KR101773362B1 - New polipeptides having an antibiotics and an antifungal activity from Osmia cornifrons - Google Patents

Abstract

The present invention relates to a novel polypeptide having antimicrobial and antifungal activity derived from horsetail scissors, a nucleic acid sequence encoding the same, and a composition for an antibacterial and antifungal agent containing the polypeptide as an active ingredient.
The novel polypeptide having antimicrobial and antifungal activity derived from the hairpin scissors according to the present invention can effectively inhibit the growth of microorganisms such as bacteria and pathogenic fungi through the inhibitory activity against chymotrypsin, subtilisin A and protease K , Medicines, detergents, herbicides, coating agents or cosmetics.

Description

TECHNICAL FIELD [0001] The present invention relates to novel polypeptides having antimicrobial and antifungal activity derived from hairy horned scabbard (New Polipeptides having an antibiotic and an antifungal activity from Osmia cornifrons)

The present invention relates to the use of Osmia < RTI ID = 0.0 > The present invention relates to a novel polypeptide having antifungal and antifungal activity derived from cornifrons , and more particularly, to a novel polypeptide comprising a cDNA gene encoding a serine protease inhibitory protein derived from a horny scallop, a nucleic acid sequence encoding the novel polypeptide, To a composition for antibacterial and antifungal use.

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 elimination.

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.

In particular, insect-derived serine protease inhibitory proteins are known to be involved in the immune response, and functions such as bee, Drosophila, Bombyx mori, mosquito (Aedes aegypti, Anopheles gambiae, Manduca sexta) It is revealed.

Korean Patent No. 10-1481902 (a novel polypeptide having antibacterial and antifungal activity) and Korean Patent No. 10-1391988 (Kunitz-type serine protease inhibitory polypeptide of a bovine serum having a fibrinolytic function and A nucleotide sequence encoding the same) and the like. However, the function of the serine protease inhibitory protein derived from the horny scallop has not been reported yet.

Therefore, the present inventors first cloned a cDNA gene coding for a serine protease inhibitory protein derived from horsetail scissors, inserted a polynucleotide encoding the cDNA into a vector, introduced it into an insect cell line, and produced recombinant proteins containing subtilisin A and protease K activity, and confirmed that the antimicrobial and antifungal effects were excellent against Gram-positive bacteria, phytopathogenic fungi, and insect pathogenic fungi, thereby completing the present invention.

It is an object of the present invention to provide a novel polypeptide having antimicrobial and antifungal activity derived from horsetail scissors.

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.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

To attain the above object, the polypeptide of the present invention having antimicrobial and antifungal activity is called Osmia cornifrons) written as the origin, it is characterized by consisting of an amino-acid sequence shown in SEQ ID NO: 1.

In one embodiment of the invention, the polypeptide may be a serine protease inhibiting protein.

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

In one embodiment of the present invention, the microorganism capable of inhibiting growth of the polypeptide is selected from the group consisting of Bacillus subtilis , Escherichia coli , and beauveria bassiana Or more.

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.

In addition, the present invention relates to the use of Osmia cornifrons ) derived polypeptides; Or an expression vector comprising a polynucleotide encoding said polypeptide as an active ingredient. The polypeptide is characterized by comprising the amino acid sequence shown in SEQ ID NO: 1.

In one embodiment of the present invention, the composition may have an effect of inhibiting growth of bacteria and fungal pathogens through inhibitory activity against any one or more of chymotrypsin, subtilisin A, and protease K.

In one embodiment of the present invention, the bacterium is selected from the group consisting of Bacillus subtilis or Escherichia It can be coli).

In one embodiment of the present invention, the fungal pathogen is beauveria bassiana ).

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 antimicrobial and antifungal activity derived from the hairpin scissors according to the present invention can effectively inhibit the growth of microorganisms such as bacteria and pathogenic fungi through the inhibitory activity against chymotrypsin, subtilisin A and protease K , Medicines, detergents, herbicides, coating agents or cosmetics.

Fig. 1 shows the homology between the serine protease inhibitory amino acid sequence derived from the hairpin scissors of the present invention and another bee-derived serine protease inhibitor amino acid sequence (asterisk: P1 region; Solid circles: cysteine residues that are conserved in each other.
Figure 2 shows the homology between the RCL region of the serine protease inhibitory protein of the hairpin scabbard of the present invention and the Serpin sequence of other insects (asterisk: P1 region; Solid circles: Cysteine residues that are conserved in each other.
Fig. 3 is a photograph showing electrophoresis results of the recombinant protein of the present invention (serine protease inhibitory protein cDNA derived from horseradish scabbard, OcSPI).
(A) Recombinant protein molecular weight of OCSPI
(B) Molecular weight of glycoprotein
Fig. 4 is a photograph showing the result of expressing the site of expression of the recombinant protein of the present invention (serine protease inhibitory protein cDNA, OcSPI derived from horseshoe shearbird) in the horned pinchers.
FIG. 5 is a graph showing the inhibitory activity of the recombinant protein of the present invention (serine protease inhibitory protein cDNA derived from horseradish peroxidase, OcSPI) to an animal-derived serine protease.
6 is a graph showing the inhibitory activity against the microbial serine protease of the recombinant protein (serine protease inhibitory protein cDNA, OcSPI derived from horseshoe crab) of the present invention.
FIG. 7 is a western blot analysis and confocal microscope photograph showing the binding ability of the recombinant protein (serine protease inhibitory protein cDNA, OcSPI) of the present invention to bacterial and fungal pathogens.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail, and a detailed description of known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted.

The present invention relates to a novel polypeptide derived from a horseshoe shearbird having antibacterial and antifungal activity, and is characterized by comprising the amino acid sequence shown in SEQ ID NO: 1.

The present inventors first cloned a cDNA gene coding for a serine protease inhibitory protein from Osmia cornifrons as a pollen-mediated insect. As a result, the cDNA gene was composed of 405 amino acid residues, And 45 to 69% homology with the protease inhibitory protein (Fig. 1). In addition, it contains a serine protease RCL (potential reactive center loop) region at the C-terminus, and it was confirmed that this region and the Drosophila-derived Serpin27A show homology of 37% (Fig. 2).

Since the gene of the present invention was first isolated from the hairy horned scallop and its characteristics have not yet been reported, the gene was registered on Apr. 17, 2015 with the approval number KR140160 (cDNA) in the NCBI GenBank database.

On the other hand, the novel polypeptide having antibacterial and antifungal activity of the present invention is a serine protease inhibitory protein, and the amino acid sequence and the nucleic acid sequence encoded thereby are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

In order to verify the activity of a novel serine protease inhibitory protein derived from the horsetail scissors, the present inventors have inserted a polynucleotide encoding the polypeptide into a recombinant vector, introduced it into an insect cell line to produce a recombinant protein, The inhibitory activity and antimicrobial activity of chymotrypsin, subtilisin A, and protease K were examined using recombinant proteins. As a result, it was confirmed that the recombinant protein inhibited the activity of chymotrypsin, subtilicin A, and protease K, and had antimicrobial and antifungal effects 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 can be prepared by directly isolating from the serine protease inhibitory protein derived from the hairpin scabbard, chemically synthesized, or obtained using gene recombination technology.

First, the novel polypeptide of the present invention can be prepared by directly isolating from a serine protease inhibitory protein derived from hairy horned scabbard. Separation and purification of the serine protease inhibiting 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 methods for synthesizing a polypeptide are described in detail in related books.

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 virus vector must meet the following criteria:

(2) the transferred gene should be able to be preserved and expressed in cells for an appropriate period of time; and (3) the vector Should 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) Day, the culture broth was collected to prepare recombinant baculovirus expressing the serine protease inhibitory protein derived from the horned scissors. Bekyul recombinant virus was grown in Sf9 cells, the recombinant protein of the present invention was isolated by using MagneHis ^TM 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 and antifungal polypeptide as an active ingredient, and the polypeptide is Bacillus subtilis subtilis ) or Escherichia In addition , it has been experimentally proved that the growth of E. coli and the growth of beauveria bassiana , widely known as an insect pathogenic fungus, can be effectively inhibited. As a result, It can be useful as a plant disease control 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 to date in order to further enhance antibacterial and antifungal activity

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes only and do not limit the scope of protection of the present invention.

Example 1 Cloning of a gene (OcSPI) encoding a serine protease inhibitory protein derived from the hairpin scabbard

1. Experimental Method

In order to clone a gene encoding a serine protease inhibiting protein derived from the hairy scissors, ESTs were analyzed after constructing a cDNA library from females of hairy horned scissors.

Full-length OcSPI gene EST was identified from the cDNA library.

Specifically, the 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 ^TM Premix Kit (Seegene, Korea), the nucleotide sequences at the 5 'and 3' ends were determined based on the identified sequences.

2. Experimental results

As a result of the above experiment, it was confirmed that the gene encoding the serine protease inhibitory protein derived from the hairpin scabbard was composed of 405 amino acid residues. The present inventors confirmed that the analyzed nucleotide sequence is a novel nucleotide sequence Respectively.

The nucleotide sequence and the nucleotide sequence of the above-identified gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The results of analysis by the BLAST program of the above sequence are shown in FIGS. 1 and 2. FIG.

As shown in FIG. 1 and FIG. 2, the serine protease inhibitory protein derived from the horned scissors identified in the present invention has a homology of 45 to 69% with the previously reported serine protease inhibitory protein (FIG. 1) -Terminal protease, RCL (potential reactive center loop) region, which is homologous to Serpin27A from Drosophila (Fig. 2), and three disulfide bond cysteines cysteine residues and PI regions were well preserved.

Example 2 Preparation of Recombinant Vector Expressing Serine Protease Inhibitory Protein Derived from Hairy Herringbone of the Present Invention and Expression in Insect Cell Lines

1. Experimental Method

In order to express the serine protease inhibitory protein of the present invention, a gene (OcSPI) cDNA encoding a serine protease inhibitory protein derived from the hairpin scabbard of Example 1 was prepared using a baculovirus system to prepare a recombinant vector After that, the recombinant protein was expressed in sf9 insect cell line and confirmed by electrophoresis.

Specifically, the serine protease inhibitory protein cDNA derived from the horned scissors was mixed with an insect baculovirus ( Autographa californica nucleopolyhedrovirus transfection vector pBAC8 (Clontech, USA), and then transfection vector (500 ng) and bacGOZA virus DNA (100 ng) were ligated together using Lipofectin (Clonetech) And co-transfected into the insect cell line Sf9 (Spodoptera frugiperda 9). After 5 days, the culture broth was collected to prepare recombinant baculovirus expressing the serine protease inhibitory protein cDNA derived from the horned scissors. Recombinant baculovirus was proliferated in Sf9 cell line. Recombinant protein (serine protease inhibition protein cDNA, OcSPI from hairy horn scallop) was isolated using MagneHis ^™ protein purification system (Promega, USA).

2. Experimental results

As a result of the above experiment, electrophoresis photographs of the isolated recombinant protein (serine protease inhibition protein cDNA from hairy horn scallop, OcSPI) are shown in FIG. 3 and FIG.

As a result, as shown in Fig. 3, it was confirmed that the recombinant protein (serine protease inhibition protein cDNA, OcSPI derived from horseshoe shear) was a protein having a molecular weight of 64 kDa on the electrophoresis (Fig. 3-A). It was also identified as a 64 kDa glycoprotein by glycoprotein assay via treatment with tunicamycin (Fig. 3-B).

4, expression of the recombinant protein (serine protease inhibition protein cDNA, OcSPI derived from horseshoe shearbird) in the horny scallop epidermis is expressed in the epidermis, the fat body, the gut, the venom gland) but no expression in muscle (Muscle).

Example 3: Verification of the activity of the recombinant protein of the present invention (serine protease inhibitory protein cDNA derived from horseshoe scissors, OcSPI)

In order to verify the characteristics of the serine protease inhibitory protein of the present invention, which has been identified in Example 1, the recombinant protein (serine protease inhibitory protein cDNA derived from the hairy horn scallop, OcSPI) against various enzymes was measured, and microbial binding ability and antimicrobial activity were confirmed.

<3-1> Identification of inhibitory activity against various enzymes

1. Experimental Method

In order to verify the properties as serine protease-inhibiting proteins, trypsin, chymotrypsin and serine protease, Subtilisin A and protease K (protease K), which are animal-derived serine proteases, K) was inhibited.

Specifically, 20 nM of alpha chymotrypsin (Sigma, USA), trypsin (Sigma, USA) was added to confirm that it inhibited serine protease chymotrypsin, trypsin, subtilisin A and protease K. [ AcCTSPI recombinant protein was added to various concentrations of 20 mM CaCl ₂ and 100 mM Tris-HCl (pH 7.4) containing 0.05% Triton X-100 in a solution of the compound A (subtilisin A, Sigma, USA) and protease K pH 8.0) buffer. (Sigma, USA), chymotrypsin, subtilisin A, and protease K substrate 0.5 mM Suc-AAPF-pNA (Sigma, USA) were added to the reaction mixture And reacted at 37 DEG C for 30 minutes.

2. Experimental results

The results of the experiment are shown in Figs. 5 and 6 and Table 1 below.

enzyme density
(nM) ^a IC ₅₀
(nM) K _i
(nM) Chymotrypsin 100 97.68 + 1.81 198.66 ± 2.27 Subtilisin A 100 274.29 + - 5.07 552.93 + - 7.31 Protease K 100 291.64 + - 8.46 488.17 ± 9.66

a: concentration of enzymes used in this experiment

As shown in FIGS. 5 and 6 and Table 1, the recombinant protein of the present invention (serine protease inhibitory protein cDNA, OcSPI) of the present invention did not inhibit trypsin, but chymotrypsin and bacterial and fungal serine protease Subtilisin A and protease K, respectively.

<3-2> Confirmation of microbial binding ability

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

1. Experimental Method

Escherichia coli , Bacillus subtilis were cultured in Luria-Bertani (LB) medium and Beauveria bassiana were cultivated in potato dextrose broth. When the culture reached 0.4 at OD600, bacteria and fungi were collected, washed with PBS, and mixed with microorganisms in 40 [mu] l containing 0.8 [micro] g of the recombinant protein of the present invention (serine protease inhibitor protein cDNA derived from horseshoe shear) 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.

2. Experimental results

As shown in FIG. 7, the recombinant protein of the present invention (serine protease inhibitory protein cDNA, OcSPI) of the present invention binds to E. coli , B. subtilis and B. bassiana I could confirm.

<3-3> Identification of antimicrobial activity

In order to confirm the antimicrobial activity of the recombinant protein of the present invention (serine protease inhibitory protein cDNA derived from horseradish scrotum, OcSPI), Gram-positive B. subtilis and Gram-negative E. coli And B. bassiana , an insect pathogen.

1. Experimental Method

The growth inhibitory activities of Gram positive B. subtilis and Gram negative E. coli were investigated. To the 96-well plate was added 200 μl of the recombinant protein of the present invention (serine protease inhibitory protein cDNA from the hairy horned scabbard) and bacterium (10 ⁵ cfu / ml) at various concentrations and incubated at 37 ° C for 24 hours at 220 rpm The cells were cultured under shaking and the bacterial growth inhibitory activity was measured at 595 nm.

To investigate the antifungal activity against insect pathogenic fungi, 200 占 퐇 of 2 占⁰⁴ conidia / ml of B. bassiana was mixed with various concentrations of the recombinant protein of the present invention and incubated at 22 占 폚 for 48 hours at a rate of 220 rpm And the fungal growth inhibitory activity was measured at 595 nm.

2. Experimental results

The results of the experiment are shown in Table 2 below.

Microorganism name MIC ₅₀ ([mu] M) Gram-positive bacteria B. subtilis 56.37 ± 3.17 Gram-negative bacteria E. coli 36.19 ± 1.87 IC ₅₀ ([mu] M) Insect pathogenic fungi B. bassiana 82.03 + 5.09

As shown in Table 2, 50% inhibitory activity of MIC ₅₀ (minimum inhibition concentration) was 56.37 μM and 36.19 μM in gram-positive and negative bacteria, respectively. In the case of insect pathogens, IC ₅₀ (inhibition concentration) Was confirmed to be 82.03 [mu] M.

As a result, it was confirmed that the recombinant protein of the present invention (serine protease inhibitory protein cDNA, OcSPI derived from hornbill scallop) has excellent inhibitory activity against chymotrypsin, subtilisin A and protease K, The growth of microorganisms such as E. coli can be effectively inhibited.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be possible. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> New polipeptides have an antibiotic and an antifungal activity          from Osmia cornifrons <130> P2015-0174 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> polypeptide <400> 1 Met Ser Ile Leu Lys Lys Ser Phe Leu Ser Val Cys Leu Val Leu Leu   1 5 10 15 Thr Met Ala Lys Ala Glu Asn Asn Thr Gln Ala Phe Arg Ala Val Ser              20 25 30 Glu Gly Ile Asn Gln Phe Ser Ser Ser Leu Tyr Gln Ser Glu Leu Glu          35 40 45 Glu Asn His Gly Asn Ile Ile Met Ser Pro Leu Ser Ala Ala Val Val      50 55 60 Leu Ala Met Ala Ala Phe Gly Ala Arg Gly Glu Glu Thr Glu Lys Gln Leu  65 70 75 80 Lys Lys Val Leu His Ile Pro Ser Pro Asp Thr Leu Gly Thr Ser Gly                  85 90 95 Tyr Gln Ser Leu Ile Glu Asn Leu Asn Asn Val Thr Val Ala Lys Leu             100 105 110 Leu Leu Ala Asn Lys Val Tyr Ala Ala Glu Lys Phe Ser Val Lys Pro         115 120 125 Ala Tyr Ile Glu Leu Thr Glu Lys Tyr Phe His Ser Val Thr Gln Leu     130 135 140 Val Asn Phe Ala Lys Ser Glu Glu Ala Ala Asn Thr Ile Asn Thr Trp 145 150 155 160 Val Gln Gln Asn Thr Asn Asp Arg Ile Lys Glu Ile Val Asn Ser Gly                 165 170 175 Asp Leu Asn Ala Met Thr Ala Met Val Leu Val Asn Ala Val Tyr Phe             180 185 190 Lys Gly Gln Trp Glu Asp Lys Phe Asp Pro Glu Asn Thr Glu Glu Arg         195 200 205 Pro Phe His Val Asn Glu Asn Thr Val Lys Asn Val Ser Thr Met Phe     210 215 220 Arg Ser Gly Ser Tyr Leu Tyr Gly Glu Leu Ser Asn Phe Asn Ala Lys 225 230 235 240 Phe Val Leu Leu Pro Tyr Lys Asp Asn Glu Leu Ser Met Ile Ile Ile                 245 250 255 Leu Pro Asn Glu Ile Asn Gly Leu Ala Glu Val Glu Lys Lys Leu Gln             260 265 270 Asn Thr Asn Ile Met Asp Ile Leu Asn Gln Gly His Glu Arg Glu Val         275 280 285 Glu Leu Tyr Leu Pro Lys Phe Lys Ile Glu Ser Lys Ile Glu Leu Asn     290 295 300 Glu Pro Leu Asn Lys Leu Gly Leu Thr Asp Met Phe Thr Ser Arg Ala 305 310 315 320 Asn Phe Ser Gly Ile Asp Asp Asn Leu Phe Val Ser Lys Val Val                 325 330 335 Gln Lys Ala Phe Ile Glu Val Asn Glu Glu Gly Gly Ser Glu Ala Ala Ala             340 345 350 Ala Thr Ala Val Ile Val Pro Phe Phe Leu Ala Val Pro Thr Glu         355 360 365 Phe Thr Val Asp Arg Pro Phe Phe Tyr Asn Ile Val Lys Leu Ser Gln     370 375 380 Gly Asp Ser Asn Ala Ala Thr Ala Thr Thr Leu Phe Thr Gly His Val 385 390 395 400 Asn Glu Pro Lys Val                 405 <210> 2 <211> 1504 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide <400> 2 gttcttccag tcgacgtttg actgccgaag aagaatacgt ggtccaataa agtattaca 60 gtatgagtat attaaagaaa agttttttga gcgtgtgcct tgttctacta acaatggcaa 120 aagcagaaaa caacacccaa gcgtttcgcg ctgtttcgga aggaataaat caattttcat 180 cctccttata tcagtccgag ctagaggaaa atcatggtaa tattataatg tctcctctta 240 gtgcagctgt tgttcttgct atggctgcat ttggagctcg tggtgaaaca gaaaaacaat 300 taaaaaaagt acttcatatt ccatcaccag atactctagg tacatctggt tatcaatcac 360 ttattgaaaa tcttaataat gttacagtgg ctaaacttct tcttgctaat aaagtttatg 420 ctgctgagaa attctctgtt aaaccagctt acattgagtt aacagagaag tattttcact 480 ctgttactca actagtaaac tttgctaaat ctgaagaagc tgccaatact attaacacat 540 gggttcaaca aaatacaaat gaccgtatta aagaaattgt taactctggt gatttgaatg 600 caatgacagc aatggtactt gtaaatgcag tatattttaa gggtcagtgg gaagataaat 660 ttgatcctga gaatactgaa gaaagaccat tccatgtcaa tgaaaatacg gtaaaaaatg 720 tctccacaat gttcagatca ggttcctatc tatatggtga actttcaaat tttaatgcta 780 aattcgtcct attgccatac aaggacaatg aactgagtat gatcataatt cttccaaatg 840 aaatcaatgg tttggcagag gttgaaaaaa aattacaaaa tacaaatatt atggatatcc 900 taaatcaggg acatgaacgc gaagtagaat tgtatctacc aaagtttaaa attgaaagta 960 aaatagagct gaacgaaccg ttaaataagc tgggcttaac cgatatgttt acttcacgag 1020 ccaacttttc tggtatcgcc gatgacaacc tgtttgttag taaggttgta caaaaagctt 1080 ttatcgaagt gaacgaagaa ggaagcgaag ctgctgccgc tactgctgtg cacatagtac 1140 cattttttct ggcagtacca acggagttta cagtagatcg accattcttc tacaacattg 1200 tcaaactatc tcagggtgat tctaatgctg caactgcaac aacactattt acaggacatg 1260 ttaatgaacc gaaagtttaa gtaaataaaa tatcgctttt taatattata taaatgtaaa 1320 cgcatcttaa ttgggattat tgaaggtata gatttatata aaaaatctat ttcaataatt 1380 catcatgcat aagagtaaaa ataaaaattt atttatcatt aaaaaaaaaa aaaaaaaaaa 1440 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500 aaaa 1504

Claims (14)

1. A polypeptide having antimicrobial and antifungal activity, characterized in that it is composed of the amino acid sequence of SEQ ID NO: 1 derived from Osmia cornifrons .
delete The method according to claim 1,
Wherein said polypeptide is a serine protease inhibiting protein.
A polypeptide having antibacterial and antifungal activity.
The method according to claim 1,
Wherein the polypeptide has an effect of inhibiting the growth of microorganisms through inhibitory activity against any one or more of chymotrypsin, subtilisin A, and protease K,
Polypeptides having antimicrobial and antifungal activity
5. The method of claim 4,
Microorganism in which the polypeptide can inhibit the growth of Bacillus subtilis (Bacillus subtilis), E. coli (Escherichia wherein the polypeptide is at least one selected from the group consisting of E. coli and beauveria bassiana .
A nucleic acid molecule comprising the polynucleotide of SEQ ID NO: 2 which codes for the polypeptide of any one of claims 1 to 5.
delete A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 derived from Osmia cornifrons ; Or an expression vector comprising a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 encoding the polypeptide, as an active ingredient.
delete 9. The method of claim 8,
Wherein the composition has an effect of inhibiting the growth of bacterial and fungal pathogens through inhibitory activity against any one or more of chymotrypsin, subtilisin A and protease K,
Compositions for antibacterial and antifungal agents.
11. The method of claim 10,
The bacterium is Bacillus subtilis subtilis ) or Escherichia coli ).
Compositions for antibacterial and antifungal agents.
11. The method of claim 10,
Characterized in that the fungal pathogen is beauveria bassiana .
Compositions for antibacterial and antifungal agents.
delete 13. The method according to any one of claims 8 to 10,
Wherein the composition is applied to medicines, detergents, plant disease control agents, coating agents or cosmetics.

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