly Phe   1 5 10 15 Arg Lys Ile Phe Lys Lys Thr Lys Ile Phe Ile Gly              20 25 <210> 9 <211> 24 <212> PRT <213> Unknown <220> <223> Python bivittatus <400> 9 Thr Arg Ser Arg Trp Arg Arg Phe Ile Arg Gly Ala Gly Arg Phe Ala   1 5 10 15 Arg Arg Tyr Gly Trp Arg Ile Ala              20

Claims (7)

A novel antimicrobial peptide comprising the amino acid sequence of any one of SEQ ID NOS: 7 to 9.
The method according to claim 1,
Wherein said amino acid sequence is isolated from python bivittatus.
a) selecting antimicrobial peptide sequence information from a database having protein sequence information of a reptile;
b) selecting a protein sequence derived from Python bivittatus which is highly similar to the antimicrobial peptide selected in a) above; And
c) selectively selecting an amino acid sequence comprising the antimicrobial activity domain of the protein sequence selected through comparison of similarity in b)
Wherein said method comprises the steps of:
The method of claim 3,
The protein sequence selected in b) above is selected from the group consisting of the cathelicidin family, the crotamine-myotoxin family, the Flavin monoamine oxidase family-FIG 1 subfamily, , Glycosyl hydrolase 22 family, Phospholipase A2 Family-Group I subfamily-D49 sub-subfamily, Phospholipase A2 family-Group I subfamily-D49 sub-subfamily, Family II subfamily, Group II subfamily, Group II subfamily, Group II subfamily, Group II subfamily, Group II subfamily-K49 sub-subfamily, subfamily-K49 sub-subfamily, Snake waprin family, and poison metalloproteinase [M12B] family-P-III sub-subfamily (Venom metalloproteinase [M12B] family- P-III subfamily y-P-IIIa sub-subfamily).
The method of claim 3,
Wherein the protein sequence selected in step b) has an e-value of 0.001 or less as a result of BLAST analysis of the reptile-derived antimicrobial peptide sequence.
The method of claim 3,
Wherein the antimicrobial activity domain of c) is located at the C-terminus.
The method of claim 3,
Wherein the antimicrobial activity domain of c) exhibits an a-helix structure during formation of a secondary structure.

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