CN106167524B - Secapin peptides from eastern bees - Google Patents
Secapin peptides from eastern bees Download PDFInfo
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- CN106167524B CN106167524B CN201610320078.0A CN201610320078A CN106167524B CN 106167524 B CN106167524 B CN 106167524B CN 201610320078 A CN201610320078 A CN 201610320078A CN 106167524 B CN106167524 B CN 106167524B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/8146—Metalloprotease (E.C. 3.4.24) inhibitors, e.g. tissue inhibitor of metallo proteinase, TIMP
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The present invention provides a cercarin peptide derived from eastern bees, and more particularly, to a peptide exhibiting plasmin-inhibiting activity, elastase-inhibiting activity, antibacterial and antifungal effects for use in a wide range of applications, such as blood coagulants, anti-inflammatory agents, antibacterial or antifungal agents.
Description
Technical Field
The present invention relates to a secapin peptide derived from the oriental bee (Apis cerana).
background
Bees are economically important insects. Bees not only carry pollen but also provide a number of products such as honey, royal jelly, propolis, pollen, beeswax, and bee venom, among others. These bee products are materials commonly used and important in oriental medicine. Recently, many investigations on the pharmacological actions of components in bee venom have been conducted. Bee venom includes various bee venom proteins or peptides, and specifically, bee venom peptide, phospholipase a2(PLA2), melimine peptide, hyaluronidase, serine protease inhibitor, and the like are known in the related art.
More specifically, such various proteins or peptides found in the venom of various organisms have attracted a high interest because they are involved in various physiological activities such as immune response, complement activity, hemostasis, coagulation mechanisms, fibrinolytic function, reduction of inflammation, and the like.
The base sequence encoding the seecapelin peptide, one of the bee venom components of the western bee Apis melifera, was first identified in 1984 [ Vlasak R & Kreil G, Eur J Biochem 145: 279-292(1984)]. Another base sequence related to hymenoptera insects including some species of ants and aedes aegypti has also been reported. However, since secateine peptide, which is known as one of bee venom components, is present in bee venom in small portions of not more than 1%, it is rather difficult to isolate and obtain it. For this reason, the number of studies on various physiological effects of raceuticals is relatively small.
Accordingly, the present inventors have made cloning of cDNA gene encoding Secaine peptide derived from the eastern bee, Apis cerana. In addition, the present inventors have found that a recombinant peptide produced by inserting a polynucleotide encoding a mature peptide among the cDNA genes into a vector and then introducing the vector into a cell line of insects can inhibit the activities of fibrinolytic enzyme, elastase, subtilisin A and proteinase K and exhibit excellent antibacterial and antifungal effects against gram-positive bacteria, gram-negative bacteria and entomopathogenic fungi, thereby completing the present invention.
[ Prior art documents ]
[ non-patent document ]
(non-patent document No.0001) Vlasak R & Kreil G, Eur J Biochem 145: 279-282(1984)
Disclosure of Invention
Accordingly, it is an object of the present invention to provide a raceutic peptide 'eastern bee derived raceutic peptide' derived from eastern bees.
It is another object of the present invention to provide a nucleotide encoding the above peptide.
In addition, another object of the present invention is to provide a recombinant expression vector comprising the above-mentioned nucleotide.
in addition, another object of the present invention is to provide a coagulant, an anti-inflammatory agent, an antibacterial agent or an antifungal agent comprising the above peptide or recombinant expression vector.
The above object of the present invention will be achieved by the following features:
(1) A Secaine peptide derived from an eastern bee having an amino acid sequence defined by SEQ ID No. 2.
(2) The cercaritin peptide according to the above-mentioned (1), wherein the peptide has a plasmin-inhibiting activity.
(3) The racecadine peptide according to (1) above, wherein the peptide has elastase inhibitory activity.
(4) The cercapene peptide according to the above (1), wherein the peptide has an inhibitory activity against at least one protein selected from the group consisting of subtilisin A and proteinase K.
(5) A polynucleotide having a base sequence defined by SEQ ID No.1 encoding the peptide according to the above (1).
(6) A recombinant expression vector comprising the polynucleotide according to (5) above.
(7) A coagulant comprising the peptide according to (1) above or the expression vector according to (6) above.
(8) An anti-inflammatory agent comprising the peptide according to the above (1) or the expression vector according to the above (6).
(9) An antibacterial or antifungal agent comprising the peptide according to the above (1) or the expression vector according to the above (6).
The present invention can identify the Secainpin peptide from oriental bee and its coded gene sequence, and can produce large amount of Secainpin peptide from oriental bee.
The cercapeptide of the present invention can exhibit plasmin-inhibiting activity, elastase-inhibiting activity, antibacterial and antifungal effects, and thus is useful for a wide range of applications including, for example, blood coagulants, anti-inflammatory agents, antibacterial and antifungal agents, and the like.
Drawings
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a graph showing homology between the Apis cerana peptide of the present invention and other Secarina peptides (underlined: signal peptide site, dotted frame: mature peptide region, and filled circle: cysteine residue forming the remaining disulfide bond);
FIG. 2 is a diagram illustrating the result of electrophoresis of recombinant proteins in which only the mature peptide site among the eastern bee-derived racecadin peptides of the present invention is expressed;
FIG. 3 is a graph showing plasmin-inhibiting activity of the recombinant peptide of the present invention (mature peptide of the Apis cerana peptide cDNA);
FIG. 4 is a graph showing elastase inhibitory activity of the recombinant peptide of the present invention (mature peptide of the eastern bee-derived Seatchin peptide cDNA);
FIG. 5 is a graph showing the inhibitory activity of the recombinant peptide of the present invention (mature peptide of the Apis cerana peptide cDNA) against subtilisin A and proteinase K; and
FIG. 6 is a Western blot analysis and confocal micrograph illustrating the binding ability of the recombinant peptide of the present invention (mature peptide of the eastern bee-derived Seatchin peptide cDNA) to bacteria and fungi.
Detailed Description
Hereinafter, the present invention will be described in more detail.
The present inventors have isolated the eastern bee-derived secateine peptide (secateine: AcScp) amino acid sequence and a polynucleotide encoding the above secateine peptide as described below.
A cDNA library was prepared using poly (A) and mRNA extracted from oriental bee insects, and then analysis of Expressed Sequence Tags (ESTs) was performed. According to the eastern bee EST analysis, the cDNA of the gene coding the eastern bee-derived Seatcaine peptide is cloned and defined by SEQ ID No. 1. In addition, the result of amino acid sequence database analysis translated from cDNA encoding the eastern bee-derived racecadin peptide is defined by SEQ ID No. 2.
The eastern bee-derived racecadin peptide gene was first isolated from eastern bees, however, its specific characteristics have not been reported. The present invention identifies the sequence of AcScp derived from the eastern honey bee, and this gene was registered in NCBIGenBank database at 5/15 of 2015 under accession No. KR 732613 (cDNA).
As a result of isolating cDNA derived from eastern honey bee AcScp according to the present invention and analyzing its base sequence, it was found that the obtained cDNA of the eastern honey bee-derived AcScp gene has the base sequence of SEQ ID No.1, which is 348bp in length up to the stop codon, wherein the protein encoded by the polynucleotide sequence of SEQ ID No.1 consists of 115 amino acids, includes a signal peptide site consisting of 24 amino acids and a mature peptide region consisting of 25 amino acids, and has two cysteine structures. The recombinant protein obtained by expressing only the mature peptide portion had a molecular weight of about 3.7kDa by electrophoresis.
The amino acid sequence encoded by the eastern bee-derived secainide peptide gene shows high homology with the amino acid sequence of secainide peptide (western bee-3, dormitopsis florida, etc.) registered in NCBI GenBank database (see fig. 1).
The peptide having the amino acid sequence of SEQ ID No.2 according to the present invention may have protein inhibitory activity against plasmin, elastase, subtilisin A, proteinase K, etc.
In addition, the present invention provides a recombinant expression vector comprising the nucleotide of SEQ ID No. 1.
In the present disclosure, the term "vector" refers to a DNA product containing the base sequence of a particular gene operably linked to appropriate control sequences for expression of the gene of interest in a suitable host. Here, the control sequence may include a promoter capable of initiating the transfer, any random operator sequence that controls the transfer, a sequence encoding an appropriate mRNA ribosome binding site, and a sequence that controls the termination of the transfer and transcription.
In the present disclosure, the phrase "operably linked" refers to a nucleic acid sequence encoding a protein of interest functionally linked to nucleic acid expression control sequences to perform a general function. For example, a promoter may be operably linked to a nucleic acid sequence that encodes a protein or RNA that affects the expression of a coding sequence. The operable linkage with the recombinant vector may be formed using genetic recombination techniques well known in the related art, and the site-specific DNA cleavage and linkage are performed using any enzymes and the like generally known in the related art.
The vector of the present invention may include, for example, a promoter, an initiation codon, a stop codon, expression control elements such as a polyadenylation signal and enhancer, a secretion signal, etc., and may be prepared in various types according to the use thereof. The start codon and stop codon should be functional to the subject to whom the genetic construct is administered and be present in the coding sequence and frame (in-frame).
The vector usable in the present invention is not particularly limited as long as it is replicable in a host, and may include any vector known in the related art. For example, a non-viral vector or a viral vector may be used.
As a representative example, the non-viral vector may include a plasmid. The plasmid expression vector is a tool for directly delivering plasmid DNA to human cells according to the FDA-certified gene transfer method, which is suitable for humans, and thus, has an advantage of being able to purify the plasmid DNA homogeneously, unlike viral vectors. The plasmid expression vector usable in the present invention may include any mammalian expression vector known in the related art. For example, but not limited thereto, pRK5 (European patent No.307,247), pSV16B (International patent publication No.91/08291), pVL1392(PharMingen), and the like are representative.
In addition, the expression vector usable in the present invention may include a viral vector. The viral vector may include, for example, retrovirus, adenovirus, adeno-associated virus, herpes virus, fowlpox virus, and the like. The viral vector should satisfy the following conditions: (1) the vector should infect the cell of interest, and therefore, a viral vector having an appropriate host range should be selected; (2) the transgene should be retained and expressed in the cell for a desired period of time; and (3) the vector should be safe in the host.
Retroviral vectors are constructed such that all viral genes are removed or altered, thereby enabling non-viral proteins to be made in cells infected with the viral vector. The main advantage of such retroviral vectors in gene therapy is the delivery of large numbers of genes in replicable cells and the correct integration of the genes transferred into the DNA of said cells, while not causing persistent infection after gene transfection. FDA-certified retroviral vectors have been manufactured using PA317 amphotropic retroviral packaging cells (Miller, A.D. and Buttimore, C., Molec.cell biol., 6: 2895-2902, 1986).
The non-retroviral vector may include, for example, an adenovirus as described above. The main advantage of adenovirus is the ability of the virus to carry large DNA fragments (36kb genome) and to infect non-replicating cells at high titers. In addition, herpes viruses can also be effectively used in human gene therapy (Wolfe, J.H., et al, Nature Genetics, 1: 379-384, 1992). In addition, suitable viral vectors known in the related art may be used.
Other viral vectors that may be used for gene transfer into cells may include, for example, Murine Leukemia Virus (MLV), JC, SV40, polyoma, Epstein-ear virus, papilloma virus, vaccinia virus, poliovirus, herpes virus, Sindbis virus, lentivirus, other human and animal viruses.
The expression vector of the present invention can be introduced into cells by any conventional method known in the related art. For example, although not limited thereto, the vector may be introduced into cells using transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene guns, and other known methods for nucleic acid influx into cells (Wu et al, J.Bio.chem., 267: 963-loop 967, 1992; Wu and Wu, J.Bio.chem., 263: 14621-loop 14624, 1988).
The vectors of the invention may also comprise a selectable marker. The selection marker is used for selecting cells into which the vector is transferred, i.e., for determining whether the gene of interest is inserted, and some markers that can confer drug resistance, nutritional requirements, tolerance to cytotoxic agents, or selectable phenotypes such as expression of surface layer proteins may be used. In the context of treatment with a selection agent, only cells expressing the selection marker may survive or exhibit the surrogate phenotype, thereby enabling selection of transformed cells.
In addition, the present invention provides a transformant transformed with the above-described vector.
The transformant of the present invention can be constructed by introducing the vector into a host cell in a specific form in which the promoter functions.
In the present disclosure, the term "transformation" refers to the introduction of DNA into a host such that the DNA can be replicated as an extrachromosomal element or by complete chromosomal integration. The transformation may include any method of introducing a nucleic acid molecule into an organism, cell, tissue or organ, and may be performed by selecting an appropriate standard technique depending on the host cell, as is well known in the relevant art.
Among these methods as described above, electroporation, CaPO 4 precipitation, CaCl 2 precipitation, microinjection, PEG method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, etc. may be included, but are not limited thereto.
Since the amount of expression and modification of the protein may vary depending on the host cell to be transferred into the expression vector, the host cell closest to the intention may be selected and used. The host cell usable in the present invention may include, for example, an insect cell line, yeast, fungus, bacterium or algae, for example, preferably using insect cell line Sf9 (Spodoptera frugiperda 9), but is not limited thereto.
In the following embodiment of the present invention, an insect baculovirus transition vector (pBAC8) comprising the polynucleotide of SEQ ID No.1 was co-transfected with insect cell line Sf9 (Spodoptera frugiperda 9), and after 5 days, culture broth was collected to prepare a recombinant baculovirus expressing the eastern bee-derived racecadin peptide. The recombinant baculovirus was propagated in Sf9 cell line, and the recombinant protein of the invention (eastern bee-derived Secat peptide) was isolated using the MagneHisTM protein purification system (Promega Co., USA).
From the above-mentioned transformant, the peptide of the present invention can be isolated. After expression of the peptide in the selected host cell, it can be isolated and purified using any conventional biochemical separation technique, such as treatment with a protein precipitant (salting out), centrifugation, ultrasonication, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like. In general, the above-described methods may be used in combination of two or more thereof in order to isolate a high-purity protein (polypeptide).
In addition, the present invention provides a coagulant, an anti-inflammatory agent and an antibacterial or antifungal agent, each of which includes an expression vector of eastern bee-derived Secat peptide having the amino acid sequence of SEQ ID No.2 as described above or a nucleotide of SEQ ID No.1 encoding the above peptide.
The eastern bee-derived racecadin peptide of the present invention may have plasmin inhibitory activity to exhibit a coagulation effect, elastase inhibitory activity to exhibit an anti-inflammatory effect, and subtilisin a and proteinase K inhibitory activities to exhibit antibacterial and antifungal effects.
More specifically, the eastern bee-derived racecadine peptide of the present invention inhibits degradation of fibrin by fibrinolysis of plasmin, a phenomenon in which solid fibrin is dissolved into a liquid state by plasmin and blood vessels are thereby re-opened due to blood clot blockage. However, the racepine peptide can inhibit the above phenomenon to act on coagulation.
In addition, elastase is a serine protease involved in inflammation. Since serpin inhibitors function as anti-inflammatory agents, the tecavir peptides as described above may have elastase inhibitory activity, thereby acting on inflammation inhibition.
With respect to antibacterial and antifungal effects, experiments have shown that the growth of gram-positive bacteria such as Bacillus subtilis or Bacillus thuringiensis is inhibited and that the growth of Fusarium graminearum, which is known as a plant pathogenic bacterium, is also effectively inhibited.
The coagulant and anti-inflammatory agent according to the present invention can be effectively applied to materials for medicines, cosmetics, and the like, while the antibacterial or antifungal agent can be used for materials for various products such as medicines, cosmetics, detergents, plant disease preventive agents, coating agents, and the like.
When the blood coagulation agent, anti-inflammatory agent, antibacterial or antifungal agent is prepared into a pharmaceutical composition, the composition may further include a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier is generally used for formulation, and may include, for example, lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but is not limited thereto. In addition to the above components, the pharmaceutically acceptable carrier may also include lubricants, moisturizers, sweeteners, flavors, emulsifiers, suspending agents, preservatives and the like. Pharmaceutically acceptable carriers and formulations suitable for use are described in detail in Remington's Pharmaceutical Sciences (nineteenth edition, 1995).
The pharmaceutical compositions of the present invention may be administered by oral or parenteral routes, preferably by parenteral routes, more preferably by topical application.
The appropriate dosage amount of the pharmaceutical composition may be determined according to various prescriptions based on various parameters such as formulation method, administration mode, age, weight, sex and state of the patient, food, administration time, administration route, excretion rate, reaction and response, etc.
The pharmaceutical composition may be prepared into a formulation using the pharmaceutically acceptable carrier and/or excipient according to any conventional method that is easily carried out by those skilled in the relevant art to which the present invention pertains. Thus, the pharmaceutical compositions may be formulated in unit dosage form or packaged in multi-dose containers to form products. In this regard, the formulation may include, for example, a solution, suspension, syrup or emulsion in an oil or aqueous medium, or an extract, dispersion, powder, granule, tablet, capsule form, or the like. In addition, the formulation may also include a dispersant or stabilizer.
Hereinafter, the present invention will be described in more detail with reference to the following examples.
[ examples ]
1. Cloning of coding gene of Apis cerana Fabricius linn
a cDNA library was prepared using Poly (A) and mRNA extracted from the eastern honey bee insects using commercially available kits, namely, Uni-ZAP vector and Gigapack III Gold packaging extract (Stratagene Co., United States), and Expression Sequence Tags (ESTs) were analyzed.
Another commercially available kit, Wizard mini prep kit (Promega co., usa) was used to extract DNA base sequences were analyzed by an automatic DNA analyzer (Applied Biosystems co., usa) the analyzed base sequences were compared using the BLAST program (http:// www.ncbi.nlm.nih.gov/BLAST) of NCBI.
According to the eastern bee EST analysis, the cDNA of said eastern bee-derived racecadin peptide gene coding for 115 amino acids is cloned and defined as SEQ ID No. 1. In addition, the analysis result of the amino acid sequence database translated from the cDNA of the above-mentioned oriental bee-derived racecadin peptide gene was defined as SEQ ID No. 2.
The results of the analysis of the above sequences by using the BLAST program are shown in FIG. 1.
As shown in fig. 1, it can be seen that the peptide of the present invention has relatively high homology with the existing racecadin peptide reported in the related art, and the cysteine residue and the mature peptide region are well retained to form a disulfide bond.
2. preparation of vector for expressing recombinant Apis cerana Secatena peptide and verification of expression in insect cell line
To express the oriental bee-derived raceutic peptide, the mature peptide site in the cDNA of the oriental bee-derived raceutic peptide was inserted into the insect baculovirus (autographa californica nuclear polyhedrosis virus) transition vector pBAC8(clontech co., usa), followed by the above transition vector (500ng) with bsacgoza virus DNA (100ng) [ Je et al, biotechnol.lett., 23: 575- & 582(2001) ] were Co-transfected into the insect cell line Sf9 (Spodoptera frugiperda) using liposomes (lipofectin) (Clonetech Co.).
After 5 days, the culture broth was collected to construct a recombinant baculovirus expressing the eastern bee-derived racecadin peptide. The recombinant baculovirus was propagated in Sf9 cell line and the recombinant AcScp was isolated using the magnehis protein purification system (promegaco, usa). FIG. 2 is an electrophoretogram illustrating the isolated Apis cerana Secatena peptide.
As shown in fig. 2, the dyeing result confirmed that the eastern bee-derived racecadin peptide has a molecular weight of about 3.7kDa by electrophoresis.
3. Activity confirmation of Apis cerana Fabricius peptide
(1) Validation of inhibitory Activity against plasmin, Elastase, subtilisin A or proteinase K
To verify whether the eastern bee-derived racecadin peptide inhibited plasmin, elastase, subtilisin a, and proteinase K, various concentrations of AcScp recombinant peptide were treated with 10nM plasmin (Sigma co., usa), 100nM human neutrophil elastase, and porcine pancreatic elastase (Sigma co., usa), 100nM subtilisin a (Sigma co., usa), or proteinase K (Sigma co., usa) in 100mM tris-HCl (pH 8.0) buffer containing 20mM CaCl 2 and 0.05% Triton X-100, respectively.
After the above solution was reacted at 37 ℃ for 30 minutes without any substrate, 0.5mM S-2251 plasmin substrate (chromogenix Co., USA), 0.5mM S4760 elastase substrate (Sigma Co., USA), and 0.5mM Suc-AAPF-pNA subtilisin A and proteinase K substrates (Sigma Co., USA) were added to the reaction product, followed by reaction at 37 ℃ for 30 minutes.
The recombinant peptide AcScp shows inhibitory activity against plasmin (fig. 3), human neutrophil elastase (fig. 4A), porcine pancreatic elastase (fig. 4B), bacterial and fungal serine proteases, namely subtilisin a (fig. 5A) and proteinase K (fig. 5B) (table 1).
As shown in Table 1, it can be seen that IC 50 inhibiting fibrinolysis of plasmin is 457.98. + -. 11.43nM, IC 50 inhibiting elastase, an inflammation-associated serine protease, is 347.81. + -. 5.79nM for human neutrophil elastase, 94.70. + -. 1.97nM for porcine pancreatic elastase, 379.20. + -. 9.42nM for subtilisin A, and 189.43. + -. 3.14nM for proteinase K, respectively.
[ Table 1]
Inhibitory Activity of recombinant peptide (mature peptide of the Apis cerana Ceramia cDNA) on plasmin, human neutrophil elastase, porcine pancreatic elastase, subtilisin A and proteinase K
| Enzyme | Concentration (nM)a | IC50(nM)b |
| Plasmin | 10 | 457.98±11.43 |
| Human neutrophil elastase | 100 | 347.81±5.79 |
| Porcine pancreatic elastase | 100 | 94.70±1.97 |
| Subtilisin A | 100 | 379.20±9.42 |
| Proteinase K | 100 | 189.43±3.14 |
a: enzyme concentrations used in this assay
Molar ratio of IC 50 to enzyme concentration
(2) Verification of binding capacity of microorganisms
To verify the possibility of using the eastern bee-derived racecadine peptides of the invention as antibacterial or antifungal microbial agents, the binding ability of AcScp was investigated.
More specifically, Bacillus thuringiensis (B.thuringiensis) and Escherichia coli (Escherichia coli) were cultured in Luria-Bertani (LB) medium, respectively, and Beauveria bassiana (Beauveria bassiana) in potato dextrose broth, when the culture broth reached 0.4 at OD 600, the bacteria and fungi were collected, washed with PBS, and mixed with 40. mu.l of a microorganism containing 0.8. mu.g of AcScp recombinant peptide, followed by reaction at room temperature for 10 minutes, after which the product was separated by centrifugation into a supernatant and a microorganism, which were again washed with PBS, and the microorganism and supernatant were subjected to 15% SDS-PAGE, respectively, followed by western blot analysis using His-tagged antibody.
AcScp binds to bacillus thuringiensis as a gram-positive bacterium, escherichia coli as a gram-negative bacterium, and beauveria bassiana as an entomopathogenic fungus (fig. 6A).
To visibly determine the binding capacity of AcScp, the microorganism was reacted with AcScp and immobilized on a slide. The reaction product was stained with a His-tag antibody and a fluorescent antibody, followed by observation and photograph taking using a confocal microscope. As a result, AcScp bound to the bacteria and fungi similarly to the results of Western blot analysis (fig. 6B).
(3) Verification of antimicrobial Activity
In order to verify the antibacterial and antifungal activity of AcScp, which shows specific inhibitory activity against bacterial and fungal serine proteases of bacterial and fungal origin, i.e. subtilisin a and proteinase K, the growth inhibitory activity against the gram-positive bacteria bacillus thuringiensis and the gram-negative bacteria escherichia coli was investigated.
In addition, to investigate antifungal activity against fungi, 2X 10 4 conidia/ml and 200. mu.l of Beauveria bassiana were mixed with various concentrations of AcScp recombinant peptide, subjected to shake culture at 220rpm at 22 ℃ for 48 hours, followed by measuring the fungal growth inhibitory activity at 595 nm.
[ Table 2]
Antibacterial and antifungal Activity of AsScp against bacteria and fungi
| Microorganisms | MIC50(M) | |
| Gram-positive bacteria | Bacillus thuringiensis | 4.21±0.49 |
| Gram-negative bacteria | Escherichia coli | 6.5±1.76 |
| IC50(M) | ||
| Fungi | Fusarium graminearum (F. graminearum) | 2.57±1.31 |
As shown in Table 2, it can be seen that the MIC 50 for the 50% growth inhibitory activity of Bacillus thuringiensis, which is a gram-positive bacterium, was 4.21. + -. 0.49M, the MIC 50 for Escherichia coli was 6.5. + -. 1.76M, and the IC 50 for the 50% growth inhibitory activity of Beauveria bassiana, which is an entomopathogen, was 2.57. + -. 1.31M, respectively.
From the results described above, it can be seen that the eastern bee-derived recombinant ticagrelor peptide has excellent inhibitory activity against subtilisin a and proteinase K and is useful for inhibiting the growth of microorganisms.
Claims (6)
1. Secaine peptide derived from eastern honey bee, consisting of the amino acid sequence defined by SEQ ID No. 2.
2. A polynucleotide consisting of the base sequence defined in SEQ ID No.1 encoding the peptide according to claim 1.
3. a recombinant expression vector comprising the polynucleotide of claim 2.
4. A coagulant comprising the peptide of claim 1 or the expression vector of claim 3.
5. An anti-inflammatory agent comprising the peptide of claim 1 or the expression vector of claim 3.
6. An antibacterial or antifungal agent comprising the peptide of claim 1 or the expression vector of claim 3.
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| KR101481902B1 (en) * | 2013-07-10 | 2015-01-14 | 동아대학교 산학협력단 | Novel polypeptide having antibacterial and antifungal activity |
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| CN1400309A (en) * | 2002-02-01 | 2003-03-05 | 浙江大学 | Large wasp sedative peptide precursor gene and its coded polypeptide and preparation method |
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| ACCESSION：AEM44814，secapin-3 precursor [Apis mellifera];GenPept;《GenPept》;20120901;全文 * |
| Secapin, a bee venom peptide, exhibits anti-fibrinolytic，anti-elastolytic, and anti-microbial activities;Kwang Sik Lee 等;《Developmental and Comparative Immunology》;20160518;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| KR101638863B1 (en) | 2016-07-13 |
| CN106167524A (en) | 2016-11-30 |
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