JPH11137261A - Def2 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject newly identified polynucleotide useful for screening a compound corresponding to an antibiotic activities, determining the roll in getting sick of infection, insufficiency or disease, and preventing, improving and treating the infection, the insufficiency or the disease. SOLUTION: This polynucleotide is the isolated polynucleotide def2 having at least 70% identity with the polynucleotide encoding def2 polypeptide having an amino acid sequence of formula I, and has a nucleic acid sequence of formula II. A DNA fragment is cloned from a deposited strain (NCIMB accession number 40794) of Streptococcus pneumoniae 0100993 strain, sequenced and subjected to a standard cloning method to obtain a complete length clone, and screening method to provide the polynucleotide of this invention encoding the def2 polypeptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This invention claims the benefit of US Provisional Patent Application 60/037535, filed February 10, 1997, and the PCT application (application number unknown), filed November 24, 1997.

[0002]

The present invention relates to newly identified polynucleotides and polypeptides, their production and use, and their variants, agonists and antagonists, and their uses. In particular, the invention relates to novel polynucleotides and polypeptides of the polypeptide deformylase family, referred to herein as "def2".

[0003]

2. Description of the Related Art Streptococci
Forms a medically important microbial genus, which in humans, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis,
It is known to cause several types of diseases, including empyema and endocarditis, especially meningitis such as, for example, infection of the cerebrospinal fluid. Since more than 100 years have passed since the isolation of the genus Streptococcus, Streptococcus pneumoniae is one of the microorganisms that has been studied in more detail. For example, in fact, much of the early view that DNA is the genetic material comes from Griffith and Avery, Mac
Affirmed in studies by leod and McCarty. Despite the vast amount of work on Streptococcus pneumoniae, many questions remain regarding the toxicity of this microorganism. It is particularly preferred to use Streptococcus genes and gene products as targets for the development of antibiotics.

[0004] The frequency of Streptococcus pneumoniae infections has increased dramatically over the past few decades. This has been attributed to the emergence of multiple antibiotic resistant strains and an increasing population of people with a compromised immune system. It is no longer uncommon to isolate Streptococcus pneumoniae strains that are resistant to some or all of the standard antibiotics. This phenomenon requires new antimicrobial agents, vaccines and diagnostic tests for this organism.

It is apparent that a need exists for an agent, such as the def2 embodiment of the present invention, which has the advantage of being useful for screening compounds for antibiotic activity. Such factors are also useful in determining their role in the pathogenesis of infection, dysfunction or disease.
To find a way to prevent, ameliorate or cure infection, dysfunction or disease, there is also a need to identify and characterize such factors and their antagonists and agonists. Certain polypeptides of the present invention have amino acid sequence homology to known fms, pdf, def proteins (GenBank accession number Z96934).

[0006]

SUMMARY OF THE INVENTION One object of the present invention is to provide an amino acid sequence shown in Table 1 [SEQ ID NO: 2 or 4],
It is to provide a polypeptide identified as a novel def2 polypeptide by homology between known amino acid sequence (s) of other proteins such as fms, pdf, def proteins. It is a further object of the present invention to provide a polynucleotide encoding a def2 polypeptide, in particular a polynucleotide encoding a polypeptide referred to herein as def2.

[0007]

In a particularly preferred embodiment of the present invention, the polynucleotide comprises a def2 polypeptide comprising the full-length gene and having the sequence shown in Table 1 [SEQ ID NO: 1 or 3]. Or a variant thereof. Another particularly preferred embodiment of the present invention is a novel def2 protein from Streptococcus pneumoniae having the amino acid sequence of Table 1 [SEQ ID NO: 2 or 4] or a variant thereof. As further aspects of the present invention, including mRNA, cDNA, genomic DNA,
def2, especially Streptococcus pneumoniae
An isolated nucleic acid molecule encoding f2 is provided. Further embodiments of the present invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.

According to another aspect of the present invention there is provided the use of a polynucleotide of the present invention for therapeutic or prophylactic purposes, particularly in genetic immunity. Particularly preferred embodiments of the present invention are naturally occurring allelic variants of def2 and the polypeptides encoded thereby. Another aspect of the invention includes a novel polypeptide of Streptococcus pneumoniae, referred to herein as def2, and biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and the like. A composition comprising: Particularly preferred embodiments of the present invention include variants of the def2 polypeptide encoded by the natural allele of the def2 gene. In a preferred embodiment of the present invention, a method for producing the def2 polypeptide is provided.

In accordance with yet another aspect of the present invention, there are provided inhibitors to such polypeptides, including, for example, antibodies, which are useful as antibacterial agents. According to certain preferred embodiments of the present invention, assess def2 expression, treat disease, assess genetic mutations, and administer def2 polypeptides or polynucleotides to organisms to control bacteria, especially Streptococcus pneumoniae bacteria. Materials, compositions, and methods for raising an immune response are provided.
According to certain preferred embodiments of this and other aspects of the invention, there are provided polynucleotides that hybridize to the def2 polynucleotide sequence, particularly under stringent conditions. In certain preferred embodiments of the present invention, d
Antibodies to ef2 polypeptides are provided.

In another embodiment of the present invention, there is provided a method of identifying a compound that binds to, or otherwise interacts with, a polypeptide or polynucleotide of the present invention and inhibits or activates its activity, comprising: The polypeptide or polynucleotide is contacted with the compound under conditions that allow the compound to be bound or otherwise interacted with the polypeptide or polynucleotide of the invention to bind or interact with the compound or polynucleotide. (Such binding or interaction is associated with a secondary component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound); Or by detecting the presence or absence of a signal resulting from binding or interaction with the polynucleotide, Or compound binds to the polypeptide or polynucleotide, and otherwise interact,
A method comprising determining whether to activate or inhibit the activity is provided.

According to yet another aspect of the present invention, def
2 Agonists and antagonists, preferably bacteriostatic or bactericidal agonists and antagonists are provided. In a further aspect of the invention there is provided a composition comprising a def2 polynucleotide or a def2 polypeptide for administration to a cell or a multicellular organism.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from a reading of the ensuing description and other portions of this specification.

[0012]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel def2 polypeptides and polynucleotides as described in more detail below. In particular, the present invention provides fms, pdf,
Novel def2 polypeptides and polynucleotides of Streptococcus pneumoniae, which are related by amino acid homology to def polypeptides. The invention particularly relates to def2 having the nucleotide and amino acid sequences shown in Table 1 as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0013]

TABLE 1 def2 polynucleotide and polypeptide sequences

(A) A sequence derived from the Streptococcus pneumoniae def2 porinkreotide sequence [SEQ ID NO: 1]

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(B) Streptococcus pneumoniae def2 deduced from the polynucleotide sequences in this table
Polypeptide sequence [SEQ ID NO: 2]

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(C) a polynucleotide sequence having a Streptococcus pneumoniae def2.ORF sequence [SEQ ID NO: 3]

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(D) Streptococcus pneumoniae deduced from the polynucleotide ORF sequences in this table
ef2 polypeptide sequence [SEQ ID NO: 4]

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Deposited Materials Deposited strains, including the Streptococcus pneumoniae 0100993 strain, were deposited on April 11, 1996, at the National Collection of Industrial and Marine Bacteria, 23 St., Matchell Drive, AB21RY, Aberdeen, Scotland. Accession number 4 to Limited (hereinafter referred to as “NCIMB”)
Deposited under 0794. The deposited strain is, at the time of deposit, Streptococcus pneumoniae 0100993
Was named. Streptococcus pneumoniae 01 in Escherichia coli on April 17, 1996
Similarly, the 00993 DNA library has accession number 408
Under 00, deposited with NCIMB. This S. pneumoniae strain deposit is referred to herein as the "deposited strain" or "DNA of the deposited strain."

The deposited strain contains the full length def2 gene. The sequence of the polynucleotide contained in the deposited strain, as well as the amino acid sequence of the polypeptide encoded thereby, will control for any inconsistency with the sequence description herein. Deposits of the deposited strains are made under the terms of the Budapest Treaty on International Recognition of Microbial Deposits for Patent Procedure. After the patent is issued, there are no restrictions or conditions, and the shares are eventually sold. The deposited strain is provided solely for the convenience of those skilled in the art and does not acknowledge that the deposit is a feasible requirement, as required under 35 USC 112. A license is required to make, use, or sell the deposited strain, but such license is not granted here.

[0020] One aspect of the present invention is to provide an isolated nucleic acid molecule encoding a mature polypeptide that can be expressed by Streptococcus pneumoniae strain 0100993 contained in the deposited strain. Furthermore, the present invention provides the def2 nucleotide sequence of the DNA in the deposited strain and the amino acid sequence encoded thereby. The present invention also provides a def2 polypeptide sequence isolated from the deposited strain and an amino acid sequence derived therefrom.

Polypeptides The polypeptides of the present invention are listed in Table 1 [SEQ ID NO: 2 or 4]
(Especially the mature polypeptide) and, in particular, has a biological activity of def2 and is at least 70% identical to the polypeptide of Table 1 [SEQ ID NO: 1 or 3] or a corresponding part thereof, preferably Table 1 [ At least 80% identity to the polypeptide of SEQ ID NO: 2 or 4], more preferably at least 90% similarity to the polypeptide of Table 1 [SEQ ID NO: 2 or 4] (more preferably at least 90% And polypeptides and fragments having even more preferably at least 95% similarity (even more preferably at least 95% identity) to the polypeptides of Table 1 [SEQ ID NO: 2 or 4]. And generally, a polypeptide comprising at least 30 amino acids, more preferably at least 50 amino acids. Also encompasses portions of such polypeptides with such portion of the peptide.

The present invention also provides

X- (R ₁ ) _m- (R ₂ )-(R ₃ ) _n -Y

Wherein X at the amino terminal is hydrogen, Y at the carboxyl terminal is hydrogen or metal, and R ₁ and
R ₃ is any amino acid residue, m is 1 to 1000
And n is an integer of 1 to 1000 or 0, and R ₂ represents the amino acid sequence of the present invention, in particular, the amino acid sequence of Table 1]. In the above formulas, R ₂ is bonded to R ₁ amino terminal residues at its left, its carboxy terminal residue is directed to bind to R ₃ in the right. When m and / or n is greater than 1, the chain of amino acid residues represented by any of the R groups may be either a heteropolymer or a homopolymer, with a heteropolymer being preferred.

A fragment is a variant polypeptide having an amino acid sequence that entirely is the same, but not all, of the amino acid sequence of the above-described polypeptides. As with def2 polypeptides, fragments are "self-supporting" or they form one part or region, most preferably a single contiguous region, a larger polypeptide forming a single larger polypeptide. It may be contained in a peptide.

Preferred fragments are described, for example, in Table 1.
Includes truncated polypeptides having some of its variants, such as the amino acid sequence of [SEQ ID NO: 2 or 4] or a series of residues including the amino terminus or a series of residues including the carboxyl terminus. I do. Also preferred is a degraded form of the polypeptide of the present invention in a host cell, especially Streptococcus pneumoniae. In addition, alpha helices and alpha helix forming regions, beta sheets and beta sheet forming regions, turns and turn forming regions, coils and coil forming regions, hydrophilic regions, hydrophobic regions, alpha amphiphilic regions, beta amphiphilic regions, variable regions Fragments characterized by structural or functional attributes, such as those comprising an interface-forming region, a substrate binding region, and a high antigen index region, are also preferred fragments.

[0026] Biologically active fragments are also preferred fragments. Biologically active fragments include those with similar or improved activity, or those with reduced undesired activity, including def2
Is a fragment that mediates the activity of In addition, animals,
Also included are those fragments that are antigenic or immunogenic, especially in humans. Particularly preferred are fragments consisting of a receptor or domain of a group of enzymes that confers a function essential to the survival of Streptococcus pneumoniae or the ability to initiate or maintain the disease in an individual, particularly a human. Variants that are fragments of the polypeptides of the present invention can be used for the production of the corresponding full-length polypeptides by peptide synthesis methods. Thus, these variants can be used as intermediates for producing the full-length polypeptides of the invention.

In addition to the standard one-letter and three-letter codes for amino acids, the term "X" or "Xaa" is also used to describe certain polypeptides of the present invention. "X" and "Xaa" mean that any of the twenty naturally occurring amino acids can be at such intended position in the polypeptide sequence.

Polynucleotides Another embodiment of the present invention relates to isolated polynucleotides encoding a def2 polypeptide having the deduced amino acid sequence of Table 1 [SEQ ID NO: 2 or 4], closely related polynucleotides and polynucleotides thereof. About the variant. Table 1 [SEQ ID NO: 1 or 3]
Standards for cloning and sequencing chromosomal DNA fragments from bacteria using Streptococcus pneumoniae 0100993 as a starting material, using the information herein, such as the polynucleotide sequences shown in Using the methods of cloning and screening, a polynucleotide of the invention encoding a def2 polypeptide can be obtained. For example, to obtain a polynucleotide sequence of the present invention, such as the sequence shown in Table 1 [SEQ ID NOs: 1 or 3], typically
Chromosomal DNA of Streptococcus pneumoniae 0100993 in Escherichia coli or other suitable host
Is probed with a radiolabeled oligonucleotide derived from the subsequence, preferably an oligonucleotide 17 times or more in length. The clones having the same DNA as the probe can then be distinguished using stringent conditions. By sequencing individual clones identified with sequencing primers designed from the original sequence, it is possible to extend the sequence in both directions and determine the full length. Conveniently, such sequencing is performed using denatured double-stranded DNA prepared from a plasmid clone. Suitable techniques are described in Maniatis, T., Fritsch, EF and Sambrook et al., MOL
ECULAR CLONING: A LABORATORYMANUAL, 2nd Ed .; Cold
Spring Harbor Laboratory Press, Cold Spring Harbo
r, New York (1989) (see, inter alia, screening by hybridization 1.90 and sequencing of denatured double-stranded DNA templates 13.70). For example, the polynucleotide shown in Table 1 [SEQ ID NO: 1 or 3] is Streptococcus pneumoniae 0
It was found in a DNA library derived from 100933.

The DNA sequence shown in Table 1 [SEQ ID NO: 1 or 3] has the approximate number of amino acid residues shown in Table 1 [SEQ ID NO: 2 or 4]. It has an open reading frame that encodes the protein it has. Nucleotide number 1
And the polynucleotide of SEQ ID NO: 1 between the stop codon starting at nucleotide number 409 of SEQ ID NO: 1 encodes the polypeptide of SEQ ID NO: 2. The def2 polypeptides of the present invention are structurally related to other proteins in the polypeptide deformylase family.

The present invention provides a polynucleotide sequence that is identical over its entire length to the coding sequence in Table 1 [SEQ ID NOS: 1 or 3]. The invention also relates to the coding sequence for the mature polypeptide or fragment thereof as well as the reading sequence in the reading frame having other coding sequences, such as coding sequences for leader or secretory sequences, pre, pro or prepro protein sequences. A coding sequence for a polypeptide or fragment is provided. Polynucleotides also include non-coding 5 'such as, for example, transcribed sequences, untranslated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequences that encode additional amino acids. And non-coding sequences, including, but not limited to, 3 'sequences. For example, it can encode a marker sequence that facilitates purification of the fusion polypeptide. In certain embodiments of the invention, the marker sequence is provided in a pQE vector (Qiagen, Inc.), Gentz et al., Proc. Natl. Acad. Sci. USA (1989) 8
6: 821-824, or a hexa-histidine peptide or an HA tag (Wilson et al., C
ell, 37: 767 (1984)). The polynucleotide of the present invention,
Includes, but is not limited to, polynucleotides consisting of structural genes and naturally occurring sequences that regulate gene expression.

A preferred embodiment of the present invention comprises nucleotides 1 to 409 shown in SEQ ID NO: 1 of Table 1.
With a nucleotide immediately upstream of or including the nucleotide.

The present invention also provides a compound of the formula:

X- (R ₁ ) _m- (R ₂ )-(R ₃ ) _n -Y

Wherein X at the 5 ′ end of the molecule is hydrogen, Y at the 3 ′ end of the molecule is hydrogen or a metal, R ₁ and R ₃ are any nucleic acid residues, and m is 1 to 3000
And n is an integer of from 1 to 3000 or 0, and R ₂ represents a nucleic acid sequence of the present invention, in particular, a nucleic acid sequence selected from Table 1]. In the above described formula of the polynucleotide, R ₂ is 5 'terminal residue is bonded to R ₁ at its left and its 3' terminal residue is, bound to R ₃ in the right. m
When and / or n is greater than 1, the chain of nucleic acid residues represented by any of the R groups may be either a heteropolymer or a homopolymer, with a heteropolymer being preferred. In a preferred embodiment, m and / or n
Is an integer between 1 and 1000.

The polynucleotide of the present invention is most preferably derived from Streptococcus pneumoniae, but is also preferably obtained from a taxonomic organism of the same genus. Also, for example, they may be obtained from the same department or order in taxonomics.
As used herein, the term "polynucleotide encoding a polypeptide" refers to a polypeptide of the present invention, in particular, a bacterial polypeptide, more specifically, an amino acid sequence shown in Table 1 [SEQ ID NO: 2 or 4]. And a polynucleotide comprising a sequence encoding the polypeptide of Streptococcus pneumoniae def2. The term includes a single contiguous or non-contiguous region encoding the polypeptide (eg, interrupted by integrated phage or inserted sequences or editing), with additional regions that may include coding and / or non-coding sequences. As a whole). The invention further relates to variants of the polynucleotides described herein that encode variants of the polypeptide having the deduced amino acid sequence of Table 1 [SEQ ID NO: 2 or 4]. Variants that are fragments of the polynucleotides of the invention can be used in the synthesis of full-length polynucleotides of the invention.

Further preferred embodiments are those in which several, a few, 5-10, 1-5, 1-3, 2 or 1 or 0 amino acid residues are substituted in any combination,
D of Table 1 [SEQ ID NO: 2 or 4] deleted or added
def2 having the amino acid sequence of ef2 polypeptide
A polynucleotide encoding a variant. Particularly preferred are silent substitutions, additions and deletions that do not alter the properties and activities of def2.

Further preferred embodiments of the present invention are shown in Table 1.
A polynucleotide having at least 70% identity over the full length of a polynucleotide encoding a def2 polypeptide having the amino acid sequence set forth in [SEQ ID NO: 2 or 4], and a polynucleotide complementary to such a polynucleotide. . Also most preferred are polynucleotides consisting of a region having at least 80% identity over its entire length to a polynucleotide encoding a def2 polypeptide and polynucleotides complementary thereto. In this regard, polynucleotides having at least 90% identity over their full length to the same are particularly preferred, especially at least 95%.
% Are particularly preferred. Furthermore, among those having at least 95% identity, those having at least 97% identity are more preferred, and those with at least 98% and at least 99% identity are particularly preferred. Those with at least 99% identity are more preferred. A preferred embodiment is a polynucleotide that encodes a polypeptide that retains substantially the same biological function or activity as the mature polypeptide encoded by the DNA of Table 1 [SEQ ID NO: 1 or 3].

[0037] The present invention further relates to polynucleotides that hybridize to the sequences described herein. In this regard, the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As used herein, the terms "stringent conditions" and "stringent hybridization conditions" refer only to the case where hybridization has at least 95%, preferably at least 97%, identity between sequences. Means what happens. As an example of stringent hybridization conditions, 50% formaldehyde, 5 × SSC (150 mM
NaCl, 15 mM trisodium citrate), 50 mM
Sodium phosphate (pH 7.6), 5x Denhart (Denh
ardt's) solution, 10% dextran sulfate and 20μ
g / ml denatured truncated salmon sperm DNA in a solution comprising
Incubation at 2 ° C. overnight, followed by washing the hybridization support with 0.1 × SSC (about 65 ° C.). Hybridization and washing conditions are known and are described in Sambrook et al., MOLECULAR CLONIN.
G: A LABORATORY MANUAL, Second Edition, Cold Sprin
g Harbor, NY (1989), especially in Chapter 11.

The present invention provides for the use of a suitable library comprising substantially the complete gene of the polynucleotide sequence set forth in SEQ ID NO: 1 under stringent hybridization conditions, A polynucleotide comprising a polynucleotide sequence obtainable by screening with a probe having the following or a fragment thereof, and isolating the DNA sequence. Fragments useful for obtaining such polynucleotides include, for example, the probes and primers described herein.

As further described herein for the analysis of the polynucleotides of the present invention, for example, the polynucleotides of the present invention as described above may be used as hybridization probes for RNA, cDNA, and genomic DNA, and def2 Can be isolated, and cDNA and genomic clones of other genes with high sequence similarity to the def2 gene can be isolated. Such a probe will generally have at least 15 bases. Preferably, such a probe has at least 30
It consists of bases and may have at least 50 bases. Particularly preferred probes have at least 30 bases and have 5
It has no more than 0 bases. For example, the coding region of the def2 gene can be isolated by screening using the DNA sequence of Table 1 (SEQ ID NO: 1 or 3) to synthesize an oligonucleotide probe. A cDNA, genomic DNA or mRNA library is then screened using a labeled oligonucleotide having a sequence complementary to the sequence of the gene of the invention to determine which members of the library will hybridize to the probe.

The polynucleotides and polypeptides of the present invention may be used, for example, as research reagents and materials for the discovery of treatments and diagnostics for diseases, especially human diseases, as further described herein with respect to polynucleotide analysis. Can be used. Polynucleotides of the invention, which are oligonucleotides derived from the sequence of Table 1 (SEQ ID NO: 1 or 2 or 3 or 4), can be used in the methods described herein, but are preferably
To determine whether the polynucleotides identified herein are transcribed in bacteria in whole or in part in infected tissues. Such sequences are also useful in diagnosing the stage and type of infection whose etiology has been reached.

The present invention also relates to a mature protein wherein an additional amino or carboxy terminal amino acid is added, or an amino acid is added inside the mature polypeptide (eg, when the mature form has one or more polypeptide chains). Provided is a polynucleotide encoding a polypeptide. Such sequences play a role, inter alia, in the processing of the protein from the precursor to the mature form, carry the protein, increase or decrease the half-life of the protein, or manipulate the protein for analysis or production. Can be facilitated. As is common in vivo, the added amino acids are processed away from the mature protein by cellular enzymes. A precursor protein having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide.
When the prosequence is removed from such an inactive precursor, it is generally activated. Some or all of the prosequence can be removed prior to activation. Generally, such precursors are called proproteins.

Standard symbols A, G, C, T for nucleic acid bases
In addition to / U, the term "N" can be used to describe certain polynucleotides of the present invention.
"N" is a base that has the effect of forming a premature terminal codon at such reading frame when reading in the correct reading frame when acting together with adjacent nucleotide positions. Unless otherwise preferred, it means that any of the four DNA or RNA bases is at that given position in the DNA or RNA sequence.

In general, the polynucleotides of the present invention are mature proteins, mature proteins with a leader sequence (also referred to as preproteins), precursors of mature proteins having one or more prosequences that are not the leader sequence of the preprotein. It encodes a preproprotein that is a precursor of a proprotein having a body, or leader sequence, and, generally, one or more prosequences that are removed during a processing step that produces an active, mature form of the polypeptide.

Vectors, Host Cells, Expression The present invention also relates to polynucleotides of the invention or vectors comprising polynucleotides, host cells genetically engineered with vectors of the invention and recombinant polypeptides of the invention. For production. Further, such a protein can be produced using a cell-free translation system and RNA derived from the DNA construct of the present invention. For recombinant production, host cells can be genetically engineered to incorporate an expression system or a portion thereof, or a polynucleotide of the present invention. Introduction of a polynucleotide into a host cell
Davis et al., BASIC METHODS IN MOLECU, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
LAR BIOLOGY (1986) and Sambrook et al., MOLECULAR CLO
NING: A LABORATORY MANUAL, 2nd Ed. Cold Spring Har
bor Laboratory Press, Cold Spring Harbor, NY (198
This can be done by methods described in several standard laboratory manuals, such as 9).

Representative examples of suitable hosts include streptococci (stre
ptococcus), staphylococcus, enterococcus, E. coli, streptomyces and Bacillus subtili
s) Bacterial cells such as cells; fungal cells such as yeast cells and Aspergillus cells; Drosophila S2 and Spodopte
ra) insect cells such as Sf9 cells; CHO, COS, HeLa, C1
27, 3T3, BHK, HEK293 and animal cells such as Bowes melanoma cells; and plant cells.

A wide variety of expression systems can be used for producing the polypeptides of the present invention. Such systems include, inter alia, chromosomal, episomal and viral derived systems,
For example, bacterial plasmid derived, bacteriophage derived, transposon derived, yeast episomal derived, insertion factor derived, yeast chromosomal factor derived, baculovirus, SV
Papova virus, vaccinia virus, such as 40,
Includes vectors derived from viruses such as adenovirus, chicken poxvirus, pseudorabies virus and retrovirus, and vectors derived from combinations thereof, such as vectors derived from plasmids such as cosmids and phagemids and bacteriophage genetic elements. . Expression system constructs may have regulatory regions that control as well as cause expression. In general, systems or vectors suitable for maintaining, amplifying or expressing the polynucleotide in the host and / or expressing the polypeptide may be used for expression in this regard. Suitable DNA sequences can be obtained, for example, from Sambrook et al., MOLECULAR CLONIN.
G, A LABORATORY MANUAL (supra), may be inserted into the expression system by any of a variety of well-known conventional techniques.

To secrete the translated protein into the endoplasmic reticulum lumen, periplasmic space or extracellular environment, an appropriate secretion signal may be inserted into the expressed polypeptide. These signals may be native to the polypeptide or they may be heterologous signals. Polypeptides of the invention include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. It can be recovered and purified from recombinant cell culture by known methods. Most preferably, high performance liquid chromatography is used for purification. The polypeptide is isolated and / or
Alternatively, if denatured during purification, the protein can be reconstituted into an active conformation using well known methods for regenerating proteins.

Diagnostic Assays The present invention also provides d-types of the invention for use as diagnostic reagents.
Also related to the use of ef2 polynucleotides. Def2 in eukaryotes, especially mammals, especially humans
Detection provides a diagnostic method for diagnosis of a disease. Detection of eukaryotes (also referred to herein as "individuals"), particularly mammals, and especially humans, at the nucleic acid level by various methods when they are or are likely to be infected by organisms containing the def2 gene. it can.

Nucleic acids for diagnosis can be obtained from cells and tissues of infected individuals, such as bone, blood, muscle, cartilage and skin. Genomic DNA can be used for direct detection or can be amplified enzymatically by using PCR or other amplification methods before subjecting it to analysis. RNA, cDNA and genomic DNA can be used in the same way. Upon amplification, the prokaryotic species and strains present in the individual can be characterized by genotyping prokaryotic genes. Deletions and insertions can be detected by a change in the size of the amplified product relative to the genotype of the control sequence. Point mutations can be identified by hybridizing the amplified DNA to a labeled def2 polynucleotide sequence. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences can also be detected by changes in the electrophoretic mobility of the DNA fragments in the gel, with or without denaturing agents, or by direct DNA sequencing. For example, Myers et al., Science, 230: 1242 (1985).
checking ... Sequence changes at specific locations can also be detected by nuclease protection assays, such as RNase and S1
It can also be revealed by protection or chemical cleavage. For example, Cotton et al., Proc. Natl. Acad. Sci., US
A, 85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the present invention can also be obtained at the DNA level by various techniques.
For example, it can be detected by serotyping. For example, mutations can be detected using RT-PCR. It is particularly preferred to use RT-PCR in combination with an automatic detection system, such as GeneScan. RNA, cDNA or genomic DNA can also be used for the same purpose by PCR or RT-PC.
R can be used. As an example, mutations can be identified and analyzed using PCR primers complementary to the nucleic acid encoding def2. Table 2 shows examples of typical primers.

[0051]

Table 2 Primers for amplification of def2 polynucleotide SEQ ID No. Primer sequence 55'-CTTGCCAGAGATTTGCAGGATAC-3 '65'-AGCACTGGGTTAAACATGACCAC-3'

The present invention also provides a compound of the formula:

X- (R ₁ ) _m- (R ₂ )-(R ₃ ) _n -Y

Wherein X at the 5 ′ end of the molecule is hydrogen, Y at the 3 ′ end of the molecule is hydrogen or a metal, R ₁ and R ₃ are any nucleic acid residues, and m is An integer of 1 to 20 or 0, n is an integer of 1 to 20 or 0, and R ₂ represents a primer sequence of the present invention, particularly a primer sequence selected from Table 2]. I do. In the above described formula of the polynucleotide, R ₂ is its 5 'terminal residue bound to R ₁ at its left and its 3' terminal residue is, bound to R ₃ in the right. When m and / or n is greater than 1, the chain of nucleic acid residues represented by any of the R groups may be either a heteropolymer or a homopolymer, and may be complementary to the polynucleotide region of Table 1. Heteropolymers are preferred. In a preferred embodiment, m and / or n is an integer between 1 and 10.

The present invention further provides these primers comprising 1, 2, 3 or 4 nucleotides removed from the 5 'and / or 3' end. These primers can be used, inter alia, to amplify def2 DNA isolated from samples derived from individuals. The primers can be used to amplify a gene isolated from an infected individual, and the gene can then be subjected to various techniques for examining DNA sequences. Thus, detecting mutations in the DNA sequence and using this to diagnose infection,
Infectious agents can be serotyped and / or classified.

The present invention further provides a method for diagnosing a disease, preferably a bacterial infection, more preferably an infection with Streptococcus pneumoniae, wherein the expression level of a polynucleotide having the sequence of Table 1 [SEQ ID NO: 1 or 3] is increased. Is determined from a sample from the individual. Increasing or decreasing expression of the def2 polynucleotide can be determined by any method known in the art for quantifying a polynucleotide, such as amplification, PCR, RT-PCR, RNase protection, Northern blotting, and other hybridization methods. Can be used to measure.

In addition, a diagnostic assay according to the invention for detecting overexpression of the def2 protein compared to a normal control tissue sample can be used, for example, to detect the presence of an infection. Assay techniques that can be used to determine levels of def2 protein, in a sample derived from a host are well-known to those of skill in the art. Such assays include radioimmunoassays, competitive binding assays, western blot analysis and ELISA assays.

Antibodies The polypeptides or variants thereof, or cells expressing them, of the present invention can be used as immunogens to produce antibodies immunospecific for such polypeptides.
As used herein, "antibody" includes monoclonal and polyclonal antibodies, chimeras, single chains, monkey and humanized antibodies, and Fab fragments, including the products of a Fab immunoglobulin expression library. Antibodies obtained by antagonizing the polypeptide of the present invention,
Fragments with polypeptides or epitopes,
An analog or cell, preferably in a non-human animal,
It can be obtained by administering using conventional protocols. For preparing monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. See, for example, Kohler, G. and Milstein, C., Nature, 256: 495-497 (1975); Kozb.
or et al., Immunology Today, 4:72 (1983); Cole et al., MON.
OCLONAL ANTIBODIES AND CANCER THERAPY, Alan R Lis
s, Inc., pp. 77-96 (1985).

Using techniques for producing single-chain antibodies (US Pat. No. 4,946,778), single-chain antibodies against the polypeptides of the present invention can be produced. Also,
Transgenic mice or other organisms, such as other mammals, can be used to express humanized antibodies. Alternatively, phage displa
y) an antibody gene that has binding activity to a polypeptide from a repertoire of PCR-amplified v genes of human-derived lymphocytes screened for retaining anti-def2 using the technique, or from an intact library; (McCafferty, J. et al., Nat
ure 348: 552-554 (1990); Marks, J. et al., Biotechnology.
10: 779-783 (1992)). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., Nature 352: 624-628 (199).
1)).

When there are two antigen binding domains,
Each domain can be directed to a different epitope, which is referred to as a "bispecific" antibody. Clones expressing the polypeptide can be isolated or identified using the antibodies described above, and the polypeptide can be purified by affinity chromatography. Thus, among others, antibodies to the def2 polypeptide can be used to treat infections, especially bacterial infections.

[0060] Polypeptide variants include antigenically, epitopically or immunologically equivalent variants that form a particular aspect of the invention. The term "antigenically equivalent derivative" as used herein, when induced against a protein or polypeptide of the invention, prevents an immediate physical interaction between the pathogen and the mammalian host. Polypeptides or equivalents thereof that are specifically recognized by certain antibodies. As used herein, the term "immunologically equivalent derivative" refers to an antibody that, when used in a suitable formulation to elicit an antibody in a vertebrate, is not immediately physical between the pathogen and the mammalian host. Includes peptides or equivalents that act to interfere with the interaction.

A polypeptide, such as an antigenically or immunologically equivalent derivative, or a fusion protein thereof, is used as an antigen to immunize a mouse or other animal, such as a rat or chicken. Fusion proteins can confer stability on a polypeptide. The antigen may be, for example, by conjugation, to produce an immunogenic carrier protein such as bovine serum albumin (BSA) or keyhole limpet haemocyanin (K).
LH). Alternatively, a multi-antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof, has sufficient antigenicity to improve immunogenicity. No need to use carrier.

It is preferred that the antibody or variant thereof be modified to reduce immunogenicity in the individual. For example, if the individual is human, the antibody is most preferably "humanized"; for example, Jones, P. et al., Nature 321: 522-525.
(1986) or Tempest et al., Biotechnology 9: 266-273.
As described in (1991), the complementarity-determining regions of a hybridoma-derived antibody have been grafted onto human monoclonal antibodies. Use of the polynucleotides of the present invention in genetic immunization is preferably by direct injection of plasmid DNA into muscle (Wolff et al., Hum. Mol. Genet 1: 363 (199).
2); Manthorpe et al., Hum. Gene Ther. 4: 419 (196
3)), delivery of DNA complexed with a specific protein carrier (Wu et al., J. Biol Chem. 264: 16985 (1989)), DNA co-precipitation using calcium phosphate (Benvenisty & Reshef,
PNAS USA 83: 9551 (1986)), DNA encapsulation in various forms of liposomes (Kaneda et al., Science 243: 375 (198).
9)), particle bombardment (Tang et al., Nature 356: 152 (199)
2); Eisenbraun et al., DNA Cell Biol. 12: 791 (1993))
And appropriate delivery methods such as in vivo infection with cloned retroviral vectors (Seeger et al., PNAS USA 81: 5849 (1984)).

Antagonists and Agonists-Assays and Molecules In addition, the polypeptides of the present invention can be used to couple small molecule substrates with ligands, for example, in cells, cell-free preparations, chemical libraries, and natural product mixtures. Binding can also be evaluated. These substrates and ligands may be natural substrates and ligands, or may be structural or functional mimetics. For example, Coligan et al., Current
Protocols in Immunology 1 (2): See Chapter 5 (1991). The present invention also provides a method of screening compounds to identify compounds that enhance (agonist) or block (antagonist) the action of def2 polypeptides or polynucleotides, especially those that are bacteriostatic and / or bactericidal. provide. Screening methods include high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mixture comprising a def2 polypeptide and a labeled substrate or ligand of such a polypeptide, a cell compartment such as a membrane, cell envelope or cell wall, or a preparation of any of these. Is incubated in the presence or absence of a candidate molecule that may be a def2 agonist or antagonist. The ability of a candidate molecule to agonize or antagonize a def2 polypeptide is reflected in reduced binding of a labeled ligand or reduced production of a product from such a substrate. Molecules that have no effect when bound, ie, d
Molecules that do not elicit the effects of an ef2 polypeptide will most likely be good antagonists. Molecules that bind well and increase the rate of product formation from the substrate are agonists. Detection of the rate or level of product formation from the substrate can be enhanced by using a reporter system. Reporter systems useful in this regard include, but are not limited to, colorimetrically labeled substrates that are converted to products, reporter genes that respond to changes in def2 polynucleotide or polypeptide activity, and binding assays known in the art. It is not limited.

Another example of an assay for def2 antagonists is to use def2 and a potential antagonist under conditions suitable for competitive inhibition assays to convert def2 binding molecules, recombinant def2 binding molecules, natural substrates or ligands, or substrates or ligands. This is a competition assay that mixes with the mimic. The def2 molecule can be labeled, for example, with a radioactive or colorimetric compound, and the number of def2 molecules bound to the binding molecule or converted to product can be accurately determined to assess the effect of the potential antagonist.

Potential antagonists are small organic molecules, peptides, which bind to the polynucleotide or polypeptide of the present invention, thereby inhibiting or extinguishing its activity.
Includes polypeptides and antibodies. Potential antagonists may also bind to the same site on the binding molecule, such as binding molecules that do not induce def2 inducing activity, such as closely related proteins or antibodies that prevent the action of def2 by eliminating def2 from binding. Small organic molecules,
It may be a peptide, polypeptide.

[0069] Potential antagonists include small molecules that bind and occupy the binding site of the polypeptide, thereby preventing binding to cellular binding molecules and preventing normal biological activity. Examples of small molecules include, but are not limited to, small organic molecules, peptides, peptide-like molecules. Other potential antagonists include antisense molecules (for a description of these molecules, see Okano, J., Neurochem. 56: 560 (1991); OLI
GODEOXYNUCLEOTIDES AS ANTISENSE INHIBTORSOF GENE E
XPRESSION, CRC Press, Bockalaton, FL (1988)). Preferred potential antagonists include compounds related to def2 and variants thereof.

The DNA sequences obtained herein can each be used for the discovery and development of antimicrobial compounds. Upon expression, the encoded protein can be used as a target for screening antimicrobial drugs. In addition, using the encoded protein or the DNA sequence encoding the amino-terminal region of the Shine-Delgarno or other individual mRNA translatability sequence to construct an antisense sequence that regulates expression of the coding sequence of interest can do.

The present invention also relates to the sequelae of infection,
Provided is the use of a polypeptide, polynucleotide or inhibitor of the invention to disrupt an initial physical interaction between a pathogen and a mammalian host. In particular, the molecules of the present invention; protection of bacteria, especially Gram-positive bacteria, from adhering to mammalian extracellular matrix proteins on internal devices, or to wound extracellular matrix proteins;
Upon initiation of mammalian tyrosine kinase phosphorylation, d
Blocking ef2 protein-mediated mammalian cell entry (Rosen
60: 2211 (1992)); Blocking bacterial adhesion between mammalian extracellular matrix proteins and the bacterial def2 protein that mediates tissue damage; Implanting indwelling devices or other surgical procedures It can be used to block the normal progression of etiology in infections initiated by other than.

[0069] The antagonists and agonists of the present invention can be used, for example, to inhibit and treat diseases. Helicobacter pylori
Bacteria (also referred to herein as H. pylori) are gastric cancer,
It affects more than one-third of the stomach of people with ulcers and gastritis worldwide (International Organization for Cancer Research)
Agency for Research on Cancer (1994)
omoses, River Flukes and Helicobacter Pylori
(International Agency for Research on Cance
r, Lyon, France; http: // www. uicc. ch / ecp / e
cp2904. htm)). In addition, the International Agency for Research on Cancer has recently recognized a causal relationship between Helicobacter pylori and gastric adenocarcinoma and has classified the bacterium as a Group I (limited) carcinogen. Preferred antibacterial compounds of the invention (de) found using the screening method provided by the invention
Agonists and antagonists of f2), especially broad spectrum antibiotics, are useful in the treatment of Helicobacter pylori infection. Such treatment reduces the appearance of Helicobacter pylori-induced cancer, for example, gastrointestinal cancer.
Such treatments also cure gastric ulcers and gastritis.

Vaccine Another aspect of the present invention is a method of eliciting an immunological response in an individual, especially a mammal, comprising def2, or a fragment thereof, suitable for generating an antibody and / or a T cell immune response. Alternatively, the present invention relates to a method comprising inoculating a variant with an individual and protecting the individual against infection, especially bacterial infection, most preferably Streptococcus pneumoniae infection. Also provided are methods of delaying bacterial replication due to such an immunological response. Yet another aspect of the present invention is a method of eliciting an immunological response in an individual, comprising expressing def2 or a fragment or variant thereof in vivo to express the def2 or a fragment or variant thereof. The targeted nucleic acid vector is delivered to the individual and, for example, cytokine production T
Elicit an immunological response to generate an antibody and / or T cell immune response, including cells or cytotoxic T cells, to elicit an individual with or without disease already established in the individual. A method comprising protecting against disease. One method of administering the gene is by administering the gene to desired cells as a coating of particles or the like. Such nucleic acid vectors may consist of DNA, RNA, modified nucleic acids or DNA / RNA hybrids.

A further aspect of the present invention is that an immunological response to def2 or a protein encoded thereby in an individual having the ability to elicit or induce an immunological response therein when introduced into the individual. An immunological composition which elicits a specific response, comprising a recombinant def2 or a protein encoded therefrom, comprising a DNA encoding and expressing the antigen of said def2 or the protein encoded thereby. Immunological responses can be used therapeutically and prophylactically, and can be derived from antibody immunization or cellular immunity, such as that arising from CTLs or CD4 + T cells.

The def2 polypeptide or fragment thereof does not itself produce antibodies, but has the ability to stabilize the first protein and produce a fusion protein which will have immunogenic and protective properties.
-Protein). Such a fusion recombinant protein preferably further solubilizes an antigenic coprotein, such as lipoprotein D, glutathione-S-transferase (GST) or beta-galactosidase from Hemophilus influenzae, protein. And consists of a relatively large coprotein that facilitates its production and purification. In addition, coproteins can act as adjuvants in providing generalized stimulation of the immune system. The coprotein may be linked to either the amino or carboxy terminus of the first protein. The present invention provides a polypeptide or polynucleotide of the present invention and Sa
to, Y. et al., Science, 273: 352 (1996). Compositions comprising immunostimulatory DNA sequences, particularly vaccine compositions and methods are provided.

The present invention also relates to the description of the invention, which has been found to encode a non-variable region of a bacterial cell surface protein in a DNA construct used in such a genetic immunization experiment in an animal model of Streptococcus pneumoniae infection. Also provided are methods of using the polynucleotides or particular fragments thereof, which are particularly useful for identifying protein epitopes capable of raising a prophylactic or therapeutic immune response. By this method, for the development of a prophylactic or therapeutic agent for a bacterial infection in an animal, especially a human, particularly a Streptococcus pneumoniae infection, producing a monoclonal antibody that is particularly useful for resistance or elimination of the infection from a necessary organ of the animal. Can be. The polypeptide can be used as an antigen to immunize a host, for example, by blocking the attachment of bacteria to damaged tissue, thereby producing specific antibodies that antagonize bacterial invasion. Examples of tissue damage include, for example, cutaneous or connective tissue wounds due to mechanical, chemical or thermal damage, or implanted indwelling devices, or wounds in the mucous membranes such as the mouth, mammary gland, uterus or vagina .

The present invention also includes a vaccine formulation comprising the immunogenic recombinant protein of the present invention and a suitable carrier. Parenteral administration (including, for example, subcutaneous, intramuscular, intravenous, intradermal, etc.) is desirable because proteins can be destroyed in the stomach. Formulations suitable for parenteral administration include antioxidants, buffers, antimicrobial agents, and their formulations in body fluids,
Aqueous and non-aqueous sterile injectable solutions, which may contain solutes which are preferably isotonic with blood; including aqueous and non-aqueous sterile suspensions, which may contain suspending or thickening agents. . Formulations may be provided in unit or multi-dose containers, for example, sealed ampules and vials;
It can be stored in a lyophilized state, requiring only the addition of a sterile liquid carrier immediately before use. Vaccine formulations may also have adjuvant systems to enhance the immunogenicity of the formulation, such as an oil-in-water system, and other systems known in the art. The dosage depends on the specific activity of the vaccine and can be readily determined by routine experimentation. The invention may be applied to certain de
Although described for the f2 protein, it is understood that the description also encompasses fragments of the native protein and similar proteins with additions, deletions, and substitutions that do not substantially alter the immunogenicity of the recombinant protein. Would.

Compositions, Kits and Administration The present invention also relates to compositions comprising the above-described polynucleotides or polypeptides or agonists or antagonists thereof. The polypeptide of the present invention
It can be used in combination with non-sterile or sterile carriers for cells, tissues or organs, such as pharmaceutical carriers suitable for administration to a subject. Such compositions comprise, for example, a vehicle-added or therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation must suit the mode of administration. The present invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more components of the composition of the invention described above.

The polypeptides and other compounds of the present invention may be used alone or in combination with other compounds, such as therapeutic compounds. The pharmaceutical composition, for example,
Topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,
It is administered in any effective and convenient manner, including subcutaneous, nasal, transdermal, and the like. In therapy and prophylaxis, the active ingredient is administered to an individual in an injectable composition, such as a sterile aqueous dispersion,
It is preferably administered as an isotonic aqueous dispersion. Also,
The composition can be a topical formulation in the form of, for example, ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwashes, impregnated bandages and sutures, and aerosols, with suitable conventional additives For example, preservatives, solvents that aid drug penetration, emollients in ointments and creams, and the like can be included. Such topical formulations may also contain compatible conventional carriers, such as cream or ointment bases, ethanol or oleyl alcohol for lotions. Such carriers can contain from about 1 to 98% by weight, usually up to about 80% by weight, based on the total formulation.

For administration to mammals, especially humans, the daily dose of active agent is from 0.01 mg / kg to 10 mg / kg.
kg, typically about 1 mg / kg. In any event, the actual dose most suitable for an individual will be determined by a physician, and will vary with the age, weight and response of the individual. The above doses are exemplary of the average case. Of course, for some individuals higher or lower dose ranges are appropriate and are also within the scope of the present invention. Indwelling devices include surgical implants, prostheses and catheters, ie, devices that are introduced into the body of an individual and remain in place for an extended period of time. For example, such devices include artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts,
Urine catheter, continuous ambulatory peritoneal dialysis (continuous ambulat)
ory peritoneal dialysis (CAPD) catheter.

The compositions of the present invention can be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of the indwelling device. Treatment can continue after surgery, as long as the device is in the body. In addition, the composition can be used to expand the perioperative cover for any surgery to prevent bacterial wound contamination, particularly of Streptococcus pneumoniae. Many orthopedic surgeons believe that humans with prosthetic joints should consider antibiotic prophylaxis before treating teeth that can cause bacteremia. Later severe infections are a serious complication, sometimes with loss of prosthetic joints and significant morbidity and mortality. Thus, the active substance can be extended to use as a replacement for prophylactic antibiotics in this situation.

In addition to the treatments described above, the compositions of the present invention may also be used as a wound healing agent to prevent bacterial attachment to matrix proteins exposed to wound tissue, antibiotic prophylaxis, or together with dental prophylaxis. Can be used for prophylactic use in the treatment of Alternatively, the compositions of the present invention can be used to bathe an indwelling device immediately prior to insertion. The active substance can be used at 1 μg / ml to 10 μg / ml when soaking wounds or internal devices.
Can be used at a concentration of ml.

The vaccine composition is conveniently in the form of an injection. Conventional immune adjuvants can be used to enhance the immune response. A suitable unit dose for the vaccine is antibody 0.5.
5-5 μg / kg, and this dose is preferably
Administer 1 to 3 times at 3 week intervals. At the indicated dose range, no detrimental toxic effects of the compounds of the present invention are observed which would prevent its administration to appropriate individuals. The contents of the references disclosed herein are hereby incorporated by reference. Any patent application for which this application claims priority is also hereby incorporated by reference.

Definitions Certain terms used frequently herein are defined below to facilitate their understanding. "Disease (s)" includes otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, empyema and endocarditis, especially meningitis such as, for example, cerebrospinal fluid infection. , A disease caused by or associated with an infection by a bacterium. A “host cell” is a cell that has been transformed or transfected, or is capable of being transformed or transfected by an exogenous polynucleotide sequence.

"Identity" is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as known in the art and determined by comparing the sequences. Also, in the art, "identity" refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by pairing between the strands of the sequences. "identity"
Alternatively, “similarity” can be easily calculated by a known method. For example, but not limited to, Comput
ational Molecular Biology, Less, AM, Oxford Un
iversity Press, New York, 1988; Biocomputing: Info
rmatics and Genome Projects, Smith, DW, Academi
c Press, New York, 1993; Computer Analysis of Sequ
ence Data, Part I, Griffin, AM and Griffin, H.
G. Ed., Humana Press, New Jersey, 1994; Sequence Ana
lysis in Molecular Biology, Von Heinje, G. Academic
Press, 1987; and Sequence Analysis Primer, Grib
skov, M. and Devereux, J. Ed., M Stockton Press, New
York, 1991; and Carlio, H and Lipman, D., SIAM
J. Applied Math., 48: 1073 (1988). The preferred method of determining identity is designed to give the largest match between the sequences tested. Methods for determining identity and similarity are combined in publicly available computer programs. 2
Preferred computer programming methods for determining identity and similarity between two sequences include, but are not limited to, for example, the GCS program package (Devere
ux, J. et al., Nucleic Acids Research (1984) 12 (1): 38
7), BLASTP, BLASTN and FASTA (A
tschul, SF et al., J. Molec. Biol. (1990) 215: 403-41.
0) is included. BLAST X program is NCBI
And other sources (BLAST Manual, Altshul, S. et al.,
NCBI NLM NIH Bethesda, MD20894; Altschu
l, S. J. et al. Mol. Biol., 215: 403-410 (1990)). As an example, a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to the nucleotide sequence of the reference SEQ ID NO: 1 is a polynucleotide whose polynucleotide sequence is that of the reference nucleotide sequence of SEQ ID NO: 1. Except that it may have up to 5 point mutations per 100 nucleotides,
It is intended that the nucleotide sequence of the polynucleotide is identical to the sequence of the reference. In other words,
At least 95% of the reference nucleotide sequence
To obtain a polynucleotide having the same nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or 5% of the total nucleotides in the reference sequence. Up to% nucleotides may be inserted into the sequence of the control. These mutations in the reference sequence are at the 5 or 3 terminal site of the reference nucleotide sequence,
Alternatively, it may occur anywhere between the terminal sites and may be interspersed individually between nucleotides in the sequence of the reference, or at one or more adjacent groups in the sequence of the reference. Similarly, a polypeptide having an amino acid sequence that has at least 95% identity to the reference amino acid sequence of SEQ ID NO: 2, for example, has 100% of each amino acid of the reference amino acid sequence of SEQ ID NO: 2 It is intended that the amino acid sequence of the polypeptide is identical to the sequence of the control, except that it may have up to 5 amino acid mutations per control. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the amino acid sequence of a reference, up to 5% of the amino acid residues in the reference sequence are deleted or replaced with another amino acid. Alternatively, up to 5% of all amino acid residues in the reference sequence may be inserted into the reference sequence. These changes in the sequence of the reference are made at the amino or carboxy terminal site of the amino acid sequence of the reference,
Alternatively, it may occur anywhere between the terminal sites and may be interspersed between residues in the sequence of the reference, or at one or more adjacent groups in the sequence of the reference.

"Isolated" means altered "by the hand of man" from its natural state, ie,
In the case of a natural product, this means that it has been changed and / or removed from its natural environment. For example, a polynucleotide or polypeptide naturally occurring in a living organism is not `` isolated, '' but the same polynucleotide or polypeptide separated from its coexisting material in its natural state is the term used herein. "Isolated" as
Things. Furthermore, by transformation, genetic manipulation,
Or a polynucleotide or polypeptide that has been introduced into an organism by any other method, while still in the organism and the organism is alive or dead,
"Isolated".

“Polynucleotide (s)” is defined as
Generally, it refers to either polyribonucleotides or polydeoxyribonucleotides, which may be unmodified RNA or DNA, or modified RNA or DNA.
"Polynucleotide" refers to single-stranded and double-stranded DNA, single-stranded and double-stranded regions or DNA that is a mixture of single-stranded, double-stranded and triple-stranded regions, single-stranded and double-stranded RNA, single-stranded and RNA, which is a mixture of double-stranded regions, and DNA and RNA, which may be single-stranded or more typically a double- or triple-stranded region or a mixture of single- and double-stranded regions But not limited thereto. In addition, "polynucleotide" as used herein refers to triple-stranded regions consisting of RNA or DNA, or both RNA and DNA. The chains in these regions may be from the same molecule or from different molecules. The region may include all one or more of these molecules, but more typically includes only some regions of the molecule. One of the molecules of the triple helix region is often an oligonucleotide. As used herein, the term polynucleotide (s) also includes those DNAs or RNAs containing one or more modified bases. That is, DNA or RNA having a backbone modified for stability or for other reasons is also the polynucleotide (s) as the term is intended herein.
Furthermore, DNA or R comprising unusual bases such as inosine or modified bases such as tritylated bases
NA (only two examples are shown) is also a polynucleotide as that term is used herein. Many types of modification are DNA
And RNA and have been used for many useful purposes, as is known to those of skill in the art. The term "polynucleotide" as used herein refers to such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as viruses and, for example, cells such as simple and complex cells. Includes characteristic DNA and RNA chemical forms. "Polynucleotide (s)" also encompasses relatively short polynucleotides, often referred to as oligonucleotide (s).

“Polypeptide (s)” refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide (s)" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 genetically encoded amino acids. "Polypeptide (s)"
It has been modified by either natural processes, such as processing and other post-translational modifications, or by chemical modification techniques. Such modifications are described in detail in the basic text and in more detailed research articles, as well as in the extensive research literature, which are well known to those skilled in the art. It will be apparent that the same type of modification may be present at the same or varying degrees at several sites in a given polypeptide. Also, a given peptide may have many types of modifications. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, amino or carboxyl termini. Modification, for example,
Acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavin, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphotidylinositol, crossover Bond, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methyl , Myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipidation, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and AD
Includes transfer RNA-mediated amino acid addition to proteins, such as P-ribosylation, selenoylation, sulfation, arginylation, and ubiquitination. For example, PROTEINS-STRUCTUR
E AND MOLECULAR PROPERTIES, 2nd edition, TE Creight
on, WH Freeman and Company, New York (1993)
And Wold, F., POSTTRANSLATIONALCOVALENT MODIFIC
ATION OF PROTEINS, Posttranslational Protein Modif
ications: Perspectives and Prospects, pgs. 1-12,
Edited by BC Johnson, Academic Press, New York (1983); S
eifter et al., Meth Enzymol. (1990) 182: 626-646, and Rattan et al., Protein Synthesis: Posttranslational M.
odifications and Aging, Ann NY Acad Sci (1992) 6
63: 48-62. Polypeptides may be branched and may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides are the result of natural post-translational processes and can likewise be synthesized in entirely synthetic ways.

The term “variant” as used herein is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not be different from the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. Variant and reference polypeptides may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis or by direct synthesis or other recombinant methods known to those of skill in the art.

[0087]

The following examples are carried out using standard techniques, which are well known and routine to those skilled in the art, except as otherwise described in detail. The examples are illustrative and do not limit the invention.

Example 1 Selection of Strain, Preparation of Library and Sequencing The polynucleotide having the DNA sequence shown in Table 1 (SEQ ID NO: 1 or 3) is a clone library of chromosomal DNA of Streptococcus pneumoniae in Escherichia coli. I got more. Using sequence data from two or more clones containing overlapping Streptococcus pneumoniae DNA, the contiguous DN of SEQ ID NO: 1 was used.
The A sequence was constructed. The library may be produced by conventional means, for example, methods 1 and 2 below. Whole cell DNA
Is isolated from Streptococcus pneumoniae 0100993 according to standard methods and size fractionated by one of two methods.

Method 1 Whole cell DNA was used for size fractionation according to standard methods.
Is mechanically sheared through a needle. DNA fragments up to 11 kbp in size are made blunt by treatment with exonuclease and DNA polymerase, and an EcoRI linker is added. Fragment
The vector is ligated to Lambda ZapII, a vector cut with EcoRI, the library is packaged by standard methods, and the packaged library is then used to infect Escherichia coli. The library is amplified by standard methods.

Method 2 One restriction enzyme (eg, RsaI, PalI, AluI, Bsh) suitable for obtaining a series of fragments for cloning total cellular DNA into a library vector.
l235I) or a combination thereof, and such fragments are size fractionated according to standard methods. The EcoRI linker is ligated to the DNA, and the fragment is then ligated to the EcoRI-cut vector, Lambda ZapII, the library is packaged by standard methods, and the packaged library is used to infect Escherichia coli. The library is amplified by standard methods.

Example 2 Characterization of def2 def2 was expressed from Streptococcus pneumoniae during infection. Recently, several novel methods have been described which are intended to effect global gene expression during infection (Chuang, S. et al. (1993); Mahan, MJ).
Et al., Science 259: 686-688 (1993); Hensel, M. et al., S.
cience 269: 400-403 (1995)). These new techniques have been demonstrated to some extent in Gram-negative pathogen infections, but not at all in Gram-positive infections. This is probably due to the very slow development of suitable vectors required for global transposon mutagenesis and methods in these organisms, see Chuang, S. et al., J. Bacteriol.
In the case of the method described in 175: 2026-2036 (1993), an appropriate amount of bacterial RNA free from mammalian RNA derived from infected tissue is isolated to obtain bacterial RNA labeled to a sufficiently high specific activity. Is difficult.

The present invention utilizes a novel method to measure gene expression in pathogenic bacteria at various stages of infection of a mammalian host. A novel aspect of the present invention is the use of appropriately labeled oligonucleotide probes that specifically anneal to bacterial ribosomal RNA in Northern blots of bacterial RNA preparations from infected tissues. More abundant ribosomes as hybridization targets
The use of RNA makes it much easier to optimize protocols to purify the appropriate size and quantity of bacterial RNA for RT-PCR from infected tissues.

A suitable oligonucleotide useful for applying this method to genes expressed in Staphylococcus aureus is 5'-GCTCCTAAAAGGTTACTCCACCGGC-3 '(SEQ ID NO: 7). Using the methods of the invention, bacterial genes transcribed during infection can be identified, and their inhibitors have utility in antimicrobial therapy. Of such gene transcription,
Or inhibitors that are specific for the subsequent translation of the resulting mRNA, or for the function of the corresponding expressed protein, would have utility in antimicrobial therapy.

[0094]

[Sequence list]

(1) General information: (i) Applicants: Lonnet, Michael Ajawaski, Deborah Diwan, Min Kosmatoka, Ana El Knowles, David Jay Sea Hodgson, John A. Black, Michael Titherfoss (Ii) Title of invention: def2 (iii) Number of sequences: 7 (iv) Contact: (A) Address: Dechart Price & Rose (B) Street: 4000 bell Atlantic
Tower, 1717 Ark Street (C) City: Philadelphia (D) State: Pennsylvania (E) Country: United States (F) Zip Code: 19103-2793 (v) Computer readable form: (A) Medium Form: Diskette (B) Computer: IBM compatible (C) Operating system: DOS (D) Software: Windows version 2.0
FastSEQ for (vi) Current application data: (A) Application number: (B) Application date: December 12, 1997 (C) Classification: (vii) Previous application data (A) Application number: 60/037535 ( B) Filing date: February 10, 1997 (A) Filing number: PCT / US / (B) Filing date: November 24, 1997 (viii) Information of agents, etc .: (A) Name: Fork, Stephan・ Tee (B) Registration number: 36,795 (C) Processing number at agent etc .: GM50011 (ix) Telecommunication information: (A) Telephone number: 215-994-2488 (B) Telefax number: 215- 994-2222 (C) Telex:

(2) Information of SEQ ID NO: 1 (i) Sequence characteristics: (A) Sequence length: 411 base pairs (B) Sequence type: nucleic acid (C) Number of chains: two (D) topology: wherein linear (xi) SEQ: SEQ ID NO 1: GTGGAAAAGA AAATTGTGAA GGATATCTTA TTTTTATCTC AAGTGTCTCA GCCGGCAAGT 60 CAGGAGGACC TTTATCTTGC CAGAGATTTG CAGGATACAC TCTTAGCAAA TCGTGATACC 120 TGTGTTGGTC TAGCTGCCAA TATGATTGGG GTGCAGAAGC GCGTGATTAT CTTTAATCTT 180 GGCTTAGTTC CCGTGGTCAT GTTTAACCCA GTGCTTCTGT CCTTTGAAGG ATCTTATGAG 240 GCAGAAGAAG GCTGTTTGTC CTTGGTAGGT GTGAGATCAA CTAAGCGTTA TGAAACCATA 300 AGGCTTGCCT ATCGTGACAG CAAGTGGCAG GAACAGACCA TTACCTTGAC AGGCTTCCCA 360 GCTCAGATTT GCCAGCATGA GCTGGATCAC TTGGAAGGAC GAATCATTTA G 411

(2) Information of SEQ ID NO: 2 (i) Sequence characteristics: (A) Sequence length: 136 amino acids (B) Sequence type: amino acids (C) Number of chains: 1 (D) Topology : Linear (xi) Sequence description: SEQ ID NO: 2: Val Glu Lys Lys Ile Val Lys Asp Ile Leu Phe Leu Ser Gln Val Ser 1 5 10 15 Gln Pro Ala Ser Gln Glu Asp Leu Tyr Leu Ala Arg Asp Leu Gln Asp 20 25 30 Thr Leu Leu Ala Asn Arg Asp Thr Cys Val Gly Leu Ala Ala Asn Met 35 40 45 Ile Gly Val Gln Lys Arg Val Ile Ile Phe Asn Leu Gly Leu Val Pro 50 55 60 Val Val Met Phe Asn Pro Val Leu Leu Ser Phe Glu Gly Ser Tyr Glu 65 70 75 80 Ala Glu Glu Gly Cys Leu Ser Leu Val Gly Val Arg Ser Thr Lys Arg 85 90 95 Tyr Glu Thr Ile Arg Leu Ala Tyr Arg Asp Ser Lys Trp Gln Glu Gln 100 105 110 Thr Ile Thr Leu Thr Gly Phe Pro Ala Gln Ile Cys Gln His Glu Leu 115 120 125 Asp His Leu Glu Gly Arg Ile Ile 130 135

(2) Information of SEQ ID NO: 3 (i) Sequence characteristics: (A) Sequence length: 396 base pairs (B) Sequence type: nucleic acid (C) Number of chains: two (D) topology: wherein linear (xi) SEQ: SEQ ID NO 3: GTGAAGGATA TCTTATTTTT ATCTCAAGTG TCTCAGCCGG CAAGTCAGGA GGACCTTTAT 60 CTTGCCAGAG ATTTGCAGGA TACACTCTTA GCAAATCGTG ATACCTGTGT TGGTCTAGCT 120 GCCAATATGA TTGGGGTGCA GAAGCGCGTG ATTATCTTTA ATCTTGGCTT AGTTCCCGTG 180 GTCATGTTTA ACCCAGTGCT TCTGTCCTTT GAAGGATCTT ATGAGGCAGA AGAAGGCTGT 240 TTGTCCTTGG TAGGTGTGAG ATCAACTAAG CGTTATGAAA CCATAAGGCT TGCCTATCGT 300 GACAGCAAGT GGCAGGAACA GACCATTACC TTGACAGGCT TCCCAGCTCA GATTTGCCAG 360 CATGAGCTGG ATCACTTGGA AGGACGAATC ATTTAG 396

(2) Information of SEQ ID NO: 4 (i) Sequence characteristics: (A) Sequence length: 131 amino acids (B) Sequence type: amino acids (C) Number of chains: 1 (D) Topology : Linear (xi) Description of sequence: SEQ ID NO: 4: Val Lys Asp Ile Leu Phe Leu Ser Gln Val Ser Gln Pro Ala Ser Gln 1 5 10 15 Glu Asp Leu Tyr Leu Ala Arg Asp Leu Gln Asp Thr Leu Leu Ala Asn 20 25 30 Arg Asp Thr Cys Val Gly Leu Ala Ala Asn Met Ile Gly Val Gln Lys 35 40 45 Arg Val Ile Ile Phe Asn Leu Gly Leu Val Pro Val Val Met Phe Asn 50 55 60 Pro Val Leu Leu Ser Phe Glu Gly Ser Tyr Glu Ala Glu Glu Gly Cys 65 70 75 80 Leu Ser Leu Val Gly Val Arg Ser Thr Lys Arg Tyr Glu Thr Ile Arg 85 90 95 Leu Ala Tyr Arg Asp Ser Lys Trp Gln Glu Gln Thr Ile Thr Leu Thr 100 105 110 Gly Phe Pro Ala Gln Ile Cys Gln His Glu Leu Asp His Leu Glu Gly 115 120 125 Arg Ile Ile 130

(2) Information of SEQ ID NO: 5 (i) Sequence characteristics: (A) Sequence length: 23 base pairs (B) Sequence type: Nucleic acid (C) Number of chains: One (D) Topology: linear (xi) Description of sequence: SEQ ID NO: 5: CTTGCCAGAG ATTTGCAGGA TAC 23

(2) Information of SEQ ID NO: 6: (i) Sequence characteristics: (A) Sequence length: 23 base pairs (B) Sequence type: nucleic acid (C) Number of chains: 1 (D) Topology: linear (xi) Description of sequence: SEQ ID NO: 6: AGCACTGGGT TAAACATGAC CAC 23

(2) Information of SEQ ID NO: 7: (i) Sequence characteristics: (A) Sequence length: 25 base pairs (B) Sequence type: nucleic acid (C) Number of chains: 1 (D) Topology: linear (xi) Description of sequence: SEQ ID NO: 7: GCTCCTAAAA GGTTACTCCA CCGGC 25

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 1/21 C12P 21/02 C C12P 21/02 21/08 21/08 C12Q 1/00 C12Q 1/00 1/68 Z 1 / 68 G01N 33/15 Z G01N 33/15 33/50 T 33/50 P 33/53 D 33/53 A61K 37/02 // (C12N 15/09 ZNA C12R 1:46) (C12N 1/21 C12R 1 : 46) (C12P 21/02 C12R 1:46) (71) Applicant 595047190 SmithKline Beecham Public Limited Company SmithKline Beecham p. l. c. Middlesex Tiedevue 8.9 EP, Brenford, New Horizons Court (no address) (72) Inventor Michael A. Lonet Victoria, Victoriaville, Pennsylvania, United States 19426 Circle 18 (72) Inventor Robert H. Reid USA 19401 East Norriton, PA, Pacer Lane 8 (72) Inventor Deborah di Jawaski United States 19382 West Chester, PA, Skills Bulba No. 1827 (72) Inventor Philip Serfos United States 19403 Norristown, PA, Yorktown North, 1907 No. 1907 (72) Inventor Ming Wan United States 19422 Blue Bel, Pennsylvania, Centennial Drive No. 302 (7 2) Inventor Michael T. Black United States 19425 Miller Way, 502, Chester Springs, PA (72) Inventor Ana El Cosmatoka United States 18901 Pipple Hill Road, Doylestown, Pennsylvania 482 ( 72) Inventor John E. Hodgson, UK No. 58, C.A. 3.0 A.F., Cumbria, Carlisle, Newfield Drive (72) Inventor David J. C. Knowles UK, Y.05.9 E.B., North Yorkshire, Borrow Bridge, York Road, Ladywell House

Claims (24)

[Claims] 1. A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and at least 7
An isolated polynucleotide consisting of a polynucleotide having 0% identity. 2. def2 contained in the deposited strain Streptococcus pneumoniae.
An isolated polynucleotide consisting of a polynucleotide having at least 70% identity to a polynucleotide encoding the same mature polypeptide expressed by the gene. 3. An isolated polynucleotide consisting of a polynucleotide encoding a polypeptide consisting of an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2. 4. An isolated polynucleotide that is complementary to the polynucleotide of claim 1. 5. The polynucleotide according to claim 1, wherein the polynucleotide is DNA or RNA. 6. The polynucleotide according to claim 1, comprising the nucleic acid sequence shown in SEQ ID NO: 1. 7. The polynucleotide according to claim 1, comprising a stop codon starting from nucleotide 1 to nucleotide number 409 shown in SEQ ID NO: 1. 8. The polynucleotide according to claim 1, which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 9. A vector comprising the polynucleotide according to claim 1. 10. A host cell comprising the vector according to claim 9. 11. The host cell according to claim 10 and its DN.
A method for producing a polypeptide, which comprises expressing the polypeptide encoded by A. 12. A method for producing a def2 polypeptide or fragment, comprising culturing the host according to claim 10 under conditions sufficient to produce the def2 polypeptide or fragment. 13. A polypeptide having an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2. 14. A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2. 15. An antibody against the polypeptide according to claim 14. 16. An antagonist that inhibits the activity or expression of the polypeptide according to claim 14. 17. A method for treating an individual in need of a def2 polypeptide, comprising administering to the individual a therapeutically effective amount of the polypeptide of claim 14. 18. A method of treating an individual in need of inhibition of a def2 polypeptide, comprising administering to the individual a therapeutically effective amount of the antagonist of claim 16. 19. A method for diagnosing a disease associated with the expression or activity of the polypeptide according to claim 14 in an individual, comprising: (a) measuring a nucleic acid sequence encoding the polypeptide in a sample derived from the individual; And / or (b) analyzing the presence or amount of said polypeptide. 20. A method for identifying a compound that interacts with the polypeptide according to claim 14 and inhibits or activates the activity of the polypeptide, wherein the compound to be screened is interacted with the polypeptide under conditions that allow the polypeptide to interact with the polypeptide. A composition comprising a peptide is contacted with the compound to assess the interaction of the compound (such interaction is a second component capable of providing a detectable signal in response to the interaction of the polypeptide with the compound). Detecting the presence or absence of a signal resulting from the interaction between the polypeptide and the compound, and measuring whether the compound interacts with the polypeptide and activates or inhibits its activity. Method consisting of. 21. A method for inducing an immune response in a mammal, comprising using the def2 polypeptide of claim 14 or an antibody and / or a fragment or variant thereof suitable for generating an immune response in a T cell. A method of inoculating an animal and protecting the animal from disease. 22. A method for inducing an immune response in a mammal, comprising the steps of:
Fragment or variant thereof for expressing an f2 polypeptide, or a fragment or variant thereof for eliciting an immunological response in vivo and generating an antibody and / or T cell immune response, is used to disease the animal. How to protect from. 23. An isolated polynucleotide comprising a polynucleotide having at least 70% identity to a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4. 24. An isolated polynucleotide comprising a polynucleotide having at least 70% identity to the polynucleotide sequence of SEQ ID NO: 3.