KR101481902B1 - Novel polypeptide having antibacterial and antifungal activity - Google Patents

Abstract

The present invention relates to a novel polypeptide having antibacterial and antifungal activity, a nucleic acid sequence encoding the same, and an antibacterial and antifungal composition containing the same polypeptide as an active ingredient. According to the present invention, the polypeptide can effectively inhibit the growth of microorganisms such as bacteria and pathogenic fungi by inhibitory activities against subtilisin A and proteinase K. Therefore, the polypeptide can be advantageously used for materials in various fields such as pharmaceutical products, detergents, plant disease preventing agents, coating agents, or cosmetics.

Description

[0001] The present invention relates to novel polypeptides having antibacterial and antifungal activity,

The present invention relates to a novel polypeptide having antibacterial and antifungal activity, a nucleic acid sequence encoding the polypeptide, and a composition for an antibacterial and antifungal agent containing the polypeptide as an active ingredient.

Bees have a bee venom, an effective defense to protect their group from invaders or predators, such as animals and insects. The bee venom is composed of several kinds of venom proteins or peptides, and melittin [Gauldie et al., Eur. J. Biochem., 61: 369-376 (1976)], phospholipase A2 (PLA2) [Six & Biophys. Acta 1488: 1-19 (2000)], apamin [Banks et al., Nature 282: 415-417 (1979)], hyaluronidase [Kreil, Protein Sci. 1666-1669 (1995)], serine protease [Winningham KM et al. J Allergy Clin Immunol 2004; 114: 928-33).

In particular, serine protease and serine protease inhibitory proteins found in various organisms are of great interest because they have various physiological functions such as immune response, complement activity, hemostasis, blood coagulation, fibrinolysis, and inflammation. The serine protease inhibitory protein is known to inhibit the activity of plasmin, trypsin, chymotrypsin, elastase or protease. For this reason, the potential for development of new drugs Has attracted much attention with its achievements.

Insect and other arthropod-derived serine protease inhibitory proteins play a multifunctional role. Spider-derived serine protease inhibitory proteins are known to inhibit trypsin, chymotrypsin, plasmin and elastase as well as potassium ion inhibition, Bombus The anti-fibrinolysis function of the Kunitz-type serine protease inhibiting protein has been reported in Y. K. et al., PLoS ONE. 3: e3414 (2008); Wan H et al., PLoS ONE 8: e53343 (2013), Comp. Biochem. Physiol. B 165: 36-41 (2013), Choo YM et al. PLoS ONE 7: e10393 (2012)].

On the other hand, the Kazal-type serine protease inhibitor (KTSPI), which is one of the serine protease inhibiting proteins, has one or several Kazal domains together with three disulfide bond structures.

The Kajal-type protease inhibitory proteins known so far have been reported to originate from insects such as blood-sucking insects, mosquitoes, silkworms, fruit flies, grasshoppers and beetles (Friedrich et al., J. Biol. Chem. 22: 16216-16222 (Watanabe et al., Biochimie 92: 933-719 (2004)), and Mende et al., Eur. J. Biochem. 266: 583-590 (1999), Insect Biochem. Mol. Biol. B39: 234-240 (2007)), [Gandhe et al., BMC Genomics 7: 184 (2006); Zheng et al., Comp Biochem Physiol. (Brillard-Bourdet et al., Biochem. J. 400: 467-476 (2006)], [Horita et al. al., J. Biol. Chem. 285: 30150-30158 (2010))), yet there has been no report on the function of the honeybee-derived kazar-type protease inhibitory protein.

Thus, the present inventors found that the oriental bees, Apis cerana) derived kajal-type serine was first cloned protease inhibitory protein The cDNA gene coding for the cDNA of kajal recombinant protein produced by introducing a polynucleotide into insect cell lines after sapin the vector encoding the domain subtilis new A and Inhibits the activity of protease K and confirms that the antimicrobial and antifungal effect is excellent against Gram-positive bacteria, phytopathogenic fungi, and insect pathogenic fungi, thereby completing the present invention.

Korean Patent Publication No. 10-2013-0049382

It is therefore an object of the present invention to provide novel polypeptides having antibacterial and antifungal activity.

It is another object of the present invention to provide a nucleic acid molecule encoding said polypeptide.

It is still another object of the present invention to provide a composition for antibacterial and antifungal agents containing the polypeptide as an active ingredient.

In order to accomplish the above object, the present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and having antifungal and antifungal activity.

In one embodiment of the present invention, the polypeptide is tojongbeol (Apis ceral protease inhibitory protein derived from the Kazal domain.

In one embodiment of the present invention, the polypeptide may have the effect of inhibiting the growth of microorganisms through the inhibitory activity against subtilisin A and protease K.

In one embodiment of the invention, the microorganism from which the polypeptide is capable of inhibiting growth is Bacillus subtilis subtilis), Bacillus Tudor ringi N-Sys (Bacillus thuringiensis , beauveria bassiana , and Fusarium graminearum . In the present invention,

The present invention also provides a nucleic acid molecule encoding said polypeptide.

In one embodiment of the present invention, the nucleic acid molecule may be a polynucleotide of SEQ ID NO: 2.

The present invention also provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 1; Or an expression vector containing a polynucleotide encoding said polypeptide as an active ingredient.

In one embodiment of the present invention, the composition may have the effect of inhibiting the growth of bacterial and fungal pathogens through the inhibitory activity against subtilisin A and protease K.

In one embodiment of the present invention, the bacterium is selected from the group consisting of Bacillus subtilis subtilis ) or Bacillus thuringiensis .

In one embodiment of the present invention, the fungal pathogen is beauveria bassiana , or Fusarium graminearum .

In one embodiment of the present invention, the composition is applicable to medicines, detergents, plant disease control agents, coating agents or cosmetics.

The novel polypeptide having antibacterial and antifungal activity according to the present invention can effectively inhibit the growth of microorganisms such as bacteria and pathogenic fungi through the inhibitory activity against subtilisin A and protease K, And can be useful as a material for various fields such as an antiseptic, a coating agent or a cosmetic.

Figure 1 shows the homology between the cotyledon-derived serine protease inhibitory protein of the present invention and another serine-type serine protease inhibiting protein (underlined: Kazal domain; An asterisk: P1 area; Solid circle: a cysteine residue that forms a conserved disulfide bond].
FIG. 2 is a photograph showing the result of electrophoresis of a recombinant protein expressing only the Kazal domain region of the Kajal-type serine protease inhibitory gene of the herbal composition of the present invention.
FIG. 3 is a western blot analysis and confocal microscope photograph showing the binding ability of the recombinant protein of the present invention (kasal domain of serine protease inhibition protein cDNA derived from native bean) to bacterial and fungal pathogens.

In one aspect, the present invention relates to a novel polypeptide having an amino acid sequence represented by SEQ ID NO: 1 or a polypeptide having 90% or more homology thereto and having antibacterial and antifungal activity.

"Kazal-type serine protease inhibitor (KTSPI)" has one or several Kazal domains with three disulfide bond structures.

The serine protease inhibitory protein of the present invention has been reported to be effective in insect, insect, mosquito, silkworm, grasshopper and beetle. The insect-derived cajal-type serine protease inhibitory protein is chymotrypsin, trypsin, It is known to inhibit the activity of plasmin, thrombin, elastase, protease and the like.

The present inventors have found that Apis cerana ) was first cloned, and as a result, it was confirmed that the cDNA gene consisted of 384 nucleotide sequences (387 termination codons) encoding 128 amino acids. I could. In addition, it was confirmed that the protein of the present invention has a P1 domain comprising 56 amino acids and a Kazal domain including 6 cysteine structures.

Since the gene of the present invention was first isolated from native beetle and its characteristics have not yet been reported, the gene was registered with NCBI GenBank database on June 11, 2013 with the approval number KF219696 (cDNA). In the present invention, the nucleotide sequence and the deduced amino acid sequence of the cDNA gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

On the other hand, the novel polypeptide having the antibacterial and antifungal activity of the present invention corresponds to the Kazal domain of the cajal-type serine protease inhibitory protein derived from such nematode ( Apis cerana ). In the present invention, the amino acid sequence and the nucleic acid sequence of the cajal domain are shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

In order to verify the activity of the novel Kajal-type serine protease inhibitory protein derived from the above-mentioned genetic markers, the present inventors have inserted a polynucleotide encoding a cajal domain portion of the protein into a recombinant vector and introduced it into an insect cell line to produce a recombinant protein Then, the subtilisin A or protease K inhibitory activity and antimicrobial activity were tested using the recombinant protein thus produced. As a result, it was confirmed that the recombinant protein (casal domain portion) inhibits the activity of subtilicin A and protease K and has an antibacterial and antifungal effect on Gram-positive bacteria, plant pathogenic and insect pathogenic fungi.

In the present invention, the novel polypeptide having antibacterial and antifungal activity may include not only a wild type but also a functional homolog having an amino acid homology of at least 90% and having an enzyme activity.

The term "homology" in the present invention is intended to indicate a degree of similarity to the amino acid sequence of a wild-type protein. The novel polypeptide of the present invention has a wild-type amino acid sequence that is at least 70% , Preferably 90% or more, and more preferably 95% or more. This comparison of homology can be performed using a visual program or a comparison program that is easy to purchase. Commercially available computer programs can calculate homology between two or more sequences as a percentage, and homology (%) can be calculated for adjacent sequences.

One of ordinary skill in the art will readily recognize that this artificial modification retains at least 90% homology and is an equivalent polypeptide as long as it possesses enzyme activity in the present invention.

The novel polypeptides of the present invention may include amino acid sequence variants thereof as long as they retain antimicrobial and antifungal activity. As used herein, a variant refers to a protein (polypeptide) having a sequence that is different from a natural amino acid sequence and one or more amino acid residues by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

Such variants include functional homologues having substantially equivalent activity to wild-type or polypeptides having modifications that increase or decrease physicochemical properties. Preferably, the physicochemical properties of the polypeptide are modified variants. For example, physical factors such as temperature, moisture, pH, electrolyte, reducing sugar, pressurization, drying, freezing, interfacial tension, lightning, repetition of freezing and thawing, high concentration conditions, and acid, alkali, neutral salt, organic solvent, , Oxidation-reduction agent, protease, and the like are increased in structural stability to the external environment. It may also be a mutant in which the enzyme activity is increased by a mutation in the amino acid sequence.

The novel polypeptides of the present invention tojongbeol (Apis cerana protease inhibitor protein derived from a cerana- derived serine protease inhibitor protein, chemically synthesized, or obtained using a gene recombinant technique.

First, the novel polypeptides of the present invention tojongbeol (Apis cerase protease inhibitory protein derived from a cerana- derived serine protease inhibitor protein. Separation and purification of the cajal-type serine protease inhibitory protein contained in the cells can be carried out by a number of known methods.

The polypeptide of the present invention can also be chemically synthesized. When chemically synthesized, it can be obtained by using a polypeptide synthesis method well known in the art. Polypeptides can be prepared using conventional stepwise liquid or solid phase synthesis, fractional condensation, F-MOC or T-BOC chemistry. In particular, the preferred method of preparing the polypeptide is by using solid phase syntheses. The novel polypeptides of the present invention can be synthesized by a condensation reaction between protected amino acids in a conventional solid phase method, starting from the C-terminus and proceeding sequentially in accordance with the sequence. After the condensation reaction, the protecting group and the carrier to which the C-terminal amino acid is linked can be removed by a known method such as acid decomposition or aminolysis. The above-mentioned polypeptide synthesis methods are described in detail in the relevant publications.

The novel polypeptide of the present invention may also be obtained using a gene recombinant technique. When a recombinant technology is used, a polynucleotide (nucleic acid) encoding a novel polypeptide of the present invention is inserted into an appropriate expression vector, and the vector is transformed into a host cell to introduce a novel polypeptide of the present invention Culturing the cells, and recovering the proteins from the host cells. The protein may be expressed in a selected host cell and then subjected to conventional biochemical separation techniques such as treatment with a protein precipitant (salting-out method), centrifugation, ultrasonic disruption, ultrafiltration, dialysis, (Gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like. In order to separate proteins (polypeptides) having high purity, they are used in combination.

In another aspect, the invention relates to a nucleic acid molecule encoding a polypeptide having said antimicrobial and antifungal activity.

The nucleic acid molecule of the present invention preferably has the nucleotide sequence shown in SEQ ID NO: 2, and may include a nucleic acid sequence having 90% or more homology thereto.

Nucleic acid molecules of the present invention include functional equivalents thereof that perform functionally equivalent functions, such as by deletion, substitution, insertion or deletion of a part of the nucleotide sequence by an artificial modification insertion or variant thereof, or functional fragments of the nucleic acid molecule which perform the same function.

As those skilled in the art will appreciate, as long as the nucleotide sequence retaining more than 70% homology by such an artificial modification retains the antimicrobial and antifungal activity activity for gene expression desired in the present invention, It is easily understood that the present invention belongs to the scope of the present invention.

In another aspect, the present invention relates to a vector comprising the nucleic acid molecule.

The nucleic acid molecule encoding the novel antibacterial and antifungal activity polypeptides of the present invention may be provided by a vector expressing it and expressed as a protein.

The term "vector" as used herein refers to a DNA construct containing a base sequence of a gene operably linked to a suitable regulatory sequence so as to be capable of expressing the gene of interest in a suitable host, said regulatory sequence being capable of initiating transcription A promoter, any operator sequence for modulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation.

In the present invention, "operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein to perform a general function. For example, a nucleic acid sequence encoding a promoter and a protein or RNA may be operably linked to affect the expression of the coding sequence. The operative linkage with the recombinant vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage are made using enzymes generally known in the art.

The vector of the present invention may include expression regulatory elements such as a promoter, an initiation codon, a stop codon, a polyadenylation signal and an enhancer, a secretion signal, and the like, and may be variously manufactured according to the purpose. The initiation codon and the termination codon must be operative in the individual when the gene construct is administered and in the coding sequence and in frame.

The vector used in the present invention is not particularly limited as long as it is replicable in a host, and any vector known in the art can be used. For example, non-viral vectors or viral vectors can be used.

As the non-viral vector, a plasmid is representative. The plasmid expression vector is a method for transferring plasmid DNA directly to human cells using an approved FDA-approved gene transfer method which can be used for human. Unlike the viral vector, the plasmid DNA has an advantage that it can be homogeneously purified. As the plasmid expression vector that can be used in the present invention, mammalian expression plasmids known in the art can be used. Examples include, but are not limited to pRK5 (European Patent No. 307,247), pSV16B (International Patent Publication No. 91/08291) and pVL1392 (PharMingen).

A viral vector can also be used as an expression vector of the present invention. Viral vectors include, for example, retroviruses, adenoviruses, adenovirus-associated viruses, herpes viruses and avipoxviruses. The viral vector must meet the following criteria: (1) it should be able to infect the desired cell, so that a viral vector with an appropriate host range has to be selected; (2) And (3) the vector must be safe for the host.

The retroviral vector is constructed such that all of the viral genes have been removed or altered so that the non-viral proteins are made in cells infected with the viral vector. The main advantage of retroviral vectors for gene therapy is that they transfer a large number of genes into the cloned cells, accurately integrate the genes transferred into the cellular DNA, and do not lead to subsequent infection after gene transfection. FDA-approved retroviral vectors were prepared using PA317 amphotropic retroviral packaging cells (Miller, A.D. and Buttimore, C., Molec. Cell Biol., 6: 2895-2902, 1986).

Non-retroviral vectors include the adenoviruses mentioned above. The main advantage of adenoviruses is the ability to carry large quantities of DNA fragments (36 kb genome) and to infect very high reverse non-cloned cells. Herpesviruses may also be useful for human gene therapy (Wolfe, J. H., et al., Nature Genetics, 1: 379-384, 1992). In addition, a known appropriate viral vector can be used.

Other viral vectors that may be used for gene delivery into cells include, but are not limited to, murine leukemia virus (MLV), JC, SV40, polyoma, Epstein-Barr virus papilloma virus, vaccinia, poliovirus, herpes virus, Viruses, other human and animal viruses.

The expression vector according to the present invention can be introduced into cells using methods known in the art. But are not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran- DEAE Dextran-mediated transfection, polybrene-mediated transfection, electropora tion, gene gun, and other notifications for introducing nucleic acids into cells (Wu et al., J. Bio. Chem., 267: 963-967, 1992; Wu and Wu, J. Bio. Chem., 263: 14621-14624, 1988) .

In addition, the vector of the present invention may further include a selection marker. The selection marker is used to select a cell transformed with a vector, that is, to confirm whether a target gene has been inserted, and to give a selectable phenotype such as resistance to drug resistance, nutritional requirement, tolerance to cytotoxic agent, or surface protein expression May be used. In the environment treated with the selective agent, only the cells expressing the selection marker survive or express different phenotypes, so that the transformed cells can be selected.

In another aspect, the present invention provides a transformant transformed with said vector. The transformant of the present invention can be constructed by introducing the vector into the host cell in such a manner that the promoter can function.

The term "transformed" in the present invention means that DNA is introduced into the host and the DNA becomes replicable as an extrachromosomal factor or by chromosome integration completion. Transformation includes any method of introducing a nucleic acid molecule into an organism, cell, tissue or organ, and can be carried out by selecting a suitable standard technique depending on the host cell as is known in the art. Such methods include electroporation, CaPO4 precipitation, CaCl2 precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposomes, and lithium acetate- DMSO, and the like, but are not limited thereto.

Since the expression amount of the protein and the expression are different depending on the host cell transformed with the expression vector, the host cell most suitable for the purpose may be selected and used. Host cells that can be used in the present invention include insect cell lines, yeast, fungi, bacteria, or algae. For example, the insect cell line Sf9 (Spodoptera frugiperda 9) is preferable, but not limited thereto.

In the following example of the present invention, an insect baculovirus transfer vector (pBAC8) comprising a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 was co-transfected into an insect cell line Sf9 (Spodoptera frugiperda 9) After 5 days, the culture broth was collected to prepare a recombinant baculovirus expressing the Kazal domain among the Kajal-type serine protease inhibitory proteins derived from the native bean. Recombinant baculovirus was proliferated in Sf9 cell line, and the recombinant protein of the present invention (kasal domain of serine protease inhibition protein cDNA derived from native clone) was isolated using MagneHis protein purification system (Promega, USA).

In another embodiment, the present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 1; Or an expression vector containing a polynucleotide encoding the polypeptide as an active ingredient.

In the present invention, not only the polypeptide wild type consisting of the amino acid sequence shown in SEQ ID NO: 1 but also the functional homolog having the same antibacterial and antifungal activity with 90% or more amino acid homology can be included.

The polynucleotide encoding the polypeptide of the present invention may be isolated from nature or artificially synthesized or modified. The nucleotide sequence encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is a nucleotide sequence in which at least one nucleotide base is substituted Deletion or insertion of the protein, and the protein expressed by such modification should not contain any significant change in its biological functionality. Such modifications include modifications to heterologous homologous genes.

Therefore, the polynucleotide encoding the polypeptide is characterized in that it is represented by the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto. The polynucleotide encoding the polypeptide may be at least 70%, preferably at least 80%, more preferably at least 90% And is represented by a nucleotide sequence having homology.

Preferably, the polynucleotide of SEQ ID NO: 2 of the present invention may be provided in the form of a recombinant expression vector operably linked to a vector expressing it. Such expression vectors include, but are not limited to, plasmids, phage, cosmid, viral vectors or other vectors known in the art. The vector may be self-replicating or integrated into the host DNA.

Since the composition of the present invention has antibacterial and antifungal use (effect), it can be usefully used as a material for products such as medicines, detergents, plant disease control agents, coating agents or cosmetics.

First, since the composition of the present invention contains an expression vector containing a polypeptide having excellent antibacterial and antifungal activity or a polynucleotide encoding the same as an active ingredient, it can be used for therapeutic or prophylactic treatment of veterinary medicine or human have. For example, since antimicrobial polypeptides generally can enhance the function of adhesion-related receptors and adhesion molecules of therapeutic cells (cell therapy agents), the use of such antimicrobial polypeptides may be beneficial for the target organs (bone marrow, Damaged tissue, lymph node, tumor tissue, etc.) can be significantly increased. Therefore, the composition of the present invention contains an antimicrobial polypeptide as an active ingredient, and can be usefully used for the prevention and treatment of various diseases that are treated using a cell therapy agent for the purpose of enhancing the engrafting function in vivo.

In addition, since the composition of the present invention contains an expression vector containing a polypeptide having excellent antibacterial and antifungal activity or a polynucleotide encoding the same as an active ingredient, the composition of the present invention can be used as a pathogen control agent, a pest repellent agent, Can be used. For example, by contacting or treating the composition of the present invention with plant cells or plant seeds, pathogens present on the surface of plant cells or plant seeds can be significantly inhibited.

In particular, the composition of the present invention contains an antibacterial polypeptide as an active ingredient, and the polypeptide is Bacillus subtilis subtilis) or Bacillus tube ringi N-Sys (at the same time as inhibiting the growth of Bacillus thuringiensis), Fusarium widely known as plant pathogens Graeme California room (Fusarium graminearum) bar hayeotneun proven experimentally that it is possible to effectively inhibit the growth of, the present invention Can be usefully used as a biological plant disease controlling agent.

In addition, since the composition of the present invention contains an expression vector containing a polypeptide having excellent antibacterial and antifungal activity or a polynucleotide encoding the same as an active ingredient, the composition can be used for the application of a coating agent to an article requiring antibacterial activity. For example, the composition of the present invention can be used as an antimicrobial tablet, an antibacterial toothpaste container, an antibacterial toothbrush pack, an antibacterial cosmetics container, an antibacterial waste basket, an antibacterial bath, an antibacterial cup, an antibacterial bottle, Antibacterial toilet seat cover, Antibacterial toilet seat cover, Antibacterial toilet seat cover, Antibacterial toilet seat cover, Antibacterial toilet seat cover, Antibacterial toilet seat cover, Antibacterial toilet seat cover, , Antimicrobial diapers, antibacterial sanitary napkins, antibacterial masks, antibacterial bandages, antibacterial bands, antimicrobial medical articles, antibacterial fruit wrapping paper, antibacterial flower wrapping paper, antimicrobial wallpaper, antimicrobial plate and antibacterial tile It can be usefully used as a material of an antimicrobial composition.

In addition, the composition of the present invention may be used in combination with other antimicrobial substances known so far to further enhance antibacterial and antifungal activity.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.

< Example  1>

Origin Kazal  A gene encoding a type serine protease inhibitory protein Cloning

In order to clone a gene coding for the Kajal type serine protease inhibition protein derived from Toyohara bean, the Uni-ZAP XR vector and Gigapack III Gold Packing Extract, which are commercially available kits using poly (A) and mRNA extracted from Apis cerana, (Stratagene, USA), and analyzed expressed sequence tags (ESTs). DNA extraction was performed using a Wizard mini-preparation kit (Promega, USA) as a commercial kit. The DNA sequence was sequenced with an automated DNA sequencer (Applied Biosystems, USA). The nucleotide sequences were compared by the BLAST program of NCBI (http://www.ncbi.nlm.nih.gov/BLAST). Using the DNA Walking Speedup ™ Premix Kit (Seegene, Korea) based on some of the identified sequences, the nucleotide sequences of the 5 'and 3' ends were all revealed.

As a result, it was confirmed that the gene coding for the chalcone-type serine protease inhibitory protein derived from canola beads was composed of 384 nucleotide sequences (387 including the termination codon) encoding 128 amino acids (see FIG. 1) Confirmed that the analyzed nucleic acid sequence was a novel nucleic acid sequence that had not been previously identified and was listed in the NCBI GENE BANK database (KF219696). The nucleotide sequence and the amino acid sequence of the above-identified gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The results of analysis by the BLAST program of the above sequence are shown in Fig.

As shown in FIG. 1, the Kazal-type serine protease inhibitory protein derived from native bean, which is identified in the present invention, has relatively high homology with the Kazal-type serine protease inhibitory proteins reported from existing bees, and has three disulfide bonds. The cysteine residues and the P1 region were well preserved.

< Example  2>

[0030] Kazal  The production of recombinant vectors expressing type serine protease inhibitory proteins and From an insect cell line  Confirmation of expression

In order to express the Kajal-type serine protease inhibitory protein of the present invention, the Kajal domain portion of the serine protease inhibitory protein cDNA derived from the genus bean was inserted into an Autographa californica nucleopolyhedrovirus transfer vector pBAC8 (Clontech, USA) (500 ng) and bAcGOZA virus DNA (100 ng) [Je et al., Biotechnol. Lett., 23: 575-582 (2001)] were co-transfected into the insect cell line Sf9 (Spodoptera frugiperda 9) using lipofectin (Clonetech). After 5 days, the culture broth was collected to prepare a recombinant baculovirus expressing the casar domain portion of the serine protease inhibitory protein cDNA derived from canola beet. Recombinant baculovirus was propagated in Sf9 cell line, and the recombinant protein (kasal domain of serine protease inhibition protein cDNA derived from bean curd) was isolated using MagneHis protein purification system (Promega, USA). An electrophoresis photograph of the isolated recombinant protein (casal domain of serine protease inhibition protein cDNA derived from native bean) is shown in FIG.

As a result, as shown in Fig. 2, it was confirmed that the recombinant protein (casal domain of the serine protease inhibition protein cDNA derived from the native bean) was a protein having a molecular weight of 10 kDa on the electrophoresis.

< Example  3>

The recombinant protein of the present invention (serine protease inhibitory protein cDNA of Kazal  Domain)

In order to verify the characteristics of the chimeric serine protease inhibitory protein derived from native rice beet according to the present invention as identified in Example 1, the recombinant protein produced in Example 2 (the chasm domain of the serine protease inhibitory protein cDNA derived from native rice beet) ) Were assayed for their inhibitory activity against various enzymes, and their microbial binding ability and antimicrobial activity were confirmed.

<3-1> Subtilisin  A or protease K inhibitory activity

In order to verify the properties as serine protease inhibiting proteins, serine protease trypsin, chymotrypsin, elastase, thrombin, tissue plasminogen activator, (Sigma, USA), 20 mM trypsin (Sigma, USA), alpha chymotrypsin (Sigma, USA), and Elastase (Sigma, USA) in order to determine whether the compounds inhibited the tPA, factor Xa, subtilisin A and protease K (Sigma, USA), protease K (Sigma, USA), thrombin (Sigma, USA), tissue plasminogen activator (Sigma, USA), Factor Xa , USA) was treated with various concentrations of AcKTSPI recombinant protein in 100 mM Tris-HCl (pH 8.0) buffer containing 20 mM CaCl 2 and 0.05% Triton X-100. (Sigma, USA), 0.5 mM BApNA (Sigma, USA), chymotrypsin, subtilicin A and protease K substrate 0.5 mM Suc-AAPF-pNA (Chromogenix, Sweden), a tissue plasminogen activator substrate 0.5 mM S2288 (Chromogenix, Sweden) and a Factor Xa substrate 0.5 mM S2222 (Chromogenix, Sweden), thrombin substrate S2238 ) Was added and reacted at 37 캜 for 30 minutes.

As a result, the recombinant protein of the present invention (kasal domain of serine protease inhibition protein cDNA derived from native bean) showed no inhibitory activity against trypsin, alpha chymotrypsin, elastase, thrombin, tissue plasminogen activator and Factor Xa, And subtilicin A and protease K which are fungal-derived serine proteases.

In detail, as shown in the following Table 1, the recombinant protein of the present invention (kasal domain of serine protease inhibitory protein cDNA) of the present invention had 50% inhibitory concentration IC ₅₀ of 67.03 nM in subtilisin A and an IC ₅₀ of protease K And 91.53 nM, respectively.

The inhibitory activity against the subtilisin A and protease K of the recombinant protein of the present invention (casal domain of the serine protease inhibitory protein cDNA derived from native bean) enzyme Concentration (nM) ^a IC ₅₀ (nM) Ratio ^b Ki (nM) Subtilisin A 10 67.03 0.67 83.39 Protease K 10 91.53 0.92 127.28

a: concentration of enzymes used in this experiment

b: mole ratio of IC ₅₀ to enzyme concentration

<3-2> Microorganisms Cohesion  Confirm

In order to confirm the possibility of using the recombinant protein of the present invention (kasal domain of serine protease inhibition protein cDNA derived from native bean) as an antimicrobial agent, the binding ability of the protein to the microorganism was confirmed.

In detail, Bacillus subtilis , B. thuringiensis , and Escherichia coli in Luria-Bertani (LB) medium and Beauveria bassiana and Fusarium Graminearum was cultured in potato dextrose broth. When the culture solution reached 0.4 at OD ₆₀₀ , the bacteria and fungi were collected, washed with PBS, and 40 μl containing 0.8 μg of the recombinant protein of the present invention (kasal domain of serine protease inhibitor protein cDNA derived from native cloned bees) And reacted at room temperature for 10 minutes. After 10 minutes, the supernatant and microorganisms were separated by centrifugation, and the microorganisms were washed again with PBS. Each microorganism and supernatant were subjected to 15% SDS-PAGE and subjected to Western blot analysis using a His-tag antibody.

As a result, as shown in Fig. 3, the recombinant protein of the present invention (kasal domain of serine protease inhibition protein cDNA derived from the native bean) was isolated from bacteria B. subtilis and B. thuringiensis except for E. coli , and B. bassiana and F. graminearum , respectively.

<3-3> Antimicrobial  Verify Active

In order to confirm the antimicrobial activity of the recombinant protein of the present invention (kasal domain of the serine protease inhibition protein cDNA derived from bean curd), the growth inhibitory activity against Gram-positive bacteria B. thuringiensis , B. subtilis and Gram-negative bacteria E. coli was examined . The 96-well plate was mixed with 200 쨉 l of bacteria (10 ⁵ cfu / ml) and various recombinant proteins of the present invention (kasal domain of serine protease inhibitory protein cDNA derived from the cotyledon) at a concentration of 220 rpm And the bacterial growth inhibitory activity was measured at 595 nm. In addition, to investigate the antifungal activity against fungi, 200 占 퐇 of 2 占⁰⁴ conidia / ml of B. bassiana and F. graminearum were mixed with various concentrations of the recombinant proteins of the present invention and incubated at 22 占 폚 for 48 hours at 220 rpm And the fungal growth inhibitory activity was measured at 595 nm.

As a result, as shown in Table 2, the gram-negative bacteria is E. coli, but did not determine the growth inhibitory activity, gram-positive bacteria of B. thuringiensis and B. subtilis of 50% growth inhibition MIC ₅₀ are respectively 53.33 and 43.83 And 50% growth inhibitory activity IC ₅₀ of insect pathogen B. bassiana and plant pathogen F. graminearum were 12.44 and 92.15, respectively.

The antimicrobial activity of the recombinant protein of the present invention (kasal domain of the serine protease inhibitory protein cDNA derived from native bean) against bacteria and fungi Kinds microbe Inhibitory activity Gram-positive bacteria B. subtilis 43.83 MIC ₅₀ ([mu] M) B. thuringiensis 53.33 MIC ₅₀ ([mu] M) Gram-negative bacteria E. coli ND mold B. bassiana 12.44 IC ₅₀ (μM) F. graminearum 92.15 IC ₅₀ ([mu] M)

ND, not detected.

As a result, it was confirmed that the recombinant protein of the present invention (kasal domain of serine protease inhibition protein cDNA derived from native bean) was excellent in inhibitory activity against subtilisin A and protease K, and the growth of microorganisms was effectively inhibited Respectively.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> Novel polypeptide having antibacterial and antifungal activity <130> PN1306-207 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 56 <212> PRT <213> Artificial Sequence <220> <223> Novel kazal domain polypeptide <400> 1 Asp Gln Cys Val Ala Thr Cys Arg Thr Thr Pro Glu Tyr Asn Pro Val   1 5 10 15 Cys Gly Thr Asp Gln Ile Asp Tyr Lys Asn Pro Gly Gln Leu Ser Cys              20 25 30 Ala Ser Met Cys Gly Lys Asp Val Ser Leu Lys His Tyr Gly Arg Cys          35 40 45 Thr Thr Lys Ile Arg Gly Arg      50 55 <210> 2 <211> 171 <212> DNA <213> Artificial Sequence <220> <223> Novel accidental domain polynucleotide <400> 2 gatcaatgtg ttgcaacatg tcggacgaca cccgaatata atcctgtatg tggaacagac 60 caaatcgact ataaaaatcc tgggcaacta agctgtgcat ctatgtgtgg aaaagatgtt 120 tcattaaaac attatggacg ttgcacgact acgaaaataa gaggaagata a 171 <210> 3 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> Kazal-type serine protease inhibitor cDNA sequence <400> 3 atgtggtttt ttctaatatt agctattgct gcgaatagga atgtaatcgc atttccacag 60 aatgacttga gcgataacaa tattgagatt caaacgctac ctaccacatt cgaagtagat 120 ggatttattt ttgatggacc agcaaataga ccattacggc aaacttccac aattataaca 180 actactacta cagcagctac agttgatcct caattcgatc aatgtgttgc aacatgtcgg 240 acgacacccg aatataatcc tgtatgtgga acagaccaaa tcgactataa aaatcctggg 300 caactaagct gtgcatctat gtgtggaaaa gatgtttcat taaaacatta tggacgttgc 360 acgactacga aaataagagg aagataa 387 <210> 4 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Kazal-type serine protease inhibitor amino acid sequence <400> 4 Met Trp Phe Phe Leu Ile Leu Ala Ile Ala Ala Asn Arg Asn Val Ile   1 5 10 15 Ala Phe Pro Gln Asn Asp Leu Ser Asp Asn Asn Ile Glu Ile Gln Thr              20 25 30 Leu Pro Thr Thr Phe Glu Val Asp Gly Phe Ile Phe Asp Gly Pro Ala          35 40 45 Asn Arg Pro Leu Arg Gln Thr Ser Thr Ile Ile Thr Thr Thr Thr Thr      50 55 60 Ala Ala Thr Val Asp Pro Gln Phe Asp Gln Cys Val Ala Thr Cys Arg  65 70 75 80 Thr Thr Pro Glu Tyr Asn Pro Val Cys Gly Thr Asp Gln Ile Asp Tyr                  85 90 95 Lys Asn Pro Gly Gln Leu Ser Cys Ala Ser Met Cys Gly Lys Asp Val             100 105 110 Ser Leu Lys His Tyr Gly Arg Cys Thr Thr Thr Lys Ile Arg Gly Arg         115 120 125

Claims (12)

1. A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 and having antibacterial and antifungal activity. The method according to claim 1,
The polypeptide may be produced by Apis wherein the polypeptide is a casal domain of a chalcone-type serine protease inhibitory protein derived from a cerana . The method according to claim 1,
Wherein said polypeptide has an effect of inhibiting the growth of microorganisms through inhibitory activity against subtilisin A and protease K. &lt; RTI ID = 0.0 &gt; 25. &lt; / RTI &gt; The method of claim 3,
Microorganisms capable of inhibiting growth of the polypeptide include Bacillus subtilis subtilis), Bacillus Tudor ringi N-Sys (Bacillus thuringiensis , beauveria bassiana , and Fusarium graminearum . The antimicrobial and antifungal activity of the polypeptide according to claim 1, A nucleic acid molecule encoding the polypeptide of claim 1. 6. The method of claim 5,
Wherein the nucleic acid molecule comprises the polynucleotide of SEQ ID NO: 2. A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; or
A composition for an antibacterial and antifungal agent comprising an expression vector containing a polynucleotide encoding said polypeptide as an active ingredient. 8. The method of claim 7,
Wherein the composition has an effect of inhibiting the growth of bacterial and fungal pathogens through inhibitory activity against subtilisin A and protease K. 9. The method of claim 8,
The bacterium is Bacillus subtilis subtilis) or Bacillus tube ringi N-Sys (composition for antimicrobial and antifungal activity, characterized in that Bacillus thuringiensis). 9. The method of claim 8,
The fungal pathogens include beauveria bassiana), or Fusarium Grammy California Room (Fusarium graminearum ). &lt; / RTI &gt; 8. The method of claim 7,
Wherein the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 2. 12. The method according to any one of claims 7 to 11,
Wherein the composition is applied to medicines, detergents, plant disease control agents, coating agents or cosmetics.

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