HK1117175B - Antifungal protein and usage thereof - Google Patents

Abstract

The present invention relates to an antifungal protein gene and its cDNA sequence, which are obtained by exploring the whole genome sequence and single gene database of Monascus pilosus.The gene can encode the antifungal protein MAFP1.A purified protein with a molecular weight of approximately 7kDa was obtained from the culture medium of Monascus purpureus and identified as MAFP1 protein through N-terminal protein sequencing and comparative analysis.The purified MAFP1 protein can inhibit the growth of pathogenic bacteria such as Paecilomyces variotii and Helminthosporium panici.Through PCR experiments, it was found that the gene for the antifungal protein is present in other species of Monascus, such as Monascus Buck (MBarkeri)、 Florida Monascus (M. floridanus), Moon Spore Monascus (M. lunisporas), Silk haired Monascus, Red Monascus (M. ruber), and so on.It has also been proven that the mafp1 gene and its cDNA of Monascus purpureus and Monascus purpureus have the same DNA sequence.

Description

Antifungal proteins and uses thereof

Technical Field

The invention relates to an antifungal protein and application thereof. Provides a preparation method of the protein. The invention also relates to sequences of antifungal proteins and DNA encoding said protein sequences, vectors containing said DNA sequences, cells transformed with said DNA sequences and methods of treating and/or controlling fungal diseases.

Background

It is known that more than 200 animal pathogenic fungi and more than 30 common plant pathogenic fungi can have a tremendous impact on human health and economy. At present, drugs for controlling human pathogenic fungi are mainly based on small molecules, such as polyenes (polyenes), azoles (azoles), fluconazole (fluconazoles), amphotericin b (amphotericin b), and the like. As the number of drug abuse increases, the resistance of fungal strains becomes more and more severe. There is an urgent need to develop new antifungal drugs (see, selitrnikoff, C.P, 2001, antimugial proteins, appl. envirion. microbiol.67, 2883-.

It is known that plants, bacteria, fungi, insects, birds and mammals produce antifungal proteins (see Kaiser, L., Oberparietiter, C., Weiler-R.Burgstaller, W., Leiter, E., and Marx, F., 2003, Characterization of the Penicillium chrysogenum anti uniform protein PAF. Arch Microbiol.180, 204-. Although these proteins have different amino acid sequences and quaternary structures, the small molecular weight, high basicity and the high amount of cysteine are the main molecular characteristics of most antifungal proteins (see Selitrennikoff et al, 2001).

A few antifungal proteins of filamentous fungi have been studied, such as the AFP protein of Aspergillus megaterium (see, Wnendt, S., Ullbrich, N. and Stahl, U.S., 1990, Cloning and nucleic acid sequence of encoding the antibacterial-protein of Aspergillus and expressing the native gene, nucleic acid Res.18, 3987; Wnendt, S.S., Ullbrich, N.and Stahl, U.S., 1994, Molecular Cloning, sequence analysis and expression of the genetic encoding protein of the genetic encoding amino protein from Aspergillus, Cu.Curr.25, 519, S.31, S.B., M.A. Pat. No. 4, M.A. of Aspergillus, S.A. Pat. No. 4, M.A. 4, M.A.A. 4, M.A. 4, M.A.A.Aicrobilol.156, 47-56; and The PAF proteins of Penicillium chrysogenum (see, Marx, F., Hass, H., Reindel, M., Stoffer, G., Lottspch ei, F. and Redl B., 1995, Cloning, structural organization and regulation of The Penicillium chrysogenum coding of The isolated gene linked to gene linked to gene linked to gene linked to; and kaiser et al, 2003) and the Anafp protein of aspergillus niger (aspergillus niger) (see, Lee, g.d., Shin, s.y., Maeng, c.y., Jin, z.z., Kim, K.L and Hahm, KS., 1999, Isolation and catalysis of a novel antibiotic peptide from aspergillus niger biochem, biophysis, res, comm.263, 646-. The antifungal proteins are secreted proteins and can inhibit the growth of various fungi, but have no influence on bacteria and yeasts. These antifungal proteins have similar molecular characteristics, but the amino acid sequences of the antifungal proteins PAF and AFP have only 42% sequence similarity (see, Kaiser et al, 2003). Antifungal proteins derived from these fungi primarily inhibit fungi of the genera Aspergillus spp and Fusarium spp (see, Theis et al, 2003; and Kaiser et al, 2003). PAF proteins may also inhibit human and animal pathogenic fungi, such as the genera Abaidia, Mortierella (Mortierella spp.), Rhizomucor (Rhizomucor spp.) and Rhizopus (Rizopus spp.). These proteins are not only useful as biocontrol agents for phytopathogenic fungi, but also have the potential to develop antifungal drugs for humans and animals (see,，L.，Papp.T.，Letter，E.Marx，F.，i, and，C.，2005，Sensitthe property of differential Zygomycetes to the Penicillium chrysogenum anti Protein (PAF). J.basic microbiological.45, 136-. In addition, it has been reported in the literature that resistance of rice to rice fever pathogenic rice blast (Magnaporthe grisea) can be enhanced by transfecting cDNA of AFP protein of Aspergillus megaterium into rice, and thus AFP protein can be used for control of rice fever diseases (see Coca, M., Bortolti, C., Rufat, M., Penas, G., Eritja, R., Tharreau, D., Del Pozo A, M., Messeguer, J. and San Seguindo, B., 2004, Transgenic plants expressing the anti-genetic AFP protein from rice possessed fermented product of the plant resistance to the plant resistance of rice, and also see, biological sample A, 14, Aspergillus strain, Aspergillus.

Paecilomyces variotii (Paecilomyces variotii) and Paecilomyces lilacinus (P.lilacinus) are the most common species in the genus of Paecilomyces (Paecilomyces) and most commonly cause human infections. Endophthalmitis and endocarditis are the two most common infections caused by paecilomyces variotii and paecilomyces lilacinus, respectively, and the prognosis is extremely poor. The failure rate for standard treatment of these infections is about 40%. The use of combination therapy or the development of novel antifungal agents would be the direction of future therapeutic development (see, Ortoneda, M., Capilla, J., Pastor, F.J., Pujol, I., Yusts, C., Serena, C., and Guarro, J. (2004) In vitro interaction of advanced and novel drugs against Paecilomyces sp. antibiotic. Agents Chemother.48, 2727-. Helminthosporium paniculatum (Helminthosporium panici) is the causative bacterium of plant ring spot. The use of biomolecular technology to achieve effective fungal infection control and reduce the loss of fungal diseases to human health, commercial crops and livestock animals is a considerable topic.

Monascus (Monascus) strains are important traditional fermentation fungi in east Asia and are used for manufacturing fermented products such as wine, red wine lees (anka), tofu (sufu), soy sauce and the like. In addition, species of Monascus also produce various metabolites and enzymes, such as monacolin K (monoacolin K) (see, Endo, A., Hashimi, K., and Negishi, S. (1985) Monacolins J and L, new inhibitors of cholesterol biosynthesis and promoter J. Antibiot. 38 (Tokyo) 420-2), citrinin (trinin) (see, Hajjaj, H., klalebe, A., Gogma, G., Blanc, P. J., Barbier, E. and Francois, J. GABA (2000) Medium-chains and intermediates of protein synthesis and yellow pigment, dye of protein, 1. C., 1. and yellow, dye of protein, 1. 12. and yellow, (see, yellow) pigment, dye of protein, 1. C., 1. and yellow, dye of pigment, 2. 12. C., 1. and yellow pigment, 2. 12. C., and yellow. 12. C., yellow. and yellow. 12. pigment, yellow pigment, dye of protein, 2. C. 12. and yellow. 12. C., 1. 7. C., 1. and 7. pigment, 2. C. pigment, 2. D, C, 2. C. pigment, 2. C. origin, 2. E. C, C. origin, 2. D. B. origin, 2. D. origin, C. D. origin, C. origin, C, shaken cut. can.j. microbiol.23 (10): 1360-72; and Tseng, y.y., Chen, m.t. and Lin, C.F (2000) Growth, fragment production and protease activity of monascus purpureus as infected by salt, sodium nitrate, polyphosphate and vacuum sources, j.appl.microbiol.88 (1): 31-7) and proteases (see, Tsai, m.s., Hseu, t.h., and Shen, Y.S (1978) Purification and characterization of an acid protease from Monascus kaoliang. int.j. protein res.12, 293-302), and thus have infinite potential in drug development and industrial enzyme applications. In these applications, citrinin is known to have bacterial growth inhibitory activity. However, there is no disclosure in the literature of monascus species having inhibitory activity on fungal growth. By completing whole genome sequencing and decoding of monascus in early days, we explored a possible antifungal protein gene, from which monascus species were presumed to have antifungal activity.

Disclosure of Invention

An object of the present invention is to provide an isolated and purified antifungal protein MAFP1 obtained from Monascus species. Preferably, the species Monascus is selected from the group consisting of Monascus bakanae (Monascus barkeris), Monascus floridanus (Monascus floridanus), Monascus lunatus (Monascus lunispora), Monascus hirsutus (monascu spilosus) and monasculus rubra (Monascus ruber). More preferably, the monascus species is selected from the group consisting of monascus ruber BCRC33309 ═ ATCC16966, monascus florida BCRC33310 ═ IMI282587, monascus lunatus BCRC33640 ═ ATCC204397, monascus hirsutus BCRC38072 (stored at the Food Industry Research and Development Institute, FIRDI) biological resource Collection and Research Center (bioresearch Collection and Research Center, BCRC) (331Shih-Pin Road, 300Taiwan)), BCRC38093 (stored in FIRDI's BCRC) and BCRC31502 ═ ATCC16363, monascus ruber BCRC 3178 ═ ATCC 163533, ATCC 31533 ═ ATCC 3316399, ATCC 3166, ATCC 31181323 ═ ATCC 33323, ATCC 3399.

Another object of the present invention is to provide an isolated and purified polynucleotide comprising a nucleotide sequence encoding the antifungal protein MAFP 1.

Another objective of the invention is to provide a recombinant vector nucleotide sequence for encoding antifungal protein MAFP 1.

It is another object of the present invention to provide a recombinant host cell comprising the recombinant vector of the present invention.

It is another object of the present invention to provide a composition comprising the antifungal protein of the present invention and a suitable carrier, wherein the amount of the protein is sufficient to provide protection from fungal disease.

It is another object of the present invention to provide a method of controlling plant fungi comprising applying to a plant an antifungal protein of the present invention in an amount sufficient to provide protection from fungal disease.

It is another object of the present invention to provide a transgenic organism incorporating in its genome a transgene encoding an antifungal protein of the present invention.

It is another object of the present invention to provide a method of treating a fungal disease in a patient, the method comprising administering to the patient an antifungal protein of the present invention in an amount sufficient to provide protection from the fungal disease.

It is a further object of the present invention to provide an isolated and purified primer pair which can amplify a nucleotide encoding an antifungal protein of the present invention.

It is still another object of the present invention to provide a PCR assay kit comprising the primer pair of the present invention.

The following sections describe the invention in detail. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

Drawings

FIG. 1 shows the amino acid sequence of the Monascus antifungal protein MAFP 1.

Fig. 2 shows the sequence alignment of the mature region (a) and full length protein (B) of the antifungal protein MAFP1 of monascus filamentosus, the AFP protein of aspergillus megaterium (a. giganteus) and the PAF protein of p.chrysogenus.

FIG. 3 shows the SFDS PAGE analysis of the purified Monascus antifungal protein MAFP 1. Lane 1: marker protein, lane 2: the MAFP1 protein was purified.

FIG. 4 shows the antifungal activity assay of the Monascus antifungal protein MAFP 1. The antifungal ability of the purified MAFP1 protein against the pathogenic fungi Paecilomyces variotii (BCRC33174) and Helminthosporium paniculatum (BCRC35004) was investigated by double culture, and the growth of the fungi was observed. (-) MAFP1 control without MAFP 1; (+) MAFP1 Experimental groups with varying amounts of MAFP1 protein. A to H represent paper discs with 0.4. mu.g, 0.2. mu.g, 0.1. mu.g, 0.05. mu.g, 0. mu.g, 0.8. mu.g, 0.64. mu.g and 0.32. mu.g of MAFP1 protein, respectively.

Detailed Description

The present invention describes a novel gene of the monascus species (hereinafter referred to as masp 1) characterized by similarity to the gene encoding the antifungal protein AFP of aspergillus megaterium and the gene encoding the PAF of penicillium chrysogenum. It has been found that the protein encoded by the novel gene (hereinafter MAFP1) has antifungal activity and is useful in the treatment and/or control of fungal diseases.

Definition of

Unless defined otherwise herein, technical and scientific terms used herein will have the meaning commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clearly understood, however, in any case ambiguous, the present invention provides a definition that takes precedence over any dictionary or extrinsic definition.

The terminology and techniques used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are generally well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific documents cited and discussed throughout the present specification. Enzymatic reactions and purification techniques were performed according to manufacturer's instructions, as commonly practiced in the art or as described herein. The nomenclature used and the laboratory procedures and techniques described herein for analytical chemistry, synthetic organic chemistry, and medicinal chemistry are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

As used in accordance with the present invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

the term "isolated and purified protein" as referred to herein means that the protein of interest is (1) at least free of certain other proteins that may normally be found with the protein, (2) substantially free of other proteins of the same origin (e.g., from the same species), (3) expressed by cells from a different species, (4) separated from at least about 50% of the polynucleotides, lipids, carbohydrates or other materials with which the protein is naturally associated, (5) not associated (by covalent or non-covalent interactions) with the portion of the protein with which the isolated protein is naturally associated, (6) operably associated (by covalent or non-covalent interactions) with the polypeptide with which it is not naturally associated, or (7) does not occur naturally. Genomic DNA, cDNA, mRNA or other RNA of synthetic origin, or any combination thereof, may encode the isolated protein. Preferably, the isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that interfere with its therapeutic, diagnostic, prophylactic, research or other use. Isolated and purified proteins may also be made substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

The term "antifungal protein" means a protein having antifungal properties, e.g., inhibiting the growth of a fungal cell, killing a fungal cell, or disrupting or delaying the progression of the fungal life cycle, such as spore germination, sporulation, and mating.

The term "amino acid sequence" means the amino acid sequence of a naturally occurring protein molecule. "amino acid sequence" and similar terms (such as "polypeptide" or "protein") are not intended to limit the amino acid sequence to the complete, native amino acid sequence associated with the referenced protein molecule. Amino acid sequences include oligopeptide, peptide, polypeptide, or protein sequences and fragments or portions thereof, as well as naturally occurring or synthetic molecules.

The term "biologically functional equivalent" refers to an equivalent with respect to an antifungal protein of the present invention that contains a sequence or portion that exhibits sequence similarity to the novel peptide of the present invention (e.g., MAFP1) and exhibits the same or similar functional properties (including antifungal activity) as the polypeptides disclosed herein. For example, there may be some variation in the amino acid sequence of the biologically functional equivalent of an antifungal protein of the present invention that is different from but substantially identical to the amino acid sequence of the protein, and that is substantially identical (only to a greater or lesser degree) to the properties of the protein as set forth herein.

The term "isolated and purified polynucleotide" as referred to herein means that the target polynucleotide is (1) not associated (covalently or non-covalently) with all or a portion of the other polynucleotides with which the target polynucleotide is naturally associated, (2) associated with molecules with which it is not naturally associated, or (3) not associated with any other polynucleotides in nature. The polynucleotide may be genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof. Preferably, the isolated and purified polynucleotides of the present invention comprise a single coding region. Although a polynucleotide includes a single coding region, it may contain other nucleotides that do not adversely affect the function of the polynucleotide. For example, the 5 'and 3' untranslated regions may contain different numbers of nucleotides. Preferably, the additional nucleotides are outside the single coding region.

The term "nucleotide sequence" means a single-or double-stranded nucleic acid polymer of at least 10 bases in length. In certain embodiments, the nucleotides comprising the polynucleotide may be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Such modifications include base modifications (e.g., bromouracil nucleoside and inosine derivatives), ribose modifications (e.g., 2 ', 3' -dideoxyribose), and internucleotide linkage modifications (e.g., diselenophosphate, phosphoroanilothioate, phosphoroaniladate, and phosphoramidate), and the like. The nucleotide sequences of the present invention may include labels for detection assays, including radioisotope labels, fluorescent labels, haptens or antigenic labels.

The term "vector" refers to any molecule (e.g., nucleic acid, plasmid, episome, or virus) used to transfer encoded information into a host cell. The term also includes "recombinant vectors", "expression vectors" or "expression constructs". The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing a nucleotide sequence that directs and/or controls (in conjunction with the host cell) the expression of one or more operably linked heterologous coding regions. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, if an intron is present, RNA splicing of a coding region to which the intron is operably linked. The vector is preferably one which is capable of autonomous replication and expression of the nucleic acid to which it is linked.

The term "host cell" means a cell that has been transformed with, or is capable of being transformed with, a nucleic acid sequence so as to express a selected gene of interest. This term includes progeny of the parent cell, whether or not the progeny are identical to the original parent cell in morphology or genetic marker (genetic marker-up), so long as the selected gene is present.

The term "transformation" means the alteration of a genetic characteristic of a cell, which is said to be transformed when the cell is modified to contain new DNA. For example, a cell is said to be transformed when it is genetically modified from its natural state by transfection, transduction, or other techniques.

The term "transgenic organism" refers to any organism in which an exogenous gene is introduced into one or more (preferably substantially all) cells of the organism. Genes are introduced into cells directly or indirectly by means of careful genetic manipulation, e.g., by microinjection or infection with recombinant vectors, into cell precursors. The term genetic manipulation encompasses not only traditional cross breeding or in vitro fertilization, but also the introduction of recombinant DNA molecules that can integrate into the chromosome or replicate the DNA extrachromosomally. Transgenic animals include animals or organs, tissues or cells derived from transgenic animals. Transgenic plants include plants, plant progeny, seeds of transgenic plants, or cells derived from transformed plant cells or protoplasts.

The term "identity" refers to a sequence relationship between two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing their sequences. "identity" measures the percentage of identical matches between the smaller of two or more sequences.

The term "similarity" is used in the art with respect to related concepts; however, in contrast to "identity," similarity is a measure of relatedness that includes both identity matches and conservative substitution matches.

Unless otherwise required by the context, singular terms include the plural and plural terms include the singular.

Monascus antifungal protein and gene thereof

It is an object of the present invention to provide an isolated and purified antifungal protein from monascus species. It is another object of the present invention to provide an isolated and purified polynucleotide comprising a nucleotide sequence encoding said antifungal protein. Through exploring the monascus whole genome database and carrying out comparative analysis, the monascus antifungal protein and gene encoding antifungal protein are found.

Tblastn was used to compare the amino acid sequences of both the antifungal protein AFP of Aspergillus megaterium (accession No.: embICAA37523.1I) and the PAF of Penicillium chrysogenum (accession No.: gbIAAA92718.1|) with the sequences of the single gene database from the Monascus pilosus BCRC38072 whole genome sequence database (the institute for food industry development (FIRDI) internal database). BLAST programs (including blastp, blastn, blastx, tblastn, tblastx, and the like) are publicly available from the National Center for Biotechnology Information, NCBI, and other sources (BLAST Manual, Altschul et al, NCB/NLM/NIH Bethesda, MD 20894). Single gene contigs (unigene contigs) with approximately 30% similarity to the protein sequences of AFP and PAF were obtained and analyzed by Vector NTI (InforMax) software for Open Reading Frame (ORF) prediction. The 279 base pair cDNA (SEQ ID NO: 1) is predicted to be recognized by the ORF and can be translated into a protein consisting of 92 amino acids (SEQ ID NO: 2). The antifungal protein was named MAFP 1. The expression of the polypeptide as shown in SEQ ID NO: 2 has a single peptide (positions 1-18), a propeptide (positions 19-34) and a mature protein (positions 35-92) (see figure 1). It is shown that the protein can be secreted by the monascus cells.

The sequences shown in SEQ ID NO: l and the sequence shown in the sequence I is compared with the Monascus purpureus whole genome database, and the genome DNA sequence (SEQ ID NO: 4) of antifungal protein is obtained and named as the mafp1 gene. The sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4, and tblastn is used to compare the sequence shown in SEQ ID NO: 2 was compared to the NCBI and Swiss-Prot protein databases to identify published sequences similar to the DNA and protein sequences of MAFP 1. The alignment program of Vector NTI software was used to align the amino acid sequences of MAFP1(SEQ ID NO: 2), AFP of Aspergillus megaterium (accession No.: embICAA37523.1I) and PAF of Penicillium chrysogenum (accession No.: gbIAAA92718.1. I) to find a highly conserved sequence of amino acid sequences (AAXGXVAXP) (see FIG. 2 (B)). It has been found that there are highly conserved regions in the single and propeptide regions. The mature protein sequence of MAFP1(SEQ ID NO: 3) shares 29% and 31% similarity with the amino acid sequences of AFP from Aspergillus megaterium and PAF from Penicillium chrysogenum, respectively. The six cysteines at positions 8, 15, 28, 36, 43, and 54 in the mature MAFP1 sequence are highly conserved residues of antifungal proteins of fungal origin. By blastn comparison, NO DNA sequence similar to that of the cDNA (SEQ ID NO: 1) and genomic DNA sequence (MAFP1, SEQ ID NO: 4) of MAFP1 was found in the DNA sequences of NCBI nr database. Thus, it can be concluded that: MAFP1 is a novel protein and masp 1 is a novel gene.

The scope of the present invention encompasses peptides, polypeptides and proteins that are biologically functional equivalents of the antifungal proteins of the present invention, including amino acid sequences that contain conservative amino acid changes in the basic sequence of the antifungal protein. In such amino acid sequences, one or more amino acids in the base sequence are replaced with other amino acids that have a charge and polarity similar to the natural amino acids, i.e., conservative amino acid substitutions that result in silent changes (silent changes).

The substituents of amino acids within the base polypeptide sequence may be selected from other members of the class to which the naturally occurring amino acids belong.

Amino acids can be divided into the following four categories: (1) acidic amino acids, (2) basic amino acids, (3) neutral polar amino acids, and (4) neutral nonpolar amino acids. Representative amino acids in the classes include, but are not limited to, (1) acidic (negatively charged) amino acids, such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids, such as arginine, histidine and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.

Conservative amino acid changes may be made in the base protein sequence by replacing one amino acid belonging to one of the classes with another amino acid belonging to the same class. Biologically functional equivalents of antifungal proteins may have 20 or fewer conservative amino acid changes, more preferably 10 or fewer conservative amino acid changes, and most preferably 5 or fewer conservative amino acid changes. Thus, the encoding nucleotide sequence (gene, plasmid DNA, cDNA or synthetic DNA) will have corresponding base substitutions such that it can encode a biologically functional equivalent form of the antifungal protein.

Thus, biologically functional equivalent peptides, polypeptides and proteins encompassed herein should have about 80% or greater sequence similarity to the base amino acid sequence of the antifungal protein of the present invention or the corresponding portion thereof, preferably 85% or greater sequence similarity, and most preferably about 90% or greater sequence similarity.

Although the antifungal polypeptide of the present invention preferably has the amino acid sequence of SEQ ID NO: 2 or a biologically functional equivalent thereof, but fragments and variants of said sequences having the same or similar antifungal activity as the antifungal polypeptide also belong to the invention. Thus, SEQ ID NO: 2 or a contiguous sequence of more than 8 amino acids may exhibit this activity.

SEQ ID NO: 2 fragments may be truncated forms in which one or more amino acids are deleted from the N-terminus, C-terminus, polypeptide middle, or combinations thereof. The fragment may be SEQ ID NO: 2, and the sequence of SEQ ID NO: 2, antifungal activity.

SEQ ID NO: 2 includes forms in which one or more amino acids have been inserted in the natural or synthetic sequence. The variant may also be SEQ ID NO: 2, and the sequence of seq id NO: 2, antifungal activity.

Combinations of the above forms, i.e., antifungal protein forms containing both amino acid deletions and additions, are also within the present invention. Amino acid substitutions may also be present.

SEQ ID NO: 2 and SEQ ID NO: 2 should preferably have a sequence similarity of about 70% or more, more preferably about 80% or more, and most preferably about 90% or more.

Other biologically functional equivalent forms of the antifungal proteins of the present invention suitable for use in the present invention include conjugates of the polypeptide or biologically functional equivalent thereof as described above with other peptides, polypeptides or proteins to form fusion products exhibiting the same, similar or higher antifungal activity as the antifungal proteins of the present invention.

Biologically functional equivalents also include peptides, polypeptides and proteins that react with (i.e., specifically bind to) antibodies (including monoclonal and polyclonal antibodies) against the antifungal proteins of the present invention and exhibit the same or similar antifungal activity.

Methods for producing biologically functional equivalents of a polypeptide or protein include any suitable method known in the art, such as direct chemical synthesis or synthesis in heterologous biological systems such as microbial, plant and animal systems; tissue culture; culturing cells; or an in vitro translation system. Methods for altering amino acid sequences are well known in the art, such as genetic engineering techniques (e.g., site-directed mutagenesis) to alter the nucleotide or amino acid sequence and expression of recombinant proteins.

The invention includes not only the nucleotide sequences as shown in SEQ ID NO: 1 or 4, and further includes biologically functionally equivalent nucleotide sequences. The phrase "biologically functionally equivalent nucleotide sequence" means a nucleotide sequence encoding a polypeptide exhibiting a sequence identical to SEQ ID NO: DNA and RNA for peptides, polypeptides and proteins of the same or similar antifungal activity, including genomic DNA, plasmid DNA, cDNA, synthetic DNA and mRNA nucleotide sequences, i.e., that when introduced into a host cell in a functionally operable manner such that it is expressed, produces peptides, polypeptides or proteins that exhibit antifungal activity at levels sufficient to be resistant to the pathogenic fungus on the cell.

Biologically functional equivalent nucleotide sequences of the invention include nucleotide sequences that encode conservative amino acid changes in the base antifungal protein sequence, resulting in silent changes therein. The nucleotide sequence contains corresponding base substitutions compared to the nucleotide sequence encoding the wild-type antifungal protein (as shown in SEQ ID NO: 2).

In addition to nucleotide sequences encoding conservative amino acid changes in the base antifungal protein sequence, biologically functional equivalent nucleotide sequences of the present invention include nucleotide sequences containing other base substitutions, additions or deletions. The nucleotide sequence comprises a nucleotide sequence comprising a nucleotide sequence identical to SEQ ID NO: 1 or 4, and which encodes a nucleotide sequence that confers upon the host cell and organism the same inherent genetic information as that contained in SEQ ID NO: 2, the same or similar fungal resistance. The nucleotide sequence may be referred to as SEQ ID NO: 1 or 4, and can be identified by the methods described herein.

Mutations in cDNA, plasmid DNA, genomic DNA, synthetic DNA or other nucleotides encoding the antifungal proteins of the present invention (e.g., SEQ ID NO: 1) preferably preserve the reading frame of the coding sequence. Furthermore, the mutations preferably do not produce complementary regions that can hybridize to produce secondary mRNA structures that adversely affect mRNA translation, such as loops or hairpins.

Although the site of mutation can be predetermined, the nature of the mutation itself need not be predetermined. For example, to select the best characteristics of a mutation at a given site, random mutations can be made at the target codon. Alternatively, mutations can be introduced at specific loci by synthesizing oligonucleotides containing mutant sequences flanked by restriction sites to allow ligation to fragments of the native cDNA sequence. Following ligation, the resulting reconstituted nucleotide sequence encodes a derivative form of the nucleic acid sequence with the desired amino acid insertion, substitution or deletion. In each case, the expressed mutants can be screened for the desired antifungal activity, for example, by the method described in example 4.

A useful genetically equivalent modified form of SEQ ID NO: 1 or 4 include those having a sequence exhibiting an amino acid sequence identical to that of SEQ ID NO: 1 or 4, or a nucleotide sequence having high sequence identity. This range of identity may be with SEQ id no: 1 or 4 or a corresponding portion thereof, has about 70% or greater sequence identity, more preferably about 80% or greater sequence identity, and most preferably about 90% or greater sequence identity.

The genetically equivalent modified forms can be readily isolated using conventional DNA-DNA or DNA-RNA hybridization techniques or by amplification using the Polymerase Chain Reaction (PCR) method. The forms should have the ability to confer resistance to pathogenic fungi when introduced into host cells with normal susceptibility to such fungi by conventional transformation techniques.

Fragments and variants of antifungal proteins (e.g., SEQ ID NO: 2) may be encoded by cDNA, plasmid DNA, genomic DNA, synthetic DNA, or mRNA. The nucleic acid has a sequence similar to the sequence having SEQ ID NO: 1 or 4 or its corresponding mRNA should have a sequence similarity of about 70% or greater, preferably about 80% or greater, and most preferably about 90% or greater.

In the present invention, the polypeptide having the amino acid sequence of SEQ ID NO: 1 or 4 includes:

DNA of altered length by natural or artificial mutation (e.g., partial nucleotide deletion, insertion, addition, etc.) is modified so that the DNA sequence shown in SEQ ID NO: 1 or 4, the biologically functional equivalent sequence has the sequence of SEQ ID NO: an approximate length of about 60% to about 120% of the length of 15, preferably about 80% to about 110% thereof; or

Nucleotide sequences containing partial (typically about 20% or less, preferably about 10% or less, more preferably about 5% or less of the full length) natural or artificial mutations such that the sequences encode different amino acids, but wherein the resulting polypeptide retains the antifungal activity of the antifungal polypeptide of the present invention (e.g., SEQ ID NO: 2). The mutated DNA produced in this way typically encodes a DNA sequence identical to SEQ ID NO: 2, or a polypeptide having about 70% or more, preferably about 80% or more, and more preferably about 90% or more sequence identity.

In the present invention, the method for generating artificial mutations is not particularly limited, and the mutations may be generated by any conventional method in the art. Biologically functional equivalents of the DNA sequences disclosed herein produced by any of the methods can be selected by analyzing the peptide, polypeptide, or protein encoded thereby using the techniques described in the examples.

Due to the degeneracy of the genetic code, i.e., more than one codon for most amino acids naturally occurring in a protein, a DNA containing substantially the same genetic information as the DNA of the invention and encoding a polypeptide identical to the polypeptide represented by SEQ ID NO: 1 or 4, or a sequence of another DNA (and RNA) sequence having an amino acid sequence substantially identical to the amino acid sequence encoded by the nucleotide sequence of 1 or 4. This principle also applies to any other nucleotide sequence discussed herein.

Biologically functional equivalent forms of the DNA of the invention also include synthetic DNA designed to enhance expression in a particular host cell. Host cells typically exhibit a preferred codon usage pattern, and thus synthetic DNA designed to enhance expression in a particular host should reflect the codon usage pattern in the host cell.

SEQ ID NO: 1 or 4 includes those modified to encode a conjugate with other peptides, polypeptides or proteins, thereby encoding a fusion product therewith.

Although one example of a nucleotide sequence encoding an antifungal protein (e.g., SEQ ID NO: 2) consists of the nucleotide sequence of SEQ ID NO: 1 or 4, but it is to be understood that the invention also includes variants that are similar to SEQ ID NO: 1 or 4 and the complement thereof and encodes a peptide, polypeptide or protein having the same or similar antifungal activity as the antifungal protein of the present invention. The nucleotide sequence is preferably identical to SEQ ID NO: 1 or 4 or the complement thereof.

If the nucleotide sequence described above encodes a nucleotide sequence identical to SEQ ID NO: 2 has a peptide, polypeptide or protein that differs by about 25% or less, then the nucleotide sequence is considered to have a sequence that differs from SEQ ID NO: 1 or 4 is substantially equivalent in biological function.

Vector and host system

It is another object of the present invention to provide a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1 or 4. To express a biologically active MAFP1, the nucleic acid sequence encoding MAFP1 or a biologically functional equivalent may be inserted into an appropriate expression vector, i.e., a vector containing the necessary elements for transcription and translation of the inserted coding sequence. In accordance with the present invention, expression vectors containing the sequence encoding MAFP1 and appropriate transcriptional and translational control elements can be constructed using methods well known to those skilled in the art. The methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo gene recombination. The vector is typically selected to function in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that gene amplification and/or gene expression can occur).

It is another object of the present invention to provide a polypeptide encoded by the nucleotide sequence as set forth in SEQ ID NO: 1 or 4 or an expression vector comprising such a sequence. A variety of host systems containing and expressing sequences encoding MAFP1 may be used in accordance with the present invention. Such host systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant phage, plasmid, or cosmid (cosmid) DNA expression vectors; yeast transformed with a yeast expression vector; insect cell systems infected with viral expression vectors; plant cell systems transformed with viral expression vectors or with bacterial expression vectors; or animal cell systems. After the vector is constructed and the nucleic acid sequence encoding MAFP1 is inserted into the appropriate site in the vector, the completed vector may be inserted into an appropriate host cell for amplification and/or polypeptide expression. Transformation of the MAFP1 protein into the selected host cells with the expression vector can be accomplished by well-known methods, including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, diethylaminodextran (DEAE-dextran) transfection, or other known techniques. The method chosen will vary, in part, with the type of host cell used. When cultured under appropriate conditions, the host cell may synthesize the MAFP1 protein, and then the MAFP1 protein may be collected from the culture medium (if the host cell secretes it into the culture medium) or directly from the host cell producing the MAFP1 protein (if it is not secreted). The choice of an appropriate host cell will depend on a variety of factors, such as the desired level of expression, the polypeptide modifications that are desired or necessary to achieve activity (e.g., glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

Utility of

According to the present invention, it has been surprisingly found that the MAFP1 protein is a secreted protein and is effective in inhibiting the growth of pathogenic fungi. Thus, the MAFP1 protein of the invention may be used in the treatment, control and/or prevention of fungal diseases in animals, plants or microorganisms. The antifungal protein can be used directly by administering the antifungal protein to a subject or by transforming a subject with a vector comprising a DNA molecule encoding the antifungal protein such that the encoded antifungal protein is expressed in the subject, thereby providing protection from fungal disease.

In addition to the described uses of the antifungal proteins of the present invention, the nucleic acid sequences encompassed herein have a variety of other uses. For example, it may also serve as a probe or primer in nucleic acid hybridization embodiments. Thus, it is contemplated to comprise a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 4, or a sequence region consisting of a contiguous sequence of at least 14 nucleotides which is complementary thereto. Longer contiguous consensus or complementary sequences, such as sequences of about 20, 30, 40, 50, 100, 200bp, and the like (including all intermediate lengths and up to and including the full-length sequence of 409 base pairs), will also be useful in certain embodiments.

The ability of the nucleic acid primers to specifically amplify or hybridize to sequences encoding antifungal proteins will allow them to be used to detect the presence or absence of sequences encoding antifungal proteins in a given sample. However, other uses are envisioned, including the use of sequence information to make mutant species or primers for use in making other genetic structures.

Antifungal composition

The present invention also provides antifungal compositions comprising the antifungal proteins of the present invention, which compositions are particularly useful in the treatment, control and/or prevention of fungal diseases. The compositions may contain suitable carriers, diluents, solvents, inerts or other additives, and optionally other antifungal actives, excipients, adjuvants, fertilizers or growth regulators.

The antifungal compositions of the present invention may be manufactured by means known in the art, for example, by means of a conventional process comprising the steps of: mixing, dissolving, granulating, dragee-making, grinding, emulsifying, encapsulating, entrapping and/or lyophilizing processes.

The antifungal compositions can be used to inhibit the growth of or kill pathogenic fungi by administration to an animal by a variety of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means, or by applying the composition directly to the plant or plant environment using standard agricultural techniques (e.g., spraying or seed treatment) to expose the composition to the pathogenic fungus.

As previously mentioned, the antifungal proteins of the present invention may be used in combination with other antifungal agents, including other peptides, polypeptides and proteins that exhibit antifungal activity, to provide a broader range of activity (i.e., control of other pathogenic bacteria) or to provide multiple modes of action for control of the same pathogenic fungi.

The antifungal compositions contemplated herein also include those in the form of host cells (e.g., bacterial and fungal cells) that produce the antifungal polypeptides of the present invention.

Transgenic organisms

The cDNA isolated in the manner described may be transferred to an appropriate transformation/expression vector for introduction into a host cell. In another aspect, the antifungal genes of the present invention can be used to produce transgenic organisms that express nucleic acid fragments encoding the novel antifungal proteins of the present invention. Methods for generating transgenic organisms are well known in the art, such as by microinjection or infection with recombinant vectors. The method generally comprises transforming a suitable host cell with a DNA fragment comprising a promoter operably linked to a coding region encoding the MAFP1 antifungal protein. The coding region is typically operably linked to a transcription termination region, such that the promoter is capable of driving transcription of the coding region in a cell, and thereby providing the cell with the ability to produce the recombinant protein in vivo.

Transgenic organisms may express genes or gene fragments encoding one or more of the novel antifungal proteins disclosed herein. Expression of the encoded antifungal protein results in the formation of an organism that exhibits resistance to fungi by transforming a suitable host cell (e.g., a plant cell) with a recombinant nucleic acid sequence encoding a MAFP1 antifungal polypeptide (e.g., SEQ ID NO: 2). The term "transgenic organism" as used herein means an organism into which has been incorporated a DNA sequence including, but not limited to, genes that may not normally be present, DNA sequences that are not normally transcribed into RNA or translated into a protein ("expressed"), and any other gene or DNA sequence that is desired to be introduced into an untransformed organism (such as a gene that may normally be present in an untransformed organism but that is desired to be genetically engineered or altered in expression).

More than one transgene will be incorporated into the genome of the transformed host plant cell. This is the case when more than one DNA fragment encoding the MAFP1 antifungal protein is incorporated into the genome of the organism. In certain instances, it may be desirable to incorporate and stably express one, two, three, four, or even more antifungal proteins (native or recombinantly engineered) in a transformed transgenic organism.

It may also be desirable to incorporate into the genome of a transgenic organism other DNA fragments that encode other antifungal proteins that are non-homologous to the disclosed antifungal proteins or various other proteins that improve product quality or organism performance. Thus, other types of proteins encoded by the DNA may include antibacterial, antiviral or insecticidal proteins.

The transgenic organism may be any convenient organism, such as a non-human mammal, plant or microorganism, e.g. for use in laboratory testing procedures, such as rodents, e.g. mice or rats, and e.g. for use in agriculture, such as rice, potato and tobacco. However, it is clear that the invention is applicable to every organism that is susceptible to a fungus that exhibits inhibition by MAFP 1.

Primer and method for producing the same

In certain embodiments, it is advantageous to use oligonucleotide primers. The sequences of the primers are designed to detect, amplify or mutate a defined segment of the antifungal protein gene using PCR techniques. Can be based on SEQ ID NO: 1 or 4, primers for PCR and probes for hybridization screening were designed. The primer should preferably not have a self-complementary sequence and also not have a complementary sequence at its 3' end, thereby preventing the formation of dimers. The primer may contain a restriction site. The primers anneal to the DNA and a sufficient number of PCR cycles are performed to produce a product that is easily visualized by gel electrophoresis and staining. The primer is generally at least 14 nucleotides in length, generally at least 20 nucleotides in length, preferably at least 24 nucleotides in length, and more preferably 28 nucleotides in length. The primer can specifically start the primer which encodes the polypeptide shown in SEQ ID NO: 2 a gene for an antifungal polypeptide or protein having the same or similar antifungal activity. Fragments of the relevant antifungal protein genes from other species may also be amplified by PCR using the primers. The amplified fragments can be purified and inserted into an appropriate vector and propagated by conventional means known in the art.

The following examples are provided to assist those skilled in the art in practicing the present invention. Even so, the examples should not be construed as unduly limiting the invention since modifications and variations to the embodiments set forth herein may be made by those of ordinary skill in the art without departing from the spirit or scope of the invention as found.

Examples of the invention

Material

The monascus strains used in the following examples were selected from monascus strains stored in taiwan biological resource collection and research center (BCRC): monascus pilosus (BCRC38072, BCRC38093 and BCRC31502), monascus barkeri (BCRC33309), monascus florida (BCRC33310), monascus lunata (BCRC33640), monascus ruber (BCRC31534, BCRC31523, BCRC31535, BCRC33314, BCRC33323 and BCRC31533), monascus kaoliang (m.kaoliang) BCRC31506 ═ CBS302.78, monascus purpureus (m.purpurpurpurpurpureus) (BCRC31541 ═ ATCC16379, BCRC33325 ═ IFO30873, BCRC31615 ═ DSM1379, BCRC31499 ═ ATCC16360 ═ ATCC26311 and BCRC31542 ═ ATCC16365 ═ ATCC16426), and monascus sanguineus (BCRC 33446).

The fungal strain used to test the antifungal activity of the monascus strain or the isolated and purified antifungal protein MAFP1 is selected from the group consisting of helminthosporium paniculatum BCRC35004 and paecilomyces variotii BCRC 33174.

The fungal strains were inoculated onto PDA (Patato Dextrose Agar, Difico Co.) plates and cultured at 25 ℃ for 7-14 days.

Example 1 masp 1 Gene distribution in Monascus species

To observe the presence or absence of the masp 1 gene (SEQ ID NO: 1) in each monascus species, primers for amplifying the masp 1 gene were designed using Primer Design software (Vector NTI (InforMax) Primer Design). The primers can be paired into 3 groups: (1) primers H160-5F (SEQ ID NO: 6) and H160-3R (SEQ ID NO: 7) can be used for amplifying the full-length sequence of the masp 1 gene; (2) primers H160-5S (SEQ ID NO: 8) and primers H160-3R, useful for amplifying a nucleotide sequence encoding a sequence comprising the propeptide and the mature MAFP1 region; and (3) primers H160-5P (SEQ ID NO: 9) and H160-3R, which can be used to amplify a nucleotide sequence encoding a sequence comprising the mature MAFP1 region.

Monascus strains were cultured in YM medium (7% glycerol, 3% glucose, 3% MSG (monosodium L-glutamate), 1.2% polypeptone (polypeptone), 0.2% NaNO at 25 deg.C₃And 0.1% MgSO₄-7H₂O, pH6.0) for 9 days. The fungus body and the culture liquid were separated by vacuum filtration through a 3M filter. Chromosomal DNA of fungi was obtained by a conventional phenol extraction method using an appropriate amount of fungi.

The presence of the masp 1 gene was detected in monascus species by PCR amplification using the 3 primer pairs mentioned in the above paragraph. 100ng of chromosomal DNA obtained from an Aspergillus oryzae strain was used as a PCR template. The PCR reaction solution contained 0.2. mu.l of 10nM dNTP, 2.5. mu.l of 10X PCR buffer, 5 picomoles (pmole) of forward and reverse primers, and 5UTaq enzyme. PCR conditions for amplification of the masp 1 gene were (1)94 ℃ for 5 minutes; (2) and (3) 30 times of circulation: 40 seconds at 94 ℃, 40 seconds at 55 ℃ and 30 seconds at 72 ℃; (3) 3 minutes at 72 ℃; and (4) maintained at 4 ℃. The PCR amplification results are shown in table 1.

TABLE 1 PCR amplification detection of the masp 1 gene in A.erythreus strain

Bacterial strains	mafp1 gene a
		Monascus pilosus BCRC38072	+
Monascus pilosus BCRC38093	+
		Monascus pilosus BCRC31502	+
Monascus ruber BCRC31523	+
		Monascus ruber BCRC31533	+
Monascus ruber BCRC31534	+
		Monascus ruber BCRC31535	+
Monascus ruber BCRC33314	+
		Monascus ruber BCRC33323	+
Monascus barkeri BCRC33309	+
		Monascus florida BCRC33310	+
Monascus lunatus BCRC33640	+
		Monascus jowar BCRC31506
Monascus purpureus BCRC31542
		Monascus purpureus BCRC31499
Monascus purpureus BCRC31541
		Monascus purpureus BCRC31615
Monascus purpureus BCRC33325
		Monascus sanguineus BCRC33446

a: "+" indicates that all three primer pairs were used to amplify the masp 1 gene fragment; "-" indicates that none of the three primer pairs were available for amplifying the masp 1 gene fragment.

The results showed the presence of the mafp1 gene in monascus barkeckii BCRC33309, monascus florida BCRC33310, monascus lunata BCRC33640, monascus hirsutus (BCRC38072, BCRC38093 and BCRC31502) and monascus ruber (BCRC31523, BCRC31533, BCRC31534, BCRC31535, BCRC33314 and BCRC 33323). No macp 1 gene was found in R.kawakamii BCRC31506, R.purpureus (BCRC31499, BCRC31542, BCRC31541, BCRC31615, and BCRC33325), and R.sanguineus BCRC 33446. It is shown that Monascus species such as Monascus barkeri, Monascus florida, Monascus lunatus, Monascus pilosus and Monascus ruber can have antifungal activity.

Example 2 sequence comparison of the masp 1 Gene of its cDNA in various Monascus species

Cloning and sequencing of the macp 1 Gene

Monascus strains were cultured in YM medium (7% glycerol, 3% glucose, 3% MSG (monosodium L-glutamate), 1.2% polypeptone, 0.2% NaNO at 25 deg.C₃And 0.1% MgSO₄-7H₂O, pH6.0) for 9 days. The fungus body and the culture liquid were separated by vacuum filtration through a 3M filter. Chromosomal DNA of fungi was obtained by a conventional phenol extraction method using an appropriate amount of fungi. PCR amplification was performed using primers H160-5F and H160-3R. Using Monascus purpureus went100ng of chromosomal DNA obtained from the strain was used as a template for PCR. The PCR reaction solution contained 0.2. mu.l of 10nM dNTP, 2.5ul of 10X PCR buffer, 5 picomolar forward and reverse primers and 5U Taq enzyme. PCR conditions for amplification of the masp 1 gene were (1)94 ℃ for 5 minutes; (2) and (3) 30 times of circulation: 40 seconds at 94 ℃, 40 seconds at 55 ℃ and 30 seconds at 70 ℃; (3) 3 minutes at 72 ℃; and (4) maintained at 4 ℃. The PCR amplified nucleotide fragment of the full-length sequence of the masp 1 gene was purified and cloned into pGEM-T vector (Promega). Plasmid DNA was extracted for sequencing.

Cloning and sequencing of the macp 1cDNA

Monascus strains were cultured in YM medium (7% glycerol, 3% glucose, 3% MSG (monosodium L-glutamate), 1.2% polypeptone, 0.2% NaNO at 25 deg.C₃And 0.1% MgSO₄-7H₂O, pH6.0) for 9 days. The fungus body and the culture liquid were separated by vacuum filtration through a 3M filter. Passing Ribopure with appropriate amount of fungus^TMYeast kit (Ambion) to obtain total RNA of fungi. Using Improm-II^TMFirst strand cDNA was prepared using reverse transcription System kit (Promega). A primer pair specific for the masp 1 gene (H160-5F and H160-3R) was used in PCR to amplify the full-length masp 1cDNA fragment. The appropriate amount of first strand cDNA was used as a PCR template. The PCR reaction solution contained 0.2. mu.l of 10nM dNTP, 2.5. mu.l of 10X PCR buffer, 5 picomoles of H160-5F and H160-3R, and 5U of Taq enzyme. PCR conditions for amplification of the masp 1cDNA were (1)94 ℃ for 5 minutes; (2) and (3) 30 times of circulation: 40 seconds at 94 ℃, 40 seconds at 55 ℃ and 1 minute at 72 ℃; (3) 3 minutes at 72 ℃; and (4) maintained at 4 ℃. The amplified PCR product was purified and cloned into pGEM-T vector (Promega). Plasmid DNA was extracted for sequencing.

The results of comparing the sequences of the masp 1 gene of its cDNA in various Monascus species are shown in Table 2.

TABLE 2 sequence similarity analysis of the masp 1 gene and its cDNA in Monascus species

The results showed that the sequences of the masp 1 gene and its cDNA in monascus pileus BCRC38093, monascus pileus BCRC31502 and monascus ruber BCRC31533 had 100% sequence similarity with the sequences of the masp 1 gene and its cDNA of monascus pileus BCRC 38072. All of these strains of A.erythraea were demonstrated to have the same masp 1 gene and the transcribed mRNA was identical. It can be concluded that the strains produced the same MAFP1 protein.

EXAMPLE 3 Dual culture analysis of pathogenic bacteria

To confirm the antifungal activity of the monascus fungi, two monascus species (monascus serrulata BCRC38072 and BCRC38093) and a circular fungal block (0.5cm diameter) of the pathogenic fungus (helminthosporium paniculatum BCRC35004) were divided on both sides of a PDA plate and cultured at 28 ℃. The growth of the pathogenic fungi was observed to be inhibited. Preliminary results show that both strains inhibit the growth of Helminthosporium paniculatum BCRC 35004.

Monascus hirsutus BCRC38093 is a mutant of monascus hirsutus BCRC 38072. Both have the same masp 1 sequence (as shown in Table 2) and have the same antifungal activity. Monascus hirsutus BCRC38093 was used in the following purification examples.

Example 4 purification, N-terminal sequencing and analysis of antifungal Activity of Monascus antifungal protein (MAFP1)

Purification of MAFP1

400mL of a culture solution of Monascus pilosus BCRC38093 after 9 days of culture at 25 ℃ was centrifuged at 4,500rpm using a 0.22 μm filter to remove impurities. The centrifuged medium is treated with a 30kDa filter and the solution containing molecules smaller than 30kDa is collected and adjusted to pH 6.6. 10ml of CM Sepharose Fast Flow (Amersham Biosciences) resin was mixed with 40ml of protein solution at room temperature for 16 hours. The mixture was packed into a hollow chromatographic column. Unbound protein was washed out. The column was eluted with 100ml of solution A (25 mM NaCl in 10mM sodium phosphate buffer, pH6.6) and the protein was eluted using solutions with different ratios of solution A to solution B (1M NaCl in 10mM sodium phosphate buffer, pH 6.6). The concentration gradient of the solution A is from 95%, 80%, 75%, 50% to 0%. 100ml of solution was used for each gradient and the eluted fractions were individually dispensed into 15ml tubes.

1ml of the solution from each tube was precipitated with TCA and analyzed by SDS-PAGE. The molecular weight of the MAFP1 protein is approximately 7 kDa. The solution containing the MAFP1 protein was collected in a single tube. The solution was centrifuged at low speed (2,000rpm) using a 3kDa filter. The solution containing proteins greater than 3kDa is collected and concentrated and desalted with a 1kDa filter. The purified MAFP1 protein was used for pathogen antagonism analysis and protein N-terminal sequencing.

N-terminal sequencing

The purified MAFP1 solution was TCA precipitated and analyzed using 15% acrylamide SDS-PAGE. Different concentrations of lysozyme were used as a protein quantification reference. Following electrophoresis, the proteins were transferred from the gel to a PVDF membrane and stained with 0.1% Coomassie Brilliant Blue R-250 (see FIG. 3). The MAFP1 protein band was excised and destained with methanol. With double distilled water (ddH)₂O) the membrane was rinsed several times and left to dry in the shade. The treated MAFP1 protein was sequenced N-terminal using an Applied Biosystems Procise model 494 sequencer.

The N-terminal sequencing result of the purified MAFP1 protein showed that the N-terminal of the purified MAFP1 protein was LSKYGGECSLQHNTC (SEQ ID NO: 5). The N-terminal sequence of the purified protein agreed with the first 15 amino acid sequences of the mature form of MAFP1 protein (SEQ ID NO: 3). The purified protein was confirmed to be the mature form of MAFP1 protein.

Antifungal Activity assay

A pathogen antagonistic dose assay was performed to confirm the antifungal activity of the purified MAFP1 protein. Fungal blocks of the pathogenic fungi (Paecilomyces variotii BCRC33174 and Helminthosporium paniculatum BCRC35004) were placed in the center of the PDA plate. 6mm paper discs with different concentrations of MAFP1 solution (containing 0 to 0.8. mu.g of purified MAFP1 protein) were placed around the fungal mass of the pathogenic fungus. The plates were incubated at 28 ℃ and observed for inhibition of growth of the pathogenic fungi. The results are shown in fig. 4. The results showed that 0.2. mu.g of MAFP1 protein significantly inhibited the growth of Paecilomyces variotii. The results also show that 0.4 μ g of MAFP1 protein significantly inhibited the growth of Helminthosporium paniculatum. It was observed that the higher the concentration of MAFP1, the stronger the inhibitory activity against the growth of pathogenic fungi. The MAFP1 protein from Monascus species was shown to be a protein with antifungal activity that inhibits the growth of human pathogens (such as Paecilomyces variotii) and plant pathogens (such as Helminthosporium paniculatum).

Claims (7)

1. An isolated and purified antifungal protein consisting of

LSKYGGECSLOHNTCTYLKGGKNQVVHCGSAANQKCKSDRHHCEYDEHHKTV

The amino acid sequence of NCQTPV.

2. A composition comprising the antifungal protein of claim 1 and a suitable carrier, wherein the protein is provided in an amount sufficient to provide protection against fungal disease.

3. The composition of claim 2, comprising an additional antifungal agent.

4. A method of controlling fungi in a plant, the method comprising applying to the plant the antifungal protein of claim 1 in an amount sufficient to provide protection against fungal disease.

5. The method of claim 4, wherein the antifungal protein is provided by the composition of claim 2 or 3.

6. Use of an antifungal protein of claim 1 for the preparation of a medicament for treating a fungal disease in a patient.

7. Use according to claim 6, wherein the antifungal protein is provided by a composition according to claim 2 or 3.