MXPA99010235A - Antimicrobial peptide - Google Patents

Abstract

The present invention relates to a novel human antimicrobial peptide which is a member of the defensin superfamily. In particular, isolated nucleic acid molecules are provided encoding the human antimicrobial peptide. Antimicrobial peptides are also provided as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for detecting disorders related to the immune system and therapeutic methods for such disorders.

Description

ANTIMICROBIAL PEPTIDE BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a novel human defensin peptide. More specifically, isolated nucleic acid molecules encoding a human antimicrobial peptide are provided. Antimicrobial polypeptides are also provided, such as vectors, host cells and recombinant methods for the production thereof. Diagnostic methods are also provided to detect disorders related to the immune system and therapeutic methods to treat such disorders.

Related Technique One of the key roles of respiratory epithelium in mammals is to form a barrier to potentially hazardous environmental threats. A number of REF. 31,953 defense mechanisms that protect the respiratory tract from airborne agents that are thought to be responsible for diseases of the respiratory tract, such as infectious agents, gases and particulate materials. (Newhouse et al., 'Respiratory Tract Defense Mechanism', Textbook of Pulmonary Disease, Little Brown and Co. (1989).) Recent identification and characterization of antimicrobial peptides from a variety of species has unveiled a new member of the defense system. of animal hosts, and believed to be involved in the defense against potential microbial pathogens.These families of newly identified antimicrobial peptides include cecropins, magainins, and defensins.Cecropins were the first well-characterized family of structurally related antimicrobial polypeptides and were found in a wide distribution of insects (Bowman et al., Ann. Rev. Of My Crobi ol., 41: 103 (1987).) In vertebrates, the family of magainin d'e antimicrobial peptides has been isolated from the glands of the skin and the gastrointestinal tract of Xenopus evi s, and is thought to form the basis for the defense system of the super mucosal fibers of amphibians against infections. (Soravia et al., FEBS Lett. 228: 337 (1988); Zasloff et al., Proc. Nati, Acad. Sci 84: 5449 (1987)). Defensins are antimicrobial peptides found in isolated phagocytic cells of various mammalian species, including man, and can be cax-acterized by eight non-variant residues within the sequence. (Gabay et al., Curr. Opin. Immunol., 1: 36 (1988); Gabay et al., Proc. Nati, Acad. Sci. 86: 5610 (1989); Ganz et al., Infec. Immun. 55: 568 (1987 Ganz et al., J. Immunol., 143: 1358 (1989), Ganz et al., J. Clin.Invest.76: 1427 (1985)). The mechanism of antimicrobial activity of d-e peptides such as defensins is via a disorganization of the selective membrane that leads to a broad characteristic spectrum of antibiotic activity. (Bowman, Ann. Rev. Of Immunol., 13: -61 (1995)). The antimicrobial spectrum of defensins includes gram positive and gram negative bacteria, mycobacteria, T. pallidum, many fungi, some of the enveloped viruses. (Bowman, Ann. Rev. Of Immunol., 13:61 (1995)). Defensins exert non-specific cytotoxic activity against a wide range of normal and malignant targets, including cells resistant to TNF-α and the NK cytolytic factor. These seem to kill the mammalian target cells and microorganisms by a common mechanism that involves the initial electrostatic interactions with the surface molecules of the target cell, negatively charged, followed by the insertion inside the cell membranes which can be permeabilized, forming regulated channels by voltage. (Lehrer et al., Ann. Rev. Of Immunol., 11: 105 (1993)). In addition to their antimicrobial and cytotoxic properties, some defensins act as opsonins, while others inhibit protein kinase C, bind specifically to the ACTH receptor and block steroidogenesis or act as selective chemoattractants for monocytes. Defensins are a newly delineated family of effector molecules whose contribution to the defense of the host cell, to inflammation, and to cytotoxicity can be considerable for humans (Lehrer et al., ann.Rev. Of Immunol., 11: 105 (1993)). Defensins are basic peptides of 30 to 34 amino acids with three disulfide bridges. Characterized defensins known for myeloid and non-myeloid tissues have all highly conserved amino acid residues within the family, including 6 non-variant cysteines. Recent studies have found that similar antimicrobial peptides are also made by certain epithelial cells suggesting an additional role in the defense of mucosal surfaces. (Diamond et al, Proc. Nati, Acad. Sci. 90: 5496 (1993); Diamond et al., Proc. Nati Acad. Sci. 88: 3952 (1991); Eisenhauer et al., Infect. Immun. 60: 3556 (1992); Jones et al., J. Biol. Chem. 267: 23216 (1992); Schonwetter et al., Science 261: 1645 (nineteen ninety five); Selsted et al., J. Cell. Biol. 118: 929 '(1992)). Tracheal antimicrobial peptide (TAP) is a 38-amino acid peptide isolated from the respiratory mucosa of cattle and was the first member of what is now recognized as a relatively large family of antimicrobial peptides, β-defensin, all of which have activity high-spectrum antimicrobial in vitro. (Diamond et al, Proc. Nati, Acad. Sci. 88: 3952 (1991)). Recently, a second family of ß-defensins of epithelial origin was isolated, the tongue antimicrobial peptide (LAP) of the bovine tongue. (Schonwetter et al., Sci ence 2 61: 1645 (1995), Selsted et al., J. Cell 1. Biol. 118: 929 (1992)). Thus, there is a need for polypeptides that function as antimicrobial agents or regulators of the immune response, since disturbances of such regulation may be involved in disorders related to infectious diseases, inflammation and immune disorders.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides isolated nucleic acid molecules comprising a polynucleotide that encodes the antimicrobial polypeptide having the amino acid sequence shown in SEQ ID NO. 2 or the amino acid sequence encoded by the cDNA clone that was deposited on April 14, 1997 in the North American Collection of Species (American Type Culture Collection), Patent Deposit, 10801 University Boulevard, Manassas, VA 20110-2209 , and to which the access number 97982 was given.

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and host cells that contain the recombinant vectors, as well as the methods for making such vectors and host cells, and for the use of them for the production of antimicrobial polypeptides or peptides by recombinant techniques. The invention further provides a human isolated, antimicrobial isolated polypeptide having an amino acid sequence encoded by a polynucleotide described herein. The invention provides a diagnostic method useful during the diagnosis of disorders of the immune system. A further aspect of the invention relates to a method for the treatment of an individual in need of an increased level of antimicrobial peptide activity in the body, comprising administering to such an individual a composition comprising a therapeutically effective amount of a peptide. antimicrobial, of the invention or an agonist thereof.

A further aspect of the invention is related to a method for the treatment of an individual in need of a decreased level of antimicrobial peptide activity in the body, comprising, administering to such an individual a composition comprising a therapeutically effective amount of a antimicrobial peptide antagonist.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the nucleotide (SEQ ID No. 1) and the deduced amino acid sequences (SEQ ID No. 2) of the human antimicrobial peptide. The protein has a forward or guiding sequence of approximately 23 amino acid residues (underlined) and a deduced molecular weight of approximately 7.3 kDa. The amino acid residues underlined in Figure 1 (for example, the first 23 amino acids) correspond to amino acids -23 to -1 in SEQ ID NO. 2. The following 41 amino acids (not underlined) in Figure 1 correspond to amino acids 1 to 41 in SEQ ID NO. 2.

Figure 2 shows the regions of similarity between the amino acid sequence of the antimicrobial peptide protein and the bovine tracheal antimicrobial peptide (SEQ ID NO 3).

Figure 3 shows an analysis of the amino acid sequence of the antimicrobial peptide. The alpha, beta, back and spiral regions are also shown; hydrophilicity and hydrophobicity; the amphipathic regions; the flexible regions; the antigenic index and the superficial probability. In the graph of 'Antigenic Index - Jameson-Wolf', the amino acid residues about 42 to about 50, and about 54 to about 64 in Figure 1, correspond to the highly antigenic regions shown by the peptide protein Antimicrobial These highly antigenic fragments in Figure 1 correspond to the following fragments, respectively, in SEQ ID NO: 2: the amino acid residues approximately 19 to about 27, and about 31 to about 41.

Figure 4 shows a schematic representation of the expression vector pHE4a (SEQ ID NO: 4). The locations of the kanamycin resistance marker gene, the linker region of the multiple cloning site, the oriC sequence, and the laclq coding sequence are also indicated.

Figure 5 shows the nucleotide sequence of the regulatory elements of the pHE4a promoter (SEQ ID NO: 5). The two operator sequences l a c, the Shine-Dalgarno sequence (S / D), and the terminal restriction sites iJindIII and Ndel (in italics) are indicated.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding an antimicrobial peptide having the amino acid sequence shown in SEQ ID NO. 2, which was determined by sequencing a cloned cDNA. The antimicrobial peptide of the present invention shares sequential homology with the bovine tracheal antimicrobial peptide (Figure 2) (SEQ ID NO. 3) . The nucleotide sequence shown in SEQ ID NO. 1 was obtained- by sequencing an ANDc clone (HLJB175), which was deposited on April 14, 1997 at the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, VA 20110-2209, and was gave accession number 97982. The deposited clone is inserted into pCMVSportl using the Sall / Notl restriction endonuclease cleavage sites.

Molybdenum of Nuclonic Acid Unless otherwise indicated, all nucleotide sequences determined by sequencing of a DNA molecule herein were determined using an automated DNA sequencer (such as Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of the polypeptides encoded by the DNA molecules determined herein, were predicted by translation of a DNA sequence determined as described above. Therefore, as is known in the art for any DNA sequence determined by this automated method, any nucleotide sequence determined herein may contain some errors. The nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the effective nucleotide sequence of the sequenced DNA molecule. The effective sequence may be more precisely determined by other methods including manual DNA sequencing methods well known in the art. As is also known in the art, a simple insertion or deletion in a given nucleotide sequence compared to the effective sequence, will cause a structural shift in translation of the nucleotide sequence, such that the predicted amino acid sequence encoded on a given nucleotide sequence. , will be completely different from the amino acid sequence effectively encoded by the sequenced DNA molecule, starting at the point of such insertion or deletion. Using the information provided herein, such as the nucleotide sequence in SEQ ID NO. 1, a nucleic acid molecule of the present invention encoding an antimicrobial peptide polypeptide can be obtained, using standard methods of cloning and selection, such as those for the cloning of ssDNA using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in SEQ ID NO. 1 was discovered in a cDNA library derived from lung tissue. The determined nucleotide sequence of the antimicrobial peptide cDNA of SEQ ID NO. 1, contains an open reading frame that encodes a protein of approximately 64 amino acid residues, a predicted leader or forward sequence of approximately 23 amino acid residues, and a deduced molecular weight of approximately 7.3 kDa.

The antimicrobial peptide shown in SEQ ID NO. 2 is approximately 44% identical and approximately 63% similar to the bovine tracheal antimicrobial peptide (Figure 2). The present invention also provides the mature form (s) of the antimicrobial peptide protein of the present invention. According to the signal hypothesis, the proteins secreted by mammalian cells have a signal sequence or secretory guide which is excised from the mature protein once the export of the growing protein chain through the endoplasmic reticulum has been initiated. rugged Most mammalian cells and even insect cells break secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not completely uniform, which results in two or more mature species on the protein. Furthermore, it has been widely known that the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the entire protein, i.e., it is inherent in the amino acid sequence of the polypeptide. Therefore, the present invention provides a nucleotide sequence coding for mature polypeptides of the antimicrobial peptide, having the amino acid sequence encoded by the cDNA clone contained in the host identified as ATCC Deposit No. 97982 and as shown in SEQ ID NO. 2. By mature antimicrobial peptide having the amino acid sequence encoded by the cDNA clone contained in the host identified as ATCC Deposit 97982, is meant the mature form (s) of the antimicrobial peptide produced by expression in mammalian cells (e.g. COS cells, as described below) of the complete open reading frame encoded by the human cDNA sequence of the clone contained in the vector in the deposited host. As indicated below, the mature antimicrobial peptide having the amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 97982 may or may not differ from the predicted 'mature' antimicrobial peptide, shown in SEQ ID NO 2 ( amino acids from about 1 to about 23) depending on the accuracy of the cleavage site predicted based on the computer analysis.The methods for predicting whether a protein has a secretory guide, as well as the cleavage point for that sequence are available. For example, the methods of McGeoch (Viurs Res. 3: 271-286 (1985)) and von Heinje (Nucl ei c Aci ds Res. 14: 4683-4690 (1986)) can be used. Precision for predicting the cleavage sites of mammalian secretory proteins, known, for each of these methods is in the range of 75 to 80% .Von Heinje, upra .. However, the two methods do not always produce the m We are predicted cleavage points for a given protein. In the present case, the predicted amino acid sequence of the complete polypeptides of the antimicrobial peptide, of the present invention, were analyzed by a CPSORT computer program ") (K. Nakai and M. Kanehisa, Genomi cs 14: 897-911 ( 1992), which is an expert system for predicting the cellular localization of a protein, based on the amino acid sequence As part of this computational prediction of localization, the McGeoch and von Heinje methods are also incorporated. through the program PSORT predicted the cleavage site between amino acids -1 and 1 in SEQ ID No. 2. After this, the complete amino acid sequences were further analyzed by visual inspection, applying a simple form of the rule (-1 , Von Heinje, -3) Von Heinje, supra, Thus, the leader or leader sequence for the antimicrobial peptide protein is predicted to consist of the approximate amino acid residues From -23 to approximately -1 in SEQ ID NO. 2, whereas the mature antimicrobial peptide protein is predicted to consist of residues from about 1 to about 41 in SEQ ID NO. 2. As a person skilled in the art could appreciate, due to the possibilities of errors in the sequencing, as well as the variability of the cleavage sites for the guides in different known proteins, the polypeptide of the antimicrobial peptide, predicted, encoded by the Deposited cDNA, comprises approximately 64 amino acids, but can be anywhere in the range of 55 to 75 amino acids; and the predicted leader sequence of this protein is about 23 amino acids, but it can be 20, 21, 22, 24 or 25 amino acids in length. As indicated, the nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA or in the form of DNA, including, for example, cDNA and genomic DNA obtained by cloning or synthetically produced. DNA can be double-stranded or single-stranded. Single-stranded DNA or single-stranded RNA can be the coding strand, also known as the strand in sense, or this can be the non-coding strand, also referred to as the antisense strand.

The "isolated" nucleic acid molecule (s) is intended to include a nucleic acid, DNA or RNA molecule, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for purposes of the present invention. Additional examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified DNA molecules. (partially or substantially) in solution. Isolated RNA molecules include the transcripts of RNA in vi vo or vi n of the DNA molecules of the present invention. The isolated nucleic acid molecules according to the present invention further include such synthetically produced molecules. The isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in SEQ ID NO. 1; the DNA molecules comprise the coding sequence for the mature antimicrobial peptide protein; and DNA molecules comprising a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still code for the antimicrobial peptide protein. Of course, the genetic code is well known in the art. In this way, it could be routine for a person skilled in the art to generate such degenerate variants. In still another aspect, the invention provides the isolated nucleic acid molecules encoding the antimicrobial peptide polypeptide, which has an amino acid sequence as encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97982 on 14 April 1997. In a further embodiment, the nucleic acid molecules encoding the mature antimicrobial peptide polypeptide or the full length antimicrobial peptide polypeptide, which lacks the N-terminal methionine, are provided. The invention also provides an isolated nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO. 1 or the nucleotide sequence of the antimicrobial peptide cDNA, contained in the deposited clone described above, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, are useful as probes for the formation of the genetic map, by hybridization in itself with chromosomes, and for the detection of the expression of the antimicrobial peptide gene in human tissue, for example, by analysis of Spotted from Northern. The present invention is further directed to the fragments of the isolated nucleic acid molecules described herein. A fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1) is intended to mean fragments of at least about 15 nucleotides, and more preferably at least about 20 nucleotides, still more preferably at least about 30 nucleotides, and even more preferably, at least about 40 nucleotides in length, which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments of 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300 or 323 nucleotides in length are also useful according to the present invention, as are the fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO. 1. For a fragment of at least 20 nucleotides in length, for example, it is intended to mean fragments that include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID. DO NOT. 1. Preferred nucleic acid fragments of the present invention include the nucleic acid molecules for the epitope-possessing portions of the antimicrobial peptide protein. In particular, such nucleic acid fragments of the present invention include the nucleic acid molecules encoding: for a polypeptide comprising the amino acid residues from about 19 to about 27 in SEQ ID NO. 2; and a polypeptide comprising the amino acid residues from about 31 to about 41 in SEQ ID NO. 2. The inventors have determined that the above polypeptide fragments are antigenic regions of the antimicrobial peptide protein. Methods for the determination of other epitope-bearing portions of the antimicrobial peptide protein are described in detail below.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide that hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, eg, the cDNA clone contained ATCC Deposit No. 97982. 'Stringent hybridization conditions "means overnight incubation at 42 ° C in a solution comprising: 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate) , 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g / ml denatured, cut salmon sperm DNA, followed by washing the filters in 0. lx SSC a about 65 ° C. A polynucleotide that hybridizes to a "portion" of a polynucleotide is understood to be a polynucleotide (either DNA or RNA) that hybridizes at least about 15 nucleotides (nt), and more preferably at least s about 20 nucleotides, still more preferably at least about 30 nucleotides, and even more preferably about 30-70 nucleotides of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above, and in more detail below. For a portion of a polynucleotide of 'at least 20 nucleotides in length' for example, we mean 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (for example the deposited cDNA or the nucleotide sequence as shown in FIG. SEQ ID No. 1) Of course, a polynucleotide that hybridizes only to a poly A sequence (such as the poly (A) 3 'terminal stretch of the antimicrobial peptide cDNA shown in SEQ ID NO. complementary section of T (or U) residues, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide could hybridize to any nucleic acid molecule that contains a stretch poly (A) or the complement thereof (e.g., virtually any double-stranded cDNA clone.) As indicated, the nucleic acid molecules of the present invention which The anti-microbial peptide polypeptides may include, but are not limited to, those that code for the amino acid sequence of the mature polypeptide, by themselves; the coding sequence for the mature polypeptide and additional sequences, such as those coding for the guiding or secretory sequence of approximately 23 amino acids, such as the pre-, or pro- or prepro-protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned coding sequence, together with the additional non-coding sequences, including for example, but not limited to introns and non-coding 5 'and 3' sequences, such as untranslated, transcribed sequences that play a role in transcription, in mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and mRNA stability; an additional coding sequence that codes for additional amino acids, such as those that provide additional functionalities. In this way, the sequence encoding the polypeptide can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the tag or label provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc, Na ti. Aca d. Sci. USA 86: 821-824 (1989), for example, hexa-histidine provides convenient purification of the fusion protein. The "HA" tag or label is another peptide useful for purification, which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37: 767 (1984). As discussed below, other such fusion proteins include the antimicrobial peptide fused to either the N-terminus or the C-terminus. The present invention further relates to the variants of the nucleic acid molecules of the present invention, which code for portions, analogs or derivatives of the antimicrobial peptide protein. Variants may appear naturally, such as a natural allelic variant. By an "allelic variant" is meant one of several alternate forms of a gene that occupies a given locus on a chromosome of an organism Genes II, Lewin, B., ed., John Wiley & amp;; Sons, New York (1985). Variants of non-natural origin can be produced using mutagenesis techniques known in the art. Such variants include those produced by substitutions, deletions or additions of nucleotides, which may involve one or more nucleotides. The variants can be altered in the coding regions, in the non-coding regions, or in both. Alterations in the coding regions can produce conservative or non-conservative amino acid substitutions, deletions or additions thereof. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the antimicrobial peptide protein or portions thereof. Also especially preferred in this regard are conservative substitutions. Additional embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence of at least 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence coding for the polypeptide having the amino acid sequence in SEQ ID NO. 2; (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO. 2, but lacking the N-terminal methionine; (c) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 1 to about 41 in SEQ ID NO. 2; (d) a nucleotide sequence encoding the polypeptide having the amino acid sequence encoded for the cDNA clone contained in the ATCC Deposit No. 97982; (e) a nucleotide sequence encoding the mature antimicrobial peptide polypeptide, having the amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 97982; or (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), or (e). For a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence that codes for an antimicrobial peptide, it is understood that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence encoding the antimicrobial peptide polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides Up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence may appear at the 5 'or 3' -terminal positions of the reference nucleotide sequence or at any site between those terminal positions, interposed either individually between nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, if any particular nucleic acid molecule is at least 95%, 96%, 97%, 98% or 99% identical, for example to a nucleotide sequence shown in SEQ ID NO. 1 or to the nucleotide sequence of the deposited cDNA clone, this can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequenced Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive , Madison, Wl 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Appli ed Ma thema ti cs 2: 482-489 (1981), to find the best homology segment between two sequences. When Bestfit or any other sequence alignment program is used to determine if a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage Identity is calculated on the full length of the reference nucleotide sequence and that clearances in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed. The present application is directed to nucleic acid molecules at least 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO. 1 or the nucleic acid sequence of the deposited cDNA, regardless of whether they code for a polypeptide having antimicrobial peptide activity. This is because even where a particular nucleic acid molecule does not code for a polypeptide having the antimicrobial peptide activity, someone skilled in the art could still know how to use the nucleic acid molecule, for example, as a probe. hybridization or a primer for polymerase chain reaction (PCR). Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having antimicrobial peptide activity include, but are not limited to, (1) isolation of the antimicrobial peptide gene or allelic variants thereof in a cDNA library.; (2) in-situ hybridization (eg, "FISH"), to metaphase chromosomal divisions to provide accurate chromosomal localization of the antimicrobial peptide gene, as described in Verma et al., Human Chromosomes: A Man ual of Ba si c Techni ques, Pergamon Press, New York (1988); and analysis of Norther spotting (Northern Blot) to detect the expression of antimicrobial peptide mRNA in specific tissues.

However, nucleic acid molecules having at least 95%, 96%, 97%, 98% or 99% sequences identical to a nucleic acid sequence shown in SEQ ID NO. or to a nucleic acid sequence of the deposited cDNA which, in fact, does code for a polypeptide having antimicrobial peptide protein activity. By "a polypeptide having antimicrobial peptide activity" are meant polypeptides that show antimicrobial peptide activity in a particular assay or biological test. For example, the activity of the antimicrobial peptide can be measured using, for example, the antimicrobial assay as described in Diamond et al., Proc. Na ti. Aca d. Sci. 88: 3952 (1991). In summary, a concentrated aliquot of the candidate antimicrobial peptide is placed as a spot or spot on an E layer. Coli and incubate overnight at 37 ° C. The minimum inhibitory concentration of the peptide is determined by a modification of the method described by Soravia et al. FEBS Le t t. 228: 337-340 (1988). Briefly, 2.5 x 104 bacteria are incubated with increasing concentrations of the peptide in 96-well static microtiter plates overnight at 37 ° C. Bacterial development is evaluated by optical density measurements at 600 nm. The control incubations in the absence of the peptide and the incubations in the absence of the bacteria serve to establish the baseline values. Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence of at least 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence of the deposited cDNA or to a nucleic acid sequence shown in SEQ ID NO. 1, will code for a polypeptide "having antimicrobial peptide protein activity." In fact, since the degenerate variants of these nucleotide sequences all code for the same polypeptide, this will be clear to a skilled person even without performing the comparison test It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also code for a polypeptide having antimicrobial peptide protein activity.

This is because the person skilled in the art is fully aware of amino acid substitutions that are either less likely or that are not likely to significantly affect the function of the protein (eg, the replacement of an aliphatic amino acid). with a second aliphatic amino acid). For example, guidance on how to make phenotypically silent amino acid substitutions is provided in Bowie, JU et al., 'Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions', Sci ence 247: 1306-1310 (1990), in where the authors indicate that the proteins are surprisingly tolerant to amino acid substitutions.

Vectors and Guest Cells The present invention also relates to vectors that include molecules isolated from DNA of the present invention, host cells that are genetically engineered with recombinant vectors, and the production of antimicrobial peptide polypeptides or fragments thereof, by recombinant techniques.

The polynucleotides can be linked to a vector that contains a selectable marker for propagation in a host. In general, a plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or into a complex with a charged lipid. If the vector is a virus, it can be packaged using a suitable packaging cell line and then transduced into host cells. The DNA insert must be operably linked to an appropriate promoter, such as the PL promoter of lambda phage, the l ac, trp and ta c promoters of E. Col i, the early and late SV40 promoters and the retroviral LTR promoters, to name a few. Other suitable promoters will be known to those of skill in the art. The expression constructs will also contain sites for the initiation and termination of the transcript, and in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include an initiation of translation at the beginning and a termination codon (UAA, UGA or UAG) appropriately placed at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for culture of eukaryotic cells and genes for resistance to tetracycline or ampicillin for E culture. col i and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as E cells. Coli, Streptomyces and Salmonell to typhimuri um; fungal cells, such as yeast cells; insect cells such as Drosophi cell to S2 and Spodopt was Sf9; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. The appropriate culture media and conditions for the host cells described above are known in the art. In addition to using the expression vectors in the practice of the present invention, the present invention further includes novel expression vectors comprising the operator elements and promoters operably linked to the nucleotide sequences encoding a protein of interest. An example of such vector is pHE4a which is described in detail below. As summarized in Figures 4 and 5, the components of the vector pHE4a (SEQ ID NO: 4) include: 1) a neomycin phosphotrans ferase gene as a selection marker, 2) an origin of replication of E. Coli, 3) a promoter sequence of phage T5, 4) two lac operator sequences, 5) an ine-Dalgarno S sequence, 6) the lactose operon repressor gene (laclq) and 7) a region binding to the cloning site multiple. The origin of replication (oriC) is derived from • pUC19 (LTl, Gaithersburg, MD). The promoter sequence and the operator sequences were synthetically prepared. The synthetic production of the nucleic acid sequences is well known in the art. The Clontech Catalog 95/96, pages 215-216, Clontech, 1020 East Meadow Circle, Palo Alto, CA 94303. The pHE4a vector was deposited with the ATCC on February 25, 1998, and given the accession number 209645 A nucleotide sequence encoding the antimicrobial polypeptide (SEQ ID No. 1), is operably linked to the promoter and pHE4a operator by restricting the vector with Ndel and either Xbal, BamHl, Xhol, or Asp718, and isolation of the largest fragment (the region of the multiple cloning site is approximately 310 nucleotides) on a gel. The nucleotide sequence coding for the antimicrobial polypeptide (SEQ ID No. 1) having the appropriate restriction sites is generated, for example, according to the PCR protocol described in Example 1, using the PCR primers having the sites of restriction for Ndel (as the 5 'primer) and any of Xbal, BamHl, Xhol, or Asp718 (as the 3' primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and the vector are ligated according to standard protocols. As noted above, vector pHE4a contains a laclq gene. Laclq is an allele of the l acl gene and confers severe regulation of the operator l ac. Amann E. et al., Gene 69: 301-315 (1988); Stark, M., Gene 51: 255-267 (1987). The laclq gene codes for a repressor protein that binds to the operant sequences l ac and blocks the transcription of the downstream sequences (e.g., 3 '). However, the laclq gene product dissociates from the lac operator in the presence of either lactose or certain lactose analogs, eg, isopropyl-BD-thiogalactopyranoside (IPTG), and thus the antimicrobial polypeptide is not produced in amounts appreciable in non-induced host cells containing the vector pHE4a. Induction of these host cells by the addition of an agent such as IPTG, however, results in the expression of the sequence encoding the antimicrobial polypeptide. The promoter / operator sequences of the vector pHE4a (SEQ ID NO: 5) comprises a promoter of phage T5 and two sequences of the operator l to c. One operator is located 5 'to the transcription start site and the other is located 3' to the same site. These operators, when present in combination with the product of the laclq gene, confer severe repression of downstream sequences in the absence of an inducer of the operon ac I, e.g., IPTG. The expression of operably linked sequences located downstream of the operators l a c can be induced by the addition of an inducer of the operon ac ac, such as IPTG. The binding of an inducer to the laclq proteins results in their release from the operant sequences l ac and the initiation of transcription of the operably linked sequences. The regulation of the l ac operon of gene expression is reviewed in Deblin, T., TEXTBOOK OF BIOCHEMISTRY WITH CLINICAL CORRELATIONS, 4th Edition (1997), pages 802-807. The vector pHE4 series contains all the components of the pHE4a vector except for the antimicrobial polypeptide coding sequence. Characteristics of the pHE4a vectors include the optimized synthetic promoter of phage T5, the operator c, and the Shine-Delagarno sequences. In addition, these sequences are also optimally spaced so that the expression of an inserted gene can be strictly regulated and a high level of expression occurs after induction. Among the known bacterial promoters, suitable for use in the production of proteins of the present invention, are included the promoters lacl and l to cZ of E. Coli, the T3 and T7 promoters, the gpt promoter, the PR and PL lambda promoters and the t_rp promoter. Suitable eukaryotic promoters include the cytomegalovirus immediate early promoter (CMV), the HSV thymidine kinase promoter, the SV40 early and late promoters, the retroviral LTR promoters, such as those of the Rous Sarcoma Virus (RSV). ), and metallothionein promoters, such as the mouse metallothionein-I promoter. The vector pHE4a contains a 5 'Shine-Delgarno sequence to the AUG start codon. The Shine-Delgarno sequences are short sequences generally located approximately 10 nucleotides upstream (for example 5 ') of the AUG start codon. These sequences essentially direct the prokaryotic ribosomes towards the AUG start codon. Thus, the present invention is also directed to the expression vector useful for the production of the proteins of the present invention. This aspect of the invention is exemplified by the vector pHE4a (SEQ ID NO: 4). Preferred vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to one skilled in the art. The introduction of the construct into the host cells can be effected by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many of the standard laboratory manuals, such as Davis et al., Ba si c Me th ods In Mol ecul ar Bi olgy (1986). The polypeptide can be expressed in a modified form, such as a fusion protein, and can include not only the secretion signals, but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, the peptide portions can be added to the polypeptide to facilitate purification. Such regions can be removed before the final preparation of the polypeptide. The addition of the peptide portions to the polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous region - derived from immunoglobulin, which is useful for solubilizing proteins. For example, EP-A-0,464, 533 (Canadian counterpart 2045869) describes fusion proteins comprising various portions of the constant region of the immunoglobulin molecules, together with another human protein or part thereof. In many cases, the Fe part in a fusion protein is completely advantageous for use in therapy and diagnosis and this, for example, results in improved pharmacokinetic properties (EP-A-0232,262). On the other hand, for some uses it would be desirable to be able to suppress the Fe part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when the Fe portion proves to be an impediment to use in therapy and diagnosis, for example, when the fusion protein is to be used as an antigen for immunizations. In drug discovery, for example, human proteins, such as the hIL-5 receptor have been fused with the Fe portions for purposes of high throughput screening assays to identify hIL-5 antagonists. See, D. Bennett et al., Journal of Molecular Recognition, Vol. 8: 52-58 (1995) and K. Johanson et al., The Journal of Biological Ogi cal Chemi s try, Vol. 270, No 16: 9459-9471 (1995). The antimicrobial peptide can be recovered and purified from recombinant cell cultures by well-known methods, including precipitation with ammonium sulfate or with ethanol, acid extraction, anionic or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is used for the purification. The polypeptides of the present invention include naturally purified products, products of chemical synthesis procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host., including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending on the host employed in a recombinant production process, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, the polypeptides of the invention may also include a modified methionine residue at the start, in some cases as a result of the processes mediated by the host.

Antimicrobial Polypeptides Human and Fragmen t os of l os M osmos The invention further provides an isolated antimicrobial peptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence in SEQ ID NO. 2, or a peptide or polypeptide comprising a portion of the above polypeptides. It will be recognized in the art that some amino acid sequences of the antimicrobial peptide can be varied without significant effect of the structure or function of the protein. If such differences in the sequence are contemplated, it must be remembered that these will be critical areas on the protein which determine the activity. Thus, the invention further includes variations in the antimicrobial peptide, which exhibit substantial antimicrobial peptide activity, or which include regions of the antimicrobial peptide such as the protein portions discussed below. Such mutants include deletions, insertions, inversions, repetitions, and typical substitutions. As indicated above, guidance on which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U. and collaborators, 'Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions, "Sci en 247: 1306-1310 (1990) Thus, the fragment, derivative or analog of the polypeptide of SEQ ID No. 2, or that encoded by the deposited cDNA, can be i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such a substituted amino acid residue can or not being one encoded by the genetic code, or ii) one in which one or more of the amino acid residues includes a substituent group, or iii) one in which the mature polypeptide is fused to another compound, such as a compound for increase the half-life of the polypeptide (eg, polyethylene glycol), or iv) one in which additional amino acids are fused to the mature polypeptide, such as a peptide from the IgG Fe fusion region or a guide or secretory, or a sequence that is employed for the purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are considered within the scope of those of skill in the art from the teachings herein. Of particular interest are amino acid substitutions charged with another charged amino acid, and with neutral or negatively charged amino acids. The latter result in proteins with reduced positive charge to improve the characteristics of the antimicrobial peptide. The prevention of aggregation is highly desirable. The aggregation of the proteins not only results in a loss of activity, but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al., Clin. Exp. Immunol., 2: 331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10: 307- 377 (1993)). As indicated, the changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 1).

TABLE 1. Conservative Amino Acid Substitutions Of course, the number of amino acid substituents that an expert can perform depends on many factors, including those described above. Generally speaking, the number of amino acid substitutions by any given antimicrobial polypeptide will be no greater than 50, 40, 30, 20, 10, 5 or 3. The amino acids in the antimicrobial peptide of the present invention that are essential for the function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells Sci en 244: 1081-1085 (1989)). The last procedure introduces simple alanine mutations in each residue in the molecule. The resulting mutant molecules are then tested for biological activity such as binding to the receptor or in vi tro, or proliferative activity in vi tro. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Bi ol. 224: 899-904 (1992) and de Vos et al. Sci en 255: 306-312 (1992)). The polypeptides of the present invention are preferably provided in an isolated form. By "isolated polypeptide" is meant a polypeptide removed from its native environment, Thus, a polypeptide produced and / or contained within a recombinant host cell is considered isolated for purposes of the present invention Also considered as an "isolated polypeptide" are polypeptides that have been purified, partially or substantially, from a recombinant host cell. For example, a recombinantly produced version of the antimicrobial peptide polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 67: 31-40 (1988). The polypeptides of the present invention include the polypeptide encoded by the deposited cDNA that includes the leader, the mature polypeptide encoded by the deposited cDNA minus the leader (e.g., the mature protein), a polypeptide comprising the amino acids from about -23 to approximately 41 in SEQ ID NO. 2; a polypeptide comprising amino acids from about -22 to about 41 in SEQ ID NO. 2; a polypeptide comprising the amino acids from about 1 to about 41 in SEQ ID NO. 2; as well as polypeptides that are at least 95% identical, still more preferably at least 96%, 97%, 98%, or 99% identical to those described above and also include portions of such polypeptides with at least 30 aminoapoids, and more preferably at least 50 amino acids. For a polypeptide having at least one amino acid sequence, for example, 95% "identical" to a reference amino acid sequence of an antimicrobial peptide, it is understood that the amino acid sequence of the polypeptide is identical to the reference sequence, except that the polypeptide sequence can include up to five amino acid alterations per 100 amino acids of the reference amino acid of the antimicrobial peptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence * can be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence can be inserted into the reference sequence. These alterations of the reference sequence can occur at the amino- or carboxyl-terminal positions of the reference amino acid sequence or at any site between those terminal positions, interspersed either individually between residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, if any particular polypeptide is at least 95%, 96%, 97%, 98% or 99% identical to, for example, the amino acid sequence shown in SEQ ID NO. 2 or the amino acid sequence encoded by the deposited cDNA clone, can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetic Computer Group, University Research Park, 575 Science Drive, Madison, Wl 53711. When Bestfit or any other sequence alignment program is used to determine if a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are adjusted , of course, such that the percent identity is calculated over the entire length of the reference amino acid sequence and that clearances in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.

The polypeptide of the present invention is useful as a molecular weight marker on SDS-PAGE gels on molecular sieve gel filtration columns using methods well known to those of skill in the art. In still another aspect, the invention provides a peptide or a polypeptide comprising a portion that possesses epitope of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide described herein. An "immunogenic epitope" is defined as a part of a protein that promotes an antibody response when the entire protein is the immunogen. On the other hand, a region of a protein molecule to which the antibody can be linked is defined as an "antigenic epitope". The number of immunogenic epitopes of a protein is generally less than the number of antigenic epitopes. See, for example, Geysen et al., Proc. Na ti. Aca d. Sci. USA 81: 3998-4002 (1983). For the selection of peptides or polypeptides that possess an antigenic epitope (e.g., that contain a region of a protein molecule to which an antibody can be linked), it is well known in the art that relatively short synthetic peptides that mimic part of a protein sequence, are routinely capable of producing an antiserum that reacts with the partially mimicked protein. See, for example, Sutcliffe, J.G., Shinnick, T.M., Green, N. and Learner, R.A. (1983) Antibodies that react with predetermined sites on proteins. Sci ence 219: 660-666. Peptides capable of producing sera reactive with the protein are often represented in the primary sequence of a protein, and can be characterized by a group of simple chemical rules, and are not confined to the immunodominant regions of the intact proteins (eg, immunogenic epitopes) or the amino or carboxyl termini. The peptides and polypeptides possessing the antigenic epitope of the invention are therefore useful for producing antibodies, including monoclonal antibodies, which specifically bind to a polypeptide of the invention. See, for example, Wilson et al., Cell 37: 767-778 (1984) at 777. Peptides and polypeptides possessing antigenic epitopes of the invention preferably contain a sequence of at least seven, more preferably at least nine and more preferably between about at least about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. Non-limiting examples of antigenic polypeptides or antigenic peptides that can be used to generate antibodies specific for the antimicrobial peptide include: a polypeptide comprising amino acid residues from about 19 to about 27 in SEQ ID NO. 2; and a polypeptide comprising the amino acid residues from about 31 to about 41 in SEQ ID NO. 2. As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the antimicrobial peptide protein. The epitope-possessing peptides and polypeptides of the invention can be produced by any conventional means. Houghten, R.A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Na ti. Acad. Sci. USA 82: 5131-5135. This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten et al. (1986). As one of ordinary skill in the art will appreciate, the antimicrobial peptide polypeptides of the present invention and the epitope-possessing fragments thereof described above, can be combined with portions of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. . These fusion proteins facilitate purification and show an increased half-life in vi. This has been shown, for example, for chimeric proteins consisting of the first two domains of the human CD4 polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Na ture 331: 84-86 (1988)). Fusion proteins that have a dimeric structure linked by disulfide bridges, due to the IgG part, can also be more efficient in the binding and neutralization of other molecules than the monomeric protein of the antimicrobial peptide or a protein fragment alone (Fountoulakis and collaborators, J. Bi och em. 270: 3958-3964 (1995)).

Diagnosis The present inventors have discovered that the antimicrobial peptide of the present invention is expressed in lymph nodes of the lung and in corneal tissues. It is believed that for a number of diseases related to the immune system, the substantially altered (increased or decreased) levels of the antimicrobial peptide gene *, the expression can be detected in the tissue of the immune system or in other cells or in body fluids ( for example, serum, plasma, urine, synovial fluid or spinal fluid), taken from a patient who has such a disorder, in relation to a level of expression of the "standard" antimicrobial peptide gene, which is the level of expression of the antimicrobial peptide. in the tissues of the immune system or in body fluids coming from an individual who does not have the disorder in the immune system. Thus, the invention provides a diagnostic method useful during the diagnosis of an immune system disorder, which involves measuring the level of expression of the gene encoding the antimicrobial peptide protein, in the tissue of the immune system or other cells or bodily fluids from an individual, and comparing the level of expression of the measured gene with a level of expression of the antimicrobial peptide gene, standard, by which an increase or decrease in the level of expression of the gene compared to the standard is indicator of an immune system disorder. In particular, certain tissues of mammals with cancer of the lymph nodes of the lung or cornea are believed to express significantly improved or reduced levels of the antimicrobial peptide protein and of the mRNA encoding the antimicrobial peptide protein when compared to a corresponding "standard" level. In addition, it is believed that increased levels of the antimicrobial peptide protein can be detected in certain body fluids. (for example, serum, plasma, urine, and spinal fluid) of mammals with such cancer, when compared to the serum of mammals of the same species that do not have cancer.

Thus, the invention provides a diagnostic method useful during the diagnosis of an immune system disorder, including cancers of this system that involve measuring the level of expression of the gene encoding the antimicrobial peptide protein in the tissue of the system. immune or in other cells or body fluids from an individual, and comparing the level of expression of the measured gene with a level of expression of the standard antimicrobial peptide gene, whereby an increase or decrease in the expression level of the gene in Comparison to the standard is an indicator of an immune system disorder. Where a diagnosis of a disorder in the immune system has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients showing decreased expression of the antimicrobial peptide gene will experience a worse prognosis. clinical in relation to patients who express the gene at a level closer to the standard level. By "assay or evaluation of the level of expression of the gene encoding the antimicrobial peptide" is meant the qualitative or quantitative measurement or the estimation of the level of the antimicrobial peptide or the level of the mRNA encoding the antimicrobial peptide in a first biological sample and either directly (for example, by determining or estimating the absolute protein level or mRNA level) or relatively (for example, by comparing the level of the antimicrobial peptide or the level of the mRNA in a second biological sample). Preferably, the level of the antimicrobial peptide protein or the level of mRNA in the first biological sample is measured or estimated and compared to a standard level of the antimicrobial peptide protein or at the mRNA level, being taken the standard of a second biological sample obtained from an individual that does not have the disorder, or that is determined by the average of the levels of a population of individuals that do not have an immune system disorder. As will be appreciated in the art, once a standard antimicrobial peptide level or a level of mRNA is known, it can be used repeatedly as a standard for comparison. By "biological sample" is meant any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source containing the antimicrobial peptide protein or the mRNA thereof As indicated, biological samples include the body fluids (such as serum, plasma, urine, synovial fluid and spinal fluid) which contain the free antimicrobial peptide, tissue of the immune system, and other tissue sources that express the complete or mature antimicrobial peptide or an antimicrobial peptide receptor. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.The present invention is useful for the diagnosis or treatment of various disorders related to the immune system in mammals, preferably humans. inflammatory and infectious conditions and any dysregulation of the function of immune cells. The total cellular RNA can be isolated from a biological sample using any suitable technique such as the one-step guanidionium-thiocyanate-phenol-chloroform method, described in Chomczynski and Sacchi, Anal. Bi och em. 162: 156-159 (1987). The levels of mRNA encoding the antimicrobial peptide are then assayed or evaluated using any appropriate method. These include Northern blot analysis, nuclease SI tracing, polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (R-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR). The evaluation of antimicrobial peptide levels in a biological sample can occur using antibody-based techniques. For example, the expression of the antimicrobial peptide in tissues can be studied with classical immunohistological methods (Jalkanen, M., et al., J. Cell, Bi ol. 101: 976-985 (1985).; Jalkanen, M. And collaborators, J. Cel l. Bi ol. 105: 3087-3096 (1987)). Other antibody-based methods, useful for the detection of the expression of the antimicrobial peptide gene, include immunoassays, such as the enzyme-linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay levels are known in the art and include enzymatic labels, such as glucose oxidase, and radioisotopes such as iodine (125 I, 121 D, carb (14C), sulfur (35S), tritium (3H), indium (• inl: zIn) and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin. In addition to the evaluation of the levels of the antimicrobial peptide in a biological sample obtained from an individual, the antimicrobial peptide can also be detected in vi vo by means of imaging. Antibody levels or markers for the imaging of the antimicrobial peptide include those detectable by X-ray, NMR or ESR. For X-ray, adequate levels include radioisotopes such as barium or cesium, which emit detectable radiation but are not manifestly harmful to the individual. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which can be incorporated into the antibody by labeling nutrients for the relevant hybridoma. An antibody specific for the antimicrobial peptide or an antibody fragment that has been labeled with an appropriate, detectable imaging portion, such as a radioisotope (eg, 131I, 112In, 99mTc), a radiopaque substance, or a material detectable by nuclear magnetic resonance, is to introduce (eg, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for the disorder of the immune system. It will be understood in the art that the size of the subject and the image formation system used will determine the amount of portions of imaging necessary to produce diagnostic images. In the case of a portion of radioisotope, for a human subject, the amount of radioactivity injected will normally be in the range of about 5 to 20 illicuriums of 99mTc. The labeled antibody or antibody fragment will then accumulate preferentially at the site of the cells containing the antimicrobial peptide. Imaging of the tumor in vi vo is described in SW Burchiel et al., 'Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments' (Chapter 13 in Tumor Imagi ng: The Radi ochemi cal Detect on of Cancer, SW Burchiel and BA Rhodes, eds ., Masson Publishing Inc. (1982)).

Terapéu ti ca The antimicrobial peptide of the present invention can be used as an antimicrobial agent for the treatment of fungal or bacterial infections. The peptide of the present invention can be used for such treatment in a topical or systemic formulation for the treatment of acne, burns, eye infections, mouthwash, deodorant or topical fungicides. In addition, C. albi cans, the common cause of mucocutaneous fungal disease in patients with AIDS, which is extremely susceptible to several ß-defensins, can be controlled in these individuals more effectively by a β-defensin-based therapy or by combination with existing drugs.

Management Modes It will be appreciated that conditions caused by a decrease in the standard or normal level of antimicrobial peptide activity in an individual can be treated by administration of the antimicrobial peptide. Thus, the invention further provides a method for the treatment of an individual in need of an increased level of antimicrobial peptide activity, comprising administering to said individual a pharmaceutical composition comprising an effective amount of an isolated antimicrobial peptide, the invention, particularly a mature form of the antimicrobial peptide, effective to increase the level of activity of the antimicrobial peptide in such an individual. As a general proposition, the total pharmaceutically effective amount of the antimicrobial peptide administered parenterally per dose will be in the range of about 1 μg / kg / day to 10 mg / kg / day of the patient's body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg / kg / day, and more preferably for humans between about 0.01 and 1 mg / kg / day for the hormone. If administered continuously, the antimicrobial peptide is typically administered at a dose rate of about 1 μg / kg / hour to about 50 μg / kg / hour, either by 1 to 4 injections per day or by continuous subcutaneous infusions, for example , using a mini pump. An intravenous bag solution may also be employed. Pharmaceutical compositions containing the antimicrobial peptide of the invention can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments, drops or transdermal patch), buccally, or as an oral or nasal spray. By "pharmaceutically acceptable carrier" is meant a filler, diluent, encapsulation, solid, semi-solid or liquid, non-toxic, or an auxiliary formulation of any type The term "parenteral" as used herein is refers to modes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection, and infusion.

Chromosome tests The nucleic acid molecules of the present invention are also valuable for the identification of chromosomes. The sequence is specifically targeted and can hybridize to a particular site on an individual human chromosome. The formation of the map of the DNAs for the chromosomes according to the present invention is an important first step in the correlation of those sequences with the genes associated with the disease. In certain preferred embodiments in this regard, the cDNA described herein is used to clone the genomic DNA of an antimicrobial peptide gene. This can be accomplished using a variety of well-known techniques and libraries, which are generally commercially available. Genomic DNA is then used to map the chromosome map in si t u, using techniques well known for this purpose. In addition, in some cases the sequences can be mapped to the chromosomes by preparing PCR primers (preferably 15-25 base pairs) from the cDNA. Computer analysis of the 3 'untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for the selection by PCR of somatic cell hybrids containing individual human chromosomes. Fluorescence in si t u hybridization ("FISH") from a cDNA clone to a metaphase chromosome split can be provided to provide a precise chromosome site in a single step. This technique can be used with probes from the cDNA as short as 50 or 60 base pairs. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basi c Techni ques, Pergamon Press, New York (1988). Once a sequence has been plotted on a map at an accurate chromosome site, the physical position of the sequence on the chromosome can be correlated with the data on the genetic map. Such data are found, for example, in V. McKusick, Mendelian Inh eri tance In Man, available on-line through Johns Hopkins University, Welch Medical Library. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (co-inheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA or in the genomic sequence between affected and unaffected individuals. If a mutation is observed in any or all affected individuals but not in any normal individual, then the mutation is likely to be the causative agent of the disease. Having generally described the invention, it will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting.

Eg emplos Example 1: Expression and Purification of the Antimicrobial Peptide in E. col i The bacterial expression vector pQE60 is used for bacterial expression in this example (QIAEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311). pQE60 codes for resistance to the antibiotic ampicillin ('Amp') and contains a bacterial origin of replication ("ori"), an inducible IPTG promoter, a ribosome binding site ("RBS"), six codons that code for histidine residues that allow affinity purification using the nickel-nitrile-triacetic acid affinity resin ("Ni-NTA"), sold by QUIAGEN, Inc., upra, and the cleavage sites by simple restriction enzyme These elements are accommodated such that an inserted DNA fragment encoding a polypeptide expresses that polypeptide with all six His residues (eg, a "6 His tag") covalently linked to the polypeptide. carboxyl end of that polypeptide. The DNA sequence encoding the desired portion of the human antimicrobial peptide lacking the hydrophobic leader sequence is amplified from the deposited cDNA clone using the oligonucleotide PCR primers which anneal to the amino-terminal sequences of the desired portion of the human antimicrobial peptide and the sequences in the construct deposited 3 'to the coding sequence of the cDNA. Additional nucleotides containing the restriction sites to facilitate cloning in the pQE60 vector are added to the 5 'and 3' sequences, respectively.

For the cloning of the mature protein, the 5 'primer has the sequence 5' GACTCCATGGGTGTTTTTGGTGGTATAGGC-3 '(SEQ ID NO: 6) containing the underlined Ncol restriction site followed by 20 nucleotides complementary to the amino-terminal coding sequence of the mature sequence of the human antimicrobial peptide, in Figure 1. One of ordinary skill in the art would appreciate, of course, that the point in the coding sequence of the protein where the 5 'primer begins can be varied to amplify a DNA segment that codes for any desired portion of the complete protein, shorter or longer than the mature form. The 3 'primer has the sequence 5'-GACTAGATCTTGGCTTTTTGCAGCATTTTG-3' (SEQ ID NO: 7) containing the BglII underlined site, followed by 20 nucleotides complementary to the 3 'end of the coding sequence immediately before the stop codon in the DNA sequence of the human antimicrobial peptide, in Figure 1, with the coding sequence aligned with the restriction site to maintain its reading structure with that of the six His codons in the vector pQE60.

The DNA fragment of the amplified human antimicrobial peptide and the vector pQE60 are digested with Ncol and BglII and the digested DNAs are then ligated together. The insertion of the DNA of the human antimicrobial peptide into the restricted pQE60 vector places the coding region of the human antimicrobial peptide protein downstream (3 ') of the IPTG-inducible promoter and within the structure with a start AUG and the six codons of histidine. The ligation mixture is transformed into E cells. competent experts using standard procedures such as those described in Sambrook et al., Mol ecul ar Cloning: a Labora t ory Manual, 2nd Ed.; Cold Sring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Strain M15 / rep4 of E. Coli, which contains multiple copies of the plasmid pREP4, which expresses the lacy repressor conferring resistance to kanamycin ("Kan"), is used to carry out the illustrative example described herein.This strain, which is only one of many that are suitable for the expression of the human antimicrobial peptide are commercially available from QIÁGEN Inc., upra .. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. resistant colonies and the identity of the cloned DNA * is confirmed by restriction analysis, PCR and DNA sequencing Clones containing the desired constructs are developed from day to day ("O / N") in LB medium of liquid culture supplemented with ampicillin (100 μg / ml) and kanamycin (25 μg / ml) The O / N culture is used to inoculate a large culture, at a dilution of approximately 1:25 to 1: 250. The cells are developed at an optical density at 600 nm ("OD600") between 0.4 and 0.6. The isopropyl-b-D-thiogalactopyranoside ("IPTG") is then added to a final concentration of 1 mM to induce transcription from the repressor sensitive promoter l a c, by inactivation of the lacl repressor. The cells are subsequently further incubated for 3 to 4 hours. The cells are then harvested by centrifugation. The cells are then shaken for 3 to 4 hours at 4 ° C in 6M guanidine hydrochloride, pH 8. The cell debris is removed by centrifugation, and the supernatant containing the human antimicrobial peptide is loaded onto a column of affinity resin. nickel-acid-nitrile-triacetic acid ("Ni-NTA") (available from QIAGEN Inc., s upra). Proteins with a 6x His tag or label bind to Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist, 1995, QUIAGEN, Inc., supra). In summary, the supernatant is loaded on the column in 6 M guanidine hydrochloride, pH 8, the column is first washed with 10 'volumes of 6 M guanidine hydrochloride, pH 8, then washed with 10 volumes of 6M guanidine hydrochloride, pH 6, and finally the human antimicrobial peptide is eluted with 6 M guanidine hydrochloride, pH 5. The purified protein is then renatured by dialysis of it against phosphate buffered saline (PBS) or 50 mM sodium acetate, pH buffer 6 more 200 mM sodium chloride. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renaturation using a linear gradient of 1 6 6M-1M urea in 500 mM sodium chloride, 20% glycerol, 20 mM Tris / HCl pH 7.4, containing protease inhibitors. The renaturation must be done in a period of 1.5 hours or more. After renaturation, the proteins can be eluted by the addition of 250 mM imidazole. The imidazole is removed by a final dialysis step against PBS or 50 mM sodium acetate buffer, pH 6 plus 200 mM sodium chloride. The purified protein is stored at 4 ° C or frozen at -80 ° C.

Example 1 (b): Expression and Purification of Antimicrobial Peptide in E. col i The bacterial expression vector pQE60 is used for bacterial expression in this example (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311). pQE60 codes for resistance to the antibiotic ampicillin ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG-inducible promoter, a ribosome binding site ("RBS"), six codons coding for histidine residues that allow affinity purification using the nickel-acid-nitrile-triacetic affinity resin ("Ni-NTA") sold by QIAGEN Inc., s upra, and restriction enzyme sites, simple, suitable. These elements are accommodated such that a DNA fragment encoding a polypeptide can be inserted in such a manner to produce that polypeptide with all six Histidine residues (eg, a "6 X His tag"), covalently linked to the carboxyl end of that polypeptide. However, in this example, the polypeptide coding sequence is inserted such that translation of the six His codons is prevented, and, therefore, the polypeptide is produced without the 6 X His tag. The DNA sequence encoding the desired portion of the mature human antimicrobial peptide lacking the hydrophobic leader sequence is amplified from the deposited cDNA clone using the oligonucleotide PCR primers which anneal to the amino-terminal sequences of the desired portion of the human antimicrobial peptide and the sequences in the 3 ** deposited construct to the coding sequence of the cDNA. Additional nucleotides containing the restriction sites to facilitate cloning in the pQE60 vector are added to the 5 'and 3' sequences, respectively.

For the cloning of the human protein, the 5 'primer has the sequence 5'-GACTCCATGGGTGTTTTTGGTGGTATAGGC-3' (SEQ ID NO: 6) containing the underlined Ncol restriction site followed by 20 nucleotides complementary to the amino-terminal coding sequence of the sequence of the human antimicrobial peptide, mature in Figure 1. One of ordinary skill in the art would appreciate, of course, that the point in the coding sequence of the protein where the 5 'primer starts can be varied to amplify a desired portion of the complete protein, shorter or longer than the mature form. The 3 'primer has the sequence 5'-GACTAGATCTTCATGGCTTTTTGCAGCATTTTG-3' (SEQ ID NO: 8) containing the BglII underlined site, followed by a stop codon and 20 nucleotids complementary to the 3 'end of the coding sequence in the DNA sequence of the human antimicrobial peptide, in Figure 1. The amplified DNA fragments of the human antimicrobial peptide, and the vector pQE60 are digested with Ncol and BglII and the digested DNAs are then ligated together. The insertion of the DNA of the human antimicrobial peptide into the restricted pQE60 vector places the coding region of the human antimicrobial peptide protein, including its associated arrest codon, downstream of the IPTG-inducible promoter and within the structure with a starting AUG . The associated stop codon prevents translation of the six histidine codons downstream of the insertion point. The ligation mixture is transformed into E cells. Competent authorities using standard procedures such as those described in Sambrook et al., Mol ecul ar Cl oning: a Labora t ory Manual, 2nd Ed.; Cold Sring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Strain M15 / rep4 of E. Col i, which contains multiple copies of the plasmid pREP4, which expresses the lacy repressor confers resistance to kanamycin ('Kanr'), is used to carry out the illustrative example described herein.This strain, which is the only one Of the many that are suitable for expression of the human antimicrobial peptide, it is commercially available from QIAGEN Inc., supra .. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin.The plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA is confirmed by restriction analysis, PCR and DNA sequencing Clones containing the desired constructions are developed overnight (O / N ") in liquid culture in LB medium supplemented with ampicillin ( 100 μg / ml) and kanamycin (25 μg / ml). The O / N culture is used to inoculate a large culture, at a dilution of about 1:25 to 1: 250. The cells are developed at an optical density at 600 nm ("OD600") between 0.4 and 0.6. The isopropyl-β-D-thiogalactopyranoside ("IPTG") is then added to a final concentration of 1 mM to induce transcription from the sensitive promoter, to the lac repressor, by inactivation of the lacl repressor. The cells are subsequently further incubated for 3 to 4 hours. The cells are then harvested by centrifugation. The cells are then shaken for 3 to 4 hours at 4 ° C in 6M guanidine hydrochloride, pH 8. The cell debris is removed by centrifugation, and the supernatant containing the human antimicrobial peptide is dialyzed against 50 mM sodium acetate buffer. , pH 6, supplemented with 200 M sodium chloride. Alternatively, the protein can be successfully refolded by dialysis thereof against 500 mM NaCl, 20% glycerol, 25 mM Tris / HCl, pH 7.4, containing protease inhibitors. After renatularization, the protein can be purified by ion exchange, hydrophobic interaction and size exclusion chromatography. Alternatively, an affinity chromatography step such as an antibody column can be used to obtain the pure human antimicrobial peptide. The purified protein is stored at 4 ° C or frozen at -80 ° C.

Example 2: Cloning and Expression of the Antimicrobial Peptide in a Baculovirus Expression System In this illustrative example, the plasmid shuttle vector pA2 is used to insert the cloned DNA encoding the entire protein, including its secretory, naturally associated, secretory signal (leader), into a baculovirus to express the mature human antimicrobial peptide, using standard methods as described in Summers and collaborators, A Manual of Me ods for Ba ck ovi rus Vec t ors and Insect Cei l Cul t ure Procedures. Texas Agricultural Experimental Station, Bulletin No. 1555 (1987). This expression vector contains the strong polyhedrin promoter of nuclear polyhedrosis virus by Au t ographa cali forni ca (AcMNPV) followed by convenient restriction sites such as BamHl and Asp718. The polyadenylation site of simian virus 40 ("SV40") is used for efficient polyadenylation. For the easy selection of the recombinant virus, the plasmid contains the beta-galactosidase gene, from E. Col i under the control of a weak Drosophi promoter in the same orientation, followed by the polyadenylation signal of the pqlihedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with the wild-type viral DNA to generate viable viruses expressing the cloned polynucleotide. Many other baculoviral vectors could be used in place of the above vector, such as pAc373, pVL941, and pAcIMl, as one skilled in the art would readily appreciate, so long as the construction provides appropriately localized signals for transcription, translation, secretion and similar, including a signal peptide and an AUG within the structure, as required. Such vectors are described for example in Luckow et al., Virol. Ogy 170: 31-39. The cDNA sequence encoding the full length human antimicrobial peptide protein in the deposited clone, including the AUG start codon and the naturally associated leader sequence shown in Figure 1 (SEQ ID NO: 2), is amplified using oligonucleotide PCR primers corresponding to the 5 'and 3' sequence of the gene. The 5 'primer has the 5' sequence GACTGGATCCGCCATCATGAGGGTCTTGTATCTCC 3 '(SEQ ID NO 9) which contains the BamHl restriction enzyme site underlined, an efficient signal for the initiation of translation in eukaryotic cells as described by Kozak, M. , J. Mol. I saw ol. 196: 947-950 (1987), followed by 19 bases of the complete human antimicrobial peptide sequence, shown in Figure 1, beginning with the AUG start codon. The 3 'primer has the sequence 5' GACTGGTACCGATGTCGCACGTCTCTGATG 3 '(SEQ ID NO: 10) containing the underlined Asp718 restriction site, followed by 20 nucleotides complementary to the 3' noncoding sequence of Figure 1. The amplified fragment is isolated from a 1% agarose gel using commercially available equipment ('Geneclean', BIO 101 Inc., La Jolla, Ca.) The fragment is then digested with BamHI and Asp718 and again purified on an agarose gel This fragment is designated in the present "Fl." The plasmid is digested with the restriction enzymes BamHl and Asp718 and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. then isolated from a 1% agarose gel using commercially available equipment ("Geneclean" BIO 101 Inc., La Jolla, Ca.) This vector DNA is desigando in the present "VI". Fl and the dephosphorylated plasmid VI are ligated together with the T4 DNA ligase. E. Coli HB101 or other suitable hosts of E. Coli such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, CA) are transformed with the ligation mixture and spread on culture plates. The bacteria that contain the plasmid with the human antimicrobial peptide gene are identified using the PCR method, in which one of the primers that is used to amplify the gene and the second primer is from the well within the vector, so that only those bacterial colonies containing the human antimicrobial peptide gene fragment will show DNA amplification. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is referred to herein as the antimicrobial peptide pBac. Five μg of the plasmid pBac antimicrobial peptide are cotransfected with 1.0 μg of commercially available linearized baculovirus DNA ("BaculoGoldMR baculovirus DNA", Pharmingen, San Diego, CA. ), using the lipofection method described by Felgner et al., Proc. Na ti.

Acad. Sci. USA 84: 7413-7417 (1987). 1 μg of the BaculoGold ™ viral DNA and 5 μg of the plasmid pBac antimicrobial peptide are mixed in a sterile well of a microtitre plate containing 50 μl of serum free Grace medium (Life Technologies Inc., Gaithersburg, MD). After this, 10 μl of Lipofectin plus 90 μl of Grace medium are added, mixed and incubated for 15 minutes at room temperature. Subsequently, the transfection mixture is added dropwise to the Sf9 insect cells (ATCC CRL 1711), seeded in a 35 mm tissue culture plate with 1 ml of Grace medium without serum. The plate is swung back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27 ° C. After 5 hours, the transfection solution is removed from the plate and 1 ml of Grace insect medium supplemented with 10% fetal calf serum is added. The plate is placed again in an incubator and the culture is continued at 27 ° C for four days. After four days the supernatant is harvested and a plaque assay is performed, as described by Summers and Smith, supra. An agar gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce plates stained blue. (A detailed description of such a "plate assay" can also be found in the user guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, pages 9-10).

After the appropriate incubation, the blue stained plates are picked up with the tip of a micropipette (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 μl of Grace medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm boxes. Four days later, the supernatants of these culture boxes are harvested and then stored at 4 ° C. The recombinant virus is called V-antimicrobial peptide. To verify the expression of the human antimicrobial peptide gene, Sf9 cells are grown in Grace medium supplemented with 10% heat-inactivated FBS. The cells are infected with the V-antimicrobial peptide of the recombinant baculovirus, at a multiplicity of infection ("MOI") of approximately 2. Six hours later it is removed and replaced with the SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, MD). If radiomarker proteins are desired, 42 hours later, 5 μCi of 35S-met ionin and 5 μCi of 35S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then these are harvested by centrifugation. The proteins in the supernatant, as well as the intracellular proteins, are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled). The microsequencing of the amino acid sequence of the amino terminus of the purified protein can be used to determine the amino-terminal sequence of the mature protein and thus the cleavage site and the peptide length of the secretory signal.

Example 3: Cloning and Expression of Human Antimicrobial Peptide in Mammalian Cells A typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of the mRNA, the coding sequence to the protein, and the signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by the donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) of Retroviruses, for example, RSV, HTLVI, HIVI and the cytomegalovirus early promoter (CMV). However, cellular elements can also be used (for example, the human actin promoter). Expression vectors suitable for use in the practice of the present invention include, for example, vectors such as PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be used include, human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1 cells, Cos 7 and CV 1, shrunken QC1-3 cells, mouse L cells and mouse cells. Chinese hamster ovary (CHO). Alternatively, the gene can be expressed in stable cells that contain the integrated gene within a chromosome. Cotransfection with a selectable marker such as dhfr, gpt, neomycin, or hygromycin, allows the identification and isolation of the transfected cells. The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR marker (dihydrofolate reductase) is useful for developing cell lines that have several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the glutamine synthase (GS) enzyme (Murphy et al., Bi och em J. 227: 277-279 (1991); Bebbington et al., Bi o / Technol ogy 10: 169-175 (1992 )). Using these markers, the mammalian cells are developed in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene (s) integrated within a chromosome. Chinese hamster ovary (CHO) and NSO cells are frequently used for protein production. The pCI and pC4 expression vectors contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Mol ecul ar and Cell ul ar Bi olgy, 438-447 (March, 1985)) plus a fragment of the enhancer. from CMV (Boshart et al., Cell 41: 521-530 (1985)). Multiple cloning sites, for example, with cleavage sites by restriction enzyme BamHl, Xbal and Asp718, facilitate cloning of the gene of interest.

The vectors also contain the 3 'intron, the polyadenylation and termination signal of the rat preproinsulin gene.

Example 3 (a): Cloning and Expression in COS Cells The expression plasmid, pAntimicrobial HA, is made by cloning a cDNA encoding the full-length human antimicrobial peptide into the pcDNAI / Amp or pcDNAIII expression vector (which can be obtained from Invitrogen, Inc.). The pcDNAI / Amp expression vector contains: (1) an origin of replication of E. Col i effective for propagation in E. Col i and other prokaryotic cells; (2) an ampicillin resistance gene for the selection of prokaryotic cells containing the plasmid; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron; (5) several codons that encode a fragment of hemagglutinin (eg, an "HA" tag or label to facilitate purification) followed by a termination codon and a polyadenylation signal arranged so that a cDNA can be conveniently placed under the control of expression of the CMV promoter and operably linked to the SV40 intron and to the polyadenylation signal, by means of restriction sites in the polylinker. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al., Cell 37: 767 (1984). The fusion of the HA tag to the target protein allows easy detection and recovery of the recombinant protein with an antibody that recognizes the HA epitope. PcDNAIII contains, in addition, the selectable neomycin marker. A DNA fragment encoding the full-length antimicrobial peptide is cloned into the vector polylinker region, so that the expression of the recombinant protein is driven by the CMV promoter. The construction strategy of the plasmid is as follows. The antimicrobial peptide cDNA of the deposited clone is amplified using primers containing convenient restriction sites, much of which is described above for the construction of vectors for the expression of the antimicrobial peptide in E. Coli Suitable primers include the following, which are used in this example. ' The 5 'primer, which contains the underlined BamHl site, a Kozak sequence, an AUG start codon and 19 nucleotides of the 5' coding region of the complete antimicrobial peptide has the following sequence: 5 ' GACTGGATCCGCCATCATGAGGGTCTTGTATCTCC 3 '(SEQ ID NO 9). Primer 3 **, which contains the underlined BglII site, and 20 base pairs of the 3 'coding sequence has the following sequence (at the 3' end): 5 'GACTAGATCTTGGCTTTTTGCAGCATTTTG 3' (SEQ ID NO: 7). The DNA fragment amplified by PCR and the vector, pcDNAI / Amp, are digested with BamHl and BglII and then ligated. The ligation mixture is transformed into E. Col i strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media which are then incubated to allow the development of resistant colonies. Ampicillin The plasmid DNA is isolated from the resistant colonies and examined by restriction analysis or other means for the presence of the fragment encoding the antimicrobial peptide. For the expression of the recombinant antimicrobial peptide, the COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRANE, as described, for example, in Sambrook et al., Mol ecul ar Cl oni ng: a Labora t ory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989). The cells are incubated under conditions for the expression of the antimicrobial peptide by the vector. The expression of the HA-antimicrobial peptide fusion protein is detected by radiolabeling and immunoprecipitation, using the methods described in, for example Harlow et al., Antibodi is: A Labora t ory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold, Spring Harbor, New York (1988). For this purpose, two days after transfection, the cells are labeled by incubation in media containing 35 S-cysteine for 8 hours. The cells and media are collected, and the cells are washed and lysed with RIPA buffer containing detergent: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH 7.5 , as described by Wilson et al., cited above. The proteins are precipitated from the cell lysate and from the culture media using a HA specific monoclonal antibody. The precipitated proteins are then analyzed by SDS-PAGE and autoradiography. An expression product of the expected size is observed in the cell lysate, which is not observed in negative controls.

Example 3 (b): Cloning and Expression in CHO Cells The pC4 vector is used for the expression of the antimicrobial peptide. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Access No. 37146). The plasmid contains the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary cells or other cells lacking the dihydrofolate activity that are transfected with these plasmids can be selected by developing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate. The amplification of DHFR genes in methotrexate-resistant cells (MTX) has been well documented (see, for example, Alt, FW, Kellems, RM, Bertino, JR, and Schimke, RT, 1978, J. Bi ol. Ch em 253: 1357-1370, Hamlin, JL and Ma, C. 1990, Bi och em et Bi ophys Ac 1097: 107-143, Page, MJ Y Sydenham, MA 1991, Bi ot echnol ogy 9: 64-68 ). Cells developed in increasing concentrations of MTX develop resistance to the drug by overproduction of the target enzyme, DHFR, as a result of the amplification of the DHFR gene. If a second gene is linked to the DHFR gene, it is usually co-amplified and overexpressed. It is known in the art that this method can be used to develop cell lines that possess more than 1,000 copies of the gene or of the amplified genes. Subsequently, when methotrexate is removed, the cell lines are obtained containing the integrated amplified gene within one or more chromosomes of the host cells. Plasmid pC4 contains for the expression of the gene of interest, the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen et al., Mol ecul ar and Cell ul al Bi olgy, March 1985: 438- 447) plus an isolated fragment of the immediate early human cytomegalovirus (CMV) enhancer (Boshart et al., Cell 41: 521-530 (1985)). Downstream of the promoter are the cleavage sites of the restriction enzymes BamHl, Xbal, and Asp718 that allow the integration of the genes. Behind these cloning sites the plasmid contains the 3 'intron and the polyadenylation site of the rat preproinsulin gene. Another high efficiency promoter can also be used for the expression, for example, the human β-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, for example, HIV and HTLVI. The Tet-Off and Tet-On gene expression systems of Clontech and similar systems can also be used to express the antimicrobial peptide in a regulated manner in mammalian cells (Gossen, M., &Bujard, H. 1992, Proc. Na ti, Acad. Sci. USA 89: 5547-5551). Other signals may also be used for the polyadenylation of the mRNA, for example, from the genes of human growth hormone or globin. Stable cell lines that possess a gene of interest integrated within the chromosomes can be selected after co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker at the beginning, for example, G418 plus methotrexate. Plasmid pC4 is digested with the restriction enzymes BamHI and Asp718, and then dephosphorylated using calf intestinal phosphatase by methods known in the art. The vector is then isolated from a 1% agarose gel. The DNA sequence encoding the complete antimicrobial peptide including its leader sequence is amplified using oligonucleotide PCR primers corresponding to the 5 'and 3' sequences of the gene. The 5 'primer has the 5' sequence GACTGGATCCGCCATCATGAGGGTCTTGTATCTCC 3 ' (SEQ ID NO: 9) which contains the BamH1 restriction enzyme site underlined, followed by an efficient signal for the initiation of translation in eukaryotes, as described by Kozak, M., J. Mol. Bi ol. 196: 947-950 (1987), and 20 bases of the antimicrobial peptide coding sequence shown in Figure 1 (SEQ ID NO: 1). The 3 'primer has the sequence 5' GACTGGTACCGATGTCGCACGTCTCTGATG 3 '(SEQ ID NO: 10) which contains the restriction site Asp718 underlined, followed by 20 nucleotides complementary to the untranslated region of the antimicrobial peptide gene shown in Figure 1 (SEQ ID NO.1). The amplified fragment is digested with the endonucleases BamHI and Asp718 and then purified again on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with the T4 DNA ligase. E: Coli HB101 or XL-1 Blue cells are then transformed and the bacteria containing the inserted fragment within the pC4 plasmid are identified using, for example, restriction enzyme analysis. Chinese hamster ovary cells lacking an active DHFR gene are used for transfection. 5 μg of the expression plasmid pC4 are cotransfected with 0.5 μg of the pSV2-neo plasmid using lipofectin (Felgner et al., Sizpra). Plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 that codes for an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg / ml of G418. After 2 days, the cells are trypsinized and seeded in cloning plates by hybridoma (Greiner, Germany) in alpha minus MEM supplemented with 10, 25 or 50 ng / ml of methotrexate plus 1 mg / ml of G418. After about 10 to 14 days the single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). The clones that develop at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure is repeated until the clones that are developed at a concentration of 100 to 200 μM are obtained. The expression of the desired gene product is analyzed, for example, by SDS-PAGE and Western blotting or by reverse phase HPLC analysis.

Example 4: Tissue Distribution of Antimicrobial Peptide mRNA Expression Northern blot analysis is carried out to examine the expression of the antimicrobial peptide gene in human tissues, using the methods described by, among others, Sambrook et al. Cited above. A cDNA probe containing the complete nucleotide sequence of the antimicrobial peptide (SEQ ID No. 1) is labeled with 32P using the rediprime ™ DNA marking system (Amersham Life Science), according to the manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN-100MR column (Clontech Laboratories, Inc.), according to the manufacturer's protocol number PT1200-1. The labeled, purified probe is then used to screen various human tissues for the antimicrobial peptide mRNA. The Northern Spotting of Multiple Tissues (MTN) containing various human tissues (H) or tissues of the human immune system (IM) are obtained from Clontech and examined with the labeled probe using the hybridization solution ExpressHybMR (Clontech) according to the protocol of the manufacturer number PT1190-1 . After hybridization and washing, the stains are mounted and exposed to film at -70 ° C overnight, and the films are developed according to standard procedures. It will be clear that the invention can be practiced otherwise than is particularly described in the description and previous examples. Numerous modifications and variations of the present invention are possible in the light of the foregoing teachings and, therefore, are within the scope of the appended claims. The full description of all publications (including patents, patent applications, journal articles, laboratory manuals, books or other documents) cited herein are incorporated by reference herein.

LIST OF SEQUENCES [D GENERAL INFORMATION: (i) APPLICANT: HUMAN GENOME SCIENCES, INC. 9410 KEY WEST AVENUE ROCKVILLE, MD 20850 UNITED STATES OF AMERICA APPLICANT / INVENTOR: OLSEN HENRIK S. RUBEN, STEVEN M. (ii) TITLE OF THE INVENTION: ANTIMICROBIAL PEPTIDE (iii) SEQUENCE NUMBER: 10 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C. (B) STREET: 1100 NEW YORK AVENUE, SUITE 600 (C) CITY: WASHINGTON (D) STATE: DC (E) COUNTRY: USA (F) POSTAL CODE: 20005 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: PC Compatible with IBM (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentin Relay # 1.0, Version # 1.30 (vi) DATA OF THE CURRENT APPLICATION: (A) ) APPLICATION NUMBER: to be assigned (B) DATE OF SUBMISSION: with this (C) CLASSIFICATION: (vii) DATA OF THE PREVIOUS APPLICATION: (A) NUMBER OF APPLICATION: US 60 / 046,415 (B) DATE OF SUBMISSION: 14-MAY0-1997 (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: STEFFE, ERIC K. (B) REGISTRATION NUMBER: 36,688 (C) REFERENCE NUMBER / CASE: 1488.093PC01 (ix) INFORMATION FOR TELECOMMUNICATION: (A) TELEPHONE: (202) 371-2600 (B) FAX: (202) 371-2540 (2) INFORMATION FOR SEQ ID NO. 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 323 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: both (D) TOPOLOGY A: both (ii) TYPE OF MOLECULE: cDNA ( ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 36..227 (ix) FEATURES: (A) NAME / KEY: sig_peptide (B) LOCATION: 36..104 (ix) FEATURES: (A) ) NAME / KEY: mat_peptide (B) LOCATION: 105.227 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 1: GACTCAGCTC CTGGTGAAGC TCCCAGCCAT CAGCC ATG AGG GTC TTG TAT CTC 53 Met Arg Val Leu Tyr Leu -23 -20 CTC TTC TCG TTC CTC TTC ATA TTC CTG ATG CCT CTT CCA GGT GTT TTT 101 Leu Phe Ser Phe Leu Phe He Phe Leu Met Pro Leu Pro Gly Val Phe -15 -10 -5 GGT GGT ATA GGC GAT CCT GTT ACC TGC CTT AAG AGT GGA GCC ATA TGT 149 Gly Gly He Gly Asp Pro Val Thr Cys Leu Lys Ser Gly Wing He Cys 1 5 10 15 CAT CCA GTC TTT TGC CCT AGA AGG TAT AAA CAA ATT GGC ACC TGT GGT 197 His Pro Val Phe Cys Pro Arg Arg Tyr Lys Gln He Gly Thr Cys Gly 20 25 30 CTC CCT GGA ACA AAA TGC TGC AAA AAG CCA TGAGGAGGCC AAGAAGCTGC 247 Leu Pro Gly Thr Lys Cys Cys Lys Lys Pro 35 40 TGTGGCTGAT GCGGATTCAG AAAGGGCTCC CTCATCAGAG ACGTGCGACA TGTAAACCAA 307 ATTAAACTAT GGTGTC 323 (2) INFORMATION FOR SEQ ID NO. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 64 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 2: Mee Arg Val Leu Tyr Leu Leu Phe Be Phe Leu Phe He Phe Leu Met -23 -20, -15 -10 Pro Leu Pro Gly Val Phe Gly Gly He Gly Asp Pro Val Thr Cys Leu -5 1 5 Lys Ser Gly Ala He Cys His Pro Val Phe Cys Pro Arg Arg Tyr Lys 10 15 20 25 Glr. He Gly Thr Cys Gly Leu Pro Gly Thr Lys Cys Cys Lys Lys Pro 30 35 40 (2) Information for SEQ ID NO. 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 63 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: not relevant (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 3: Met Arg Leu His His Leu Leu Leu Ala Leu Leu Phe Leu Val Leu Ser 1 5 10 15 Wing Trp Ser Gly Phe Thr Gln Gly Val Gly Asn Pro Val Ser Cys Val 20 25 30 Arg Asn Lys Gly He Cys Val Pro He Arg Cys Pro Gly Ser Met Lys 35 40 45 Gln He Gly Thr Cys Val Gly Arg Ala Val Lys Cys Cys Arg Lys 50 55 60 (2) INFORMATION FOR SEQ ID NO. 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH ': 3974 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: both (D) TOPOLOGY: both (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 4: GAGTA CTAAG TGAGTAGGGC GTCCGATCGA CGGACGCCTT TTTTTTGAAT TCGTAATCAT 60 GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG 120 CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG 180 CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA 240 TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT TCCTCGCTCA 300 CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG 360 TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC 420 AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTTCCAT AGGCTCCGCC 480 CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC 540 TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC 600 TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA 660 C-CTCAC3CTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC 720 ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA 780 ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG 840 CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA 900 GAAGAACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG 960 GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC 1020 AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT 1080 CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCGTCGA 1140 CAATTCGCGC GCGAAGGCGA AGCGGCATGC ATTTACGTTG ACACCATCGA ATGGTGCAAA 1200 ACCTTTC3CG GTATGGCATG ATAGCGCCCG GAAGAGAGTC AATTCAGGGT GGTGAATGTG 1260 AAACCAGTAA CGTTATACGA TGTC3CAGAG TATGCCGGTG TCTCTTATCA GACCGTTTCC 1320 CGCGTGGT3A ACCAGGCCAG CCACGTTTCT GCGAAAACGC GGGAAAAAGT GGAAGCGGCG 1380 ATGGCGG .-. GC TGAATTACAT TCCCAACCGC GTGGCACAAC AACTGGCGGG CAAACAGTCG 1440 TTGCTGATTG GCGTTGCCAC CTCCAGTCTG GCCCTGCACG CGCCGTCGCA AATTGTCGCG 1500 GCGATTAAAT CTCGCGCCGA TCAACTG.GGT GCCAGCGTGG TGGTGTCGAT GGTAGAACGA 1560 AGCGGCG7CG AAGCCTGTAA AGCGGCGGTG CACAATCTTC TCGCGCAACG CGTCAGTGGG 1620 CTGATCAT7A ACTATCCGCT GGATGACCAG GATGCCATTG CTGTGGAAGC TGCCTGCACT 1680 AATGTTCC3G CGTTATTTCT TGATGTCTCT GACCAGACAC CCATCAACAG TATTATTTTC 1740 TCCCATGAAG ACGGTACGCG ACTGGGCGTG GAGCATCTGG TCGCATTGGG TCACCAGCAA 1800 ATCGCGCTGT TAGCGGGCCC ATTAAGTTCT GTCTCGGCGC GTCTGCGTCT GGCTGGCTGG 1860 CATAAAT.-.TC TCACTCGCAA TCAAATTCAG CCGATAGCGG AACGGGAAGG CGACTGGAGT 1920 GCCATGTCCG GTTTTCAACA AACCATGCAA ATGCTGAATG AGGGCATCGT TCCCACTGCG 1980 ATGC GGTTG CCAACGATCA GATGGCGCTG GGCGCAATGC GCGCCATTAC CGAGTCCGGG 2040 CTGCGCGTTG GTGCGGATAT CTCGGTAGTG GGATACGACG ATACCGAAGA CAGCTCATGT 2100 TATA CCCC-C CGTTAACCAC CATCAAACAG GATTTTCGCC TGCTGGGGCA AACCAGCGTG 2160 GACCGCTTGC TGCAACTCTC TCAGGGCCAG GCGGTGAAGG GCAATCAGCT GTTGCCCGTC 2220 TCACTGG7GA AAAGAAAAAC CACCCTGGCG CCCAATACGC AAACCGCCTC TCCCCGCGCG 2280 TTGGCCG.-.TT CATTAATGCA GCTGGCACGA CAGGTTTCCC GACTGGAAAG CGGGCAGTGA 2340 GCGCAACGCA ATTAATGTAA GTTAGCGCGA ATTGTCGACC AAAGCGGCCA TCGTGCCTCC 2400 CCACTCCTGC AGTTCGGGGG CATGGATGCG CGGATAGCCG CTGCTGGTTT CCTGGATGCC 2460 GACGGATTTG CACTGCCGGT AGAACTCCGC GAGGTCGTCC AGCCTCAGGC AGCAGCTGAA 2520 CCAACTCGCG AGGGGATCGA GCCCG3GGTG GGCGAAGAAC TCCAGCATGA GATCCCCGCG 2580 CTGGAGGATC ATCCAGCCGG CGTCCCGGAA AACGATTCCG AAGCCCAACC TTTCATAGAA 2640 GGCGGCGGTG GAATCGAAAT CTCGTGATGG CAGGTTGGGC GTCGCTTGGT CGGTCATTTC 2700 GAACCCCAGA GTCCCGCTCA GAAGAACTCG TCAAGAAGGC GATAGAAGGC GATGCGCTGC 2760 GAATCGGGAG CGGCGATACC GTAAAGCACG AGGAAGCGGT CAGCCCATTC GCCGCCAAGC 2820 TCTTCAGCAA TATCACGGGT AGCCAACGCT ATGTCCTGAT AGCGGTCCGC CACACCCAGC 2880 CGGCCACAGT CGATGAATCC AGAAAAGCGG CCATTTTCCA CCATGATATT CGGCAAGCAG 2940 GCATCGCCAT GGGTCACGAC GAGATCCTCG CCGTCGGGCA TGCGCGCCTT GAGCCTGGCG 3000 AACAGTTCGG CTGGCGCGAG CCCCTGATGC TCTTCGTCCA GATCATCCTG ATCGACAAGA 3060 CCGGCTTCCA TCCGAGTACG TGCTCGCTCG ATGCGATGTT TCGCTTGGTG GTCGAATGGG 3120 CAGGTAGCCG GATCAAGCGT ATGCAGCCGC CGCATTGCAT CAGCCATG .-. T GGATACTTTC 3180 TCGGCAGGAG CAAGGTGAGA TGACAGGAGA TCCTGCCCCG GCACTTCGCC CAATAGCAGC 3240 CAGTGCCTTC CCGCTTCAGT GACAACGTCG AGCACAGCTG CGCAAGGAAC GCCCGTCGTG 3300 GCCAGCCACG ATAGCCGCGC TGCCTCGTCC TGCAGTTCAT TCAGGGCACC GGACAGGTCG 3360 GTCTTGACAA AAAGAACCGG GCGCCCCTGC GCTGACAGCC GGAACACGGC GGCATCAGAG 3420 CAGCC3ATTG TCTGTTGTGC CCAGTCATAG CCGAATAGCC TCTCCACCCA AGCGGCCGGA 3480 GAACCTGCGT GCAATCCATC TTGTTCAATC ATGCGAAACG ATCCTCATCC TGTCTCTTGA 3540 TCAGATCTTG ATCCCCTGCG CCATCAGATC CTTGGCGGCA AGAAAGCCAT CCAGTTTACT 3600 TTGCAGGGCT TCCCAACCTT ACCAGAGGGC GCCCCAGCTG GCAATTCCGG TTCGCTTGCT 3660 GTCCATAAAA CCGCCCAGTC TAGCTATCGC CATGTAAGCC CACTGCAAGC TACCTGCTTT 3720 CTCTTTGCGC TTGCGTTTTC CCTTGTCCAG ATAGCCCAGT AGCTGACA T CATCCGGGGT 3780 CA3CACCGTT TCTGCGGACT GGCTTTCTAC GTGTTCCGCT TCCTTTAGCA GCCCTTGCGC 3840 CCTGAGTGCT TGCGGCAGCG TGAAGCTTAA AAAACTGCAA AAAATAGTTT GACTTGTGAG 3900 CGGATAACAA TTAAGATGTA CCCAATTGTG AGCGGATAAC AATTTCACAC ATTAAAGAGG 3960 AG AAAT ACA TATG 3974 INFORMATION FOR SEQ ID NO. 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 112 base pairs (B) TYPE: nucleic acid (C) TI PO OF HEBRA: both (D) TOPOLOGY: both (ii) TYPE OF MOLECULE: cDNA ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 5: AAGCTTAAAA AACTGCAAAA AATAGTTTGA CTTGTGAGCG GATAACAATT AAGATGTACC 60 CAATTGTGAG CGGATAACAA TTTCACACAT TAAAGAGGAG AAATTACATA TG 112 (2) INFORMATION FOR SEQ ID NO. 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) 'LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 6: GACTCCATGG GTGTTTTTGG TGGTATAGGC 30 (2) INFORMATION FOR SEQ ID NO. 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 7: GACTAGATCT TGGCTTTTTG CAGCATTTTG 30 (2) INFORMATION FOR SEQ ID NO. 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 8: GACTAGATCT TCATGGCTTT TTGCAGCATT TTG 33 (2) INFORMATION FOR SEQ ID NO. 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 9: GACTGGATCC GCCATCATGA GGGTCTTGTA TCTCC 35 (2) INFORMATION FOR SEQ ID NO. 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 10: GACTGGTACC GATGTCGCAC GTCTCTGATG 30 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following claims is claimed as property:

Claims (15)

1. An isolated nucleic acid molecule, characterized in that it comprises a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a nucleotide sequence encoding amino acids of about -23. at about 41 in SEQ ID NO. 2; (b) a nucleotide sequence encoding amino acids from about -22 to about 41 in SEQ ID NO. 2; (c) a nucleotide sequence encoding amino acids from about 1 to about 41 in SEQ ID NO. 2; (d) a nucleotide sequence encoding a polypeptide having the amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 97982; (e) a nucleotide sequence encoding the mature antimicrobial peptide having the amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 97982; and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), or (e).

2. An isolated nucleic acid molecule, characterized in that it comprises a polynucleotide that codes for the amino acid sequence of a portion that possesses epitope of the antimicrobial polypeptide of SEQ ID NO. 23.

The isolated nucleic acid molecule according to claim 2, characterized in that the epitope-possessing portion is selected from the group consisting of: amino acids from about 19 to about 27 in SEQ ID NO. 2; and the amino acids from about 31 to about 41 in SEQ ID NO. 2.

4. An isolated nucleic acid molecule, characterized in that it comprises a polynucleotide having a sequence selected from the group consisting of: (a) the nucleotide sequence of a fragment of the sequence shown in SEQ ID NO. 1, wherein the fragment comprises at least 20 contiguous nucleotides of SEQ ID NO. 1; and (b) a nucleotide sequence complementary to the nucleotide sequence in (a).

5. A method for the preparation of a recombinant vector, characterized in that the method comprises the insertion of the isolated nucleic acid molecule according to claim 1, within a linkage vector operable to a promoter.

6. A recombinant vector, characterized in that it is produced by the method according to claim 5.

7. A method for the preparation of a recombinant host cell, characterized in that it comprises introducing the recombinant vector according to claim 6 into a host cell.

8. A recombinant host cell, characterized in that it is produced by the method according to claim 7.

9. A recombinant method for producing a polypeptide, characterized in that it comprises culturing the recombinant host cell according to claim 8, under conditions such that the polypeptide is expressed and said polypeptide is recovered.

10. An isolated antimicrobial polypeptide, characterized in that it comprises amino acids at least 95% identical to amino acids selected from the group consisting of: (a) amino acids from about -23 to about 41 in SEQ ID NO. 2; (b) amino acids from about -22 to about 41 in SEQ ID NO. 2; (c) amino acids from about 1 to about 41 in SEQ ID NO. 2; and (d) amino acids comprising a portion that possesses epitope of any of the polypeptides of (a), (b) or (c).

11. An isolated polypeptide comprising an epitope-possessing portion of the antimicrobial protein of SEQ ID NO. 2, characterized in that the portion is selected from the group consisting of: amino acids from about 19 to about 27 in SEQ ID NO. 2; and the amino acids from about 31 to about 41 in SEQ ID NO. 2.

12. An isolated antibody, characterized in that it binds specifically to the antimicrobial polypeptide according to claim 10.

13. An isolated nucleic acid molecule, characterized in that it comprises a polynucleotide that encodes an antimicrobial polypeptide wherein, except for at least one conservative amino acid substitution, the polypeptide has a sequence selected from the group consisting of: (a) the amino acids of about the -23 to approximately 41 in the .SEQ ID NO. 2; (b) amino acids from about -22 to about 41 in SEQ ID NO. 2; (c) amino acids from about 1 to about 41 in SEQ ID NO. 2; (d) the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97982; (e) the amino acid sequence of the mature antimicrobial polypeptide encoded by the cDNA clone contained in the ATCC Deposit No. 97982; and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d) or (e).

14. An isolated antimicrobial polypeptide, characterized in that, except for at least one conservative amino acid substitution, the polypeptide comprises a sequence selected from the group consisting of: (a) amino acids from about -23 to about 41 in SEQ ID NO. 2; (b) amino acids from about -22 to about 41 in SEQ ID NO. 2; (c) amino acids from about 1 to about 41 in SEQ ID NO. 2; (d) the amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 97982; (e) the amino acid sequence of the mature antimicrobial polypeptide encoded by the cDNA clone contained in the ATCC Deposit No. 97982; and (f) the amino acid sequence of a portion that possesses epitope of any of the polypeptides (a), (b), (c), (d) or (e).

15. An isolated antimicrobial polypeptide, characterized in that it comprises at least 95% amino acids identical to the amino acids encoded by the cDNA clone contained in the ATCC Deposit No. 97982.