JPH1169982A - New divib - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new polynucleotide, having a high identity to a base sequence capable of coding a Div1b polypeptide composed of a specific amino acid sequence and useful for the production or the like of a factor useful for screening a compound for activities of an antimicrobial substance. SOLUTION: This new isolated polynucleotide comprises a polynucleotide having at least 70% identity to a polynucleotide capable of coding a Div1b polypeptide composed of an amino acid sequence represented by the formula, a polynucleotide having at least 70% identity to a polynucleotide capable of coding the same mature polypeptide as that expressible by a Div1b gene contained in Staphyloccocus aureus which is a deposited strain, a polynucleotide capable of coding a polypeptide composed of an amino acid sequence having at least 70% identity to the amino acid sequence represented by the formula, a polynucleotide or the like complementary thereto and is used for the production or the like of a factor useful for screening a compound for activities of an antimicrobial substance.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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, in these and other respects, the present invention relates to novel polynucleotides and polypeptides of the div (cell division) family, referred to herein as "Div1b".

[0002]

BACKGROUND OF THE INVENTION It is particularly preferred to use the Staphylococcus gene and its gene product as targets for developing antibiotics. Staphylococcus constitutes a group of medically important genes of microorganisms. They are known to result in two forms, invasive and toxogenic. Invasive infections are usually characterized by the formation of abscesses both on the skin surface and in deep tissue. Staphylococcus aureus secondary induces the cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis, and acute bacterial endocarditis are also relatively common. From the characteristics of toxic occurrence of Staphylococcus,
At least three clinical conditions occur. Elucidation of these diseases comes from the effects of exotoxins on tissue invasion and bacteremia. These conditions are staphylococcal food poisoning, burn skin syndrome and toxic shock syndrome, including:

[0003] The frequency of Staphylococcus aureus infections has increased dramatically over the past two 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 Staphylococcus aureus strains that are resistant to some or all of the standard antibiotics. This calls for both new antimicrobial agents and diagnostic tests for this organism.

[0004] Divlb (in Escherichia coli, ft
In sQ, Bacillus subtilis, it is named divlb. ) Is an essential gene in bacterial cell division. Escherichia coli FtsQ has been identified as a homologue of DivlB with 18% homology and 4% similarity.
4% (Harry et al., (1994) Gene 147: 85-89). F
tsQ has been shown in Escherichia coli to be essential in various places throughout the process of septum formation during cell division. FtsQ has about 2 in the cytoplasmic domain.
A simple cell membrane protein with one amino acid residue, 25 amino acid residues in the cell membrane and about 230 amino acid residues in the periplasmic domain (Carson et al., (1994) J. Bac
teriol. 173: 2187-2195). FtsQ is about 2 per cell
It is calculated that there are 0 (Carson et al., (1994) J. Bacte
riol. 173: 2187-2195). DivIB of Bacillus subtilis 168 is essential for bacterial survival above 37 ° C. and is required at all temperatures at which cell division occurs at normal rates (Beall and Lutkenhaus et al., (1989) J. Ba.
cteriol. 171: 6821-6834). Where the cytoplasmic and membrane spanning regions of FtsQ have been replaced by similar domains of MalG, the FtsQ-MalG fusion protein is known to be the complement of FtsQ temperature-sensitive mutants. This suggests the following. The cytoplasmic and membrane spanning regions of FtsQ may not be necessary for protein function (Dai et al., (1996) 178: 1328-1334).
However, a portion of the FtsQ membrane spanning region is required for protein transport into the periplasm. On the other hand, Guzm
An et al. 1997 (J. Bacteriol. 179: 5094) reported that ft
Only the membrane-spanning segment of sQ was replaced, suggesting that the cytoplasmic domain is essential for function. Descotea
ux and Drapeau et al. 1987 (J. Bacteriol. 169: 1938)
Suggest that FtsQ interacts with FtsZ. The biochemical function of FtsQ is not known.

It is apparent that a need exists for factors, such as the novel compounds of the present invention, which have 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. There is also a need to identify and characterize such factors and their antagonists and agonists that may play a role in the prevention, amelioration or cure of infection, dysfunction or disease.
The polypeptide of the present invention may be a known Escherichia coli F
Has amino acid sequence homology to tsQ and Bacillus subtilis Div1b protein.

[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], an Escherichia coli FtsQ and a Bacillus subtilis Div.
Due to the homology between the known amino acid sequence (s) of other proteins such as the 1b protein,
It is to provide a polypeptide identified as a v1b polypeptide. A further object of the present invention is to provide Div1
It is an object of the present invention to provide a polynucleotide encoding a polypeptide, in particular, a polynucleotide designated Div1b herein.

[0007]

In a particularly preferred embodiment of the present invention, the polynucleotide comprises a Div having the sequence shown in Table 1 [SEQ ID NO: 1], including the entire gene.
It comprises a region encoding a 1b polypeptide, or a variant thereof. Another particularly preferred embodiment of the present invention is a novel Div1b protein from Staphylococcus aureus having the amino acid sequence of Table 1 [SEQ ID NO: 2] or a variant thereof. According to another aspect of the present invention, there is provided an isolated nucleic acid molecule encoding a mature polypeptide that can be expressed by Staphylococcus aureus strain WCUH29 contained in the deposited strain. In a further aspect of the invention, including mRNA, cDNA, genomic DNA,
Div1b, especially Staphylococcus aureus Di
Provided is an isolated nucleic acid molecule encoding v1b. 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 invention are the natural allelic variants of Div1b and the polypeptides encoded thereby. In another aspect of the present invention, a novel polypeptide of Staphylococcus aureus, referred to herein as Div1b, and biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and the like. There is provided a composition comprising: A particularly preferred embodiment of the invention is Div1b encoded by the natural allele of the Div1b gene.
A variant of a polypeptide. In a preferred embodiment of the present invention, there is provided a method for producing the Div1b polypeptide.

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. In a preferred embodiment of the invention, assaying Divlb protein expression, diseases such as: upper respiratory infections (eg otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis),
Lower respiratory infections (eg, abscess, lung abscess), heart (eg, infective endocarditis), gastrointestinal tract (eg, secretory diarrhea, spleen abscess, retroperitoneal abscess), central nervous system (brain abscess), eye (Blepharitis, conjunctivitis, keratitis, endophthalmitis, anterior cellulitis, orbital cellulitis, lacrimal inflammation), kidney and urinary tract (eg epididymitis, intrarenal and perirenal abscess, toxin shock syndrome) ), Treating skin (eg impetigo, folliculitis, skin abscess, cellulitis, wound infection, bacterial myositis) bones and joints (eg septic arthritis, osteomyelitis), testing for genetic diversity Provided are materials, compositions and methods for administering and administering Divlb polypeptides or polynucleotides to an organism to produce an immunological response against bacteria, particularly Staphylococcus aureus. . According to certain preferred embodiments of this and other aspects of the invention, Div
Provided are polynucleotides that hybridize to the 1b polynucleotide sequence, particularly under stringent conditions. In certain preferred embodiments of the present invention, there are provided antibodies against Div1b polypeptides.

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, a Div
Provided are 1b agonists and antagonists, preferably bacteriostatic or bactericidal agonists and antagonists. In a further aspect of the invention there is provided a composition comprising a Div1b polynucleotide or Div1b polypeptide for administration to a cell or 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]

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions Certain terms used frequently herein are defined below to facilitate their understanding. 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" or "similarity" can be easily calculated by a known method.
For example, but not limited to, COMPUTATIONAL MOLE
CULAR BIOLOGY, Lesk, AM, Oxford University Pre
ss, New York, 1988; BIOCOMPUTING: INFORMATICS AND G
ENOME PROJECTS, Smith, DW, Academic Press, New
York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PA
RT I, Griffin, AM and Griffin, HG, Humana P
ress, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLEC
ULAR BIOLOGY, Von Heinje, G. Academic Press, 1987;
And SEQUENCE ANALYSIS PRIMER, Gribskov, M. and D
evereux, J. Ed., M Stockton Press, New York, 1991; and Carllio, H. and Lipman, D., SIAM J. Applied Mat.
h., 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. Preferred computer programming methods for determining identity and similarity between two sequences include, but are not limited to, for example,
GCS program package (Devereux, J. et al., Nucleic
Acids Research (1984) 12 (1): 387), BLAST
P, BLASTN and FASTA (Atschul, SF et al.,
J. Molec. Biol. (1990) 215: 403-410).
The BLASTX program is available from NCBI and other sources (BL
AST Manual, Altshul, S.M. Et al., NCBI NLM N
IH Bethesda, MD20894; Altschul, S .; J. et al. Mol. B
iol., 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. It is intended that the nucleotide sequence of the polynucleotide be identical to that of the control, except that it may have up to 5 point mutations per 100 nucleotides. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to the nucleotide sequence of the reference, up to 5% of the nucleotides in the sequence of the reference have been deleted or replaced with another nucleotide. Alternatively, up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
These variations in the reference sequence may occur at the 5 or 3 terminal sites of the reference nucleotide sequence, or anywhere between those terminal sites, individually between nucleotides in the reference sequence, or May be interspersed with one or more adjacent groups in the sequence of 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 can occur at or between the amino or carboxy terminal sites of the amino acid sequence of the reference, individually between residues in the sequence of the reference, or It may be interspersed with one or more adjacent groups in the standard sequence.

"Isolated" means altered "by the hand of man" from its natural state, that is,
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 an 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.

"Polynucleotide (s)" is defined as
Generally refers to either polyribonucleotides or polydeoxyribonucleotides, which are unmodified RNA
Alternatively, it may be DNA, or modified RNA or DNA. "Polynucleotide" refers to single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, single-stranded and RNA, which is a mixture of double-stranded regions,
And hybrid molecules comprising DNA and RNA, which may be single-stranded or more typically a double-stranded or triple-stranded region or a mixture of single-stranded and double-stranded regions, including Not limited. In addition, “polynucleotide” as used herein refers to RNA or DN.
A or a triple-stranded region composed of 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.
In addition, DNA or RNA comprising only unusual bases such as inosine, or modified bases such as tritylated bases (only two examples are shown) are also polynucleotides as the term is used herein. is there. Numerous modifications have been made to DNA and RNA and used for many useful purposes, as will be known to those skilled 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. Characteristic DNA and R
Includes chemical forms of NA. "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 together 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-STRUCT
URE AND MOLECULAR PROPERTIES, 2nd edition, TE Creig
hton, WH Freeman and Company, New York (199
3) and Wold, F., POSTTRANSLATIONAL COVALENT MOD
IFICATION OF PROTEINS, Posttranslational Protein M
odifications: Perspectives and Prospects, pgs.1-1
2. Edited by BC Johnson, Academic Press, New York (198
3); Seifter et al., Meth Enzymol. (1990) 182: 626-646;
And Rattan et al., “Protein Synthesis: Posttranslatio
nal Modifications and Aging ”, Ann NY Acad Sci
(1992) 663: 48-62. Polypeptides may be branched and may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides are:
It is the result of a natural post-translational process, which can likewise be synthesized in a totally synthetic manner.

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.

The present invention relates to novel Div1b polypeptides and polynucleotides as described in more detail below. In particular, the present invention relates to Escherichia coli Ft
Novel Div1b polypeptides and polynucleotides of Staphylococcus aureus, which are related by amino acid homology to sQ and Bacillus subtilis Div1b polypeptide. The present invention specifically provides D
iv1b and the Div1b nucleotide sequence of the DNA of the deposited strain and the amino acid sequence encoded thereby.

[0018]

Table 1 Div1b polynucleotide and polypeptide sequences (A) Sequence derived from Staphylococcus aureus Div1b polynucleotide sequence [SEQ ID NO: 1]

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(B) Div1b polypeptide sequence [SEQ ID NO: 2] deduced from the polynucleotide sequences in this table

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(C) Specific example of polynucleotide sequence [SEQ ID NO: 1]

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(D) Specific example of polypeptide sequence [SEQ ID NO: 2]

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(E) Staphylococcus aureus D
Sequence from iv1b polynucleotide ORF sequence [SEQ ID NO: 3]

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(F) Div1b polypeptide sequence deduced from the polynucleotide ORF sequences in this table [SEQ ID NO: 4]

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Deposited Materials Deposited strains, including Staphylococcus aureus WCUH29 strain, were deposited on September 11, 1996 in Scotland, Scotland.
AB2 1RY, Aberdeen, Machidrive 23
Accession number 407 to the National Collection of Industrial and Marine Bacteria Limited (referred to herein as "NCIMB") of St.
Deposit under 71. The deposited strain was named Staphylococcus aureus WCUH29 at the time of deposit. The deposited Staphylococcus aureus strain is referred to herein as "deposited strain" or "DNA of the deposited strain."

The deposited strain contains the full-length Div1b 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.

Polypeptides The polypeptides of the present invention include the polypeptides of Table 1 [SEQ ID NO: 2] (especially mature polypeptides) and, in particular,
Having a biological activity of Div1b and at least 7% of the polypeptide of Table 1 [SEQ ID NOS: 2 and 4] or a corresponding portion thereof
0% identity, preferably Table 1 [SEQ ID NOs: 2 and 4]
At least 80% identity, more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of Table 1 [SEQ ID NOs: 2 and 4], and More preferably, it includes polypeptides and fragments having at least 95% similarity (even more preferably, at least 95% identity) to the polypeptides of Table 1 [SEQ ID NOs: 2 and 4], and further generally, Also encompassed are portions of such polypeptides that have a portion of the polypeptide that includes at least 30 amino acids, more preferably at least 50 amino acids.

The present invention also relates to Table 1 (D) wherein amino terminal X is hydrogen, carboxyl terminal Y is hydrogen or metal, R ₁ and R ₂ are any amino acid residues, and n is an integer of 1 to 1000. The polypeptide of the formula shown in [SEQ ID NO: 2] is also included. When R 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. D
As with iv1b polypeptides, the fragments are "self-supporting" or they form a 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 NOs: 2 and 4] or a series of residues including the amino terminus or a series of residues including the carboxyl terminus. . Host cells, especially
Also preferred are degraded forms of the polypeptides of the invention in Staphylococcus aureus. 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.

[0029] Biologically active fragments are also preferred fragments. Biologically active fragments include Div1 that have similar or improved activity, or that have reduced undesirable activity.
b is a fragment that mediates the activity of b. Also included are fragments that are antigenic or immunogenic in an animal, particularly a human. Particularly preferred are functions or individuals essential for the survival of Staphylococcus aureus,
In particular, fragments consisting of receptors or domains of enzymes that confer the ability to initiate or maintain the disease in humans. 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.

Polynucleotides Another aspect of the present invention relates to isolated polynucleotides encoding Div1b polypeptides having the deduced amino acid sequences of Table 1 [SEQ ID NOs: 2 and 4, polynucleotides closely related thereto, and polynucleotides thereof. About the variant.
Using information herein, such as the polynucleotide sequences shown in Table 1 [SEQ ID NOs: 1 and 3], using Staphylococcus aureus WCUH29 as a starting material,
Using standard cloning and screening methods to clone and sequence chromosomal DNA fragments from bacteria and subsequently obtain full-length clones, Di
A polynucleotide of the invention encoding a v1b 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 and 3], typically a Staphylococcus aureus WCUH29 in Escherichia coli or other suitable host is provided. Is probed with a radiolabeled oligonucleotide derived from the partial sequence, preferably a 17-fold or more oligonucleotide. Then, the same DNA as the probe
Clones can 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 full length. Conveniently, such sequencing is performed using denatured double-stranded DNA prepared from a plasmid clone. A suitable technique is Maniatis, T., Fritsc
h, EF and Sambrook et al., MOLECULAR CLONING: A LABOR
ATORY MANUAL, 2nd Ed .; Cold Spring Harbor Laborato
ry Press, Cold Spring Harbor, New York (1989) (especially screening by hybridization 1.
Sequencing of 90 and denatured double stranded DNA templates 13.70
). For example, Table 1
The polynucleotide shown in [SEQ ID NO: 1] is a DNA derived from Staphylococcus aureus WCUH29.
It was found in the library.

The DNA sequence of Table 1 [SEQ ID NO: 1]
It has an open reading frame encoding a protein having the approximate number of amino acid residues shown in [SEQ ID NO: 2] and having an estimated molecular weight that can be calculated from the molecular weight of known amino acid residues. Nucleotide numbers 1 to 1317
The polynucleotide of SEQ ID NO: 1 between SEQ ID NO: 2
Encoding the polypeptide of Stop codon is SEQ ID NO: 1
Starting with the next nucleotide number after nucleotide 1318 Div1b of the present invention is structurally compatible with the deposited strain Div.
Linked to other proteins of the div (cell division) family, which are found as a result of sequencing the DNA encoding v1b. The protein shows the greatest homology to the Escherichia coli FtsQ and Bacillus subtilis Div1b proteins among known proteins. Div1b in Table 1 [SEQ ID NO: 2]
Has about 27.9% identity over its entire length and about 52.5% similarity over its entire length with the amino acid sequence of Bacillus subtilis Div1b polypeptide.

The present invention provides a polynucleotide sequence that is identical over its entire length to the coding sequence in Table 1 [SEQ ID NO: 1]. 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 may 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) 86: 821.
A hexa-histidine peptide or an HA tag as described in -824 (Wilson et al., Cell, 3
7: 767 (1984)). Polynucleotides of the present invention include, but are not limited to, polynucleotides consisting of structural genes and naturally occurring sequences that regulate gene expression.

A preferred embodiment of the present invention is a polynucleotide encoding Div1b polypeptide consisting of nucleotides 1-1317 or 1320 shown in SEQ ID NO: 1 of Table 1. The invention also provides that X at the 5 ′ end of the molecule is hydrogen, Y at the 3 ′ end of the molecule is hydrogen or metal, R ₁ and R ₂ are any nucleic acid residues, and n is 1 to
Includes polynucleotides of the formula shown in Table 1 (C), which are integers of 1000. When R is more than 1, the chain of the nucleic acid residue represented by any of the R groups may be a heteropolymer or a homopolymer, and a heteropolymer is preferable. As used herein, the term "polynucleotide encoding a polypeptide" refers to a polypeptide of the present invention, in particular, a bacterial polypeptide, and more specifically, Table 1 [SEQ ID NO: 2].
And a polynucleotide comprising a sequence encoding a polypeptide of Staphylococcus aureus Div1b having the amino acid sequence shown in Table 1. This term, together with additional regions that may include coding and / or non-coding sequences,
Includes polynucleotides comprising a single contiguous or non-contiguous region encoding the polypeptide (eg, integrated phage or interrupted sequences or interrupted by editing). 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]. Variants that are fragments of the polynucleotides of the invention can be used in the synthesis of full-length polynucleotides of the invention.

More 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.
Div1b of Table 1 [SEQ ID NO: 2] deleted or added
A polynucleotide having the amino acid sequence of the polypeptide and encoding a Div1b variant. Particularly preferably,
Silent substitutions, additions and deletions that do not alter the properties or activity of Div1b. A further preferred embodiment of the present invention is a polynucleotide having at least 70% identity over the full length of a polynucleotide encoding a Div1b polypeptide having the amino acid sequence shown in Table 1 [SEQ ID NOs: 2 and 4], and so forth. Is a polynucleotide that is complementary to the normal polynucleotide. Also,
Most preferred are polynucleotides consisting of a region having at least 80% identity over its full length with the polynucleotide encoding the Div1b polypeptide of the deposited strain, and polynucleotides complementary thereto.
In this regard, polynucleotides having at least 90% identity over their full length with the same are particularly preferred, especially those with at least 95% identity. 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 in Table 1 [SEQ ID NO: 1].

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 20 μg
/ Ml in a solution comprising denatured cleaved salmon sperm DNA.
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 a suitable library comprising substantially the entire gene of the polynucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 under stringent hybridization conditions. A polynucleotide comprising a polynucleotide sequence obtainable by screening with a probe having the nucleotide sequence or a fragment thereof and isolating the DNA sequence is provided. 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
Used as a hybridization probe for NA and genomic DNA, full-length cDNA and genomic clones encoding Div1b were isolated and
cDNA and genomic clones of other genes with high sequence similarity to the b gene can be isolated. Such a probe will generally have at least 15 bases. Preferably, such a probe consists of at least 30 bases and may have at least 50 bases. Particularly preferred probes have at least 30 bases and no more than 50 bases. For example,
The coding region of the Div1b gene is the DNA of SEQ ID NO: 1.
It can be isolated by screening using the sequence to synthesize an oligonucleotide probe. Then
A cDNA, genomic DNA or mRNA library is 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. The polynucleotide of the present invention, which is an oligonucleotide derived from the sequence of SEQ ID NO: 1 and / or 2, can be used for the method described herein, but is preferably used for PCR and the polynucleotide identified herein. Is 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 additional amino or carboxy terminal amino acids are added or amino acids are 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 may, among other things, play a role 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 produce a protein for analysis or production. Can be easily operated. 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.

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 leader sequences 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 or vectors comprising polynucleotides of the present invention, host cells genetically engineered with vectors of the present invention and polypeptides of the present invention by recombinant techniques. 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 invention. Introduction of the polynucleotide into the host cell can be performed by calcium phosphate transfection, DEA
E-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation,
Davis et al., BASIC METHODS IN, such as transduction, scrape loading, ballistic introduction and infection
MOLECULAR BIOLOGY (1986) and Sambrook et al., MOLECUL
AR CLONING: A LABORATORY MANUAL, 2nd Ed. Cold Spri
ng Harbor Laboratory Press, Cold Spring Harbor, N.
This can be done by methods described in several standard laboratory manuals, such as Y. (1989).

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, H
animal cells such as eLa, C127, 3T3, BHK, 293 and 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 are, inter alia, chromosomal, episomal and viral derived systems, such as bacterial plasmid derived, bacteriophage derived, transposon derived, yeast episomal derived, insertion factor derived, yeast chromosomal factor derived, baculovirus, papovavirus such as SV40, vaccinia. Vectors derived from combinations thereof, such as viruses, adenoviruses, chicken poxviruses, vectors derived from viruses such as pseudorabies virus and retrovirus, and vectors derived from plasmids such as cosmids and phagemids and genetic elements of bacteriophage. Include. 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 are provided, for example, in Sambrook et al., MO.
LECULAR CLONING, A LABORATORY MANUAL (supra) may be inserted into the expression system by any of a variety of well-known conventional techniques.

In order 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. The polypeptides of the present 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 active conformation may be regenerated using well known methods for regenerating proteins.

Diagnostic Assays The present invention also relates to the Ds of the present invention for use as diagnostic reagents.
It is also related to the use of iv1b polynucleotides.
Div in eukaryotes, especially mammals, especially humans
Detection of 1b provides a diagnostic method for diagnosis of a disease. Eukaryotes (also referred to herein as "individuals"), particularly mammals, and particularly humans, can be detected at the nucleic acid level by various methods when they are infected with an organism that contains the Div1b gene. 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 PCR prior to analysis.
Alternatively, it can be amplified enzymatically by using other amplification methods. RNA or cDNA 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. The point mutation was performed using Div1b labeled amplified DNA.
It can be identified by hybridizing to a polynucleotide sequence. The perfectly matched sequence is RNase
Mismatched duplexes can be distinguished by digestion or by differences in melting temperatures. DNA sequence differences can also be detected by changes in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. For example, Myers et al., Science, 230:
See 1242 (1985). Sequence changes at specific locations may also be detected by nuclease protection assays, eg, RNas
It can also be revealed by e and S1 protection or chemical cleavage methods. For example, Cotton et al., Proc. Natl. Ac
ad.Sci., USA, 85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the invention can also be detected at the DNA level by various techniques, for example by serotyping.
For example, mutations can be detected using RT-PCR. RT-PCR is automatically detected by a system such as GeneSc.
It is particularly preferred to use in combination with an or the like. RNA or cDNA can also be used for PCR or RT-PC for the same purpose.
R can be used. As an example, mutations can be identified and analyzed using PCR primers complementary to the nucleic acid encoding Div1b. Examples of representative primers are shown in Table 2 below.

[0046]

Table 2 Primers for Amplification of Div1b Polynucleotide SEQ ID NO: Primer Sequence 5 5′-atgggataaaaacgaagaa-3 ′ 65′-ttattcttacttgatttgttt-3 ′ The present invention was further removed from the 5 ′ and / or 3 ′ end. These primers containing 1, 2, 3 or 4 nucleotides are provided. Div1 isolated from samples derived from individuals using these primers, among others
b DNA can be amplified. 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. In this way, mutations in the DNA sequence can be detected and used to diagnose infection and serotype and / or classify infectious agents.

The invention further relates to diseases, preferably bacterial infections, more preferably infections by Staphylococcus aureus, and most preferably upper respiratory infections (eg otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis) ), Lower respiratory infections (eg, abscess, lung abscess), heart (eg, infectious endocarditis), gastrointestinal tract (eg, secretory diarrhea, spleen abscess, retroperitoneal abscess), central nervous system (brain abscess) , Eyes (blepharitis, conjunctivitis, keratitis, endophthalmitis, anterior cellulitis, orbital cellulitis, lacrimal inflammation), kidneys and urinary tract (eg epididymitis,
Intrarenal and perirenal abscess, toxic shock syndrome), skin (eg impetigo, folliculitis, skin abscess, cellulitis, wound infection,
Bacterial myositis) bones and joints (eg septic arthritis, osteomyelitis)
And the like, wherein an increase in the expression level of a polynucleotide having the sequence of Table 1 [SEQ ID NO: 1] is determined from a sample derived from an individual. Increasing or decreasing the expression of Div1b polynucleotide can be determined by any method known in the art for quantifying a polynucleotide, for example, amplification, PCR, RT.
-Can be measured using PCR, RNase protection, Northern blotting and other hybridization methods.

In addition, a diagnostic assay according to the present invention for detecting overexpression of Div1b protein compared to normal control tissue samples can be used to detect the presence of an infection, for example. Div1 in a host-derived sample
Assay techniques that can be used to determine levels of a protein, 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 of the invention, or cells expressing them, can be used as immunogens to produce antibodies immunospecific for such polypeptides.
As used herein, "antibody" includes monoclonal and polyclonal antibodies, chimeric, single chain, monkey and humanized antibodies, and Fab fragments, including the products of a Fab immunoglobulin expression library. Antibodies raised against the polypeptides of the invention can be obtained by administering fragments, analogs or cells bearing the polypeptide or epitope, preferably to a non-human animal, using conventional protocols. In preparing monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. For example, Kohler, G. and Mi
lstein, C., Nature, 256: 495-497 (1975); Kozbor et al., I.
mmunology Today, 4:72 (1983); Cole et al., Monoclonal.
Antibodies and Cancer Therapy, Alan R Liss, Inc., 77
-96 (1985).

Techniques for producing single-chain antibodies (US Pat. No. 4,946,778) can be used to produce single-chain antibodies to the polypeptides of the present invention. 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-Div1b using the technique, or from an intact library; (McCafferty, J. et al., N
ature 348: 552-554 (1990); Marks, J. et al., Biotechnolo.
gy 10: 779-783 (1992)). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., Nature 352: 624-628 (1
991)). If two antigen binding domains are present, 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, inter alia, infections, especially bacterial infections, especially upper respiratory infections (eg otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory infections, using antibodies against Div1b polypeptides. (Eg, abscess, lung abscess), heart (eg, infective endocarditis), gastrointestinal tract (eg, secretory diarrhea, spleen abscess, retroperitoneal abscess), central nervous system (brain abscess), eye (blepharitis, conjunctivitis) , Keratitis, endophthalmitis,
Anterior nasal cellulitis, orbital cellulitis, lacrimal inflammation), kidney and urinary tract (eg, epididymitis, intrarenal and perirenal abscess, toxic shock syndrome), skin (eg, impetigo, folliculitis, Skin abscesses, cellulitis, wound infections, bacterial myositis) bones and joints (eg, septic arthritis, osteomyelitis) and the like can be treated. Polypeptide variants form an antigenic, which form a particular aspect of the invention,
Epitopeically or immunologically equivalent variants are included.
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.

The polypeptide, eg, 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.

Preferably, the antibody or variant thereof is modified to reduce immunogenicity in the individual. For example, if the individual is a human, the antibody is most preferably "humanized"; for example, Jones, P. et al., Nature 321: 522-525 (19
86) or Tempest et al., Biotechnology 9: 266-273 (199).
As described in 1), the complementarity determining region of the antibody derived from the hybridoma has been transplanted into a human monoclonal antibody. 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 (1992); Ma).
nthorpe et al., Hum. Gene Ther. 4: 419 (1963)), Delivery of DNA complexed with specific protein carriers (Wu et al., J. Am.
Biol Chem. 264: 16985 (1989)), DNA co-precipitation using calcium phosphate (Benvenisty & Reshef, PNAS USA 8).
3: 9551 (1986)), DN into various forms of liposomes.
A encapsulation (Kaneda et al., Science 243: 375 (1989)), particle bombardment (Tang et al., Nature 356: 152 (1992); Eisenbraun)
12: 791 (1993)) and in vivo infection with cloned retroviral vectors (Seeger
Et al., PNAS USA 81: 5849 (1984)).

Antagonists and Agonists-Assays and Molecules In addition, using the polypeptides of the present invention,
For example, the binding of a small molecule substrate to a ligand in a mixture of cells, cell-free preparations, chemical libraries, and natural products can be assessed. 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): Chapter 5 (19
See 91). The invention also relates to the action of Div1b polypeptides or polynucleotides, in particular bacteriostasis and / or
Or enhance the action of bactericidal compounds (agonists)
Alternatively, methods for screening compounds to identify compounds that block (antagonist) are provided. Screening methods include high-throughput techniques. For example, to screen for agonists or antagonists, synthetic reaction mixtures comprising Div1b polypeptides and labeled substrates or ligands of such polypeptides, cell compartments such as membranes, cell envelopes or cell walls, or preparations of any of them. Is incubated in the presence or absence of a candidate molecule that may be a Div1b agonist or antagonist. Candidate molecule is D
The ability to agonize or antagonize the iv1b polypeptide is reflected in reduced binding of the labeled ligand or reduced production of product from such substrates. Molecules that bind and have no effect, ie, do not elicit the effects of Div1b 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. A useful reporter system in this regard is a colorimetrically labeled substrate that is converted to a product, Di
Reporter genes that respond to changes in v1b polynucleotide or polypeptide activity, and include, but are not limited to, binding assays known in the art.

Another example of a Div1b antagonist assay is Div1b under conditions suitable for a competitive inhibition assay.
b and a potential antagonist are mixed with a Div1b binding molecule, a recombinant Div1b binding molecule, a natural substrate or ligand, or a substrate or ligand mimetic. Div1b is labeled, eg, with a radioactive or colorimetric compound, and attached to a binding molecule;
Alternatively, the number of Div1b molecules converted to product can be accurately determined to assess the effects of potential antagonists.
Potential antagonists include small organic molecules, peptides, polypeptides, and antibodies that bind to a polynucleotide or polypeptide of the present invention, thereby inhibiting or extinguishing their activity. Potential antagonists also
Small organic molecules, peptides, such as closely related proteins or antibodies, that bind to the same site on the binding molecule, such as a binding molecule that does not induce Div1b-inducing activity, and prevent the action of Div1b by eliminating Div1b from binding. May be a polypeptide.

Potential antagonists include small molecules that bind and occupy the binding site of the polypeptide, thereby preventing binding to a cellular binding molecule 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 Inhibitorsof Gene
Expression, CRC Press, Boca Raton, FL (1988)). Preferred potential antagonists include compounds related to Div1b and variants thereof.

The DNA sequences obtained herein are each
Can 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, the encoded protein or Shine-Delgarno
Alternatively, a DNA sequence encoding the amino-terminal region of the translation-enhancing sequence of another individual mRNA can be used to construct an antisense sequence that regulates the expression of the coding sequence of interest. The invention also provides for the use of a polypeptide, polynucleotide or inhibitor of the invention to disrupt the initial physical interaction between a pathogen and a mammalian host involved in the sequelae of infection. In particular, the molecules of the present invention; protection of bacteria, especially Gram-positive bacteria, from attachment to mammalian extracellular matrix proteins on an internal device or to wound extracellular matrix proteins;
For example, initiation of mammalian tyrosine kinase phosphorylation blocks Div1b protein-mediated mammalian cell entry (Rosenshine et al., Infect. Immunon. 60: 2211 (1992)); mediates mammalian extracellular matrix proteins and tissue damage Blocking bacterial attachment to and from bacterial Div1b proteins that can be used to block the normal progression of etiology in infections initiated by other than implantation of an indwelling device or other surgical procedure.

The antagonists and agonists of the present invention can be used, for example, to treat upper respiratory infections (eg, otitis media,
Bacterial tracheitis, acute epiglottitis, thyroiditis, lower respiratory infections (eg, pustules, lung abscess), heart (eg, infective endocarditis), gastrointestinal tract (eg, secretory diarrhea, splenic abscess) Peritoneal abscess), central nervous system (brain abscess), eyes (blepharitis, conjunctivitis,
Keratitis, endophthalmitis, anterior cellulitis, orbital cellulitis, lacrimal inflammation), kidney and urinary tract (eg, epididymitis, intrarenal and perirenal abscess, toxic shock syndrome), skin (eg, pus) Crusts, folliculitis, skin abscess, cellulitis, wound infection, bacterial myositis) bones and joints (eg, septic arthritis, osteomyelitis) and the like can be treated.

Vaccine Another aspect of the invention is a method of eliciting an immunological response in an individual, especially a mammal, wherein the Div1b, or a fragment thereof, suitable for generating an antibody and / or a T cell immune response. Alternatively, it relates to a method comprising inoculating a variant with an individual and protecting the individual against infection, especially bacterial infection, most preferably Staphylococcus aureus infection. Also provided are methods for slowing bacterial replication by such immunological responses. Yet another aspect of the present invention is a method of eliciting an immunological response in an individual, the method comprising expressing Div1b or a fragment or variant thereof in vivo to express said Div1b or a fragment or variant thereof. A nucleic acid vector directed to the individual to elicit an immunological response to generate an antibody and / or T cell immune response, including, for example, cytokine producing T cells or cytotoxic T cells, to the individual. And protecting the individual from the disease, whether or not the disease has already been established. 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 are DNA, R
It may consist of NA, modified nucleic acid or DNA / RNA hybrid.

A further aspect of the present invention is that an immunological response to Div1b or a protein encoded therefrom when introduced into an individual having or capable of eliciting an immunological response therein. Composition which elicits an objective response, said composition comprising a recombinant Div1b or a protein encoded therefrom, comprising a DNA encoding and expressing an antigen of said Div1b or a protein encoded therefrom.
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 Div1b polypeptide or a fragment thereof does not itself produce antibodies, but has the ability to stabilize the first protein and to produce a fusion protein that will have immunogenic and protective properties (co-Protein). ) Can be fused. Such a fusion recombinant protein is preferably further provided with a Hemophilus influenzae.
From lipoprotein D, glutathione-S-transferase (GST) or beta-galactosidase, a relatively large coprotein that solubilizes the protein and 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
A polypeptide or polynucleotide of the invention and
Provided are compositions comprising immunostimulatory DNA sequences, particularly vaccine compositions and methods, as described in Sato, Y. et al., Science, 273: 352 (1996).

The present invention has also been shown to encode non-variable regions of bacterial cell surface proteins in the DNA constructs used in such genetic immunization experiments in animal models of Staphylococcus aureus infection. Also provided are methods of using the described polynucleotides or particular fragments thereof, which are particularly useful for identifying protein epitopes capable of raising a prophylactic or therapeutic immune response. This method produces a monoclonal antibody that is particularly useful for the resistance or elimination of infection from the required organs of an animal for the development of a prophylactic or therapeutic agent for a bacterial infection in an animal, especially a human, especially a Staphylococcus aureus infection. Can be done. 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 that 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 present invention relates to certain Di
Although described for the v1b 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. In-dwelling 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 Staphylococcus aureus. 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 be used in general to replace wound healing agents, antibiotic prophylaxis to prevent bacterial adhesion to matrix proteins exposed to wound tissue, or both. 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 injectable form. Conventional immune adjuvants can be used to enhance the immune response. A suitable unit dose for the vaccine is 0.5-5 μg / kg of antibody, and this dose is administered one to three times, preferably at intervals of one to three weeks. 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.

[0069]

EXAMPLES The following examples are carried out using standard techniques 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 Strain Selection, Library Preparation and Sequencing A polynucleotide having the DNA sequence shown in SEQ ID NO: 1 was obtained from a clone library of chromosomal DNA of Staphylococcus aureus in Escherichia coli. . Duplicate Staphylococcus aureus DN
Using the sequence data from two or more clones containing A, a contiguous DNA sequence of SEQ ID NO: 1 was constructed. The library may be produced by conventional means, for example, methods 1 and 2 below. Total cellular DNA is isolated from Staphylococcus aureus WCUH29 according to standard methods and size fractionated by one of two methods.

Method 1 For size fractionation according to standard methods, whole cell DN
A is mechanically sheared through the 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. The fragment was digested with EcoRI, the vector Lambda ZapI.
L. and package the library by standard methods, and then infect Escherichia coli with the packaged library. The library is amplified by standard methods.

Method 2 One restriction enzyme (eg, RsaI, PalI, AluI, Bs) suitable for obtaining a series of fragments for cloning the whole cell DNA into a library vector.
hl235I) or a combination thereof, and such fragments are size fractionated according to standard methods. A vector in which an EcoRI linker is ligated to the DNA and the fragment is then cut with EcoRI;
Ligate to Lambda ZapII, package the library by standard methods, and infect Escherichia coli with the packaged library. The library is amplified by standard methods.

Example 2 Divlb Characterization Escherichia coli FtsQ, Haemophilus influenza FtsQ, Bacillus subtilis Divlb and Staphylococcus aureus Divlb have similar hydrophobicities. In all four proteins, there is a highly hydrophobic region of about 20 amino acid residues that suggests a membrane spanning region. These regions are located on the N-terminal side of the mature peptide. Dai et al. On homologues of Escherichia coli., 1996 (J. Bacteriology 178, 1328-
According to the study of 1334), the cytoplasmic and membrane-bound region of StaphDivB is not necessarily required. Therefore, removal of the cytoplasmic and membrane-extending regions of Staph DivlB allows the evaluation of this protein in the following assays.

The interaction of FtsQ with other cell division proteins (eg, Fts A, Z, N, W, Y) is utilized in assays to identify DivlB agonists or antagonists. Methods for measuring the interaction of DivlB protein with added proteins and peptides in a lipid-based cell membrane system or in solution are provided as potential assays. Heterogeneous assays, including the use of immunoassays and surface coating techniques, with radiolabeled or visually labeled proteins and components can be used. The interaction of unlabeled DivlB with other polypeptides can be observed directly using surface plasmon resonance technology in a visualization biosensor device. This method is particularly useful for measuring interactions with large peptides (> 5 kDa) and is easily adapted to select inhibitors of protein-protein interactions. Solution homogeneous assays using fluorescently labeled components can be set up to show changes in fluorescence intensity, fluorescence anisotropy, fluorescence energy transfer, and reciprocal variations in fluorescence intensity as a result of binding. it can.
Binding proteins useful in these assays include, for example, the visualization biosensor method (Bartley et al., (1994) Nature 36).
8: 558), and bacterial extracts are subjected to affinity capture or chromatography on immobilized DivlB. The protein bound to the immobilized Div1B is eluted by salt, pH change or chaotropic reagent, and the eluted protein can be separated using a method with good resolving power such as reversed phase high performance liquid chromatography.
Individual peptides can then be characterized by N-terminal amino acid sequencing or mass mapping (mass spectrometry combining ionic molecular weights compatible with protein databases). Yeast two hybrid and phage display techniques are also used to identify ligands that bind to DivlB. The interaction of DivlB with the ligand, identified by the methods described above and other methods, is based on DivlB.
It will be the basis for assays to select for agonists and inhibitors of B.

The purified DivlB protein is used to raise antibodies in mouse, rabbit and other suitable animal hosts. Standard techniques using materials such as gold-antibody conjugates, fluorescently labeled antibodies, and GFP can be used to determine the location of DivlB protein and to examine the effect of the mixture.

[0076]

[Sequence list]

(1) General information: (Ii) Title of invention: New Div1b (iii) Number of sequences: 6 (iv) Contact: (A) Address: Dechart Price & Rose (B) Street: 4000 Bell Atlantic Tawa, 1717 arc・ Street (C) City name: Philadelphia (D) State name: Pennsylvania (E) Country name: United States (F) Zip code: 19103 (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: (C) Classification: (vii) Previous application data: (A) Application number: (B) Application date: (viii) Information on agents: (A) Name: Dickinson, Todd Kyu (B) Registration number: 28,354 (C) Processing number in agents: P50592-1 (ix) Telecommunication information: (A) Telephone Number: 215-994-2252 (B) Telefax number: 215-994-2222 (C) Telex:

(2) Information of SEQ ID NO: 1 (i) Sequence characteristics: (A) Sequence length: 1316 base pairs (B) Sequence type: nucleic acid (C) Number of chains: 2 (D ) topology: wherein the linear (xi) SEQ: SEQ ID NO: l: ATGGATGATA AAACGAAGAA CGATCAACAA GAATCAAATG AAGATAAAGA TGAATTAGAA 60 TTATTTACGA GGAATACATC TAAGAAAAGA CGGCAAAGAA AAAGGTCAAA GGCTACACAT 120 TTTTCTAATC AAAATAAAGA TGATACATCT CAACAAGCTG ATTTTGATGA AGAAATTTAC 180 TTGATAAATA AAGACTTCAA AAAAGAAGAA AGCAATGATG AAAATAATGG TTCTGCTTCT 240 AGTCATGCGA ATGATAATAA TATCGATGAT TCTACAGACT CTAATATTGA AAATGAGGAT 300 TATAGATATA ATCAAGAAAT TGACGACCAA AATGAATCGA ATGGAATTGC AGTCGCCAAC 360 GAACAACCTC AATCAGCTCC TAAAGAACAA AATAGCGACT CGAATGATGA GGAAACAGTA 420 ACGAAAAAAG AGCGAAAAAG TAAAGTAACA CAATTAAAGC CATTAACACT TGAAGAAAAG 480 CGGAAGTTAA GACGTAAGCG ACAAAAACGA ATCCAATACA GTGTTATTAC AATATTAGTA 540 TTGTTGATTG CTGTTATATT AATTTACATG TTTTCACCAC TTAGTAAAAT TGCGCATGTA 600 AATATAAATG GAAATAA TCA CGTTAGTACT TCAAAGATAA ACAAAGTTTT AGGTGTTAAA 660 AATGATTCGA GGATGTATAC GTTTAGTAAA AAAAATGCTA TTAATGATCT CGAAGAGGAT 720 CCATTAATCA CAAGTGTTGA GATACACAAG CAATTCCCAA ACACATTAAA CGTAGATATC 780 ACAGAAAATG AAATTATTGC TTTAGTGAAA TATAAAGGTA AATATTTACC TTTATTAGAA 840 AATGGTAAAT TGCTTAAAGG TTCAAATGAT GTCAAAATTA ATGATGCACC TGTCATGGAT 900 GGTTTCAAAG GTACAAAAGA AGATGATATG ATTAAGGCGT TATCTGAAAT GACACCTGAA 960 GTTAGACGAT ATATTGCCGA AGTGACATAC CCCCCAAGTA AAAACAAACA TAGCAGAATT 1020 GAATTGTTTA CGACAGATGG ACTTCAAGTA ATCGGTGATA TTTCGACGAT ATCTAAGAAA 1080 ATGAAATATT ATCCGCAGAT GTCACAATCA TTATCAAGGG ATAGTTCGGG TAAACTAAAA 1140 ACAAGAGGCT ATATTGATTT ATCAGTCGGT GCTTCATTTA TCCCATACCG TGGAAACACG 1200 TCTAGTCAAT CAGAAAGCGA TAAAAATGTG ACTAAATCAT CTCAAGAGGA AAATCAAGCA 1260 AAAGAAGAAT TACAAAGCGT TTTAAACAAA ATTAACAAAC AATCAAGTAA GAATAA 1316

(2) Information of SEQ ID NO: 2: (i) Sequence characteristics: (A) Sequence length: 439 amino acids (B) Sequence type: amino acids (C) Number of chains: one (D) Topology: linear (xi) Description of sequence: SEQ ID NO: 2: Met Asp Asp Lys Thr Lys Asn Asp Gln Gln Glu Ser Asn Glu Asp Lys 1 5 10 15 Asp Glu Leu Glu Leu Phe Thr Arg Asn Thr Ser Lys Lys Arg Arg Gln 20 25 30 Arg Lys Arg Ser Lys Ala Thr His Phe Ser Asn Gln Asn Lys Asp Asp 35 40 45 Thr Ser Gln Gln Ala Asp Phe Asp Glu Glu Ile Tyr Leu Ile Asn Lys 50 55 60 Asp Phe Lys Lys Glu Glu Ser Asn Asp Glu Asn Asn Gly Ser Ala Ser 65 70 75 80 Ser His Ala Asn Asp Asn Asn Ile Asp Asp Ser Thr Asp Ser Asn Ile 85 90 95 Glu Asn Glu Asp Tyr Arg Tyr Asn Gln Glu Ile Asp Asp Gln Asn Glu 100 105 110 Ser Asn Gly Ile Ala Val Ala Asn Glu Gln Pro Gln Ser Ala Pro Lys 115 120 125 Glu Gln Asn Ser Asp Ser Asn Asp Glu Glu Thr Val Thr Lys Lys Glu 130 135 140 Arg Lys Ser Lys Val Thr Gln Leu Lys Pro L eu Thr Leu Glu Glu Lys 145 150 155 160 Arg Lys Leu Arg Arg Lys Arg Gln Lys Arg Ile Gln Tyr Ser Val Ile 165 170 175 Thr Ile Leu Val Leu Leu Ile Ala Val Ile Leu Ile Tyr Met Phe Ser 180 185 190 Pro Leu Ser Lys Ile Ala His Val Asn Ile Asn Gly Asn Asn His Val 195 200 205 Ser Thr Ser Lys Ile Asn Lys Val Leu Gly Val Lys Asn Asp Ser Arg 210 215 220 Met Tyr Thr Phe Ser Lys Lys Asn Ala Ile Asn Asp Leu Glu Glu Asp 225 230 235 240 Pro Leu Ile Thr Ser Val Glu Ile His Lys Gln Phe Pro Asn Thr Leu 245 250 255 Asn Val Asp Ile Thr Glu Asn Glu Ile Ile Ala Leu Val Lys Tyr Lys 260 265 270 Gly Lys Tyr Leu Pro Leu Leu Glu Asn Gly Lys Leu Leu Lys Gly Ser 275 280 285 Asn Asp Val Lys Ile Asn Asp Ala Pro Val Met Asp Gly Phe Lys Gly 290 295 300 Thr Lys Glu Asp Asp Met Ile Lys Ala Leu Ser Glu Met Thr Pro Gnu 305 310 315 320 Val Arg Arg Tyr Ile Ala Glu Val Thr Tyr Pro Pro Ser Lys Asn Lys 325 330 335 His Ser Arg Ile Glu Leu Phe Thr Thr Asp Gly Leu Gln Val Ile Gly 340 345 350 Asp Ile Ser Thr Ile Ser Lys Lys Met Lys T yr Tyr Pro Gln Met Ser 355 360 365 Gln Ser Leu Ser Arg Asp Ser Ser Gly Lys Leu Lys Thr Arg Gly Tyr 370 375 380 Ile Asp Leu Ser Val Gly Ala Ser Phe Ile Pro Tyr Arg Gly Asn Thr 385 390 395 400 400 Ser Ser Gln Ser Glu Ser Asp Lys Asn Val Thr Lys Ser Ser Gln Glu 405 410 415 Glu Asn Gln Ala Lys Glu Glu Leu Gln Ser Val Leu Asn Lys Ile Asn 420 425 430 Lys Gln Ser Ser Lys Asn Asn 435

(2) Information of SEQ ID NO: 3 (i) Sequence characteristics: (A) Sequence length: 990 base pairs (B) Sequence type: nucleic acid (C) Number of chains: 2 (D ) topology: wherein the linear (xi) SEQ: SEQ ID NO: 3: ATGAATCGGA ATGGAATTGC AAGTCGGCAA CCGGACCAAC CTCAATCAGC TCCTAAAGAA 60 CAAAATAGCG ACTCGAATGA TGAGGAAACA GTAACGAAAA AAGAACGAAA AAGTAAAGTA 120 ACACAATTAA AGCCATTAAC ACTTGAAGAA AAGCGGAAGT TAAGACGTAA GCGACAAAAA 180 CGAATCCAAT ACAGTGTTAT TACAATATTA GTATTGTTGA TTGCTGTTAT ATTAATTTAC 240 ATGTTTTCAC CACTTAGTAA AATTGCGCAT GTAAATATAA ATGGAAATAA TCACGTTAGT 300 ACTTCAAAGA TAAACAAAGT TTTAGGTGTT AAAAATGATT CGAGGATGTA TACGTTTAGT 360 AAAAAAAATG CTATTAATGA TCTCGAAGAG GATCCATTAA TCAAAAGTGT TGAGATACAC 420 AAGCAATTAC CAAACACATT AAACGTAGAT ATCACAGAAA ATGAAATTAT TGCTTTAGTG 480 AAATATAAAG GTAAATATTT ACCTTTATTA GAAAATGGTA AATTGCTTAA AGGTTCAAAT 540 GATGTCAAAA TTAATGATGC ACCTGTCATG GATGGTTTCA AAGGTACAAA AGAAGATGAT 600 ATGATTAAGG CGTTATCTGA AATGACACCT GAAGTTAGAC GATATATTGC CGAAGTGACA 660 TACGCCCCAA GTAAAAACAA ACATAGCAGA ATTGAATTGT TTACGACAGA TGGACTTCAA 720 GTAATCGGTG ATATTTCGAC GATATCTAAG AAAATGAAAT ATTATCCGCA GATGTCACAA 780 TCATTATCAA GGGATAGTTC GGGTAAACTA AAAACAAGAG GCTATATTGA TTTATCAGTC 840 GGTGCTTCAT TTATCCCATA CCGTGGAAAC ACGTCTAGTC AATCAGAAAG CGATAAAAAT 900 GTGACTAAAT CATCTCAAGA GGAAAATCAA GCAAAAGAAG AATTACAAAG CGTTTTAAAC 960 AAAATTAACA AACAATCAAG TAAGAATAAT 990

(2) Information of SEQ ID NO: 4: (i) Sequence characteristics: (A) Sequence length: 330 amino acids (B) Sequence type: amino acids (C) Number of chains: one (D) Topology: linear (xi) Description of sequence: SEQ ID NO: 4: Met Asn Arg Asn Gly Ile Ala Ser Arg Gln Pro Asp Gln Pro Gln Ser 1 5 10 15 Ala Pro Lys Glu Gln Asn Ser Asp Ser Asn Asp Glu Glu Thr Val Thr 20 25 30 Lys Lys Glu Arg Lys Ser Lys Val Thr Gln Leu Lys Pro Leu Thr Leu 35 40 45 Glu Glu Lys Arg Lys Leu Arg Arg Lys Arg Gln Lys Arg Ile Gln Tyr 50 55 60 Ser Val Ile Thr Ile Leu Val Leu Leu Ile Ala Val Ile Leu Ile Tyr 65 70 75 80 Met Phe Ser Pro Leu Ser Lys Ile Ala His Val Asn Ile Asn Gly Asn 85 90 95 Asn His Val Ser Thr Ser Lys Ile Asn Lys Val Leu Gly Val Lys Asn 100 105 110 Asp Ser Arg Met Tyr Thr Phe Ser Lys Lys Asn Ala Ile Asn Asp Leu 115 120 125 Glu Glu Asp Pro Leu Ile Lys Ser Val Glu Ile His Lys Gln Leu Pro 130 135 140 Asn Thr Leu Asn Val Asp Ile Thr Glu Asn G lu Ile Ile Ala Leu Val 145 150 155 160 Lys Tyr Lys Gly Lys Tyr Leu Pro Leu Leu Glu Asn Gly Lys Leu Leu 165 170 175 Lys Gly Ser Asn Asp Val Lys Ile Asn Asp Ala Pro Val Met Asp Gly 180 185 190 Phe Lys Gly Thr Lys Glu Asp Asp Met Ile Lys Ala Leu Ser Glu Met 195 200 205 Thr Pro Glu Val Arg Arg Tyr Ile Ala Glu Val Thr Tyr Ala Pro Ser 210 215 220 Lys Asn Lys His Ser Arg Ile Glu Leu Phe Thr Thr Asp Gly Leu Gln 225 230 235 240 Val Ile Gly Asp Ile Ser Thr Ile Ser Lys Lys Met Lys Tyr Tyr Pro 245 250 255 Gln Met Ser Gln Ser Leu Ser Arg Asp Ser Ser Gly Lys Leu Lys Thr 260 265 270 Arg Gly Tyr Ile Asp Leu Ser Val Gly Ala Ser Phe Ile Pro Tyr Arg 275 280 285 Gly Asn Thr Ser Ser Gln Ser Glu Ser Asp Lys Asn Val Thr Lys Ser 290 295 300 Ser Gln Glu Glu Asn Gln Ala Lys Glu Glu Leu Gln Ser Val Leu Asn 305 310 315 320 Lys Ile Asn Lys Gln Ser Ser Lys Asn Asn 325 330

(2) Information of SEQ ID NO: 5 (i) Sequence characteristics: (A) Sequence length: 20 base pairs (B) Sequence type: Nucleic acid (C) Number of chains: One (D) Topology: linear (xi) Description of sequence: SEQ ID NO: 5: ATGGATGATA AAACGAAGAA 20

(2) Information of SEQ ID NO: 6: (i) Sequence characteristics: (A) Sequence length: 20 base pairs (B) Sequence type: nucleic acid (C) Number of chains: 1 (D ) Topology: linear (xi) Description of sequence: SEQ ID NO: 6: TTATTCTTAC TTGATTGTTTT
20

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 48/00 A61K 48/00 ABA ABA C07K 2/00 C07K 2/00 14/31 14/31 C12N 1/21 C12N 1/21 C12P 21/02 C C12P 21/02 C12Q 1/68 A C12Q 1/68 C12P 21/08 // C12P 21/08 A61K 37/02 ADZ (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1 : 19) (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 Kenneth H. Pears Jr., Phoenix Building, Pennsylvania, United States 19426 Anderson Avenue 7 (72) Inventor John Edward Hodgson Lap Road 260, Malvern, Pennsylvania, United States 19355 United States David John Paine United States 19460 Waterfall Way, Phoenixville, Pennsylvania, United States 19460 No. 618 (72) Inventor Stuart Campbell Pearson High Point Drive 75, Birwin, PA 19212, U.S.A.

Claims (20)

[Claims] (1) a polynucleotide having at least 70% identity to a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; (b) a deposited strain of Staphylococcus aureus (Stap)
hylococcus aureus), a polynucleotide having at least 70% identity to the polynucleotide encoding the same mature polypeptide as expressed by the Div1b gene contained in the Div. 1b gene; 7
A polynucleotide encoding a polypeptide consisting of an amino acid sequence having 0% identity; (d) a polynucleotide complementary to the polynucleotide of (a), (b) or (c); and (e) An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a polynucleotide consisting of at least 15 consecutive bases of the polynucleotide of (a), (b), (c) or (d). 2. The polynucleotide according to claim 1, wherein the polynucleotide is DNA. 3. The polynucleotide according to claim 1, wherein the polynucleotide is RNA. 4. The polynucleotide according to claim 2, comprising the nucleotide sequence of SEQ ID NO: 1. 5. The polynucleotide according to claim 2, consisting of nucleotides 1 to 1317 shown in SEQ ID NO: 1. 6. The polynucleotide according to claim 2, which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 7. A vector comprising the polynucleotide according to claim 1. 8. A host cell comprising the vector according to claim 7. 9. A DNA from the host cell according to claim 8.
A method for producing a polypeptide, comprising expressing the polypeptide encoded by the polypeptide. 10. A method for producing a polypeptide or fragment according to claim 8, wherein the host is cultured under conditions sufficient to produce a Div1b polypeptide or fragment. 11. A polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2. 12. A polypeptide comprising the amino acid sequence of SEQ ID NO: 2. 13. An antibody against the polypeptide according to claim 11. 14. An antagonist which inhibits the activity or expression of the polypeptide according to claim 11. 15. A method for treating an individual in need of a Div1b polypeptide, comprising administering to the individual a therapeutically effective amount of the polypeptide of claim 11. 16. A method for treating an individual in need of inhibiting Div1b polypeptide, comprising administering to the individual a therapeutically effective amount of the antagonist of claim 14. 17. A method for diagnosing a disease associated with the expression or activity of the polypeptide according to claim 11 in an individual, comprising measuring (a) 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. 18. A method for identifying a compound that interacts with the polypeptide according to claim 11 and inhibits or activates the activity of the polypeptide, wherein the compound to be screened is capable of interacting 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 from the interaction between the polypeptide and the compound to determine whether the compound interacts with the polypeptide and activates or inhibits its activity. Method consisting of. 19. A method for inducing an immune response in a mammal, comprising using the Div1b polypeptide of claim 11 or a fragment or variant thereof suitable for generating an antibody and / or T cell immune response. A method of inoculating an animal and protecting the animal from disease. 20. A method for inducing an immune response in a mammal, comprising the steps of:
Fragments or variants thereof for expressing an iv1b polypeptide or eliciting an immunological response in vivo and delivering the fragment or variant to produce an antibody and / or T cell immune response can be used to deliver the animal. How to protect from disease.