JPH11506022A - Polynucleotide and amino acid sequences from Staphylococcus aureus - Google Patents

Abstract

  (57) [Summary] The present invention relates to Staphylococcus polynucleotides, polypeptides encoded by the polynucleotides, such polynucleotides and polypeptides, and the production of such polynucleotides and polypeptides, transforming the polynucleotides. To a recombinant host cell. The present invention also relates to the use of the inhibitors in inhibiting and treating the biosynthesis or action of the polynucleotides and polypeptides.

Description

DETAILED DESCRIPTION OF THE INVENTION Polynucleotide and amino acid sequences from Staphylococcus aureusField of the invention   The present invention relates to the production of newly identified polynucleotides and polypeptides. As well as recombinant host cells transformed with the polypeptide. It relates to polynucleotides and polypeptides. The present invention relates to the use of such polypeptides. It relates to inhibiting biosynthesis or action and the use of inhibitors in therapy.Background of the Invention   The genus Staphylococcus is a medically important microorganism genus. They are known to produce two types of diseases, invasive and toxinogenic . Invasive infections are commonly tumors that affect both skin surface and deep tissues Has the characteristic of forming Staphylococcus aureus warns of bacteremia in cancer patients Next cause. Osteomyelitis, septic arthritis, septic thrombophlebitis and And acute bacterial endocarditis are also relatively common. Staphylococcus venom At least three clinical conditions are caused by the intrinsic properties. Manifestation of these diseases Are the result of the action of exotoxins as against tissue infiltration and bacteremia. This These conditions include: Staphylococcus food poisoning, burn-like skin syndrome and toxic Queshock syndrome and the like.   Several staphylococcal proteins associated with virulence have been identified, For example, coagulase, hemolysin leucocidin and exo and enteroki Syn is a gene for bacterial pathogenesis during infection and disease progression in mammalian hosts Very little is known about the transient expression of Set of bacterial genes The findings are likely to manifest different stages of the infection, and specifically when the infection has been established At the stage, by identifying possible, previously unrecognized targets, Significance for the search and characterization of novel antibiotics that can inhibit development Provide important information.   In recent years, several new claims claiming to help comprehensive gene expression during infection The approach has been described [Chung, S. et al. [1993] Global Regulation of Gene  Expression in Escherichia coli, J. Bacteriol. 175, 2026-2036, Mahan M. J.  [1993] Selection of Bacterilal Virulence Genes That Are Specifically I nduced in Host Tissues, SCIENCE 259,686-688. Hensel, M. et al. [1995] Simultan eous Identification of Bacterial Virulence Genes by Negative Selection, SCIENCE 269,400-403]. These new technologies are still in use for Gram-negative pathogens today. But no gram-positive infection has probably been Comprehensive transposers required for these strategies in these organisms Possibly because the zon mutagenesis and the development of suitable vectors are very slow . And Chuang, S .; In the case of the process described by A. et al. Difficulties in isolating the appropriate amount of bacterial RNA free of milk RNA can be attributed to infected tissue. Derive from labeling the bacterial RNA for sufficiently high specific activity. Departure Ming et al. To determine the gene expression of a pathogen at different stages of infection in a mammalian host. Use new technology.Detailed description of the invention   A novel aspect of the present invention is a northern block of bacterial RNA preparations from infected tissue. Appropriately labeled oligonucleotides that specifically anneal to bacterial ribosomal RNA in Related to the use of oligonucleotide probes. As a target for hybridization The use of more abundant ribosomal RNA will result in the appropriate size and Facilitates optimization of protocols for purifying bacterial RNA for RT-PCR in low and high amounts. Purify RNA of Staphylococcus aureus from bacteria growing in vitro Techniques for use in making have been reported in the scientific literature.   In a first aspect, therefore, the present invention relates to the use of RT-PCR Genes transcribed in organs in infected host tissues by identifying RNA For selectively damaging mammalian RNA, and at the same time, RT- Appropriate bacterial disruption techniques to obtain sufficient quantities of bacterial RNA for PCR Is it possible to enrich bacterial RNA to obtain total RNA from infected tissues? Thus, a bacterial mRNA preparation was obtained, wherein the conditions to selectively enrich bacterial RNA were: Probe with oligonucleotide probes specific for bacterial ribosomal RNA Is determined by   This optimization process preferably determines conditions that give optimal levels of bacterial RNA. Bacterial Ribozos Used in Northern Experiments to Determine Experimental RNA Preparations A unique labeled oligonucleotide probe for the RNA is used. Bacteria Ribosomal RNA is present in bacterial mRNA species on the order of 2-4 in quantity. As a result, bacterial ribosomal RNA is not detected in infected tissues. The presence and quality of bacterial RNA in the presence of larger and higher levels of mammalian RNA Provide appropriate sensitivity suggestions. This detection system has a 1%   Combine with total RNA visualization by ethidium bromide staining of agarose gel May be used. On these gels, mammalian ribosomal RNA is It can be identified because it moves at a different rate than ribosomal RNA. Surprisingly, The disruption state that was found to lead to mammalian RNA deletions was Gives the best preparation of bacterial RNA as judged by experimentation. Stafiloko Application of this method for genes expressed in C. aureus A suitable oligonucleotide useful for this purpose is 5-gctcctaaaaggtta. ctccaccggc-3 '[SEQ ID NO: 91].   Using the technology of the present invention, it is possible to identify bacterial genes that are transcribed during infection, The inhibitor would have utility in antimicrobial therapy. Such gene transcription, Subsequent translation of the resulting mRNA or inhibition of the function of the corresponding expressed gene. Harmful agents may have utility in antimicrobial treatment.   The present invention relates to any of the sequences in Table 1 or the sequence 1 in Table 1 [SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 4 3, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76] A DNA sequence selected from the group consisting essentially of: We also offer matching. The present invention further relates to S. aureus W A polynucleotide encoding the protein of CUH 29 is provided, wherein the sequence 1 of Table 1 [ SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34 , 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70 , 73, 76]. It is characterized by that. Sequence 1 of Table 1 [SEQ ID NOs: 1, 4, 7, 10, 13, 16] , 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52 , 55, 58, 61, 64, 67, 70, 73, 76]. The polynucleotide having the DNA sequence obtained in a row is E. coli. Of a library of clones of chromosomal DNA of S. aureus WCUH 29 Obtained from sequencing.   Es Aureus WCUH 29 is the Nati of Aberdeen, Scotland onal Collection of Industrial and Marine Bacteria Ltd. (NCIMB) Deposited under NCIMB 40771 on September 11, 5th.   The present invention also relates to the sequence 1 of Table 1 [SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76] To be obtained by expression of a gene characterized in that it consists of a given DNA sequence. New from Staphylococcus aureus Provide specific proteins or their fragments, analogs or derivatives You.   The present invention further relates to the sequence shown in Sequence 2 of Table 1 or the sequence 2 [ Column numbers: 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90], the amino acid obtained in any of the sequences selected from the group consisting essentially of: Staphylococcus aureus WCUH29 characterized by having a sequence The present invention relates to a novel protein, fragment, analog or derivative thereof.   The present invention also relates to SEQ ID NO: 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90]. Polypeptide fragment of a protein having an amino acid sequence For conductors.   As used herein, the term polypeptide refers to proteins and fragments thereof, and analogs. Or derivative.   In another embodiment of the invention, a polypeptide encoding such a polypeptide is provided. A nucleotide (DNA or RNA) is provided.   The invention also relates to a staphylo identified herein in whole or in part. To determine whether the C. aureus gene is transcribed in infected tissues Act as PCR primers in the process described herein Sequence 1 of Table 1 [SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34.37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76] [SEQ ID NO: 3] : 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38 , 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74 , 77] and 4 [SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78]. For regular oligonucleotides. Such sequences can also be used at the stage of infection and Would have utility in diagnosing the type of infection that the pathogen achieves.   Each DNA sequence provided herein provides for the discovery and development of antimicrobial compounds. Can be used in departure. The protein encoded upon expression is Can be used as targets for training. In addition, the encoded protein DNA sequence of the white matter-coding region, Shine-Delgano sequence Or other corresponding mRNA transcription-promoting sequences may also be of interest. Can be used to construct antisense sequences to control expression of . Further, many of the sequences disclosed herein are upstream of the encoding sequence. Or provide a downstream area. These sequences are used to control bacterial gene expression. Useful as a source of control elements. Such sequences can be conveniently prepared by the action of restriction enzymes. single Released or chemically synthesized, for example, introduced into a promoter-identifying strain. You. These strains contain a reporter gene if an active promoter is inserted. Reporter structural gene located downstream from the restriction enzyme site that will be expressed including.   Although each sequence can be used as described above, the present invention provides particularly useful target genes. Some means are also provided for identifying offspring. The first of these approaches Requires searching a suitable database for sequence matches. Therefore, if a homolog is present, the Staphylococcus-like form of this gene Shape is likely to play a similar role. For example, to the cell surface in another organism Staphylococcal proteins identified as homologous are valuable vaccine candidates. Would be for Such homology is identified as the sequences disclosed herein To the extent, they are reported along with the encoding sequence.   Encodes the protein using any of the DNA sequences given in SEQ ID NO: 1 Typically, E. coli or several polynucleotides to obtain Cloning of a chromosomal DNA clone of S. aureus WCUH29 in another suitable host Libraries are released from subsequences, preferably 17 mer or longer, Probe with radiolabeled oligonucleotide. Has the same DNA as the probe Clones can therefore be distinguished using a high stringent wash. Original array Individual clones identified with sequencing primers designed from Expands the sequence in both directions to determine the entire gene sequence It is possible. Conveniently, such sequencing is performed from plasmid clones. This is performed using the prepared denatured double-stranded DNA. Suitable technologies are Maniatis, T. and  Frisch, E .; F., MOLECULAR CLONING, A Laboratory Manual 1989 [Part 2, Cold  Spring Harbor Laboratory. Screening By Hybridization 1.90 and Sequenc ing Denatured Double-Stranded DNA Templates 13.70.] Have been.   The polynucleotide of the invention may be in the form of RNA or DNA, NA includes cDNA, genomic DNA, and synthetic DNA. DNA is two It can be single-stranded or single-stranded, and if single-stranded, the coding strand or non-core. Leading (anti-sense) strand. Encodes a polypeptide The coding sequence is the sequence 1 in Table 1 [SEQ ID NOs: 1, 4, 7, 10, 13, 16]. , 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52 , 55, 58, 61, 64, 67, 70, 73, 76] Or as a result of duplication or condensation of the genetic code , Different coding sequences encoding the same polypeptide.   The present invention relates to a polypeptide of the invention, in particular sequence 2 [SEQ ID NO: 79] of Table 1. , 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90]. A specific polypeptide sequence characterized by the deduced amino acids represented by each Fragments, analogs and derivatives of the polynucleotides described above. Including variants. A variant of the polynucleotide is a naturally occurring version of the polynucleotide. Allelic variants or variants of a polynucleotide that are not naturally occurring Is a seed.   Thus, the present invention relates to polynucleotides encoding the same polypeptide of the present invention. In which the variant comprises a polypeptide fragment, derivative or analog. As with such polynucleotide variants encoding the log, sequence 2 [SEQ ID NO: Column numbers: 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90]. Such a nu Variants of nucleotides include deletion variants, substitution variants, addition or insertion variants.   The polynucleotide has the sequence 1 [SEQ ID NO: 1, 4, 7, 10, 13, 16, 1]. 9, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 5 5, 58, 61, 64, 67, 70, 73, 76]. A naturally occurring algorithm for coding sequences characterized by any DNA sequence. It has a coding sequence that is a relic variant. As is known in the art, Rick variants have one or more nucleotide substitutions, deletions or additions, Does not substantially alter the function of the encoded polypeptide.   The polynucleotide encoding the mature polypeptide may be a copy of the mature polypeptide. Coding sequence of the mature polypeptide, Further coding, such as der or secretory or proprotein sequences Contains an array.   Thus, the term "polynucleotide encoding a polypeptide" , A polynucleotide comprising additional coding and / or non-coding sequences Polynucleotides containing only coding sequences for polypeptides, similar to otides Includes tide.   The present invention thus provides that the coding region of a mature polypeptide is a host cell. Polynucleotide sequences that assist in the expression and secretion of these polypeptides, e.g., A leader sequence that functions as a secretory sequence that controls the transport of polypeptide from the vesicle You may fuse them into the same reading frame. The polypeptide having the leader sequence is a preprotein That are cleaved from the host cell to form the mature form of the polypeptide. And a polynucleotide that may have a leader sequence. Polype with leader sequence Peptides are preproteins that are used by the host to form the mature form of a polypeptide. Has a leader sequence that is cleaved from the cell. Polynucleotide is a mature protein plasmid It also encodes a proprotein that is an additional 5 'amino acid residue. Components with professional sequence Mature proteins are proproteins and are inactive forms of the protein. Pro sequence cut This leaves the active mature protein.   Therefore, the polynucleotide of the present invention may be a mature protein or a protein having a prosequence. Coat white matter or proteins that have both prosequence and presequence (leader sequence). May be loaded. Further, the amino acid sequence provided herein has the NH_Two-Terminal Shows a methionine residue. However, during the post-translational modification of the peptide, this residue Is preferably deleted. Thus, the present invention contemplates the use of both sequences You.   The expression vector contains the specific coding sequence along with the appropriate regulatory sequences in the vector. Position and orientation of the coding sequence with respect to the control sequence. Is transcribed under the control of the control sequence (ie, DNA RNA polymerase that binds to the molecule transcribes the coding sequence). Coordination I Modification of the signaling sequence may be desirable to achieve this purpose. For example, go In some cases, the control sequence can be attached in an appropriate orientation, ie, the reading frame It may be necessary to modify the sequence to maintain it. Control sequences and other adjustments Prior to insertion into a vector, such as the cloning vector described above, Can be linked to a coding sequence. Alternatively, the coding sequence is already controlled It can be cloned directly into an expression vector containing the sequence and appropriate restriction sites.   Generally, recombinant expression vectors contain an origin of replication and a transformant for the host cell. Selectable markers that allow for mation, such as E. coli and Ampicillin resistance of the TRP1 gene of S. cerevisiae Promotes from highly expressed genes to direct transcription of gene and downstream structural sequences Data. Heterologous structural sequences include translation initiation and termination sequences. In addition, preferably in the periplasmic space or extracellular medium of the translated protein Assemble at the appropriate stage with a leader sequence that allows for direct secretion into Desired Depending on the heterologous sequence, the desired properties, for example, the stability of the expressed recombinant product, can be improved. Fusion proteins, including N-terminal identification peptides, that provide for simplified or simplified purification Can encode white matter.   Vectors containing the appropriate DNA sequences are compatible with the appropriate promoter or control sequences. As described above, a suitable host is used for expressing a protein in a host. Can be used to transform   More specifically, the invention relates to a recombinant comprising one or more of the sequences described broadly above. It also consists of constructs. The construct may be a plasmid or a viral vector. Into which the sequence of the invention is inserted in a forward or reverse orientation. You. In a preferred embodiment of this embodiment, the construct further comprises, for example, (operable It consists of regulatory sequences, including a promoter operably linked to the sequence. Multiple fit Various vectors and promoters are known in the art and are commercially available. is there. The following vectors are provided as examples. Bacterial: pET-3 vector ( Stratadine (Stratagene), pQE70, pQE60, pQE-9 (Qiagen (Q iagen)), pbs, pD10, phagescript, psiX 174, pbluescript SK (pbluescript SK), pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); pt rc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pBlueBacIII (in vitro Invitrogen), pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSV L (Pharmacia). However, any other plasmid or Vectors may be used as long as they are replicable in the host and visible.   Recombinant DNA vectors for cloning and their transformation Examples of possible host cells include bacteriophage λ (E. coli), pBR 322 (E. coli), pACYC177 (E. coli), pKT230 (gram) Negative bacteria), pGV1106 (Gram negative bacteria), pLAFR1 (Gram negative cells) Bacteria), pME290 (Gram-negative bacteria not E. coli), pHV14 (E. coli) Coli and Bacillus subtilis, pBD9 (Bacillus subtilis). illus)), pIJ60 (Streptomyces), pUC6 (Streptomyces) Leptomyces), YIp5 (Saccharomyces), baculo The virus-infected insect cell system, YCp19 (Saccharomyces). General Specifically, "DNA cloning": Volumes I and II, edited by Glover et al., IRLPress  Oxford (1985) (1987) and; Maniatis et al., "Molecular Cloning", Cold S. See the Spring Harbor Laboratory (1982). Methionine of each protein Amino-terminal variants containing and free of methionine are disclosed herein .   The polypeptides of the present invention may also include a marker sequence at the 5 'or 3' end of the gene. And a coding sequence fused in frame. Thus, the polypeptide of the present invention can be purified. The marker sequence is In some cases, to provide purification of the polypeptide fused to the marker, the pQE series Vectors (commercially available from Qiagen Inc.) Hex-histidine labeling as described above.   The present invention further provides that if between sequences at least 50%, and preferably 7%, A polynucleotide that hybridizes to the sequence described above if there is 0% identity About. The present invention specifically relates to the stringent polynucleotides described above. Staphylococcus polynucleotides that hybridize About As used herein, the term "stringent conditions" refers to the sequence If there is at least 95% and preferably 97% identity between them, It means that hybridization can occur. In a preferred embodiment, The polynucleotide that hybridizes to the above-described polynucleotide is the present invention. Polypeptide having substantially the same biological function or activity as the polypeptide of the invention Code. Preferred embodiments of the present invention include SEQ ID NOs: 79, 80, 81, 82 , 83, 84, 85, 86, 87, 88 and 89 consisting essentially of Or any combination of these amino acid sequences 70%, 80%, 9% Polynucleotides having 0% or 95% identity.   The deposit referred to herein relates to the international recognition of the deposit of microorganisms in patent proceedings. Will be maintained in the sense of the Budapest Treaty. These deposits are made by those skilled in the art. Provided only for deposits of 35 U.S. S. C. Required under Chapter 112 I do not admit that. To the polypeptide contained in the deposited material As with the encoded amino acid sequence, The sequence of the nucleotides is incorporated herein by reference, and the sequences herein If there is a collision with any of the above statements, control is performed. Create and use deposited materials Requires a license to use and sell, but such a license is not Not given in the book.   When referring to a polypeptide of the present invention, “fragments”, “derivatives” and The term "analog" refers to the same biological function or activity as such a polypeptide. Retain the nature. Thus, analogs produce only active mature polypeptides. And proproteins that can be activated by cleavage of the proprotein portion.   The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. A polypeptide, preferably a recombinant polypeptide.   Fragments, derivatives or analogs of the polypeptides of the present invention may comprise (i) Wherein one or more amino acid residues is a retained or unretained amino acid Substituted with an acid residue (preferably a conserved amino acid residue) Noic acid residues may or may not be encoded in the genetic code Or (ii) wherein one or more amino acid residues have a substituent Or (iii) wherein the polypeptide is a compound of the polypeptide To increase the half-life, another compound (eg, polyethylene glycol ) Or (iv) within which a leader or secretory sequence is present. Or sequences used for purification of polypeptide or proprotein sequences Would be fused to additional amino acids to the polypeptide. Such a hula Fragments, derivatives and analogs are within the scope of those skilled in the art, given the teachings herein. Seems to be in the enclosure.   The polypeptides and polynucleotides of the present invention are preferably in isolated form. Provided and preferably purified to homogeneity.   The term “isolate” refers to a substance that is removed from its original environment (eg, If it occurs naturally, it means the natural environment). For example, living movement The naturally occurring polynucleotide or polypeptide present in the product is not isolated. But the same polyisolated from some or all co-existing materials in natural systems The nucleotide or polypeptide is isolated. Such a polynucleotide is May be part of a vector and / or such a polynucleotide or The polypeptide can be part of a composition, and can be a vector or composition What is isolated in an object is not part of its natural environment.   The present invention relates to a vector containing the polynucleotide of the present invention, and Host cells designed with vectors, production of polypeptides of the invention by recombinant techniques Related to life.   In a further embodiment of the present invention, a polypeptide encoding a polypeptide in a host is provided. By recombinant techniques by producing nucleotides and recovering the expressed product. And methods for producing the polypeptides of the present invention. As an alternative, the po Ripeptides can be produced synthetically by conventional peptide synthesizers.   A host cell can be, for example, a cloning vector or an expression vector. Genetically designed with the vector of the invention (either transduced or Sculpted or transfected). Vectors, for example, plus It may be in the form of a mid, cosmid, phage and the like. The designed host cells are Cultivation in a normal nutrient medium, modified as appropriate for Select a formant or amplify the gene. Culture conditions such as temperature and pH Are those previously used with the host selected for expression and will be apparent to those of skill in the art. Will.   Suitable expression vectors include chromosomal, non-chromosomal and synthetic DNA sequences, e.g. Bacterial plasmid; phage DNA; baculovirus; yeast plasmid; And vectors derived from combinations of DNA and phage DNA. However, no Other vectors may be used as long as they are replicable and viable in the host. Can be   The appropriate DNA sequence may be inserted into the vector by various methods. general Alternatively, the DNA sequence may be converted to a suitable restriction endonuclease by methods known in the art. Inserted into the site.   The DNA sequence in the expression vector is ligated with an appropriate expression control vector for direct mRNA synthesis. Operably linked to a row (promoter). Such a promoter Representative examples may be mentioned here: LTR or SV40 promoter ー, E. coli lac or trp, phage lambda P_LPromoter or eukaryote, Other prokaryotic or viral genes known to control gene expression promoter. The expression vector contains this ribosome binding site for translation initiation and And a transcription terminator. The vector also contains the appropriate sequences for expression amplification. Including.   Further, the expression vector is preferably a dihydrofolate for eukaryotic cell culture. Original enzyme or neomycin resistance, or coli tetracycline or Traits for selection of transformed host cells, such as resistance to ampicillin And one or more selectable marker genes to provide genetic characteristics.   A gene is a vector in which the DNA sequence encoding the desired protein contains the expression construct. So that it is transcribed into host cell RNA transformed by the Motor, a ribosome binding site (for bacterial expression) and, additionally, Adjustment of the operator (collectively referred to herein as the “adjustment” element) Put it down. The coding sequence may include a signal peptide or leader sequence. It may or may not be out. The polypeptide of the present invention can be, for example, E. coli. coli tac Using a promoter or protein A gene (spa) and a signal sequence. Can be manifested. Leader sequence is a bacterium in post-translational processing It can be removed by the host. For example, U.S. Pat. No. 4,431,739; See 4,425,437; 4,338,397. Promoter territory The region is CAT (chloramphenicol transferase) vector or selection Use any other vector with a specific marker to select from any desired gene. You. Two suitable vectors are PKK232-8 and PCM7. Especially fingers The defined bacterial promoters are lacI, lacZ, T3, T7, gpt, lambda P_R, P_L And trp. Eukaryotic promoters include CMV immediate, HSV thymidine Kinases, early and late SV40, LTRs from retrovirus, and And mouse metallothionein-I. Choosing the right vector and promoter Is within the level of ordinary skill in the art.   In addition to regulatory sequences, allows for regulation of protein sequence expression compared to host cell growth It may be desirable to add a regulatory sequence that: Regulatory sequences are known to those skilled in the art. For example, chemical or physical expression, including the presence of a regulatory compound, Includes things that wake up or cease in response to a stimulus.   In some cases, secretion of the polypeptide from the host organism and subsequent It may be desirable to add a sequence that causes cleavage of the subsequent secretion signal U.   The polypeptide can be expressed in host cells under the control of a suitable promoter. Wear. Cell-free translation systems use such RNAs from the DNA constructs of the invention. It could also be used to produce proteins. Eukaryotic and nuclear facilities Suitable cloning and expression vectors for primary use are described in Sambrook et al., Mo. lecular Cloning: A Laboratory Manual, Vol. 2, Cold Spring Harbor, N.Y. (1 989), the disclosure of which is incorporated herein by reference.   Transformation of appropriate host strains and transformation of host strains to appropriate cell densities Subsequent to growth, the selected promoter is treated by appropriate means (eg, temperature shift or Is induced by chemical induction) and the cells are cultured for an additional period.   Cells are typically collected by centrifugation and disrupted by physical or chemical means. The broken and the resulting crude extract was retained for further purification.   Microbial cells used in protein expression include freeze-thaw cycling, sonication, Processing, mechanical disruption, or any convenient method, including the use of cytolytic agents. However, such methods are well known to those skilled in the art.   Depending on the expression system and host selected, the polypeptides of the invention may be of interest Under the conditions under which the polypeptide is expressed, the trans It can be produced by growing a formed host cell. Polypep The tide is then isolated from the host cell and purified. If the expression system grows the polypeptide If secreted into the medium, the polypeptide can be purified directly from the medium. if If the polypeptide is not secreted, it can be isolated from cell lysates or Recover from the alveolar fraction. If the polypeptide is present on the cell surface, the whole cell Use of the body or isolated membrane as an analytical source of the desired gene product it can. E. Polypeptides expressed in bacterial hosts such as E. coli Would require isolation and regeneration from the fusion body. Mature protein, If you have very hydrophobic regions that lead to overexpressed insoluble products, It is desirable to express a deleted protein with a deleted region. Choosing appropriate growth conditions And methods of recovery are within the skill of the art.   Ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange Chromatography, phosphocellulose chromatography, hydrophobic interaction Chromatography, affinity chromatography, hydroxyapatite By methods including chromatography and lectin chromatography, The polypeptide can be recovered and purified from the recombinant cell culture. The protein regeneration method is Can be used as needed to complete the configuration of the mature protein It is. Finally, high performance liquid chromatography (HPLC) is the final purification step Available for floors.   As can be used in recombinant production methods, the polypeptides of the invention may be glycosylated. It may be silylated or unglycosylated. The peptide of the invention is the first It may contain a methionine residue.   A "replicon" is a functioning autonomous unit of DNA replication in vivo. That is, any genetic element that is replicable under its own control (eg, Sumids, chromosomes, viruses).   A "vector" contains another D such that the attached segment replicates. Plasmid, phage or cosmid to which the NA segment can attach This is a replicon.   A “double-stranded DNA molecule” is a double-stranded DNA molecule in both a relaxed and supercoiled state. Lix, polymerized deoxyribonucleotides (adenine, guanine, thymidine Or cytosine base). This term refers to the primary and secondary structure of a molecule. Mean and not limited to any particular form. Therefore, this term is especially , Linear DNA molecules (eg, restriction fragments), viruses, plasmids, and dyes. Contains double-stranded DNA found in chromosomes. Discuss the structure of a specific double-stranded DNA molecule In discussing, the untranscribed strand of DNA (ie, having sequence homology to mRNA) According to the usual convention of giving only sequences in the 5 'to 3' direction along The sequence may be described herein.   Encoding a specific protein "coding sequence" or a specific protein " Nucleotide sequence '' is transcribed when placed under the control of appropriate regulatory sequences. A DNA sequence that is translated into a polypeptide.   "Promoter sequence" refers to DNA capable of binding RNA polymerase in a cell. A regulatory region that initiates transcription of the downstream (3 'direction) coding sequence. The present invention For the purpose of the definition of By a don (e.g., ATG) to the 3 'end and above background Minimum number of bases or elements required to initiate transcription at detectable levels To expand upstream (5 'direction). Causes of RNA polymerase binding Nuclease S1 as well as the protein binding domain (consensus sequence) (Conveniently defined by mapping in). U. Eukaryotic promoters are often, but not always, "TATA Box and a "CAT" box. Prokaryotic promoters are -10 And a Shine-Dalgarno sequence in addition to the -35 consensus sequence.   DNA "control sequences" include a promoter sequence, a ribosome binding site, and boriadeni. Host cell, such as a cleavage site, transcription termination sequence, upstream regulatory domain, enhancer Collectively provide expression (ie, transcription and translation) of coding sequences therein Collectively means things.   The control sequence binds to the promoter sequence by RNA polymerase and binds to the coding sequence. The sequence is transcribed into mRNA, which is then transcribed by the coding sequence "Directs expression" of a coding sequence in a cell when translated into a repeptide.   A “host cell” is defined as a transform or transform by an exogenous DNA sequence. Transfected or transformed or transfected Cells that can be   A cell is "transformed" when exogenous DNA has been introduced into the cell membrane. Exogenous DNA is integrated (shared) into the chromosomal DNA that makes up the genome of the cell. (Combined) or not. In prokaryotes and yeast, for example, The native DNA may be maintained as an episomal element, such as a plasmid. Eukaryotic Vesicles are stably transformed or transfected The cells contain foreign DNA that is passed on to daughter cells through chromosomal replication. It is integrated into the chromosome so that it breaks. This stability allows exogenous DNA Eukaryotic to establish cell lines or clones, consisting of expansion of daughter cells, including Indicated by the ability of the cell.   A "clone" is a cell that is derived from a single cell or a common ancestor by mitosis Is a group. A “cell line” is a cell line that is stable in vitro for many generations. Primary cell clones capable of growth.   The “heterologous” region of a DNA construct is a natural term for assembling with other molecules. In or attached to another DNA molecule that is not It is a definable DNA segment.   According to a further aspect of the present invention, there is provided a method for treating or preventing a polypeptide of the present invention. For use as antibacterial agents or vaccines.   In another embodiment of the present invention, the polynucleotide of the present invention is used for therapeutic purposes or Provide use for prophylactic purposes, especially in genetic immunization.   In yet another embodiment of the present invention, such a poiy useful as an antimicrobial agent. An inhibitor for a polypeptide is provided. In particular, for such polypeptides An antibody is provided.   Another embodiment of the present invention provides a polypeptide, polynucleotide as described above of the present invention. Alternatively, it is a pharmaceutical composition comprising an inhibitor and a pharmaceutically acceptable carrier thereof.   In a specific embodiment of the present invention, there is provided the use of the inhibitor of the present invention as an antibacterial agent. I do.   The invention further relates to the manufacture of a medicament for such use.   The polypeptide is used as a host vaccine to produce specific antibodies with antimicrobial activity. It can be used as an antigen for chin injection.   Polypeptides or cells expressing them produce antibodies against them It can be used as an immunogen for These antibodies are, for example, polyclones It can be a null or monoclonal antibody. The term antibody refers to Fab fragment Chimeric, single chain, and humanized antibodies, or Fab expression lines Includes Berry products. Various methods known in the art may be used for such antibodies and It may be used for the production of fragments.   Antibodies raised against a polypeptide of the present invention can be used to inject the polypeptide directly into animals. Or administering the polypeptide to an animal, preferably a non-human animal. Obtained by: The antibody thus obtained is the polypeptide itself Will bind to In this method, only fragments of the polypeptide are The encoding sequence results in an antibody that binds to the whole naturally occurring polypeptide. Can be used for Such antibodies may be from tissues that express the polypeptide. It can be used to isolate these polypeptides.   A polypeptide derivative may be an antigenic or immunological form that forms an aspect of the invention. And derivatives equivalent to   As used herein, “antigenically equivalent derivative” refers to a derivative according to the present invention. When promoted to a protein or polypeptide, it can cause pathogen and mammalian host Will be specifically recognized by certain antibodies that will inhibit the interaction between Polypeptides or their equivalents.   As used herein, an "immunologically equivalent derivative" refers to a mammalian anti-derivative. Disease equivalent when used in an appropriate formulation to elevate the body Includes antibodies that act to inhibit the interaction between the native and mammalian host.   Slightly longer or slightly longer than the native protein or polypeptide of the invention In certain derivatives, the fragments of the invention may be used. In addition to this Polypeptides in which one or more amino acid residues have been modified may also be used. Such a pe Peptides may be obtained, for example, by substitution, addition, or transfer of an amino acid or chemical modification thereof. Can be adjusted. All such substitutions and modifications are generally made in peptide chemistry. It is well known to those skilled in the art.   A polypeptide or a fusion protein thereof, such as an antigenically or immunologically equivalent derivative; White matter immunizes mice or other animals such as rats or chickens. Used as an antigen for Fusion proteins confer stability to polypeptide Will. The antigen may be, for example, bovine serum albumin (BSA) or keyhole. Immunological carrier proteins such as limpet (Keyhole limpet) hemocyanin (KLH) Can be assembled by compounding with quality. Alternatively, a large number of proteins or polypeptides Multi-antigen protein peptide consisting of multiple copies of, or its antigenic or immunological Equivalent polypeptides enhance immunogenicity by eliminating the need for use as carriers. Will have sufficient antigenicity to spread.   Produced by continuous cell line culture for preparation of monoclonal antibodies Any technique that provides an antibody can be used. For example, the human monoc Hybridoma technology for producing lonal antibodies [Kohler and Milstein, 197 5, Nature, 256: 495-497], trioma technology, human B-cell hybrid. Doma Technology [(Kozbor) et al., 1983, Immunology Today 4:72] and EBV-Hybrid Doma technology [Cole et al., 1985, Monoclonal Antibody and Cancer Therapy, Alan R . Liss, Inc., pp. 77-96].   The technology described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) Applied to produce single chain antibodies against the immunogenic polypeptides of the invention. Can be   Using the method of Kohler and Milstein (above (1975)), anti- Fusing body-containing cells with myeloma cells to secrete monoclonal antibodies Make bridoma cells.   Hybridomas are identified as having high binding affinity and one or more original polypeptides. Other staphylococcus species (staphylococcus) using peptides and / or fusion proteins occal species) to select cell lines with favorable cross-reactivity. Training. The selected cell line is cultured to obtain the desired Mab.   The hybridoma cell line that secretes the monoclonal antibody is another of the present invention. It is one aspect.   As an alternative, phage display technology can be implemented by having anti-Fbp. Repertoire of cleaned, human amplified lymphocyte PCR amplified v-genes Or binding activity to polypeptides from a simple library Could be used to select antibody genes [McCafferty, J. et al. (1990), Nature 348, 552-554; Marks, J et al., (1992), Biotechnology 10, 779-. 783]. The affinity of these antibodies is based on chain shuffling. ng) [Clackson, T et al. (1991) Nature 352, 6]. 24-628].   Antibodies have high affinity for polypeptides and / or fusion proteins Should be screened again for   As described above, the final antibody fragment can be prepared.   The antibody is M_rAbout 150,000 intact antibodies or derivatives thereof, for example, Skerra, A and Pluckthum, A, interact with any of (1988) Science 240 1038-1040 Will be. If there are two antigen binding domains, each domain Antibodies are directed against different epitopes and are called "bispecific" antibodies. Would.   The antibodies of the present invention can be used, for example, by establishing established monoclonal antibody technology [Kohler, G. et al. And Milstein, C .; (1975)] or prepared by ordinary means, or For example, as described in Huse, W.D. et al. (1989), Science 246,1275-1281], It can be prepared using replacement means, for example, using a recombinant library.   Preferably, the antibody is as described above for expression of a polypeptide of the invention. Prepared by expression of a DNA polymer encoding the antibody in a suitable expression system I do. Selection of the vector for the expression system is, in part, based on E. coli ( Prokaryotic cells such as B strain) or Streptomyces sp. Vesicles, mouse C127, mouse myeloma, human HeLa, tea Hamster ovary, mold or single-cell mold, or insect cells Sometimes it will be determined by the host, which can be a eukaryotic cell. The host can also be shaped Transgenic animals or transformed plants [eg, Hiatt, A et al., (1989), Nature 34, 76-78]. It may be. Plasmids, bacteriophages, cosmids and recombinant viruses Suitable vectors including, for example, baculovirus and vaccinia virus Origin.   Fab fragments also cleave Fab sites from, for example, Fc sites. Of the parent monoclonal antibody by enzyme treatment with papain Can be prepared.   Preferably, the antibody or derivative thereof makes it more immunogenic in the patient. Is modified as follows. For example, if the patient is human, the antibody is most preferably "human Wherein the complementarity-determining region of the hybridoma-derived antibody is, for example, Jo nes, P. et al., Nature 321, 522-525 or Tempest et al., (1991) Biotechnology 9,266-27. 3] was transplanted into a human monoclonal antibody.   Modifications need not be limited to those which are "humanized"; other important sequences [e.g. Newman, R. et al., 1992, Biotechnology, 10, 1455-1460] are also used. It is.   Humanized monoclonal antibody, or a fragment thereof having binding activity Form an aspect of the invention.   The present invention provides for the screening of drugs to identify those that inhibit the proteins herein. Provide a method for measuring the inhibition of protein activity by a test agent. Consists of For example, if the protein has the ability, appropriate purification and After formulation and formulation, the activity of the enzyme can be tracked by its ability to convert natural substrates. You. Different chemically synthesized test compounds or natural products can be used for enzyme activity analysis. Detects additives that compete with natural substrates or inhibit enzyme activity It is possible.   The present invention also relates to the inhibitors identified thereby.   The use of a polynucleotide of the present invention in genetic immunization is preferably Injection of rasmid DNA directly into muscle [Wolff et al., Hum Mol Genet 1992, 1: 363, Manth orpe et al., Hum. Gene Ther. 1963: 4, 419], the transfer of DNA complexed with a specific protein carrier. [Wu et al. Biol. Chem 1989: 264,16985], DNA sharing with calcium phosphate. Precipitation [Benvenisty & Reshef, PNAS, 1986: 83, 9551], D to various forms of liposomes Encapsulation of NA [Kaneda et al., Science 1989: 243, 375], Particle bombardment [ Tang et al., Nature 1992, 356: 152, Eisenbraun et al., DNA Cell Biol 1993, 12: 791] and In vivo infection with cloned retroviral vectors [Seeger et al., PNAS  1984: 81, 5849]. Muscle transfection Suitable promoters for CMV, RSV, SRa, actin, MCK , Alpha globulin, adenovirus and dihydrofolate reductase.   In treatment or as prophylaxis, the active substance is provided as an injectable composition, e.g. It can be administered to a patient preferably as an isotonic, sterile aqueous dispersion.   Alternatively, the composition may be for topical application, for example, ointments, creams, lotions, eye ointments Eye drops, ear drops, mouthwashes, impregnated bandages and sutures, and air It can be formulated in the form of a sol or the like, and contains appropriate conventional additives such as preservatives and drug penetration. Ointments and creams may contain an emollient. No. Such topical formulations may also contain compatible conventional carriers, such as creams or Contains ointment base and lotion contains ethanol or oleyl alcohol May be. Such carriers may be from about 1% to about 98% by weight of the formulation; Typically it will be up to about 80% by weight of the formulation.   For administration to human patients, the daily dose of active ingredient is 0.01 m g / kg to 10 mg / kg, typically about 1 mg / kg. The doctor In each case, determine the actual dosage which is most suitable for the individual, and determine the age, weight and In particular, it is changed according to the response of the individual. The above dosages are exemplary of the average case It is. Of course, there are individual cases where the high and low dose ranges fit, It is within the scope of the present invention.   Conveniently, the vaccine composition is in an injectable form. Conventional adjuvants Can be used to enhance the immune response.   A suitable unit dose for vaccination is 0.5-5 μg / kg of antigen, Such a dose is preferably administered one to three times at intervals of one to three weeks.   In the stated dosage range, the compounds of the invention may prevent administration to appropriate patients. No adverse toxicological effects are observed. Example   To facilitate understanding of the following examples, certain frequently found methods and And / or terms will be described.   "Plasmid" is preceded by uppercase letters and / or numbers, or It is defined by the lower p following. The starting plasmid herein is Whether it is commercially available, publicly available on unrestricted principles, or It can be constructed from available plasmids that are consistent with the methods opened. this In addition, plasmids equivalent to those described are known in the art and It will be obvious.   "Digestion" of DNA refers to DNA digestion with a restriction enzyme that acts only at certain sequences in the DNA. Means the catalytic cleavage of The various restriction enzymes used herein are commercially available. Available and their reaction conditions, cofactors and other requirements are determined by one skilled in the art. Used as known to the public. For analytical purposes, typically 1 μg plus The amide or DNA fragment is combined with about 2 units of enzyme in about 20 μl of buffer. Use in buffer solution. For the purpose of isolating DNA fragments from plasmid constructs For this purpose, typically 5 to 50 μg of DNA are added to 20 to 250 units. Digest in larger volumes with enzymes. Buffers and buffers appropriate for the particular restriction enzyme And the amount of substrate is specified by the manufacturer. Incubation at 37 ° C for about 1 hour Solution times are usually used, but will vary according to the provider's instructions. Extinction After the reaction, the reaction is directly electrophoresed on a polyacrylamide gel to obtain the desired fragment. obtain.   Size separation of the cleaved fragments is described by Goeddel, D. et al., (1980) Nucleic Acids.  Res. 8: 4057 using an 8 percent polyacrylamide gel as described in It is.   An "oligonucleotide" is a single-stranded polynucleotide or two complementary polynucleotides. A deoxynucleotide chain, which can be chemically synthesized. Such synthesis Oligonucleotides do not have a 5 'phosphate and will be released with ATP in the presence of a kinase. Will not ligate to another oligonucleotide without adding acid . A synthetic oligonucleotide will ligate to a fragment that is not dephosphorylated. Would.   "Linking" forms a phosphodiester bond between double-stranded nucleic acid fragments Means process (Maniatis, T et al., Ibid., P146). Unless otherwise specified The ligation is about 0.5 μm, which is approximately an equimolar amount of the DNA fragment to be ligated. Known buffer with 10 units of T4 DNA ligase ("ligase") per gram liquid And conditions. Example 1 Isolation of DNA from S. Aureus WCUH29   A polynucleotide having the DNA sequence set forth in SEQ ID NO: 1 is Of a clone of the chromosomal DNA of S. aureus WCUH29 in E. coli Obtained from Ibrary. In some cases, duplicate S. aureus WC Sequencing data from two or more clones containing UH29 DNA was [SEQ ID NOs: 1, 4, 10, 13, 16, 19, 22, 25, 28, 31, 34] , 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70 , 73, 76] were used to construct adjacent DNA sequences in the sequence shown in . Libraries can be prepared by mechanical methods, for example: Methods 1 and 2   Whole cell DNA was prepared by standard methods using Staphylococcus aureus. occus aureus) strain WCUH29 (NCIMB 40771). Size fractionate by any of the methods. Method 1.   Mechanically shear through a needle to size fractionate total cellular DNA by standard methods. Refuse. DNA fragments up to 11 kbp in size were Stain with DNA polymerase and DNA polymerase and add EcoRI linker I do. The fragment was ligated into Lambda ZapII, a vector cut with EcoRI. Concatenated, packaged in a library using standard methods, and packaged Infect E.coli in the library. The library is amplified by standard methods You. Method 2.   Whole cell DNA was combined with four restriction enzymes (RsaI, PalI, Alu and And Bsh1235I) and partially hydrolyzed by standard methods. Draw. An EcoRI linker is ligated to the DNA and the fragment is then The vector was ligated into a vector, Lambda ZapII, which had been cut with I. Packaged into a library by the method, and use E.coli with the packaged library. Infect. The library is amplified by standard methods. Example 2 Determination of expression during infection of a gene from Staphylococcus aureus WCUH29 Set   Necrosis from 4-day inguinal infection of Staphylococcus aureus in mice Efficient destruction of adipose tissue, presence of chaotropic drugs and RNase inhibitors Processing to obtain a mixture of animal and bacterial RNA. Stable preparation of bacterial RNA Optimal conditions for disruption and processing to obtain high yields are Aureus 16S RNA-specific radioactive label on the kit By use of hybridization to a recognition oligonucleotide. RNase The resulting RN without DNAase, without DNA and without protein Preparation A contains the respective sequences of Staphylococcus aureus WCUH29. Transcription PCR (RT-PCR) using unique primer pairs designed by Are suitable. a) Staphylococcus aureus WCUH2 from a mouse animal model of infection Isolation of tissue infected with 9   10 ml sterile nutrient broth (No.2 Oxoid) isolated from agar culture plate Individual colonies of Staphylococcus aureus WCUH29. The culture is incubated aerobically (static culture) at 37 ° C. for 16-20 hours. 4 Week-old mice (female, 18-22 g, strain MF1) were each 0.5 ml of this broth culture of S. aureus WCUH29 (about 1 in broth). 0⁸cfu / ml) into the anterior right lower quadrant (inguinal region) subcutaneously. Infect by entering. The mice were infected for the first 24 hours after infection and then at the end of the experiment. Should be monitored regularly until the end of the day. Signs of systemic infection, lethargy Closely monitor animals that may be disturbed and leave the group, If the signs develop moribund, the animals should be killed immediately.   Visible external signs of disease progression may be found 24-48 hours after infection. U. Examination of animal abdomen will show raised contour of abscess under skin . Locally restricted obstructions should remain in the lower right quadrant, but occasionally on the left Will expand in preference to the lower quadrant and chest. Occasionally, the abscess overlies Will rupture through the skin layer. In such cases, if possible If so, the affected animal should be killed immediately and a tissue sample taken. Killing animals Failure, the necrotic skin tissue overlying the abscess falls off, exposing the abdominal muscle wall Results.   Approximately 96 hours after infection, animals are killed by carbon dioxide asphyxiation. Death and tissue processing Mice should be killed one at a time rather than in groups to minimize delays during storage It is. Place the dead animal with its back down and fur roughly 70% alcohol Wipe with. Make the first incision with scissors through the lower left quadrant and preferentially Proceed to and then on the chest. The incision is completed by cutting the lower left part of the abdomen down. Tie. Care should be taken not to penetrate the abdominal wall. Tweezers skin flap And gently peel off the skin from the abdomen. Cover the peritoneal wall, but generally Causes exposed abscesses that do not completely penetrate the meat sheet and do not puncture the internal organs Be careful.   Cut the abscess / muscle sheet and other infected tissue before snap freezing in liquid nitrogen. Must be cut into pieces, which makes the plastic collection vial more Easy storage will be possible. b) Staphylococcus aureus WCUH29 RNA from infected tissue sample Isolation   4-6 week infected tissue samples in 2 ml screw cap tubes (approximately 0.5-0.7 g) from storage at -80 ° C and place in a dry ice ethanol bath. It is. Destroy samples individually in a microbiologically safe cabinet while remaining Keep the sample cold in an anhydrous ice ethanol bath. Destroy bacteria in tissue samples For this, add 1 ml Trizol reagent (Gibco BRL, Life Technologies) Then almost fill the tube with enough 0.1 mm zirconia / silica beads. Screw lid to seal well and remove aerosol formation. Carefully replace the beads so that they do not enter the beads. The sample is then mini-BeadBeate r Homogenize in type BX-4 (Biospec Products). Achieve bacterial lysis For this purpose, necrotic adipose tissue is processed at 5000 rpm for 100 seconds. Grow in vivo Bacteria are destroyed by a 30 second bead-beat in vivo growing S. aureu Requires longer processing than WCUH29.   After bead-beating, the heat generated during destruction breaks down TRIzol, Cool the tube on ice to release the anide and then place in a fume hood. Open with.   200 microliters of chloroform are then added and the tube is manually placed Shake for 2 seconds to ensure thorough mixing. After 2-3 minutes at room temperature, the tube was Centrifuged at 4 ° C. for 15 minutes and the RNA extract was then The method provided by the manufacturer of the reagent, ie, about 0.6 ml of aqueous layer Transfer to a fungus eppendorf tube and add 0.5 ml isopropanol. After 10 minutes at room temperature, the sample is centrifuged at 12,000 × g at 4 ° C. for 10 minutes. Supernatant And discard, then wash the RNA pellet with 1 ml 75% ethanol You. Before centrifugation at 7,500 xg for 5 minutes at 4 ° C, use a short bottle to mix the sample. Use lutex. Remove the ethanol and remove the RNA pellet in just 5 minutes Dry under reduced pressure. Samples were replicated in 100 microliters of DEPC-treated water. Repeat pipetting, then resuspend for 5-10 minutes at 55 ° C. Final , Add 200 units of RNasin (Promega) after leaving on ice for 1 minute Continue with   Store the RNA preparation at -80 ° C for up to one month. For longer term storage The RNA precipitate is washed in 75% ethanol during the washing step of the protocol. It can be stored at -20 ° C for at least one year.   The quality of the isolated RNA was determined by running the sample on a 1% agarose gel. Evaluation. 1x TBE gel stained with ethidium bromide, total RNA yield visible Used for To demonstrate the isolation of bacterial RNA from infected tissues, 1 × MO PS, electrophoresed on 2.2M formamide gel and vacuum blown to Hybond-N (Amersham) Tsu To The blot was then subjected to S. aureus 16s rRNA specific³²P Hybridize with a labeled oligonucleotide probe [K. Greisen, M .; Loe ffelholz, A.S. Purohit and D.H. Leong., J. Clin. (1994) Microbiol. 32, 335-351 ].   5'-gctcctaaaaaggttactccaccgggc-3 '[sequence number No .: 91] Is used as a probe. Hybridized van Size was isolated from S. aureus WCUH29 in a Northern blot. Compare with that of control RNA. Bacterial 16s rRNA band in total RNA sample , Which, when visualized on a TBE gel, covers a wide range of mammalian RNA. Decomposition in c) Removal of DNA from RNA derived from Staphylococcus aureus WCUH   DNA was purified from a 73 microliter RNA sample to a final volume of 90 microliters. In a buffer supplied by the addition of 200 units of Rnasin (Promega) in torr. Unit DNAase I, amplification grade (Gibco BRL, Life Technologie Remove by treatment for 15 minutes on ice in s).   The DNAase was purified with TRIzol LS reagent (Gibco B RL, Life Technologies) to inactivate and remove. DNAase treatment The RNA was treated with 73 microliters of DEPC by adding Rnasin described in Method 1. Resuspended again in water. d) Preparation of cDNA from RNA sample from infected tissue   10 microliters of DNAase-treated RNA sample was prepared according to the manufacturer's instructions. SuperScript Preamplification System for First Strand cDNA Synthesisk Reverse transcription is performed using it (Gibco BRL, Life Technologies). 1 nanogram random Hexamers are used to prime each reaction. SuperScriptII revers Controls without added etranscriptase also run. Before following the PCR reaction, Treat both / -RT samples with RNaseH. e) Use of PCR to determine the presence of bacterial cDNA species   The PCR reaction is used by adding the following components in a 0.2 ml tube. I mean:           45 microliters of PCR SUPERMIX (Gibco BRL, Life  Technologies)           1 microliter of 50 mM to adjust the final concentration to 2.5 mM MgCl_Two           1 microliter of PCR primers (optimally 18-25 Base pairs and are designed to have similar annealing temperatures). Initial concentration of each primer at 10 mM           2 microliters of cDNA   The PCR reaction was run on a Perkin Elmer Gene Amp PCR System 9600 as follows. Is done.           5 minutes at 95 ° C., then each of 94 ° C., 42 ° C. and 4 ° C. Hold temperature at 4 ° C. for 3 minutes at 72 ° C., followed by 50 cycles of 30 seconds at 50 ° C. (The number of cycles is the number of cycles required to determine the presence or absence of the PCR product. Optimally, 30-50, if the starting amount of cDNA from the RT reaction is estimated Optimally, if done, 8-30 cycles)   10 microliters, then smell with PCR product, if present Run on a 1% 1 × TBE gel stained with ethidium bromide, size 100 bpD Estimate by comparing with NA ladder (Gibco BRL, Life Technologies). As an alternative The PCR product is then labeled with a labeled PCR primer (e.g., labeled at the 5 'end of the dye). And the appropriate amount of the PCR product is polyacrylic. Run on a gel sequencing gel and determine its presence and quality by appropriate gel scanning. Scanning system (eg GeneScan provided by Perkin Elmer)^TMsoft ABI Prism using ware^TM377 Sequencer).   RT / PCR controls were +/- reverse transcriptase reaction, 16s   rRNA primer or non-transcribed S. aureus WCUH29 Geno DNA-specific primers designed to produce PCR products derived from DNA sequences May include pairs.   To test the efficiency of the primer pairs, they were combined with WCUH29 total DNA. Both are used in DNA PCR. PCR reaction is performed with 1 microliter instead of cDNA. Perform as above with 35 cycles of PCR using grams of DNA.   Produce predicted size products in DNA PCR or RT / PCR Primer pairs that failed to perform were PCR failures and Does not give knowledge. Although giving the correct size product in DNA PCR Among them, two classes are distinguished in RT / PCR:   1. Genes that are not reproducibly transcribed in vivo have been identified in RT / PCR No product is given.   2. Genes transcribed in vivo with reproducibility are positive in RT / PCR. Give a product of the correct size, and the signal (if present) in the -RT control A stronger signal is shown in the ROR + RT sample.   The following nucleotide sequence (SEQ ID NO: 1, 4, 7, 1 0, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 4 6, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76]. Was identified in the above test to be transcribed in vivo. each The set of sequences is associated with separate genes (gene numbers). Putative amino acids The sequence was replaced with Sequence 2 in Table 1 [SEQ ID NOs: 79, 80, 81, 82, 83, 84, 85]. , 86, 87, 88, 89, 90]. Give where. The PCR primer pairs used to identify the genes SEQ ID NO: 2 [SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 2] 9, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 6 5, 68, 71, 74, 77] and sequence 4 [SEQ ID NOs: 3, 6] , 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78] As shown in the sequence. Once the homology to a known gene is determined, Given at that time, represent the putative identification of the function of each gene in Table 1.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 16/12 C12N 1/19 C12N 1/19 1/21 1/21 C12P 21/02 C C12P 21/02 G01N 33/15 Z G01N 33/15 33/50 T 33/50 A61K 39/395 N // A61K 39/395 C12P 21/08 C12P 21/08 A61K 37/02 ADZ (C12N 15/09 ZNA C12R 1: 445) (72) Invention Hodgson, John Edward United States 19426-0989 College Building, Pennsylvania P.O. Box 5089, South Collegeville Road 1250 Smithkline Beecham Pharmaceuticals

Claims (1)

[Claims]   1.   (A) SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25 and 28 Encoding a polypeptide having an amino acid sequence selected from the group consisting of: Having at least 70% homology to a polynucleotide of interest Tide;   (B) a polynucleotide complementary to the polynucleotide of (a); and   (C) a poly comprising at least 15 consecutive bases of (a) or (b) nucleotide; An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of: Creotide.   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. SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25 and 28 3. The polynucleotide of claim 2 having a nucleotide sequence selected from the group consisting of: Creotide.   5.   (A) expressing the expressed polypeptide contained in NCIMB deposit no. Have at least 70% homology to the encoding polynucleotide; Group consisting of column numbers 1, 4, 7, 10, 13, 16, 19, 22, 25 and 28 A polynucleotide selected from:   (B) a polynucleotide complementary to the polynucleotide of (a); and   (C) a polynucleotide comprising at least 15 bases of (a) or (b) Tide; An isolated polynucleotide comprising a member selected from the group consisting of:   6. A vector comprising the DNA according to claim 2.   7. A host cell having the vector according to claim 6.   8. The polypeptide encoded by the DNA is converted from the host cell according to claim 7. A method for producing a polypeptide, which is expressed.   9. A method for producing a cell expressing a polypeptide, wherein the cell is contained in a vector. Claims to express a polypeptide encoded by the cDNA contained therein. 6. Transforming or transfecting a cell with the vector according to 6. How to sign.   10. A method for producing a polypeptide or fragment of the present invention, comprising: 7 under conditions sufficient to produce said polypeptide or fragment. A method comprising culturing.   11. Substantially 79, 80, 81, 82, 83, 84, 85, 86, 87 and And a polypeptide having an amino acid sequence selected from the group consisting of   12. An antibody against the polypeptide of claim 11.   13. An antagonist that inhibits the activity of the polypeptide according to claim 11.   14． Administering to the individual a therapeutically effective amount of the polypeptide of claim 11. A method of treating a solid, having a need for a polypeptide of the present invention.   15. Providing DNA encoding the polypeptide to an individual, wherein the polypeptide Is administered in vivo to administer the therapeutically effective amount of the polypeptide. The method of claim 14, wherein:   16. Administering to the individual a therapeutically effective amount of the antagonist of claim 13. A method for treating an individual having a need to inhibit the polypeptide of the present invention, characterized in that: .   17． A method for diagnosing a disease associated with expression of the polypeptide according to claim 11. And determining a nucleic acid sequence encoding said polypeptide. .   18. Analyzing the presence of the polypeptide of claim 11 in a sample derived from the host. And a diagnostic method characterized by the following.   19. Identification of a compound that binds to the polypeptide of claim 11 and inhibits the activity The method;   Under conditions that allow binding to the binding site, the binding site for the polypeptide is Contacting cells expressing the surface with the compound to be screened (this Here, the binding site is a signal detectable in response to binding of the compound to the binding site. Which binds to a second component capable of providing   The presence or absence of a signal resulting from the interaction of the compound with the binding site Upon detection, the compound binds to the binding site to activate or inhibit Deciding what to do; A method characterized by the following.   20. Sufficient polyprotein of the invention to produce antibodies that protect the mammal from disease. Inoculating a mammal with a peptide or fragment or variant thereof. A method for eliciting an immunological response in a mammal.   21. By gene therapy, the polypeptides of the invention or their To express a variant, the polypeptide fragment or variant of the present invention is modified. Delivering the encoding gene and eliciting an immunological response to produce antibodies, A method of eliciting an immunological response in a mammal that protects against disease.   22. Coding the polynucleotide of the present invention or the protein encoded thereby. An immunological composition comprising DNA to be expressed and expressed, said DNA being introduced into a mammal. The polynucleotide or a polynucleotide encoded thereby in a mammal. An immunological composition that elicits an immunological response that produces a protein.   23. Under stringent hybridization conditions, a polynucleotide arrangement of the present invention may be used. An appropriate library containing the complete gene sequence is inserted into the polynucleotide sequence or its Screening with a probe having the sequence of the Polynucleotides Essential for DNA Sequence Obtained by Isolating a Sequence .