JP2003511013A - BASB132 polypeptides and polynucleotides from Moraxella catarrhalis - Google Patents

Abstract

(57) SUMMARY The present invention provides BASB132 polypeptides and polynucleotides encoding BASB132 polypeptides and methods for producing such polypeptides by recombinant techniques. The invention further provides diagnostic, prophylactic, and therapeutic uses.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to polynucleotides (referred to herein as "BASB132 polynucleotides") and polypeptides encoded thereby (herein "BASB132" or "B ASB132").
ASB132 polypeptide "), a recombinant material and a method for producing the same. In another aspect, the invention relates to methods of using the polypeptides and polynucleotides, including vaccines against bacterial infections. In a further aspect the invention relates to a diagnostic assay for detecting infection of a particular pathogen.

BACKGROUND OF THE INVENTION Moraxella catarrhalis; (also referred Moraxella catarrhalis (Branhamella catarrhalis)) (Moraxella catarrhalis M.catarrhalis) is a Gram-negative bacterium frequently isolated from the human upper respiratory tract. This bacterium is responsible for several pathologies, mainly otitis media in infants and children, and pneumonia in the elderly. It is also the cause of sinusitis, nosocomial infections, and rare invasive diseases.

Otitis media is a childhood disease that is important both in terms of the number of cases and its possible sequelae. More than 3.5 million cases are recorded each year in the United States, with 80% of children having 3
It is estimated that he has had otitis at least once before his age (Kl
ein. JO (1994) Clin. Inf. Dis 19: 823). Left untreated or chronic, the disease can cause hearing loss that can be transient (if fluid builds up in the middle ear) or permanent (if the auditory nerves are damaged). There is. In infants, such deafness can also cause language learning delays.

Three types of bacteria are mainly isolated from the middle ear of children with otitis media. Streptococcus pneumoniae (S.pneumoniae),
Haemophilus influenzae (NTHi), and M. catarrhalis. These are present in 60-90% of cases. A review of recent studies has shown that S. pneumoniae and NTHi account for about 30% and M. catarrhalis for about 15% of cases of otitis media (Murphy, TF (1996) Microbiol. Rev. .60
: 267). Other bacteria may also be isolated from the middle ear (H. influenzae B
Type, S. pyogenes, etc.), much less frequently (less than 2% of cases).

Epidemiological data on pathogens found in the middle ear indicate that colonization of the upper respiratory tract is an absolute prerequisite for the development of otitis. However, other events are also required for disease to occur (Dickinson, DP et al. (1988) J. In.
fect. Dis. 158: 205, Faden, HL et al. (1991) Ann. Otorhinol. Laryngol. 100: 612
). They are of great importance because they trigger the migration of bacteria through the Eustachian tube to the middle ear, which in turn initiates the inflammatory process. Until now, those factors are unknown. It has been postulated that transient abnormalities of the immune system following viral infection may cause, for example, uncontrollability of airway colonization (Faden, HL et al. (1994) J
Infect. Dis. 169: 1312). Another explanation is that exposure to environmental factors causes more severe colonization in some children. This is because the continued presence of middle ear pathogens makes these children subsequently more susceptible to otitis media (Murphy, TF (1996) Microbiol. Rev. 60).
: 267).

The immune response to M. catarrhalis is not well characterized. Analysis of strains isolated from the infant's nasopharynx over time, tracking from 0 to 2 years of age, shows that infants often acquire and eliminate new strains. This indicates that an effective immune response against this bacterium is provided by colonized children (Faden, HL et al. (1994) J. Infect. Dis. 169: 1312).

Bactericidal antibodies have been identified in most of the adults examined (Chapman, AJ et al. (1985).
J. Infect. Dis. 151: 878). Strains of M. catarrhalis vary in their ability to tolerate serum bactericidal activity. In general, isolates from affected individuals are more resistant than those from simply colonized individuals (Hol, C et al. (1993) Lancet 341).
: 1281, Jordan, KL et al. (1990) Am. J. Med. 88 (Supplement 5A): 28S). Therefore, serum resistance can be considered as one of bacterial virulence. Opsonizing activity is found in the sera of children recovering from otitis media.

The antigen targeted by these various immune responses in humans is OMP B1.
, UspA1 and UspA2 (Chen D. et al. (1999), Infect. Immun. 67: 1310). This OMP B1 is a 84 kDa protein whose expression is regulated by iron, and the serum of a patient suffering from pneumonia (Sethi. S. et al. (1995) infect. Immun.
63: 1516) or serum of UspA1 and UspA2 (Chen D. et al. (1999), Infect. Immun. 6
7: 1310).

A few other membrane proteins present on the surface of M. catarrhalis have been characterized using biochemical techniques or for their potential relevance in the induction of protective immunity ( For a review, see Murphy, TF (1996) Microbiol. Rev. 60: 267). In the mouse pneumonia model, the presence of antibodies raised against some of them (UspA, CopB) favors faster clearance of lung infections. Another polypeptide (OMP CD) is highly conserved among M. catarrhalis strains and also P. aeruginosa (P
The homology with the porin of (seudomonas aeruginosa) has been shown, and the efficacy against this bacterium has been shown in an animal model.

The frequency of Moraxella catarrhalis infections has risen dramatically over the last few decades. This is due to the emergence of strains that are resistant to multiple antibiotics and an increasing number of populations with weakened immune systems. It is no longer unusual to isolate Moraxella catarrhalis strains that are resistant to some or all standard antibiotics. This phenomenon has created an unprecedented need and demand for pharmaceuticals for new antimicrobial agents, vaccines, drug screening methods, and diagnostic tests for these organisms.

SUMMARY OF THE INVENTION The present invention relates to BASB132, particularly BASB132 polypeptides and BASB132 polynucleotides, recombinant materials and methods of producing the same. In another aspect, the invention relates to methods of using said polypeptides and polynucleotides, especially including the prevention and treatment of microbial diseases. In a further aspect, the invention provides a diagnostic assay for detecting diseases and conditions associated with such infections, such as BA.
An assay for detecting the expression or activity of an SB132 polynucleotide or polypeptide.

Various changes and modifications within the spirit and scope of the disclosed invention will be readily apparent to those skilled in the art from reading the following description and from reading other parts of the disclosure. Become.

DESCRIPTION OF THE INVENTION The present invention relates to BASB132 polypeptides and polynucleotides, as described in more detail below. In particular, the present invention relates to Moraxella catarrhalis BASB13.
2 of polypeptides and polynucleotides. These are related to the hypothetical outer membrane protein YtfN of Escherichia coli by sequence homology. The invention is particularly directed to SEQ ID NO: 1 or 3 and SEQ ID NO: 2 or 4 respectively.
To BASB132 having the nucleotide and amino acid sequences shown in. One of the embodiments of the present invention is that the sequences listed as “DNA” in the sequence listing below are understood by those skilled in the art to be effectively used for polynucleotides including ribopolynucleotides in general. It will be understood that this is only an example of.

Polypeptides In one embodiment of the invention, the Moraxella catarrhalis polypeptides referred to herein as "BASB132" and "BASB132 polypeptides", and biological, diagnostic, prophylactic, clinical or therapeutic. Useful variants thereof, as well as compositions containing them, are provided.

Furthermore, the present invention provides (a) at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity to the amino acid sequence shown in SEQ ID NO: 2 or 4. Most preferably an isolated polypeptide comprising an amino acid sequence having at least 97-99% identity or being completely identical, (b) to the polynucleotide sequence set forth in SEQ ID NO: 1 or 3, At least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, even more preferably at least 97-99% identity over the entire length shown in SEQ ID NO: 1 or 3, respectively. A polynucleotide encoded by an isolated polynucleotide containing a polynucleotide sequence that has or is completely identical. Peptide, at least 85% identity to the amino acid sequence of (c) SEQ ID NO: 2 or 4,
Preferably comprising at least 90% identity, more preferably at least 95% identity, even more preferably at least 97-99% identity or a polynucleotide sequence encoding a polypeptide which is completely identical. A polypeptide encoded by the isolated polynucleotide is provided.

The BASB132 polypeptide shown in SEQ ID NO: 2 or 4 is a BASB132 polypeptide obtained from the strain Mc2931 of Moraxella catarrhalis (ATCC 43617).

The present invention also provides an immunogenic fragment of a BASB132 polypeptide, ie, a sequence of said polypeptides which has the same or substantially the same immunogenic activity as the BASB132 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4. Provide the part you did. That is, the fragment, optionally conjugated to a carrier, is capable of eliciting an immune response that recognizes the BASB132 polypeptide. Such immunogenic fragments include, for example, BASB132 polypeptides lacking an N-terminal leader sequence and / or a transmembrane domain and / or a C-terminal anchor domain. In a preferred embodiment, the immunogenic fragment of BASB132 of the invention has at least 85% identity, preferably at least 90% identity to the polypeptide sequence of SEQ ID NO: 2 or 4 over the entire length of SEQ ID NO: 2 or 4. , More preferably at least 95% identity, and most preferably at least 97-99% identity to substantially all extracellular domains of the polypeptide.

A fragment is a polypeptide having an amino acid sequence that is partially but not entirely identical to any amino acid sequence of any polypeptide of the invention.
With respect to BASB132 polypeptides, a fragment may be "freestanding," or may be a single polypeptide in a larger polypeptide, most preferably a single larger polypeptide, in which the fragment forms part or region thereof. May be included as a continuous region of.

A preferred fragment is a truncation polypeptide having, for example, a portion of the amino acid sequence of SEQ ID NO: 2 or 4 or a variant thereof (eg, a contiguous series of residues including amino-terminal and / or carboxy-terminal amino acid sequences). Including. Degraded forms of the polypeptides of the invention produced by or within the host cell are also preferred. More preferred are α-helix and α-helix forming region, β
Sheet and β sheet forming region, turn and turn forming region, coil and coil forming region, hydrophilic region, hydrophobic region, α amphiphilic region, β amphiphilic region, flexible region, surface forming region, substrate binding region, And high antigenicity index (high anti
A fragment characterized by structural and functional properties, such as a fragment containing the genic index) region.

Further preferred fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids derived from the amino acid sequence of SEQ ID NO: 2 or 4. Or at least 15 truncated or deleted from the amino acid sequence of SEQ ID NO: 2 or 4
, An isolated polypeptide comprising an amino acid sequence of 20, 30, 40, 50 or 100 contiguous amino acids.

Fragments of the polypeptides of the present invention can be used to produce the corresponding full-length polypeptide by peptide synthesis. Therefore, these fragments can be used as intermediates to produce the full-length polypeptides of the invention.

Particularly, a mutant in which several, 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 amino acids are substituted, deleted or added in any combination is preferable. .

The polypeptide or immunogenic fragment of the invention may be in the form of a “mature” protein or may be part of a larger protein such as a precursor or fusion protein. Often, it is advantageous to include additional amino acid sequences, which may include secretory or leader sequences, prosequences, sequences useful for purification such as multiple histidine residues, or during recombinant production. Additional sequences are included to ensure stability. In addition, the addition of foreign polypeptides or lipid tails or polynucleotide sequences to increase the immunogenic potential of the final molecule is also contemplated.

In one embodiment, the present invention provides a polypeptide of the present invention or a fragment thereof, and various portions of the constant region of the H chain or L chain of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). And a genetically engineered soluble fusion protein comprising: As an immunoglobulin, human IgG, particularly IgG1
The constant region of the H chain of is preferred, in which case the fusion occurs in the hinge region. In a particular example, the Fc portion can be easily removed by incorporating a cleavage sequence that can be cleaved by blood coagulation factor Xa.

Furthermore, the present invention relates to methods of genetically engineering these fusion proteins and their use in drug screening, diagnosis and therapy. Also, a further aspect of the present invention relates to a polynucleotide encoding such a fusion protein. Examples of fusion protein technology are international patent applications WO94 / 29458 and WO94 / 22914
Can be found in.

By chemically linking the protein or expressing it as a recombinant fusion protein, the protein is produced in the expression system at increased levels relative to the non-fusion protein. The fusion partner helps to provide a T helper epitope, preferably a T helper epitope recognized by humans (immunological fusion partner), or helps to express said protein in a higher yield than the original recombinant protein. (Expression enhancer). Preferably, the fusion partners are both immunological fusion partners and expression enhancer partners.

The fusion partner may be Haemophilus influenza.
e) derived protein D and influenza virus derived non-structural protein NS
Contains 1 (hemagglutinin). Another fusion partner is the protein known as LytA. Preferably, the C-terminal part of the molecule is used. LytA is N-acetyl
-L-alanine amidase amidase LytA (encoded by the lytA gene [G
ene. 43 (1986) page 265-272]), that is, Streptococcus pneumoniae that synthesizes an autolytic enzyme that specifically decomposes a specific bond in the peptidoglycan skeleton.
(Streptococcus pneumoniae). The C-terminal domain of the LytA protein is involved in affinity for choline or several choline analogues such as DEAE. This property was used for the development of an E. coli C-LytA expression plasmid useful for expressing the fusion protein. Purification of a hybrid protein containing the C-LytA fragment at its amino terminus is described in Biotechnology: 10. (1992) pages 795-798. The repeat portion found at the C-terminus of the LytA molecule beginning at residue 178 (
For example residues 188-305) can be used.

The present invention also includes variants of the above polypeptides, ie, polypeptides that differ from the reference polypeptide by conservative amino acid substitutions (where one residue is replaced by another residue of similar nature). include. Typical such substitutions are Ala, Val, Le
Between u and Ile; between Ser and Thr; between acidic residues Asp and Glu; between Asn and Gln; between basic residues Lys and Arg; or between aromatic residues Phe and Tyr.

The polypeptides of the present invention can be produced by any suitable method. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Be done. Means for producing such polypeptides are well understood in the art.

Most preferably, the polypeptide of the invention is from Moraxella catarrhalis, but can preferably be obtained from other organisms within the same taxonomic genus. The polypeptides of the invention can also be obtained from organisms of the same taxonomic family or class, for example.

Polynucleotides It is an object of the present invention to provide a polynucleotide encoding a BASB132 polypeptide, particularly a polynucleotide encoding the polypeptide herein designated BASB132.

In a particularly preferred embodiment of the invention said polynucleotide comprises a region encoding a BASB132 polypeptide comprising the sequence set forth in SEQ ID NO: 1 or 3, including full length genes or variants thereof. .

The BASB132 polynucleotide shown in SEQ ID NO: 1 or 3 is a BASB132 polynucleotide derived from the strain Mc2931 of Moraxella catarrhalis (ATCC 43617).

In a further aspect of the invention there is provided an isolated nucleic acid molecule encoding and / or expressing a BASB132 polypeptide and polynucleotide, particularly a Moraxella catarrhalis BASB132 polypeptide and polynucleotide. Is, for example, unprocessed RNA, ribozyme RNA, mRNA, c
DNA, genomic DNA, B- and Z-DNA are included. Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and variants thereof, and compositions containing them.

Another aspect of the invention is BASB13 having the deduced amino acid sequence of SEQ ID NO: 2 or 4.
It relates to an isolated polynucleotide containing at least one full-length gene encoding a 2 polypeptide, and a polynucleotide closely related to the polynucleotide and variants thereof.

Another particularly preferred embodiment of the invention is a BASB132 polypeptide from Moraxella catarrhalis comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or 4, or a variant thereof.

Using the information provided herein, polynucleotides of the invention encoding BASB132 polypeptides, such as the polynucleotide sequences set forth in SEQ ID NO: 1 or 3, may be labeled using standard cloning and screening methods. , For example, using the Catlin cells of Moraxella catarrhalis as the starting material, the
A fragment can be obtained by a method such as cloning and sequencing, and then obtaining a full-length clone. For example, to obtain a polynucleotide sequence of the invention, such as the polynucleotide sequences set forth in SEQ ID NOs: 1 or 3, typically a chromosomal DNA clone of Moraxella catarrhalis catrin in E. coli or some other suitable host is used. The library is probed with radiolabeled oligonucleotides, preferably those of 17-mer or longer, derived from a partial sequence. Then, the DNA of the probe
Clones carrying the same DNA as can be identified using stringent hybridization conditions. Therefore, individual clones identified by hybridization can be sequenced using sequencing primers designed from the original polypeptide or polynucleotide sequence to subsequently extend the polynucleotide sequence in both directions to produce the full length gene sequence. It is possible to determine Such sequencing is conveniently carried out using denatured double-stranded DNA prepared, for example, from plasmid clones. Suitable techniques are Maniatis, T., Fritsch, EF and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Second Edition; Cold Spri.
ng Harbor Laboratory Press, Cold Spring Harbor, New York (1989) (especially Screening By Hybridization 1.90 and Sequencing Denatured Do.
See uble-Stranded DNA Templates 13.70). In addition, direct genomic DNA
The full length gene sequence can also be obtained by sequencing. Each polynucleotide shown in SEQ ID NO: 1 or 3 which is a specific example of the present invention was found in a DNA library derived from Moraxella catarrhalis.

Furthermore, the DNA sequence shown in SEQ ID NO: 1 or 3 is a rough approximation of the amino acid sequence shown in SEQ ID NO: 2 or 4 having an estimated molecular weight that can be calculated using molecular weight values of amino acid residues well known to those skilled in the art. It contains an open reading frame that encodes a protein with amino acid residues.

The polynucleotide set forth in SEQ ID NO: 1 (between the start codon at nucleotide number 1 and the stop codon beginning at nucleotide number 5017 of SEQ ID NO: 1) encodes the polypeptide of SEQ ID NO: 2.

The polynucleotide set forth in SEQ ID NO: 3 (between the start codon at nucleotide number 2042 and the stop codon beginning at nucleotide number 5014 of SEQ ID NO: 3) encodes the polypeptide of SEQ ID NO: 4.

In a further embodiment, the present invention provides an isolated polynucleotide comprising the polynucleotide sequence of (a) or (b) below, or consisting of the polynucleotide sequence of (a) or (b) below: (A) at least 85% identity, preferably at least 90% identity, and more preferably over the entire length shown in SEQ ID NO: 1 or 3, to the polynucleotide sequence shown in SEQ ID NO: 1 or 3, respectively. A polynucleotide sequence which has at least 95% identity, more preferably at least 97-99% identity or is completely identical, or (b) to the amino acid sequence of SEQ ID NO: 2 or 4, respectively, At least 85% identity over the entire length of SEQ ID NO: 2 or 4, preferably at least 90
A polynucleotide sequence encoding a polypeptide having a% identity, more preferably at least 95% identity, even more preferably at least 97-99% identity or 100% exact identity.

A polynucleotide encoding a polypeptide of the invention, including homologues and orthologs from species other than Moraxella catarrhalis, is SEQ ID NO: 1 or 3.
Under a stringent condition (for example, 4
Screening appropriate libraries (using temperatures ranging from 5 to 65 ° C. and SDS concentrations of 0.1 to 1%) and isolating full-length genes and / or genomic clones containing the polynucleotide sequences. Can be obtained.

The present invention provides a polynucleotide sequence that is identical over its entire length to the coding sequence (open reading frame) in SEQ ID NO: 1 or 3.
Also according to the present invention, the coding sequence of the mature polypeptide or fragment thereof alone as well as another coding sequence (eg, leader or secretory sequence, pre- or pro-).
Or a sequence encoding the prepro-protein sequence) is provided in its reading frame for the mature polypeptide or fragment thereof. Polynucleotides of the invention also include, for example and without limitation, at least one 5'and 3'non-coding sequence, eg, a transcribed but untranslated sequence, a termination signal (eg, rho-dependent and rho-dependent. (Independent termination signal), a ribosome binding site, Kozak sequences, mRNA stabilizing sequences, introns, and polyadenylation signals. The polynucleotide sequence may also include additional coding sequences that encode additional amino acids. For example, a marker sequence that facilitates purification of the fusion polypeptide can be encoded. In a particular embodiment of the invention, the marker sequence is provided by the pQE vector (Qiagen, Inc.) and Gentz et al., Proc. Natl. Acad. Sci. U.
Hexa-histidine peptide, or HA peptide tag (Wilson et al., Cell 37: 767 (1984)), as described in SA 86: 821-824 (1989), both polypeptide sequences fused to them. May be useful in purifying The polynucleotide of the present invention also includes, but is not limited to, a polynucleotide containing a structural gene and a gene expression control sequence to which the gene is related in its natural state.

The nucleotide sequence encoding the BASB132 polypeptide of SEQ ID NO: 2 or 4 is the polypeptide coding sequence contained in nucleotide numbers 1 to 5016 shown in SEQ ID NO: 1 or nucleotide numbers 1 to 5016 shown in SEQ ID NO: 3. May be identical to the respective polypeptide coding sequences contained in. Alternatively, it may also be the sequence encoding the polypeptide of SEQ ID NO: 2 or 4 as a result of the redundancy (degeneracy) of the genetic code.

As used herein, the term “polynucleotide encoding a polypeptide” refers to a polypeptide of the invention, particularly a bacterial polypeptide, more particularly SEQ ID NO: 2.
Or a polypeptide of Moraxella catarrhalis having the amino acid sequence shown in 4
BASB132, including a polynucleotide comprising a sequence that encodes BASB132. The term also refers to a single continuous or discontinuous region encoding the polypeptide (e.g., integrated phage, integrated insert sequence, integrated vector sequence,
A polynucleotide that has been interrupted by an integrated transposon sequence or by RNA editing or genomic DNA rearrangement), along with additional regions that may also contain coding and / or non-coding sequences.

The present invention further relates to variants of the polynucleotides described herein that encode variants of the polypeptide having the deduced amino acid sequence of SEQ ID NO: 2 or 4. Fragments of the polynucleotides of the invention can be used, for example, to synthesize full length polynucleotides of the invention.

In a further particularly preferred embodiment, a polynucleotide encoding a BASB132 variant having the amino acid sequence of the BASB132 polypeptide of SEQ ID NO: 2 or 4, comprising several, 2-3, 5-10, There are polynucleotides in which 1-5, 1-3, 2, 1, or 0 amino acid residues are substituted, modified, deleted and / or added in any combination. Of these, particularly preferred is BASB132.
Silent substitutions, additions and deletions that do not alter the properties and activities of the polypeptide.

In a further preferred embodiment of the present invention, a polynucleotide having at least 85% identity over its entire length to a polynucleotide encoding a BASB132 polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or 4. , As well as polynucleotides complementary to such polynucleotides. Or BASB
Most highly preferred are polynucleotides that comprise a region of at least 90% identity over its entire length to a polynucleotide encoding a 132 polypeptide and its complementary polynucleotide. In this regard, as above, polynucleotides having at least 95% identity over their entire length are particularly preferred. Further, those having at least 97% identity among polynucleotides having at least 95% identity are more preferred, those with at least 98% and at least 99% identity are particularly preferred, and at least 99%. Those with% identity are most preferred.

In a preferred embodiment, a polynucleotide encoding a polypeptide having substantially the same biological function or activity as the mature polypeptide encoded by the DNA shown in SEQ ID NO: 1 or 3. There is.

In accordance with certain preferred embodiments of the invention, there are provided polynucleotides that hybridize under particularly stringent conditions to BASB132 polynucleotide sequences, such as the polynucleotides set forth in SEQ ID NO: 1 or 3.

The invention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein. In this regard, the present invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As used herein, the terms "stringent conditions" and "stringent hybridization conditions" refer to sequences having at least 95% identity, and preferably at least 97% identity. It means that hybridization occurs only in. Specific examples of stringent hybridization conditions include: 50% formamide, 5 ×
SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.
6) Incubate overnight at 42 ° C. in a solution containing 5 × Denhardt's solution, 10% dextran sulfate and 20 μg / ml denatured and sheared salmon sperm DNA, then hybridize the support in 0.1 × SSC at about 65 ° C. It is to wash. Hybridization and wash conditions are well known, see Sambrook et al., Molecular Clonin.
g: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY, (1989), especially chapter 11. Also, solution hybridization can be used with the polynucleotide sequences provided by the invention.

The present invention also uses a probe having the polynucleotide sequence shown in SEQ ID NO: 1 or 3 or a sequence thereof, and a suitable library containing the complete gene under stringent hybridization conditions. Is screened for the polynucleotide sequence shown in SEQ ID NO: 1 or 3, and a polynucleotide consisting of or containing the polynucleotide sequence obtained by isolating the polynucleotide sequence is provided. Fragments useful for obtaining such polynucleotides include, for example, the probes and primers fully described elsewhere herein.

As discussed elsewhere herein with respect to the polynucleotide assays of the present invention, for example, the polynucleotides of the present invention are used as hybridization probes for RNA, cDNA and genomic DNA to encode BASB132. Full length cDNA and genomic clones can be isolated, or cDNA and genomic clones of another gene with high identity, especially high sequence identity to the BASB132 gene can be isolated. Such probes usually contain at least 15 nucleotide residues or base pairs. Preferably, such probes have at least 30 nucleotide residues or base pairs, and may have at least 50 nucleotide residues or base pairs. Particularly preferred probes are those having at least 20 nucleotide residues or base pairs and less than 30 nucleotide residues or base pairs.

The coding region of the BASB132 gene can be isolated by screening using the DNA sequence shown in SEQ ID NO: 1 or 3 to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to the sequence of the gene of the present invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which member of the library the probe hybridizes to. You can decide.

There are several methods well known and available to those of skill in the art for obtaining full length DNA or for extending short DNA, for example, the cDNA terminal rapid amplification method (RA).
CE) based methods (eg Frohman et al., PNAS USA 85, 8998-9002, 1
988). Recent improvements in the above techniques, eg, as exemplified by Marathon ^™ technology (Clontech Laboratories Inc.), have greatly facilitated the search for longer cDNAs. With Marathon ^TM technology, mRNA extracted from a given tissue
CDNA is made from and the "adapter" sequences are ligated to each end. Nucleic acid amplification (PCR) is then carried out using a combination of gene-specific and adapter-specific oligonucleotide primers to amplify the "deletion"5'end of the DNA. Then, a "nested" primer, i.e., a primer designed to anneal within the amplification product (typically an adapter-specific primer and a selected gene that anneal further 3'to the adapter sequence). The PCR reaction is repeated using a gene-specific primer that anneals to the 5 ′ side of the sequence. Then, the product of this reaction was analyzed by DNA sequencing, and the product was analyzed by
Full-length DNA can be constructed by directly binding to A to form a complete sequence or by performing another full-length PCR using new sequence information for 5 ′ primer design.

The polynucleotides and polypeptides of the present invention can be used as described further herein for polynucleotide assays, eg, for discovering therapeutic and diagnostic agents for diseases, particularly human diseases, It can be used as a research reagent as well as a material.

A polynucleotide of the invention, which is an oligonucleotide derived from the sequence of SEQ ID NO: 1 or 3-8, is used in the methods described herein, preferably PCR, to identify a polynucleotide of the invention. It can be confirmed whether or not all or part of the bacterium is transcribed by bacteria in the infected tissue. It will also be appreciated that such sequences will be useful in diagnosing the stage of infection and the type of infection the pathogen has reached.

The invention also adds additional amino- or carboxy-terminal amino acids to the mature protein, or (for example, if the mature form has more than one polypeptide chain) within the mature protein. A polynucleotide encoding the polypeptide is provided. Such sequences play some role in processing the protein from its precursor to its mature form, permitting transport of the protein, extending or shortening the half-life of the protein, or especially for assay or production. The manipulation of proteins can be facilitated. In vivo, in general, the additional amino acids can be removed from the mature protein by intracellular enzymes.

For each and every polynucleotide of the invention there is provided a polynucleotide complementary thereto. These complementary polynucleotides are preferably sufficiently complementary to each of the polynucleotides with which they are complementary.

The precursor protein may be an inactive form of the polypeptide when the mature form of the polypeptide is fused to one or more prosequences. Removal of the prosequence typically activates such inactive precursors. Some or all of the prosequences may be removed prior to activation. Generally, such precursors are called proproteins.

In addition to the standard symbols A, G, C, T / U for nucleotides, the term “N” can also be used in describing certain polynucleotides of the invention. "N"
Means that any of the four DNA or RNA nucleotides is found at its indicated position in the DNA or RNA sequence. However, when used in combination with adjacent nucleotide positions or read in the correct reading frame, N is preferably not a nucleic acid that has the effect of producing an early termination codon within such reading frame.

In summary, the polynucleotide of the present invention is a mature protein, a mature protein to which a leader sequence is added (also referred to as a preprotein), or a precursor of a mature protein having one or more prosequences which are not the preprotein leader sequence. , Or a preproprotein (usually a precursor of a proprotein having one or more prosequences removed during the processing step that produces the active and mature forms of the polypeptide, and a leader sequence). You may

According to an aspect of the invention there is provided the use of the polynucleotides of the invention for therapeutic or prophylactic purposes, in particular in genetic immunization.

The use of the polynucleotides of the invention in genetic immunization preferably involves direct intramuscular injection of plasmid DNA (Wolff et al., Hum Mol Genet (1992) 1: 363,
Manthorpe et al., Hum. Gene Ther. (1983) 4: 419), delivery of DNA complexed with a particular protein carrier (Wu et al., J Biol Chem. (1989) 264: 16985), DNA and calcium phosphate. Coprecipitation (Benvenisty and Reshef, PNAS USA, (1986) 83: 9551), D
Encapsulation of NA in various forms of liposomes (Kaneda et al., Science (1989) 243: 375), particle bombardment (Tang et al., Nature (1992) 356: 152, Eisenbraun.
Et al., DNA Cell Biol (1993) 12: 791) as well as in vivo infection with cloned retroviral vectors (Seeger et al., PNAS USA (1984) 81: 5849).

Vector, Host Cell, Expression System The present invention relates to a vector containing the polynucleotide of the present invention, a host cell genetically engineered with the vector of the present invention, and production of the polypeptide of the present invention by a recombinant method. Cell-free translation systems can also be used to produce proteins of this type using RNA derived from the DNA constructs of the invention.

Recombinant polypeptides of the invention can be prepared from genetically engineered host cells containing expression systems by techniques well known to those of skill in the art. Therefore, in a further aspect, the invention relates to expression systems comprising the polynucleotides of the invention, host cells genetically engineered with such expression systems, as well as the production of the polypeptides of the invention by recombinant methods.

For recombinant production of the polypeptides of the invention, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
Introduction of polynucleotides into host cells is described, for example, in Davis et al., BASIC METHODS IN
MOLECULAR BIOLOGY (1986) and Sambrook et al., MOLECULAR CLONING: A LABORAT
ORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Ha
Methods described in many standard laboratory manuals, such as rbor, NY (1989),
For example, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic i.
ntroduction) and infection.

As a representative example of a suitable host, bacterial cells (eg Streptococcus, Staphylococcus, Enterococcus, Escherichia coli, Streptomyces, Cyanobacteria, Bacillus subtilis, Neisseria meningitidis and Moraxella catarrhalis), Fungal cells (eg yeast Kluveromyces
ces), Saccharomyces, and basidiomycetes, Candida albican.
s) and Aspergillus), insect cells (eg Drosophila S2, Spodoptera Sf9), animal cells (eg CHO, COS, HeLa, C127, 3T3, BH).
K, 293, CV-1 and Bowes melanoma cells) and plant cells (eg cells of angiosperms or angiosperms).

A wide variety of expression systems can be used to produce the polypeptides of the invention. As such a vector, in particular, a vector derived from chromosome, episome and virus, for example, bacterial plasmid derived, bacteriophage derived, transposon derived, yeast episome derived, insertion factor derived, yeast chromosome element derived, virus (eg: baculovirus, Vectors derived from papovaviruses such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, picornavirus, retroviruses and alphaviruses), and vectors derived from combinations thereof, such as cosmids and phagemids. Some are derived from different plasmids and genetic elements of bacteriophage. Expression system constructs may include control regions that cause as well as regulate expression. In general, any system or vector suitable for maintaining, increasing or expressing a polynucleotide in a host, and / or any system or vector suitable for expressing a polypeptide is for expression in this regard. Can be used for For example, Sa
The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as those described in mbrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (supra).

In the case of recombinant expression systems in eukaryotic cells, appropriate secretion signals are expressed in order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. It can be incorporated into a polypeptide. These signals may be endogenous or heterologous to the polypeptide.

To recover and purify the polypeptides of the present invention from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity Well known methods can be used including chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, ionic metal affinity chromatography (i
on metal affinity chromatography (IMAC) is used for purification. When the polypeptide is denatured during intracellular synthesis, isolation and / or purification, well known techniques for refolding proteins can be used to restore the active conformation.

The expression system may also be a recombinant live microorganism such as a virus or bacterium. The gene of interest can be inserted into the genome of a recombinant live virus or bacterium. By inoculation or in vivo infection with this survival vector, i
It leads to n vivo expression and induction of immune responses. Examples of viruses and bacteria used for this purpose include poxvirus (e.g., vaccinia virus, fowlpox virus, canarypox virus), alphavirus (sindbis virus, semliki forest virus, Venezuelan equine encephalitis virus), adenovirus. There are viruses, adeno-associated virus, picornavirus (poliovirus, rhinovirus), herpesvirus (varicella-zoster virus, etc.), Listeria, Salmonella, Shigella, Neisseria, BCG. These viruses and bacteria can be toxic or attenuated by various methods to obtain live vaccines. Such live vaccines also form part of the present invention.

[0073] Diagnostic assays, prognostic assays, serum typing (serotyping) assay and mutation assays The present invention also relates to the use as diagnostic reagents BASB132 polynucleotides and polypeptides of the present invention. The detection of BASB132 polynucleotides and / or polypeptides in eukaryotes (especially mammals, especially humans) results in diseases,
Diagnostic methods are provided for diagnosing the stage of a disease or the response of an infectious organism to a drug. Eukaryotes (especially mammals, especially humans, especially humans infected with or suspected of having been infected with organisms containing the BASB132 gene or protein) can be isolated at the nucleic acid level by a variety of well-known techniques and methods shown herein. Alternatively, it can be detected at the amino acid level.

Polypeptides and polynucleotides for prognosis, diagnosis or other analysis can be obtained from suspected infected individuals and / or biomaterials of infected individuals. Polynucleotides from any of these sources, especially DNA or RNA, may be used directly for detection or may be enzymatically amplified using PCR or other amplification methods prior to analysis. . RNA (particularly mRNA), cDNA and genomic DNA can also be used in the same manner. Amplification can be used to characterize the species or strain of an infectious or resident organism present in an individual by genotyping the selected polynucleotides of the organism. Deletions and insertions are related organisms (preferably organisms of the same genus and of different species or of the same species and of different strains)
It can be detected by the change in the size of the amplification product when compared with the genotype of the reference sequence selected from. Point mutations can be identified by hybridizing amplified DNA to labeled BASB132 polynucleotide sequences. Perfectly or significantly matched sequences and incompletely or more significantly mismatched duplexes detect differences in melting temperature or refolding kinetics for DNA or RNA, respectively, by DNase or RNase digestion. You can distinguish by doing. Differences in the polynucleotide sequence can also be detected by changes in the electrophoretic mobility of the polynucleotide fragment on the gel when compared to the reference sequence. This can be done with or without denaturing agents. Polynucleotide differences can also be detected by direct DNA or RNA sequencing (eg, Myers et al., Science 23).
0: 1242 (1985)). Sequence changes at specific positions can also be confirmed by nuclease protection assays (eg, RNase, V1 and S1 protection assays) or chemical cleavage methods (Cotton et al., Proc. Natl. Acad. Sc).
i. USA 85: 4397-4401 (1985)).

In another embodiment, an array of oligonucleotide probes comprising BASB132 nucleotide sequences or fragments thereof is constructed to provide efficient screening for, eg, genetic mutations, serotypes, taxonomic classifications or identifications. It can be carried out. Array techniques are well known and generally applicable, and can be used to address questions in molecular genetics including gene expression, genetic linkage, and genetic diversity (eg, Chee et al., Science, 274: 610 (1996)).

Thus, in another embodiment, the present invention provides (a) the polynucleotide of the present invention (preferably the nucleotide sequence shown in SEQ ID NO: 1 or 3) or a fragment thereof, (b) the nucleotide sequence of (a) A complementary nucleotide sequence, (c) a polypeptide of the invention (preferably a polypeptide of SEQ ID NO: 2 or 4) or a fragment thereof, or (d) a polypeptide of the invention (preferably of SEQ ID NO: 2 or 4) An antibody against a polypeptide).

It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial constituent. Such a kit is useful for diagnosing a disease or susceptibility to a disease.

The present invention also relates to the use of the polynucleotide of the present invention as a diagnostic agent. A polynucleotide of the invention, preferably SEQ ID NO: 1, associated with a disease or pathogenicity
Alternatively, the detection of the polynucleotide variant shown in 3 may be carried out to determine the disease caused by underexpression, overexpression or modified expression of the polynucleotide, the prognosis of the progression of the disease, the stage of the disease, or the susceptibility to the disease. It will provide a diagnostic tool that can add to or make a diagnosis. Organisms with mutations in the polynucleotide, particularly infectious disease organisms, can be detected at the polynucleotide level by a variety of techniques described elsewhere herein.

Cells from organisms having mutations or polymorphisms (allelic variations) in the polynucleotides and / or polypeptides of the invention can also be detected at the polynucleotide or polypeptide level by various techniques. It can be, for example, serotyped. For example, using RT-PCR, RNA
Mutations in can be detected. Particularly preferably, RT-PCR is used in combination with an automatic detection system such as GeneScan. RNA, cDNA or genomic DNA can also be used for PCR for the same purpose. For example, using PCR primers complementary to the polynucleotide encoding the BASB132 polypeptide,
Mutations can be identified and analyzed.

The invention further provides that 1, 2, 3 or 4 removed from the 5'end and / or the 3'end.
A primer having 4 nucleotides is provided. These primers are especially
BASB132 DNA and / or RNA isolated from samples derived from individuals such as biomaterials
Can be used to amplify The primers can be used to amplify a polynucleotide isolated from an infected individual, which polynucleotide can then be subjected to various techniques for elucidation of the polynucleotide sequence. Thus, a mutation in a polynucleotide sequence is detected and used to diagnose and / or prognose an infection or the stage or progression of the infection,
Alternatively, the infectious agent can be serotyped and / or classified.

The invention further provides a method of diagnosing a disease, preferably a bacterial infection, more preferably an infection caused by Moraxella catarrhalis. The method includes measuring an increase in the expression level of the polynucleotide having the sequence shown in SEQ ID NO: 1 or 3 from a sample derived from an individual such as a biomaterial. BASB132
Increasing or decreasing the expression of the polynucleotide can be carried out by any method known in the art, such as a method for quantifying the polynucleotide, such as amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrophotometry, and other hybridization methods. It can be measured by one or the other.

Further, the diagnostic assays of the invention for detecting overexpression of BASB132 polypeptide as compared to a normal control tissue sample may be used to detect, for example, the presence or absence of infection. Those of skill in the art are familiar with assay methods that can be used to determine the level of BASB132 polypeptide in a sample (such as biomaterial) obtained from a host. Such assay methods include radioimmunoassays, competitive binding assays, Western blot analysis, antibody sandwich assays, antibody detection, ELISA assays and the like.

The polynucleotides of the present invention can be used as a component of a polynucleotide array, preferably a high density array or grid. These high density arrays are particularly useful for diagnostic and prognostic applications. For example, one set of spots, each containing a different gene, further comprising a polynucleotide of the invention, is probed, such as by hybridization or nucleic acid amplification, using probes obtained from or derived from the biological sample. Can be used to determine the presence of a particular polynucleotide sequence or related sequences in an individual. The presence of such sequences may indicate the presence of pathogens, especially Moraxella catarrhalis, and may be useful in diagnosing and / or prognosing a disease or disease progression. Sequence number 1
Or a grid comprising several variants of the polynucleotide sequence shown in 3 is preferred. Also preferred is a grid comprising some variants of the polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 2 or 4.

Antibodies The polypeptides and polynucleotides of the invention, or variants thereof, or cells expressing them are used as immunogens to produce antibodies immunospecific for the polypeptides or polynucleotides, respectively. be able to. The term "immunospecific" means that the affinity of an antibody for a polypeptide of the invention is substantially higher than the affinity for other related polypeptides of the prior art.

In certain preferred embodiments of the invention there are provided antibodies against BASB132 polypeptides or polynucleotides.

Antibodies generated against the polypeptides or polynucleotides of the invention can be prepared in animals (preferably non-humans) using the conventional protocols, and / or the polypeptides and / or polynucleotides of the invention. Alternatively, it can be obtained by administering cells expressing both epitope-containing fragments, either or both analogs, or either or both. For preparation of monoclonal antibodies, any technique known in the art to produce antibodies in continuous cell line cultures can be used. For example, the method of Kohler, G. and Milstein, C. (Nature 256: 495-497 (1975)), the method of Kozbor et al. (Immunology Today 4:72 (1)
983)) and the method of Cole et al. (Monoclonal Antibodies and Cancer Therapy, pg77-
96, Alan R. Liss, Inc. (1985)).

In order to produce a single chain antibody against the polypeptide or polynucleotide of the present invention, a method for preparing a single chain antibody (US Pat. No. 4,946,778) can also be applied. Also, transgenic mice or animals such as other organisms or other mammals can be utilized to express humanized antibodies immunospecific for the polypeptides or polynucleotides of the invention.

Alternatively, a PCR-amplified v gene repertoire of human-derived lymphocytes screened for retention of anti-BASB132 using the phage display method, or a natural library (McCafferty et al., (1990) Nature 348, 552- 554; Marks et al. (1992) Biotechnology 10, 779-783), an antibody gene having binding activity to the polypeptide of the present invention can be selected. The affinity of these antibodies can be determined, for example, by chain shuffling: Clackson et al. (1991).
) Nature 352: 628).

The antibodies described above can also be used to isolate or identify clones expressing the polypeptide or polynucleotide of the invention, or to purify the polypeptide or polynucleotide, for example by affinity chromatography.

Accordingly, among others, antibodies to BASB132 polypeptides or BASB132 polynucleotides may be used to treat infections, particularly bacterial infections.

Polypeptide variants include equivalent variants that antigenically, epitopically or immunologically form a particular aspect of this invention.

Preferably, the antibody or variant thereof is modified so that it is less immunogenic in the individual. For example, if the individual is human, the antibody is most preferably "humanized", in which case the complementarity-determining regions of the hybridized antibody are described, for example, in Jones et al. (1986) Nature 321, 522. -525, or Tempest et al. (19
91) Introduced into human monoclonal antibodies as described in Biotechnology 9, 266-273.

Antagonists and Agonists-Assays and Molecules Also using the polypeptides and polynucleotides of the invention, eg, cells,
Binding of small molecule substrates to ligands can be assessed in cell-free preparations, chemical libraries and natural product mixtures. These substrates and ligands may be natural substrates and ligands, or may be structurally or functionally mimetic. For example, Coligan et al., Current Protocols in Immunology 1 (2
): See Chapter 5 (1991).

In the screening method, the binding of the candidate compound to the polypeptide or polynucleotide of the present invention, or a cell or membrane carrying the polypeptide or polynucleotide, or a fusion protein of the polypeptide is carried out by binding the candidate compound to the candidate compound. It can be easily measured using a label which is directly or indirectly bound. Alternatively, the screening method may use competition with a labeled competitor. In addition, such screening methods determine whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide,
It can be tested using a detection system suitable for cells containing the polypeptide or polynucleotide. In general, inhibitors of activation are assayed in the presence of a known agonist to observe the effect of the presence of the candidate compound on activation by the agonist. In a screening method for an anti-agonist or an inhibitor by examining whether or not the candidate compound suppresses the activation of the polypeptide or polynucleotide in the absence of an agonist or an inhibitor, in some cases, constitutively active Polypeptides and / or constitutively expressed polypeptides and polynucleotides are used. In addition, these screening methods combine a candidate compound with a solution containing a polypeptide or polynucleotide of the invention to form a mixture, measure BASB132 polypeptide and / or polynucleotide activity in the mixture, and BASB 132 in mixture
It may simply include the steps of comparing the polypeptide and / or polynucleotide activity with a standard. High-throughput screening assays for identifying antagonists of the polypeptides of the invention, as well as phylogenetically and / or functionally related polypeptides, include those prepared from Fc moieties and BASB132 polypeptides as described above. Fusion proteins can also be used (D. Benn
ett et al., J. Mol. Recognition, 852-58 (1995) and K. Johanson et al., J. Biol.
Chem., 270 (16): 94599471 (1995)).

In addition, using a polynucleotide, polypeptide and antibody that bind to and / or interact with the polypeptide of the present invention, intracellular mRNA
And / or screening methods can be assembled to detect the effect of added compounds on the production of the polypeptide. For example, monoclonal or polyclonal antibodies can be used by standard methods known in the art to construct an ELISA assay for measuring the level of secretion or cell binding of a polypeptide, which can be used to engineer appropriately engineered cells. Or substances that suppress or enhance the production of polypeptide from tissues (also called antagonists or agonists, respectively)
Can be used to search for.

The present invention also provides compounds for enhancing (agonist) or blocking (antagonist) compounds, in particular bacteriostatic and / or bactericidal compounds, of the action of BASB132 polypeptides or polynucleotides. Provide a screening method. The screening method may involve high throughput techniques. For example, to screen for agonists or antagonists, BA
Synthetic reaction mixture, cell component (membrane, cell envelope, or cell wall) containing SB132 polypeptide and a labeled substrate or ligand for the polypeptide
Alternatively, any preparation thereof is incubated in the absence or presence of a candidate molecule that may be an agonist or antagonist of BASB132. The ability of the candidate molecule to agonize or antagonize the BASB132 polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from the substrate. Molecules that bind blindly, ie, without inducing the action of BASB132 polypeptides, are the most promising and effective antagonists. Molecules that bind well and optionally increase the rate of production of product from the substrate, enhance signal transduction, or increase chemical channel activity are agonists. In some cases, the generation of product from the substrate, signal transduction, or detection of the rate or level of chemical channel activity can be enhanced with a reporter system. Reporter systems that may be effective in this regard include, but are not limited to, colorimetric labeled substrates that are converted to products, reporter genes that respond to changes in BASB132 polynucleotide or polypeptide activity, and in the art. Known binding assays are included.

Other examples of assays for BASB132 agonists have BASB132 and BASB132 binding molecules, recombinant BASB132 binding molecules, natural substrates or ligands, or substrate or ligand mimetics under conditions suitable for competitive inhibition assays. A competitive assay that binds potential agonists. BASB132 can be labeled with a radioactive or colorimetric compound, etc., thereby accurately determining the number of BASB132 molecules bound to the binding molecule or converted to product to assess the efficacy of potential antagonists. You can.

Other potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polynucleotides and / or polypeptides of the invention, thereby suppressing or abrogating its activity or expression. included. Potential antagonists also bind to the same site on a binding molecule, such as a binding molecule, without inducing BASB132-inducing activity, thereby eliminating binding of BASB132 polypeptides and / or polynucleotides to BASB132 poly-nucleotides. It may be a polypeptide such as a small organic molecule, peptide, closely related protein or antibody that interferes with the action or expression of the peptide and / or polynucleotide.

Potential antagonists interfere with normal biological activity by binding to and occupying the binding site of the polypeptide, thereby preventing it from binding to the cellular binding molecule. Small molecules are included. Examples of small molecules include, but are not limited to, small organic molecules, peptides or peptide-like molecules. Other potential agonists include antisense molecules (a description of these molecules is
Okano, J. Neurochem. 56: 560 (1991); Oligodeoxynucleotides as Antisense I
nhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Preferred potential antagonists include compounds related to BASB132 and variants of BASB132.

In a further embodiment, the invention provides a polypeptide of the invention or a fragment thereof and various portions of the constant regions of the heavy or light chain of immunoglobulins of different subclasses (IgG, IgM, IgA, IgE). And a soluble fusion protein produced by genetic engineering. As the immunoglobulin, the constant region of the H chain of human IgG, particularly IgG1, is preferable, and in that case, fusion occurs at the hinge region. In certain embodiments, the Fc portion can be easily removed by incorporating a cleavage sequence that can be cleaved by blood coagulation factor Xa. Furthermore, the present invention relates to methods of genetically engineering these fusion proteins and their use in drug screening, diagnosis and therapy. Yet a further aspect of the invention relates to a polynucleotide encoding such a fusion protein. Examples of fusion protein technology are described in International Patent Applications WO94 / 29458 and WO94 / 22914.

Each of the polynucleotide sequences provided herein can be used in the discovery and development of antibacterial compounds. Upon expression, the encoded protein can be used as a target for antibacterial drug screening. In addition, polynucleotide sequences encoding the respective mRNA-encoding proteins or the amino-terminal regions of Shine-Dalgarno or other translation-promoting sequences are used to construct antisense sequences and express the desired coding sequences. Can be controlled.

The present invention also provides for disruption of the first physical interaction between the pathogen (s) and the eukaryotic (preferably mammalian) host responsible for the sequelae of infection. Of the polypeptides, polynucleotides, agonists or antagonists of the invention. In particular, the molecules of the invention may be used to eukaryotic bacteria (especially Gram-positive and / or Gram-negative) eukaryotic (preferably mammalian) extracellular matrix proteins on indwelling devices or extracellular matrix in wounds. Interfering with attachment to proteins, blocking bacterial attachment between eukaryotic (preferably mammalian) extracellular matrix proteins and bacterial BASB132 proteins that mediate tissue damage and / or retention It is possible to block the normal progression of pathogenesis of infections initiated by other than device implantation or other surgical techniques.

Yet another aspect of the invention provides BASB132 agonists and antagonists, preferably bacteriostatic or bactericidal agonists and antagonists.

The antagonists and agonists of the invention may be used, for example, to prevent, inhibit and / or treat disease.

In a further aspect the invention provides a mimotope of a polypeptide of the invention.
) Concerning. A mimotope is a peptide sequence that is sufficiently similar (sequentially or structurally) to a natural peptide and can be recognized by an antibody that recognizes the natural peptide, or the natural peptide when bound to a suitable carrier. It is possible to raise an antibody that recognizes.

Peptide mimotopes can be designed for the specific purpose by addition, deletion or substitution of selected amino acids. Therefore, the peptide may be modified to facilitate binding to protein carriers. For example, for some chemical attachment methods it is preferred to include a terminal cysteine. Furthermore, for peptides bound to a protein carrier, it is preferable to include a hydrophobic end at a position remote from the binding end of the peptide so that the non-binding end of the peptide remains bound to the surface of the carrier protein. It presents the peptide in a conformation that most closely resembles that of the peptide as found in the case of intact natural molecules. For example, the peptide can be modified to contain an N-terminal cysteine and a C-terminal hydrophobic amidated tail. Alternatively, additions or substitutions of D stereoisomers of one or more amino acids can be made to produce useful derivatives, eg, that improve the stability of the peptide.

Alternatively, techniques such as the phage display method (EP 0 552 267 B1) can be used to identify peptide mimotopes with antibodies that are themselves capable of binding the polypeptides of the invention. . This method produces a number of peptide sequences that mimic the structure of the native peptide, and thus the sequence has the ability to bind anti-natural peptide antibodies, but which itself is a significant sequence of the native polypeptide. It is not necessary to share homology.

Vaccine Another aspect of the invention relates to a method of inducing an immunological response in an individual, particularly a mammal, preferably a human, which method produces an antibody and / or T cell immune response. B sufficient to protect the individual from infections, specifically bacterial infections, most particularly Moraxella catarrhalis infections.
Comprising inoculating said individual with ASB132 polynucleotides and / or polypeptides or fragments or variants thereof. Also provided are methods of delaying bacterial replication by such an immunological response. Yet another aspect of the invention is a method of inducing an immunological response in an individual, wherein the BASB132 polynucleotide and / or polypeptide, or fragment or variant thereof, is expressed in vivo for expression of the BASB132 polynucleotide and / or Alternatively, a nucleic acid vector, sequence or ribozyme that directs expression of the polypeptide or fragment or variant thereof is delivered to the individual to induce an immunological response (e.g., antibody and / or T cell immune response (e.g., (Providing cytokine-producing T cells or cytotoxic T cells) to protect the individual, preferably a human, from a disease (whether or not the disease has already been established in the individual). Concerning how to be. Gene administration is performed, for example, by coating the gene on particles or other and rapidly injecting it into the desired cells. Such nucleic acid vectors can include DNA, RNA, ribozymes, modified nucleic acids, DNA / RNA hybrids, DNA-protein complexes or RNA-protein complexes.

A further aspect of the invention is that when introduced into an individual, preferably a human, having the ability to induce an immunological response, the BASB132 polynucleotide and / or the polynucleotide encoded by said polynucleotide in such an individual. An immunological composition for inducing an immunological response to a polypeptide which comprises a recombinant BASB132 polynucleotide and / or a polypeptide encoded by said polynucleotide, And / or comprises DNA and / or RNA that encodes and expresses an antigen of the BASB132 polynucleotide of the invention, the polypeptide encoded by the polynucleotide, or other polypeptides. This immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and / or cellular immunity, such as cellular immunity resulting from CTL or CD4 + T cells.

The BASB132 polypeptide or fragment thereof stabilizes the first protein, whether or not it produces a co-protein or chemical moiety (which may or may not itself be an antibody) and is antigenic and / or Immunogenic, preferably fusion proteins with protective properties or modified proteins may be produced). Therefore,
The fusion recombinant protein is preferably an antigenic accessory protein (e.g. lipoprotein D from Haemophilus influenzae, glutathione-S-
It further comprises a transferase (GST) or β-galactosidase), or any other relatively large accessory protein that solubilizes the protein and facilitates its production and purification. In addition, accessory proteins may act as adjuvants in the sense of providing generalized stimulation to the immune system of the organism to which they are administered. The accessory protein may be attached to the amino-terminus or the carboxy-terminus of the first protein.

In the vaccine composition of the invention, BASB132 polypeptides and / or polynucleotides, or fragments, mimotope or variants thereof, are also vectors such as the above-described live recombinant vectors such as live bacterial vectors. May be present inside.

Non-viable vectors for BASB132 polypeptides are also suitable and include, for example, bacterial outer membrane vesicles or “blebs”. OM bleb is derived from the outer membrane of Gram-negative bacterial bilayer membranes and is associated with many Gram-negative bacteria (Ct
rachomatis and C. psittaci) (Zhou, L et al., 1998, FEM)
S Microbiol. Lett. 163: 223-228). Examples of bacterial pathogens that have been reported to produce Brebb include Bordetella pertussis and Lyme disease (B
orrelia burgdorferi), Maltese fever (Brucella melitensis), Brucella obis (
Brucella ovis), Escherichia coli, Haemophilus in
fluenza), Legionella pneumophila, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria mening
Itides), Pseudomonas aeruginosa, and Yersinia enterocolitica, but not all.

Brebbs have the advantage of presenting outer membrane proteins in their native conformation and are particularly useful in vaccines. Bacteria can also be engineered to modify expression of one or more molecules in the outer membrane to improve blebs for vaccine applications. Thus, for example, the desired immunogenic protein on the outer membrane
Expression (eg, BASB132 polypeptide) can be introduced or upregulated (eg, by modifying the promoter, etc.). Alternatively or additionally, unrelated outer membrane molecules (such as unprotected antigens or immunodominant but variable proteins) or harmful outer membrane molecules (e.g., toxic molecules such as LPS). , Or those that may induce an autoimmune response) and the like. These techniques are discussed in more detail below.

The non-coding flanking region of the BASB132 gene contains regulatory elements important in the expression of the gene. This control occurs at both transcriptional and translational levels. The sequence of this region, either upstream or downstream of the open reading frame of the gene, can be obtained by DNA sequencing. With this sequence information,
Different promoter elements, termination sequences, inducible sequence elements, repressors, elements responsible for phase mutations, shine-dalgarno sequences, regions that may have secondary structures involved in regulation, as well as other types It allows the determination of potential regulatory motifs, such as regulatory motifs or sequences of.

The sequence information allows for regulation of the natural expression of the BASB132 gene. Upregulation of the gene expression can occur by modifying the promoter, Shine-Dalgarno sequence, potential repressor or operator elements, or any other element of interest. Similarly, down-regulation of expression can be achieved by similar types of modifications. Alternatively, the expression of the gene can be put under the control of phase mutation by changing the phase mutation sequence, or can be removed from this control. In other approaches, gene expression can be placed under the control of one or more inducible elements that allow for regulated expression. Examples of such control include induction by temperature shift, induction by the addition of inducible substrates such as selective hydrocarbons or their derivatives, trace elements, vitamins, cofactors, metal ions, etc. , But not limited to.

The modifications as described above can be introduced by various means. Modification of sequences involved in gene expression can be performed in vivo by random mutagenesis followed by selection of the desired phenotype. Another approach is to isolate the region of interest and modify it by random mutagenesis or site-specific substitutions, insertions or deletions. The modified region is then introduced into the bacterial genome by homologous recombination and the effect on gene expression can be measured. In other approaches, one may substitute or delete all or part of the natural regulatory sequences by knowing the sequence of the region of interest. In this case, the control region of interest is isolated and contains a control region derived from another gene, a combination of control elements derived from a different gene, a synthetic control region or any other control region, or It is modified to delete a selected portion of the wild type control sequence. These modified sequences can then be introduced into the bacterium via homologous recombination into the genome. Examples of preferred promoters that can be used for upregulation of gene expression include porA,
porB, lbpB, tbpB, p110, lst, hpuAB (from Neisseria meningitidis or Neisseria gonorrhoeae); ompCD, co
pB, lbpB, ompE, UspA1; UspA2; TbpB (from Moraxella catarrhalis); p
1, p2, p4, p5, p6, lpD, tbpB, D15, Hia, Hmw1, Hmw2 (from Haemophilus influenzae), but not all.

In one example, gene expression can be regulated by replacing the promoter with a strong promoter (isolating the upstream sequence of the gene, modifying the sequence in vitro, and re-expressing by homologous recombination). Incorporate into the genome). Up-regulation is obtained both in bacteria as well as in outer membrane vesicles shed (or made by) bacteria.

In another example, the techniques described above can be used to produce recombinant bacterial strains with improved properties for vaccine applications. These are attenuated strains, strains with increased expression of selected antigens, strains that knock out genes that impair the immune response (or have decreased expression of the genes), and regulate expression of immunodominant proteins. Strains, modified detachment of outer membrane vesicles, and the like, but are not limited thereto.

Accordingly, the present invention also provides a modified upstream region of the BASB132 gene, which contains a heterologous regulatory element that modifies the expression level of the BASB132 protein located in the outer membrane. The upstream region of such aspects of the invention comprises sequences upstream of the BASB132 gene.
The upstream region generally begins immediately upstream of the BASB132 gene and continues from the start codon of ATG to approximately 1000 bp upstream of the gene. Polycistronic sequence (
For genes located in the operon), the upstream region may begin immediately before the gene of interest, or it may begin before the first gene in the operon. Preferably, the modified upstream region in such an embodiment of the invention contains a heterologous promoter at a position 500-700 bp upstream of ATG.

Accordingly, the present invention provides BASB132 polypeptides that are in modified bacterial blebs. The present invention further provides modified host cells capable of producing non-viable membrane-based Breb vectors. The invention further provides a nucleic acid vector containing the BASB132 gene having a modified upstream region containing a heterologous regulatory element.

Further provided herein are methods for providing the host cells and bacterial blebs of the invention.

The invention also provides compositions comprising the polypeptides and / or polynucleotides of the invention and immunostimulatory DNA sequences such as those described in Sato, Y et al. Science 273: 352 (1996). Articles, especially vaccine compositions, as well as methods.

The present invention also provides that a known polynucleotide, or a specific fragment thereof, known to encode a non-variable region of a bacterial cell surface protein may be used to generate such a gene in an animal model infected with Moraxella catarrhalis. Also provided is a method for use in a polynucleotide construct used in an immunization experiment according to. Such experiments are particularly useful in identifying protein epitopes that can elicit a prophylactic or therapeutic immune response. By this approach, the essential of animals that have subsequently succeeded in not being infected or successfully eliminating the infection for the development of a preventive or therapeutic treatment for bacterial infections, especially Moraxella catarrhalis infection, in mammals, especially humans. It is believed that it becomes possible to prepare a specific-valued monoclonal antibody derived from the organs of.

The invention also provides a vaccine formulation comprising an immunogenic recombinant polypeptide and / or polynucleotide of the invention and a suitable carrier such as a pharmaceutically acceptable carrier. Since the polypeptides and polynucleotides may be degraded in the stomach, it is preferable to administer both parenterally (eg by subcutaneous, intramuscular, intravenous or intradermal injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostatic compounds and solutes which make this formulation isotonic with the recipient's body fluids, preferably blood. , And aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. 1 such formulation
It can be provided in a single-dose or multi-dose container (eg, sealed ampoules and vials), or it can be stored in lyophilized form with the addition of a sterile liquid carrier just prior to use.

The vaccine formulation of the present invention may also include an adjuvant system to enhance the immunogenicity of the formulation. Preferably, the adjuvant system preferentially produces a TH1-type response.

The immune response can be broadly divided into two extreme categories: the humoral or cell-mediated immune response [conventionally characterized by antibody and cell effector defense mechanisms, respectively]. . These response categories are called the TH1-type immune response (cellular response) and the TH2-type immune response (humoral response).

The extreme TH1-type immune response is characterized by the generation of antigen-specific, haplotype-restricted cytotoxic T lymphocytes and the response of natural killer cells. In mice, TH1-type responses are often characterized by the production of antibodies of the IgG2a subtype, whereas in humans they correspond to IgG1-type antibodies. The TH2-type immune response is characterized by the generation of a wide range of immunoglobulin isotypes, including mouse IgG1, IgA and IgM.

The forces driving the development of these two types of immune responses implicitly are thought to be cytokines. High concentrations of TH1-type cytokines tend to preferentially induce a cell-mediated immune response against a given antigen, whereas high concentrations of TH2-type cytokines preferentially induce a humoral immune response against said antigens Tend.

The distinction between TH1 and TH2-type immune responses is not absolute. In fact, some people support an immune response as described as predominantly TH1 or predominantly TH2.
However, in most cases, what was described by Mosmann and Coffman for a mouse CD4 + ve T cell clone (Mosmann, TR and Coffmann, RL (1989) TH1
and TH2 cells: different patterns of lymphokine secretion lead to differen
t functional properties. Annual Review of Immnology, 7, p145-173),
It is convenient to consider the family of cytokines. By convention, TH1 type response is T
It is associated with INF-γ and IL-2 cytokine production by lymphocytes. Other cytokines often directly involved in the induction of TH1-type immune responses are not produced by T cells such as IL-12. In contrast, TH2-type responses are associated with the secretion of IL-4, IL-5, IL-6 and IL13.

It is known that certain vaccine adjuvants are particularly suitable for stimulating cytokine responses of either TH1 or TH2 type. By convention, the best indicator of TH1: TH2 equilibrium in the immune response after vaccination or infection is that after antigen restimulation.
Direct measurement of TH1 or TH2 cytokine production by T lymphocytes in vitro and / or measurement of the IgG1: IgG2a ratio of the antigen-specific antibody response may be mentioned.

Thus, TH1-type adjuvants stimulate preferentially isolated T-cell populations when restimulated in vitro with antigen to produce high levels of TH1-type cytokines and CD8
+ Promotes the development of cytotoxic T lymphocytes and antigen-specific immunoglobulin responses associated with TH1-type isotypes.

An adjuvant capable of preferentially stimulating a TH1 cell response is International Patent Application No. WO94 / 00.
153 and WO 95/17209.

3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. This is known from GB2220211 (Ribi). Chemically, the adjuvant is a mixture of 3 De-O-acylated monophosphoryl lipid A and 4, 5 or 6 acylated chains, manufactured by Ribi Immunochem. Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid A is EP 0 689 454 B1 (SmithKline
Beecham Biologicals SA).

Preferably, the particles of 3D-MPL are small enough to be sterile filtered through a 0.22 micron membrane (EP 0 689 454). 3D-MPL is 10 μg per dose
It is present in the range ˜100 μg, preferably 25-50 μg. In this case, the antigen will normally be present in the range of 2-50 μg per dose.

Another preferred adjuvant comprises QS21 (Hplc purified non-toxic fraction obtained from the bark of Quillaja Saponaria Molina). It may optionally be mixed with 3 De-O-acylated monophosphoryl lipid A (3D-MPL), optionally with a carrier.

[0136]   A method of making QS21 is disclosed in US Pat. No. 5,057,540.

Non-reactive adjuvant formulations containing QS21 have been previously described (WO96 /
33739). Such formulations containing QS21 and cholesterol have been shown to be good TH1-stimulating adjuvants when formulated with antigen.

Other adjuvants that are preferential stimulators of TH1 cell responses include immunomodulatory oligonucleotides such as the unmethylated CpG sequences disclosed in WO96 / 02555.

Combinations of different TH1 stimulating adjuvants such as those mentioned above may also be used for TH1
It is considered in providing adjuvants that are preferential stimulators of cellular responses. For example, QS21 can be formulated with 3D-MPL. QS21: 3D-MPL ratio is usually 1: 1
It is from 0 to 10: 1, preferably from 1: 5 to 5: 1 and often substantially 1: 1.
The preferred range for optimal synergy is 2.5: 1 to 1: 1 3D-MPL: QS21.

[0140] Preferably, a carrier is also present in the vaccine composition of the invention. The carrier may be an oil-in-water emulsion or an aluminum salt such as aluminum phosphate or aluminum hydroxide.

Preferred oil-in-water emulsions include metabolisable oils such as squalene, α-tocopherol and Tween80. In a particularly preferred embodiment, the antigen in the vaccine composition of the invention is combined with QS21 and 3D-MPL in such an emulsion. Furthermore, the oil-in-water emulsion may comprise Span 85 and / or lecithin and / or tricaprylin.

Generally, for human administration, QS21 and 3D-MPL will be present in the vaccine in the range of 1 to 200 μg per dose, eg 10 to 100 μg, preferably 10 to 50 μg. Usually, oil-in-water emulsions contain 2-10% squalene, 2-10% α-tocopherol and 0.3-3% Tween 80. Preferably, the squalene: α-tocopherol ratio is 1 or less, which provides a more stable emulsion. Also, span 85 may be present at a concentration of 1%. In some cases, it may be beneficial for the vaccine of the present invention to further comprise a stabilizing agent.

Non-toxic oil-in-water emulsions preferably comprise a non-toxic oil (eg squalane or squalene), an emulsifier (eg Tween 80) in an aqueous carrier. The aqueous carrier can be, for example, phosphate buffered saline.

A particularly effective adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

The invention also provides multivalent vaccine compositions comprising the vaccine formulations of the invention in combination with other antigens, particularly those useful in the treatment of cancer, autoimmune diseases and related conditions. Such a multivalent vaccine composition may include a TH1 inducing adjuvant as described above.

Although the present invention has been described with respect to particular BASB132 polypeptides and polynucleotides, the present invention is substantially directed to the immunogenicity of its native polypeptides and polynucleotides, as well as recombinant polypeptides or polynucleotides. It should be understood to include similar polypeptides and polynucleotides with additions, deletions or substitutions that have no deleterious effect.

Compositions, Kits and Administration In a further aspect of the invention, BASB132 for administration to cells or multicellular organisms.
Compositions are provided that include a polynucleotide and / or a BASB132 polypeptide.

The present invention also relates to compositions comprising the polynucleotides and / or polypeptides discussed herein or agonists or antagonists thereof. The polypeptides and polynucleotides of the present invention may be used with nonsterile or sterile carriers, such as pharmaceutical carriers suitable for administration to an individual, for use in cells, tissues or organisms. Such compositions include, for example, a media additive or a therapeutically effective amount of a polypeptide and / or polynucleotide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol,
Ethanol and combinations thereof may be mentioned. The formulation should suit the mode of administration. The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more components of the above compositions of the invention.

The polypeptides, polynucleotides and other compounds of the invention may be used alone or with other compounds such as therapeutic compounds.

The pharmaceutical composition may be administered in any effective and convenient manner, such as topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, among others. Administration, or by the transdermal route.

In treatment or prevention, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably an isotonic solution.

In a further aspect, the invention provides a therapeutically effective amount of a polypeptide and / or polynucleotide (eg, a soluble form of the polypeptide and / or polynucleotide of the invention), an agonist peptide / antagonist peptide, or There is provided a pharmaceutical composition comprising a low molecular compound in combination with a pharmaceutically acceptable carrier or excipient. Such carriers include saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof,
It is not limited to these. The invention further provides one of the compositions of the invention described above.
The present invention relates to a pharmaceutical pack and kit comprising one or more containers filled with one or more components. The polypeptides, polynucleotides and other compounds of the invention may be used alone or in combination with other compounds such as therapeutic compounds.

The pharmaceutical composition may be adapted to the route of administration, eg the systemic or oral route of administration. Suitable forms for systemic administration are infusion, typically intravenous.
Other injection routes such as subcutaneous, intramuscular or intraperitoneal can also be used. Another means of systemic administration is transmucosal and transdermal administration using penetrants such as bile salts, fusidic acids and other surfactants. Furthermore, oral administration is possible provided that the polypeptide of the present invention or other compound can be formulated as an enteric solution or capsule. These compounds may be administered locally and / or localized in the form of ointments, pastes, gels, solutions, powders and the like.

For administration to mammals, especially humans, the daily dose of active agent is from 0.01 mg / kg to 10 mg / kg,
It is typically expected to be about 1 mg / kg. The physician in any situation will determine the actual dosage which will be most suitable for an individual and it will vary with the age, weight and response of the particular individual. The above dosages are typical of the average case.
Of course, there may be cases where higher or lower dose ranges are beneficial.
Such cases are also included in the scope of the present invention.

The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the patient's condition, and the judgment of the treating physician. However, suitable dosages are in the range of 0.1-100 μg / kg of patient weight.

The vaccine composition is conveniently in injectable form. The immune response can be enhanced by using conventional adjuvants. A suitable unit dose for vaccination is 0.5 to 5 micrograms / kg of antigen, such dose being 1 to
It is preferable to administer 1 to 3 times at 3 week intervals. When the compound of the present invention is used in the above dose range, no harmful toxic effect which makes administration to an appropriate individual impossible is observed.

However, given the wide variety of compounds available and the varying efficiencies of administration routes, it is expected that the required dosage will vary widely. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Such variation in dosage levels can be adjusted using standard empirical optimization procedures, as is well understood in the art.

Sequence databases, sequences in tangible medium, and algorithms
Sequence of rhythm polynucleotides and polypeptides to determine their 2-dimensional structures and three-dimensional structure, form a valuable information resource with which to identify further sequences of similar homology. These techniques involve storing sequences in computer readable media and then using the stored data in known macromolecular structure programs or to search sequence databases in known search tools such as the GCG program package. Promoted to the maximum.

The invention also provides a method for analyzing a character sequence or sequence, in particular a gene sequence or an encoded protein sequence. Preferred sequence analysis methods include, for example, sequence homology analysis methods such as identity and similarity analysis, DNA, RNA and protein structural analysis methods, sequence assembly methods, branching analysis methods, sequence motif analysis methods, open reading frame determination. Method, nucleobase calling method, codon usage analysis method,
Nucleobase trimming methods, and sequencing chromatogram peak analysis methods are included.

A computer-based method for performing homology confirmation, the method comprising providing in a computer-readable medium a first polynucleotide sequence comprising a sequence of a polynucleotide of the invention, and the first polynucleotide sequence. Is compared to at least one second polynucleotide or polypeptide sequence to confirm homology.

A computer-based method for performing homology confirmation, the method comprising providing a first polypeptide sequence comprising a sequence of a polypeptide of the invention in a computer readable medium and the first polypeptide sequence. Also provided is the above method comprising the step of confirming homology by comparing to at least one second polynucleotide or polypeptide sequence.

Publications and references cited herein, such as but not limited to patents and patent applications, are specifically as if each publication or reference was incorporated herein by reference in its entirety. And individually, the entire contents of which are hereby incorporated by reference. Any patent application to which this application claims priority is incorporated herein by reference in its entirety as set forth above for publications and references.

Definitions “Identity,” as known in the art, is an affinity between two or more such sequences, as the case may be, as determined by comparison of the polypeptide or polynucleotide sequences. In the art, "identity" also means the degree of sequence similarity between such sequences, as the case may be, as determined by the match between the strands of the polypeptide or polynucleotide sequences. To do. "Identity" can be calculated easily by a known method, and as such a method, for example, Computation
al Molecular Biology, Lesk, AM, Oxford University Press, New York, 1
988; Biocomputing: Informatics and Genome Projects, Smith, DW Edition, Acad
emic Press, New York, 1993; Computer Analysis of Sequence Data, Part I,
Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequ
ence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987
; Sequence Analysis Primer, Gribskov, M. and Devereux, J. Ed., M Stockton
Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Appli
ed Math., 48: 1073 (1988), but not limited thereto. Methods to determine identity are designed to give the largest match between the sequences considered. Methods to determine identity are codified in publicly available computer programs. A computer program method for determining the identity between two sequences includes the GAP program (Devere) in the GCG program package.
ux, J. et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLA.
STN (Altschul, SF et al., J. Molec. Biol. 215: 403-410 (1990)) and F
ASTA (Pearson and Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (19
88) but not limited to these. BLAST program family is NCBI
And publicly available from other sources (BLAST Manual, Altschul, S. et al.,
NCBI NLM NIH Bethesda, MD 20894; Altschul, S. et al., J. Mol. Biol. 215: 403-
410 (1990)). The well known Smith Waterman algorithm can also be used to determine identity.

Parameters for comparing polypeptide sequences include: Algorithm: Needleman & Wunsch, J. Mol. Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62, Hentikoff and Hentikoff, Proc. Natl. Aca
d. Sci. USA, 89: 10915-10919 (1992) Gap penalty: 8 Gap length penalty: 2 Effective programs using these parameters are Genetics Computer Group.
(Madison WI), generally available as the "gap" program. The above parameters are the default parameters for peptide comparison (default paramet
er) (no end gap penalty).

Parameters for comparing polynucleotide sequences include: Algorithm: Needleman & Wunsch, J. Mol. Biol. 48: 443-453 (1970) Comparison matrix: match = + 10, mismatch = 0 gap. Penalty: 50 Gap length penalty: 3 Programs that are useful with these parameters are Genetics Computer G
It is publicly available as "gap" program from roup (Madison WI). These are the default parameters for nucleic acid comparisons.

Preferred meanings of "identity" for polynucleotides and polypeptides are optionally given in (1) and (2) below.

(1) An embodiment of the polynucleotide is a polynucleotide sequence having at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence set forth in SEQ ID NO: 1. Further included is an isolated polynucleotide comprising. Here, the polynucleotide sequence is the same as the reference sequence shown in SEQ ID NO: 1 or may contain up to a certain integer number of nucleotide mutations with respect to the reference sequence. Such mutations are selected from the group consisting of deletions, substitutions (including transitions and transversions) or insertions of at least one nucleotide, such mutations being the 5'or 3'terminal position of the reference nucleotide sequence, or these It may be anywhere between the terminal positions, interspersed between the nucleotides in the reference sequence individually or as one or more contiguous groups within the reference sequence. The number of such nucleotide mutations is obtained by adding an integer indicating% identity to the total number of nucleotides shown in SEQ ID NO: 1.
It can be determined by multiplying by a value divided by 100, and then subtracting the product from the total number of nucleotides shown in SEQ ID NO: 1, that is, the following formula: n _n ≦ x _n − (x _n · y). In the formula, n _n is the number of nucleotide mutations, x _n is the total number of nucleotides shown in SEQ ID NO: 1, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, and 80% for 70% Is 0.80 and 85% is 0.85
, 90% 0.90, 95% 0.95, 97% 0.97 or 100%
Is 1.00, and is a symbol of the multiplication operator, and the product of non-integers of x _n and y is rounded down to the nearest integer before subtracting the product from x _n . Modification of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 results in nonsense, missense or frameshift mutations in the coding sequence, thereby altering the polypeptide encoded by the polynucleotide after such mutations. be able to.

By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence shown in SEQ ID NO: 1, ie have 100% identity to the reference sequence or have a% identity of 100%. It may contain up to a certain integer number of nucleic acid mutations with respect to the reference sequence so as to be less than. Such mutations are selected from the group consisting of deletions, substitutions (including transitions and transversions) or insertions of at least one nucleic acid, such mutations being at the 5'or 3'terminal position of the reference polynucleotide sequence, or Can be anywhere between the terminal positions of, and are interspersed among the nucleic acids in the reference sequence, either individually or as one or more contiguous groups within the reference sequence. The number of nucleic acid mutations for a given% identity is determined by multiplying the total number of nucleic acids set forth in SEQ ID NO: 1 by an integer indicating the% identity value divided by 100 and then multiplying the product by It can be obtained by subtracting from the total number, that is, by the following formula: n _n ≦ x _n − (x _n · y). In the formula, n _n is the number of nucleic acid mutations, x _n is the total number of nucleic acids shown in SEQ ID NO: 1, and y is 0.70 for 70% and 0 for 80%, for example.
For 80% and 85%, 0.85, etc., is a symbol of the multiplication operator, and the product of non-integers of x _n and y is rounded down to the nearest integer before subtracting the product from x _n .

(2) A polypeptide embodiment is a polypeptide having at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the polypeptide reference sequence of SEQ ID NO: 2. Further included is an isolated polypeptide comprising. Here, the polypeptide sequence may be the same as the reference sequence of SEQ ID NO: 2 or may contain up to a certain integer number of amino acid mutations with respect to the reference sequence. Such mutations are selected from the group consisting of deletions, substitutions (including conservative and non-conservative substitutions) or insertions of at least one amino acid, such mutations being at the amino or carboxy terminal position of the reference polypeptide sequence, or It may be anywhere between these terminal positions, interspersed among the amino acids in the reference sequence, individually, or as one or more contiguous groups within the reference sequence. The number of such amino acid mutations is determined by multiplying the total number of amino acids shown in SEQ ID NO: 2 by the integer showing the percent identity divided by 100, and then subtracting the product from the total number of amino acids shown in SEQ ID NO: 2. , i.e., the following equation: n _a ≦ x _a - can be obtained by (x _a · y). In the formula, n _a is the number of amino acid mutations, x _a is the total number of amino acids shown in SEQ ID NO: 2, y is 0.50 for 50% and 0 for 60%.
0.70 for 60, 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1 for 100%
.00, · is a symbol for the multiplication operator, and the product of non-integers of x _a and y rounds down to the nearest integer before subtracting the product from x _a .

By way of example, a polynucleotide sequence of the invention may be identical to the reference sequence of SEQ ID NO: 2, ie have 100% identity to the reference sequence or have a% identity.
May contain up to a certain integer number of amino acid mutations with respect to the reference sequence so that is less than 100%. Such mutations are selected from the group consisting of deletions, substitutions (including conservative and non-conservative substitutions) or insertions of at least one amino acid, such mutations being at the amino or carboxy terminal position of the reference polypeptide sequence, or It may be anywhere between these terminal positions, interspersed among the amino acids in the reference sequence, individually, or as one or more contiguous groups within the reference sequence. The number of amino acid mutations for a given% identity is calculated by multiplying the total number of amino acids shown in SEQ ID NO: 2 by the integer showing the% identity value divided by 100, and then multiplying the product by by subtracting from the total number, i.e., the following equation: n _a ≦ x _a - can be obtained by (x _a · y). In the formula, n _a is the number of amino acid mutations, x _a is the total number of amino acids shown in SEQ ID NO: 2, y is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, etc. , Are symbols of the multiplication operator, and the product of non-integers of x _a and y is rounded down to the nearest integer before subtracting the product from x _a .

“Individual” as used herein with respect to an organism is a multicellular eukaryote, such as, but not limited to, a metazoan, a mammal, an ovid, a bovid.
, Simian, primate, human etc.

“Isolated” means altered “by the hand of man” from the natural state, ie, if the “isolated” composition or substance is naturally occurring. It is either altered or isolated from its original environment, or both. For example, a polynucleotide or polypeptide naturally occurring in the body of a living organism is not "isolated", but a polynucleotide or polypeptide separated from its naturally occurring coexisting materials is As used in the text, it is "isolated". Further, the polynucleotide or polypeptide introduced into the organism by transformation, genetic engineering or any other recombinant method is present in the organism, which may or may not be alive. Is also "isolated".

“Polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which is unmodified RNA or DNA, including single-stranded and double-stranded regions, or modified RNA. Alternatively, it can be DNA.

A "variant" is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains essential properties.
Typical polynucleotide variants differ in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of this variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, deletions, additions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. Typical polypeptide variants differ in amino acid sequence from a reference polypeptide. Generally, the sequence of the reference polypeptide and the sequence of the variant are generally similar and limited to differences that are identical in many regions. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, deletions, additions in any combination. The amino acid residues to be substituted or inserted may or may not be encoded by the genetic code. Variants of polynucleotides or polypeptides may be naturally occurring variants, such as allelic variants, or variants not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis or direct synthesis.

“Disease” means any disease caused by or associated with a bacterial infection. Such diseases include, for example, otitis media in infants and children, pneumonia in the elderly, sinusitis, nosocomial infections and invasive diseases, deafness, fluid accumulation in the middle ear, auditory nerve damage, delayed language learning, and above. Chronic otitis media with infection of the respiratory tract and inflammation of the middle ear.

[0176] EXAMPLE The following examples, unless specifically described in detail, was performed by standard methods well-known conventional to those skilled in the art. These examples are illustrative and not limiting of the invention.

Example 1: DNA sequencing of BASB132 gene from Moraxella catarrhalis ATCC 43617 strain A: Moraxella catarrhalis strain BASB132 from Moraxella catarrhalis ATCC 43617 strain (also called MC2931 strain)
The DNA sequence of the BASB132 gene is shown in SEQ ID NO: 1. The translation product of the BASB132 polynucleotide sequence is shown in SEQ ID NO: 2.

B: BASB132 of Moraxella catarrhalis strain 43617 The sequence of BASB132 gene was confirmed in Moraxella catarrhalis ATCC 43617 strain. For this purpose, a truncated BA from Moraxella catarrhalis ATCC 43617 strain
For plasmid DNA containing the gene region encoding SB132 (corresponding to the last 2977 nucleotides at the 3'end) (see Example 2A), the Big Dyes kit (Applied biosystems
) Is used to determine the DNA sequence, and an ABI373 / A DNA sequencer is used to assay Moraxella catarrhalis oli 5 specific for the BASB132 gene under the conditions indicated by the manufacturer.
YtfN (5'-CCC ACC GTT TGC CCT TCA AT-3 ') (SEQ ID NO: 5) and oli 6 YtfN (5'-C
AT TTT TCC CGA GCA TTC AAA C-3 ') (SEQ ID NO: 6) and oli 7 YftN (5'-AAT CAG
CCA CTT ATC GCC AC-3 ') primer and vector-specific M13 universal sequence primer (5'-GTA AAA CGA CGG CCA GT-3') (SEQ ID NO: 8) and M13 reverse sequence primer (5'-CAG GAA ACA GCT ATG AC-3 ') (SEQ ID NO: 9). As a result, a polynucleotide sequence and a deduced polypeptide sequence (designated as SEQ ID NO: 3 and SEQ ID NO: 4, respectively) were obtained. The MegAlign program from the DNASTAR software package was used to align the polynucleotide sequences of SEQ ID NOs: 1 and 3. The alignment is shown in Figure 1; a pair-wise comparison of identities shows that the two BASB132 polynucleotide gene sequences are 100% identical in the overlapping regions. With the same MegAlign program,
The polypeptide sequences of SEQ ID NOs: 2 and 4 were aligned. The alignment is shown in Figure 2; a pair-wise comparison of identities shows that the two BASB132 protein sequences are 100% identical in the overlapping regions.

Example 2: Construction of a plasmid expressing recombinant truncated BASB132 A: Cloning of truncated BASB132 The restriction enzyme sites for BspHI and BglII were oli1 YtfN (5'-TCA TGA ATG ACT CAG GCA AAG-
3 ') [SEQ ID NO: 10] forward amplification primer and oli2 YtfN (5'-AGA TCT
Introduced into the reverse amplification primer of AAA CTT CCA ACG ATA AAT C-3 ') [SEQ ID NO: 11] so that the truncated BASB132 protein can be expressed as a fusion protein containing (His) 6 affinity chromatography tag at the C-terminus The PCR product was directionally cloned into the E. coli expression plasmid pQE60. Due to the large size of the gene, two PCR experiments were performed simultaneously in parallel to amplify the entire coding sequence. In the first amplification, oli1 YtfN (5'-TCA TGA ATG ACT CAG GCA
AAG-3 ') [SEQ ID NO: 10] and oli3 (AAA CAA ATC GCA CCC ACG CC-3') [SEQ ID NO: 12]
, And in the second amplification, oli2 YtfN (5'-AGA TCT AAA CTT CCA ACG ATA A
AT C-3 ') [SEQ ID NO: 11] and oli4 YtfN (5'-ACA AAT TGC AGC GCA TTG TTG G-3') [
Sequence number 13] was used. Both amplified fragments shared a common region with a single BstXI restriction site. The BASB132 PCR product was first introduced into pCRII TOPO cloning vector (Invitrogen) using Top10 bacterial cells as per the manufacturer's instructions. The realization of this intermediate construct facilitated further cloning into expression vectors. Transformants containing the BASB132 DNA insert were selected by restriction enzyme analysis. Following digestion, approximately 20 μl aliquots of the reaction were analyzed by agarose gel electrophoresis (0.8% agarose in Tris-acetic acid-EDTA (TAE) buffer). DNA fragments were visualized by UV emission after gel electrophoresis and ethidium bromide staining. A DNA molecular weight standard (1 kb ladder, Life Technologies) was run in parallel with the test sample and used to estimate the size of the DNA fragment. For each cloning, the purified plasmid from the selected transformants was followed by BspH.
Sequential complete digestion was performed with I and BstXI or BstXI and BglII restriction enzymes as recommended by the manufacturer (Life Technologies). The digested DNA fragment was purified again using a silica gel-based spin column and then ligated with the pQE60 plasmid.

B: Expression Analysis of PCR-Positive Transformants To prepare the expression plasmid pQE60 for ligation, it was also digested to completion with both NcoI and BglII and then the manufacturer to prevent self-ligation. Were treated with calf intestinal phosphatase (CIP, approximately 0.02 units per pmol of 5'end, Life Technologies) as instructed in. The ligation reaction was set up using about 5-fold molar excess of the digested fragment to the prepared vector. A standard about 20 μl ligation reaction (about 16 ° C. for about 16 hours) was prepared using T4 DNA ligase (about 2.0 units / reaction, Life Technologies) using methods well known in the art.
Was done using. Using an aliquot of this ligation (approximately 5 μl),
Electrocompetent cells were transformed according to methods well known in the art.
Transformed cells were plated on LB agar plates containing ampicillin (100 μg / ml) following a growth time of about 2-3 hours at 37 ° C. in about 1.0 ml LB broth. Antibiotics were included for selection. The plates were incubated at 37 ° C for about 16 hours and overnight. Individual Ap
Pick R colonies with a sterile toothpick and use them to fresh LB ApR plates.
Patch ”inoculation, as well as about 1.0 ml of LB AnR broth culture. Both patch plates and broth cultures were grown overnight at 37 ° C in a standard incubator (for plates) or a shaking water bath. Then, restriction enzyme analysis was performed to confirm that the transformant had the BASB 132 DNA insert. Following digestion, approximately 20 μl aliquots of the reaction were subjected to agarose gel electrophoresis (0.
8% agarose in Tris-acetate-EDTA (TAE) buffer). After gel electrophoresis and ethidium bromide staining, DNA fragments were visualized by UV irradiation. A DNA molecular size standard (1 kb ladder, Life Technologies) was aligned with the test sample, electrophoresed, and used to estimate the size of the PCR product. Transformants producing a PCR product of the expected size were identified as strains containing the BASB132 expression construct. The expression plasmid containing strains were then analyzed for inducible expression of recombinant BASB132.

C: Expression Analysis of PCR-Positive Transformants Aliquots (1 μl) of recombinant plasmid DNA preparations were made into competent M15 (pREP4
) Transformed into bacterial cells by methods well known to the person skilled in the art. Following a growth period of about 2-3 hours in about 1,0 ml of LB medium, transformant cells were treated with ampicillin (100 μg / ml).
) And kanamycin (30 μg / ml) on LB agar plates.
Antibiotics were included in the selection. The plates were incubated overnight at 37 ° C for about 16 hours. Individual
ApR KmR colonies were picked with a sterile toothpick and inoculated into approximately 5.0 ml LB ApR KmR culture medium. Liquid cultures were incubated overnight at 37 ° C with shaking (about 250 rpm). An aliquot of overnight seed culture (about 1.0 ml) was inoculated into about 25 ml LB Ap broth in a 125 ml Erlenmeyer flask until the turbidity of the culture reached an OD600 value of about 0.5, ie, mid-log phase. until reaching the mid-log phase (usually about 1.5 to 2.0
The cells were grown with shaking (about 250 rpm) at 37 ° C. for an hour. At this point, about half of the culture (about 12.5 ml) was transferred to a second 125 ml flask and a final concentration of 1.0 mM was added.
Expression of recombinant BASB132 protein was induced by adding IPTG (1.0 M stock prepared in sterile water, Sigma) to Both the IPTG-induced culture medium and the non-induced culture medium were continuously cultured for another 4 hours at 37 ° C. with shaking. After the induction time, samples (about 1.0 ml) of both induced and uninduced cultures were removed and cells were harvested by centrifugation in a microcentrifuge at room temperature for about 3 minutes. Suspend each cell pellet in approximately 50 μl sterile water, then
-The protein was denatured by mixing with an equal volume of 2x Laemelli SDS-PAGE sample buffer containing mercaptoethanol and placing in a boiling water bath for about 3 minutes. Equal volumes (approximately 15 μl) of both IPTG-induced crude cell lysate and uninduced crude cell lysate were repeated in duplicate on a 12% Tris / glycine polyacrylamide gel (1 mm thick Mini-gels, Novex). Loaded Induced and non-induced lysate samples were electrophoresed using standard SDS / Tris / glycine running buffer (BioRad) with prestained molecular weight markers (SeeBlue, Novex) under conventional conditions. After electrophoresis, one gel was stained with Coomassie Brilliant Blue R250 (BioRad) and then destained to visualize the novel BASB132 IPTG inducible protein. The second gel was a BioRad Mini-Protean II blotting device and Towbin's methanol (
20%) transfer buffer using PVDF membrane (0.45 micron pore size,
Novex) and electroblotted at 4 ° C. for about 2 hours. Membrane blocking and antibody incubation were performed according to methods well known in the art. Monoclonal anti-RGS (His) 3 antibody followed by a second rabbit anti-mouse antibody conjugated to HRP (QiaGen) was used to express and identify the BASB132 recombinant protein.
ty) was confirmed. The reaction pattern of anti-His antibody could be visualized using ABT insoluble substrate or using Hyperfilm with Amersham ECL chemiluminescence system.

Example 3: Production strain of recombinant BASB132 For purification of recombinant protein, a recombinant expression strain of Escherichia coli M15 (PREP4) containing a plasmid (pQE60) encoding BASB132 derived from Moraxella catarrhalis was used. Produced a cell mass of. This expression strain was treated with 100 μg / ml ampicillin (“Ap”) and
Cultures were performed on LB agar plates containing 30 μg / ml kanamycin (“Km”) to ensure retention of pQE60 and pREP4. This strain was grown in LB broth containing the same concentration of antibiotics as above for cryopreservation at -80 ° C and then 30% (w / v
) Mixed with an equal volume of LB broth containing glycerol.

[0183] The fermentation medium used for the culture recombinant protein production, 100 [mu] g / ml of Ap and 30
It consisted of 2X YT broth (Difco) containing Km of μg / ml. Antifoam (Anti
Foam 204, Sigma) was added to the fermentor medium at a concentration of 0.25 ml / L. B
IPTG (isopropyl β-D-thiogalactopyranoside) was added to the fermentor to induce expression of ASB132 recombinant protein (final concentration 1 mM).

Fermentation A 500 ml seedling Erlenmeyer flask containing 50 ml working volume was inoculated with 0.3 ml of a rapidly thawed frozen culture or several colonies obtained from a selective agar plate culture, at 150 rpm. It was cultured at 37 ± 1 ° C. for about 12 hours while shaking the sample table (Innova 2100, New Brunswick Scientific). This seed culture was then used to inoculate a 5 L real-volume fermentor containing 2X YT broth as well as both Ap antibiotics. The fermentor (Bioflo 3000, New Brunswick Scientific) was operated in a Rushton impeller at 37 ± 1 ° C., 0.2-0.4 VVM air sparging, 250 rpm. No pH was adjusted in the flask seed culture or in the fermenter. During fermentation, the pH in the fermenter went up and down in the range of 6.5-7.3. IPTG (1.0 M stock prepared in sterile water) was added to the fermentor when the culture reached the mid-log phase (approximately 0.7 units at OD600). Cells were induced for 2-4 hours and then harvested by centrifugation using either a 28RS Heraeus (Sepatech) or RC5C ultra high speed centrifuge (Sorvall Instruments). The cell paste was stored at -20 ° C until processed.

Chemicals and Materials Imidazole and Triton X-100 were purchased from Merck. Guanidine hydrochloride is Flu
It was made by ka company. Aprotinin was obtained from Sigma Chemical Company. Urea and AEBSF were from ICN-Biochemicals. All other chemicals were reagent grade or better. Ni-NTA Superflow resin and Penta-His antibody, BSA free, were obtained from QiaGen. MicroBCA assay was obtained from Pierce. Ami
The con 3 filter was manufactured by Millipore. The dialysis membrane (MWCO12-1400) was manufactured by MFPI, USA. The molecular weight marker (BenchMark ladder) was manufactured by Life-technology.

Example 4 Purification of Recombinant BASB132 from E. coli Extraction / Purification Cell paste from 2750 ml of IPTG induction medium (about 4 hours, OD620 = 0.5) was added to 100 mM NaH ₂ PO ₄ , 10 mM containing 6 M guanidinium chloride. It was resuspended in Tris-HCl buffer, pH 8 (buffer A), left at room temperature for 1 hour and then centrifuged for 20 minutes at 10,000 g. The supernatant was applied to Ni-NTA superflow resin equilibrated with buffer A and immediately recovered. Resin is 100 mM NaH ₂ PO ₄ containing 8 M urea, 10 mM Tris-HCl buffer, pH 6
Washed twice with .3 (buffer C). Elution was with 4x 100 mM Na containing 8M urea
Using H ₂ PO ₄ , 10 mM Tris-HCl buffer, pH 5.9 (buffer D), followed by 4 × 100 mM NaH ₂ PO ₄ , 10 mM Tris-HCl buffer, pH 4.5 (buffer E) containing 8M urea went. Fractions were neutralized with 25% volume of 200 mM phosphate buffer, pH 7.5. BASB
Fractions containing 132 protein were pooled and 100M containing 8M urea, 4M urea, then 2M urea.
Dialyze against mM NaH ₂ PO ₄ buffer, pH 7.4 and finally contain 0.1% Triton-X100
It was dialyzed 3 times against PBS buffer, pH 7.4.

The solution was cloudy (solution A) due to precipitation during the dialysis process. Two centrifugation steps (
After 10 minutes at 3000 rpm) the solution became clear (solution B). Solutions A and B of purified BASB132 protein were quantified using MicroBCA assay reagents. Solution A is 170μ
It was quantified as g / ml. 1.65 mg purified and clarified protein (Solution B) at 75 μg / ml
Was obtained at a final concentration of.

As shown in Figure 4-A, the purified BASB132 protein appeared in SDS-PAGE as a major band migrating to approximately 100 kDa (assessed as relative molecular weight). Purity was estimated to exceed 70%. The BASB132 protein was reactive with a mouse monoclonal antibody raised against the 6-histidine motif (Figure 4B).

Example 5: Production of antiserum against recombinant BASB132 By vaccinating rabbits with the purified recombinant BASB132 protein,
A multivalent antiserum against the BASB132 protein is produced.

Multivalent antisera against BASB132 protein are also generated by vaccinating mice with purified recombinant BASB132 protein. Animals are bled prior to the first immunization ("pre-bleeding") and after the final vaccination.

The titer of anti-BASB132 protein was determined by ELISA using purified recombinant BASB132 protein.
To measure. Titers are mid-poi calculated using a 4-parameter logistic model using XL Fit software.
nt titer).

Antisera are also used as the first protein to identify proteins in Western blots as described in Example 7 below. Western blots can show the presence of anti-BASB132 antibody in the sera of immunized animals.

Example 6: Immunological characterization: ELIS using whole cells of Moraxella catarrhalis killed with surface-exposed formalin of BASB132.
A measures the titer of anti-BASB132 protein. Titers are defined as midpoint titers calculated with a 4-parameter logistic model using XL Fit software.

Example 7: Western Blot Analysis ATCC 43617 and several strains of M. catarrhalis, including clinical isolates from various geographical regions, were grown on Muller Hinton agar plates for 24 hours at 36 ° C. Let Inoculate the liquid medium with several strains. The culture is grown to an A620 of about 0.6 and harvested by centrifugation. The cells are then concentrated and solubilized in PAGE sample buffer. The solubilized cells are then fractionated on a 4-20% polyacrylamide gel and the separated proteins are electrotransferred to PVDF membranes. The PVDF membrane is then pretreated with saturated buffer. All subsequent incubations are performed with this pretreatment buffer.

PVDF membranes are incubated with pre-immune serum, rabbit or mouse immune serum for 1 hour at room temperature. Next, the PVDF membrane is washed. PVDF membranes are incubated with biotin-labeled sheep anti-rabbit or anti-mouse Ig. The PVDF membrane is then washed 3 times with wash buffer and incubated with streptavidin-peroxidase. Then, this PVDF membrane was washed with a washing buffer three times, and 4-chloro-1-
Color with naphthol.

Example 8 Immunological Characterization: Bactericidal Activity The complement-mediated cytotoxic activity of anti-BASB132 antibody was determined by the method of BASB132 prepared as described above.
Test to see the potential of protein antisera as vaccines. Preimmune serum and anti-BASB in complement-mediated killing of Moraxella catarrhalis
132 Measure antiserum activity.

Strains of Moraxella catarrhalis are grown on plates. Add a few colonies to the liquid medium. The culture is grown to an A620 of approximately 0.4. After one wash step, the pellet is suspended and diluted.

Pre-immune serum and anti-BASB132 serum are placed in the first well of a 96-well plate and serial dilutions are placed in other wells of the same row. Subsequently, live Moraxella catarrhalis is added and the mixture is incubated. Complement is added to each well at an effective dilution factor previously determined in the toxicity test.

Each test included complement controls (wells containing an active or inactivated complement source but no serum), positive controls (bactericidal antibody to a known titer). , Wells), culture controls (wells without serum and complement), and serum controls (wells without complement).

The bactericidal activity of rabbit or mouse antisera (killing 50% of homologous strains) is determined.

Example 9: Presence of Antibodies to BASB132 in Convalescent Human Serum Western blots of purified recombinant BASB132 using a pool of human serum from M. catarrhalis-infected children as the first antibody preparation. Except that the procedure is as described in Example 7 above.

Example 10: Efficacy of BASB132 Vaccine; M. catarrhalis Lungs in Mice
Acceleration of realism This mouse model is based on analyzing lung invasion of M. catarrhalis following standard intranasal challenge of mice after vaccination.

Groups of BALB / c mice are immunized with the BASB132 vaccine. After the booster, the mice are challenged under anesthesia by injecting the bacterial suspension into the nostrils. Mice are killed 30 minutes to 24 hours after challenge and lungs are aseptically excised and individually homogenized. The log10 weighted average number of CFU per lung is determined by counting the colonies grown on agar plates after plating dilutions of the homogenate. L of CFU per lung
The arithmetic mean and standard deviation of the og10 weighted average number are calculated for each group.

[0204]   Analyze the results statistically.

In this experiment, groups of mice are inoculated with either BASB132 or a killed whole cell (kwc) preparation of the M. catarrhalis strain, or mock inoculated.

Example 11: Inhibition of M. catarrhalis adhesion to cells by anti-BASB132 antiserum This assay measures the ability of anti-BASB serum to inhibit the adhesion of Moraxella bacteria to epithelial cells. This activity prevents nasopharyngeal colonization by Moraxella.

One volume of bacteria is incubated on ice with one volume of preimmune or anti-BASB132 immune serum dilution. This mixture is then added to the wells of a 24-well plate containing confluent cell cultures that have been washed once with culture medium to remove traces of antibiotic. The plate is then centrifuged and incubated. Then wash each well gently. After the final wash, sodium glycolate is added to the wells. After incubation, the cell layer is detached and homogenized. Plate dilutions of homogenate on agar plates and incubate. The number of colonies on each plate is counted and the number of bacteria present in each well is calculated.

Deposits Deposits containing Catrin strains of Moraxella catarrhalis are American Type Cultur
It was deposited in the e Collection ("ATCC") on June 21, 1997, and the accession number 43617 was obtained. This deposit was described as Branhamella catarrhalis (Frosch and Kolle) and was lyophilized to 1.5, which was constructed from M. catarrhalis isolates obtained from transtracheal aspirates of coal miners suffering from chronic bronchitis. ~ 2.9 kb insert library. This deposit is described in Antimicrob. Agents Chemother. 21: 506-508 (1982).

The deposits of the Moraxella catarrhalis strains are referred to herein as “deposited strains” or “deposited strains”.
The DNA of the deposited strain ".

[0210]   The deposited strain contains the full length BASB132 gene.

The deposit of vector pMC-D15 consisting of Moraxella catarrhalis DNA inserted in pQE30 was deposited on February 12, 1999 in the American Type Culture Collection (ATCC), and was designated as Accession No. 207105.

In the event of a conflict with the sequence description herein, the sequence of the polynucleotide contained in the deposited strain / clone, as well as the amino acid sequence of any polypeptide encoded thereby, will prevail.

Deposited strains were deposited under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure. This deposited strain cannot be changed and will be openly disclosed to the public upon issuance of a patent without limitation or condition. The deposited strains are provided solely for the convenience of those of ordinary skill in the art and do not acknowledge that the deposited deposits are necessary to meet the enablement requirements as required by section 112 of the U.S.C. Absent.

[0214]

Sequence Information BASB132 Polynucleotide and Polypeptide Sequence SEQ ID NO: 1 Polynucleotide sequence of Moraxella catarrhalis BASB132 from ATCC43617 strain. SEQ ID NO: 2 Moraxella catarrhalis BA deduced from the polynucleotide shown in SEQ ID NO: 1
SB132 polypeptide sequence SEQ ID NO: 3 Polynucleotide sequence of Moraxella catarrhalis BASB132 derived from ATCC43617 strain Moraxella catarrhalis BA deduced from the polynucleotide shown in SEQ ID NO: 4.
SB132 polypeptide sequence SEQ ID NO: 5 CCC ACC GTT TGC CCT TCA AT SEQ ID NO: 6 CAT TTT TCC CGA GCA TTC AAA C SEQ ID NO: 7 AAT CAG CCA CTT ATC GCC AC SEQ ID NO: 8 GTA AAA CGA CGG CCA GT SEQ ID NO: 9 CAG GAA ACA GCT ATG AC sequence No. 10 TCA TGA ATG ACT CAG GCA AAG SEQ ID No. 11 AGA TCT AAA CTT CCA ACG ATA AAT C SEQ ID No. 12 AAA CAA ATC GCA CCC ACG CC SEQ ID No. 13 ACA AAT TGC AGC GCA TTG TTG G

[Sequence list]

[Brief description of drawings]

[Figure 1]   Alignment of BASB132 polynucleotide sequences.   The identity to SEQ ID NO: 2 is indicated by a dot. Gaps are indicated by dashes.

[Fig. 2]   Alignment of BASB132 polypeptide sequences.   The identity to SEQ ID NO: 2 is indicated by a dot. Gaps are indicated by dashes.

FIG. 3A: Small scale expression analysis of BASB132: Coomassie staining of SDS-polyacrylamide gels. 3B: Small scale expression analysis of BASB132: Western blot (anti-His antibody).

[Figure 4]   4A: Coomassie staining of SDS-polyacrylamide gel of purified BASB132.   4B: Western blot of purified BASB132 (anti-His antibody).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 A61P 31/04 4H045 A61P 31/04 C07K 14/195 C07K 14/195 16/12 16/12 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 C12P 21/02 C 5/10 C12N 15/00 ZNAA C12P 21/02 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, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM , AZ, Y, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F terms (reference) 4B024 AA01 AA13 BA31 BA50 CA01 GA11 HA15 4B064 AG27 AG31 CA19 CC24 DA01 DA15 4B065 AA01Y AB01 AC14 BA01 CA24 CA25 CA45 CA46 4C084 AA07 AA13 ZB35 4C085 AA03 AA 08 AA13 AA14 BA16 BB11 BB31 CC21 EE01 4H045 AA10 AA11 AA30 BA10 CA11 DA76 DA86 EA29 EA31 EA52 FA74

Claims (26)

[Claims] 1. An amino acid sequence represented by SEQ ID NO: 2 or 4, respectively,
An isolated polypeptide comprising an amino acid sequence having at least 85% identity over the entire length of SEQ ID NO: 2 or 4. 2. The amino acid sequence has at least 95% identity over the entire length of SEQ ID NO: 2 or 4 to the amino acid sequence shown in SEQ ID NO: 2 or 4.
The isolated polypeptide of claim 1. 3. The polypeptide according to claim 1, which comprises the amino acid sequence shown in SEQ ID NO: 2 or 4. 4. An isolated polypeptide as set forth in SEQ ID NO: 2 or 4. 5. An immunogenic fragment of the polypeptide according to any one of claims 1 to 4, wherein the immunogenic activity of the immunogenic fragment is the polypeptide of SEQ ID NO: 2 or 4. The above fragment that is substantially identical to the original activity. 6. The polypeptide is part of a larger fusion protein,
The polypeptide according to any one of claims 1 to 5. 7. An isolated polynucleotide encoding the polypeptide of any one of claims 1-6. 8. The amino acid sequence shown in SEQ ID NO: 2 or 4, respectively,
An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having at least 85% identity over the entire length of SEQ ID NO: 2 or 4.
Or an isolated polynucleotide consisting of a nucleotide sequence complementary to the isolated polynucleotide. 9. An isolated polynucleotide comprising a nucleotide sequence having at least 85% identity over its entire coding region to a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2 or 4, or An isolated polynucleotide consisting of a nucleotide sequence complementary to the isolated polynucleotide. 10. An isolated polynucleotide comprising a nucleotide sequence having at least 85% identity over the entire length of SEQ ID NO: 1 or 3 to the nucleotide sequence shown in SEQ ID NO: 1 or 3, respectively, or An isolated polynucleotide consisting of a nucleotide sequence complementary to the isolated polynucleotide. 11. The isolated polynucleotide of any one of claims 7-10, which has at least 95% identity to the sequence set forth in SEQ ID NO: 1 or 3. 12. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2 or 4. 13. An isolated polynucleotide comprising the polynucleotide set forth in SEQ ID NO: 1 or 3. 14. SEQ ID NO: 2 or 4 obtained by screening an appropriate library with a labeled probe having the sequence shown in SEQ ID NO: 1 or 3 or a fragment thereof under stringent hybridization conditions. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide set forth in. 15. An expression vector or a recombinant live microorganism comprising the isolated polynucleotide according to any one of claims 7 to 14. 16. A host cell containing the expression vector of claim 15, or an isolated poly-comprising an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 2 or 4. A cell fraction or membrane of the host cell expressing the peptide. 17. A method for producing the polypeptide according to any one of claims 1 to 6, wherein the host cell according to claim 16 is cultivated under conditions sufficient to produce the polypeptide. And recovering the polypeptide from the culture medium. 18. A method for expressing a polynucleotide according to any one of claims 7 to 14, wherein the host cell is transformed with an expression vector containing at least one of the polynucleotide, and the host cell is A method as described above, comprising culturing under conditions sufficient to express any one of said polynucleotides. 19. A vaccine composition comprising an effective amount of the polypeptide of any one of claims 1-6 and a pharmaceutically acceptable carrier. 20. A vaccine composition comprising an effective amount of the polynucleotide of any one of claims 7-14 and a pharmaceutically effective carrier. 21. At least one other Moraxella catarrhalis (Moraxel)
21. A vaccine composition according to claim 19 or 20, which comprises a (la catarrhalis) antigen. 22. An antibody immunospecific for the polypeptide or immunological fragment of any one of claims 1-6. 23. The polypeptide according to any one of claims 1 to 6 or the polypeptide according to any one of claims 1 to 6, which is present in a biological sample derived from an animal suspected of being infected with Moraxella catarrhalis. A method for diagnosing Moraxella catarrhalis infection, which comprises identifying an immunospecific antibody. 24. Use of a composition comprising an immunologically effective amount of a polypeptide according to any one of claims 1-6 in the preparation of a medicament for use in eliciting an immune response in an animal. . 25. Use of a composition comprising an immunologically effective amount of a polynucleotide according to any one of claims 7-14 in the preparation of a medicament for use in eliciting an immune response in an animal. . 26. A human suffering from a Moraxella catarrhalis infection comprising at least one antibody directed against the polypeptide of any one of claims 1-6 and a suitable pharmaceutical carrier. A therapeutic composition useful for the treatment of.