JP2002253245A - Fused protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a membrane-active protein having improved specificity and activities of a target cell. SOLUTION: This fused protein has a first peptide site acting on the cell surface layer of the target cell, and the second peptide site capable of bonding to a main constituent component of the cell surface layer of the target cell, and fused therewith. As a result, the specificity is increased, and thereby the activity of the first peptide site is enhanced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a technique for increasing the specificity of a peptide acting on the cell surface to a target cell, and particularly to a peptide having a binding property to a component of the cell surface such as a cell membrane or a cell wall of the target cell. The present invention relates to a fusion protein used as a ligand for obtaining cell specificity.

[0002]

2. Description of the Related Art Many antibacterial peptides are known. Among them, there are so-called membrane-acting peptides which act on the membrane of susceptible cells. Such a peptide is considered to exert an antibacterial action in the following two steps. That is, first, it binds to the cell surface. Then
Based on its special structure, this protein penetrates the cell membrane to form ion channels,
The target cell is killed by causing TP production to stop or the like.

[0003] Such membrane-acting peptides include bacteria,
There may or may not be differences in cytotoxicity between fungi, plant cells and animal cells. In recent years, various approaches have been attempted to enhance the antibacterial properties against bacteria, especially in order to expand the application of membrane-acting antibacterial proteins to bacteria. For example, (1) use a hydrophobic amino acid to make it amphiphilic; (2) introduce a basic amino acid to enhance its binding to the negatively charged cell surface; Have been reported.
However, even with these techniques, not all membrane-acting peptides can be improved in specificity to bacteria.

[0004] It has also been reported that the antimicrobial activity of hybrid antimicrobial peptides in which membrane-acting peptides having different antimicrobial activities are linked has changed. In other words, Dong Gun Lee et al., Biotechnology Letters, 2
According to 0 (3) 211-214 (1998), it has strong antibacterial activity, but has weak antifungal activity and has no hemolytic activity.
A peptide in which 8 amino acid residues at the terminal and 12 amino acid residues at the N-terminal of melittin having strong antibacterial activity, antifungal activity and hemolytic activity are linked maintains the same antifungal activity as melittin, Hemolytic activity is reported to be weaker than melittin. However, this technique of coupling peptides having different antibacterial activities is not applicable to all membrane-acting peptides. In addition, hybrid proteins in which peptide fragments such as hormones and cytokines capable of binding to the surface of the toxin-sensitive cell have been produced have been produced. However, such techniques are aimed at targeting differentiated or specialized cells, e.g., that express a particular receptor in mammalian cells, to increase the specificity by recognizing differences between cells of different species Not even a thing.

[0005]

SUMMARY OF THE INVENTION In view of the above-mentioned problems, the present invention has been made to increase the specificity of a membrane-active peptide capable of acting on the surface of a cell for a specific target cell by increasing its specificity. Its purpose is to increase the action of

[0006]

Means for Solving the Problems The present inventors have specifically developed proteins capable of acting on various cells such as animal cells such as bacteria, fungi, mammals and the like, or plant cells such as plant cells and the like, specifically for surface components of target cells. The present inventors have found that by linking proteins to be bound, it is possible to change the cell specificity of the membrane-acting protein and to increase the action of the membrane-acting protein on target cells. That is, according to one aspect of the present invention, the method includes a first peptide site capable of acting on a cell surface of a target cell and a second peptide site having a binding property to a component of the cell surface of the target cell. A fusion protein is provided. According to another aspect, there is provided a DNA construct having a DNA sequence encoding the first peptide site and the second peptide site, and a vector including the DNA sequence. According to still another aspect, the D
Provided are a transformed cell and a transformed plant that retain the NA sequence so that it can be expressed. Also provided is a method for producing a fusion protein, comprising a step of culturing such a transformed cell. According to another aspect of the invention, the first peptide site has cell killing properties and the second peptide site is a target cell (eg, a fungus) that is sensitive to the first peptide. By using a transformed cell that produces a fusion protein that is a peptide capable of binding to the surface component (in the case of fungi, preferably chitin), the organism that can be damaged by the target cell is: A method for imparting resistance to the target cell is provided. According to these inventions, specificity for a target cell can be imparted or enhanced to a peptide that can act on the target cell. This allows the peptide to act effectively on the target cell.

[0007]

Embodiments of the present invention will be described below in detail. The fusion protein of the present invention includes a first peptide site capable of acting on the cell surface of a target cell, and a second peptide site capable of binding to a component of the target cell. In the present invention, the target cells include, but are not limited to, cells of all types and forms, and include various prokaryotic cells such as bacteria, eukaryotic cells such as animal cells including fungi, yeast and mammals, and plant cells. , Animals and plants, eukaryotic cells in the animals and plants, and tissues and organs that are part of the eukaryotic cells. It also includes virus particles. Since a large number of membrane-acting proteins that specifically act on the cells have not been obtained, preferably, target cells are crustacean cells such as fungi or insects. In addition, the cell surface of the target cell includes a cell wall and an outer skin in addition to a cell membrane held by all cells. Preferably, it is an outer coat (sugar coat) present on the outermost layer, an outer membrane of bacteria, etc., or a cell wall, which contains a large amount of polysaccharides.

[0008] In the present invention, the peptide in the case of the peptide site or peptide includes oligopeptides and polypeptides (proteins). Hereinafter, peptides characterized by the first peptide site are collectively referred to as membrane-active peptides. As used herein, the term "membrane-active peptide" refers to any action on the surface of or within a membrane (including a cell wall) on the surface of a target cell by itself or by interaction with the membrane. Or a peptide that imparts some function to the cell. For example, the action on the target cell is a change in the osmotic pressure adjustment function of the membrane, ATP production ability, endocytosis function and the like. The functions of the protein itself include enzymes, antigenicity, antibodies, receptors and the like.

[0009] In the present specification, a peptide capable of finally killing a target cell by damaging the target cell or inhibiting the proliferation ability of the cell by any action on the target cell, a cell-killing peptide It is assumed that. In particular, peptides that show killing properties on the cell surface are called membrane-acting antimicrobial peptides. As such a cell-killing peptide, various known antibacterial proteins can be used. Such peptides include, for example, human or rat CAP18 (James et al., Anti-microbia
l Activity of Human CAP18 Peptides, Immunotechnolo
gy 1 (1995), 65-72), Andropin, BLP-1, Bombinin, Bo
mbolitin, Cecropins, Ceratotoxin A, Clavanin, CRA
MP, Dermaseptin 1, Enbocinm, FALL-39, Lycotoxin
I, Magainin I, Melittin, Misgrin, PGLa, Pleurocidi
n 、 Seminalplasmin 、 Styelin 、 Abaecin 、 Apidaecin I
A, Bactenecin 5, Diptericin, Drosocin, Enkelytin,
Formaecinsin 1, Histatin I, Indolicidin, Lebocin
1, Mectchnikowin, PR-39, Prophenin, Pyrrhocorici
n, Tenenecin, Bovine dodecapeptide, Brevinin-1, Brev
inin-1E, Esculentin, Pipinin, Ranalexin, Thanati
n, 11-kD polypeptide (Andru and Rivas, Animal a
ntimicrobial peptides, Bipolymers (Peptide Scienc
e) Vol. 47, 415-433 (1998)), Thionins, Defensins, L
ipid Transfer Proteins (Plant Defense PeptidesGar
acia-Olemedoet al., Bipolymers (Peptide Science) Vo
l.47,479-491 (1998)), antibacterial peptide derived from Bombina Genus Amphibia, antibacterial peptide derived from Xenopus Laevis, antibacterial peptide derived from Rana Genus Amphibia, antibacterial derived from Phyllonedusa Genus Amphibia Peptides, antimicrobial peptides derived from Australian frogs (Antimicrobial Peptides from Amphibian Skin: Wha
t Do They Tell Us ?, simmaco et al., Bipolymers (Pe
ptide Science) Vol. 47, 435-450 (1998)), Cecropins, Magainins, Dermaseptins, Alamethicins, Parda
xins (Oren et al., Mode of Action of Linear Amphipathi
c α-Helical Antimicrobial Peptides, Biopolymers
(Peptide Science) Vol.47,451-463 (1998)), Insect De
fensin, Drosomycin, Tachyplesin, Thanatin, A
ndroctonin, Lysozyme, Androctonin, Proteglin
, Penaeidins, Tachycitins, Mytilin, Mytimycin
(Dimarcq et al., Cystein-Rich, Antimicroial Peptides in
Invertebrates Biopolymers (Peptide Science) Vol.4
7,465-477 (1998)).

In the present invention, human or rat-derived CAP18, magainins, melittins, and alamethicins are used.
s), polymixins (Polymixins), secrobins (C
ecropins), tachyplesins, dermaseptins, bombinins
s), thionins (Thionins), or their killing active sites, or derivatives thereof can be preferably used.

[0011] The first peptide site in the present invention is not particularly limited as long as it can act on or impart a function to a target cell, and can be used irrespective of the whole or a part of the above-mentioned membrane-active peptide. . For example, only a part of the peptide exhibiting a function such as antibacterial activity can be used as the first peptide site in the present invention. Derivatives thereof can also be used. The first peptide site may be a peptide functionally equivalent to the above-mentioned peptide or its membrane-active site. For example, a peptide in which one or more of the amino acids of the control peptide is changed by deletion, substitution, addition, and / or insertion by another amino acid, and has the same membrane activity as the control peptide. Peptides can also be used. Also, the first
As long as it has at least one kind of membrane-active peptide part, a plurality of one kind of peptide part may be used, or different kinds of peptide parts may be used. The first peptide site can be used irrespective of whether it is obtained from nature or synthesized by an artificial process including genetic manipulation and chemical synthesis.

The second peptide moiety has a binding property to a component of the cell surface of the target cell. Here, it is preferable that the component of the cell surface layer is a component that is not temporarily expressed in the cell membrane and the cell wall but is constantly expressed. Examples of such a component having a peptide having a binding property to such a component include lipid, protein, saccharide (including polysaccharide), glycoprotein, lipopolysaccharide, and glycolipid. In the present invention, it is preferable that the components of the cell wall have a binding property. In addition, it is preferable to target the sugar chain of the component regardless of whether it is a polysaccharide, a glycoprotein, a glycolipid, a lipopolysaccharide, or the like. The sugar chain is at least one selected from the group consisting of acidic polysaccharides such as teichoic acid or tichronic acid, peptidoglycan, chitin, cellulose, hemicellulose, lignin, pectin, and cholesterol. For example, chitin is not universally present on many types of cell surfaces, but is specifically and / or abundantly present in fungi such as filamentous fungi and insects and crustaceans. It is preferable to improve or impart specificity. In addition, peptidoglycan is preferable in that it effectively acts on Gram-positive bacteria.

[0013] The term "having a binding property with a component" means that the component has a certain affinity with the component irrespective of whether it is a covalent bond or a noncovalent bond, and whether it is reversible or irreversible. Means that Preferably, it is non-covalent. Examples of such a second peptide having a binding property to a cell surface component include various lectins, agglutinins, enzymes, antibacterial peptides and the like. For example, lectin, hevein, chitinase, Ac-AMP, potato WIN and the like can be mentioned as those having a binding property to chitin. Specifically, wheat germ agglutinin isolectins, barley lectin (Barley lection), rice lectin (rice lecti)
n), Uritica Dio squid agglutinin (Uritica Dio
ica agglutinin, potato lectin (potato lecti)
n), a potato-derived tryptic peptide (tryptic pe
ptide from potato lectin, hevein, potato chitinase-derived wound-induced protein (wound-induced)
proteins from potato chitinase, WIN1, WIN2), bean basic chitinase, tobacco basic chitinase, l
ection, poplar injury-inducing chitinase (poplarwound-indu
ced chitinase, Arabidopusis thaliana basic chitinas
e), rice basic chitinas (rice basic chitinas)
e), bean acidic pathogenesis-
related 4 chitinase, maize seed chitinase (maiz
e seed chitinase, Amaranth Scaudatus (Amar)
anthus caudatus) Antimicrobial peptide (Ac-AMP) (Lee et al., Stru
cture and Functions of chitin-binding proteins, An
nu.Rev.Plant.Physiol.Plant.Mol.Biol.1993.44: 591-61
5) There is. In addition, peptidoglycan recognition protein (pe
ptidoglycan recognition protein (PGRP) (Proc.N
atl.Acad.Sci.USA Vol. 95, issue 17, 10078-10082, Aug
ust 18, 1998). In addition, known transmembrane proteins, GPI anchors, and the like can also be used as the component-binding peptide.

The second peptide site in the present invention may be any one as long as it has a binding property to a component on the surface of the target cell. Therefore, it is sufficient that the second peptide site contains a peptide portion having binding activity, and the second peptide site can be used irrespective of all or a part of the above-mentioned various peptides. In addition, derivatives thereof can also be used. Preferably, only sites with binding activity are used. The second peptide site may be a peptide functionally equivalent to the above-described peptide or a site having a binding activity thereof. For example, a peptide in which one or more amino acids of a control peptide are deleted, substituted, added, and / or inserted by another amino acid, and exhibit the same binding activity as the control peptide. Can also be used.
As the second peptide site, only one type may be used, or two or more types may be used in combination. In addition, a plurality of one type may be used. The second peptide site can be used irrespective of whether it is obtained from nature or synthesized by an artificial process including genetic manipulation and chemical synthesis.

[0015] The fusion protein of the present invention has the first peptide site and the second peptide site. These peptide moieties are, for example, linked to each other so as to function effectively so that a specific secondary structure or tertiary structure is required when their functions are required. The form does not matter. Preferably, they are linked via peptide bonds. More preferably, they are linked only via a peptide chain. Each of the first peptide site and the second peptide site may also contain one or more. Between the first peptide site, the second
Between the peptide sites, or between the first and second peptide sites
May be directly connected via a peptide bond. In this case, the N-terminus and the C-terminus of these peptide sites are directly linked by a peptide bond.

Further, between these various peptide sites,
A spacer having an appropriate length and / or an appropriate function may be provided so that each peptide site preferably functions. Preferably, the spacer is also formed of a peptide, and the spacer and various peptide sites are connected by a peptide bond. As the peptide as the spacer, a peptide mainly composed of glycine is preferable. Examples of such a peptide for spacer include G
A GGGS (G: glycine, S: serine) chain or a chain in which this chain is repeated about 2 to about 5 can be mentioned. Also, Patrick's reference (J. Mol. Bio
l. (1990) 211, 943-958), a peptide consisting only of serine (S), threonine (T) and / or glycine (G) (type I (STG type)), serine, threonine and / or glycine And one of aspartic acid (D), lysine (K), glutamine (Q), asparagine (N), and alanine (A)
Alternatively, a peptide consisting of proline (P) (type I
I), a combination consisting of STG type I and II amino acid residues (type III (STGDKQNA type)), a type I and type II peptide having a plurality of prolines (type IV (Pro type)), a type III is an arbitrary combination of amino acid residues contained in III, a peptide having at least 8 amino acid residues (type V), GSGSG, SGGSG, NGGGG, E
GKSSGSGSESKST, SKSTS, PVPST
PPTPSPSTPPTPSM and the like.

In particular, a plurality of first or second peptide sites are linked to form a first peptide site group or a second peptide site.
When forming the peptide site group of the above, the same kind of peptide sites may be connected by the above-mentioned spacer. Further, the first peptide site and the second peptide site can be connected by a freely rotatable spacer.

Next, a method for producing the fusion protein of the present invention will be described. Such a fusion protein can be obtained by chemical synthesis, but is preferably obtained by culturing a protein-producing cell obtained by genetic manipulation. Get each peptide site,
After that, the fusion protein may be linked. However, in order to obtain the fusion protein as a single unit, a DNA capable of expressing the fusion protein having the DNA sequences corresponding to the first peptide and the second peptide is used. Preferably, the vector containing the sequence is introduced into a cell suitable for expressing the protein, transformed and produced.

The DNA encoding the fusion protein includes at least one of the first peptide site and one
And a second peptide site. The order of the first peptide and the second peptide does not matter, and is selected as needed. These DNA sequences may be of natural origin or artificially synthesized. Further, the usage of codons can be modified and selected as necessary.

When there is a spacer between these coding sequences, in addition to the sequences corresponding to these two kinds of peptides, it has a DNA sequence corresponding to the peptide. Further, a secretory signal gene corresponding to the type of the host cell may be provided. By having a secretory signal gene, it is easy to be transported out of the cell, and it is easy to localize the protein on the cell surface or to secrete the protein out of the cell. The secretory signal gene is preferably located upstream of the peptide corresponding sequence.

Further, a DNA sequence for expressing the DNA of the fusion protein can be provided at the same time.
Specifically, a promoter sequence arranged upstream of a sequence encoding a peptide, a termination sequence arranged downstream of a coding sequence, and a polyadenylation signal. The promoter sequence is not particularly limited.
What can express the coding sequence in the host cell may be used, but various promoter sequences can be used if necessary. It can also include enhancers.
Further, it is preferable that a marker gene for selecting a cell having the fusion DNA is also introduced at the same time. The selectable marker gene sequence may be directly linked to the fusion DNA or may be introduced by co-transfection.

[0022] Such a DNA sequence may be incorporated and retained in various vectors suitable for the host cell to be used. Such a vector constitutes a vector for expressing the fusion protein of the present invention. An appropriate vector is selected depending on the type of the host cell and the gene transfer method. Generally, various plasmid vectors, viral vectors, YAC, BAC, MAC
Etc. can be used. Preferred prokaryotic, eukaryotic, animal and plant cell vectors are well known in the art.

Once the fusion DNA or a vector containing the fusion DNA is constructed, transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun, calcium phosphate precipitation, direct micro It is introduced by any of a variety of suitable means, such as an injection method. After introduction of the vector or the like, the recipient cells are cultured in a selection medium.

By selecting a cell into which the DNA encoding the fusion protein has been introduced by an appropriate method, a transformed cell producing the fusion protein can be obtained.
The transgene in the transformed cell may be retained within or outside the chromosome, and the expression of the trait may be transient or stable. By culturing and growing the transformed cells under appropriate culture conditions, the transformed cells produce the fusion protein intracellularly, on the cell surface, or extracellularly. This fusion protein can be recovered and purified by an appropriate method. It can be recovered and purified by utilizing the affinity (binding property) to the specific component inherently obtained in the fusion protein.
For example, by bringing a fraction containing the fusion protein into contact with a separation stationary phase in which a component of the target cell surface is immobilized, the affinity between the component and the second peptide of the protein can be improved. Depending on the nature, the fusion protein can be adsorbed to the stationary phase, and the adsorbed fusion protein can be separated from the stationary phase in a suitable manner. For example, it is possible to prepare a column of the component itself or a packing material provided on the surface thereof, load the column with a cell-soluble fraction, and then elute the fusion protein using an appropriate eluent. The transformed cells in the present specification include, but are not limited to, cells of all types and forms, various prokaryotic cells such as bacteria, eukaryotic cells such as fungi, yeast, animal and plant cells, and animal and plant cells. Includes individuals, eukaryotic cells that can constitute the animals and plants, and tissues and organs that are part thereof. It also includes virus particles. It also includes its propagation medium (seed), which is part of a plant individual.

The thus obtained fusion protein accesses a target cell having a component having a binding property to the peptide site based on the second peptide site, and is bound to the target cell. The second peptide site acts as an anchor on the target cell surface,
Are retained on the target cell surface. Thereby, the original function of the first peptide is specifically and effectively exerted on the target cell.

The first peptide site is a cell-killing peptide having a killing effect on a plurality of types of cells, and the second peptide site is a component constituting a surface layer of a specific cell type among a number of sensitive cells. When the fusion protein has binding property to the specific cell type, the fusion protein specifically binds to the specific cell type, exerts an antibacterial property on a surface layer of the cell type, and kills the cell. Thereby, growth inhibition on a specific target cell can be achieved at a lower concentration. That is,
The minimum inhibitory concentration of the killing peptide for a particular target cell can be reduced. Further, by selecting the second peptide site based on the distribution of the constituents among the cell types, the antimicrobial spectrum of the killing peptide can be changed.

The first peptide site has a killing property for a plurality of cell types, and the second peptide site has increased specificity for a specific cell, and as a result, kills bacteria and / or fungi. In the case of high potency and low killing property for plant cells and / or animal cells, such a fusion protein can be applied, for example, as a pesticide to a cultivated plant or the like by spraying, coating, or the like. The antifungal agent can be an external preparation or a drug (therapeutic agent) that can be administered to mammals including humans via various administration routes. In particular, when a fungus such as a filamentous fungus is used as a target cell (for example, when a peptide that binds to chitin is used as the second peptide site), a pesticide or a drug is used as a more specific, effective and safe antifungal agent. (Therapeutic agent). Further, when the first peptide site has a killing property against two or more types of cells including insects, and the second peptide site has a binding property to a component of the surface layer of insect cells (for example, chitin When a binding peptide is used as the second peptide site), it has high specificity to insects and the like, and exhibits effective insecticidal activity even in a small amount. Such a fusion protein can be used as an insecticide by directly applying or spraying on the habitat of the pest or on them. Also, such a fusion protein can be included in insect baits or attractants.

The obtained fusion protein can be subjected to secondary processing to further enhance its effectiveness. For example, after obtaining from a transformed cell, modification can be performed so that an active secondary structure or tertiary structure can be obtained.
Further, control of electric charge is also possible. Also, other peptides can be bound.

On the other hand, transformed cells producing such a fusion protein can be used in various forms. That is,
Wherein the first peptide moiety has cell killing properties and the second
Using a transformed cell that produces a fusion protein having a binding site to a component of the surface of a target cell that is sensitive to the first peptide, to an organism that can be damaged by the target cell, Methods for conferring resistance to target cells are also provided. First, a transformed cell itself that produces a fusion protein containing a first peptide site that is harmful to pathogenic bacteria and pests (particularly preferably a secretory type) is used as an agricultural or therapeutic agent, livestock, or the like. Can be used as feed additives. In this case, various kinds of transformed cells such as bacteria and yeast can be adopted as the transformed cells.

When the transformed cells are plant cells, the cultured cells of the transformed cells can be differentiated, and then the individual plants can be regenerated. Also, by mating,
It is possible to easily obtain seeds that are the propagation medium and plants in which the introduced gene is homozygous. Furthermore, modification by crossing and cell fusion techniques is also possible. By cultivating such a plant, a large amount of fusion protein can be produced. In this transformed cell, the first peptide site has a killing property for a plurality of cell types, and the second peptide has enhanced specificity for a specific cell (for example, the chitin-binding peptide is replaced with the second peptide). As a result, it produces a fusion protein that is highly killing against bacteria and / or fungi and has little or no negligible killing on plant and animal cells. If so, the resulting plant body has its antimicrobial peptide encapsulated in the plant cell itself, and has a surface layer, or secretes it, thereby improving resistance to diseases caused by invasion of bacteria and the like, Without spraying pesticides, etc., it will be protected from diseases. In addition, plant cells or animal cells that are capable of producing a fusion protein with high killing ability against pathogenic bacteria that can cause disease to mammals such as livestock are highly or not killing plant cells or animal cells by the second peptide, It is a preferred feed ingredient for livestock.

When the transformed cell is an animal cell, the first peptide site has a killing property for a plurality of cell types, and the second peptide site enhances specificity for a specific cell. (Eg, when a peptide that binds to chitin is used as the second peptide site), resulting in high killing of bacteria and / or fungi and little or no killing of mammalian cells If the fusion protein is produced as low as possible, the cell itself can be used as a drug (therapeutic agent). Further, for example, when such animal cells are dermal or epidermal cells, they are proliferated by appropriate means to form a sheet (skin-organized).
By doing so, an antibacterial agent for topical application can be obtained.

[0032]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to the following examples. In addition, methods necessary for general gene recombination such as DNA cleavage, ligation, yeast transformation, and gene base sequence determination are described in commercially available reagents used for each operation, instructions attached to mechanical devices, and the like. In addition, experiment books (for example, "Mo
rectangular Cloning (Maniatis
T. et al. Cold Spring Harbor
Laboratory Press).

Example 1 Chitin-binding peptide-CAP18 (L104-V135) fusion
Construction of DNA Fragment A DNA fragment encoding a fusion protein of the N-terminal chitin-binding region of wheat germ agglutinin-A and human-derived CAP18 (L104-V135) was prepared by chemical synthesis and PCR. This DNA fragment and the shuttle vector pYES2 manufactured by Invitrogen were subjected to a restriction enzyme EcoRI according to a conventional method.
And XhoI, and the digested DNA fragment was subjected to GFX ^™ PCR D
NA and Gel Band Purification Kit (Pharmacia)
After purification, both DNA fragments were ligated according to a conventional method. The ligated DNA was introduced into E. coli JM109 according to a conventional method. After extracting a plasmid from the obtained transformant, the nucleotide sequence was examined to confirm that the fused DNA fragment was correctly constructed. SEQ ID NO: 1 shows the nucleotide sequence of this DNA fragment. wheat germ agglutini
The chitin-binding region of nA has a nucleotide sequence at positions 22 to 150, and CAP18 (L104 to V135)
It has the nucleotide sequence at position 246. The base sequence GAATTC at positions 7 to 12 is a cleavage site by the restriction enzyme EcoR1, and the base sequence CTCG at positions 253 to 246.
AG is the site of cleavage by XhoI. SEQ ID NO: 2 shows the amino acid sequence of the protein encoded by this DNA fragment.

Example 2 Chitin-binding peptide- (GGGGS) -CAP18
(L104-V135) Construction of fusion DNA fragment N-terminal chitin-binding region of wheat germ agglutinin-A and amino acids GGGGS) G: glycine, S: serine)
Fragment and a DNA fragment encoding a fusion protein with human-derived CAP18 (L104-V135) were synthesized by chemical synthesis and PCR.
It was produced by the method. This DNA fragment and the shuttle vector pYES2 manufactured by Invitrogen were subjected to a restriction enzyme EcoR according to a conventional method.
I and XhoI, and digested DNA fragment was subjected to GFX ^™ PCR
DNA and Gel Band Purification Kit (Pharmacia)
After purification, both DNA fragments were ligated according to a conventional method. The ligated DNA was introduced into E. coli JM109 according to a conventional method. After extracting a plasmid from the obtained transformant, the nucleotide sequence was examined to confirm that the fused DNA fragment was correctly constructed. SEQ ID NO: 3 shows the nucleotide sequence of this DNA fragment. wheat germ agglutini
The chitin-binding region of nA has a nucleotide sequence at positions 22 to 150, and encodes the amino acid sequence GGGGS.
The A region has a nucleotide sequence at positions 151 to 165,
AP18 (L104 to V135) has a nucleotide sequence at positions 166 to 261. Nucleotide sequence GAATTC at positions 7 to 12
The sequence is a cleavage site by the restriction enzyme EcoR1, and the nucleotide sequence CTCGAG at positions 268 to 273 is Xho.
This is the site of cleavage by I. In addition, this D
2 shows the amino acid sequence of the protein encoded by the NA fragment.

Example 3 Chitin-binding peptide- (GGGGS) ₂ -CAP18
(L104-V135) Construction of fusion DNA fragment N-terminal chitin-binding region of wheat germ agglutinin-A, amino acids GGGGSGGGGS, human-derived CA
D encoding fusion protein with P18 (L104-V135)
NA fragments were produced by a chemical synthesis method and a PCR method. This DNA fragment and Invitrogen shuttle vector pYE
S2 is cleaved with restriction enzymes EcoRI and XhoI in a conventional manner,
The cleaved DNA fragment was subjected to GFX ^™ PCR DNA and Gel Band Pu
After purification using rification Kit (Pharmacia),
According to a conventional method, both DNA fragments were ligated. The ligated DNA was introduced into E. coli JM109 according to a conventional method. After extracting a plasmid from the obtained transformant, the nucleotide sequence was examined to confirm that the fused DNA fragment was correctly constructed. SEQ ID NO: 5 shows the nucleotide sequence of this DNA fragment. The chitin-binding region of wheat germ agglutinin-A has a nucleotide sequence at positions 22 to 150, the DNA region encoding the amino acid sequence GGGGSGGGGS has a nucleotide sequence at positions 151 to 180, and CAP1
8 (L104 to V135) has a nucleotide sequence at positions 181 to 276. The base sequence GAATTC at the 7th to 12th positions is a cleavage site by the restriction enzyme EcoR1;
The nucleotide sequence CTCGAG at positions 3 to 288 is a cleavage site by XhoI. In addition, SEQ ID NO: 6, this DNA
2 shows the amino acid sequence of the protein encoded by the fragment.

Example 4 Chitin-binding peptide- (GGGGS) ₃ -CAP18
( L104-V135) Construction of Fusion DNA Fragment A DNA fragment encoding a fusion protein of the N-terminal chitin-binding region of wheat germ agglutinin-A, amino acids GGGGSGGGGGSGGGGS, and human-derived CAP18 (L104-V135) is chemically synthesized. It was prepared by the PCR method and the PCR method. This DNA fragment and the shuttle vector pYES2 manufactured by Invitrogen were subjected to restriction enzymes EcoRI and Xho
I, and the cleaved DNA fragment was subjected to GFX ^™ PCR DNA and
After purification using a Gel Band Purification Kit (Pharmacia), both DNA fragments were ligated according to a conventional method. The ligated DNA was introduced into E. coli JM109 according to a conventional method. After extracting a plasmid from the obtained transformant, the nucleotide sequence was examined to confirm that the fused DNA fragment was correctly constructed. In SEQ ID NO: 7, this DN
2 shows the nucleotide sequence of fragment A. The chitin-binding region of wheat germ agglutinin-A has a nucleotide sequence at positions 22 to 150, the DNA region encoding the amino acid sequence GGGGSGGGGGSGGGGS has a nucleotide sequence at positions 151 to 195, and CAP18 ( L104 ~ V135) is 196th ~
It has the nucleotide sequence at position 291. The base sequence GAATTC at positions 7 to 12 is a cleavage site by the restriction enzyme EcoR1, and the base sequence CTCG at positions 298 to 303.
AG is the site of cleavage by XhoI. SEQ ID NO: 8 shows the amino acid sequence of the protein encoded by this DNA fragment.

(Comparative Example) Construction of DNA Fragment Encoding Chitin-Binding Peptide A DNA fragment encoding the N-terminal chitin-binding region of wheat germ agglutinin-A was prepared and ligated in a shuttle vector pYES2 manufactured by Invitrogen. Restriction enzyme EcoR
The DNA fragment was cloned by inserting it between the 1-XhoI cleavage sites. SEQ ID NO: 9 shows the nucleotide sequence of this DNA fragment. The chitin-binding region of wheat germ agglutinin-A has a nucleotide sequence at positions 22 to 150. 7th ~
The base sequence GAATTC at position 12 is identical to the restriction enzyme Eco.
It is a cleavage site by R1, and the nucleotide sequence CTCGAG at positions 157 to 162 is a cleavage site by XhoI. SEQ ID NO: 10 shows the amino acid sequence of the protein encoded by this DNA fragment.

Example 5 Expression of Fusion Gene in Yeast The vectors constructed in Examples 1 to 4 and Comparative Example 1 were used to transform yeast INVSc1 manufactured by Invitrogen, respectively. Selection was carried out using a selection marker carried by the vector to obtain each transformant. All the cultures of the transformants obtained by the genes of Examples 1 to 4 and Comparative Example 1 were used for 20
0 ml of YP (2% Bacto peptone, 1%
Bacto Yeast Extract) was added to a medium supplemented with 2% raffinose, followed by shaking culture at 30 ° C. When the OD600 reaches about 1, 20 ml of 2
After adding 0% galactose and shaking culture for further 24 hours, the cells were collected by centrifugation. The cells were washed twice with saline, and 300 μl of extraction buffer (50 mM T
ris-HCl (pH 7.5), 10 mM β-mercaptoethanol, 2 mM EDTA, 1 mM PMSF,
10 μl / laprotinin, 10 μl / l leupeptin, 1 μ
l / l pepstatin), an equal amount of glass beads (0.45-0.5 mm in diameter) was added, and the cells were disrupted by vigorous stirring (4 ° C) for 15 minutes with a vortex mixer. Further, 300 μl of an extraction buffer was added, and a soluble fraction from which unmilled cells were removed was obtained by centrifugation (1000 g, 5 minutes).

Example 6 Purification of Fusion Protein Various soluble fractions derived from each of the transformants obtained in Example 5 were subjected to a 10 mM sodium phosphate buffer (pH 7.4) containing 0.15 M NaCl (PBS). ) Was dialyzed. The various soluble fractions after dialysis were applied to a column filled with chitin powder (manufactured by Seikagaku Corporation) equilibrated with PBS. After washing with PBS containing 1M NaCl, 0.1% NaCl was added.
The protein bound to chitin was eluted with 5N acetic acid to obtain a fusion protein fraction derived from each transformant.
For each fraction, the eluted protein concentration was measured using a protein assay kit (manufactured by Bio-Rad).

(Example 7) Evaluation of antibacterial activity of fusion protein Next, the minimum inhibitory concentration (MIC) of the five proteins obtained in Example 6 against filamentous fungi, Escherichia coli and CHL cells was determined. As a control, CAP18 (L104-V135) produced by chemical synthesis was similarly MI
C was determined. (Filamentous fungi) 30 μl of a solution prepared by diluting 5 types of proteins to predetermined concentrations in each well of a 96-well microplate
l, 200 mM MES buffer, pH 6.0 (2- (N
-Morpholino) etahnesulfoic
acid) 10 μl, sweet potato black spot fungus (Ceratocyc)
Conidiospore suspension of tes Fimbriata IFO 30501 (1 x 1)
0 ⁵ spores / ml, 80% Potato Dextrose Broth) 6
0 μl was added and the cells were cultured at 26 ° C. OD after 48 hours using a microplate reader (manufactured by Bio-Rad)
415 was measured to determine the MIC.

(Escherichia coli) Escherichia coli was inoculated into a bouillon medium (manufactured by Nissui Pharmaceutical Co., Ltd.).
After culturing at 7 ° C., the mixture was diluted with a bouillon medium to an OD660 of 0.01. 5 μl of this bacterial solution, 5 parts of broth medium
After adding 10 μl of a solution diluted to each concentration with respect to each of the five types of proteins, the cells were cultured for 16 hours at 37 ° C. After 16 hours, the OD660 of each culture was measured to determine the MIC.

(CHL cells) CHL / IU cells (Dainippon
MEM medium containing 10% CS (calf serum)
With 5% CO_TwoIncubate at 37 ° C for 4 days in an atmosphere.
4.4 × 10 cells in medium^FourPrepare suspension / ml
Made. Five types of proteins in a 96-well microplate
10 μl of a solution diluted to a predetermined concentration per
A culture test solution containing 90 μl of the suspension was prepared, and 5% CO 2
_TwoThe cells were cultured at 37 ° C for 3 days under an atmosphere. After culturing for 3 days,
Add 50 μl of XTT mixed standard solution (Boehringer) and add
% CO_TwoIncubate at 37 ° C for 2 hours in an atmosphere.
Measure absorbance (OD490-655) A with a rate reader
And the MIC was determined.

Table 1 shows the MIC of each protein for each cell.

[Table 1]

As shown in Table 1, the fusion protein prepared in Examples 1 to 4 was CAP18 (L104-V13
Compared to 5), the MIC against the filamentous fungus was about one tenth, and the antifungal activity was enhanced by a factor of 10 or more. The effects of the low antibacterial activity of the chitin-binding protein were not observed in the fusion proteins of Examples 1 to 4. On the other hand, the antibacterial activity of the fusion proteins of Examples 1 to 4 against Escherichia coli was equivalent to that of CAP18 (L104-V135) before fusion, and no reduction in activity due to fusion of the chitin-binding protein was observed. In addition, for CHL cells, the fusion protein of any of the examples was CAP18 (L104-V13
5) and equivalent to the chitin-binding protein of the comparative example,
No particular effect was observed. From the above results, it was revealed that a protein that binds to a major component on the cell surface of a target cell or that is a fusion protein of a protein and a membrane-acting antibacterial protein has a strong specific antibacterial effect on a target cell. .

[0045]

According to the present invention, it is possible to increase the specificity of a membrane-active peptide capable of acting on the surface layer of a cell for a specific target cell, and to increase the effect on a target cell.

[0046]

[Sequence List] SEQUENCE LISTING <110> KABUSHIKI KAISHA TOYOTA CHUO KENKYUSHO <120> Hybrid Protein <160> 10 <210> 1 <211> 264 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> (16) .. (246) <220> <223> Description of Artificial Sequence: Hybrid DNA <400> 1 gaagctgaat tcagt atg gcc cag agg tgc ggc gag caa ggc agc aac atg 51 Met Ala Gln Arg Cys Gly Glu Gln Gly Ser Asn Met 1 5 10 gag tgc ccc aac aac ctc tgc tgc agc cag tac ggc tac tgc ggg atg 99 Glu Cys Pro Asn Asn Leu Cys Cys Ser Gln Tyr Gly Tyr Cys Gly Met 15 20 25 ggc ggc gac tac tgc ggc ag cag aac ggc gcc tgc tgg acc 147 Gly Gly Asp Tyr Cys Gly Lys Gly Cys Gln Asn Gly Ala Cys Trp Thr 30 35 40 agc ctt ctt ggt gat ttc ttc aga aaa tct aaa gag aag att gga aaa 195 Ser Leu Leu Gly Asp Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys 45 50 55 60 gag ttt aaa aga att gtc cag aga atc aag gat ttt ttg aga aat ctt 243 Glu Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu 65 70 75 gta tgatagctcg agccgcgg 264 Val <210> 2 <211> 77 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: Hybrid Protein <400> 2 Met Ala Gln Arg Cys Gly Glu Gln Gly Ser Asn Met Glu Cys Pro Asn 1 5 10 15 Asn Leu Cys Cys Ser Gln Tyr Gly Tyr Cys Gly Met Gly Gly Asp Tyr 20 25 30 Cys Gly Lys Gly Cys Gln Asn Gly Ala Cys Trp Thr Ser Leu Leu Gly 35 40 45 Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg 50 55 60 Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val 65 70 75 <210> 3 <211> 279 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> (16) .. (261 ) <220> <223> Description of Artificial Sequence: Hybrid DNA <400> 3 gaagctgaat tcagt atg gcc cag agg tgc ggc gag caa ggc agc aac atg 51 Met Ala Gln Arg Cys Gly Glu Gln Gly Ser Asn Met 1 5 10 gag tgc ccc aac aac ctc tgc tgc agc cag tac ggc tac tgc ggg atg 99 Glu Cys Pro Asn Asn Leu Cys Cys Ser Gln Tyr Gly Tyr Cys Gly Met 15 20 25 ggc ggc gac tac tgc ggc aag ggc tgc cag ag gac tac 147 Gly Gly Asp Tyr Cys Gly Lys Gly Cys Gln Asn Gly Ala Cys Trp T hr 30 35 40 agc ggt ggt ggt ggt tct ctt ctt ggt gat ttc ttc aga aaa tct aaa 195 Ser Gly Gly Gly Gly Ser Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys 45 50 55 60 gag aag att gga aaa gag ttt aaa aga att gtc cag aga atc aag gat 243 Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile Val Gln Arg Ile Lys Asp 65 70 75 ttt ttg aga aat ctt gta tgatagctcg agccgcgg 279 Phe Leu Arg Asn Leu Val 80 <210> 4 <211> 82 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: Hybrid Protein <400> 4 Met Ala Gln Arg Cys Gly Glu Gln Gly Ser Asn Met Glu Cys Pro Asn 1 5 10 15 Asn Leu Cys Cys Ser Gln Tyr Gly Tyr Cys Gly Met Gly Gly Asp Tyr 20 25 30 Cys Gly Lys Gly Cys Gln Asn Gly Ala Cys Trp Thr Ser Gly Gly Gly 35 40 45 Gly Ser Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly 50 55 60 Lys Glu Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn 65 70 75 80 Leu Val <210> 5 <211> 294 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> (16) .. (276) <220> <223> Description of Artificial Sequence : Hybrid DNA <400> 5 gaagctgaat tcagt atg gcc cag agg tgc ggc gag caa ggc agc aac atg 51 Met Ala Gln Arg Cys Gly Glu Glu Gln Gly Ser Asn Met 1 5 10 gag tgc ccc aac aac ctc tgc tgc agcgc gc ca tgc ggg atg 99 Glu Cys Pro Asn Asn Leu Cys Cys Ser Gln Tyr Gly Tyr Cys Gly Met 15 20 25 ggc ggc gac tac tgc ggc aag ggc tgc cag aac ggc gcc tgc tgg acc 147 Gly Gly Asp Tyr Cys Gly Lys G Asn Gly Ala Cys Trp Thr 30 35 40 agc ggt ggt ggt ggt tct ggt ggt ggt ggt tct ctt ctt ggt gat ttc 195 Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Leu Gly Asp Phe 45 50 55 60 ttc aga aaa tct aaa gag aag att gga aaa gag ttt aaa aga att gtc 243 Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile Val 65 70 75 cag aga atc aag gat ttt ttg aga aat ctt gta tgatagctcg agccgggg 294g Asp Phe Leu Arg Asn Leu Val 80 85 <210> 6 <211> 87 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: Hybrid Portein <400> 6 Met Ala Gln Arg Cys Gly Glu Gln Gly Ser Asn Met Glu Cys Pro Asn 1 5 10 15 Asn Leu Cys Cys Ser Gln Tyr Gly Tyr Cys Gly Met Gly Gly Asp Tyr 20 25 30 Cys Gly Lys Gly Cys Gln Asn Gly Ala Cys Trp Thr Ser Gly Gly Gly 35 40 45 Gly Ser Gly Gly Gly Gly Ser Leu Leu Gly Asp Phe Phe Phe Arg Lys Ser 50 55 60 Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile Val Gln Arg Ile Lys 65 70 75 80 Asp Phe Leu Arg Asn Leu Val 85 <210> 7 <211> 309 < 212> DNA <213> Artificial Sequence <220> <221> CDS <222> (16) .. (291) <220> <223> Description of Artificial Sequence: Hybrid DNA <400> 7 gaagctgaat tcagt atg gcc cag agg tgc ggc gag caa ggc agc aac atg 51 Met Ala Gln Arg Cys Gly Glu Gln Gly Ser Asn Met 1 5 10 gag tgc ccc aac aac ctc tgc tgc agc cag tac ggc tac tgc ggg atg 99 Glu Cys Pro Asn Asn Leu Cys Tyr Gly Tyr Cys Gly Met 15 20 25 ggc ggc gac tac tgc ggc aag ggc tgc cag aac ggc gcc tgc tgg acc 147 Gly Gly Asp Tyr Cys Gly Lys Gly Cys Gln Asn Gly Ala Cys Trp Thr 30 35 40 agc ggt ggt tct ggt ggt ggt ggt tct ggt ggt ggt ggt ctc 195 Ser Gly Gly Gly Gly Gly Ser Gly G ly Gly Gly Ser Gly Gly Gly Gly Leu 45 50 55 60 ctt ctt ggt gat ttc ttc aga aaa tct aaa gag aag att gga aaa gag 243 Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 65 70 75 ttt aaa aga att gtc cag aga atc aag gat ttt ttg aga aat ctt gta 291 Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val 80 85 90 tgatagctcg agccgcgg 309 <210> 8 <211> 92 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: Hybrid Protein <400> 8 Met Ala Gln Arg Cys Gly Glu Gln Gly Ser Asn Met Glu Cys Pro Asn 1 5 10 15 Asn Leu Cys Cys Ser Gln Tyr Gly Tyr Cys Gly Met Gly Gly Asp Tyr 20 25 30 Cys Gly Lys Gly Cys Gln Asn Gly Ala Cys Trp Thr Ser Gly Gly Gly 35 40 45 Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Leu Leu Leu Gly Asp 50 55 60 Phe Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 65 70 75 80 Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val 85 90 <210> 9 <211> 168 <212> DNA <213> Wheat germ <220> <221> CDS <222> (16) .. (150) <400> 9 gaagctgaat tcagt at g gcc cag agg tgc ggc gag caa ggc agc aac atg 51 Met Ala Gln Arg Cys Gly Glu Gln Gly Ser Asn Met 1 5 10 gag tgc ccc aac aac ctc tgc tgc agc cag tac ggc tac tgc ggg atg Asn Cylu Leu Cys Cys Ser Gln Tyr Gly Tyr Cys Gly Met 15 20 25 ggc ggc gac tac tgc ggc aag ggc tgc cag aac ggc gcc tgc tgg acc 147 Gly Gly Asp Tyr Cys Gly Lys Gly Cys Gln Asn Gly Ala Cys Trp Thr 30 agc tgatagctcg agccgcgg 168 Ser 45 <210> 10 <211> 45 <212> PRT <213> Wheat germ <400> 10 Met Ala Gln Arg Cys Gly Glu Gln Gly Ser Asn Met Glu Cys Pro Asn 1 5 10 15 Asn Leu Cys Cys Ser Gln Tyr Gly Tyr Cys Gly Met Gly Gly Asp Tyr 20 25 30 Cys Gly Lys Gly Cys Gln Asn Gly Ala Cys Trp Thr Ser 35 40 45

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/415 C07K 19/00 14/47 C12N 1/15 19/00 1/19 C12N 1/15 1 / 21 1/19 15/00 ZNAA 1/21 A61K 37/02 5/10 C12N 5/00 A (72) Inventor Masanori Hirai 41-cho, Yokomichi, Oku-cho, Nagakute-cho, Aichi-gun, Aichi Prefecture 1 Toyota Toyota Chuo Kenkyu Co., Ltd. In-house F-term (reference) 4B024 AA01 AA07 AA10 BA80 CA03 CA05 CA07 CA10 DA12 EA04 GA11 GA19 GA27 HA03 4B065 AA72X AA89Y AA93Y AB01 AC14 BB01 BB16 BB17 BC03 BC26 BD01 BD14 BD15 BD17 BD50 CA24 CA08 BA08 BA23 BA08 ZB 4H011 AA01 AA03 BB19 DH11 4H045 AA10 AA20 BA21 BA41 CA32 CA40 DA83 EA05 EA29 FA74 GA01 GA10 GA15 GA26

Claims (8)

[Claims] 1. A fusion protein comprising: a first peptide site capable of acting on the cell surface of a target cell; and a second peptide site capable of binding to a component of the cell surface of the target cell. 2. The components of the cell surface are bacteria, fungi,
The fusion protein according to claim 1, which is a cell wall component of any of animal cells and plant cells. 3. The cell surface component comprises an acidic polysaccharide,
3. The fusion protein according to claim 1, comprising at least one selected from the group consisting of chitin, cellulose, hemicellulose, lignin, pectin, and cholesterol. 4. The fusion protein according to claim 1, wherein said first peptide site comprises a cell-killing peptide, a part thereof, or a derivative thereof. 5. The method according to claim 5, wherein the first peptide site is CAP18,
5. A peptide selected from the group consisting of magainins, melittins, alamethicins, polymyxins, cecrobins, tachybresins, dermaseptins, bombinsins, and thionins, including a part thereof or a derivative thereof. A fusion protein as described. 6. A DN encoding a first peptide site capable of acting on the cell surface of a target cell, and a second peptide site having a binding property to a component of the cell surface of the target cell.
A DNA construct having an A sequence. 7. A transformed cell which carries the DNA sequence of claim 6 in an expressible manner. 8. A fusion protein having a first peptide site and a second peptide site, wherein the first peptide site has cell killing properties and the second peptide site has a chitin-binding property. A method for imparting fungal resistance to an organism that can be damaged by a fungus using a transformed cell that produces a fusion protein, the method comprising: