JPH11137273A - New compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new polynucleotide having high identity with a nucleic acid sequence encoding an HPMBQ91 polypeptide of a specific amino acid sequence, useful for treating and diagnosing diseases related to the expression and activity of the polypeptide. SOLUTION: This new nucleotide contains a nucleotide sequence having at least 85% identity with a nucleotide sequence along its whole length, encoding an HPMBQ91 polypeptide of the formula or a nucleotide sequence having complementarity to the polynucleotide sequence and is useful for producing a neuropeptide precursor and the HPMBQ91 polypeptide being the peptide product of the precursor and for treating and diagnosing diseases related to the expression and activity of the HPMBQ91 polypeptide. The polypeptide is obtained from a cDNA library derived from an mRNA of human placenta cell, fatal hepatolienal cell or lymphonodus merker cell by using a standard cloning and screening and an expression sequence tag analysis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to newly identified polynucleotides, polypeptides encoded thereby, and the use of such polynucleotides and polypeptides, and their production. More specifically, the polynucleotides and polypeptides of the present invention
It relates to a neuropeptide precursor and its peptide product, hereinafter referred to as HPMBQ91. The invention also relates to inhibiting or activating the action of the polynucleotides and polypeptides.

[0002]

BACKGROUND OF THE INVENTION It is well established that neuropeptides are derived from the proteolytic processing of the larger preprohormone precursor protein. Peptides within the preprohormone typically include, for example, Lys-Ar
g (KR), Arg-Arg (RR), Lys-Lys
Pairs of basic residues such as (KK) or Arg-Lys (RK) are adjacent. Neuropeptides also undergo post-translational modifications, including disulfide bond formation, glycosylation, C-terminal alpha-amidation, phosphorylation and sulfation
(VYH Hook et al., FASEB J, 8: 1269-1278, 1994
and refs therein). It is not uncommon for preprohormone processing to result in one or more neuropeptides (eg, proopiomelanocortin)
(H.Takahashiet al., FEBS Lett., 1981, 135: 97-10
2). Active cleaved neuropeptides exert their biological effects by interacting with the membrane protein superfamily of G-protein coupled receptors (Lefko
witz, Nature, 1991, 351: 353-354). G-protein coupled receptors contain approximately 7 to 20 amino acids of these 7
It is characterized by containing two conserved hydrophobic chains and binding to at least eight different hydrophilic loops. The G-protein family of coupled receptors includes
Includes dopamine receptors that bind to neuroleptic drugs used to treat psychiatric and neurological disorders. Other examples of this family include calcitonin, adrenergic agonists, endothelin, cAMP, adenosine, muscarinic agonists, acetylcholine, serotonin, histamine,
These include, but are not limited to, thrombin, kinin and follicle stimulating hormone, opsin, endothelial cell differentiation gene-1, rhodopsin, odorant, cytomegalovirus receptor.

[0003] Neurokinin B (also known as neuromedin K) is a neuropeptide known to excite nerves and to contract smooth muscle. Neurokinin B has been shown to be involved in vasodilation, feeding responses, and other behavioral responses. Mouse, bovine and rat orthologs have all been cloned and published (mouse neurokinin B precursor; K. Kako et al., Biomed. Res. 14: 253-259,
1993; Bovine neuromedin K precursor; KHHoshimaru
et al., Proc. Natl. Acad. Sci. 83: 7074-7078, 198
6; Rat neurokinin B precursor; TI Bonner et a
l., Brain Res. Mol. Brain Res. 2: 243-249, 1987),
Human sequences were not known prior to the disclosure of the present invention.

[0004]

SUMMARY OF THE INVENTION In one aspect, the invention is directed to an HPMB.
The invention relates to Q91 polypeptides, recombinant materials and methods for their production. In a further aspect, the invention relates to an HPM
It relates to one or more peptides formed by enzymatic processing of a BQ91 polypeptide. Another aspect relates to such HPMBQ91 polypeptides and polynucleotides, and the use of such peptides formed by enzymatic processing of the HPMBQ91 polypeptide. Such uses include, among others, nervous system disorders including pain, CNS-related disorders, gastrointestinal disorders, cardiovascular disorders,
Treatment of metabolic disorders including diabetes and obesity, smooth muscle disorders, inflammatory disorders including asthma, adenomas, leiomyomas, liposarcomas, lipomas, melanomas, pulmonary cartilage hamartomas, cancers including lung cancer, prostate cancer and breast cancer Is included. In yet another aspect, the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and methods for treating conditions associated with HPMBQ91 imbalance with the identified compounds. Yet another aspect of the invention relates to a diagnostic assay for detecting a disease associated with inappropriate HPMBQ91 activity or level. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate activity or levels of peptides resulting from HPMBQ91.

[0005]

DETAILED DESCRIPTION OF THE INVENTION Definitions The following definitions are provided to facilitate understanding of terms commonly used herein. "HPMBQ91 activity or H
"PMBQ91 polypeptide activity" or "HPMBQ9
Biological activity of 1 or HPMBQ91 polypeptide "
The term refers to the metabolic or physiological function of the HPMBQ91 polypeptide, including peptides derived from the HPMBQ91 polypeptide. The definition includes those activities with similar or improved activity or with reduced undesirable side effects. Also included are the antigenic and immunogenic activities of the HPMBQ91 polypeptide and peptides derived therefrom.

[0006] As used herein, "antibody" includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including Fab products or other immunoglobulin expression libraries. "Isolated" means altered "by the hand of man" from its natural state. When an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide that has been separated from its coexisting material in the natural state is described herein. The term "isolated" has been used in the textbook.

[0007] "Polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" refers to single-stranded and double-stranded D
NA, DNA that is a mixture of single- and double-stranded regions,
RNA that is single- and double-stranded RNA, and a mixture of single- and double-stranded regions, single-stranded or more typically double-stranded, or a mixture of single- and double-stranded regions. Including, but not limited to, hybrid molecules including DNA and RNA that may be used. In addition, "polynucleotide" refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases, as well as DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications have been made to DNA and RNA. Thus, a “polynucleotide” is typically a chemically, enzymatically or metabolically modified form of a polynucleotide as found in nature, as well as DNA and RNA in chemical forms characteristic of viruses and cells. Is included. Also,
"Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

[0008] A "polypeptide" comprises a peptide or protein comprising two or more amino acids linked together by peptide bonds or two or more amino acids linked together by modified peptide bonds. Peptide or protein, ie
Refers to peptide isostere. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. The polypeptide is a gene encoded by 2
It may contain an amino acid different from zero amino acids.
A "polypeptide" contains an amino acid sequence that has been modified by natural steps, such as post-translational processing, or by chemical modification techniques well known in the art. Such modifications can be found in basic texts and in more detailed research papers.
And in the extensive research literature. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains and the amino or carboxyl terminus. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, branched or unbranched, and cyclic. Cyclic, branched and branched cyclic polypeptides may result from natural post-translational processes or may be produced by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation,
Flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphotidylinositol covalent bond, cross-linking, cyclization, disulfide bond formation, demethylation, cross-linking Cross-link covalent bond formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation,
Transfer RNA mediated such as glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation Includes amino acid addition to proteins, as well as ubiquitination. For example, PROTEI
NS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed.,
TE Creighton, WH Freeman and Company, New Yo
rk, 1993 and Wold, F., Posttranslational Protei
n Modifications: Perspectives and Prospects, pgs.
1-12 in POSTTRANSLATIONALCOVALENT MODIFICATION OF
PROTEINS, BC Johnson, Ed., Academic Press, New
York, 1983; Seifter et al., "Analysis for protein
modifications and nonprotein cofactors ", Meth Enzym
ol (1990) 182: 626-646 and Rattan etal., "Protei
n Synthesis: Posttranslational Modifications and A
ging ", AnnNY Acad Sci (1992) 663: 48-62.

[0009] As used herein, the term "variant" is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not be different from the amino acid sequence of the polypeptide encoded by the control polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the control polypeptide and the variant are closely similar overall and, in many regions, identical.
Variant and control polypeptides may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The amino acid residue to be substituted or inserted may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a natural product, such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides are produced by mutagenesis techniques.
Alternatively, it can be produced by direct synthesis.

[0010] "% identity" refers to a measure of the relatedness of two polypeptide sequences or two polynucleotide sequences, as known in the art, as determined by comparing the sequences. Generally, the two sequences to be compared are positioned so as to provide the greatest correlation between the sequences. Examine the sequence of the two sequences to determine the number of positions where the amino acids or nucleotides between the two sequences exactly match, divide this by the total number of residues in the sequence, multiply by 100 and calculate the% identity value. obtain. This% identity value may be determined over the entire length of the sequences being compared (this is particularly suitable for sequences of the same or very similar length and for highly homologous homologues); Alternatively, a shorter specific length may be determined (this is more appropriate when the lengths are unequal or at low levels of homology).

"% Similarity" refers to a further degree of relationship between two polypeptide sequences, as is known in the art. The two sequences to be compared are arranged such that a maximum correlation between the sequences is obtained. The sequence of the two sequences is examined at each position, and a score is determined by the chemical and / or physical properties of the amino acid being compared at that position. Such chemical and / or physical properties include:
Includes amino acid side chain charge, size and hydrophobicity.

[0012] Methods for comparing the identity and similarity of two or more sequences are well known in the art. Thus, for example, Wisconsin Sequence Analysis
Package, version 9.1 (Devereux J et al, Nucleic Ac
ids Res, 12, 387-395, 1984, Genetics Computer Grou
p, available from Madison, Wisconsin, USA), using the programs BESTFIT and GAP, for example,% identity between two polynucleotides and% identity and% similarity between two polypeptide sequences. Sex may be determined. BESTFIT is Smith and Wat
Erman's "local homology" algorithm (Advances in Applied Mathematics, 2, 482-489, 1
981) to find the single best region of homology between the two sequences. BESTFIT is more suitable for comparing two polynucleotides or two polypeptide sequences of dissimilar length, and the program assumes that the shorter sequence represents a portion of the longer sequence. In contrast, GAP is based on Neddleman and Wunsch (J Mol Bio
l, 48, 443-453, 1970)
Find "maximum similarity" and arrange the two sequences GA
P is approximately the same length and is more suitable for comparing sequences that are considered to span the entire length. Preferably, the parameters used in each program, "Gap Weight" and "Length Weight" are 50 and 3 for the polynucleotide sequence and 50 for the polypeptide sequence, respectively. Are 12 and 4. Preferably, 2
When the two compared sequences are optimally positioned, the percent identity and similarity are determined.

Other programs for determining identity and / or similarity between sequences are also known in the art, for example, the BLAST family of programs (Altschul
S Fet al, J Mol Biol, 215, 403-410, 1990, Altschul
SF et al, Nucleic Acids Res., 25: 389-3402, 1997,
the National Center for Biotechnology Information
(NCBI), available from Bethesda, Maryland, USA and
NCBI at www.ncbi.nlm.nih.gov (accessible from the website) and FASTA (Pearson WR and Lipman D
J, Proc Nat Acad Sci USA, 85, 2444-2448, 1988, Wisc
onsin Sequence Analysis Package). Preferably, a BLOSUM62 amino acid substitution matrix (Henikoff S and Henikoff JG, Proc.
d Sci. USA, 89, 10915-10919, 1992) are used in polypeptide sequence comparisons, including when the nucleotide sequence is first translated into an amino acid sequence prior to comparison. Preferably, the program BESTFIT relates to a polynucleotide or polypeptide sequence of the invention to determine the% identity of the polynucleotide or polypeptide sequence to be analyzed, the optimally located% identity of the sequence to be analyzed and the control sequence. Set the program parameters to their default values.

Polypeptides of the Invention In one aspect, the present invention relates to HPMBQ91 polypeptides. The HPMBQ91 polypeptide includes a polypeptide of SEQ ID NO: 2; and a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; and at least 80% identity over its entire length to the amino acid sequence of SEQ ID NO: 2, more preferably SEQ ID NO: 2. At least 90 for 2
Polypeptides comprising amino acid sequences having a percent identity, more preferably at least 95% identity, are included. Furthermore, amino acid sequences that are at least 97-99% are highly preferred. The HPMBQ91 polypeptide also has at least 80% identity over its entire length to a polypeptide having the amino acid sequence of SEQ ID NO: 2, more preferably at least 90% to SEQ ID NO: 2.
Polypeptides having an amino acid sequence having a percent identity, more preferably at least 95% identity, are included. Furthermore, those with at least 97-99% are particularly preferred.

[0015] The polypeptide of SEQ ID NO: 2 is believed to be a human neurokinin B (neuromedin K) precursor. SEQ ID NO: 1 shows the human neurokinin B precursor c
SEQ ID NO: 2 is the amino acid sequence of the human neurokinin B precursor polypeptide. The precursor has a putative signal sequence of amino acid residues 1 to 20, which is believed to be cleaved between glycine 20 and alanine 21 based on homology to mouse, rat and bovine sequences. Preferably, the HPMBQ91 polypeptide exhibits at least one biological activity of HPMBQ91.

The HPMBQ91 polypeptide may be in the form of the "mature" protein, or may be part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include additional amino acid sequences, including secretory or leader sequences, prosequences, sequences that facilitate purification such as multiple histidine residues, or additional sequences for stability during recombination. is there.

In a further aspect, the present invention relates to an HPMB
For the Q91 peptide. Such peptides are HPMB
An amino acid sequence of SEQ ID NO: 3 produced by an enzyme from Q91, and a peptide comprising the amino acid sequence of SEQ ID NO: 3; and at least 80% identity to the amino acid sequence of SEQ ID NO: 3 over its entire length, more preferably SEQ ID NO: 3 Peptides comprising amino acid sequences having at least 90% identity to 3, more preferably at least 95% identity, but are not limited thereto. Furthermore, those with at least 97-99% are particularly preferred. The HPMBQ91 peptide also has at least 80% identity over its entire length to a polypeptide having the amino acid sequence of SEQ ID NO: 3, more preferably at least 90% to SEQ ID NO: 3.
Peptides having an amino acid sequence having a percent identity, even more preferably at least 95% identity, are included. Furthermore, those with at least 97-99% are particularly preferred.

SEQ ID NO: 3 is the amino acid sequence of a novel peptide derived from human neurokinin B precursor. The sequence of the peptide shown in SEQ ID NO: 3 shows the position of the putative N-terminal and C-terminal peptide cleavage sites (K
R and KR). Further peptides are
It is a known peptide of SEQ ID NO: 4, a human neurokinin B peptide. In the neurokinin B cleavage product, C
-Terminal glycine functions as an amide donor to the adjacent methionine and does not form part of the final active peptide. Preferably, the HPMBQ91 peptide exhibits at least one HPMBQ91 biological activity.

[0019] Fragments of the HPMBQ91 polypeptide are also included in the present invention. The fragment is the HPM
A polypeptide having an amino acid sequence that is identical to some but not all of the amino acid sequence of a BQ91 polypeptide. For HPMBQ91 polypeptides, the fragments may be "independent" or may be comprised within a larger polypeptide, in which the fragment forms part or a region, most preferably a single contiguous region. Included as Typical examples of the polypeptide fragment of the present invention include, for example, amino acid numbers of about 1 to 20, 21 to 40, 41 to 41 of HPMBQ91 polypeptide.
And fragments from 60, 61-80, 81-100 and 101 to the end. In this sense, "about" includes, on one or both ends, a range that is more or less than 5, 5, 4, 3, 2, or 1 more than the specified number.

Preferred fragments include, for example, a series of residues containing the amino terminus, or a series of residues containing the carboxyl terminus, or two series containing one amino terminus and the other carboxyl terminus. And truncated polypeptides having the amino acid sequence of the HPMBQ91 polypeptide, except that the following residues are deleted: Also, alpha helix and alpha helix forming region, beta sheet and beta sheet forming region, turn and turn forming region, coil and coil forming region, hydrophilic region, hydrophobic region, alpha amphiphilic region, beta amphiphilic region, variable region , Surface forming area,
Also preferred are fragments characterized by structural or functional attributes, such as fragments having a substrate binding region and a high antigenic index region. Other preferred fragments are biologically active fragments. Biologically active fragments are those that mediate HPMBQ91 activity, including those with similar or improved activity, or with reduced undesirable activity. Also included are fragments that are antigenic or immunogenic in animals, especially humans.

[0021] Preferably, all of these polypeptide fragments retain the biological activity of HPMBQ91, including the antigenic activity. Variants of the defined sequences and fragments also form part of the invention. Preferred variants are those that differ from the control by conservative amino acid substitutions;
That is, one residue is replaced by another residue having similar characteristics. A typical such replacement is
Between Ala, Val, Leu and Ile: between Ser and Thr; between acidic residues Asp and Glu; between Asn and Gln; and between basic residues Lys and Arg; or aromatic residues Phe and Tyr Between. Several, 5 to 10
Variants wherein one, one to five, or one or two amino acids are substituted, deleted or added in any combination are particularly preferred.

The HPMB of the present invention may be prepared in any suitable manner.
Q91 polypeptides and peptides can be produced. Such polypeptides include isolated natural polypeptides, polypeptides produced by recombinant techniques, polypeptides produced by synthetic methods, or polypeptides produced by a combination of these methods. Means for producing such polypeptides and peptides are well understood in the art.

Polynucleotides of the Invention Another aspect of the present invention relates to HPMBQ91 polynucleotides. HPMBQ91 polynucleotides include HP
Included are isolated polynucleotides encoding MBQ91 polypeptides and fragments thereof, as well as polynucleotides closely related to the polynucleotide. More specifically, the HPMBQ91 polynucleotide includes a polynucleotide having a nucleotide sequence having at least 85% identity over its entire length to the nucleotide sequence encoding the HPMBQ91 polypeptide of SEQ ID NO: 2, the polynucleotide of SEQ ID NO: 1 Polynucleotides that are at least 85% identical over their entire length. In this regard, polynucleotides that are at least 90% identical are particularly preferred, and polynucleotides that are at least 95% identical are more preferred. Furthermore, those with at least 97% are more preferred, those with at least 98-99% are most preferred, and those with at least 99% are even most preferred. HPMBQ91 polynucleotides include, in particular, polynucleotides having the sequence of SEQ ID NO: 1, sequences encoding the HPMBQ91 polypeptides contained in SEQ ID NO: 1. Also included in the nucleotide sequence contained in SEQ ID NO: 1 or in the cDNA insert in the plasmid deposited under ATCC Accession No. 97375, for hybridization under conditions that can be used for amplification or as a probe or marker. Nucleotide sequences having sufficient identity to the nucleotide sequence are also included in the HPMBQ91 polynucleotide. In addition, the HPMBQ91 polynucleotide is a HPMBQ91 polynucleotide that is expressed by a cDNA insert deposited with the ATCC under accession number 9375.
A nucleotide sequence that has at least 80% identity to the nucleotide sequence encoding the polypeptide, and a nucleotide sequence that includes at least 15 contiguous nucleotides of such a cDNA insert. In this regard, polynucleotides that are at least 90% identical are particularly preferred, and polynucleotides that are at least 95% identical are more preferred. In addition, at least 9
More preferably, it is at least 98-99%.
% Is most preferred, and at least 99% is most preferred. The present invention also provides polynucleotides that are complementary to all HPMBQ91 polynucleotides described above.

A deposited strain containing human HPMBQ91 cDNA was obtained on December 8, 1995 by American Type Culture.
Collection (ATCC), 12301 Park Lawn Drive, Rockvill
e, Maryland 20852, USA, ATCC Accession No. 9
7375. The deposited material (clone) is full length H
It contained PMBQ91 cDNA and was referred to on deposit as "DNA plasmid, 769547 (HPMBQ91)". The nucleotide sequence of the polynucleotide contained in the deposited material, as well as the amino acid sequence of the polypeptide encoded thereby, will dominate in any discrepancy with the sequence description herein. Deposits of the deposited strains are made under the terms of the Budapest Treaty on International Recognition of Microbial Deposits for Patent Procedure. After the patent is issued, there are no restrictions or conditions, and the shares are eventually sold. The deposited strain is provided solely for the convenience of those skilled in the art and has a 35 USC.
It does not acknowledge that a deposit is a feasible requirement, as required under Article 12.

The HPMBQ91 of the present invention is a human HPMB
It is structurally related to other neuropeptides, as shown in the results of sequencing the cDNA of SEQ ID NO: 1 encoding Q91. The cDNA sequence of SEQ ID NO: 1
Includes an open reading frame encoding a polypeptide of 21 amino acids (nucleotide numbers 1-360). The amino acid sequence of SEQ ID NO: 2 is derived from mouse neurokinin B precursor (K. Kako
et al., Biomed. Res. 14: 253-259, 1993), bovine neuromedin K precursor (KH Hoshimaru et al., Proc. Nat.
I. Acad. Sci. 83: 7074-7078, 1986) and rat neurokinin B precursor (TI Bonner et al., Brain Re.
s. Mol. Brain Res. 2: 243-249, 1987). The nucleotide sequence of SEQ ID NO: 1 is derived from mouse neurokinin B precursor (K. Kako et al., Biomed. Res. 1
4: 253-259, 1993), bovine neuromedin K precursor (K.
H. Hoshimaru et al., Proc. Natl. Acad. Sci. 83: 7074-
7078, 1986) and rat neurokinin B precursor
(TI Bonner et al., Brain Res.Mol. Brain Res.
2: 243-249, 1987).

CDNA derived from mRNA in human placenta, fetal liver spleen, and lymph node Merker cells
Expression sequence tag (EST) analysis using standard cloning and screening from the library (Adams, M .;
D., et al. Science (1991) 252: 1651-1656; Adams, M.
D. et al., Nature, (1992) 355: 632-634; Adams, M.
D., et al., Nature (1995) 377 Supp: 3-174) may be used to obtain a polynucleotide of the invention encoding HPMBQ91. The polynucleotides of the present invention can be obtained from natural sources, such as genomic DNA libraries, or can be synthesized using well-known and commercially available methods. The nucleotide sequence encoding the HPMBQ91 polypeptide of SEQ ID NO: 2 is SEQ ID NO: 1
(SEQ ID NO: 1)
To 360), or as a result of the redundancy (degeneracy) of the genetic code, may be a sequence encoding the polypeptide of SEQ ID NO: 2.

[0027] The polynucleotide of the present invention is HPMBQ9
When used for recombinant production of one polypeptide, the polynucleotide may itself comprise the coding sequence of the mature polypeptide or a fragment thereof;
In the reading frame, includes the coding sequence for the mature polypeptide or fragment, with other coding sequences, such as encoding a leader or secretory sequence, pre, pro, or preproprotein sequence, or other fusion peptide portion. There may be. For example, a marker sequence that facilitates purification of the fusion polypeptide can be encoded. In a particularly preferred embodiment of this aspect of the invention,
Marker sequences are provided in the pQE vector (Qiagen, Inc.) and Gentz et al., Proc Natl Acad Sci USA
(1989) 86: 821-824, or a HA-tag. Polynucleotides may also contain non-coding 5 'and 3' sequences, such as transcribed untranslated sequences, splice and polyadenylation signals, ribosome binding sites and m.
It may contain a sequence or the like that stabilizes RNA.

More preferred specific examples are several, 5 to 10
HPMBQ comprising the amino acid sequence of SEQ ID NO: 2, wherein 1 to 5, 1 to 3, 1 to 2, 1 or 1 amino acid residues are substituted, deleted or added in any combination.
Polynucleotides encoding 91 variants. The present invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention particularly relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides. As used herein, the term “stringent conditions” means that hybridization occurs only when there is at least 95%, preferably at least 97%, identity between the sequences.

The nucleotide sequence contained in SEQ ID NO: 1 or a fragment thereof, or the ATCC has accession number 9
A polynucleotide of the invention that is identical or sufficiently identical to the cDNA insert or fragment thereof in the plasmid deposited at 7375 is used as a hybridization probe for cDNA and genomic DNA, and encodes the full-length HPMBQ91 polypeptide. CDNA and genomic clones were isolated
CDNA and genomic clones of other genes with high sequence similarity to the BQ91 gene can be isolated. Such hybridization techniques are known to those skilled in the art. Typically, these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to the control nucleotide sequence.
Generally, probes contain at least 15 nucleotides. Preferably, such probes have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes range from 30 to 50 nucleotides.

The HPMBQ91 polypeptide-encoding polynucleotide is screened under stringent hybridization conditions with a labeled probe having SEQ ID NO: 1 or a suitable library thereof, using the full length c-DNA containing the polynucleotide sequence.
DNA and genomic clones may be obtained by isolation. Thus, in another embodiment, the HPMBQ91 polynucleotide of the invention further includes a nucleotide sequence comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence having SEQ ID NO: 1 or a fragment thereof. In addition, peptides containing the amino acid sequence encoded by the nucleotide sequence obtained under the above hybridization conditions are also HP
Included in the MBQ91 polypeptide. Such hybridization techniques are well known to those skilled in the art. Stringent hybridization conditions are as described above, or alternatively, 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.
6) 5x Denhardt's solution, 10% dextran sulfate and 20 microgram / ml denatured cut salmon sperm DN
Incubate overnight at 42 ° C. in the solution containing A, then wash the filter at about 65 ° C. with 0.1 × SSC. The polynucleotides and polypeptides of the present invention may be used as research reagents and materials to find treatment and diagnosis of animal and human diseases.

Vectors, Host Cells, Expression The present invention also relates to vectors comprising the polynucleotide or polynucleotides of the invention, host cells genetically engineered with the vectors of the invention and recombinant polypeptides of the invention. Related to manufacturing. Such a protein can be produced using a cell-free translation system and RNA derived from the DNA construct of the present invention. To produce a recombinant,
The host cells can be genetically engineered to incorporate an expression system for a polynucleotide of the invention, or a portion thereof. The introduction of the polynucleotide into the host cell is
vis et al., BASIC METHODS IN MOLECULARBIOLOGY (198
6) and Sambrook et al., MOLECULAR CLONING: A LA
BORATORYMANUAL, 2nd Ed., Cold Spring Harbor Labora
tory Press, Cold Spring Harbor, NY (1989), and many other standard laboratory manuals, including calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, and the like. Cationic lipid-mediated transfection, electroporation, transduction, scraping, ballistic transfer or infection, and the like.

Representative of suitable hosts are bacterial cells such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtili.
s) cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells, such as Drosophila S2 and Spodoptera Sf
9 (Spodoptera Sf9) cells; animal cells such as CHO,
COS, HeLa, C127, 3T3, BHK, HEK
293 and Bows melanoma cells; and plant cells.

[0033] A variety of expression systems can be used. Such systems include, inter alia, chromosomal, episomal and virus-derived systems, such as from bacterial plasmids, from bacteriophages, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from baculoviruses, papovaviruses, etc. And vectors derived from viruses such as vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus, and vectors derived from combinations thereof, such as plasmids such as cosmids and phagemids and vectors from bacteriophage genetic elements. . The expression system may contain regulatory regions that control and cause expression. In general, any system or vector suitable for holding, extending or expressing a polynucleotide and producing the polypeptide in a host may be used. Various well-known and conventional techniques, for example, Sambrook et al., MOLECULAR CLONING, A
By the technique described in LABORATORY MANUAL (supra),
A suitable nucleotide sequence may be inserted into the expression system.

To secrete the translated protein into the endoplasmic reticulum lumen, peripheral lumen or extracellular environment, an appropriate secretion signal can be incorporated into the desired polypeptide. These signals may be signals unique to the polypeptide, or may be heterologous signals. H
PMBQ91 polypeptides may be expressed for use in screening assays. The polypeptide may be produced at the cell surface, in which case the cells may be collected prior to use in a screening assay. Alternatively, the HPMBQ91 polypeptide or polypeptides are secreted into the medium, and the medium is then recovered to recover and purify the polypeptide or polypeptides.
If produced intracellularly, the cells must first be lysed and then the polypeptide recovered.

[0035] HPMBQ91 polypeptides include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Can be recovered and purified from recombinant cell culture by well known methods. Most preferably, high performance liquid chromatography is used for purification. If a polypeptide denatures during isolation and / or purification, it can be reconstituted into an active conformation using well known methods for regenerating proteins.

Diagnostic Assay The present invention also provides HPMBQ for use as a diagnostic reagent.
It also relates to the use of 91 polynucleotides. Detection of a mutant form of the HPMBQ91 gene involved in dysfunction is determined by HPM
Diagnostic means are provided in addition to or to determine a diagnosis of a disease or susceptibility to a disease resulting from underexpression, overexpression or altered expression of BQ91. HPM
Individuals having a mutation in the BQ91 gene can be detected at the DNA level by various methods.

The nucleic acid to be used for diagnosis can be obtained from cells of a subject, for example, blood, urine, saliva, tissue biopsy or autopsy material. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification methods prior to analysis.
RNA or cDNA can also be used in the same way. Deletions and insertions can be detected by changes in the size of the amplification product relative to the normal genotype. Point mutations can be identified by hybridizing the amplified DNA to a labeled HPMBQ91 nucleotide sequence.
Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion, by differences in melting temperatures or by HPLC. DNA sequence differences can also be detected by changes in the electrophoretic mobility of the DNA fragments in the gel, with or without denaturants, or by direct DNA sequencing. For example, Myers et al., Science (1
985) 230: 1242. Sequence changes at specific locations can also be achieved by nuclease protection assays, such as RNas
e and S1 protection or chemical cleavage can also reveal. Cotton et al., Proc Natl Acad
See Sci USA (1985) 85: 4397-4401. In another embodiment, an array of oligonucleotide probes comprising the nucleotide sequence of HPMBQ91 or a fragment thereof can be constructed, for example, to provide effective screening for genetic variation. Array techniques are well-known and widely applicable, and can be used to address various issues in molecular genetics, including gene expression, genetic linkage and genetic alterations (eg,
M. Chee et al., Science, Vol 274, pp 610-613 (1996)
checking).

The diagnostic assay was performed using the HP
By detecting mutations in the MBQ91 gene, nervous system disorders including pain, CNS-related disorders, gastrointestinal disorders, cardiovascular disorders, metabolic disorders including diabetes and obesity, among others,
Methods for diagnosing or examining susceptibility to smooth muscle disorders, inflammatory disorders including asthma, adenoma, leiomyoma, liposarcoma, lipoma, melanoma, pulmonary cartilage hamartoma, lung cancer, prostate cancer and breast cancer including breast cancer I will provide a.

In addition, nervous system disorders including pain, CNS-related disorders, gastrointestinal disorders, cardiovascular disorders, metabolic disorders including diabetes and obesity, smooth muscle disorders, inflammatory disorders including asthma, adenomas, leiomyomas, Liposarcoma, lipoma,
Cancers, including melanoma, pulmonary cartilage hamartoma, lung cancer, prostate cancer and breast cancer, have HPMBQ91 polypeptides or HPMB abnormally reduced or increased compared to samples from subjects.
Diagnosis can be by a method that includes examining the level of Q91 mRNA. The decreased or increased expression can be determined at the RNA level using methods well known in the art for quantification of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. it can. Assays that can be used to determine levels of a protein, such as an HPMBQ91 polypeptide, in a sample derived from a host are well-known to those of skill in the art. Such assays include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays.

Chromosome Assay The nucleotide sequences of the present invention can also be used in chromosome localization. The sequences can specifically target and hybridize to specific locations on individual human chromosomes. Mapping related sequences to chromosomes according to the present invention is an important first step in linking these sequences to gene-related diseases. Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be associated with genetic map data. Such data is
For example, V. McKusick, Mendelian Inheritance in Man
(Johns Hopkins University Welch Medical Library
Available online). The relationship between the disease and the gene mapped to the same chromosomal region is then identified by linkage analysis (simultaneous inheritance of physically adjacent genes).

The HPMBQ91 gene has markers D12S1632 and D12S35 corresponding to 12q14.
It maps to chromosome 12 between five. 12a14-
At 15 there is a cleavage point (breapoint) region, resulting in a high incidence of chromosomal changes, which is associated with various cancers.
Differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If the mutation is observed in some or all of the affected individuals and not in normal individuals, then the mutation may be responsible for the disease.

The nucleotide sequences of the present invention can also be used in tissue localization. Using such techniques, HP
By detecting the mRNA encoding the MBQ91 polypeptide, the expression pattern of the HPMBQ91 polypeptide in the tissue can be determined. These techniques include in situ hybridization techniques, and nucleotide amplification techniques (eg, PCR). Such techniques are well-known to those skilled in the art. The results of these studies suggest a normal function of the polypeptide in the organism. In addition, the normal expression pattern of HPMBQ91 mRNA and mRN encoded by HPMBQ91 gene
Comparison of the expression pattern of A with the mutant HP
Role of MBQ91 polypeptide or normal HPM
Useful for insight into the role of inappropriate expression of BQ91 polypeptide. Such inappropriate expression may be of a temporal, spatial, or simply quantitative nature.

Antibodies Immunospecific antibodies to the HPMBQ91 polypeptide can also be obtained using the polypeptides and peptides of the present invention or their fragments or analogs thereof, or cells expressing them, as immunogens.
The term "immunospecific" means that the antibody has a substantially greater affinity for the polypeptides of the present invention than for other related polypeptides in the prior art.

Antibodies raised against HPMBQ91 polypeptides and peptides can be obtained by administering the polypeptide or epitope-bearing fragment, analog or cell to an animal, preferably a non-human animal, using conventional laboratory techniques. Can be. For preparing monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. For example, the hybridoma method (Kohler, G. and Milstein, C.,
Nature (1975) 256: 495-497), trioma method, human B-
Cell hybridoma method (Kozbor et al., Immunology To
day (1983) 4:72) and the EBV-hybridoma method (Col.
e et al., MONOCLONAL ANTIBODIES ANDCANCER THERAPY,
pp. 77-96, Alan R. Liss, Inc., 1985).

The techniques used for producing single-chain antibodies (US Pat. No. 4,946,778) can be applied to produce single-chain antibodies against the polypeptides of the present invention. Also, other organisms, including transgenic mice or other mammals, can be used to express humanized antibodies. Using the above-described antibodies, clones expressing the polypeptide may be isolated or identified, or the polypeptide may be purified by affinity chromatography. H
Using antibodies to the PMBQ91 polypeptide, nervous system disorders including pain, CNS-related disorders, gastrointestinal disorders, cardiovascular disorders, metabolic disorders including diabetes and obesity, among others.
Smooth muscle disorders, inflammatory disorders including asthma, adenomas, leiomyomas, liposarcoma, lipomas, melanomas, pulmonary cartilage hamartomas, cancers including lung, prostate and breast cancers may be treated.

Vaccine Another aspect of the invention is a method of eliciting an immunological response in a mammal comprising the step of eliciting an antibody and / or a HPMBQ91 polypeptide or peptide or fragment thereof sufficient to generate a T cell immune response. Inoculated in mammals, especially nervous system disorders including pain, CNS related disorders, gastrointestinal disorders, cardiovascular disorders, metabolic disorders including diabetes and obesity, smooth muscle disorders, inflammatory disorders including asthma, adenomas, leiomyomas , Liposarcoma, lipoma, melanoma, pulmonary cartilage hamartoma, lung cancer, prostate cancer and breast cancer. Another aspect of the invention is a method of inducing an immunological response in a mammal, comprising delivering an HPMBQ91 polypeptide or peptide in vivo via a vector that directs expression of the HPMBQ91 polynucleotide, Eliciting such an immunological response, producing antibodies, and protecting the animal from disease.

A further aspect of the present invention is an immunological / vaccine formulation (composition) that, when introduced into a mammalian host, induces an immunological response to the HPMBQ91 polypeptide or peptide in the mammal. (The composition comprises a HPMBQ91 polypeptide, a peptide,
Or including the HPMBQ91 gene). The vaccine formulation may further include a suitable carrier. HP
Because the MBQ91 polypeptide or peptide may degrade in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the patient; It includes aqueous and non-aqueous sterile suspensions which may contain a thickening agent. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, or stored in a lyophilized condition, which only requires the addition of a sterile liquid carrier immediately before use. Vaccine formulations may also include adjuvant systems to enhance the immunogenicity of the formulation, such as oil-in-water and other systems known in the art. The dosage depends on the specific activity of the vaccine and can be readily determined by routine experimentation.

Screening Assays HPMBQ91 polypeptides and peptides of the invention can be used in screening methods for compounds that activate (agonist) or inhibit (referred to as antagonists or inhibitors) the activity of HPMBQ91 polypeptides or peptides of the invention. May be used. Thus, the polypeptides or peptides of the present invention may be used, for example, to identify agonists or antagonists from a mixture of cells, cell-free preparations, chemical libraries and natural products. These agonists or antagonists may be natural substrates, ligands, receptors, etc. of the polypeptide or peptide of the present invention, or may mimic the structural or functional properties of the polypeptide of the present invention. Coligan et al., Current Protocolsi
n See Immunology 1 (2): Chapter 5 (1991).

[0049] HPMBQ91 polypeptides and peptides are involved in one or more biological functions, including one or more pathogenicity. Accordingly, it is desirable to find compounds and agents that stimulate HPMBQ91 polypeptides or peptides on the one hand and compounds or agents that can inhibit the function of HPMBQ91 polypeptides or peptides on the other hand. In general, agonists are nervous system disorders including pain, CNS-related disorders, gastrointestinal disorders, cardiovascular disorders, metabolic disorders including diabetes and obesity,
For the treatment and prevention of symptoms such as smooth muscle disorders, inflammatory disorders including asthma, adenoma, leiomyoma, liposarcoma, lipoma, melanoma, pulmonary cartilage hamartoma, lung cancer, cancer including prostate cancer and breast cancer. Used. Antagonists are nervous system disorders including pain, CNS-related disorders, gastrointestinal disorders, cardiovascular disorders, metabolic disorders including diabetes and obesity,
Various treatment and prevention of symptoms such as smooth muscle disorders, inflammatory disorders including asthma, adenoma, leiomyoma, liposarcoma, lipoma, melanoma, pulmonary cartilage hamartoma, lung cancer, cancer including prostate cancer and breast cancer Available to

In general, such a screening operation involves H
This involves using suitable cells that express the PMBQ91 polypeptide or that respond to the HPMBQ91 polypeptide of the invention. Such cells include cells from mammals, yeast, Drosophila or E. coli. Cells expressing the HPMBQ91 polypeptide (or cell membrane with the expressed polypeptide) or responding to the HPMBQ91 polypeptide are then contacted with a test compound to observe binding or stimulation or inhibition of a functional response. For HPMBQ91 activity, the ability of cells contacted with the candidate compound is compared to the same cells without contact.

The assay may be performed using a label directly or indirectly bound to the candidate compound, or in an assay involving competition with a labeled competitor, by testing for adhesion to cells bearing the HPMBQ91 polypeptide. Compound binding can be easily tested. In addition, it was determined whether a candidate compound produced a signal generated by activation of the HPMBQ91 polypeptide.
These assays may be tested using a detection system suitable for cells having a Q91 polypeptide. In general,
Inhibitors of activation are assayed in the presence of a known agonist and observe the effect of the presence of the candidate compound on activation by the agonist. Standard methods for performing such assays are well-known in the art.

Examples of potential HPMBQ91 polypeptides or peptide antagonists are antibodies or, in some cases, oligonucleotides or proteins closely related to the ligands, substrates, receptors, etc., of the HPMBQ91 polypeptide, eg, ligands, substrates, Includes small molecules that bind to a polypeptide of the invention, but do not elicit a response, such that the activity of the receptor fragment or polypeptide is prevented.

Methods of Prevention and Treatment The present invention relates to nervous system disorders, including pain, associated with both excessive or insufficient amounts of HPMBQ91 polypeptide.
NS-related disorders, gastrointestinal disorders, cardiovascular disorders, metabolic disorders including diabetes and obesity, smooth muscle disorders, inflammatory disorders including asthma, adenomas, leiomyomas, liposarcomas, lipomas, melanomas, pulmonary cartilage hamartomas, lung Methods for treating abnormal conditions, such as cancer, including cancer, prostate cancer and breast cancer.

If the activity of the HPMBQ91 polypeptide or peptide is in excess, several methods are available. One method is to use an inhibitory compound (antagonist) in an amount effective to inhibit the function of the HPMBQ91 polypeptide, for example, by blocking the binding of ligands, substrate elements, etc., or by inhibiting a second signal. ) With a pharmaceutically acceptable carrier to the subject, thereby ameliorating the abnormal condition. In another alternative, the endogenous HPMBQ91
A soluble form of the HPMBQ91 polypeptide capable of binding to ligands, substrates and the like in competition with the polypeptide may be administered. Typical examples of such competitors include fragments of the HPMBQ91 polypeptide.

In yet another alternative, expression of the gene encoding endogenous HPMBQ91 polypeptide can be inhibited using expression blocking techniques. Known such methods include the use of internally generated or exogenously administered antisense sequences (eg, O'Connor, J Neuroc.
hem (1991) 56: 560 in Oligodeoxynucleotides as Anti
sense Inhibitors of Gene Expression, CRC Press, Bo
ca Raton, FL (1988)). Alternatively, oligonucleotides that form a triple helix ("triplex") with the gene can be provided (eg, Lee et al., Nucleic Acids Res (1979) 6: 3073; C
ooney et al., Science (1988) 241: 456; Dervan et a
l., Science (1991) 251: 1360). These oligomers can be administered per se, or the relevant oligomers can be expressed in vivo. Synthetic antisense or triplex oligonucleotides may contain modified bases or backbones. Examples of the latter include methyl phosphate, phosphorothioate or peptide nucleic acid backbones. Such scaffolds are incorporated into antisense or triplex oligonucleotides to protect them from degradation by nucleases, which are well known in the art. Antisense and triplex molecules synthesized with these or other modified scaffolds also form part of the invention.

In addition, expression of the HPMBQ91 polypeptide may be prevented by use of a ribozyme specific for the HPMBQ91 mRNA sequence. Ribozymes are enzymatically active RNAs that can be natural or synthetic (eg, Usman, N, et al., Curr. Opin. Struct. Bi.
ol (1996) 6 (4), 527-33). Synthetic ribozymes can be designed to specifically cleave HPMBQ91 mRNA at selected locations. Ribozymes may be synthesized with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively, the ribozyme may be synthesized on a non-natural backbone, such as, for example, 2'-O-methyl RNA, to protect it from ribonuclease degradation and to include modified bases.

Several additional methods are available for treating abnormal conditions associated with an under-expression of HPMBQ91 and its activity. The first method is HPMBQ9
Administering to the subject a therapeutically effective amount of a compound that activates one polypeptide (ie, an agonist described above) with a pharmaceutically acceptable carrier, thereby ameliorating the abnormal condition. Alternatively, gene therapy may be used to effect the endogenous production of HPMBQ91 by relevant cells in the subject. For example, as described above, the polynucleotides of the invention may be engineered to be expressed in a replication defective retroviral vector. The retroviral expression construct is then isolated and introduced into packaging cells transduced with an RNA-containing retroviral plasmid vector encoding the polypeptide of the present invention so that the packaging cells contain the desired gene containing the gene of interest. Viral virus particles may be generated. These producer cells may be administered to a subject to manipulate the cells in vivo and to express the polypeptide in vivo. For an overview of gene therapy, see Chapter 2
0, Gene Therapy and other Molecular Genetic-based
Therapeutic Approaches, (and references therein) i
n Human Molecular Genetics, T Strachan and AP Rea
d, see BIOS Scientific Publishers Ltd (1996). An alternative is to administer a therapeutic amount of HPMBQ91 polypeptide in combination with a suitable pharmaceutical carrier.

Formulation and Administration HPMBQ91 polypeptides and peptides, agonists and antagonist peptides or small molecules may be formulated in combination with a suitable pharmaceutical carrier. Such formulations comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. Such carriers include saline, buffered saline, dextrose,
It includes, but is not limited to, water, glycerol, ethanol, and combinations thereof. The formulation should suit the route of administration and is well known to those skilled in the art. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more components of the composition of the invention described above.

The polypeptides and other compounds of the present invention may be used alone or in combination with other compounds, such as therapeutic compounds. A preferred form of systemic administration of the pharmaceutical composition typically involves injection by intravenous injection. Other injection routes, such as subcutaneous, intramuscular or intraperitoneal, can also be used. Another means for systemic administration involves transmucosal or transdermal administration with penetrants or other surfactants, such as bile salts or fusidic acid. In addition, if properly formulated in enteric or capsule formulations, oral administration is also possible. The administration of these compounds may be topical and / or localized, in the form of salves, pastas, gels and the like.

The required dosage range depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the condition of the subject, and the judgment of the consulting physician. However, the appropriate dose is
The range is from 0.1 to 100 μg / g. However, given the varying potency of the different compounds available and the different routes of administration, various changes in the required dosage are conceivable. For example, oral administration may require higher doses than intravenous injection. Variations in these dosage levels can be made using standard empirical routines for optimization well known in the art.

In the above-mentioned therapeutic methods, often referred to as “gene therapy”, the polypeptide used for the treatment can be obtained endogenously in the subject. Thus, for example, cells of interest can be engineered with polynucleotides such as DNA or RNA using a retroviral plasmid vector to encode the polypeptide ex vivo. Next, the cells are introduced into the subject. Except for describing the following examples in particular detail, they were performed using standard techniques well known and conventional to those skilled in the art. The examples are illustrative and do not limit the invention.

Example HPMBQ91 was first identified in a cDNA library from human placenta. Clones were identified as novel human proteins with homology to several previously described neurokinin precursor proteins. For example, HPMBQ91 has an open reading frame (nucleotide number 1) encoding a polypeptide of 121 amino acids.
３360), which has about 70% identity at 121 amino acid residues to mouse neurokinin B precursor (using Smith-Waterman) (K. Kako et al., Bi.
omed. Res. 14: 253-259, 1993). The sequence of putative neuropeptide precursors suggested that the cDNA clone was full length. Subsequent extended sequencing of both the sense and antisense strands allows for the production and purification of the HPMBQ91 protein, the full length cDNA of HPMBQ91.
Copies were subcloned into bacterial and baculovirus expression vectors, pQE-9 (Qiagen, Inc., Chatsworth, CA) and pA2GP, respectively.

[0063] SEQ ID NO: 1 -132 CTCCACTCGGTTTCTCTCTTTGCAGGAGCACCGGCAGCACCAGTGTGTGAGGGGAGCAGG -72 CAGCGGTCCTAGCCAGTTCCTTGATCCTGCCAGACCACCCAGCCCCTGGCACAGAGCTGC -12 TCCACAGGCACCATGAGGATCATGCTGCTATTCACAGCCATCCTGGCCTTCAGCCTAGCT 49 CAGAGCTTTGGGGCTGTCTGTAAGGAGCCACAGGAGGAGGTGGTTCCTGGCGGGGGCCGC 109 AGCAAGAGGGATCCAGATCTCTACCAGCTGCTCCAGAGACTCTTCAAAAGCCACTCATCT 169 CTGGAGGGATTGCTCAAAGCCCTGAGCCAGGCTAGCACAGATCCTAAGGAATCAACATCT 229 CCCGAGAAACGTGACATGCATGACTTCTTTGTGGGACTTATGGGCAAGAGGAGCGTCCAG 289 CCAGACTCTCCTACGGATGTGAATCAAGAGAACGTCCCCAGCTTTGGCATCCTCAAGTAT 349 CCCCCGAGAGCAGAATAGGTACTCCACTTCCGGACTCCTGGACTGCATTAGGAAGACCTC 409 TTTCCCTGTCCCAATCCCCAGGTGCGCACGCTCCTGTTACCCTTTCTCTTCCCTGTTCTT 469 GTAACATTCTNGTGCTTTGACTCCTTCTCCATCTTTTCTACCTGACCCTGGTGTGGAAAC 529 TGCATAGTGAATATCCCCAACCCCAATGGGCATTGACTGTAGAATACCCTAGAGTTCCTG 589 TAGTGTCCTACATTAAAAATATAATGTCTCTCTCTATTCCTCAACAAATAAAGGATTT

SEQ ID NO: 2 1 MRIMLLFTAILAFSLAQSFGAVCKEPQEEVVPGGGRSKRDPDLYQLLQRLFKSHSSLEGL 61 LKALSQASTDPKESTSPEKRDMHDFFVGLMGKRSVQPDSPTDVNQENVPSFGILKYPPRA 121 E

SEQ ID NO: 31 DPDLYQLLQRLFKSHSSLEGLLKALSQASTDPKESTSPE 39

SEQ ID NO: 4 1 DMHDFFVGLM 10

[0067]

[Sequence list]

(1) General information (i) Applicant: SmithKline Beecham Public Limited Company and Human Genome Sciences (ii) Title of invention: Novel compound (iii) Number of sequences: 4 (iv) Contact : (A) Name: SmithKline Beecham, Corporate Intellectual Property (B) Street: New Horizons Court (C) City: Brenford (D) State: Middlesex (E) Country: United Kingdom (F) Postal code: TW8 9EP (v) Computer readable form: (A) Medium form: Diskette (B) Computer: IBM compatible (C) Operating system: DOS (D) Software: Windows version 2.0
Fast SEQ for

(2) Information on SEQ ID NO: 1 (i) Sequence characteristics: (A) Sequence length: 778 base pairs (B) Sequence type: nucleic acid (C) Number of strands: single strand (D ) Topology: linear (ii) Molecular type: cDNA (xi) Sequence description: SEQ ID NO: 1 TGGTTCCTGG CGGGGGCCGC 240 AGCAAGAGGG ATCCAGATCT CTACCAGCTG CTCCAGAGAC TCTTCAAAAG CCACTCATCT 300 CTGGAGGGAT TGCTCAAAGC CCTGAGCCAG GCTAGCACAG ATCCTAAGGA ATCAACATCT 360 CCCGAGAAAC GTGACATGCA TGACTTCTTT GTGGGACTTA TGGGCAAGAG GAGCGTCCAG 420 CCAGACTCTC CTACGGATGT GAATCAAGAG AACGTCCCCA GCTTTGGCAT CCTCAAGTAT 480 CCCCCGAGAG CAGAATAGGT ACTCCACTTC CGGACTCCTG GACTGCATTA GGAAGACCTC 540 TTTCCCTGTC CCAATCCCCA GGTGCGCACG CTCCTGTTAC C CTTTCTCTT CCCTGTTCTT 600 GTAACATTCT NGTGCTTTGA CTCCTTCTCC ATCTTTTCTA CCTGACCCTG GTGTGGAAAC 660 TGCATAGTGA ATATCCCCAA CCCCAATGGG CATTGACTGT AGAATACCCT AGAGTTCCTG 720 TAGTGTCCTA CATTAAAAAT ATAATGTCTCATCTCCTCATC

(2) Information on SEQ ID NO: 2 (i) Sequence characteristics: (A) Sequence length: 121 amino acids (B) Sequence type: amino acids (C) Number of chains: one (D) Topology : Linear (ii) molecular type: protein (xi) description of sequence: SEQ ID NO: 2: Met Arg Ile Met Leu Leu Phe Thr Ala Ile Leu Ala Phe Ser Leu Ala 1 5 10 15 Gln Ser Phe Gly Ala Val Cys Lys Glu Pro Gln Glu Glu Val Val Pro 20 25 30 Gly Gly Gly Arg Ser Lys Arg Asp Pro Asp Leu Tyr Gln Leu Leu Gln 35 40 45 Arg Leu Phe Lys Ser His Ser Ser Leu Glu Gly Leu Leu Lys Ala Leu 50 55 60 Ser Gln Ala Ser Thr Asp Pro Lys Glu Ser Thr Ser Pro Glu Lys Arg 65 70 75 80 Asp Met His Asp Phe Phe Val Gly Leu Met Gly Lys Arg Ser Val Gln 85 90 95 Pro Asp Ser Pro Thr Asp Val Asn Gln Glu Asn Val Pro Ser Phe Gly 100 105 110 Ile Leu Lys Tyr Pro Pro Arg Ala Glu 115 120

(2) Information on SEQ ID NO: 3 (i) Sequence characteristics: (A) Sequence length: 39 amino acids (B) Sequence type: amino acids (C) Number of chains: one (D) Topology : Linear (ii) Type of molecule: Peptide (xi) Description of sequence: SEQ ID NO: 3 Asp Pro Asp Leu Tyr Gln Leu Leu Gln Arg Leu Phe Lys Ser His Ser 1 5 10 15 Ser Leu Glu Gly Leu Leu Lys Ala Leu Ser Gln Ala Ser Thr Asp Pro 20 25 30 Lys Glu Ser Thr Ser Pro Glu 35

(2) Information on SEQ ID NO: 4 (i) Sequence characteristics: (A) Sequence length: 10 amino acids (B) Sequence type: amino acids (C) Number of chains: 1 (D) Topology : Linear (ii) molecular type: peptide

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/00 ABE A61K 48/00 AED ADP C07K 14/48 ADS 16/22 39/395 ABJ C12P 21/02 CA ADU C12Q 1 / 68 A 45/00 ACJ G01N 33/50 48/00 AED A61K 37/02 AAB C07K 14/48 AAH 16/22 AAR C12P 21/02 ABE C12Q 1/68 ADP G01N 33/50 ADS (71) Applicant 597034358 Human・ Genom Sciences, Inc. HUMAN GENOME SCIENCE ES, INC. Key West Avenue 9410, Rockville, MD 20850-3338 United States of America 9410 (72) Inventor David Malcolm Duckworth, United Kingdom -M 19.5 ADB, Essex, Harlow, Third Avenue, New Frontiers Science Park South, SmithKline Beecham Pharmaceuticals (72) Inventor Greg A Hastings United States 20850 Rockville, Maryland, Key West Ave 9410, Human Genome Sciences (72) Inventor Stephen M. Leuven United States 20850 Rockville, Maryland, Key West Ave 9410, Human Genom・ Sciences

Claims (22)

[Claims] 1. A nucleotide sequence having at least 85% identity over its entire length to a nucleotide sequence encoding the HPMBQ91 polypeptide of SEQ ID NO: 2; or an isolated nucleotide sequence having nucleotide complementarity to said nucleotide sequence. Polynucleotide. 2. The polynucleotide of claim 1, wherein the nucleotide sequence comprises a sequence encoding an HPMBQ91 polypeptide contained in SEQ ID NO: 1. 3. An isolated polynucleotide comprising a nucleotide sequence whose nucleotide sequence is at least 85% identical to the nucleotide sequence contained in SEQ ID NO: 1. 4. The polynucleotide according to claim 3, which is the polynucleotide of SEQ ID NO: 1. 5. The polynucleotide according to claim 1, which is DNA or RNA. 6. When present in a compatible host cell,
A DNA or RNA molecule comprising an expression system capable of producing a polypeptide comprising an amino acid sequence having at least 85% identity to the polypeptide of SEQ ID NO: 2. 7. A host cell comprising the expression system according to claim 6. 8. The host according to claim 7, wherein the host is HPMBQ91.
Cultured under conditions sufficient to produce the polypeptide;
A method for producing an HPMBQ91 polypeptide, comprising recovering a PMBQ91 polypeptide from a culture. 9. A method for producing a cell that produces an HPMBQ91 polypeptide, wherein the host cell is produced by the expression system according to claim 6, such that the host cell produces the HPMBQ91 polypeptide under appropriate culture conditions. A method comprising transforming or transfecting. 10. An HPMBQ91 polypeptide comprising an amino acid sequence that is at least 75% identical over its entire length to the amino acid sequence of SEQ ID NO: 2. 11. The polypeptide according to claim 10, comprising the amino acid sequence of SEQ ID NO: 2. 12. The polypeptide according to claim 10, which has the amino acid sequence of SEQ ID NO: 2. 13. An HP derived from the polypeptide of SEQ ID NO: 2.
MBQ91 peptide (excluding the peptide sequence of SEQ ID NO: 4). 14. The HPMBQ91 peptide according to claim 13, which comprises the sequence of SEQ ID NO: 3. 15. The method according to claim 13, which has the sequence of SEQ ID NO: 3.
The described HPMBQ91 peptide. 16. An antibody immunospecific for the HPMBQ91 polypeptide of claim 10. 17. A method for treating a subject in need of enhancing the activity or expression of the HPMBQ91 polypeptide according to any one of claims 10 to 15, or a peptide derived therefrom, comprising: (a) the subject Administering a therapeutically effective amount of an agonist to said polypeptide; and / or (b) producing an HPMBQ91 polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 in such a form as to produce said polypeptide or peptide in vivo. Providing to the subject a nucleotide sequence that has at least 85% identity over its entire length to the nucleotide sequence encoding the peptide, or a nucleotide sequence that has complementarity to the nucleotide sequence. Including methods. 18. A method for treating a subject in need of inhibiting the activity or expression of the HPMBQ91 polypeptide according to any one of claims 10 to 15, or a peptide derived therefrom, comprising: Administering to said subject an effective amount of an antagonist to said polypeptide; and / or (b) administering to said subject a nucleic acid molecule that inhibits expression of a nucleotide sequence encoding said polypeptide; and / or (c) administering to said subject A method comprising administering to said subject a therapeutically effective amount of a polypeptide that competes with said polypeptide for a ligand, substrate or receptor. 19. The HPM of claim 10 in a subject.
A method for diagnosing a disease or susceptibility to a disease associated with the expression or activity of a BQ91 polypeptide, comprising: (a) the presence or absence of a mutation in the nucleotide sequence encoding the HPMBQ91 polypeptide in the genome of the subject; And / or (b) H in a sample from said subject
A method comprising analyzing for the presence or amount of expression of a PMBQ91 polypeptide. 20. HPM according to claim 10
A method for identifying a compound that inhibits (antagonizes) or promotes a BQ91 polypeptide or a peptide according to any one of claims 13 to 15, comprising: (a) a cell expressing HPMBQ91 polypeptide (or HPMBQ91); Contacting a cell membrane expressing the polypeptide) or a cell responsive to the HPMBQ91 polypeptide with the candidate compound; (b) observing binding or stimulation or inhibition of a functional response; or cells contacted with the candidate compound ( Or HPMBQ91 of the same cell not in contact with
Comparing the ability to polypeptide activity, or (c) contacting cells (or cell membranes expressing the receptor) expressing the receptor for the peptide according to claims 11 to 14 with a candidate compound, and (d) binding of the peptide. Or observing the stimulation or inhibition of a functional response or the stimulation or inhibition of a functional response. 21. An agonist or antagonist identified by the method of claim 19. 22. (a) Accession number 97375 at the ATCC
Expressed by the cDNA insert deposited at
A nucleotide sequence having at least 85% identity to the nucleotide sequence encoding the MBQ91 polypeptide; and (b) a nucleotide sequence selected from the group consisting of a nucleotide sequence having complementarity to the nucleotide sequence of (a). An isolated HPMBQ91 polynucleotide.