JP2003512828A - Beta-defensin - Google Patents

Abstract

  (57) [Summary] The present invention relates to polynucleotide and polypeptide molecules of zamp3 and zamp4, which are novel members of the β-defensin family. Polypeptides, and the polynucleotides that encode them, are useful in the study and treatment of microbial infections. The invention also encompasses zamp3 or zamp4 polypeptides.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION Biological defense strategies have evolved to protect organisms from invasion by other species. The microbial infection response system involves oxidative and non-oxidative mechanisms that utilize compounds that are enzymatically synthesized in cells and peptides that are single gene products.

[0002] Antimicrobial peptides constitute the oxygen-dependent host defense found in organisms containing a large number of taxonomic families. One major class of antimicrobial peptides is sequenced by the conserved cysteine residue pattern and is designated defensin. Mammalian defensins are derived from the skin, lungs and intestines and exhibit antibiotic activity against a wide variety of pathogens, including Gram-positive and Gram-negative bacteria, fungi (eg, Candida species) and viruses. For example,
: Poter et al., Infect. Immunol. 65 (6): 2396-401, 1997.

The amphipathic properties of defensin peptides appear to be a clue to the general mechanism of microbial attack, ie, attack by creating a hole or “punching” through the cell wall. In addition, Daher et al., J. Virol. 60 (3): 1068-
74, 1986, among others, enveloped viruses, including herpes simplex 1 and 2, cytomegalovirus and influenza virus (A / WSN), were inactivated by incubation with human neutrophil peptide (HNP-1). , And speculated that binding of the defensin molecule to the virus impairs the ability of the virus to infect cells.

The defensin family of antimicrobial peptides is divisible into two major subclasses based on two distinct consensus sequences. See, for example: Martin et al., J. Leuko. Bio. 58: 128-36, 1995. The first defensin subclass, classical defensins, represented by HNPs, is stored within the so-called large azurofil particles of neutrophils and macrophages and attacks microbes phagocytosed by these cells. The amino acid sequence of HNP is consistent with the predicted disulfide bridge that distinguishes it from that of the β-defensin subclass.
Also, epithelial cells can be the source of defensins, and these cells secrete these peptides into the external, extracellular environment.

In mice, for example, Paneth cells in the small intestine and the proximal colon secrete defensin-like cells called cryptidine into the lumen. See, for example: Ouellette and Selsted, The FASEB Journal 10: 1280-9, 1996.
. Support for defensin's defense function of the endogenous pathogen was found using matrilysin homozygous knockout mice. Mice lacking matrilysin, a metalloproteinase required to regulate α-defensin activity, were found to have reduced antimicrobial activity and were more susceptible to pathogens (Wilson et al., Science 286: 133-7, 1999).

Β-Defensins, the second major defensin subclass, are peptides found in bovine lung (eg, BNBD bovine neutrophilic β-defensins) as well as secreted forms (TAP-tracheal antimicrobials). Sex peptides). For example,
See, Selsted et al., J. Biol. Chem. 268: 6641-8, 1993.
. Two human β-defensins have been reported. SAP-1 (HBD1) was isolated from human psoriatic skin and hBD-1 was found at low concentrations in human blood filtrate. See, for example: Bensch et al., FEBS Lett. 368: 331-5, 1995). HB
D2, Hardner et al., Nature 387: 861, 1997. The amino acid sequences of these human β-defensins are most similar to bovine BNDP and TAP.

For example, see the following references: Harder et al., Nature 387: 861, 1997, where SAP-1 is designated as hBD-2. Murine β-defensins include: MBD1, Huttner et al., FEBS Lett. 413: 45-9, 1997, Bals et al., Infect. Imm.
unol. 67: 42-7, 1997; MBD2, Morrison et al., FEBS Lett. 422: 112-6, 1999.
MBD3, Bals et al., Infect. Immunol. 67: 3542-7, 1999; and MBD4, Jia et al., J. Biol. Chem. 1 [equb ahead of print], 2000.

Except for conserved cysteine residues, the defensin family is highly sequenced. It may be related to the site or state of activity or to the spectrum of pathogens attacked by a particular defensin. In addition to antimicrobial activity, certain defensins exhibit metabolically sensitive cytotoxic activity (Lichtenstein et al., Blood 68: 1407-10, 1986 and Sheu et al., Antimicrob.
Agents Chemother. 28: 626-9, 1993) and modified the response of adrenal cortical cells to ACTH (Zhu et al., Proc. Natl. Acad. Sci. (USA) 85: 592-6, 1988) and to human monocytes. Has specific chemotactic activity (Territo et al., J. Clin. Invest
.).

The recruitment of monocytes by neutrophils may be mediated in part by neutrophil defensins, suggesting proinflammatory activity against these peptides in addition to their antimicrobial effects. Also, a decrease in defensin mRNA levels was demonstrated in SPG (specific granular disease). For example, see the following reference: Tamu
ra et al., Japan. Int. Hematol. 59: 137-42, 1994. Higazi et al., J. Biol. Ch
Em. 271: 17650-5, 1996 suggested that fibrin-bound plasminogen in the presence of defensins may be less sensitive to activation by tPA.

Parts of defensins with antimicrobial, immunostimulatory, proinflammatory and other properties have been sought. The present invention provides polypeptides for these uses and other uses that will be apparent to those skilled in the art from the teachings herein.

Brief Summary of the Invention The present invention provides novel defensin proteins, designated "zamp2", "zamp3" and "zamp4". The invention also provides zamp3 and zamp4 variant polypeptides and zamp3 and zamp4 fusion proteins, as well as nucleic acid molecules encoding such polypeptides and proteins, and methods of using these nucleic acid molecules and amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION Before describing the present invention in detail, it will be helpful to the understanding of the present invention to define the following terms: The term "affinity label" is used herein. Used to represent a segment of a polypeptide that can bind to a peptide, either to provide purification or detection of the polypeptide, or to provide a binding site for the polypeptide to a substrate. In principle, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity label.

Affinity labels include: polyhistidine tract, protein A (N
ilsson et al., EMBO J. 4: 1075, 1985; Nilsson et al., Methods Enzymol. 198: 3.
, 1991), glutathione S transferase (Smith and Johnson, Gene 67).
: 31, 1988), substance P, Flag ^TM peptide (Hopp et al., Biotechnology 6: 1).
204-10, 1988, Eastman Kodak Co., New Haven, CT), streptavidin binding peptides, or other antigenic epitopes or binding domains. In general, Ford et al., Protein Expression and Purificati
on 2: 95-107, 1991. DNA encoding the affinity tag is available from commercial suppliers (eg, Pharmacia Biotech, Piscataway, NJ).

The term “allelic variant” is used herein to describe any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation occurs naturally due to mutations and can result in phenotypic polymorphism within a population. The mutation in the gene may be silent (no change in the encoded polypeptide) or may encode a polypeptide having an altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.

The terms “amino terminal” or “carboxyl terminal” are used herein to describe a position within a polypeptide. Where this relationship allows, these terms are used with reference to a particular sequence or portion of a polypeptide to describe proximity or relative position. For example, certain sequences located carboxy-terminal to a reference sequence within a peptide are located close to the carboxy terminus of the reference sequence but are not necessarily at the carboxy terminus of the complete polypeptide. The term "complement of a polynucleotide molecule" is a polynucleotide molecule that has a complementary base sequence and has an opposite orientation compared to a reference sequence. For example, array 5 '
ATGCACGGG 3'is complementary to 5'CCCGTGCAT 3 '.

The term “degenerate nucleotide sequence” refers to a nucleotide sequence that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain triplets of different nucleotides, but encode the same amino acid residue (ie, each of the GAU and GAC triplets is As.
code p).

The term “expression vector” is used to describe a linear or circular DNA molecule consisting of a segment encoding the polypeptide in question operably linked to an additional segment that provides its transcription. It Such additional segments include promoter and terminator sequences and optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. be able to. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

The term “isolated”, when applied to a polynucleotide, removes the polynucleotide from its natural genetic environment, thus free of other extra or unwanted coding sequences, and genetically engineered. It represents a suitable form for use in the production system of the obtained protein. Such isolated molecules are those that have been separated from their natural environment and include cDNA and genomic clones.

The isolated DNA molecules of the present invention are free of other genes with which they are normally associated, but include naturally occurring 5'and 3'untranslated regions, such as promoters and terminators. You can Identification of associated regions will be apparent to those of skill in the art (eg, Dynan and Tijan, Nature 316: 774-.
78, 1985). Another example of an isolated nucleic acid molecule is a chemically synthesized nucleic acid molecule that does not integrate into the organism's genome. An isolated nucleic acid molecule isolated from a chromosome of a particular species is smaller than the complete DNA molecule of that chromosome.

An “isolated” polypeptide or protein is a polypeptide or protein found in conditions other than its natural environment, such as blood and animal tissues. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptide in a highly purified form, ie greater than 95% pure, more preferably greater than 99% pure. As used in this context, the term "isolated" does not exclude the presence of another physical form of the same polypeptide, such as a dimer, or a glycosylated or derivatized form.

The term “operably linked” when referring to DNA segments is such that the transcription is initiated at the promoter and the coding segment is such that the segments function in concert for their intended purpose. Indicates that the segment is arranged to progress through to the terminator. The term "ortholog" refers to a polypeptide or protein obtained from one species that is the functional counterpart of the polypeptide or protein from a different species.
Sequence differences between orthologs are the result of speciation. The term "paralog" is a distinct, structurally related protein made by an organism. Paralogs are thought to occur through gene duplication. For example, α
-Globin, β-globin, and myoglobin are paralogs of each other.

The term “polynucleotide” is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read in the 5 ′ → 3 ′ end direction.
Polynucleotides, including RNA and DNA, can be isolated from natural sources, synthesized in vitro, or produced from a combination of naturally occurring and synthetic molecules. The size of a polynucleotide depends on the base pair (abbreviation “bp”), nucleotide (
“Nt”) or kilobases (“kb”).

When allowed by this relationship, the latter two terms can describe polynucleotides that are single-stranded or double-stranded. When this term is applied to a double-stranded molecule, it will be used to describe overall length and will be understood to be equivalent to the term "base pairs". As one of skill in the art will recognize, the two strands of a double-stranded polynucleotide may differ slightly in length, and their ends may be staggered as a result of enzymatic cleavage; thus, a double-stranded polynucleotide molecule All nucleotides in may be unpaired. Such unpaired ends generally do not exceed 20 nucleotides in length.

A “polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides smaller than about 10 amino acid residues are commonly referred to as "peptides". The term "promoter" is used herein in its art-recognized meaning to refer to the portion of a gene that contains a DNA sequence that provides for the binding of RNA polymerase and initiates transcription. Promoter sequences are commonly, but not always, found in the 5'non-coding region of genes.

The term “receptor” refers to a cell associated protein that binds a bioactive molecule (ie, a ligand) and mediates the action of the ligand on the cell. Membrane bound receptors are characterized by a multi-peptide structure consisting of an extracellular ligand binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of the ligand to the receptor results in a conformational change in the receptor that causes an interaction between the effector domain and one or more other molecules in the cell. This interaction subsequently leads to alterations in cellular metabolism.

Metabolic events involved in receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increased cyclic AMP production, cellular calcium mobilization, membrane lipid mobilization, cell adhesion, It includes the hydrolysis of inositol lipids and the hydrolysis of phospholipids. Most nuclear receptors also exhibit a multi-domain structure, including an amino-terminal trans-acting domain, a DNA binding domain and a ligand binding domain. Generally, receptors are membrane-bound, cytosolic or nuclear receptors; monomeric receptors (eg thyroid stimulating hormone receptors, beta-adrenergic receptors) or multimeric receptors (eg
PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” refers to a DNA sequence that encodes a polypeptide (“secretory peptide”), which is a component of the larger polypeptide and is the secretory pathway of the cell in which the larger polypeptide is synthesized. Direct the polypeptide through.
Usually, the larger polypeptide is cleaved during transit through the secretory pathway to remove the secretory peptide.

“Soluble receptor” is a receptor polypeptide that does not bind to cell membranes. Most commonly, the soluble receptor is a ligand-binding receptor polypeptide lacking the transmembrane and cytoplasmic domains. Soluble receptors can comprise additional amino acid residues, for example, an affinity label that provides for purification of the polypeptide or provides a binding site for the polypeptide to a substrate. Many cell surface receptors have naturally occurring, soluble counterparts that are either proteolytically produced or translated from alternatively spliced mRNAs. Receptor polypeptides, when lacking sufficient portions of these segments to provide membrane anchorage or signal transduction, respectively,
It is said to be substantially free of transmembrane and intracellular polypeptide segments.

It will be understood that the molecular weights and lengths of polymers determined by inaccurate analytical methods (eg gel electrophoresis) are approximations. When expressing such a value as "about" X or "approximately" X, the stated value of X will be understood to be accurate to ± 10%. The present invention is based, in part, on the discovery of a novel DNA sequence that encodes a polypeptide having homology to the β-defensin family of proteins. That is, the zamp2, zamp3, and zamp4 polypeptides of the invention exhibit the conserved motif shown in SEQ ID NO: 4 and here: C (X) 6C (X) 3-4C (X) 7GXC (X) 6CC,
Here, “(X)” is the number of preferably non-cysteine amino acid residues between specific amino acids. The position and spacing of cysteines are characteristic of the β-defensin family. Figure 1 provides the three disulfide bonds of the conserved β-defensin motif.

The zamp3 polypeptide (Defb5) is a mouse homolog of calf intestinal β-defensin EPA (Gallager et al., Mamm. Genome 6: 554-6, 1995 and Traver et al., Infect.
Immunol. 66: 1045-56, 1998). The nucleotide sequence of the zamp3 polypeptide is set forth in SEQ ID NO: 1, its deduced amino acid sequence is set forth in SEQ ID NO: 2, and within the naturally occurring genomic sequence of the pseudogene encoding murine zamp3. Originally obtained by solving the mutation found (SEQ ID NO: 3
). Further analysis of the genomic sequence (SEQ ID NO: 19) shows that there is a second intron / exon splice site that does not result in a frameshift of zamp3 and that translation of zamp3 can occur without the aforementioned repairs.

The zamp3 polypeptide includes a signal sequence, amino acid residues 1-22 of SEQ ID NO: 2, nucleotides 10-198 of SEQ ID NO: 1. Thus, the mature polypeptide ranges from amino acid residue 23 to amino acid residue 63 of SEQ ID NO: 2, nucleotides 76 to 198 of SEQ ID NO: 1. The defensin cysteine motif C (X) 6C (X
) 3-4C (X) 7GXC (X) 6CC (amino acid residues 31-60 of SEQ ID NO: 2), where "(X)" is the number of preferably non-cysteine amino acid residues between specific amino acids. Is. Figure 2 provides an alignment of zamp3 with murine defensins 3 and 4 (SEQ ID NOs: 11 and 12).

The present invention provides an isolated polypeptide comprising amino acid residues 31-60 of SEQ ID NO: 2, an isolated polypeptide comprising amino acid residue 1-amino acid residue 63 of SEQ ID NO: 2. Peptide, and amino acid residues 31-60 of SEQ ID NO: 2, amino acid residue 31 of SEQ ID NO: 2
~ 63, amino acid residues 23-60 of SEQ ID NO: 2, amino acid residues 23-63 of SEQ ID NO: 2, amino acid residues 1-60 of SEQ ID NO: 2, and amino acid residues 1-63 of SEQ ID NO: 2. Provide a polypeptide. The present invention also provides amino acid residues 1-324 of SEQ ID NO: 1,
SEQ ID NO: 1 amino acid residues 10-198, SEQ ID NO: 1 amino acid residues 10-189, SEQ ID NO:
An isolated poly-comprising amino acid residues 76-198 of 1; amino acid residues 76-189 of SEQ ID NO: 1; amino acid residues 100-198 of SEQ ID NO: 1; and amino acid residues 100-189 of SEQ ID NO: 1. Provide nucleotides.

The nucleotide sequence of the zamp4 polypeptide is set forth in SEQ ID NO: 5, and its deduced amino acid sequence is set forth in SEQ ID NO: 6. The zamp4 polypeptide comprises amino acid residues 1-44 of SEQ ID NO: 6, nucleotides 1-132 of SEQ ID NO: 5.
The additional 5'sequences are considered to be part of the additional exons and can be found using methods known in the art. Defensin cysteine motif C (X) 6C (X) 3-4C (X) 7GXC (X) 6CC (SEQ ID NO: 6 in the zamp4 polypeptide
Amino acid residues 12-41) are present, where "(X)" is the number of preferably non-cysteine amino acid residues between specific amino acids.

The present invention provides isolated polypeptides and amino acid residues 12-41 of SEQ ID NO: 6,
Provided is a polynucleotide encoding a polypeptide consisting of amino acid residues 12-44 of SEQ ID NO: 6, amino acid residues 1-41 of SEQ ID NO: 6, and amino acid residues 1-44 of SEQ ID NO: 6. Further, the present invention, nucleotides 1-296 of SEQ ID NO: 5, nucleotides 1-132 of SEQ ID NO: 5, nucleotides 1-123 of SEQ ID NO: 5, nucleotides of SEQ ID NO: 5
34-123, and nucleotides 34-132 of SEQ ID NO: 5 are provided.

The zamp2 polypeptide is the human homologue of murine β-defensin 1 (Huttner et al., FEBS Lett. 413: 45-9, 1997). There is 100% sequence identity between mouse and human at the amino acid level and a single base pair difference at the nucleotide level. The nucleotide sequence of zamp2 polypeptide is set forth in SEQ ID NO: 7, and its deduced amino acid sequence is set forth in SEQ ID NO: 8. Defensin cysteine motif C (X) 6C (X) 3-4C (X) 7G in the mature polypeptide
There is XC (X) 6CC (amino acid residues 37-67 of SEQ ID NO: 8), where "(X)" is the number of preferably non-cysteine amino acid residues between the specific amino acids. Expression of zamp2 in bone marrow, placenta, skeletal muscle, small intestine and uterus was found using reverse transcription PCR (RT-PCR).

Standard Nezminozan blot tissue distribution of mRNA corresponding to the novel DNA of zamp3 revealed no expression. The murine embryo blot was similar. Mouse RNA master blot revealed faint positive signals corresponding to thyroid, ovary and epididymis. Normal tissue levels of zamp3 polypeptide mRNA appear to be just below or below the sensitivity of Northern blot detection. Such observations are in line with knowledge in the field of defensins: that defensins are constitutively expressed at low levels but highly inducible upon infection.

Another aspect of the invention includes fragments of zamp3 and zamp4 polypeptides. Preferred fragments include a leader sequence ranging from amino acid residues 1-22 of SEQ ID NO: 2. Such leader sequences can be used to direct the secretion of other polypeptides. Such fragments of the invention can be used as follows: a selective secretory leader fragment is formed as a fusion protein with a selective protein selected for secretion; directs expression of the fusion protein. A plasmid carrying a possible regulatory region is introduced into the test cells; and protein secretion is monitored.

Additional preferred fragments include the cysteine domain, amino acid residues 31-60 of SEQ ID NO: 2, amino acid residues 12-41 of SEQ ID NO: 4. The fragments provided by the invention are useful as described herein, where administration of zamp3 and zamp4 is beneficial, for example as an antimicrobial agent (Thennarasu and Nagaraj, Biochem.
Biophys. Res. Commun. 254: 281-3, 1999).

The present invention also provides a fusion construct incorporating a zamp3 or zamp4 polypeptide selected from the group consisting of the following sequences: (a) amino acid residues 31-60 of SEQ ID NO: 2; (b) Amino acid residues 12 to 41 of SEQ ID NO: 6; (c) SEQ ID NO: 2; (d) SEQ ID NO: 6;

A mammalian species homologue or human paralog of (a) or (b); a mature polypeptide region of another defensin molecule; and, optionally, a polypeptide linker therebetween. It is expected that fusion constructs containing a defensin molecule will exhibit a broader range of antimicrobial efficacy when the defensin molecule has a completely dissimilar spectrum of pathogens. A polypeptide linker is used when it is necessary to separate the component polypeptides of the fusion or to allow the flexibility of the fusion protein, thereby preserving the antimicrobial activity of each defensin component of the fusion protein. It is preferable. A person skilled in the art can design such a linker.

The highly conserved amino acids in the consensus domain of zamp3 and zamp4 polypeptides can be used as a tool to identify new family members. For example, reverse transcription-polymerase chain reaction (RT-PCR) can be used to amplify conserved motif-encoding sequences from RNA obtained from various tissue sources. For more details, use the probe below to zamp3 like or zam
A p4-like β-defensin can be identified. Preferred embodiments of this aspect of the invention include the following:

(A) SEQ ID NO: 2, (b) SEQ ID NO: 6, (c) amino acid residues 23-63 of SEQ ID NO: 2, (d) amino acid residues 31-60 of SEQ ID NO: 2, and (e) sequence Amino acid residues 12-41 of number 6. In particular, highly degenerate primers designed from the above sequences are effective for this purpose.

The invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the zamp3 and zamp4 polypeptides disclosed herein. As those skilled in the art will readily appreciate, considerable sequence variation is possible between these polynucleotide molecules, given the degeneracy of the genetic code. SEQ ID NO: 9 is a degenerate polynucleotide sequence that includes all polynucleotides encoding the zamp3 polypeptide of SEQ ID NO: 2. SEQ ID NO: 10 is a degenerate polynucleotide sequence that includes all polynucleotides encoding the zamp4 polypeptide of SEQ ID NO: 4.

Thus, the polynucleotides of SEQ ID NO: 9 and SEQ ID NO: 10 that encode zamp3 and zamp4 polypeptides are included in the present invention. As will be appreciated by those in the art, the degenerate sequences of SEQ ID NO: 9 and SEQ ID NO: 10 also provide all RNA sequences encoding SEQ ID NO: 2 and SEQ ID NO: 6 by replacing U with T. The one-letter codes used within SEQ ID NOs: 9 and 10 to represent degenerate nucleotide positions are listed in Table 1. The "solution" is the nucleotide represented by the code letter. "Complement"
A code for one or more complementary nucleotides. For example, the code Y represents C or T, and its complement R represents A or G, where A is complementary to T,
And G is complementary to C.

[0045]

[Table 1]

The degenerate codons used in SEQ ID NO: 9 and SEQ ID NO: 10, including all possible codons for a given amino acid, are listed in Table 2.

[0047]

[Table 2]

As will be appreciated by those in the art, some ambiguity is introduced when determining degenerate codons representative of all possible codons encoding each amino acid. For example, a degenerate codon for serine (WSN) can encode arginine (AGR) in some circumstances, and a degenerate codon for arginine (MGN) can encode serine (AGY) in some circumstances. You can A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by a degenerate sequence can encode a variant amino acid sequence, but those of skill in the art can refer to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6 as such. Different mutant sequences can be easily identified. Variant sequences can be easily tested for functionality as described herein.

Also, as one of ordinary skill in the art will appreciate, different species can exhibit “preferred codon usage”. See generally the following references: Grantham et al., Nucl. Acids Re.
s. 8: 1893-912, 1980; Haas et al., Curr. Biol. 6: 315-24, 1996; Wain-Ho.
bson et al., Gene 13: 355-64, 1981; Grosjean and Fiers, Gene 18: 199-209.
, 1982; Holm, Nucl. Acids Res. 14: 3075-87, 1986; Ikemura, J. Mol.
Biol. 158: 573-97, 1982. As used in the present invention, the term "preferred codon usage" or "preferred codon" is most often used in certain cells, thus representing one or only a few of the possible codons that encode each amino acid. Is a term in the art that refers to protein translation codons that are commonly used (see Table 2).

For example, the amino acid threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells, ACC is the most commonly used codon; other species, For example, in insect cells, yeast, viruses or bacteria, different Thr codons can be predominant. Preferential codons of a particular species can be introduced into the polynucleotides of the invention by various techniques known in the art. Introduction of preferential codons into recombinant DNA can enhance protein production by, for example, streamlining translation of the protein within a particular cell type or species.

Accordingly, the degenerate codon sequences disclosed in SEQ ID NO: 9 or SEQ ID NO: 10 are of the polynucleotides commonly used in the art and disclosed herein in various cell types and species. Serves as a template for optimizing expression. Sequences containing preferential codons can be tested for expression in various species, optimized for expression, and tested for functionality, as disclosed herein.

As mentioned above, the isolated polynucleotides of the present invention include DNA and RNA. Methods of making DNA and RNA are well known in the art. Generally, RNA is isolated from cells or tissues that produce large amounts of RNA. Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Ac
ad. Sci. USA 77: 5201, 1980). Total RNA can be produced by guanidinium isothiocyanate extraction and subsequent centrifugation of a CsCl gradient (Chirgwin et al.,
Biochemistry 18: 52-94, 1979).

Poly (A) ⁺ RNA was prepared from total RNA by the method of Aviv and Leder (Proc. Natl. Acad. Sc
i. USA 69: 1408-12, 1972). Complementary DNA (cDNA) is poly (
A) ⁺ RNA is produced by a known method. Alternatively, genomic DNA can be isolated. The polynucleotide encoding the zamp3 or zamp4 polypeptide is then identified and isolated, eg, by hybridization or PCR.

The polynucleotide sequences of zamp3 or zamp4 disclosed herein also include zamp3
It can be used as a probe or primer for cloning the 5'non-coding region of the 3 or zamp4 gene. Promoter elements from the zamp3 or zamp4 genes can be used to direct tissue-specific expression of heterologous genes, eg, in transgenic animals or in patients treated with gene therapy.
Also, cloning of the 5'flanking sequence facilitates production of the zamp3 or zamp4 protein by "gene activation" as disclosed in US Pat. No. 5,641,670.

Briefly, an endogenous zamp3 or zamp4 in a cell is introduced by introducing into the zamp3 or zamp4 locus a DNA construct comprising at least targeting sequences, regulatory sequences, exons and unpaired splice donor sites. Change gene expression. The targeting sequence is the 5'non-coding sequence of zamp3 or zamp4, allowing homologous recombination of the construct with the endogenous zamp3 or zamp4 locus, which allows the sequence within the construct to interact with the endogenous zamp3 or zamp4 coding sequence. It becomes possible to chain. In this way, endogenous zamp3 or zam
The p4 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.

The zamp3 and zamp4 polynucleotide probes can be RNA or DNA. The DNA can be cDNA or genomic DNA. Polynucleotide probes are single- or double-stranded DNA or RNA, especially synthetic oligonucleotides, but which can be generated from cloned cDNA or genomic species, generally at least 16 nucleotides, and more often 17 nucleotides. -25 or more nucleotides, sometimes 40-60 nucleotides, and in some cases za
It may comprise a substantial portion, domain or even the entire mp3 or zamp4 gene or cDNA. Typical probes and primers are nucleotides 1-324, 10-198, 10-189, 76-198, 76-189, 100-198, 100-189 of SEQ ID NO: 5.
, 1-296, 1-132, 1-123, 34-123, and 34-132 of SEQ ID NO: 5.

Probes and primers are generally synthetic oligonucleotides, but can be generated from cloned cDNA or genomic sequences or their complements. Analytical probes are generally at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used. PCR primers are at least 5 nucleotides in length, preferably 15 or more nucleotides in length,
More preferably, it is 20 to 30 nucleotides. Short polynucleotides can be used when targeting small regions of a gene for analysis. To analyze the entire gene, the polynucleotide probe can comprise all exons or more.

The probe may be labeled to give a detectable signal, wherein labeling is according to techniques well known in the art, eg enzymes, biotin, radionuclides, fluorophores, chemiluminescence. The body, promising particles and others can be used and they are commercially available from a number of sources (eg Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL). .

The preferred regions used to construct the probe are regions having homology to other thymopoietins and emerins, as described herein, ankyrin-like regions, calcime binding protein-like regions, signal sequences, and Including others.
Techniques for developing polynucleotide probes and hybridization techniques are known in the art, see for example: Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley and
Sons, Inc., NY, 1991.

The zamp3 and zamp4 polypeptides can be used in a diagnostic system to detect the presence of zamp3 or zamp4, respectively. The information derived from such detection methods will provide insights into the implications of zamp3 and zamp4 polypeptides in various diseases and will serve as a diagnostic tool for diseases in which alterations in zamp3 or zamp4 levels are significant. Altered levels of zamp3 or zamp4 polypeptides may indicate microbial infection and inflammation. In a basic assay, single-stranded probe molecules were isolated from biological samples by RN
Incubate A with conditions of temperature and ionic strength that promote base pairing between the probe and the target zamp3 or zamp4 RNA species. After separating the unbound probe from the hybridized molecules, the amount of hybrid is detected.

Well-established hybridization methods for RNA detection include Northern analysis and dot / slot blot hybridization (see, eg: Ausubel, supra, and Wu et al. (Eds.), “Analysis”). of Gene E
xpression at the RNA Level ″, Methods in Gene Biotechnology, pp.
225-239 (CRC Press, Inc., 1997). The nucleic acid probe can be detectably labeled with a radioisotope, eg, ³² P or ³⁵ S.

Alternatively, zamp3 or zamp4 RNA can be detected by non-radiative hybridization methods (see, eg, the following references: Isaac (editor), Proto).
cols for Nucleic Acid Analysis by Nonradioactive Probes, Humana
Press, Inc., 1993). Non-radioactive detection is typically accomplished by chromogenic or enzymatic conversion of chemiluminescent substrates. Exemplary non-radioactive molecules include biotin, fluorescein, and digoxigenin.

The zamp3 or zamp4 oligonucleotide probe is also effective in in vivo diagnosis. As an example, an ¹⁸ F-labeled oligonucleotide can be administered to a subject and visualized by positron emission tomography (Tavitian et al., Nature Me.
dicine 4: 467, 1998).

Many diagnostic procedures use the polymerase chain reaction (PCR) to increase the sensitivity of detection methods.
) Is used. Standard techniques for performing PCR are well known (in general, see: Mathew (editor), Protocols in Human Molecular Genetic.
s (Humana Press, Inc., 1991), White (editor), PCR Protocols: Current
Methods and Applications (Humana Press, Inc., 1993), Cotter (editor)
, Molecular Diagnosis of Cancer (Humana Press, Inc., 1996), Hanause
k and Walaszek (editor), Tumor Maker Protocols (Humana Press, Inc., 1
998), Lo (editor), Clinical Applications of PCR (Humana Press, Inc.
, 1998), and Meltzer (editor), PCR in Bioanalysis (Humana Press, In
c., 1998). As described herein, PCR primers can be designed to amplify sequences that encode homology, eg, specific zamp3 or zamp4 domains or regions that have a cysteine motif.

One variant of the diagnostic assay is reverse transcriptase-PCR (RT-PCR). In the RT-PCR technique, RNA is isolated from a biological sample, reverse transcribed into cDNA, and the cDNA zamp3 or
Incubate with zamp4 primer (see for example: Wu
Others (editor), "Rapid Isolation of Specific cDNAs or Genes by PCR
″, Methods in Gene Biotechnology, CRC Press, Inc., pp. 15-28, 199
7). PCR is then performed and the products analyzed by standard techniques.

By way of illustration, RNA is isolated from a biological sample, for example, by the guanidinium-thiocyanate cell lysis method described above. Alternatively, solid phase techniques are used to isolate mRNA from cell lysates. Isolated RNs using random oligonucleotides, short homopolymers of dT, or zamp3 or zamp4 antisense oligomers
A can prime the reverse transcription reaction. The use of oligo-dT primers has the advantage that various mRNA nucleotide sequences can be amplified and provide control target sequences. Typically at least 5 bases long, 2
The zamp3 or zamp4 sequences are amplified by polymerase chain reaction using two flanking oligonucleotide primers.

A variety of approaches can be used to detect PCR amplification products. For example, PCR products can be fractionated by gel electrophoresis and visualized by ethidium bromide staining. Alternatively, the fractionated PCR product can be transferred to a membrane, hybridized with a detectably labeled zamp3 or zamp4 probe, and examined by autoradiography. Additional selective approaches include the use of digoxigenin labeled deoxyribonucleic acid triphosphatase to obtain chemiluminescent detection, and the C-TRAK colorimetric assay.

Another approach is real-time quantitative PCR (Perkin-Elmer Cetus, Norwalk, CT). The fluorogenic probe consists of an oligonucleotide with both a reporter and a quencher dye attached, which anneals specifically between the forward and reverse primers. Using the 5'endonuclease activity of Taq DNA polymerase, the reporter dye is separated from the quencher dye, generating a sequence-specific signal, which increases as amplification increases.
Fluorescence intensity is continuously monitored and quantified during the PCR reaction.

Another approach to detecting zamp3 or zamp4 expression is the cycling probe technology (CPT), in which a single-stranded DNA target binds to excess DNA-RNA DNA chimeric probe to form a complex, RNA probes are cleaved with RNase H to detect the presence of cleaved chimeric probes (see, eg, Begg et al., J. Clin. Microbiol. 34: 2985, 1996 and Bekkaoui et al., Biotechniques.
20: 240, 1996). Other methods of detecting zamp3 or zamp4 sequences can utilize approaches such as: Nucleic acid sequence-based amplification (NASBA), cross-hybridization co-amplification (CATCH), and ligase chain reaction (LCR) ( See, for example: Marshall et al., US Pat. No. 5,686,272 (1997).
, Dyer et al., J. Virol. Methods 60: 161, 1996; Ehricht et al., Eur. J. Bioch.
em. 243: 358, 1997 and Chadwick et al., J. Virol. Methods 70:59, 1998).
. Other standard methods are known in the art.

The zamp3 or zamp4 probes and primers can also be used to detect and localize the expression of the zamp3 or zamp4 genes in tissue samples.
Such in situ hybridization methods are well known in the art (see, for example: Choo (editor), In Situ Hybridizat.
ion Products, Humana Press, Inc., 1994; Wu et al. (Editor), "Analysis of
Cellular DNA or Abundance of mRNA Radioactive In Situ Hybridiza
(RISH) ″, Methods in Gene Biotechnology, CRC Press, Inc., pp.
259-278, 1997 and Wu et al. (Editor), "Localization of DNA or Abundan.
ce of mRNA by Fluorescence In Situ Hybridization (RISH) ″, Metho
ds in Gene Biotechnology, CRC Press, Inc., pp. 279-289, 1997).

Various additional diagnostic approaches are well known in the art (eg,
See references: Mathew (editor), Protocols in Human Molecular.
Genetics, Humana Press, Inc., 1991; Coleman and Tsongalis, Molecular
Diagnostics, Humana Press, Inc., 1996 and Elles, Molecular Diagnosi
s of Genetic Disease, Humana Press, Inc., 1996).

The invention further provides counterpart polypeptides and polynucleotides (orthologs) from other species. These species include, but are not limited to, mammals, birds, amphibians, reptiles, fish, insects and other vertebrates and invertebrates. Of particular interest are zamp3 and za polypeptides from other mammalian species, such as human, rat, pig, sheep, cow, dog, cat, horse, and other primate polypeptides. Species homologues of human proteins can be cloned using the information and compositions provided by the invention in combination with conventional cloning techniques. For example, mRNA can be cloned using mRNA from tissue and cell types that express the protein.

Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. The library can then be prepared from positive tissue or cell line mRNA.
The cDNAs encoding zamp3 and zamp4 are then isolated by various methods, such as by probing with full or partial human cDNAs or with one or more sets of degenerate probes based on the disclosed sequences. Can be separated.

The cDNA can also be cloned by the polymerase chain reaction using primers designed from the sequences disclosed herein or by PCR (Mullis, US Pat. No. 4,683,202). In an additional method, the cDNA library can be used to transform or transfect host cells, and the expression of the cDNA of interest can be detected with antibodies to the zamp3 and zamp4 polypeptides. Similar techniques can also be applied to the isolation of genomic clones.

Polypeptides of another species of zamp3 and zamp4 may have therapeutic importance. It has been demonstrated that in some cases non-natural proteins, ie proteins from different species, may be more potent than native proteins. For example, salmon calcitonin has been shown to be significantly more effective in arresting bone resorption than human forms of calcitonin. Several hypotheses exist as to why salmon calcitonin is more effective than human calcitonin in treating osteoporosis.

These hypotheses include: 1) salmon calcitonin is more resistant to degradation; 2) salmon calcitonin has a lower metabolic clearance rate (MCR); and 3) salmon calcitonin It has a slightly different conformation resulting in higher affinity for bone receptor sites. Other examples are found in the β-endorphin family (Ho et al., Int. J. Pept.
ide Protein Res. 29: 521-24, 1987). Studies have demonstrated that the peripheral opioid activity of camel, horse, turkey and ostrich β-endorphins is greater than that of human β-endorphin when the ileum of isolated guinea pigs is electrically stimulated and contractions are measured. It was

Vas deferens from rats, mice and rabbits were also assayed. Camel and horse β-endorphins showed the highest relative potency in the rat vas deferens model. Rat synthesized relaxin was as active as human and porcine relaxin in the mouse pubic bone binding assay (Bullesbach and
Schwabe, Eur. J. Biochem. 241: 533-7, 1996). Thus, the murine zcys4 molecule of the invention may have greater potency than human endogenous molecules in human cells, tissues and recipients.

As will be appreciated by one of skill in the art, the sequences disclosed in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 19 represent single alleles of the human zamp3 gene, SEQ ID NO: 5 and SEQ ID NO: 5.
The sequence of 6 represents a single allele of the human zamp4 gene, and allelic variants and alternate splicing are expected to occur. Allelic variants can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Sequence number 1
, SEQ ID NO: 19, and allelic variants of the DNA sequences shown in SEQ ID NO: 5, including those containing silent mutants and those in which the mutation alters the amino acid sequence, are within the scope of the invention.

In a preferred embodiment of the invention, the isolated nucleic acid molecule is, under stringent conditions, a nucleic acid molecule having a portion of the nucleotide sequence of SEQ ID NO: 1 or 5 or a nucleotide sequence complementary to those sequences. Can be hybridized to a nucleic acid molecule having Generally, stringent conditions are about 5 ° C above the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH.
Selected to be low. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA, can hybridize if the nucleotide sequences have some degree of complementarity. The hybrid can tolerate mismatched base pairs in the double helix, but the stability of the hybrid is affected by the degree of mismatch. The Tm of mismatched hybrids is reduced by 1 ° C for every 1-1.5% base pair mismatch. Varying the stringency of the hybridization conditions can control the degree of mismatch present in the hybrid. The degree of stringency increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases.

Stringent hybridization conditions are approximately above the Tm of the hybrid.
Includes hybridization buffer with 5-25 ° C lower temperature and Na ^{+ up} to 1M. A higher degree of stringency at lower temperatures can be achieved by the addition of formamide. Formamide lowers the hybrid's Tm by about 1 ° C. for 1% formamide in buffer. Generally, such stringent conditions include temperatures of 20-70 ° C. and hybridization buffer containing SSC up to 6 × and 0-50% formamide. The higher degree of stringency is that SSCs up to 4x and 0-50 at temperatures of 40-70 ° C.
It can be achieved using a hybridization buffer containing% formamide.

Highly stringent conditions are typically at temperatures of 42-70 ° C. and 1 ×
Up to SSC and 0-50% formamide in the hybridization buffer. Different degrees of stringency can be used during hybridization and washing to achieve maximal specific binding to the target sequence. Post-hybridization washes are typically performed at increasing degrees of stringency to remove unhybridized polynucleotide probes from the hybridized complex.

The above conditions are meant to serve as a guide, and one of skill in the art can adapt these conditions for use with a particular polypeptide hybrid.
The Tm of a particular target sequence is the temperature (under defined conditions) at which 50% of the target sequence hybridizes to a perfectly matched probe sequence. Conditions that affect the Tm include the size and base pair content of the polynucleotide probe, the ionic strength of the hybridization solution, and the presence of destabilizing factors in the hybridization solution.

A number of equations for calculating Tm are known in the art and are specific for varying lengths of DNA, RNA and DNA-RNA hybrid and polynucleotide probe sequences (see, eg, See also: Molecular Cloni
ng: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Press 1989); A
usubel et al. (Editor), Current Protocols in Molecular Biology (John Wile
y & Sons, Inc. 1987); Berger and Kimmel (editor), Guide to Molecula
r Cloning Techniques (Academic Press, Inc. 1987); and Wetmur, Cri.
t. Rev. Biochem. Mol. Biol. 26: 227 (1990)).

Sequence analysis software, such as OLIGO 6.0 (LSR; Long Lake, Minnesota) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, Calif.), As well as sites on the Internet, analyze predetermined sequences and provide user-defined criteria. Is an available tool for calculating Tm based on. Further, such a program can analyze a predetermined sequence under defined conditions to identify an appropriate probe sequence. Typically, hybridization of longer polynucleotide sequences of> 50 base pairs is about 20 less than the calculated Tm.
Perform at temperatures as low as -25 ° C. For smaller probes <50 base pairs, hybridization is typically carried out at a temperature lower by Tm or 5-10 ° C. This can maximize the hybridization rate for DNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence affects the rate and stability of hybridization. Smaller probe sequences <50 base pairs rapidly reach equilibrium with complementary sequences but may form less stable hybrids. Incubation times anywhere from minutes to hours can be used to form hybrids. Longer probe sequences equilibrate more slowly, but form more stable complexes even at lower temperatures. The incubation can be allowed to proceed overnight or longer. In general, the incubation is carried out for a period equal to 3 times the calculated Cot time. Cot time is
The time it takes for a polynucleotide sequence to reassociate and can be calculated for a particular sequence by methods known in the art.

The base pair composition of the polynucleotide sequence will affect the thermal stability of the hybrid complex and thereby the hybridization temperature and ionic strength of the hybridization buffer. The AT pair is less stable than the GC pair in aqueous solutions containing sodium chloride. Therefore, the higher the G-C content,
The hybrid will be more stable. The even distribution of G and C residues within the sequence also positively contributes to hybrid stability. In addition, the base pair composition can be manipulated to change the Tm of a given sequence. For example, 5-methyldeoxycytidine can be replaced with deoxycytidine, 5-bromodeoxyuridine with thymidine to increase Tm, whereas 7-deaz-2'-deoxyguanosine can be replaced with guanosine. And the dependence on Tm can be reduced.

[0088]   The ionic concentration of the hybridization buffer also determines the stability of the hybrid.
Affect. Hybridization buffers generally include blocking agents,
For example, Denhardt's solution (Sigma Chemical Co., St. Louis, Missouri),
Denatured salmon sperm DNA, tRNA, milk powder (BLOTTO), heparin or SDS, and Na ⁺ Source, eg, SSC (1 x SSC: 0.15M sodium chloride, 15 mM sodium citrate)
Or SSPE (1 x SSPE: 1.8M NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 7.7).
Contains.

The stability of the hybrid is increased by reducing the ionic concentration of the buffer. Hybridization buffers typically contain 10 mM to ¹ M Na ⁺ . A destabilizing or denaturing agent such as formamide, a tetraalkylammonium salt, a guanidinium cation or a thiocyanate cation is added to the hybridization solution to alter the Tm of the hybrid. Typically, formamide can be used at a concentration of up to 50% to carry out the incubation at a more convenient, lower temperature. Formamide also acts to reduce non-specific background when using RNA probes.

By way of illustration, 50% formamide at 42 ° C., 5 × SSC (1 × SSC: 0.15 M sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH
7.6), 5 x Denhardt's solution (100 x Denhardt's solution: 2% (w / v) Ficoll 40
In a solution containing 0, 2% (w / v) polyvinylpyrrolidone, and 2% (w / v) bovine serum albumin), 10% dextran sulfate, and 20 μg / ml denaturing, sheared salmon sperm DNA. And a polynucleotide encoding a mutant zamp3 or zamp4 polypeptide can be hybridized overnight with a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 or 5 (or their complements).

Those skilled in the art can devise modifications of these hybridization conditions. For example, the hybridization mixture can be incubated in a formamide-free solution at higher or lower temperatures, eg, about 65 ° C. Moreover, premix hybridization solutions are available (eg, EXPRESSHYB hybridization solutions, CLONTECH Laborato
ries, Inc.) and hybridization can be performed according to the manufacturer's instructions.

After hybridization, the nucleic acid molecules are washed under stringent conditions or under highly stringent conditions to remove non-hybridized nucleic acid molecules. Typical stringent wash conditions include washing at 55-65 ° C in a solution of 0.5x-2x SSC and 0.1% sodium dodecyl sulfate (SDS).
That is, a nucleic acid molecule encoding a variant zamp3 or zamp4 polypeptide hybridizes under stringent wash conditions with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or 5 (or their complement), where wash stringent Equals 0.5 × -2 × SSC and 0.1% SDS at 50-65 ° C. and includes 0.5 × SSC and 0.1% SDS at 55 ° C. or 2 × SSC and 0.1% SDS at 65 ° C. One skilled in the art can easily devise equivalent conditions, for example by replacing SSC in the wash solution with SSPE.

Typical highly stringent wash conditions are 0.1 × to 0.2 at 50-65 ° C.
X Washing in a solution of SSC and 0.1% sodium dodecyl sulfate (SDS). In other words, the polynucleotide encoding the mutant zamp3 or zamp4 polypeptide hybridizes with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or 5 (or their complement) under highly stringent wash conditions,
The wash stringent here is 0.1 x ~ 0.2 x SSC and 0.1% at 50 ~ 65 ° C.
Equivalent to SDS, 0.1 x SSC and 0.1% SDS at 50 ° C, or 0.2 at 65 ° C
Includes SSC and 0.1% SDS.

The present invention also includes zamp3 or zamp4 variant nucleic acid molecules that can be identified using two criteria: an encoded polypeptide having the amino acid sequence of SEQ ID NO: 2 or 6, as described above. Of similarity between, and hybridization assay. Such zamp3 or zamp4 variants include the following nucleic acid molecules: (
1) a nucleic acid molecule that hybridizes under stringent wash conditions with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or 5 (or their complements),
Here, wash stringency is 0.5 × to 2 × SSC, 0.1% SDS at 50 to 65 ° C.
And (2) at least 80 for the amino acid sequence of SEQ ID NO: 2 or 6
%, At least 90%, at least 95%, or a nucleic acid molecule encoding a polypeptide having greater than 95% sequence identity.

Alternatively, a zamp3 or zamp4 variant is (1) a nucleic acid that hybridizes under highly stringent wash conditions with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or 5 (or their complement). Molecules, where wash stringency is 0.1 × to 0.2 × SSC at 50-65 ° C., equal to 0.1% SDS, and (2) at least 80%, at least 90% relative to the amino acid sequence of SEQ ID NO: 2 or 6. , A nucleic acid molecule encoding a polypeptide having at least 95% or greater than 95% sequence identity.

The present invention also provides an isolated zamp3 or zamp4 polypeptide that is substantially similar to the polypeptides of SEQ ID NO: 2 or SEQ ID NO: 6 and their species homologs / orthologs. The term "substantially similar" is used herein to mean 50% relative to the sequence set forth in SEQ ID NO: 2 or 6 or their orthologs or paralogs,
Preferably used to represent polypeptides having 60%, more preferably at least 80% sequence identity. Such polypeptides have most preferably at least 90%, most preferably at least 95% or greater sequence identity to the sequences shown in SEQ ID NO: 2 or 6 or their orthologs or paralogs.

The percentage sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992. Briefly, 10 gap opening penalties, 1 gap extension penalties, and Henikoff and Henikoff (supra) “blosum” as shown in Table 3 (amino acids are indicated by the standard one-letter code).
A 62 "scoring matrix is used to align the two amino acid sequences to optimize the alignment score. Percentage identity is then calculated as follows:

[0098]

[Equation 1]

[0099]

[Table 3]

Sequence identity of polynucleotide molecules is determined in a similar manner using the ratios disclosed above. As one of ordinary skill in the art will recognize, there are numerous algorithms that are effective for aligning two amino acid sequences. Pearson and Lipman's “FASTA” similarity search algorithm uses the amino acid sequence disclosed herein and the putative variant zamp3 or zam.
A suitable protein alignment method that tests the level of identity shared by the amino acid sequences of p4. The FASTA algorithm is described in: Pearson and Li.
pman, Proc. Natl. Acad. Sci. USA 85: 2444, 1988, and Pearson, Met.
h. Enzymol. 183: 63, 1990.

Briefly, first, without considering conservative amino acid substitutions, insertions, or deletions, the sequence in question (eg, SEQ ID NO: 2 or 6) and the highest density of identities (kt
FASTA characterizes sequence similarity by identifying regions shared by test sequences with up variables (if 1) or pairs of identities (if ktup = 2). The 10 regions with the highest densities of identity were then rescored by comparing the similarities of all paired amino acids using an amino acid substitution matrix, and included only those residues that contributed to the highest score. First, "trim" the ends of the region.

If some regions have a score greater than the “cutoff” value (calculated by a previously determined formula based on the length of the sequence and the ktup value), then the first region trimmed is examined. , To determine if the regions can be joined to form an approximate alignment with gaps. Finally, a modification of the Needleman-Wunsch-Sellers algorithm that allows the insertion or deletion of amino acid sequences (Needleman and Wun
sch, J. Mol. Biol. 48: 444, 1970; Sellers, SIAM J. Appl. Math. 26.
: 787, 1974) to align the highest scoring regions of two amino acid sequences.

Examples of parameters for FASTA analysis are as follows: ktup = 1, gap opening penalty = 10, gap extension penalty = 1, and substitution matrix = BLOSUM62. Pearson, Meth. Enzymol. 183: 63, 1990.
Scoring matrix file ("SMATR
IX ") can be modified to introduce these parameters into the FASTA program. Also, to determine the sequence identity of nucleic acid molecules using the ratios disclosed above,
FASTA can be used. Ktup value of 1 for comparing nucleotide sequences
It can range from ~ 6, preferably 4-6.

The present invention includes nucleic acid molecules that encode a polypeptide having one or more "conservative amino acid substitutions" as compared to the amino acid sequence of SEQ ID NO: 2 or 6. Conservative amino acid substitutions can be based on the chemical properties of amino acids. That is, a variant containing one or more amino acid substitutions of SEQ ID NO: 2 or 6 can be obtained, wherein the alkyl amino acid is replaced with an alkyl amino acid in the zamp3 or zamp4 amino acid sequence and the aromatic amino acid is Substituted for aromatic amino acids in the zamp3 or zamp4 amino acid sequence, sulfur containing amino acids are zamp3 or zamp4
The amino acid sequence is replaced by a sulfur-containing amino acid, the hydroxy-containing amino acid is replaced by a hydroxy-containing amino acid in the zamp3 or zamp4 amino acid sequence, and the acidic amino acid is replaced by an acidic amino acid in the zamp3 or zamp4 amino acid sequence. , Basic amino acids are substituted with basic amino acids in the zamp3 or zamp4 amino acid sequence, or dibasic monocarboxylic acid amino acids are zamp3 or zamp4
Substituted with a dibasic monocarboxylic acid amino acid in the amino acid sequence.

For example, between common amino acids, “conservative amino acid substitutions” are exemplified by substitutions between amino acids within each of the following groups: (1) glycine, alanine, valine,
Leucine and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.

The BLOSUM62 table is an amino acid substitution matrix derived from approximately 2,000 local multiple alignments of segments of protein sequences, representing highly conserved regions of more than 500 groups of proteins of interest (Henikoff and Henikoff. , Proc. N
atl'l. Acad. Sci. USA, 89: 10915, 1992). Therefore, in order to define conservative amino acid substitutions that can be introduced into the amino acid sequences of the present invention, BLOSUM
62 replacement frequencies can be used.

Although amino acid substitutions can be designed based on their chemical properties only (as described above), the term "conservative amino acid substitutions" is preferably BLOSUM6 greater than -1.
Means the substitution represented by a binary value. For example, an amino acid substitution is conservative if the substitution is characterized by 0, 1, 2, or 3. According to this system, preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (eg, 1, 2 or 3), while more preferred conservative amino acid substitutions are BLOSUM62 values of at least 2 (eg, 2 or 3). Characterized by:

Conservative amino acid changes in the zamp3 or zamp4 genes can be introduced by substituting the nucleotides set forth in SEQ ID NO: 1 or 5 with nucleotides. Such "conservative amino acid" variants can be obtained, for example, by oligonucleotide-directed mutagenesis, linker-scan mutagenesis, mutagenesis using the polymerase chain reaction, and others (see below). References: Ausubel (1995) pp. 8-10-8-22; and McPherson (editor), Direc.
ted Mutagenesis: A Practical Approach (IRL Press 1991)). The ability of such variants to promote energy balance, as well as other properties of wild-type proteins, can be determined using standard methods, eg, the assays described herein. Alternatively, due to its ability to specifically bind to anti-zamp3 or zamp4 antibodies,
Mutant zamp3 or zamp4 polypeptides can be identified.

Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes preferably have minor attributes, ie have the following naturalities: conservative amino acid substitutions and other substitutions that do not substantially affect the folding or activity of the protein or polypeptide; minor deletions, typical Typically from 1 to about 30 amino acids deleted;
And amino- and carboxyl-terminal extensions, such as amino-terminal methionine residues, small linker peptides of up to about 20-25 residues, or small extensions that facilitate purification (affinity labeling),

For example, poly-histidine tract, protein A (Nilsson et al., EMBO J. 4
: 1075, 1985; Nilsson et al., Methods Enzymol. 198: 3, 1991), glutathione.
S-transferase (Smith and Johnson, Gene 67:31, 1988), maltose binding protein (Kellerman and Ferenci, Methods Enzymol. 90: 459-
463, 1982; Guan et al., Gene 67: 21-30, 1987), thioredoxin, ubiquitin, cellulose binding protein, T7 polymerase, or other antigenic epitopes or binding domains. See generally the following references: Ford et al., Protein E.
xpression and Purification 2: 95-107, 1991.

DNAs encoding affinity tags are available from commercial suppliers (eg Pharm.
acia Biotech., Piscataway, NJ; New England Biolabs
, Beverly, Massachusetts). A polypeptide comprising an affinity tag is zamp
It may further comprise a proteolytic cleavage site between the 3 or zamp4 polypeptide and the affinity label. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites. The proteins of the invention can also include non-naturally occurring amino acid residues.

Non-naturally occurring amino acids include, without limitation, the following: trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methyl-glycine, allo-threonine, methylthreonine, hydroxyethylcystein, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4 -Azaphenyl-alanine, 4-fluorophenylalanine, 4-hydroxyproline,
6-N-methyllysine, 2-aminoisobutyric acid, isovaline and α-methylserine.

Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be used that uses chemically aminoacylated suppressor tRNA to suppress nonsense mutations. Methods of synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of the plasmid containing the nonsense mutation is performed in a cell-free system comprising E. coli S30 extract and commercially available enzymes and other reagents.

The protein is purified by chromatography. See, for example: Robertson et al., J. Am. Chem. Soc. 113: 2722, 1991; Ellman et al., Me.
thods Enzymol. 202: 301, 1991; Chung et al., Science 259: 806-09, 1993;
And Chung et al., Proc. Natl. Acad. Sci. USA 90: 10145-49, 1993. No. 2
In the method described above, xenopse oocytes (Xe) were isolated by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNA.
translation in nopus oocytes (Turcatti et al., J. Biol. Chem. 271: 1
9991-98, 1996).

In the third method, in the absence of the naturally occurring amino acid to be replaced (eg, phenylalanine) and the desired non-naturally occurring amino acid (s) (
For example, E. coli in the presence of 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine or 4-fluorophenylalanine.
i) Culture the cells. The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See Koide et al., Biochem. 33: 7470-76, 1994. in
In vitro chemical modifications can convert naturally occurring amino acid residues to non-naturally occurring species. Combining chemical modification with site-directed mutagenesis,
The range of substitutions can be further extended (Wynn and Richards, Protein Sci
.2: 395-403, 1993).

A limited number of non-conservative amino acids, amino acids not encoded by the genetic code,
Non-naturally occurring amino acids and unnatural amino acids can be substituted for amino acid residues in the zamp3 or zamp4 polypeptide. "Unnatural amino acids" have been modified after protein synthesis and / or have a chemical structure in their one or more side chains that differs from that of standard amino acids. Unnatural amino acids are either chemically synthesized or are preferably commercially available and include pipecolic acid, thiazolinecarboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline. Include.

Essential amino acids in the zamp3 or zamp4 polypeptides of the invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science
244: 1081-5, 1989). In the latter technique, a single alanine mutation is introduced at every residue in the molecule and the resulting mutant molecule is tested for biological activity (eg, antimicrobial activity) to determine the activity of the molecule. Identifies amino acid residues that are critical to. See also: Hilton et al., J. Biol.
Chem. 271: 4699-708, 1996.

The site of ligand-receptor or other biological interaction can also be determined by physical analysis of the structure, such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling. Can be determined by a combination of various techniques and mutation of amino acids at putative contact sites. For example, see the following references: de Vos et al., Science 255: 306-12, 1992; Smith et al., J. Mol. Bio.
224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992. Identification of essential amino acids can also be inferred from homology with related β-defensins.

Multiple amino acid substitutions can be made and tested using known mutagenesis and screening methods, such as those described in the following references:
Reidhaar-Olson and Sauer, Science 241: 53-7, 1988 or Bowie and Sa.
Uer, Proc. Nat. Acad. Sci. USA 86: 2152-6, 1989. In short,
These authors randomized two or more positions in the polypeptide simultaneously, selected for functional polypeptides, and then sequenced the mutated polypeptides to generate a spectrum of acceptable substitutions at each position. A method of determining is disclosed. Other methods that can be used include the following: Phage display (
For example, Lowman et al., Biochem. 30: 10832-7, 1991; Ladner et al., US Patent No. 5,22.
No. 3,409; Huse, WIPO publication WO 92/06204) and region-directed mutagenesis (Derb
Yshire et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1988).

Also, variants of the disclosed zamp3 or zamp4 DNA and polypeptide sequences are
It can be generated by DNA shuffling as disclosed in: Stemmer, Nature 370: 389, 1994, Stemmer, Proc. Nat. Acad. Sc.
i. USA 91: 10747, 1994 and International Publication WO 97/20078. Briefly, parent D
The NA is randomly fragmented and then reassembled by PCR to generate randomly introduced point mutations to generate mutant DNA molecules by in vitro homologous recombination.

A family of parental DNA molecules, eg, allelic variants or DNA from different species
Molecules can be used to modify this technique by introducing additional variability into the process. Select or screen for the desired activity,
The mutagenesis and assay is then further repeated to rapidly "evolve" the sequence by selecting for the desired mutation while selecting for deleterious changes.

The above-described mutagenesis method can be combined with high throughput automated screening methods to detect the activity of cloned and mutated polypeptides in host cells. Mutated DNA molecules encoding active polypeptides (eg, antimicrobial activity) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in the polypeptide in question and can be applied to polypeptides of unknown structure.

Using the methods described above, one of skill in the art would be substantially homologous to residues 1-63 of SEQ ID NO: 2 or residues 1-43 of SEQ ID NO: 6 or allelic variants thereof. And various polypeptides can be identified and / or prepared that retain the antimicrobial properties of the wild-type protein. Such polypeptides may include affinity tags and additional amino acids from others. Such polypeptides can also include additional polypeptide segments, generally as described above.

The polypeptides of the present invention, including full-length proteins, active fragments thereof and fusion proteins, can be produced in genetically engineered host cells according to conventional techniques. However, some care must be taken in selecting host cells as a result of the antimicrobial activity of the molecules of the invention. For example, a zamp3 or zamp4mp4 polypeptide, fragment, fusion protein, antibody,
Since agonists or antagonists can kill host cells as part of their antimicrobial function, any cell culture-based system must be evaluated.

The zamp3 or zamp4mp4 polypeptides have a size small enough to allow their preparation by PCR or by other protein chemistry techniques that avoid potential cytotoxicity problems. Alternatively, the native or engineered precursor protein is inactive prior to post-translational cleavage to give the mature zamp3 or zamp4mp4 polypeptide,
This limits the cytotoxicity of the host cell prior to lysosomal packaging. See, for example: Lehrer et al., Cell 229-30, 1991. Thus, zamp3
Alternatively, the precursor protein for the zamp4mp4 polypeptide can be produced in microbial cell culture.

Suitable host cells are types of cells that can be transformed or transfected with exogenous DNA and can be grown in culture, and include bacterial, fungal, and cultured higher eukaryotic cells. Includes. Eukaryotic cells, especially cultured cells of multicellular microorganisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into various host cells are described in the following references: S
ambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Sprin
g Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausu
bel et al. (Editor), Current Protocols in Molecular Biology, John Wiley
and Sons, Inc., NY, 1987.

Generally, a DNA sequence encoding a zamp3 or zamp4 polypeptide is operably linked to other necessary genetic elements for its expression, which generally include transcription promoters and terminators within the expression vector. Vectors also usually contain one or more selectable markers and one or more origins of replication, but one of skill in the art will appreciate that the selectable markers in certain systems will be distinct. Exogenous DN provided on the vector and integrated into the genome of the host cell.
You can get a copy of A. Selection of promoters, terminators, selectable markers, vectors and other factors is a matter of routine design within the level of ordinary skill in the art. Many such factors are described in the literature and are available from commercial suppliers.

To direct the zamp3 or zamp4 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. Secretory signal sequence is zamp3 or zamp4
It can be that of a polypeptide, or can be derived from other secreted proteins (eg, t-PA), or can be de novo synthesized. The secretory signal sequence is joined in correct reading frame to the DNA sequence encoding the zamp3 or zamp4 polypeptide. Secretory signal sequences are commonly placed 5'to the DNA sequence encoding the polypeptide of interest, although some secretory signal sequences may be located anywhere within the DNA sequence of interest ( For example, Welch et al.
U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).

The present invention provides an expression vector comprising the following operably linked factors: a transcription promoter; a DNA segment encoding a zamp3 or zamp4 protein described herein; and a transcription terminator. The present invention also provides a DNA segment further encoding a secretory signal sequence operably linked to the protein, wherein the secretory signal sequence has amino acid residue 1 to amino acid residue 22 of SEQ ID NO: 2. It is a polypeptide.

[0130] Alternatively, secretory signal sequences contained within the polypeptides of the invention can be used to direct other polypeptides into the secretory pathway. The present invention provides such fusion polypeptides. Signal fusion polypeptides are known in the art and are present in It can be made by the method disclosed in the specification.

Preferably, the secretory signal sequence contained in the fusion polypeptide of the invention is amino terminally fused to the additional peptide to direct the additional peptide into the secretory pathway. Such constructs have numerous uses known in the art. For example, fusion constructs of these novel secretory signal sequences can direct the secretion of, for example, the active components of proteins that are not normally secreted, such as receptors. Such fusions can be used in vivo or in vitro to direct the peptide through the secretory pathway.

Cultured mammalian cells are also suitable hosts in the present invention. Extrinsic
Methods for introducing DNA into mammalian host cells include the following methods: calcium phosphate mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsa
ro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van.
der Eb, Virology 52: 456, 1973), electroporation (Neumann et al., EMBO J. 1: 841-5, 1982), DEAE-dextrin mediated transfection (Ausubel et al., Current Protocols in Molecular Biology, John Wiley).
and Sons, Inc., NY, 1987), liposome-mediated transfection (Hawley)
-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993), and viral vectors (Miller and Rosman, BioTechniques 7: 980-90, 198).
9; Wang and Finer, Nature Med. 2: 714-16, 1996).

The production of recombinant polypeptides in cultured mammalian cells is described, for example, in the following patent documents: Levinson et al., US Pat. No. 4,713,339; Hagen et al.
US Pat. No. 4,784,950; Palmiter et al., US Pat. No. 4,579,821; and Ringold.
U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells include: COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BH.
K570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J.
Gen. Virol. 36: 59-72, 1977) and Chinese hamster ovary (eg, CHO-K1; ATCC No. CRL 61) cell lines.

Additional suitable cell lines are known in the art, and are public depository institutions, such as the American Type Culture Collection.
Culture Collection) (Rockville, Maryland).
In general, strong transcription promoters, such as those from SV-40 or cytomegalovirus, are preferred. See, for example, U.S. Pat. No. 4,956,288. Another suitable promoter is the promoter from the metallothionein gene (US Pat.
9,821 and US Pat. No. 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cells that have had foreign DNA inserted. Such cells are commonly referred to as "transfectants." Cells that have been cultured in the presence of the selection factor and are able to transfer the gene of interest to their progeny are termed "stable transfectants". A preferred selectable marker is the gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or other.

[0136] A method called "amplification" in which a selection system can also be used to increase the expression level of the gene of interest. Amplification is performed by culturing the transfectants in the presence of low levels of the selection factor and then increasing the amount of the selection factor to select for cells that produce high levels of the product of the introduced gene. . A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.

Other drug resistance genes (eg hygromycin resistance, multidrug resistance, puromycin acetyltransferase) can also be used. Alternate markers that introduce an altered phenotype by such means as FACS sorting or magnetic bead separation techniques, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, class I MHC, placenta. Alkaline phosphatase can be used to sort transfected cells from untransfected cells.

Other higher eukaryotic cells can also be used as host cells, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells is reviewed in Sinkar et al., J. Biosci. (Bangalore) 11: 47-58, 1987. There is. Transformation of insect cells and production of foreign polypeptides therein is described by Guarino et al., US Pat. No. 5,162,222 and WIPO Publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV).

DNA encoding a zamp3 or zamp4 polypeptide was AcN-transfected by one of two methods.
Instead of the PV polyhedrin gene coding sequence. Insert into the baculovirus genome. First, zamp3 flanked by wild-type AcNPV and AcNPV sequences.
Or the traditional method of homologous DNA recombination between transfer vectors containing zamp4. Infecting appropriate insect cells, such as SF9 cells, with wild-type AcNPV, and AcNPV
Transfection with a transfer vector consisting of a polyhedrin gene promoter, a terminator, and a zamp3 or zamp4 polypeptide operably linked to flanking sequences.

See References: King, LA and Possee, RD The Bacul.
ovirus Expression System: A Laboratory Guide, London, Chapman & Ha
ll, O'Reilly, DR and others, Baculovirus Expression Vectors: A Laboratory
Manual, New York, Oxford University Press, 1994; and Richardson,
CD, Baculovirus Expression Protocols, Methods in Molecular
Biology, Totowa, NJ, Humana Press, 1995. Natural recombination in insect cells
It will result in a recombinant baculovirus containing zamp3 or zamp4 driven by the polyhedrin promoter. Recombinant virus strains are produced by methods commonly used in the art.

A second method of making recombinant baculovirus utilizes the transposon-based system described by Luckow (Luckow, VA, et al., J. Virol. 67: 4566-.
79, 1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD). This system is a transfer vector, pFastBac1 ^™ (Life
Technologies, where pFastBac1 ^™ is a DNA encoding a zamp3 or zamp4 polypeptide in the baculovirus genome maintained in E. coli as a large plasmid called the "bacmid". Contains a Tn7 transposon to drive

The pFastBac1 ^™ transfer vector utilizes the AcNPV polyhedrin promoter to drive expression of the gene of interest, in this case zamp3 or zamp4. However, pFastBac1 ^™ can be modified to a large extent. The polyhedrin promoter is removed, and a baculovirus-based protein promoter (also
(Known as the Pcor, p6.9 or MP promoter), where this promoter was previously expressed in baculovirus infection and has been shown to favor expression of secreted proteins. .

See references: Hill-Perkins, MS and Possee, RD, J.
Gen. Virol. 71: 971-6, 1990; Bonning, BC, et al., J. Gen. Virol.
75: 1551-6, 1994; and Chazenbalk, GD and Rapoport, B., J.
Biol. Chem. 270: 1543-9, 1995. Short or long versions of the basic protein promoter can be used in such transfer vector constructs. Moreover, transfer vectors can be constructed that replace the native zamp3 or zamp4 secretion signal sequence with a secretion signal sequence derived from an insect protein.

For example, a secretory signal sequence from ecdysteroid glucosyltransferase (EGT), bee melittin (Invitrogen, Carlsbad, Calif.), Or baculovirus gp67 (PharMingen, San Diego, Calif.) Was used in the construct to The zamp3 or zamp4 secretion signal sequence can be replaced. In addition, the transfer vector can include an in-frame fusion with an epitope tag at the C-terminus or N-terminus of the expressed zamp3 or zamp4 polypeptide, eg, DNA encoding the Glu-Glu epitope tag (Grussenme).
Yer, T., et al., Proc. Natl. Acad. Sci. 82: 7952-4, 1985).

The transfer vector containing zamp3 or zamp4 was transformed into E. coli using techniques known in the art, and the interrupted lacZ gene displaying recombinant baculovirus was expressed. To screen for bacmids containing Bacmid DNA containing the recombinant baculovirus genome is isolated according to conventional techniques and transfected into Spodoptera frugiperda cells, eg Sf9 cells. Recombinant virus expressing zamp3 or zamp4 is subsequently produced. Recombinant virus strains are generated by methods commonly used in the art.

A host cell, typically Spodoptera frugiperda (Spodoptera)
recombinant virus is used to infect cell lines derived from frugiperda). In general, see: Glick and Pasternak, Molecular.
Biotechnology: Principle and Applications of Recombinant DNA, ASM
Press, Washington, DC, 1994. Other suitable cell lines are Trichoderma
High FiveO ^™ cell line (Invitrogen) derived from Trichoderma ni (US Pat. No. 5,300,435). Using commercially available serum-free medium,
Grow and maintain cells.

Suitable media are Sf900 II ^™ (Life Technologies) or ESF for Sf9 cells.
921 ^™ (Expression System); and for Trichoderma ni (T.ni) cells Ex-cellO450 ^™ (JRH Bioscience, Lenexa, Kans.) Or Express Fi
veO ^TM (Life Technologies). Cells from a seeding density of approximately 2-5 x 10 ⁵ cells
Grows to a density of 1-2 × 10 ⁶ cells, at which time recombinant virus strains are added at a multiplicity of infection (MOI) of 0.1-10, more typically about 3. Cells infected with the recombinant virus typically produce recombinant zamp3 or zamp4 polypeptide 12-72 hours after infection and secrete it into the medium with varying efficiency. Cultures are usually harvested 48 hours post infection.

Cells are separated from the medium (supernatant) using centrifugation. Supernatants containing zamp3 or zamp4 polypeptides are filtered through a micropore filter, usually a 0.45 μm pore size filter. The procedure used is described in publicly available laboratory manuals (King, LA and Possee, RD, supra; O'Reilly, DR.
, Et al., Supra; Richardson, CD, supra). Subsequent purification of zamp3 or zamp4 polypeptide from the supernatant can be performed according to the methods described herein.

Fungal cells, including yeast cells, can also be used in the present invention. Yeast species of particular interest in this regard are Saccharomyces c
erevisiae), Pichia pastoris, and Pichia metanolica. Methods for transforming S. cerevisiae with exogenous DNA and producing recombinant polypeptides therefrom are described, for example, in the following patent documents: Kawasaki, US Pat.
599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008.
Welch et al., US Pat. No. 5,037,743; and Murry et al., US Pat. No. 4,845,075.

Transformed cells are selected by phenotype determined by a selectable marker, normal drug resistance or the ability to grow in the absence of a particular nutrient (eg, leucine). Saccharomyces cerevisiae
A preferred vector system for use in Kawasaki et al. (US Pat. No. 4,931,
No. 373), which allows the selection of transformed cells by growth in medium containing glucose.

Suitable promoters and terminators for use in yeast include glycolytic enzyme genes (see Kawasaki, US Pat. No. 4,599,311; Ingsman et al., US Pat. No. 4,615,974; and Bitter, US Pat. No. 4,977,092, see. ) And alcohol dehydrogenase genes. Also, U.S. Pat.
6; U.S. Pat. No. 5,063,154; U.S. Pat. No. 5,139,936 and U.S. Pat. No. 4,661,4
See No. 54.

Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis (
Kluyveromyces lactis), Kluyveromyces fragilis (Kluyveromyces)
frgilis, Ustilago maydis, Pichia pastoris, Pichia metanolica, Pichia guillermondii and Candida maltosa.
Transformation systems for other yeasts, including andida maltosa) are known in the art.

For example, Gleeson et al., J. Gen. Microbiol. 132: 3459-3465, 1986 and Cr.
See egg, US Pat. No. 4,882,279. Aspergillus cells can be utilized according to the method of McKnight et al., US Pat. No. 4,935,349. Acremonium chrysogenum is disclosed in Sumino et al., US Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed in Lambowitz, US Pat. No. 4,486,533.

As a host for the production of recombinant proteins, Pichia menorica
tanolica) can be used for WIPO publication WO 97/17450, WO 97/17451, WO
It is disclosed in 98/02536 and WO98 / 02565. Pichia methanolica (
The DNA molecules used in the transformation of P. metanolica are commonly produced as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
For the production of the protein in P. methanolica, the promoter and terminator in the plasmid are Pichia methanolica (P. menolica).
tanolica), for example, that of the alcohol utilization gene (AUG1 or AUG2) of P. metanolica.

Other useful promoters include the promoters of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. It is preferable to have the entire expression segment of the plasmid flanked at both ends by the host DNA sequence to facilitate integration of the DNA into the host chromosome. A preferred selectable marker for use in Pichia metanolica is P. metanoli.
Ca) ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which propagates ade2 host cells in the absence of adenine.

For large-scale industrial methods that seek to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) have been deleted. Host cells lacking the vacuolar protease genes (PEP4 and PRB1) are preferred for the production of secreted proteins. A plasmid containing the DNA encoding the polypeptide in question was ligated to P. methanolica.
3.) Use electroporation to facilitate introduction into cells. Use an exponentially decaying, pulsed electric field with a field strength of 2.5-4.5 kV / cm, preferably about 3.75 kV / cm, and a time constant (τ) of 1-40 msec, most preferably about 20 msec. Preferably, P. metanolica cells are transformed by electroporation.

Prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillus and other genera, are also useful in the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, eg, Sambrook et al., Supra). E. coli (
When expressing a zamp3 or zamp4 polypeptide in a bacterium such as E. coli), the polypeptide can be retained in the cytoplasm, typically as an insoluble particle, or periplasmic by a bacterial secretory sequence. Can be turned into the Mick space.

In the former case, the cells are lysed, the particles are harvested and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide is then refolded and by dilution of the denatured product, for example, by dialysis against a solution of urea and a combination of reduced and oxidized glutathione and subsequent dialysis against buffered saline. It can be dimerized. In the latter case, the polypeptide is disrupted (eg, by sonication or osmotic shock) to release the contents of the periplasmic space, thereby eliminating the need for denaturation and refolding. Can be recovered from the periplasmic space in a soluble, functional form.

Transformed or transfected host cells are cultured according to conventional procedures in a medium containing nutrients and other components necessary for the growth of the selected host cells.
A variety of suitable media, including defined media and complex media, are known in the art and generally include carbon sources, nitrogen sources, essential amino acids, vitamins and minerals. The medium can also contain components such as growth factors or serum. Generally, growth medium is selected for cells containing exogenously added DNA.

This selection is carried out, for example, by drug selection or lack of essential nutrients, which are either carried on the expression vector or supplemented by a selectable marker cotransfected into the host cell. R. The P. metanolica cells are cultured at a temperature of about 25 ° C to 35 ° C in a medium containing appropriate sources of carbon, nitrogen and micronutrients. The liquid medium is well aerated by conventional means, such as shaking a small flask or sparging the fermentor. A preferred medium for P. methanolica is YEPD (2% D-Glucose, 2% Bacto ^™ Peptone (Difco Laboratories, Detroit, MI), 1% Bacto ^™ Yeast Extract (Difco Laboratories). , 0.004% adenine and 0.006% L-leucine).

Expressed recombinants using fractionation and / or conventional purification methods and media
zamp3 or zamp4 polypeptide (or chimeric zamp3 or zamp4 polypeptide)
Can be purified. Ammonium sulfate precipitation and acid or chaotrope extraction can be used for fractionation of the sample. Typical purification steps can include hydroxyapatite, size exclusion chromatography, FPLC and reverse phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextran, agarose, cellulose, polyacrylamide, specialty silicas, and others. PEI, DEAE, QAE and Q derivatives are preferred.

Typical chromatographic media are media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl Butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose and others; or polyacrylic resins such as Amberchrom CG 71 (Toso Haas) and others. Suitable solid supports include glass beads, silica-based resins, cellulose resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins, and the like, under the conditions in which they are used. It is insoluble.

These supports can be modified with amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or reactive groups that allow the attachment of proteins by carbohydrate moieties. An example of chemical coupling is the activation of cyanogen bromide,
It includes carboxyl and amino derivatives for N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carbodiimide chemical coupling.

These and other solid media are well known and widely used in the art and are available from commercial suppliers. Methods for coupling receptor polypeptides to solid media are well known in the art.
The choice of a particular method is a matter of routine design and will be determined in part by the nature of the support chosen. For example, Affinity Chromatography: Principle & Method
See Pharmacia LKB Biotechnology, Uppsala, Switzerland, 1988.

The present invention provides a cultured cell into which the expression vector described herein has been introduced; the cultured cell expresses zamp3 or zamp4 encoded by the DNA segment. The invention also provides a method of producing a protein; the method comprises culturing cells into which an expression vector as described herein has been introduced, whereby the cultured cells are encoded by a DNA segment. zamp3 or za
expressing mp4 and recovering the expressed polypeptide.

The polypeptides of the present invention can be isolated by taking advantage of certain properties. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those consisting of polyhistidine labels. Briefly, gels are first loaded with divalent metal ions to form chelating agents (Sulkowsiki, Trends in Biochem. 3: 1-7).
, 1985). Depending on the metal ion used, adsorb a histidine-rich protein onto this matrix with different affinities and elute the adsorbed protein using competitive elution, reduced pH, or strong chelating agents. .

Other purification methods include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods
in Enzymol., Vol. 182, "Guide to Protein Purification", M. Deu
tscher (editor), Academic Press, San Diego, 1990, pp. 529-39). Within the scope of additional aspects of the invention, a polypeptide of interest and an affinity tag (eg,
Fusions with maltose binding proteins, immunoglobulin domains) can be constructed to facilitate purification.

Protein refolding (and optionally reoxidation) procedures may be conveniently used. Protein> 80% pure, more preferably> 90%
Is preferred, even more preferably> 95% pure, and particularly preferably the protein is in pharmaceutically pure form, ie 99.9% with respect to contaminating macromolecules, especially other proteins and nucleic acids. It is of higher purity and contains no infectious or pyrogenic factors. Preferably, the purified protein is substantially free of other proteins, especially other proteins of animal origin.

Also, zamp3 or zamp4 polypeptides or fragments thereof can be produced by chemical synthetic methods. The zamp3 or zamp4 polypeptide can be a monomer or a multimer; glycosylated or non-glycosylated; PEGylated or non-PEGylated, amidated or non-amidated; sulfated or non-sulfated; and an initial methionine amino acid residue. May or may not be included.

For example, zamp3 or zamp4 polypeptides may be synthesized by proprietary solid phase synthesis, partial solid phase methods,
It can be synthesized by fragment condensation or classical solution synthesis. The polypeptide is preferably, for example, Merrifield, J. Am. Chem. Soc. 85: 2149, 19
63, by solid phase peptide synthesis. Alpha-
This synthesis is performed using amino-terminal protected amino acids. Trifunctional amino acids with labile side chains are also protected with suitable groups to prevent undesired chemical reactions during polypeptide assembly. The alpha-amino protecting group is selectively removed so that subsequent reactions occur at the amino terminus.
Conditions that remove the alpha-amino protecting group do not remove the side chain protecting group.

Alpha-amino protecting groups are groups known to be effective in the field of stepwise polypeptide synthesis. Acyl-type protecting groups (eg, formyl, trifluoroacetyl, acetyl), aryl-type protecting groups (eg, biotinyl), aromatic urethane-type protecting groups [eg, benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl and 9-fur Orenylmethyloxy-carbonyl (Fmoc)],
Included are aliphatic urethane protecting groups [eg t-butoxycarbonyl (tBco), isopropyloxycarbonyl, cyclohexyloxycarbonyl] and alkyl type protecting groups (eg benzyl, triphenylmethyl). Preferred protecting groups are
tBoc and Fmoc.

The side chain protecting groups selected must remain intact during coupling and must not be removed during deprotection of the amino terminal protecting group or during coupling conditions. Also, side chain protecting groups must be removed after the synthesis is complete using reaction conditions that do not alter the finished polypeptide. 3 in tBoc chemistry
The side chain protecting groups of functional amino acids are largely benzyl based. In Fmoc chemistry, side chain protecting groups are mostly based on butyl or trityl.

In tBoc chemistry, preferred side chain protecting groups are tosyl for arginine, cyclohexyl for aspartic acid, 4-methylbenzyl (and acetamidomethyl) for cysteine, benzyl for glutamic acid, serine and threonine, and benzyloxymethyl for histidine (and dinitro). Phenyl)
, 2-Cl-benzyloxycarbonyl for lysine, formyl for tryptophan and formyl 2-bromobenzyl for tyrosine. In Fmoc chemistry, the preferred side chain protecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) or 2,2,4,6,7-pentamethyldihydrobenzofuran-5 for arginine. Trityl for sulfonyl (Pbf), asparagine, cysteine, glutamine and histidine, t-butyl for aspartic acid, glutamic acid, serine, threonine and tyrosine, tBoc for lysine and tryptophan.

For the synthesis of phosphopeptides, direct or post-assembly incorporation of phosphate groups is used. In the direct incorporation method, the phosphate group on serine, threonine or tyrosine can be protected with methyl, benzyl, or t-butyl in Fmoc chemistry or with methyl, benzyl or phenyl in tBoc chemistry. Direct incorporation of phosphotyrosine without phosphate protection can be used in Fmoc chemistry. In the post-assembly integration method, the unprotected hydroxyl group of serine, threonine or tyrosine is di-
Derivatization with t-butyl-, dibenzyl- or dimethyl-N, N'-diisopropyl-phosphoamidite followed by oxidation with t-butyl hydro-peroxide.

Solid phase synthesis is usually performed from the carboxyl terminus by coupling an alpha-amino protected (side chain protected) amino acid to a suitable solid support.
When attached to chloromethyl, chlorotrityl or hydroxymethyl resins,
The ester bond will form and the resulting polypeptide will have a free carboxyl group at the C-terminus. Alternatively, when using an amide resin, such as benzhydrylamine or p-methylbenzhydrylamine resin (for tBoc chemistry) and Rink amide or PAL resin (for Fmoc chemistry), an amide bond is formed, The resulting polypeptide will then have a carboxamide group at the C-terminus.

These resins are polystyrene- or polyamide-based, with or without a handle or linker, with or without a bound first amino acid, or polyethylene glycol-grafted. And commercially available, and their manufacture is described in: Stewart, et al., “Solid Phase Peptide Synthesis.
(2nd Edition), (Pierce Chemical Co., Rockford, IL, 1984) and Bayer and Rapp, Chem. Pept. Prot. 3: 3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach. , IRL Press
, Oxford, 1989.

Optionally protected at the side chain and at the alpha-amino group,
The C-terminal amino acid is attached to the hydroxymethyl resin using various activators such as dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DIPCDI) and carbonyldiimidazole (CDI). . It can be attached directly to the chloromethyl or chlorotrityl resin in the form of its cesium tetramethylammonium salt or in the presence of triethylamine (TEA) or diisopropylethylamine (DIEA). The attachment of the first amino acid to the amide resin is identical to the formation of the amide bond during the coupling reaction.

After attachment of the resin to the support, various reagents are used to remove the alpha-amino protecting group, depending on the protection chemistry (eg tBoc, Fmoc). The degree of Fmoc removal is 30
It can be monitored at 0-320 nm or by a conductive cell. After removal of the alpha-amino protecting group, the remaining protected amino acids are stepwise coupled in the required order to give the desired sequence.

Various activators can be used in the coupling reaction, for example: DCC, DIPCDI, 2-chloro-1,3-dimethylimidium hexafluorophosphate (CIP), benzotriazole- 1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate (BOP) and its pyrrolidine analogues (PyBOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBrOP), O- (benzotriazole- 1-yl) -1,1,3,3-
Tetramethyl-uronium hexafluorophosphate (HBTU) and its tetrafluoroborate analogue (TBTU) or its pyrrolidine analogue (HBPyU), O- (
7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyl-uronium hexafluorophosphate (HATU) and its tetrafluoroborate analogue (
TATU) or its pyrrolidine analogue (HAPyU).

The most common catalyst additives used in coupling reactions are 4-dimethylaminopyridine (DMAP), 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbtt). ), N-hydroxybenzotriazole (BOBt) and 1
-Hydroxy-7-azabenzotriazole (HOAt). Each protected amino acid is used in excess (> 2.0 equivalents) and the coupling is usually carried out in N-methylpyrrolidone (NMP) or in DMF, CH2Cl2 or mixtures thereof. At each stage, for example, Kaiser et al., Anal. Biochem. 34: 595, 1970,
The degree of completion of the coupling reaction can be monitored by the ninhydrin reaction, as described in.

After the desired peptide has been fully assembled, the peptide-resin is cleaved off using a reagent with an appropriate scavenger. The Fmoc peptide is usually cleaved and deprotected by TFA with a scavenger (eg H ₂ O, ethanedithiol, phenol and thioanisole). The tBoc peptide is usually cleaved with liquid HF for 1-2 hours at -5 to 0 ° C and deprotected, thereby cleaving the polypeptide from the resin and removing most of the side chain protecting groups. Scavengers such as anisole, dimethyl sulfide and p-thiocresol are usually used with liquid HF to prevent cation formation from alkylation and acylation of amino acid residues present in the polypeptide during cleavage. To do.

It is necessary to remove the formyl group of tryptophan and the dinitrophenyl group of histidine in DMF prior to HF cleavage by piperidine and thiophenyl, respectively. The acetamidomethyl group of cysteine is mercury (II) acetate
Or by iodine, thallium (III) trifluoroacetate or silver tetrafluoroborate, which simultaneously oxidizes cysteine to cystine. Other strong acids used for cleaving and deprotecting the tBoc peptide include trifluoromethanesulfonic acid (TFMSA) and trimethylsilyltrifluoroacetate (TMSOTf).

Zamp3 or zamp4 binding polypeptides can also be used to purify the ligand. Immobilizing a polypeptide on a solid support, such as agarose, cross-linked agarose, glass, cellulosic resin, silica-based resin, polystyrene, cross-linked polyacrylamide, or a bead of similar material that is stable under the conditions of use. To do. Methods of attaching polypeptides to solid supports are known in the art and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. To do. The resulting medium is generally shaped into a column and the fluid containing the ligand is passed through the column one or more times to bind the ligand to the ligand binding polypeptide. Then salt concentration, chaotropic factor (guanidine HCl
), Or a change in pH that disrupts the ligand-receptor binding elutes the ligand.

Ligand binding receptors (or antibodies, members of one of the complement / anti-complement pairs) or binding fragments thereof, and commercially available biosensor instruments (BIAcore ^™ , Pharmacia Biosensor, NJ). An assay system using Piscataway, Ill. May be conveniently used. Such receptors, antibodies, complement / anti-complement pair members or fragments are immobilized on the surface of the receptor chip. The use of this instrument is disclosed in the following references: Karlsson, J. Immunol. Methods 145: 229-40, 1991 and Cunning.
Ham and Wells, J. Mol. Biol. 234: 554-63, 1993.

Amine or sulfhydryl chemistry is used to covalently attach the receptor, antibody, member or fragment to a dextran fiber bound to a thin gold film in the flow cell. Pass the test sample through the cell. If an opposite member of the ligand, epitope, or complement / anti-complement pair is present in the sample, it binds respectively to the immobilized receptor, antibody or member, altering the refractive index of the medium,
This is detected as a change in plasmon resonance on the surface of the gold thin film. This system
It allows evaluation of on and off rates, and binding stoichiometry, from which binding affinity can be calculated.

The term “complement / anti-complement pair” as used herein refers to non-identical moieties that form a non-covalently associated stable pair under appropriate conditions. For example, biotin and avidin (or streptavidin) are complementary /
Members of the anti-complement pair prototype. Other exemplary complement / anti-complement pairs include receptor / ligand pairs, antibody / antigen (or hapten or epitope) pairs, sense / antisense polynucleotide pairs, and the like. If subsequent dissociation of the complement / anti-complement pair is desired, the complement / anti-complement pair preferably has a binding affinity of <10 ⁹ / M.

Ligand binding receptor polypeptides can also be used in other assay systems known in the art. Such systems use Scatchard analysis (Scatchard, Ann. NY) to measure binding affinity.
Acad. Sci. 51: 660-72, 1949) and colorimetric assay (Cunningham et al.,
Science 253: 545-48, 1991 and Cunningham et al., Science 245: 821-25, 19
91).

The present invention also provides anti-zamp3 or anti-zamp4 antibodies. For example, the product of a zamp3 or zamp4 expression vector, or zamp3 or zamp4 isolated from natural sources can be used as an antigen to obtain antibodies to zamp3 or zamp4. zamp
Anti-zamp3 antibodies that "bind specifically" to 3 and anti-zamp4 antibodies that "bind specifically" to zamp4 are particularly effective. The binding affinity (Ka) of the antibody is 10 ⁶ / M or higher, preferably 10 ⁷ / M or higher, more preferably 10 ⁸ / M or higher, most preferably 10 ⁹ / M or higher. At zamp3 or zamp4
An antibody is considered to specifically bind when it binds to a polypeptide, peptide or epitope of. The binding affinity of an antibody can be easily determined by, for example, Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51: 660, 1949). Suitable antibodies include antibodies that bind zamp3 in a particular domain and antibodies that bind zamp4 in a particular domain.

Antigens zamp3 epitope-bearing peptides and polypeptides and zamp4 epitope-bearing peptides and polypeptides can be used to produce anti-zamp3 or anti-zamp4 antibodies. The antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least 9, preferably 15 to about 30 amino acids contained within SEQ ID NO: 2 or 4. However, the polypeptide of the invention comprises a larger portion of the amino acid sequence of the invention, contains amino acids of any length up to 30-50 amino acids, or the entire amino acid sequence of the polypeptide of the invention, and A peptide or polypeptide that includes the entire amino acid sequence of zamp3 or zamp4
Is effective to induce antibodies that bind to.

It is desirable to select the amino acid sequence of the epitope-bearing peptide so as to provide substantial solubility in an aqueous solvent (ie, the sequence contains relatively hydrophilic residues but avoids hydrophobic residues). Preferably). Hydrophilic peptides can be predicted from the hydrophobicity plots, see for example: Hopp and Wo.
ods, Proc. Nat. Acad. Sci. USA 78: 3824-8, 1981 and Kyte and Doo.
little, J. Mol. Biol. 157: 105-142, 1982. Moreover, amino acid sequences containing proline residues may also be desirable for antibody production.

Recombinant zamp3 or zamp4 protein or zamp3 or z isolated from natural sources
Polyclonal antibodies against amp4 can be produced using methods well known in the art. For example, see the following references: Green et al.,
“Production of Polyclonal Antisera”, Immunochemical Protocols (Man
Son, editor), pp. 1-5 (Humana Press 1992), and Williams et al., “Expre
ssion of foreign proteins in E. coli using plasmid vectors and
purification of specific polyclonal antibodies ”, DNA Cloning 2
: Expression Systems, 2nd Edition, Glover et al. (Editor), p. 15 (Oxford Universi
ty Press 1995).

Adjuvants such as alum (aluminum hydroxide) or Freund's complete or incomplete Freund's adjuvant are used to zamp3 or zamp4.
The immunogenicity of the polypeptide can be increased. Immunizing effective polypeptides also include fusion polypeptides, such as fusions of zamp3 or zamp4 or a portion thereof with an immunoglobulin or maltose binding protein.
The polypeptide immunogen can be a full length molecule or a portion thereof. When a portion of the polypeptide is "hapten-like", such portion is conveniently a polymeric carrier for immunization (eg, keyhole limpet hemocyanin (KLH)).
, Bovine serum albumin (BSA) or tetanus toxoid).

Polyclonal antibodies are typically raised in animals such as horses, cows, dogs, chickens, rats, mice, rabbits, hamsters, guinea pigs, goats or sheep, although the anti-zamp3 or anti-zamp4 antibodies of the invention are It can be derived from an ape antibody. General techniques for generating diagnostically and therapeutically effective antibodies in baboons are described, for example, in the following references: Goldenberg, International Patent Publication No. WO.
91/11465, and Losman et al., Int. J. Cancer 46: 310, 1990. Antibodies can also be raised in transgenic animals, such as transgenic sheep, cows, goats or pigs, and expressed in modified form in yeast and fungi and in mammalian and insect cells. it can.

Alternatively, monoclonal anti-zamp3 or anti-zamp4 antibodies can be generated. Rodent monoclonal antibodies to specific antigens can be obtained by methods known in the art (see, for example, the following references: Kohler
Et al., Nature 256: 495, 1975; Coligan et al. (Editor), Current Protocols in I.
munology, Vol. 1, pp. 2.5.1-2.6.7 (John Wiley & Sons 1991); Pick
sley et al., “Production of monoclonal antibodies against proteins exp.
ressed in E. coli ”, DNA Cloning: Expression Systems, Second Edition, Glover
Other (editor), p. 63 (Oxford University Press 1995)).

Briefly, monoclonal antibodies can be obtained as follows. za
The composition comprising the mp3 or zamp4 gene product was injected into mice and antibody production was confirmed by removing serum samples, removing the spleen to obtain B lymphocytes, and fusing the B lymphocytes with myeloma cells. To produce hybridomas, clone the hybridomas, select positive clones producing antibodies to the antigen, cultivate clones producing antibodies to the antigen, and isolate the antibodies from the hybridoma culture.

Furthermore, the anti-zamp3 or anti-zamp4 antibodies of the invention can be derived from human monoclonal antibodies. Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigen challenge. In this technique, human heavy and light chain locus factors are introduced into mice derived from an embryonic stem cell line containing targeted disruption of endogenous heavy and light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described, for example, in the following references: Green et al., Nature Genet. 7:13,
1994; Lonberg et al., Nature 368: 856, 1994; and Taylor et al., Int. Immun. 6
: 579, 1994.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography using Protein A Sepharose, size exclusion chromatography, and ion exchange chromatography (see, for example, the following references: Coligen, pp. 2.7.1). -2.7.12 and pp. 2.9.1-2.9.3; Ba
ines et al., “Purification of Immunoglobulin G (IgG)”, Methods in Mol
ecular Biology, Vol. 10, pp. 79-104 (The Humana Press, Inc. 1992
)).

For particular applications, it may be desirable to produce fragments of anti-zamp3 or anti-zamp4 antibodies. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies according to conventional methods. By way of illustration, an antibody fragment can be produced by enzymatically digesting the antibody with pepsin to form the 5S fragment designated F (ab) '2. This fragment can be further cleaved using a thiol reducing agent to produce a 3.5S Fab 'monovalent fragment.

If desired, the cleavage reaction can be carried out using a blocking group of sulfhydryl groups resulting from the cleavage of the disulfide bond. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent Fab and Fc fragments. These methods are described, for example, in the following references: Gold
Nenberg, US Pat. No. 4,331,647; Nisonoff et al., Arch. Biochem. Biophys. 89.
: 230, 1960; Porter, Biochem. J. 73: 119, 1959; Edelman et al., Methods in
Enzymology, Vol. 1, p. 422 (Academic Press 1967); and Coligan,
Ibid.

Other methods of cleaving the antibody so long as the fragment binds to the antigen recognized by the intact antibody, eg, separating the heavy chains to form a monovalent light-heavy chain fragment, further cleaving the fragment Methods, or other enzymatic, chemical or genetic techniques can also be used. For example, the Fv fragment comprises an association of VH and VL chains. This association can be non-covalent, as described in the following references: Inbar et al., Proc. Nat. Acad. Sci. USA 69: 2659, 1972. Alternatively, the variable chains can be linked by intercellular disulfide bonds or cross-linked by chemicals such as glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotch. 12: 437, 1992).

Fv fragments can comprise VH and VL chains connected by a peptide linker. These single chain antigen binding proteins (scFv) are produced by constructing structural genes that comprise DNA sequences encoding VH and VL domains connected by oligonucleotides. Insert the structural gene into an expression vector and then insert the expression vector into a host cell such as E. coli.
To introduce in. These recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. The method of producing scFv is
See, for example, Whitlow et al., Methods: A Companion to.
Methods in Enzymology 2:97, 1991; also Bird et al., Science 242: 423.
1988; Ladner et al., U.S. Pat. No. 4,946,778; Pack et al., Bio / Technology 11:12.
71, 1993; and Sandhu, supra.

By way of illustration, scFv can be obtained by exposing lymphocytes in vitro to zamp3 or zamp4 polypeptides and selecting antibody display libraries in phage or similar vectors (eg, immobilization or Use labeled zamp3 or zamp4 proteins or peptides). Genes encoding peptides with potential zamp3 or zamp4 polypeptide binding domains can be screened for random peptide libraries displayed on phage (phage display) or on bacteria such as E. coli. Can be obtained by Nucleotide sequences that encode a polypeptide can be obtained in a number of ways, including random mutagenesis and random polynucleotide synthesis.

These random peptide display libraries can be used to screen for peptides that interact with known targets. The target can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or an organic or inorganic substance. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Pat. No. 5,223,409, Ladne.
r et al., U.S. Pat.No. 4,946,778, Ladner et al., U.S. Pat.No. 5,403,484, Ladner et al.,
U.S. Pat.No. 5,571,698, and Kay et al., Phage Display of Peptides and P.
roteins (Academic Press, Inc. 1996).

And random peptide display libraries and kits for screening such libraries are described, for example, in Clontech (Palo Alto, Calif.).
, Invitrogen Inc. (San Diego, CA), New England Biolab
s, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnolo.
Commercially available from gy Inc. (Piscataway, NJ).
The zamp3 or zamp4 sequences disclosed herein can be used to screen random peptide display libraries to identify proteins that bind to zamp3 or zamp4.

Another form of antibody fragment is a peptide that encodes a single complementarity determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing the gene encoding the CDR of the antibody in question. Such a gene is produced, for example, by PCR for synthesizing a variable region from RNA of antibody-producing cells (see, for example, the following document: Larrick et al., Methods: A Companion to Meth.
ods in Enzymology 2: 106, 1991); Courtenay-Luck, “Genetic Manipul.
ation of Monoclonal Antibodies ”, Monoclonal Antibodies: Production
, Engineering and Clinical Application, Ritter et al. (Editor), p. 166 (
Cambridge University Press 1995); and Ward et al., “Genetic Manipulat
ion and Expression of Antibodies ”, Monoclonal Antibodies: Principl
es and Applications, Birch et al. (Editor), p. 137 (Wiley-Liss, Inc. 19
95)).

Alternatively, anti-zamp3 or anti-zamp4 antibodies can be derived from "humanized" monoclonal antibodies. Humanized monoclonal antibodies are produced by transferring the mouse complementarity determining regions from the heavy and light variable chains of mouse immunoglobulin into the human variable domain. Typical residues from human antibodies are then replaced in the framework regions of the murine counterpart. The use of antibody components derived from human monoclonal antibodies eliminates the potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable regions are described, for example, in the following references: Orlandoi et al., Proc. Nat. Acad. Sci. USA 86: 3833, 19
89.

Techniques for producing humanized monoclonal antibodies are described, for example, in the following references: Jones et al., Nature 321, 522, 1986; Carter et al., Proc. Nat. Acad. S.
ci. USA 89: 4285, 1992; Sandhu, Crit. Rev. Biotch. 12: 437, 1992; S.
inger et al., J. Immun. 150: 2844, 1993; Sudhir (editor), Antibody Engineeri
ng Protocols (Humana Press, Inc. 1995); Kelley, “Engineering Thera
peutic Antibodies ”, Protein Engineering: Principles and Practice, C
Leland et al. (editor), pp. 399-434 (John Wiley & Sons, Inc. 1996); and Queen et al., US Pat. No. 5,693,762 (1997).

[0208] Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-zamp3 or anti-zamp4 antibodies or antibody fragments according to standard techniques. See, for example: Green et al., “Production of Pol.
yclonal Antisera ”, Methods In Molecular Biology: Immunochemical Pr
otocols, Manson (editor), pp. 1-12 (Humana Press 1992). Also, Coliga
n, supra, see pp. 2.4.1-2.4.7.

Alternatively, monoclonal anti-idiotypic antibodies can be produced according to the techniques described above using anti-zamp3 or anti-zamp4 antibodies or antibody fragments as immunogens. Alternatively, humanized anti-idiotype antibodies or apes anti-idiotype antibodies can be produced according to the techniques described above. Methods for producing anti-idiotypic antibodies are described, for example, in the following references: Irie, US Pat.
208,146, Greene et al., US Pat. No. 5,637,677, and Varthakavi and Minoch.
a, J. Gen. Virol. 77: 1875, 1996.

On a phage (phage display) or on a bacterium such as E. coli (E.
li) By screening the random or oriented libraries displayed above, one can obtain genes encoding polypeptides, "binding proteins" with potential zamp3 or zamp4 polypeptide binding domains. Nucleotide sequences that encode a polypeptide can be obtained in a number of ways, including random mutagenesis and random polynucleotide synthesis. Alternatively, a restricted phage display library can be produced. These peptide display libraries can be used to screen for peptides that interact with known targets.

The target here can be a protein or polypeptide, for example a ligand or receptor, a biological or synthetic macromolecule, or an organic or inorganic substance. Techniques for making and screening such peptide displays are known in the art (Ladner et al., US Pat. No. 5,223,409; Ladner et al., US Pat. No. 4,946,778; Ladner et al., US Pat. No. 5,403,484 and Ladner et al.
US Pat. No. 5,571,698) and peptide display libraries and kits for screening such libraries are described, for example, in Clontech, Palo Alto, Calif., Invitrogen, San Diego, Calif., New England Biolabs. Incorporated (New England Biolabs, Inc., Beberley, Mass.) And Pharmacia LKB Biotechnology Incorporated.
chnology, Inc., Piscataway, NJ). The zamp3 or zamp4 sequences disclosed herein can be used to screen peptide display libraries to identify proteins that bind to zamp3 or zamp4. These "binding proteins" that interact with the zamp3 or zamp4 polypeptides can be used essentially like antibodies.

In another aspect of the invention there is provided a pharmaceutical composition comprising a purified zamp3 or zamp4 polypeptide and a pharmaceutically acceptable vehicle. Such pharmaceutical compositions are used in the treatment of conditions associated with the infection of pathological microorganisms such as bacteria, fungi and viruses. Antimicrobial applications of zamp3 or zamp4 polypeptides include situations where pathogens become resistant to standard treatments. For example, hospital sepsis is an increasing problem as Staphylococcus strains become resistant to commonly used antibodies.

In general, the antimicrobial activity of zamp3 or zamp4 polypeptides, fragments, fusions, antibodies, agonists and antagonists can be assessed by techniques known in the art. More specifically, antimicrobial activity can be assayed by assessing the sensitivity of the microbial cell culture to the test factor and assessing the protective effect of the test factor on infected mice. See, eg, Musiek et al., Antimicrob. Agents Chemother. 3:40, 1973. In addition, protection of mammalian cell cultures can be evaluated for antiviral activity.

Known techniques for assessing antimicrobial activity include, for example: Barsum et al., Eu.
r. Respir. J. 8 (5): 709-14, 1995; Sandovsky-Losica et al., J. Med. V.
et. Mycol. (England) 28 (4): 279-87, 1990; Mehentee et al., J. Gen. Micr.
obiol. (England) 135 (Pt.8): 2181-8, 1989; Segal and Savage, Journal.
of Medical and Veterinary Mycology 24: 477-479, 1986 and others. Known assays specific for antiviral activity are described, for example, by Daher et al., J. Vir.
ol. 60 (3): 1068-74, 1986.

Further, contract laboratories provide services for assessing antimicrobial properties. For example, Panlabs, Inc. of Bothell, Washington
Provides in vitro or in vivo tests for the following microorganisms: bacteria,
Gram-negative bacteria (Enterobactor cloacae, Escherichia coli, Klebsiella pneumon)
iae), Proteus vulgaris, Pseudomonas aeruginosa, Salmonell's Salmonell
a typhimurium) and Serratia marcescens)
, Gram-positive bacteria (Bacillus subtilis), Brevebacterium ammoniagenes,

Corynebacterium minutissimum,
Micrococcus luteus, Mycobacterium ranae, Staphylococcus strains and Streptococcus strains and anaerobes (Actinomyces viscosus), Bacteroides Fragilis (Bac
teroides fragilis), Clostidium sporog
enes), Corynebacterium acnes,

Helicobacter pylori and Porphyromonas
Porphyromonas gingivalis) and protozoa (Trichomonas foetus) and fungi (eg, Candida albicans), Epidermophyton flocsum (Epiderm)
ophyton floccosum), Exophiala Jean Selmay (Exophiala jeans
elmei), Microsporum strain, Trchophyton
Shares and others). Also, Molecular Probes of O
regon) has commercially available fluorescent technology for use in bacteriology.

If desired, the performance of zamp3 or zamp4 polypeptides, fragments, fusion proteins, agonists, antagonists or antibodies in this regard is known to be functional in this regard, eg proline-rich proteins. , Lysozyme, histatin, lactoperoxidase or others. In addition, zamp3 or zamp4 polypeptides, fragments, fusion proteins, antibodies, agonists and antagonists can be evaluated in combination with one or more antimicrobial agents to identify synergistic effects.

Defensin polypeptides comprising a cysteine motif are commercially available as antibiotic peptides, monocytes and dendritic chemotactic peptides. Commercial suppliers include: Research Diagnostics, Inc., Flanders, NJ,
Peninsula Laboratorie (Peninsula Laboratorie
s, Inc., Belmont, CA), American Peptide Company (A
America Peptide, Co., Sumvale, CA). A zamp3 polypeptide comprising amino acid residues 31-60 of SEQ ID NO: 2, and amino acid residue 12 of SEQ ID NO: 6
A zamp4 polypeptide comprising-41 would be effective for this purpose.

Defensins are associated with human ocular tissues and secretions and are effective in diseases involving ocular microbes (Cullor et al., Arch. Ophthalmol. 108: 861-4, 1990; M).
uphy et al., US Pat. No. 5,242,902; Hattenbach et al., Antimicrob. Agents Chemot
her. 42: 323, 1998; Haynes et al., Lancet 354: 451-2, 1998 and Haynes et al.,
Br. J. Ophthalmol. 83: 737-41, 199). Addition of the defensin polypeptide reduced microbial contamination of the corneal preservation medium. Such factors are effective in reducing infectious postoperative complications associated with such transplants (Schwab et al., Cornea
11: 370-5, 1992).

Thus, zamp3 or zamp4 polypeptides, their agonists or antagonists, are therapeutically effective in the treatment of eye-related infections and inflammation.
To demonstrate the existence of this ability in the zamp3 or zamp4 polypeptides, agonists or antagonists of the invention, such zamp3 or zamp4 polypeptides, agonists or antagonists, according to procedures known in the art, to their antimicrobials. Evaluate for activity and chemotaxis activity. If desired
The zamp3 or zamp4 polypeptide in this regard can be labeled with other α and β defensins,
It can be compared against rabbit neutrophil defensin and others. In addition, zamp3 or zamp4 polypeptides, their agonists or antagonists
It can be evaluated in combination with one or more antimicrobial molecules to identify synergistic effects.

Similarly, the zamp3 or zamp4 polypeptides of the invention are also therapeutically effective in treating ear-related infections and inflammation. The present invention provides methods of treating diseases associated with microorganisms. The method comprises administering to the mammal a therapeutically effective amount of a zamp3 or zamp4 polypeptide, whereby the polypeptide ameliorates the disease. Preferably, such a polypeptide comprises amino acid residues 31-6 of SEQ ID NO: 2.
0, amino acid residues 23-63, or amino acid residues 12-41 of SEQ ID NO: 6. 1
One such application is for diseases associated with eye related microorganisms, such as conjunctivitis.

The present invention also provides a method of contraception, wherein a therapeutically effective amount of the polypeptide of the present invention is administered to a mammal. Such administration would be effective in preventing embryo implantation and / or development. Defensins were also found to activate the complement cascade (Prohas
zka et al., Mol. Immunol. 34: 809-16, 1997 and Prohaszka and Fust, Lance.
t 352: 1152, 1998). The complement component Clq plays a role in host defense against infectious agents such as bacteria and viruses. Clq is known to exhibit some specialized functions.

For example, Clq triggers the complement cascade through interaction with bound antibody or C-reactive protein (CRP). Clq also interacts directly with certain bacteria, RNA viruses, mycoplasmas, urate crystals, the fat A component of bacterial endotoxins and the membranes of certain intracellular organelles. Clq binding to Clq receptors is thought to promote phagocytosis. Clq also appears to enhance the antibody-forming surface of the host defense system. For example, Johnston, Pediatr. Infect. Dis. J. 12: 933-41.
, 1993. Thus, complement activated defensins act as enhanced antimicrobial agents, facilitating lysis or phagocytosis of infectious agents.

The antimicrobial activity of defensins is also effective in contraceptive applications (Sawicki and Mystkovska, Lancet 353: 464-5, 1999). Also, when proinflammatory activity is desired, the pharmaceutical composition of the present invention can be used.
Applications of such pro-inflammatory activity include treatment of chronic tissue damage, particularly in areas with limited or damaged vascular systems, eg, damage in the extremities associated with diabetes. In contrast, antagonists to zamp3 or zamp4 polypeptides may be effective as anti-inflammatory agents.

The zamp3 or zamp4 polypeptide pharmaceutical compositions of the present invention can also be used in the treatment of conditions in which stimulation of immune responsiveness is desired. Treatment of such symptoms
It includes treatment of patients with a dysfunctional immune system, eg, patients with AIDS or individuals undergoing chemotherapy, radiation therapy or otherwise. Human B-defensins (HBD1 and HBD2) show chemotactic activity on immature dendritic cells (Yang et al., Science 2
86: 525-8, 1999). Chemotactic activity suggests that defensins are immunostimulatory in addition to their antimicrobial activity. Chemotacticity is mediated through CCR6 receptors found on the surface of immature dendritic cells as well as memory T cells.

Zamp3 and zamp4 will be effective in mediating adaptive immune responses. In particular, such β-defensin peptides can be used to promote an immune response in immunocompromised individuals. Inversely modified peptides derived from beta-defensin can be used to block the interaction of endogenous immunostimulatory molecules with their receptors and thereby achieve immunosuppressive and anti-inflammatory effects. Ah

Other members of β-defensins are found in bronchial epithelial tissue, cystic fibrosis is characterized by frequent microbial infections, and pharmaceutical compositions containing zamp3 or zamp4 polypeptides also contain cysts. Considered for use in the treatment of lung infections associated with sexual fibrosis. Also included in the invention are engineered zamp3 or zamp4 polypeptides, which are characterized by a decrease in activity with respect to salt concentration. Reduced sensitivity to high salt concentrations will preserve the antimicrobial activity of engineered zamp3 or zamp4 polypeptides in the environment of high salt concentration, eg, the lung airways of patients with cystic fibrosis. In this way, pharmaceutical compositions containing engineered zamp3 or zamp4 polypeptides formulated for pulmonary delivery can be used to treat pulmonary infections associated with cystic fibrosis.

Radioactive hybrid mapping is a stem cell genetic technique developed to construct high-resolution, contiguous maps of mammalian chromosomes (Cox et al., Scien.
ce 250: 245-50, 1990). Partial or complete knowledge of the gene sequence allows the design of suitable PCR primers for use with chromosomal radioactive hybrid mapping panels. Commercially available radioactive hybrid mapping panels covering the entire human genome, eg Stanford G3 RH Panel and G
The eneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, Alab.) is commercially available.

These panels show rapid, PCR-based chromosomal localization and ordering of genes, sequence labeling sites (STS), and other non-polymorphic and polymorphic markers within the region of interest. Make decisions possible. This involves establishing a directly proportional physical distance between the newly discovered gene of interest and the previously mapped marker. Accurate knowledge of gene location is useful for a number of purposes, including the following: 1) determining if a sequence is part of an existing contig and adding additional surrounding genetic sequences to various Morphology, eg YAC, BAC or cDNA clone; 2) Preparing a candidate gene for a hereditary disease that exhibits linkage to the same chromosomal region; and 3) What function a particular gene has. Cross-reference model organisms, such as mice, that can facilitate the determination of

T31 Whole Genome Radiation Hybrid (WGRH) Panel (Research Genetics, Inc.
, Huntsville, Alab.) And the Map Manager QT linkage analysis program was used to map the murine zamp3 gene to chromosome 8 in mice. At P = 0.0001, murine zamp3 bound to the marker D8Mit141 with an LOD score of 5.6. D8Mit141 mapped to 6.0 cM on mouse chromosome 8. Murine zamp4 was mapped to chromosomes in mice using the commercially available mouse T31 whole genome radiation hybrid (WGRH).

The results show that the murine zamp4 maps to the 25.88cR_3000 end of the framework marker D14Mit156 on the mouse chromosome 14WICGR radiation hybrid map. The proximal and distal framework markers were D14Mit156 and D14Mit31, respectively. Other defensins did not map to positions other than mouse chromosome 8. This region is a known conserved region of homology to human chromosome 8p21-p23, which overlaps with the position of the human defensin cluster.

The present invention also provides reagents for use in diagnostic applications. For example, zamp
A probe comprising 3 genes, zamp3 DNA or RNA or their subsequences can be used to determine if the zamp3 gene is present on mouse chromosome 8 or if a mutation has occurred. Detectable chromosomal abnormalities at the zamp3 locus include, but are not limited to, aneuploidy, gene copy number alterations, insertions, deletions, restriction site alterations and rearrangements. According to molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short vertical repeat (STR) analysis using PCR techniques, and other genetic linkage analysis techniques known in the art. Polynucleotides of the invention can be used to detect such chromosomal abnormalities (Sambrook et al., Supra; Ausubel et al., Supra; Marian, Chest 108: 255-265, 1).
995).

Another aspect of the invention is a cell culture or patient serum sample or tissue biopsy that is evaluated for SPG, Chediak-Higashi syndrome or other conditions characterized by altered defensin concentrations. Zamp3 or zamp4 inside
Includes detection of polypeptides. Immunoassay techniques and antibodies capable of recognizing zamp3 or zamp4 polypeptide epitopes can be used to detect zamp3 or zamp4 polypeptides. More specifically, the present invention includes the following steps:
Methods for detecting mp3 or zamp4 polypeptides include:

A solution or sample or cell culture lysate that may contain a zamp3 or zamp4 polypeptide is exposed to an antibody bound to a solid support, where the antibody is the first epitope of the zamp3 or zamp4 polypeptide. To the second antibody that binds to the immobilized antibody-polypeptide to remove unbound contaminants and binds the immobilized antibody-polypeptide to the second epitope of the zamp3 or zamp4 polypeptide. Upon exposure, the second antibody associates with the detectable label and detects the detectable label.

A zamp3 or zamp4 polypeptide concentration that differs from the control concentration may indicate SPG, the Chediak-Higashi syndrome or other symptoms characterized by altered defensin concentrations. Furthermore, expression of zamp3 or zamp4 can be monitored in patients with cystic fibrosis as a predictor of the onset of infection crisis. Also, high levels of defensins, e.g., zamp3 or zamp4 polypeptides, have been associated with cytotoxic effects in the lung, and levels of zamp3 or zamp4 polypeptides can be directly treated to avoid or treat such cytotoxicity. Indicates that can be used. For example, an antibody directed against the zamp3 or zamp4 polypeptide can be administered to inactivate the zamp3 or zamp4 polypeptide.

In an additional aspect of the invention, antibodies or synthesized binding proteins that specifically bind to the aforementioned zamp3 or zamp4 polypeptides (eg, generated by phage display, E. coli Fab, and others). Provided). Such antibodies are useful in preparing anti-idiotype antibodies, among other uses described herein. Commercially available kits, eg Ph.D ^™ Phage Display Peptide Library Kits
(New England Biolabs, Inc., Beverley, Mass.) Can be used to produce the synthesized binding protein by phage display. Phage display technology is described, for example, in US Pat. Nos. 5,223,409 and 5,403,4.
84 and 5,571,698.

An additional aspect of the invention provides a method of identifying an agonist or antagonist of a zamp3 or zamp4 polypeptide disclosed above, which agonist or antagonist may have valuable properties as further discussed herein. In one embodiment, a method of identifying an agonist of a zamp3 or zamp4 polypeptide is provided, the method comprising preparing a cell responsive thereto, culturing the cell in the presence of a test compound, and responsiveness of the cell Compared to cells cultured in the presence of zamp3 or zamp4 polypeptide and selecting a test compound for which the cell response is of the same type.

In another embodiment, a method of identifying an antagonist of a zamp3 or zamp4 polypeptide is provided, the method comprising preparing a cell that is responsive to a zamp3 or zamp4 polypeptide, the presence of the zamp3 or zamp4 polypeptide. Incubate a first portion of cells underneath and incubate a second portion of cells in the presence of zamp3 or zamp4 polypeptide and the test compound.
Culturing the portion and detecting a decreased cellular response of the second portion of the cell relative to the first portion of the cell.

A further aspect of the invention there is provided a method of studying the chemoattractant properties of monocytes in cell culture, which method comprises a zamp3 or zamp4 polypeptide, fragment, fusion protein, antibody, agonist or antagonist. Incubating the monocytes in a medium comprising the to study or assess the chemoattractant properties of the monocytes. Such evaluations can be performed using methods known in the art, such as those described in Territo et al., Supra.

The melanocortin receptor is a G-coupled protein receptor that activates adenylate cyclase and causes calcium flux. The agoroprotein, which contains a 36 amino acid domain that is toxin-like,
It is believed to inhibit the binding of MSH-alpha to l and MC4. Furthermore, agouchi protein is thought to be a calcium channel antagonist,
And some toxins are thought to modulate ion flux.
Experimental evidence arises, these suggest that defensins can block calcium channels.

A further aspect of the invention provides a method of studying the activity of the melanocortin family of receptors in cell culture, which method comprises a ligand or a putative ligand and a zamp3 or zamp4 polypeptide, fragment, fusion protein. Cells that endogenously carry such a receptor (eg, ACTH receptor or others), or cells engineered to carry such a receptor, in a medium comprising an antibody, an agonist or an antagonist. Incubate,
It involves studying or assessing the binding or / and the modulation or modulation of ion flux of a ligand or a putative ligand. Such an evaluation can be performed using methods known in the art, such as those described in Zhu et al., Supra.

A further aspect of the invention provides a method of studying ion flux in cell culture, which method comprises in a medium comprising a zamp3 or zamp4 polypeptide, fragment, fusion protein, antibody, agonist or antagonist. And incubating cells capable of forming an ion flux, such as calcium flux, sodium flux, potassium flux or otherwise, to study or evaluate the regulation or modulation of ion flux.

A further aspect of the invention provides a method of studying cell-killing activity against mammalian cells, eg tumor cells, in cell culture, which method comprises zamp3 or zamp4 polypeptides, fragments, fusion proteins, antibodies. , Incubating such cells in a medium comprising an agonist or antagonist at high test factors and low cell concentrations to study or assess cell killing activity.
Such evaluations are methods known in the art, such as Lichtenstein et al., Blood 68: 1407-10, 1986 and Sheu et al., Antimicrob. Agents Chemother.
28: 626-9, 1993.

A further aspect of the invention is the use of zamp3 or zamp4 polypeptides, fragments, fusion proteins or agonists as cell culture reagents in the in vitro study of exogenous microbial infections, eg bacterial, viral or fungal infections. Include doing. An additional aspect of the invention is to study defensin induction of epithelial cells in cell culture. In this aspect of the invention, epithelial cells are cultured and exposed to pathogen stimuli. The induction of zamp3 or zamp4 polypeptide production by epithelial cells is then measured.

The polynucleotides and polypeptides of the present invention will be used as educational tools in laboratory hands-on courses for courses related to genetics and microbiology, protein chemistry, and antibody production and analysis. Molecules of zamp3 or zamp4 can be used as standards or "unknown" for testing purposes because they have unique polynucleotide and polypeptide sequences. For example, zamp3 or zamp4 polynucleotides can be used, for example, as a teaching aid for the following purposes: expression for bacterial, viral, or mammalian expression, where zamp3 or zamp4 is the gene to be expressed. To teach students how to prepare constructs, eg, fusion constructs; to determine restriction endonuclease cleavage sites for polynucleotides; to determine mRNA and DNA localization of zamp3 or zamp4 polynucleotides in tissues To identify (ie, by Northern and Southern blotting and polymerase chain reaction); and to identify related polynucleotides and polypeptides by nucleic acid hybridization.

By way of illustration, students found that AlwI digestion of a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 1 provided two fragments of approximately 13 and 187 base pairs, and Cv
You will find that the iRI digest yields fragments of approximately 58 base pairs, doublets at 54 base pairs, 30 base pairs, and 12 base pairs.

The zamp3 or zamp4 polypeptides can be used as an adjunct material to teach: antibody preparation; protein identification by Western blotting; protein purification; zamp3 or expressed as a ratio of total expressed protein. Measurement of weight of zamp4 polypeptide; identification of peptide cleavage site; coupling of amino and carboxyl terminal tags; amino acid sequence analysis and in vitr
Monitor biological activity (ie, protease inhibition) of both native and tagged proteins in o and in vivo, without limitation.

A zamp3 or zamp4 polypeptide can be used as an adjunct material to teach: antibody preparation; protein identification by Western blotting; protein purification; zamp3 or protein produced as a ratio to total protein produced. Measurement of weight of zamp4 polypeptide; identification of peptide cleavage site; coupling of amino and carboxyl terminal tags; amino acid sequence analysis and in vitr
Monitor biological activity of both native and tagged proteins in o and in vivo, without limitation.

The zamp3 or zamp4 polypeptides can also be used to teach the following: analytical techniques, such as mass spectrometry, in particular the circular alpha dichroism, which determines the conformation of the four alpha helices, the atoms. To determine the three-dimensional structure of a detailed description X
Line crystallography, nuclear magnetic resonance spectroscopy to reveal protein structure in solution. For example, a kit containing zamp3 or zamp4 can be given to students for analysis. Since the teacher knows the amino acid sequence, he or she can give the student the protein as a test to determine or develop the student's skill, and then the teacher will know if the student has correctly analyzed the polypeptide. Since all polypeptides are unique, the educational use of zamp3 or zamp4 is unique in their own right.

Antibodies that specifically bind to zamp3 or zamp4 can be used as educational aids by preparing an affinity chromatography column for purifying zamp3 or zamp4, and competing with a polynucleotide encoding the antibody to determine the sequence. Can be used to teach, and as a hands-on course, teach students how to design humanized antibodies. The reagent company can then package the zamp3 or zamp4 gene, polypeptide, or antibody and sell it to an educational facility so that the student gains skills in the field of microbiology. Because each of the genes and proteins is unique, each of the genes and proteins creates a unique assignment and learning experience for the student in the lab hands-on course. Educational kits containing such zamp3 or zamp4 genes, polypeptides, or antibodies are considered to be within the scope of the invention.

The invention in one aspect is: a) amino acid residues 31-60 of SEQ ID NO: 2; and b)
There is provided an isolated protein comprising a polypeptide selected from the group consisting of amino acid residues 12-41 of SEQ ID NO: 2. In one embodiment, the protein is a
B) a polypeptide consisting of the sequence of amino acid residue 1 to amino acid residue 63 of SEQ ID NO: 2; and b) a polypeptide consisting of the sequence of amino acid residue 23 to amino acid residue 63 of SEQ ID NO: 2; Comprising a polypeptide having the sequence: In another embodiment, the protein comprises a polypeptide consisting of the sequence of amino acid residue 1 to amino acid residue 43 of SEQ ID NO: 4.

The present invention also includes a) a polypeptide having a sequence from amino acid residue 1 to amino acid residue 63 of SEQ ID NO: 2, wherein the polypeptide is amino acid residue 31, 32, 38 of SEQ ID NO: 2.
, 42, 52, 59 and 60; b) a polypeptide having a sequence from amino acid residue 23 to amino acid residue 60 of SEQ ID NO: 2, wherein the polypeptide is the amino acid residue of SEQ ID NO: 2. Having cysteine residues corresponding to groups 31, 32, 38, 42, 52, 59 and 60; and c) a polypeptide having a sequence from amino acid residue 1 to amino acid residue 43 of SEQ ID NO: 2, wherein the polypeptide Is amino acid residues 12, 1 of SEQ ID NO: 6
Having a cysteine residue corresponding to 9, 24, 34, 40 and 41; providing an isolated protein comprising a peptide having at least 80% identity to a polypeptide selected from the group consisting of: To do. In one embodiment, ktup = 1, gap opening penalty = 10, gap extension penalty = 1.
, And the substitution matrix = BLOSUM62, FASTA with other parameters set as defaults to determine percent amino acid identity.

[0254] Also, a) amino acid residue 31 to amino acid residue 60 of SEQ ID NO: 2; and b) SEQ ID NO: 6
A polypeptide selected from the group consisting of amino acid residue 12 to amino acid residue 41; The present invention also provides the above-mentioned polypeptide in combination with a pharmaceutically acceptable vehicle. In another aspect, the invention comprises inoculating an animal with a polypeptide as described above, wherein the polypeptide induces an immune response in the animal to produce an antibody; and the antibody is isolated from the animal. Methods of producing antibodies to peptides are provided. The present invention also provides an antibody that specifically binds to the aforementioned protein.

Also provided are anti-idiotypic antibodies of antibodies that specifically bind to the aforementioned proteins. In another aspect, the invention encodes a protein comprising a polypeptide selected from the group consisting of amino acid residues 31-60 of SEQ ID NO: 2; and b) amino acid residues 12-41 of SEQ ID NO: 6. An isolated polynucleotide is provided. In one embodiment, the protein is a) a polypeptide consisting of the sequence of amino acid residue 1 to amino acid residue 63 of SEQ ID NO: 2; and b) amino acid residue 23 to amino acid residue of SEQ ID NO: 2.
A polypeptide consisting of 63 sequences; a polypeptide having a sequence selected from the group consisting of: In another embodiment, the protein comprises a polypeptide consisting of the sequence of amino acid residue 1 to amino acid residue 43 of SEQ ID NO: 4. In other embodiments, the polynucleotide remains hybridized to the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5 or components thereof after stringent wash conditions.

The invention also includes a) a polypeptide having a sequence from amino acid residue 1 to amino acid residue 63 of SEQ ID NO: 2, wherein the polypeptide is amino acid residue 31, 32, 38 of SEQ ID NO: 2.
, 42, 52, 59 and 60; b) a polypeptide having a sequence from amino acid residue 23 to amino acid residue 63 of SEQ ID NO: 2, wherein the polypeptide is the amino acid residue of SEQ ID NO: 2. Having cysteine residues corresponding to groups 31, 32, 38, 42, 52, 59 and 60; and c) a polypeptide having a sequence from amino acid residue 1 to amino acid residue 43 of SEQ ID NO: 2, wherein the polypeptide Is amino acid residues 12, 1 of SEQ ID NO: 6
Having a cysteine residue corresponding to 9, 24, 34, 40 and 41; providing an isolated protein comprising a peptide having at least 80% identity to a polypeptide selected from the group consisting of: To do.

In one embodiment, ktup = 1, gap opening penalty = 10, gap extension penalty = 1, and substitution matrix = BLOSUM62,
FASTA with other parameters set as defaults is used to determine percent amino acid identity. In other embodiments, the difference between the peptide and SEQ ID NO: 2 or 6 is due to a conservative amino acid substitution. In other embodiments, the polynucleotide remains hybridized to the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5 or components thereof after stringent wash conditions.

The invention also includes: a) amino acid residue 31 to amino acid residue 60 of SEQ ID NO: 2; and b
3.) A polynucleotide encoding a polypeptide selected from the group consisting of amino acid residue 12 to amino acid residue 41 of SEQ ID NO: 6; Also, a) nucleotide 1 to nucleotide 324 of SEQ ID NO: 1; b) nucleotide 10 to nucleotide 198 of SEQ ID NO: 1; c) nucleotide 76 to nucleotide 1 of SEQ ID NO: 1.
98; d) nucleotides 100-189 of SEQ ID NO: 1; e) nucleotides 1-296 of SEQ ID NO: 5
F) nucleotides 1-132 of SEQ ID NO: 5; and g) nucleotides 34-34 of SEQ ID NO: 5.
An isolated polynucleotide selected from the group consisting of: 123;

In another aspect, the invention also provides an expression vector comprising the following operably linked factors: a transcription promoter; a DNA segment encoding the aforementioned protein; and a transcription terminator. In one embodiment, the DNA segment further encodes a secretory signal sequence operably linked to the protein. In another embodiment, the secretory signal sequence is a polypeptide having the sequence from amino acid residue 1 to amino acid residue 22 of SEQ ID NO: 2.

The present invention also provides a cultured cell into which the aforementioned expression vector has been introduced and which expresses the polypeptide encoded by the DNA segment. The present invention further comprises culturing cells into which the above-described expression vector has been introduced, whereby the cells express the polypeptide encoded by the DNA segment; and recover the expressed polypeptide; A method of manufacturing is provided.

In another aspect, the present invention provides a method of treating a disease associated with a microorganism comprising administering to a mammal a therapeutically effective amount of a polypeptide selected from the group consisting of the following polypeptides: Provided: a) a polypeptide of SEQ ID NO: 2; b) a polypeptide of SEQ ID NO: 6; c) a polypeptide consisting of amino acid residues 23 to 63 of SEQ ID NO: 2; d)
A polypeptide consisting of amino acid residues 31-60 of SEQ ID NO: 2; and e) a polypeptide consisting of amino acid residues 12-40 of SEQ ID NO: 6; whereby the polypeptide reduces disease. In one embodiment, the microbial related disease is associated with the eye. In other embodiments, the microbial-related disease is conjunctivitis. In other embodiments, the microbially-related disease is associated with the ear.

Also provided according to the invention is a method of contraception comprising administering to a mammal a therapeutically effective amount of a polypeptide selected from the group consisting of the following polypeptides:
a) a polypeptide of SEQ ID NO: 2; b) a polypeptide of SEQ ID NO: 6; c) a polypeptide of amino acid residues 23-63 of SEQ ID NO: 2; d) a polypeptide of amino acid residues 31-60 of SEQ ID NO: 2 A peptide; and e) a polypeptide consisting of amino acid residues 12-40 of SEQ ID NO: 6. The invention is further described by the following non-limiting examples.

Example 1 Tissue Distribution Mouse multiple tissue Northern blots (MTN ^™ ; Clontech, Palo Alto, Calif.) Were probed to determine the tissue distribution of murine zamp3 expression. Almost 214bp
PCR-derived probe (SEQ ID NO: 18) was amplified from mouse genomic DNA. Oligonucleotide primers ZC22848 (SEQ ID NO: 13) and ZC22847 (SEQ ID NO: 14) were used as primers. The probe was used in PCR as follows: 1 cycle, 94 ° C for 30 seconds; 30 cycles, 94 ° C for 15 seconds and 68 ° C for 1 minute; then 1 cycle, 68 ° C for 3 minutes.

The resulting DNA fragment was loaded onto a 1.5% agarose gel (OmniPure Agrose, EM Scie
nce, Gibbstown, NJ), and Q
Purified according to the IAquick ^™ method (Qiagen, Chatsworth, CA), the sequence was confirmed by sequence analysis. Primer II Random Prime Labeling System, Amersham according to the manufacturer's instructions.
, Arlington Heights, Ill.) Was used to radiolabel the probe.
The probe was purified using a NUCTRAP push column (Stratagene, La Jolla, CA).

ExpressHyb ^™ (Clontech) solution was used for prehybridization and as a hybridization solution for Northern blots.
Hybridization was performed at 65 ° C. using 1 × 10 ⁶ cpm / ml labeled probe. Blots were then washed at room temperature in 2 × SSC, 0.1% SDS for 1 hour and then at 50 ° C. in 0.1 × SSC, 0.1% SDS for 1 hour. No expression was observed. Thus, normal tissue levels of zamp3 polypeptide mRNA appear to be less than the sensitivity of Northern blot detection. Such observations are consistent with knowledge in the field of several defensins: defensins are constitutively expressed at low levels but are highly inducible upon infection.

Mouse embryo multiple tissue Northern blots (MTN ^™ ; Clontech) were probed and hybridized as previously described. No expression was observed. Mouse RNA Master Blot ^™ was probed as described above. Expression was detected as a faint signal in thyroid, ovary and epididymis.

Example 2 Chromosome Assignment and Placement of Murine zamp3 Commercially Available Mouse T31 Whole Genome Radiation Hybrid (WGRH) Panel (R
Mouse zamp3 was mapped to chromosome 8 in mice using the esearch Genetics, Inc., Huntsville, Ala. and Map Manager QT linkage analysis program. At P = 0.0001, the rat zamp3 is 5
It bound to the marker D8Mit141 with an LOD score of .6. D8Mit141 is on mouse chromosome 8.
Mapped to 0 cM.

The T31 WGRH panel consisted of 100 radiation hybrid clones, plus 2 control DNA (12
9aa donors and A23 recipients). For mapping murine zamp3 with a T31 WGRH panel, 20 μl of reaction was constructed in a 96-well microtiter plate (Stratagene, La Jolla, Calif.) And used in a RoboCycler Gradient 96 thermal cycler (Stratagene).

Each of the 102 PCR reactions contained 2 μl of 10 × KlenTaq PCR reaction buffer (Clontech Labo
ratories, Inc., Palo Alto, Calif., 1.6 μl dNTP mixture (2.5 mM
Each of Perkin-Elmer, Foster City, Calif.), 1 μl sense primer, ZC19960, (SEQ ID NO: 15), 1 μl antisense primer, Z
C19961, (SEQ ID NO: 16), 2 μl of RediLoad (Research Genetics, Inc.), 0.4
It consisted of μl of 50 × Advantage KlenTaq polymerase mixture (Clontech), 25 ng of individual hybrid clones or controls and ddH ₂ O for a total volume of 20 μl.

The reaction was overlaid with an equal amount of mineral oil and sealed. PCR cycle conditions were as follows: initial 1 cycle 5 min denaturation at 94 ° C, 35 cycles 94 ° C.
45 s denaturation at 60 ° C., 45 s annealing at 60 ° C. and 1 min extension at 72 ° C., followed by 7 min extension at 72 ° C. for the last cycle. Reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, Md.).

As described above, the commercially available mouse T31 whole genome radiation hybrid (W
GRH) was used to map murine zamp4 to chromosome 14 in mice. Sense primer ZC25,260 (SEQ ID NO: 17) and antisense primer ZC
25,261 (SEQ ID NO: 18) was used in the reaction. This result shows that mouse chromosome 14
25.88cR of framework marker D14Mit156 on WICGR radiation hybrid map
At the _3000 end, the mouse zamp4 indicates to map. The proximal and distal framework markers were D14Mit156 and D14Mit31, respectively. As will be appreciated, although particular aspects of the invention have been described for purposes of illustration,
Various changes and modifications may be made without departing from the spirit and scope of the invention.
Therefore, the present invention is not limited except by the appended claims.

[Sequence list]

[Brief description of drawings]

FIG. 1 illustrates the structure of the three disulfide bonds of the conserved β-defensin motif.

FIG. 2 shows murine β-defensin 3 (Mbdef3) (Bals et al., Infect. Immunol. 6).
7: 3542-7, 1999) (SEQ ID NO: 11) and murine β-defensin 4 (Mbdef4) gene bank accession No. AAD38852 (SEQ ID NO: 12). The position of a conserved cysteine residue within the cysteine domain is indicated by an asterisk "*".
".

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) C12N 1/15 C12N 1/21 1/19 C12P 21/02 C 1/21 C12N 15/00 ZNAA 5/10 5/00 A C12P 21/02 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS) , MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU , AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW , MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Gao, Zeren, Washington, USA 98052, Redmond, One Hundred Seventeenth Place Northeast 9502 # 3 (72) Inventor Holloway, James El. Washington, USA 98115, Theatre, Northeast 8th Street 835 (72) Inventor Presnell, Scott Earl. North Puget Sound Avenue 2902 F term (Reference) 4B024 AA01 BA31 CA04 DA06 EA04 GA11 HA01 4B064 AG31 CA02 CA10 CA19 CC24 DA01 4B065 AA26X AA90Y AA99Y AB01 AC14 BA02 CA24 CA44 4C084 A01 A02 BA20 BA23 CA25 DA42 NA14 ZB322 ZB352 4H045 AA10 AA11 AA30 BA10 CA40 DA86 EA29 FA74

Claims (17)

[Claims] 1. An isolated polypeptide comprising amino acid residues 31-60 of SEQ ID NO: 2. 2. An isolated polypeptide comprising amino acid residue 1 to amino acid residue 63 of SEQ ID NO: 2. 3. A polypeptide selected from the group consisting of the following polypeptides: a) a polypeptide consisting of amino acid residues 31-60 of SEQ ID NO: 2, b) consisting of amino acid residues 31-63 of SEQ ID NO: 2 A polypeptide consisting of amino acid residues 23 to 60 of SEQ ID NO: 2, a polypeptide consisting of amino acid residues 23 to 63 of SEQ ID NO: 2, e) consisting of amino acid residues 1 to 60 of SEQ ID NO: 2 A) consisting of amino acid residues 1 to 63 of SEQ ID NO: 2 g) a polypeptide consisting of amino acid residues 12 to 41 of SEQ ID NO: 6 h) amino acid residues 12 to 44 of SEQ ID NO: 6 I) a polypeptide consisting of amino acid residues 1-41 of SEQ ID NO: 6, and j) a polypeptide consisting of amino acid residues 1-44 of SEQ ID NO: 6. 4. The polypeptide of claim 1, in combination with a pharmaceutically acceptable vehicle. 5. Inoculating an animal with the polypeptide of claim 1, wherein the polypeptide elicits an immune response in the animal to produce an antibody and the antibody is isolated from the animal. , A method for producing an antibody against a polypeptide. 6. An antibody that specifically binds to the protein of claim 1. 7. An anti-idiotype antibody that is an antibody that specifically binds to the protein of claim 1. 8. An isolated polynucleotide encoding a polypeptide comprising amino acid residues 31-60 of SEQ ID NO: 2. 9. An isolated polynucleotide encoding a polypeptide comprising amino acid residue 1 to amino acid residue 63 of SEQ ID NO: 2. 10. An isolated polynucleotide encoding a polypeptide selected from the group consisting of the following polypeptides: a) a polypeptide consisting of amino acid residues 31-60 of SEQ ID NO: 2, b) SEQ ID NO: 2 A) a polypeptide consisting of amino acid residues 31-63 of c), c) a polypeptide consisting of amino acid residues 23-60 of SEQ ID NO: 2, d) a polypeptide consisting of amino acid residues 23-63 of SEQ ID NO: 2, e) a SEQ ID NO: 2) a polypeptide consisting of amino acid residues 1-60, f) a polypeptide consisting of amino acid residues 1-63 of SEQ ID NO: 2, g) a polypeptide consisting of amino acid residues 12-41 of SEQ ID NO: 6, h) a sequence A polypeptide consisting of amino acid residues 12-44 of number 6; i) a polypeptide consisting of amino acid residues 1-41 of SEQ ID NO: 6; and j) a polypeptide consisting of amino acid residues 1-44 of SEQ ID NO: 6. 11. An isolated polynucleotide encoding a polynucleotide selected from the group consisting of the following polynucleotides: a) A polynucleotide consisting of nucleotides 1-324 of SEQ ID NO: 1. b) a polynucleotide consisting of nucleotides 10 to 198 of SEQ ID NO: 1 c) a polynucleotide consisting of nucleotides 10 to 189 of SEQ ID NO: 1 d) a polynucleotide consisting of nucleotides 76 to 198 of SEQ ID NO: 1 e) SEQ ID NO: 1 F) a polynucleotide consisting of nucleotides 100 to 198 of SEQ ID NO: 1, g) a polynucleotide consisting of nucleotides 100 to 189 of SEQ ID NO: 1, h) a nucleotide of 1 to 296 of SEQ ID NO: 5 I) a polynucleotide consisting of nucleotides 1-132 of SEQ ID NO: 5, j) a polynucleotide consisting of nucleotides 1-123 of SEQ ID NO: 5, k) a polynucleotide consisting of nucleotides 34-123 of SEQ ID NO: 5, And l) a polynucleotide consisting of nucleotides 34 to 132 of SEQ ID NO: 5. 12. An expression vector comprising the following operably linked factors: a transcription promoter, a DNA segment encoding the protein of claim 1, and a transcription terminator. 13. The expression vector of claim 12, wherein the DNA segment further encodes a secretory signal sequence operably linked to the protein. 14. The expression vector according to claim 13, wherein the secretory signal sequence is a polypeptide having a sequence of amino acid residues 1 to 22 of SEQ ID NO: 2. 15. The DN vector into which the expression vector according to claim 12 has been introduced.
A cultured cell expressing the polypeptide encoded by the A segment. 16. Culturing cells into which the expression vector of claim 12 has been introduced, whereby the cells express the polypeptide encoded by the DNA segment and recover the expressed polypeptide. A method for producing a protein, comprising: 17. A method of treating a microbial-related disease, comprising administering to a mammal a therapeutically effective amount of a polypeptide selected from the group consisting of the following polypeptides: a) SEQ ID NO: 2 B) the polypeptide of SEQ ID NO: 6, c) the polypeptide of amino acid residues 23-63 of SEQ ID NO: 2, d) the polypeptide of amino acid residues 31-60 of SEQ ID NO: 2, and e) A polypeptide consisting of amino acid residues 12-40 of SEQ ID NO: 6, whereby the polypeptide reduces the disease.