JP2002529069A - Chlamydia pneumoniae genome sequence - Google Patents

Abstract

  (57) [Summary] Analysis of C. pneumoniae genomic sequences and encoded polypeptides and RNA is provided. C. pneumoniae gene nucleic acid compositions identify homologous or related proteins and DNA sequences encoding such proteins; manufacture compositions that modulate protein expression or function; and study related physiological pathways Used in In addition, modulation of gene activity in vivo is used for prophylactic and therapeutic purposes, such as for expression-based identification of cell types and the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to Chlamydia pneumoni.
ae) nucleic acids and polypeptides, and It relates to the use in the diagnosis, prevention and treatment of diseases associated with Pneumoniae.

BACKGROUND OF THE INVENTION [0002] Chlamydiaceae are a family of intracellular obligate parasites that have an affinity for epithelial cells lining the mucosa. Bacteria have two morphologically distinct forms, an "elementary body" and a "reticulate body". Elementary bodies are an infectious form and have a rigid cell wall, mainly cross-linked outer membrane proteins.
The reticulated structure is a metabolically active form within the cell. The unique developmental cycle between these two forms characterizes Chlamydia growth.

[0003] C. Pneumoniae is a human respiratory pathogen that causes acute respiratory illness and is about 10% of out-of-hospital infectious pneumonia. According to the antibody morbidity survey, virtually everyone noticed that C.I. It is clear that infection with Pneumoniae is common, and reinfection is common. In addition to respiratory disease, studies have shown that this microorganism is also involved in coronary artery disease. Atherosclerotic disease of the aorta and coronary arteries has been demonstrated by immunocytochemistry and polymerase chain reaction (Kuo
et al. , (1993) J. Infect. Dis . , 167
(4): 841-849).

[0004] According to a recent report, the wall of an abdominal aortic aneurysm is C. It has further been shown that pneumoniae is present (Juvonen et al., (1997) J Vasc Surg 25 (3): 499-505). Abdominal aortic aneurysms are often associated with atherosclerosis, and inflammation can be an important factor in aneurysmal dilation. C. Pneumonia may play a role in maintaining inflammation and causing the development of aortic aneurysms.

[0005] Muhlstein et al. , (1996) JACC 27:
1555-61 reported another case of Chlamydia species in the coronary artery wall of patients with atherosclerosis compared to patients with other forms of cardiovascular disease. The expected infection rate is very low in patients with normal coronary artery or coronary artery disease, whereas the expected infection rate in patients with symptomatic atherosclerotic disease is very high. This provides evidence of a direct link between the atherosclerotic process and Chlamydia infection. Because the history of Chlamydia infection is widespread in the population, causality issues remain. At the level of physiology and pathology, abnormal interactions between endothelial cells, platelets, macrophages, and lymphocytes may trigger a cascade of events leading to acute endothelial damage, thrombosis, and repair; It may also cause atherogenesis in blood vessels more chronically.

C. Pneumoniae is associated with other Chlamydia species, but with a relatively low level of sequence similarity. Despite being an important human pathogen, little is known about the biology of this microorganism. Structural relationships and allelic diversity between certain genes of Chlamydia species are described in Kaltenboeck et al.
, (1993) J Bacteriol 175 (2): 487-50.
2, Gaydos et al. , (1992) Infect Immu n 60 (12): 5319-5323, Everett et al. ,
(1997) Int J Syst Bacteriol 47 (2):
461-473, and Pudjiamoko et al. , (1997
) Int J Syst Bacteriol 47 (2): 425-4.
31.

Several studies have been published, and C.I. Describes methods for detecting Pneumoniae and discriminating between Chlamydia species. Such a method is
PCR detection (Rasmussen et al., (1992) Mol Cell Probes 6 (5): 389-384, Holland et al.
al. , (1990) J Infect Dis 162 (4): 9
84-987), a simplified polymerase chain reaction enzyme immunoassay (Wilson
et al. , (1996) J Appl Bacteriol 80.
(4): 431-438), sequencing and restriction endonuclease cleavage (He
rrmann et al. , (1996) J Clin Microbi ol 34 (8): it contains 1897-1902).

[0008] Different C.I. The antigenicity and molecular analysis of Pneumoniae strains is described in Jantos et a
l. , (1997) J Clin Microbiol 35 (3):
620-623. C. Several pneumoniae genes have been isolated and sequenced. These, along with some ribosomes and other genes, are the Gro E operon (Kikuta et al., (1)
991) Infect Immun 59 (12): 4665-4669.
), Major outer membrane proteins (Perez et al., (1991) Infe
ct Immun 59 (6): 2195-2199, DnaK protein homolog (Kornak et al., (1991) Infect Imm.
un 59 (2): 721-725). SUMMARY OF THE INVENTION The present invention provides a genomic sequence of Chlamydia pneumoniae. Sequence information is useful for various diagnostic and analytical methods. The genomic sequence may be embodied in a variety of media, including in computer readable form, or may be embodied as nucleic acids containing selected fragments of the sequence. Such fragments are generally composed of open reading frames, transcription or translation regulators, or fragments derived therefrom. The protein encoded by the open reading frame is useful for diagnostic purposes, as well as for its enzymatic or structural activity. Definitions The term "amino acid" is intended to mean natural or synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic information, as are the amino acids that are later modified, eg, hydroxyproline, γ-acarboxyglutamate, and O-phosphoserine. Amino acid analogs have the same basic chemical structure as a natural amino acid,
That is, carbon bonded to oxygen, a carboxyl group, an amino group, and homoserine,
It means a compound having an R group such as norleucine, methionine sulfoxide, methionine methylsulfonium and the like. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical structures that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids are commonly known as three letter symbols or IUPAC-IUB
Biochemical Nomenclature Commission
Will be mentioned here by any of the single letter symbols recommended by Nucleotides will also be referred to by their generally accepted one-letter code.

An “amplification” primer is an oligonucleotide composed of natural or analog nucleotides that serve as a basis for the amplification of a selected nucleic acid sequence. Such primers include, for example, polymerase chain reaction primers and ligase reaction oligonucleotides. "Antibodies" are immunoglobulin molecules capable of binding a specific epitope on an antigen. Antibodies are polyclonal mixtures or monoclonals. Antibodies are intact immunoglobulins of natural or recombinant origin, and are immunoreactive sites of intact immunoglobulins. Antibodies, as well as single chains, may exist in various forms, such as Fv, F _ab , and F (ab) ₂ . Single chain antibodies in which the heavy and light chain genes are combined into a single coding sequence may also be used.

An “antigen” is a molecule that is recognized and bound by an antibody, for example, a peptide, a carbohydrate, an organic molecule, or a more complex molecule such as a glycoprotein or glycolipid. The portion of the antigen that is targeted for antibody binding is an antigenic determinant, and the small functional group corresponding to a single antigenic determinant is called a hapten. "Biological sample" means any sample obtained from a living or dead microorganism. Examples of biological samples include biological fluids and tissue specimens. Such a biological sample is C.I. It is prepared for analysis of a Pneumonie nucleic acid, a protein, or an antibody that specifically reacts with the protein.

“C. pneumoniae gene” is a specific C. pneumoniae gene. It should be intended to mean an open reading frame encoding a Pneumoniae polypeptide, extending up to about 2 kb from the coding region and further involved in regulation of expression which may be in any direction. The same is true for 5 'and 3' non-coding nucleotide sequences. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or integration into the host genome.

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant means a nucleic acid that encodes the same or essentially identical amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, essentially the same sequence. In particular, degenerate codon substitutions may be achieved by generating a sequence in which the third position of one or more selected (or all) codons has been replaced by a mixed base and / or deoxyinosine residue. (Batzer e
t al. , Nucleic Acid Res. 19: 5081 (1
991), Ohtsuka et al. , J. et al. Biol. Chem.
260: 2605-2608 (1985), Rossolini et a.
l. , Mol. Cell. Probes 8: 91-98 (1994
)). Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, and GCU
All encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon is changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid mutations are "silent mutations" and are a type of conservatively modified mutation. Every nucleic acid sequence in a polypeptide encoding describes every possible silent variation of the nucleic acid. One of skill in the art will appreciate that modifying each codon of a nucleic acid (except for AUG, which is usually only one codon for methionine, and TGG, which is usually only one codon for tryptophan), produces functionally identical molecules. Will recognize that Thus, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

As with amino acid sequences, those skilled in the art will appreciate that nucleic acids that alter, add, or delete a small portion of a plurality of amino acids or a single amino acid in the encoded sequence;
Each substitution, deletion, or addition to a peptide, polypeptide, or protein sequence is a "conservatively modified variant" in which the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are alleles and interspecies homologs of the present invention, in addition to or without excluding polymorphic variants.

The following groups each contain amino acids that are conservative substitutions for one another. That is,
1) alanine (A), glycine (G), 2) serine (S), threonine (T), 3) aspartic acid (D), glutamic acid (E), 4) asparagine (N), glutamine (Q), 5 Cysteine (C), methionine (M), 6) arginine (R), lysine (K), histidine (H), 7) isoleucine (I), leucine (L), valine (V), and 8) phenylalanine ( F), tyrosine (5), tryptophan (W).

See, for example, Creighton, Proteins (1984). The term "identity" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to the comparison of one of the following sequence comparison algorithms, or according to measurements by manual alignment and visual inspection: Two or more sequences or subsequences having the same or a specified ratio of amino acid residues or nucleotides when compared and aligned for maximum correlation over a window. This definition also refers to the complementarity of a test sequence, such that if the test sequence has specified or substantial identity to a control sequence, the test sequence is designated as a specified percent sequence or subsequence complementarity. Having. For example, when aligning for maximum correlation over a comparison window using one of the following comparison algorithms or following measurements by manual alignment and visual inspection, the specified amino acid identity is 95% A sequence or subsequence having at least about 95% amino acid identity is meant.
Such sequences will thus be said to be substantially identical, or substantially identical to one another. Preferably, the sequences have at least about 70% identity, more preferably 80% identity, even more preferably 90-95% identity and more. Preferably, the percent identity is at least about 2 in length.
It is present for a region of the sequence that is 5 amino acids, more preferably for a region that is 50-100 amino acids in length.

[0017] The ratio of sequence identity is used for proteins or peptides, and it is recognized that non-identical residues often differ by conservative amino acid substitutions. In this case, a conservative amino acid substitution does not change the functional properties of the molecule, since the amino acid residue is replaced with a number of amino acid residues having similar chemical properties (eg, charge or hydrophobicity). If the sequences differ by conservative substitutions, the percent identity of the sequences will be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those skilled in the art. Generally, this involves recording conservative substitutions as part of a rather than a complete mismatch, thereby increasing sequence percent identity. Thus, for example, a score of 1 for the same amino acid and a score of zero for a non-conservative substitution will give a score between zero and one for a conservative substitution. Scoring of conservative substitutions can be performed, for example, by using the program PC / GENE (Intelligenetics, Mountain
e.g., Meyers & Miller, Computer A, as implemented in InView, California, USA).
pplic. Biol. Sci. 4: Calculated based on 11-17 (1988).

In sequence comparison, typically one sequence acts as a control sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and control sequences are entered into a computer, subsequence coordinates are specified, and, if necessary, further sequence algorithm program parameters. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the control sequence based on the designated or default program parameters.

[0019] The comparison window may be for a segment of any one of consecutive position numbers selected from the group consisting of 25 to 600, usually about 50 to about 200, and more usually about 100 to about 150. Including controls. After the two sequences have been optimally aligned, the control sequence and the test sequence at the same consecutive position number are compared.
Methods for alignment of sequences for comparison are well-known in the art. Aligning sequences optimally for comparison can be accomplished, for example, by using Smi
th & Waterman, Adv. Appl. Math. 2: 4
82 (1981), by the homology algorithm of Needleman.
& Wunsch, J .; Mol. Biol. 48: 443 (197
Pearson & Lipman, Pr.
oc. Nat'l. Acad. Sci. USA 85: 2444 (
1988) by computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in Winsconsin Genetics Software
re Package, Genetics Computer group,
575 Science Dr. , Madison, WI) or by manual alignment and visual inspection (eg, Ausubel et al.).
al. (See above).

One example of a useful algorithm is PILEUP. PILEUP generates multiple sequence alignments from a group of related sequences using progressive and pairwise alignments and shows correlations and percent sequence identity. Also,
It plots a tree or dendogram showing the cluster relationships used to generate the alignment. PILEUP, Feng & Doolit
tle, J .; Mol. Evol. 35: 351-360 (1987
2) The simplification of the progressive alignment method is used. The method used was Hi
ggins & Sharp, CABIOS 5: 151-153 (19
89). The program can align up to 300 sequences, each consisting of up to 5,000 nucleotides or amino acids. The multiple alignment method begins with a pairwise alignment of the two sequences that are most similar, creating a cluster of two aligned sequences.
This cluster is then aligned to the next closest sequence or cluster of sequences. The two sets of sequence clusters are aligned by a simple extension of the pairwise alignment of the two individual sequences. The final alignment is achieved by a series of progressive pairwise alignments. The program is
It is performed by designating a particular sequence for the region of sequence comparison and the coordinates of the amino acids or nucleotides of the sequence and by designating the program parameters. Using PILEUP, a control sequence is compared to other test sequences to determine percent sequence identity using the following parameters. That is, a default gap weight (3.00), a default gap length weight (0.10), and a weighted end gap. PILEUP is a GCG sequence analysis software package, such as version 7.0 (Devereaux et al., Nuc.
Acid. Res. 12: 387-395 (1984)).

Another example of an algorithm suitable for determining the percentage of percent sequence identity (ie, substantial similarity or identity) is described in Altschul et al. , J. et al.
Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyzes is available at the National Center for Biotechnology Information (National Cen.).
ter for Biotechnology Information (h
http: // www. ncbi. nlm. nih. Gov /) is publicly available. The algorithm involves first identifying a high scoring sequence pair (HSP) by identifying a short word of length W in the query sequence, and aligns it with the same length word in the database sequence. In this case, the threshold score T matches or satisfies a certain positive threshold score T. T is referred to as the proximity word score threshold (Altschul et al.,
Above). Their initial neighbor word hits are longer than those containing H
Serves as a seed to initiate a search to find an SP. Next, word hits extend in both directions along each sequence as long as the cumulative alignment score increases. Cumulative scores are calculated for nucleotide sequences using the parameters M (reward score for a pair of matching residues, always> 0) and N (penalty score for no residue match, always <0). You. For amino acid sequences, a cumulative score is calculated using a scoring matrix. If the cumulative alignment score deviates from the maximum achieved by an amount X, if the accumulation of one or more negative scoring residue alignments results in a cumulative score of zero or less, or When the end is reached, the expansion of word hits in each direction is interrupted. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. BLASTN program (
For nucleotide sequences), the word length (W) is 11, the expected value is 10, M
= 5, N = 4, and a comparison of both strands is used as default. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation of 10, and a BLOSUM62 scoring matrix as default (
Henikoff & Henikoff, Proc. Natl. Aca
d. Sci. ScL USA 89: 10915 (1989)).

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (eg, Karlin & Altschul, Proc. Nat'l. A.
cad. Sci. ScL USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest total probability (P (N)), which gives an indication of the probability that a match between two nucleotide or amino acid sequences may occur by chance. Provided.
For example, the smallest total probability of comparing the control nucleic acid and the test nucleic acid is less than about 0.1,
More preferably, the nucleic acid is similar to the control sequence when it is less than about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides are substantially identical is
The polypeptide encoded by the first nucleic acid may be a second polypeptide, as described below.
Cross-reacts immunologically with the antigen obtained against the polypeptide encoded by the nucleic acid of the invention. Thus, for example, where the two peptides differ only by conservative substitutions, the polypeptide will generally be substantially identical to the second polypeptide. Another indication that two nucleic acid sequences are substantially identical is 2
Two molecules or their complements will hybridize to each other under stringent conditions, as described below.

Another indication that polynucleotide sequences are substantially identical is when the two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence-dependent and will be different in different circumstances.
Generally, stringent conditions are selected to be about 5 ° C. below the thermal melting point (Tm) for a particular sequence at a given ionic strength and pH. Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe (
Under a given ionic strength and pH). Generally, stringent conditions for Southern blot protocols include hybridization in a buffer. This buffer is subjected to hybridization in a buffer containing 5 × SSC and 1% SDS at 65 ° C. or hybridization at 42 ° C. in a buffer containing 5 × SSC and 1% SDS, followed by washing with 0.2 × SSC and 0.1% SDS. 65 ℃
Need cleaning.

A “label” is a composition that can be detected by spectroscopy, photochemistry, biochemistry, immunochemistry, or chemical means. For example, useful labels include ³² P, fluorescent dyes, electron density reagents, enzymes (eg, enzymes commonly used in ELISA), biotin, dioxygenin, or haptens and proteins for which antisera or monoclonal antibodies are available. included.

The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and their polymers in either single-stranded or double-stranded form. The term encompasses nucleic acids having known nucleotide analogs or modified backbone residues or linkages, which are synthetic, natural and non-natural, and have similar binding properties as control nucleic acids. And is metabolized similarly to the control nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates,
Phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O
-Methylrubonucleotide, peptide-nucleic acid (PNA).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants (eg, condensed codon substitutions) and complementary sequences, as well as those sequences explicitly indicated. . The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. As used herein, a "nucleic acid probe or oligonucleotide" binds to a target nucleic acid of complementary sequence via one or more chemical bonds, usually complementary base pairing, usually hydrogen bond formation. Are defined as possible nucleic acids. As used herein, a probe can be a natural (ie, A, G, C, or T) or modified base (7
-Deazaguanosine, inosine, etc.). Further, the base contained in the probe may be one bonded by a linkage other than a phosphodiester bond as long as it does not interfere with hybridization. Therefore,
For example, the probe may be a peptide nucleic acid in which the constituent bases are linked by peptide bonds rather than phosphodiester bonds. It will be appreciated by those skilled in the art that depending on the stringency of the hybridization conditions, it may bind to a target sequence that lacks complete complementarity with the probe sequence. The probe is preferably labeled directly with a radioisotope, chromophore, lumiphor, chromogen, or indirectly, such as with biotin, to which the streptavidin complex will later bind. To assay for the presence or absence of a probe, the presence or absence of a selected sequence or subsequence can be detected.

The labeled nucleic acid probe or oligonucleotide is covalently, via a linker, or ionically, such that the presence of the probe is detectable by detecting the presence of a label attached to the probe. , Van der Waals or hydrogen bonds to the label. "Pharmaceutically acceptable" refers to a material that is not biological, or that is not inherently unnecessary, i.e., the material does not have any undesirable biological effects or It can be administered to an individual with a chlamydia antigen without adversely interacting with any of the other components.

The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein and refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of the corresponding naturally occurring amino acid, as well as a naturally occurring amino acid polymer.

The phrase “specifically or selectively hybridizes to” refers to a probe that binds substantially only to a target sequence when the target is in a heterogeneous mixture of polynucleotides and other compounds. Hybridization between the probe and the target sequence to form a hybridization complex. Such hybridization is a determining factor in the presence of the target sequence. The probe may bind to other unrelated sequences, but at least 90%, preferably 95%, or most of the hybridization complex formed is binding to the target sequence.

The term “recombinant”, when used in reference to a cell or nucleic acid or vector, indicates that the cell or nucleic acid or vector has been modified by the introduction of a heterologous nucleic acid or the modification of a natural nucleic acid. Or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell may express a gene that is not found in a cell in its native (non-recombinant) state, either in an expressed state or not at all, or otherwise abnormally expressed. To express the native gene.

The phrase “specifically immunoreacts” when referring to a protein or peptide refers to the binding reaction between a protein and an antibody, and refers to the binding reaction of a protein in the presence of a heterogeneous population of proteins and other components. It decides existence. Thus, under designated immunoassay conditions, certain antibodies bind to certain proteins and do not bind in significant amounts to other proteins present in the sample. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. Various immunoassay formats are available
It may be used for selection of an antibody that specifically immunoreacts with a specific protein,
They are described in detail below.

The phrase “practically pure” or “isolated” when referring to a Chlamydia peptide or protein, means that it is free of other quasicellular components of the Chlamydia microorganism. Generally, a monomeric protein is substantially pure when at least about 85% or more of the sample represents a single polypeptide backbone. Minor variants or chemical modifications will generally share the same polypeptide sequence. Depending on the purification method, a purity of 85%, preferably greater than 95% is possible. Protein purity or homogeneity can be determined by several means well known in the art, such as performing polyacrylamide gel electrophoresis of the protein, followed by silver staining to visualize a single polypeptide band on the polyacrylamide gel. Can be shown by For certain purposes, high resolution is required and H
PLC or similar means is utilized for purification. DETAILED DESCRIPTION The present invention is intended to provide C
. Nucleotide sequence of the Pseudomonas genome, SEQ ID NO: 1 (SEQ ID N
o. 1) or a typical fragment thereof is provided. As used herein, a "typical fragment" of the nucleotide sequence set forth in SEQ ID No. 1 refers to any protein not currently represented in publicly available databases Is to be mentioned. Preferred exemplary fragments of the present invention include open reading frames, expression-modified fragments, incorporation-modified fragments, and C.I. Fragment used to diagnose the presence of Pneumoniae. By utilizing the information provided in this application in conjunction with routine cloning and sequencing methods, one of skill in the art will be able to obtain a broad range of C.I. It will be possible to clone and sequence all "typical fragments" of interest, including the open reading frame (ORF) encoding the Pneumoniae protein. Non-limiting identification of such preferred widespread exemplary fragments is shown in Tables 2 and 3. C. Diagnostic Use of Pneumonie Nucleic Acids Hybridization-Based Assays Using the nucleic acids disclosed herein, one skilled in the An assay based on nucleic acid hybridization (hybridization assay) for detecting Pneumoniae can be designed. Any of several well-known techniques for the specific detection of a target nucleic acid can be used. Representative hybridization assays include, but are not limited to, traditional "direct probe" methods, such as Southern blots, dot blots, in situ hybridization (eg, FISH), PCR, and the like. The methods can be used in a wide range of formats, including but not limited to substrate (eg, membrane or glass) binding methods or array-based approaches described below. As noted, the invention also includes a method for detecting the presence of Chlamydia DNA or RNA from a biological sample. Those sequences can be used to detect the presence of Chlamydia in a biological sample from a suspected patient. There are various methods for measuring specific DNA and RNA using the nucleic acid hybridization method, which are known to those skilled in the art (Sambrook et al.
See above).

In situ hybridization methods are well known (eg, An
gerer (1987) Meth. Enzymol 152: 649)
. In general, the in situ hybridization method consists of the following main steps. That is, (1) fixation of a tissue or biological structure to be analyzed,
2) pre-hybridization to biological structures for the purpose of increasing the contactability of the target DNA and reducing non-specific binding; (3) hybridization of the nucleic acid mixture to nucleic acids in biological structures or tissues (4) washing after hybridization for the purpose of removing nucleic acid fragments not bound by hybridization, and (5) detection of hybridized nucleic acid fragments. The reagents and conditions used in each of these steps will vary depending on the particular application.

In a typical in situ hybridization assay, cells are fixed to a solid support, usually a glass slide. If the nucleic acid is probed,
Generally, cells are denatured with heat or alkali. Next, the cells are contacted with a hybridization solution at an appropriate temperature to anneal a labeled probe specific to the nucleic acid sequence encoding the protein. Next, the target (eg, cell)
Is generally washed at a predetermined stringency or at an increased stringency until a suitable signal to noise ratio is obtained.

[0036] The nucleic acids of the invention are highly suitable for array-based hybridization formats. An array is a number of different “probe” or “target” nucleic acids (or other compounds) attached to one or more surfaces (eg, a solid phase, a membrane, or a gel).
In a preferred embodiment, multiple nucleic acids (or other components) are attached to a single continuous surface or multiple surfaces parallel to each other.

In an array format, many different hybridization reactions can be performed essentially “in parallel”. This allows several hybridizations to be evaluated quickly and essentially simultaneously in one "experiment". Methods for performing hybridization reactions in an array-based format are well known to those of skill in the art (eg, Pastinen (1997) Genom).
e Res. 7: 606-614, Jackson (1996) Nat.
ure Biotechnology 14: 1685, Chee (199
5) Science 274: 610, see WO 96/17958).

[0038] Arrays, especially nucleic acid arrays, can be produced based on a wide variety of methods well known to those skilled in the art. For example, in a simple embodiment, a "low density" array is one in which different nucleic acids are spotted (eg, by hand with a pipette) at different locations on a solid support (eg, a glass surface, a membrane, etc.). It can be easily made.

This simple spotting approach has been automated to produce densely spotted arrays (eg, US Pat. No. 5,807,52).
See No. 2). This patent describes the use of an automated system in which a microcapillary is tapped against a surface to attach a small volume of a biological sample. This process is repeated to produce a high density array. Arrays can also be made using oligonucleotide synthesis. Thus, for example, US Pat. Nos. 5,143,854 and PCT Patent Publication Nos. WO 90/15070 and 92/10092 teach the use of light-directed combinatorial synthesis of high-density oligonucleotide arrays.

Many methods for immobilizing nucleic acids on various solid surfaces are known in the art.
As with other materials, a wide variety of natural and synthetic organic and inorganic polymers can be used as the solid surface material. Specific solid phase surfaces include, for example, nitrocellulose, nylon, glass, quartz, diazotized film (paper or nylon), silicone, polyformaldehyde, cellulose, and cellulose acetate. Further, plastics such as polyethylene, polypropylene, and polystyrene can also be used. Other materials that can be used include paper, ceramics, metals, metalloids, semiconductor materials, cermets, and the like. Gel-forming substances can also be used. Such materials include, for example, proteins (gelatin),
Lipopolysaccharides, silicates, agarose, and polyacrylamide. When the solid surface is porous, various pore sizes can be used depending on the nature of the system.

In preparing the above surfaces, a plurality of different materials may be used, particularly as a laminate, to obtain various properties. For example, preventing non-specific binding, simplifying covalent binding,
A mixture of proteins (eg, bovine serum albumin) or macromolecules (eg, Denhardt's solution) can be used to enhance signal detection and the like. If a covalent bond between the compound and the surface is desired, the surface will typically be polyfunctional or capable of being multifunctional. The polyfunctional groups that are believed to be present on the surface and are used for bonding can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylene groups, hydroxyl groups, mercapto groups, and the like. Methods for attaching a wide variety of compounds to various surfaces are well known and are well illustrated in the literature.

For example, a method for immobilizing a nucleic acid by introducing various functional groups into a molecule is known (Bischoff (1987) Anal Biochem., 1988).
164: 336-344, Kremsky (1987) Nucl. A
cids. Res. 15: 2891-2910). The modified nucleotides can be placed on the target using PCR primers containing the modified nucleotides or by enzymatic end labeling with the modified nucleotides. The use of glass or membrane supports (eg, nitrocellulose, nylon, polypropylene) for the nucleic acid arrays of the present invention can be achieved using manual and automated methods for aligning targets at relatively high component densities. This is advantageous because it is a technology that has been developed.
Such membranes are commonly available and protocols and equipment for hybridization to the membrane are well known.

Various sizes of target elements ranging from 1 mm to 1 μm in diameter can be used. Smaller target elements containing low concentrations of enriched and immobilized probe DNA are used for high complexity comparative hybridizations. This is because the total amount of sample available for binding to each target element is limited. Therefore, it is advantageous to have a small array target element that contains a small amount of probe DNA that is concentrated so that the resulting signal is highly localized and shiny. Such small array target elements are commonly used in arrays having a density greater than 10 ⁴ cm / cm ² . A relatively simple approach has been described that allows for quantitative fluorescence imaging of a 1 cm ² area that allows acquisition of data from a large number of target elements in one image (eg, Witrup (1)
994) Cytometry 16: 206-213).

If a fluorescently labeled nucleic acid sample is used, solid-phase substrates with much lower fluorescence than the membrane, eg, arrays on glass, quartz, or small beads, will have better sensitivity. Can be achieved. Glass or quartz glass is distinguished by providing a very low fluorescent substrate and a highly efficient hybridization environment. Covalent attachment of the target nucleic acid to glass or synthetic quartz glass can be achieved based on several well-known techniques (described above). Nucleic acids can be conveniently bound to glass using commercially available reagents. For example, materials for the preparation of silanized glasses with some functional groups are commercially available or can be prepared by standard techniques (eg, Gait
(1984) Oligonucleotide Synthesis: A
Practical Approach. IRL Press, Wash
. , D. C. See). Quartz cover glasses having at least 10 times lower fluorescence than glass can also be silanized.

Alternatively, probes can also be immobilized on commercially available coated beads or other surfaces. For example, a biotin end-labeled nucleic acid can be bound to commercially available avidin-coated beads. Streptavidin or anti-digoxigenin antibodies are also, for example,
Standard protocols (eg, Smith (1992) Science 2)
58: 1122-1126) can be deposited on silane-treated glass slides by protein-mediated coupling using protein A or the like. Biotin or digoxigenin end-labeled nucleic acids can be prepared according to standard techniques. Hybridization of nucleic acids attached to beads
They are suspended in the hybridization mixture, which is subsequently placed on a glass substrate for analysis after washing. Alternatively, paramagnetic particles, with or without avidin coating, can be used, for example iron oxide particles.

A variety of other nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in Hames and H
iggins (1985) Nucleic Acid Hybridiza
, A Practical Approach, IRL Pres
s, Gall and Parduc (1969) Proc. Natl.
Acad. Sci. USA 63: 378-383, and John.
et al. (1969) Nature 223.

[0047] Sandwich assays are commercially useful hybridization methods for detecting or isolating nucleic acid sequences. Such an assay covalently immobilizes a "captured" nucleic acid covalently immobilized on a solid support and a labeled "signal" nucleic acid in solution. The sample will provide the target nucleic acid. "Capture" and "signal" nucleic acids are
Hybridize with the target nucleic acid to form a "sandwinch" hybridization complex. Most effectively, the signal nucleic acid should not hybridize to the capture nucleic acid.

[0048] Detection of the hybridization complex may require binding of the signal-generating complex to the target and to the duplex of the probe polynucleotide or nucleic acid. Generally, such binding occurs via ligand and anti-ligand interactions, such as between a ligand-conjugated probe and an anti-ligand conjugated to a signal. The sensitivity of the hybridization assay can be increased by using a nucleic acid amplification system that amplifies the target nucleic acid being detected. Examples of such systems include, for example, the polymerase chain reaction (PCR) system and the ligase chain reaction (
LCR) system. Other methods recently described in the art include nucleic acid sequence-based amplification (NASBAO, Cangene, Mississa).
uga, Ontario) and the Q beta replicase system.

Nucleic acid hybridization simply involves providing a denatured probe and a target nucleic acid under conditions that allow the probe and its complementary target to form a stable hybrid duplex via complementary base pairing. including. The nucleic acids that do not form hybrid duplexes are then washed away, leaving the hybridized nucleic acids to be detected, generally through detection of a bound detectable label. It is generally accepted that denaturation of nucleic acids may occur by increasing the temperature, or by decreasing the salt concentration of the buffer containing the nucleic acids, by adding chemicals, or by increasing the pH. Under conditions of low stringency (eg, low temperature and / or high salt and / or
Or a high target concentration) at a hybrid duplex (eg, DNA: DNA, RNA: R
NA, or RNA: DNA) is formed even if the annealed sequence has incomplete complementarity. Thus, the specificity of hybridization is reduced for those of lower stringency. Conversely, at high stringency (eg, high temperature or low salt) effective hybridization requires even less mismatch.

One of skill in the art will readily appreciate that hybridization conditions may be chosen to provide any degree of stringency. In a preferred embodiment, hybridization is performed at low stringency, thereby ensuring hybridization, and subsequent washing at high stringency to remove mismatched hybrid duplexes. Washing may be performed continuously at a higher stringency until the desired level of hybridization specificity is obtained (eg, 37 ° C. to 7 ° C.).
Reduce as low as 0.25 X SSPE-T at 0 ° C). Stringency is
It can also be increased by adding a reagent such as formamide. Hybridization specificity may be assessed by comparing hybridization to various control groups that can be presented with hybridization to a test probe.

In general, there is a trade-off between hybridization specificity (stringency) and signal strength. Thus, in a preferred embodiment, washing is performed at the highest stringency that produces consistent results and provides a signal strength that is greater than about 10% of the background signal strength. Thus, in a preferred embodiment, the hybridized array is sequentially washed with a higher stringency solution and read between each wash. Analysis of the data set thus obtained will reveal wash stringency in which the hybridization pattern is not properly altered and which provides an appropriate signal for the particular probe of interest.

Methods for optimizing hybridization conditions are well known to those skilled in the art (eg, Tijssen (1993) Laboratory Technology).
equals in Biochemistry and Molecular
Biology. Vol. 24: Hybridization With
Nucleic Acid Probes, Elsevier, N.W. Y
. ). Labeling and Detection of Nucleic Acids In a preferred embodiment, the hybridized nucleic acids are detected by detecting one or more labels bound to the sample or probe nucleic acid. The label can be incorporated by any of several means known to those skilled in the art.
Means for attaching the label to the nucleic acid include, for example, nick translation or terminal nicking in which the label (eg, a fluorophore) is attached to the nucleic acid of the sample by kinase treatment of the nucleic acid followed by attachment (ligation) of a nucleic acid linker. Label (eg, label RN
A). A wide variety of linkers are known for attaching labels to nucleic acids. Also, the insertion of dyes and fluorescent nucleotides can be used.

Suitable detectable labels for use in the present invention include, for example, spectroscopic, photochemical,
Biochemical, immunological, electrical, optical, or chemical means may be used. Labels useful in the present invention include biotin for staining with labeled streptavidin conjugates, magnetic beads (eg, Dynabeads®), fluorescent dyes (eg, fluorescein, Texas Red, rhodamine, green fluorescein protein, etc.) For example, Molecular Probes, Euge
ne, Oregon, USA), radioactive labels (eg, ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³² P), enzymes (eg, horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISAs). Colorimetric labeling of colloidal gold or the like (eg, 40-80 nm diameter gold particles that scatter green light with high efficiency) or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) Of beads are included. Patents teaching the use of such markers include, for example, U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, Nos. 4,277,437, 4,275,149, and 4,366,241.

[0054] Fluorescent labels are preferred. This is because fluorescent labels provide a very strong signal while having low background. In addition, it can be arbitrarily detected with high resolution and sensitivity in a scanning manner. All nucleic acid samples can be labeled with a single label, eg, a single fluorescent label. Alternatively, in another embodiment, different nucleic acid samples are simultaneously hybridized, with each nucleic acid sample having a different label. For example, if one target has a green fluorescent label, a second target can have a red fluorescent label. The scanning step will distinguish binding sites for the red label from binding sites for the green fluorescent label. Each nucleic acid sample (legal nucleic acid) is analyzed separately from each other.

Suitable chromogens used absorb light in the specific wavelength range in which the color is observed,
Or molecules and compounds that emit light upon irradiation with radiation of a particular wavelength or range of wavelengths, such as fluorescent agents. Desirably, the fluorescer should absorb light above about 300 nm, preferably above 350 nm, more preferably above about 400 nm, and generally emit wavelengths about 10 nm higher than the wavelength of the absorbed light. It should be pointed out that the absorption and emission properties of the bound dye are different from the unbound dye. Therefore,
When referring to the various wavelength ranges and properties of the dye, it is meant the dye used, rather than any unconjugated, solvent-specific dye.

[0056] Fluorescent agents are generally preferred. This is because a plurality of light emissions can be obtained by irradiating the fluorescent agent with light. Thus, a single label can provide multiple measurable events. Detectable signals can also be provided by chemiluminescent and bioluminescent sources. Chemiluminescent sources include compounds that initiate electronic excitation by a chemical reaction and subsequently emit or energize light that is used as a detectable signal to a fluorescent receptor. Also, luciferin can be used with luciferase or lucigenin to provide bioluminescence. Spin labels are provided by reporter molecules with unpaired electron spins that can be detected by electron spin resonance (ESR) spectroscopy. Representative spin labels include organic free radicals, transition metal complexes, particularly vanadium, copper, iron, manganese, and the like. Representative spin labels include nitroxide free radicals.

The label may be added to the target (sample) nucleic acid prior to or after hybridization. A so-called "direct label" is a detectable label that is directly bound or incorporated into a target (sample) nucleic acid prior to hybridization. In contrast, a so-called "indirect label" is added to the hybrid duplex after hybridization. Often, the indirect label is added to the binding site binding to the target prior to hybridization. Thus, for example, the target nucleic acid may be biotinylated prior to hybridization. After hybridization, the avidin-conjugated fluorophore binds to the biotin-loaded hybrid duplex, providing a label that is easily detected. For a detailed review on labeling nucleic acids and methods of labeled hybridization nucleic acids, see the Laboratory.
technologies in Biochemistry and Molec
ullar Biology, Vol. 24: Hybridization
With Nucleic Acid Probes, P.M. Tijssen
, Ed. Elsevier, N .; Y. , (1993).

[0058] Fluorescent labels are easily added during the in vitro transcription reaction. Thus, for example, fluorescently labeled UTP and CTP can be used for TN produced by in vitro transcription.
A. Labels can be added directly or via a linker site. In general, the label site or linker label attachment site is not limited to any particular location.
For example, a label can be attached to a nucleoside, nucleotide, or analog thereof at any location, which does not interfere with the detection or hybridization already described. For example, certain label-on reagents from Clontech (Palo Alto, Calif.) Have labels scattered throughout the phosphate backbone of the oligonucleotide, and 3 ′ and 5 ′ termini. Provide a termination indicator. As shown here by way of example, the label is attached at a position on the ribose ring, or the ribose is modified and optionally removed. Useful labeling reagent base moieties include those that are natural or modified so as not to interfere with the purpose for which they are placed. Modified bases include, but are not limited to, 7-deaza A and G, 7-deaza-8-aza A and G, and another heterocyclic moiety.

It will be appreciated that fluorescent labels are not limited to a single type of organic molecule, but also include inorganic molecules, mixtures of multiple molecules of organic and / or inorganic molecules, crystals, heteropolymers, and the like. Therefore, for example, CdSe- encapsulated in a silica shell
CdS core-shell nanocrystals are easily derived for coupling to biological molecules (Bruchez et al. (1998) Scie).
nce, 281: 2013-2016). Similarly, highly fluorescent quantum dots (hi
Gly fluorescent quantum dots (zinc sulfide-capped cadmium selenium compounds) are covalently linked to biomolecules for use in ultrasensitive biological detection (Warren and Nie (1998) S
science, 281: 2016-2018). Amplification-Based Assays In another embodiment, an amplification-based assay (amplification assay) can be used to detect nucleic acids. In such amplification assays, the nucleic acid sequence acts as a template for the amplification reaction (eg, polymerase chain reaction (PCR). Detailed protocols for quantitative PCR are described in Innis et al. (1990) PCR Pr.
otocols. A Guide to Methods and Appl
ications, Academic Press, Inc. N. Y.
Is provided in).

Other suitable amplification methods include, but are not limited to, the ligase chain reaction (LCR).
) (Wu and Wallace (1989) Genomics 4;
560, Landegren et al. (1988) Science
241, 1077, and Earringer et al. (1990)
Gene 89: 117), transcription amplification (Kwoh et al. (1989).
) Proc. Natl. Acad. Sci. USA 86: 117
3), and autonomous sequence replication (Guatelli et al. (1990)
Proc. Nat. Acad. Sci. USA 87: 1874). C. Detection of pneumoniae gene expression It can also be used for pneumoniae detection gene transcription.
Methods of detecting and / or quantifying gene transcription using nucleic acid hybridization techniques are known to those of skill in the art (see Sambrook et al., Supra). Northern transfer can be used to directly detect the desired mRNA. Briefly, mRNA is, for example, acidic guanidinium-phenol-
It is isolated from a given cell sample using the chloroform extraction method. The mRNA is then subjected to electrophoresis to separate the mRNA species and the mRNA is transferred from the gel to a nitrocellulose membrane. As with Southern blots, labeled probes are used to identify and / or quantify target mRNA.

In another preferred embodiment, gene transcripts can be measured using amplification (eg, PCR) -based methods as described above to directly assess copy number of the target sequence. C. Expression of Pneumonier Proteins The nucleic acids disclosed herein are used for recombinant expression of proteins. In these methods, a nucleic acid encoding the protein of interest is introduced into a suitable host cell, and the cell is subsequently induced to produce large amounts of the protein. The present invention relies on routine techniques in the field of recombinant genetics, which are well known to those skilled in the art. A basic book disclosing the general methods used in this invention can be found in Sambrook et al.
al. , Molecular Cloning, A Laboratory
y Manual (2nd ed. 1989).

Prokaryotic, mammalian, yeast, or insect cell lines expressing high levels of the desired polypeptides are produced using standard transfection methods and purified using standard techniques (eg, Colley et al.). , J. Biol.
m. 264: 17619-17622, 1989: Guide to
See Protein Purification, supra).

[0063] The nucleotide sequences used to transfect host cells can be modified to produce Chlamydia polypeptides having various desired proteins. For example, a polypeptide can differ from a native sequence at the primary structural stage by amino acids, insertions, substitutions, deletions, and the like. These modifications can be used in several combinations to produce the final modified protein chain.

Amino acid sequence variants can be prepared for a variety of intended purposes to facilitate purification and preparation of recombinants. Modified polypeptides are also useful for altering plasma half-life, improving the efficacy of treatment, and reducing the severity and occurrence of side effects during therapeutic use. Amino acid sequence variants are generally defined variants that are not found in nature, but exhibit immunogenic activity similar to the native protein. In general, modification of the polypeptide-encoding sequence can be readily accomplished by a variety of well-known methods, such as site-directed mutagenesis (eg, Gillima).
n & Smith, Gene 8: 81-97 (1979), Robert
ts et al. , Nature 328: 731-734 (1987).
). One skilled in the art will readily appreciate that the effects of many mutations are difficult to predict. Thus, many modifications are evaluated by routine screening, using assays appropriate for the desired properties. For example, the effect of various modifications on the ability of a polypeptide to elicit a protective immune response can be readily determined using in vitro assays. For example, for a polypeptide:
Testing can be performed for the ability to induce lymphocyte proliferation, T cell cytotoxicity, or cytokine production using standard techniques.

[0065] The particular method used to introduce the genetic material into the host cell to express the polypeptide is not particularly critical. Any of the well-known methods for introducing foreign nucleotide sequences into host cells can be used. They introduce calcium phosphate transfection, spheroplasts, electroporation, liposomes, microinjections, plasmid vectors, viral vectors, as well as cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into host cells. Or any of the other well known methods (see Sambrook et al., Supra). It is only necessary that the particular method employed be able to successfully introduce at least one gene into a host cell capable of expressing the gene.

[0066] Any of a number of well-known cells and cell lines can be used to express the polypeptides of the invention. For example, prokaryotic cells, such as E. coli
Can be used. Eukaryotic cells include yeast, Chinese hamster ovary (CHO) cells, COS cells and insect cells. The particular vector used to carry the genetic information to the cell is also not particularly critical. Any conventional vector used for expression of recombinant proteins in prokaryotic and eukaryotic cells can be used. Expression vectors for mammals typically include regulatory regions from a eukaryotic virus.

[0067] Expression vectors typically include a transcription unit or expression cassette that contains all the elements required for expression of the polypeptide DNA in the host cell. A typical expression cassette contains a promoter operably linked to the DNA sequence encoding the polypeptide and the signals required for efficient polyadenylation of the transcript. The term "operably linked" as used herein refers to the binding of a promoter upstream from a DNA sequence, such that the promoter mediates transcription of the DNA sequence. The promoter is preferably located at a distance from the heterologous transcription start site that is about the same as the transcription start site in its natural setting. However, as is known in the art, some change in this distance can be accounted for without losing promoter function.

After growth of the recombinant cells and expression of the polypeptide, the culture medium is harvested for purification of the secreted protein. The medium is typically cleaned by centrifugation or filtration to remove cells and cell debris, and proteins are adsorbed to any suitable resin or using ammonium sulfate fractionation, polyethylene glycol precipitation. Or by ultrafiltration. Other routine means known in the art may be equally suitable. Standard techniques such as affinity chromatography, ion exchange chromatography, size exclusion (sizin
g) Chromatography, His ₆ tagging and Ni-agarose chromatography (as described in Dobeli et al., Mol. and Biochem. Parasit. 41: 259-268 (1990)) or homogeneity. By other protein purification techniques to obtain
Further purification of the polypeptide can be performed. The purified protein is then:
As described below, it is used to produce a pharmaceutical composition.

[0069] An alternative method of producing a recombinant polypeptide useful as a vaccine involves the use of a recombinant virus (eg, vaccinia). Vaccinia virus, as described by Macket et al. In DNA cloning Vol. II: A practical approach, pp. 191-211 (Glover), Propagated in suitable cultured mammalian cells, such as HeLa S3 spinner cells. Antibody Production The proteins of the present invention can be used to produce antibodies specifically reactive with the C. pneumoniae antigen. If separated proteins are used, they are produced recombinantly or separated from Chlamydia (Chlamydia) cultures. Synthetic peptides made using this protein sequence can also be used.

[0070] Methods for producing polyclonal antibodies are known to those skilled in the art. Briefly, an immunogen,
Preferably, the purified protein is mixed with an adjuvant and the animal is immunized. Once a suitably high titer of antibodies to the immunogen has been obtained, blood is drawn from the animal,
Prepare antiserum. Further fractionation of the antisera can be performed, if desired, to be enriched for antibodies reactive with chlamydia proteins (Harlow & Lane, Antibodies: Laboratory Manual (Antibodies: A Laborator
y Manual) (1988)).

For example, Anderson et al., JAVA 198: 241 (1991) and Barr.
Et al., Vet. Pathol. 28: 110-116 (1991) using polyclonal antisera to identify and characterize chlamydia in tissue of patients using in situ techniques and immunoperoxidase assays. Is used. Monoclonal antibodies can be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from animals immunized with the desired antigen are immortalized, usually by fusion with myeloma cells (Kohler & Milstein
(Milstein), Eur. J. Immunol. 6: 511-519 (1976)). Alternative methods of immortalization include transformation with Epstein-Barr-virus, oncogenes or retroviruses, or other methods well known in the art. 1
Colonies arising from the two immortalized cells are screened for the production of antibodies with the desired specificity and affinity for the antigen, and the yield of monoclonal antibodies produced by such cells is transferred to the peritoneal cavity of a vertebrate host. It can be increased by a variety of techniques, including injection.

Monoclonal antibodies produced in such a manner can be used in vaccine development, protein isolation, for in vitro evaluation of spirochetes invasion, eg, in ELIZA diagnostic tests, immunoperoxidase tests, immunohistochemical tests. Used to select candidate antigens for screening, and to screen genomic and cDNA libraries for selecting appropriate gene sequences. Immunodiagnostic Detection of C. pneumoniae Infection The invention also provides a method for detecting the presence or absence of C. pneumoniae or an antibody reactive therewith in a biological sample. For example, antibodies specifically reactive with chlamydia can be detected using the chlamydia proteins or isolates described herein. Proteins and isolates can also be used to increase specific antibodies (monoclonal or polyclonal) for detecting antigens in a sample. Further, the nucleic acids disclosed and claimed herein can be used to detect chlamydia-specific sequences using standard hybridization techniques.

In general, for an overview of immunological and immunoassay procedures, see Basic and Clinical Immunology (Stites & Tel).
rr), 7th edition, 1991). The immunoassay of the present invention can be performed in any of a number of configurations, including Enzyme Immunoassay (Ed. Maggio, 1980); Tijssen, Laboratory Techniques in Bioc.
hem. stry and Molecular Biology (1985). For example, the proteins and antibodies disclosed herein are conveniently used in ELIZA, immunoblot analysis and agglutination assays.

[0074] Briefly, an immunoassay for measuring anti-Chlamydia antibodies or antigens can be a competitive or non-competitive binding assay. In a competitive binding assay, a sample analyte (eg, an anti-Chlamydia antibody) is labeled for a specific binding site on a capture agent (eg, an isolated chlamydia protein) bound to a solid surface (eg, an anti-Chlamydia antibody). -Chlamydia monoclonal antibody). The concentration of the labeled analyte bound to the capture agent is inversely proportional to the amount of free analyte present in the sample.

A non-competitive assay is typically a sandwich assay, wherein a sample analyte is bound between two analyte-specific binding reagents. One of the binders is used as a capture agent and binds to the solid surface. The second binding agent is labeled and the resulting complex is used to measure or detect by visual or instrumental means. Numerous combinations of capture agents and labeled binders can be used. For example, an isolated chlamydia protein or culture can be used as a capture agent,
A labeled anti-human antibody specific for the constant region of a human antibody can be used as a label binding agent. Goats, sheep and other non-human antibodies specific for human immunoglobulin constant regions (eg, γ or μ) are well known in the art. Alternatively, the anti-human antibody can be a capture agent and the antigen can be labeled.

Various components of the assay, including antigens, anti-Chlamydia antibodies or anti-human antibodies, can be bound to a solid surface. Many methods for immobilizing biomolecules on various solid surfaces are
It is known in the art. For example, a solid surface can be a membrane (eg, nitrocellulose).
, A microtiter dish (eg, PVC or polystyrene) or beads. The desired components can be covalently attached or non-covalently attached by non-specific binding.

Alternatively, the immunoassay can be performed in the liquid phase and various separation techniques can be used to separate bound labeled components from unbound labeled components. These methods are known to those skilled in the art and include immunoprecipitation, column chromatography, adsorption, addition of binder-coated magnetizable particles and other similar techniques. Immunoassays can also be performed in the liquid phase without separation. A variety of homogeneous immunoassays are currently being applied to immunoassays for protein analytes. In these methods, binding of the binding agent to the analyte causes a change in the signal emitted by the label, so that binding can be measured without separating the conjugate from unbound labeled components.

[0078] Western blot (immunoblot) analysis can also be used to detect the presence of antibodies to chlamydia in a sample. This technique is a reliable way to confirm the presence of antibodies to a particular protein in a sample. This technique generally involves separating proteins by gel electrophoresis based on molecular weight, transferring the separated proteins to a suitable solid support (eg, a nitrocellulose filter, a nylon filter or a derivatized nylon filter), and
Incubating the sample with the separated proteins. This causes each protein to bind to the specific target antibody present in the sample. The target antibody is then detected using a labeled anti-human antibody.

The immunoassay formats described above use labeled assay components. The signs are
It can be linked directly or indirectly to the desired components of the assay according to methods well known in the art. A wide range of labels can be used. The components can be labeled by any one of several methods. Conventionally, ³ H, ¹²⁵ I
, Radiolabeled were used to incorporate ³⁵ S, ¹⁴ C or ³² P. Non-radioactive labels include ligands that bind to the labeled antibody, fluorophores, chemiluminescent agents, enzymes, and antibodies that can serve as specific binding pairs for the labeled ligand. The choice of label will depend on the sensitivity required, ease of conjugation with the compound, stability requirements and available equipment.

Enzymes of interest as labels are mainly hydrolases, especially phosphatases, esterases and glycosidases, or oxidoreductases, especially peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone and the like. Chemiluminescent compounds include luciferin and 2,3-dihydrophthalazinedione such as luminol. See US Pat. No. 4,391,904 for a review of the various labels or signal generation systems that can be used. This patent is incorporated herein by reference.

[0081] Non-radioactive labels are often applied by indirect means. Generally, a ligand molecule (eg, biotin) is covalently attached to the molecule. The ligand then binds to an anti-ligand molecule (eg, streptavidin), which is essentially detectable or covalently linked to a signal system (eg, a detectable enzyme, fluorescent or chemiluminescent compound). Is done. Numerous ligands and anti-ligands can be used.
If the ligand has a natural anti-ligand, such as biotin, thyroxine and cortisol, it can be used with a labeled naturally occurring anti-ligand. Alternatively, any hapten or antigenic compound can be used in combination with the antibody.

Some assay formats do not require the use of a labeling component. For example, an agglutination assay can be used to detect the presence of a target antibody. In this case, the particles coated with the antigen are aggregated by the sample containing the target antibody. In this format, none of the components need be labeled, and the presence of the target antibody is detected by simple visual inspection. Pharmaceutical Compositions The peptides or antibodies (typically monoclonal antibodies) of the present invention and pharmaceutical compositions thereof are useful for treating and / or preventing Chlamydia infection in mammals,
It is particularly useful for administration to humans. A suitable formulation is available from Remington's Pharmaceutical Sciences (Rem
ington's Pharmaceutical Sciences), Mac Publishing Company (Ma
ck Publishing Company), Philadelphia, PA, 17th Edition (1985).

The immunogenic peptides or antibodies of the invention are administered prophylactically or to an individual already suffering from the disease. The peptide composition is administered to the patient in an amount sufficient to elicit an effective immune response against Chlamydia. An effective immune response is one that blocks infection. An amount sufficient to accomplish this is defined as "therapeutically effective dose" or "immunogenically effective dose." The effective amount for this use will depend on, for example, the peptide composition, the mode of administration, the stage and severity of the disease being treated, the weight and general state of the patient, and the judgment of the prescribing physician, but For cropping (ie, for therapeutic or prophylactic administration), it generally ranges from about 0.1 mg to about 1.0 mg per 70 kg patient weight, more usually from about 0.5 mg to about 0.75 mg per 70 kg patient weight. . Boosting doses typically range from about 0.1 mg to about 0.5 mg, using additional boosting regimens over weeks to months, depending on the response and condition of the patient. Is a peptide. Suitable protocols include injections at weeks 0, 4, 2, 6, 10 and 14 followed by additional injections at weeks 24 and 28.

For therapeutic use, administration should begin at the first sign of infection.
This is followed by an additional dose at least until the symptoms are substantially alleviated and for a period thereafter. In some cases, a loading dose and then an additional dose may be required. The resulting immune response helps to cure or at least partially arrest the symptoms and / or complications. A vaccine composition comprising the peptide is administered prophylactically to a patient susceptible to or otherwise at risk of infection.

[0085] The pharmaceutical compositions (including peptides or antibodies) are intended for parenteral or oral administration. Preferably, the pharmaceutical compositions are administered parenterally, for example, subcutaneously, intradermally or intramuscularly. Thus, the present invention provides a composition for parenteral administration,
The composition comprises a solution of the immunogenic polypeptide dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, for example, water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solution, as is or lyophilized,
Can be packaged for use. The lyophilized preparation is combined with a sterile solution before administration. The compositions may contain pharmaceutically acceptable auxiliary substances required for approximate physiological conditions, such as buffers, tonicity adjusting agents, wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride. , Calcium chloride, sorbitan monolaurate, triethanolamine oleate and the like.

[0086] The compositions can also include a carrier to increase the immune response. Useful carriers are well known in the art and include, for example, KLH, thyroglobulin, albumin such as human serum albumin, tetanus toxoid, polyamino acids such as poly (lysine: glutamic acid), influenza, hepatitis B virus core protein, Hepatitis B virus recombinant vaccine and the like are included.

For solid compositions, conventional non-toxic solid carriers can be used, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, carbonate Magnesium and the like. For oral administration, any of the commonly used excipients, for example, the aforementioned carriers and generally 10-95% of the active ingredient, ie one or more of the peptides of the present invention, is preferably added to the composition in an amount of 25-75%. By incorporating at a% concentration, a pharmaceutically acceptable non-toxic composition is formed.

As mentioned above, the peptide composition is intended to elicit an immune response against Chlamydia. Thus, compositions and methods of administration suitable to maximize the immune response are preferred. For example, the peptides can be introduced into a host (including humans), bound to a carrier, or as a homopolymer or heteropolymer of active peptide units from various Chlamydia proteins disclosed herein. Alternatively, a "cocktail" of polypeptides can be used. A mixture of one or more polypeptides will have an increased mean immunological response and, if different peptides are used to make the polymer, have the additional ability to elicit antibodies to multiple epitopes. Have.

[0089] The compositions also include an adjuvant. Numerous adjuvants, as used herein, are well known to those skilled in the art. Suitable adjuvants include incomplete Freund's adjuvant, alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L -Alanyl-D-isoglutamine (CGP 11637, nor-MDP
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP
19835A, referred to as MTP-PE) and RIBI (in 3% squalene / Tween 80 emulsion, contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) ]. The effectiveness of an adjuvant can be determined by measuring the amount of antibody directed against the immunogenic peptide.

The concentration of the immunogenic peptides of the present invention in a drug formulation can vary widely. That is,
It varies from less than about 0.1% by weight, usually from about 2% by weight or more to 20-50% by weight or more, depending on the particular mode of administration chosen, mainly by fluid volume, viscosity and the like. The peptides of the present invention can also be expressed by an attenuated viral host, such as vaccinia or fowlpox. This approach involves using vaccinia virus as a vector to express a nucleotide sequence encoding a peptide of the invention. When introduced into a host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response. Vaccinia vectors and methods useful in immunization protocols are described, for example, in US Pat. No. 4,722,848. Another vector is BCG (Bacille Calmet
te Guerin)). The BCG vector was obtained from Stover et al. (Nature, 351: 456-4).
60 (1991)). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, such as Salmonell typhi (Salmonell
a typhi) vectors and the like will be apparent to those skilled in the art from the description herein.

[0091] DNA encoding one or more of the peptides of the present invention can also be administered to a patient. This approach is described, for example, in Wolff et al., Science 247: 1465-1468 (1990) and U.S. Patent Nos. 5,580,859 and 5,589,466. To increase serum half-life, the peptide can also be encapsulated, introduced into the lumen of the liposome, manufactured as a colloid, or other conventional methods that increase the serum half-life of the peptide. Techniques can be used. For example, as described in Szoka et al., Ann. Rev. Biophys.Bioeng. 9: 467 (1980), U.S. Pat. Various methods are available. EXAMPLES The following examples are provided to illustrate, but not limit, the claimed invention. Example 1 This example demonstrates that the genome of C. pneumoniae disclosed herein and the genome of C. trachomatis previously sequenced (Stephens et al., Science 28
2: 754-759 (1998)).

[0092] Despite similar cell structures and developmental cycles, C.
CHOMATIS) and a distinctly lower degree of DNA homology between C. pneumoniae (Campbell et al., J. Clin. Microbiol. 25: 1911-1916 (1987)) indicate that comparative analysis of the two genomes indicates that both Expect to significantly enhance understanding of pathogens. The identification of genes that are present in one species but not the other is particularly important for their incompatible biotoxicity and pathogenic potential. The identification of genes shared between the two species has evolved over their long-term relationship with mammalian host cells to reduce metabolic capacity while optimizing the survival, growth and transmission of these unique pathogens We strongly support the need for these capabilities in established biological systems.

The genome of C. trachomatis previously sequenced contains 1,042,519 nucleotides and 875 potential protein-encoding genes. The pursuit of similarity suggests that the disclosed allocation of 636 (60%) and 251 (23%) gene sequences disclosed herein is a hypothesis for other bacterial organisms, including those of C. trachomatis. It was found to be similar to the gene. The remaining 186 (17%) genes are from GenBan
Not homologous to the sequence deposited in k). Seventy C. trachomatis genes have not been shown in the C. pneumoniae genome. These include 2-17 genes and 19
Is contained in a block consisting of one gene. Of the 70 C. trachomatis genes without homologs in C. pneumoniae, 60 are classified as encoding hypothetical proteins. The remaining genes not shown in C. pneumoniae consist of the tryptophan operon (trpA, B, R), trpC, two predicted thiol protease genes, and four genes assigned to the phospholipase-D superfamily.

Since an ortholog for the C. trachomatis gene was identified for 859 (80%) of the predicted coding sequences for C. pneumoniae, high levels of C. pneumoniae and C. trachomatis between It is clear that there is functional preservation.
The level of similarity for each encoded protein can be determined by the broad spectrum (
(22-95% amino acid identity) with an average of 62% amino acid identity between orthologs from the two species. The percent amino acid identity between orthologous chlamydia proteins is similar between functional groups, highest for proteins involved in translation, whose function in chlamydia has not been characterized, and for proteins encoded by other organisms. Lowest for unrelated proteins. The gene order of the homologous set of genes in C. pneumoniae indicates a rearrangement to the C. trachomatis genome, but there is a high level of synteny for the two genomes. We have identified 39 blocks of two or more genes whose gene organization is collinear with homologs to C. trachomatis (although some of them are reversed). C. Pneumoniae code sequence 0130-0300
The distribution of genomic rearrangements is not evenly distributed on chromosomes because the regions in between contain rearrangements that are substantially larger than other regions of the genome. This region is consistent with the predicted chromosome replication end.

The present inventors have identified orthologs of enzymes characterized in other bacteria that describe essential requirements for DNA replication, repair, transcription and translation,
Two predicted D of the Swi2 / Snf2 family found in C. trachomatis
Including NA helicase. Similar to C. trachomatis, alternative sigma subunits for RNA polymerase (σ ²⁸ and σ ⁵⁴ ) are the anti-σ regulatory system factors RsbV, RsbW
-Like single-region histidine kinases and RsbU-like protein phosphatases have been identified. These findings suggest that the underlying mechanism of transcriptional regulation is conserved between Chlamydia. A C. trachomatis protein containing a SET and SWIB region and a SWIB region fused to the C-terminus of chlamydia topoisomerase I (not identified except in eukaryotes) has been found in C. pneumoniae, Supports their possible role in the chromatin condensation-decondensation characteristic of the biologically unique chlamydia development cycle.

The central metabolic pathway deduced from the C. pneumoniae genomic sequence is the same as that identified for C. trachomatis. C. pneumoniae has a glycolytic and linked tricarboxylic acid cycle and appears functional, but is incomplete because genes for citrate synthase, aconitase, and isocitrate dehydrogenase have not been identified. It is. C. pneumoniae has a complete glycogen synthesis and degradation system, which supports a role for glycogen synthesis and the use of glucose derivatives in chlamydial metabolism. There are genes encoding essential functions in aerobic respiration, and the flow of electrons is controlled by pyruvate, succinate,
It can be supported by glycerol-3-phosphate and NADH dehydrogenase, NADH-ubiquinone oxidoreductase and cytochrome oxidase. C.
Pneumoniae is also a V (with vacuole)-found in C. trachomatis.
Includes the type ATPase operon and two ATP translocases.

[0097] Three chromosome localization processes (locs) are required for invasion by some pathogenic bacteria.
ationsis) in the C. trachomatis genome is also present in the C. pneumoniae genome. Each of the components is preserved, and their relative genomic background is preserved. Genes, such as the predicted serine / threonine protein kinase and other genes physically linked to genes encoding structural components of the type-III secretion apparatus, also have no identified homology, but have two homologs. There is a high degree of similarity between these, suggesting that the functional role in modifying cell biology is essentially conserved.

[0098] Although not found in Chlamydia organisms, the intracellular Chlamydia
The Chlamydia-encoded protein, localized to the embrane, is a unique intracellular biology and inclusion morphology that is likely to be observed among Chlamydia species.
orphology) is probably essential. Several such proteins (referred to as IncA, B & C) have been characterized for C. psittaci strains (Rockey et al., Mol. Microbiol. 15: 617-626 (1995); Rocky ( Rockey) et al., Infect. Immun. 62: 106-112 (1994)). C. pneumoniae and C. trachomatis encode orthologs for C. psittaci IncB and IncC, and C. trachomatis also includes orthologs for IncA. C. pneumoniae contains two genes (CPn0186 and CPn0585) that encode proteins with a low degree of homology but with similarity to IncA, suggesting similar or altered functions.

The tryptophan biosynthetic operon identified in C. trachomatis (trpA,
trpB, trpR) and trpC are significantly missing in the genome of C. pneumoniae. This represents the entire repertoire of genes associated with tryptophan biosynthesis identified in C. trachomatis. Seventeen genes flanking the C. trachomatis tryptophan operon were also not found in the genome of C. pneumoniae. This region is one of the largest deletions in the linked genomic segment and encompasses the 4HKD superfamily-encoding gene, including a family of proteins related to endonucleases and phospholipase D. These findings are important for the ability of Chlamydia to persist in the host and cause disease by inducing a potentially focused and persistent inflammatory response that may be essential for virulence. obtain.

The genome of C. pneumoniae contains 187,711 additional nucleotides compared to the genome of C. trachomatis and is not found in C. trachomatis.
The four coding sequences account for most of the increased genome size. 88 of these genes are found in blocks of> 10 genes (11-30 genes / block), 41 are single genes and the rest are paired with at least one other gene. ing. ~ 7 of all C. pneumoniae genes except C. trachomatis
Based on the observation that 0% have identifiable homologs in GenBank, more than 150 of the 214 genes should have homologs in GenBank,
Many are expected to be associated with functions. However, only 28 coding sequences have similarity with genes from other organisms. Thus, most of the genes (186 of 214) mutually exclusive with C. trachomatis and 60 of 70 C. trachomatis genes lacking identifiable homologues in C. pneumoniae
It has no detectable homologs for genes from other organisms. We expect that most unique genes are essential for the specific properties that define the unique biology, affinity and pathogenicity of C. trachomatis and C. pneumoniae. Furthermore, this suggests that C. pneumoniae has a more unique biological (ie, toxic) potential than C. trachomatis. The ability of C. pneumoniae to be more invasive than C. trachomatis and to survive a wider range of host cell types is consistent with this hypothesis. Not all differences in biological competence can be associated with mutually exclusive genes. Although there is no identifiable ortholog in other organisms, one explanation for the significantly lower degree of homology between orthologous protein sequences of C. pneumoniae and C. trachomatis is that this set of proteins is In addition to being associated with the specific biological requirements for Chlamydia, this polymorphism can explain the unique biology between the two species. Determination of the genomic sequence from a representative of the C. psittaci group accurately represents those genes that are mutually exclusive and specific for each species.

The major functionally identifiable addition to the C. pneumoniae genome is a large extension of the gene encoding a new family of Chlamydia polymorphic membrane proteins (Pmps), 22% of the increased coding capacity. Only shows. The C. trachomatis gene has nine pmp genes, while, surprisingly, the C. pneumoniae genome contains 21 pmp genes. Most of these genes have three independent (stand-
alone) It appears to be amplified in two regions of the genome with the gene. Interestingly, one of the independent genes is most closely related to the C. trachomatis pmpD, the only independent pmp gene in the C. trachomatis genome, which is located with the same relative genomic background. This suggests an essential and conserved function for this paralog. The six Pmp encoding genes are probably not functional. 5 include expected code frame-shifts and one is truncated. The amplification of this gene family and confidently predicted frame-shifts suggest specific molecular mechanisms to increase function or antigen diversity. C. related to this protein family.
At least one protein in psittaci has been exposed to the Chlamydia surface, but the biological role of this protein family remains enigmatic.

While it is not possible to assign functions to most of the unique C. pneumoniae genes, some have significant similarities to genes from other organisms. Functions could be assigned for genes encoding GMP synthetase, IMP dehydrogenase, UMP synthase, uridine kinase, biotin synthase pathway protein, methylthioadenosine nucleosidase, DNA glycosylase and aromatic amino acid hydroxylase. Thus, a complete pathway for biotin biosynthesis has been identified. Additional purine and pyrimidine recycling pathway genes reflect metabolic limitations in one of the cell types, possibly infecting C. pneumoniae or causing differences in the ability to carry C. pneumoniae precursor nucleosides or nucleotides .

The addition of aromatic amino acid hydroxylases in C. pneumoniae is of particular interest in view of the lack of tryptophan biosynthesis genes and the inability to synthesize other amino acids, including phenylalanine. Aromatic amino acid hydroxylases include three distinct enzymes that function to receptively oxidize phenylalanine to tyrosine, tyrosine to dopa, and tryptophan to 5-hydroxytryptophan and serotonin. Chlamydia proteins are similar to this family of proteins and are very closely related to tryptophan hydroxylase, but their specific function could not be predicted with certainty.
We hypothesize that it may be related to the toxicity of C. pneumoniae. Tryptophan hydroxylase has not been previously identified in bacteria, and the origin of the Chlamydia gene appears to be from eukaryotes. The functional role of aromatic amino acid hydroxylases for C. pneumoniae is related to the unique intracellular biology of this organism and may represent a key contribution to C. pneumoniae persistence and virulence.

The examples and embodiments described herein are for illustrative purposes only, and various modifications or variations in light of them will be suggested to those skilled in the art which are It is understood that the scope is to be included within the scope as well as the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Table 1 provides a functional assignment of genomic sequences encoding C. pneumoniae non-proteins. Table 2 provides a functional assignment of the protein coding sequence. Table 3 provides the amino acid sequence of the protein corresponding to the coding sequence.

[0106]

[Table 1]

[0107]

[Table 2]

[0108]

[Table 3]

[0109]

[Table 4]

[0110]

[Table 5]

[0111]

[Table 6]

[0112]

[Table 7]

[0113]

[Table 8]

[0114]

[Table 9]

[0115]

[Table 10]

[0116]

[Table 11]

[0117]

[Table 12]

[0118]

[Table 13]

[0119]

[Table 14]

[0120]

[Table 15]

[0121]

[Table 16]

[0122]

[Table 17]

[0123]

[Table 18]

[0124]

[Table 19]

[0125]

[Table 20]

[0126]

[Table 21]

[0127]

[Table 22]

[0128]

[Table 23]

[0129]

[Table 24]

[0130]

[Table 25]

[0131]

[Table 26]

[0132]

[Table 27]

[0133]

[Table 28]

[0134]

[Table 29]

[0135]

[Table 30]

[0136]

[Table 31]

[0137]

[Table 32]

[0138]

[Table 33]

[0139]

[Table 34]

[0140]

[Table 35]

[0141]

[Table 36]

[0142]

[Table 37]

[0143]

[Table 38]

[0144]

[Table 39]

[0145]

[Table 40]

[0146]

[Table 41]

[0147]

[Table 42]

[0148]

[Table 43]

[0149]

[Table 44]

[0150]

[Table 45]

[0151]

[Table 46]

[0152]

[Table 47]

[0153]

[Table 48]

[0154]

[Table 49]

[0155]

[Table 50]

[0156]

[Table 51]

[0157]

[Table 52]

[0158]

[Table 53]

[0159]

[Table 54]

[0160]

[Table 55]

[0161]

[Table 56]

[0162]

[Table 57]

[0163]

[Table 58]

[0164]

[Table 59]

[0165]

[Table 60]

[0166]

[Table 61]

[0167]

[Table 62]

[0168]

[Table 63]

[0169]

[Table 64]

[0170]

[Table 65]

[0171]

[Table 66]

[0172]

[Table 67]

[0173]

[Table 68]

[0174]

[Table 69]

[0175]

[Table 70]

[0176]

[Table 71]

[0177]

[Table 72]

[0178]

[Table 73]

[0179]

[Table 74]

[0180]

[Table 75]

[0181]

[Table 76]

[0182]

[Table 77]

[0183]

[Table 78]

[0184]

[Table 79]

[0185]

[Table 80]

[0186]

[Table 81]

[0187]

[Table 82]

[0188]

[Table 83]

[0189]

[Table 84]

[0190]

[Table 85]

[0191]

[Table 86]

[0192]

[Table 87]

[0193]

[Table 88]

[0194]

[Table 89]

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12P 21/02 C5 / 10 C12Q 1/68 A C12P 21/02 C12P 21/08 C12Q 1 / 68 C12N 15/00 ZNAA // C12P 21/08 5/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT , LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM) , KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM , HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA , ZW (72) Inventor Kalman, Sue United States of America, California 95070, Saratoga, Sixth Street 14761 (72) Inventor of Davis, Ronald United States of America, California 94305, Palo Alto, Kingsley Avenue 433 F-term (reference) 4B024 AA01 AA13 BA08 BA11 BA38 CA02 CA09 HA14 HA19 4B063 QA11 QA19 QQ08 QQ43 QR39 QR48 QR56 QR77 QS34 QX02 4B064 AG01 AG27 CA02 CA19 CA20 DA01 DA13 DA15 4B065 AA01Y AB01 CA44 CA46 4H010 AA11A76A

Claims (9)

[Claims] 1. An isolated nucleic acid encoding a C. pneumoniae protein shown in Table 3. 2. The isolated nucleic acid of claim 1, wherein said nucleic acid has the nucleotide sequence of the open reading frame of SEQ ID NO: 1. A probe comprising a hybridizable fragment of the isolated nucleic acid according to claim 2. 4. An isolated nucleic acid which hybridizes under stringency conditions to the nucleic acid sequence of claim 2. 5. A transcription initiation region functional in an expression host, a nucleic acid having the sequence of the isolated nucleic acid according to claim 1 under the transcriptional control of said transcription initiation region, and transcription functional in said expression host. An expression cassette containing a termination region. 6. A cell containing the expression cassette according to claim 5, which is integrated into the genome of the host cell as a part of an extrachromosomal element or as a result of introducing the expression cassette into the host cell, and the host. Cell progeny of the cell. 7. A method for producing a C. pneumoniae protein, comprising growing the cell of claim 6 so that the C. pneumoniae protein is expressed; and wherein the C. pneumoniae protein is free of other proteins. A method comprising isolating a Pneumoniae protein. 8. A purified polypeptide composition comprising at least 50% by weight of a protein present as a C. pneumoniae protein comprising the amino acid sequence of claim 1. 9. A monoclonal antibody that specifically binds to the polypeptide according to claim 8.