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WO1999036525A1 - Cbfblh12: gene fortement associe au gene ci-kfyi bovin pour complexe d'ubiquinone oxyreductase - Google Patents

Cbfblh12: gene fortement associe au gene ci-kfyi bovin pour complexe d'ubiquinone oxyreductase Download PDF

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Publication number
WO1999036525A1
WO1999036525A1 PCT/CN1998/000006 CN9800006W WO9936525A1 WO 1999036525 A1 WO1999036525 A1 WO 1999036525A1 CN 9800006 W CN9800006 W CN 9800006W WO 9936525 A1 WO9936525 A1 WO 9936525A1
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Prior art keywords
polypeptide
cbfblh12
seq
nucleotide sequence
polynucleotide
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PCT/CN1998/000006
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English (en)
Inventor
Gang Fu
Juan Zhou
Mao Mao
Kai-Li He
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Shanghai Second Medical University
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Priority to PCT/CN1998/000006 priority Critical patent/WO1999036525A1/fr
Publication of WO1999036525A1 publication Critical patent/WO1999036525A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • CBFBLH12 A Gene Highly Related to Bovine CI-KFYI Gene for Ubiquinone Oxireductase
  • This mvention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production More particularly, the polynucleotides and polypeptides of the present invention relate to the gene family including NADH-ubiquinone oxireductase, hereinafter referred to as CBFBLH12 The invention also relates to inhibiting or activatmg the action of such polynucleotides and polypeptides
  • NADH-ubiquinone oxireductase is the first enzyme in the respiratory electron transport chain of mitochondna, and is a membrane-bound multi-subumt assembly
  • the CI-KFYI is one of the nuclear encoded subunits, and participates in cellular respiration This indicates that the gene family including NADH-ubiquinone oxireductase has an established, proven history as therapeutic targets Clearly there is a need for identification and characterization of further members of the gene family including NADH- ubiquinone oxireductase family which can play a role n preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to. cardiovascular disease, kidney disease, metabo c- associated disorders, and autoimmune diseases
  • the mvention relates to CBFBLH12 polypeptides and recombinant matenals and methods for their production
  • Another aspect of the invention relates to methods for using such CBFBLH12 polypeptides and polynucleotides
  • Such uses include the treatment of cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases, among others
  • the invention relates to methods to identify agonists and antagonists usmg the matenals provided by the mvention, and treating conditions associated with CBFBLH12 imbalance with the identified compounds
  • CBFBLH12 refers, among others, generally to a polypeptide having the ammo acid sequence set forth m SEQ ID NO 2 or an allehc variant thereof "CBFBLH12 activity or CBFBLH12 polypeptide activity” or "biological activity of the
  • CBFBLH12 or CBFBLH12 polypeptide refers to the metabolic or physiologic function of said CBFBLH12 including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and lmmunogenic activities of said CBFBLH12 "CBFBLH12 gene” refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO 1 or allehc variants thereof and/or their complements
  • Antibodies as used herein mcludes polyclonal and monoclonal antibodies, chime ⁇ c, smgle chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library
  • Isolated means altered “by the hand of man” from the natural state If an "isolated” composition or substance occurs m nature, it has been changed or removed from its original environment, or both
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is “isolated”, as the term is employed herein
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of smgle- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of smgle- and double-stranded regions
  • polynucleotide refers to triple-stranded regions comp ⁇ smg RNA or DNA or both RNA and DNA
  • polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include, for example
  • Polypeptide refers to any peptide or protein comp ⁇ sing two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, l e , peptide isosteres
  • Polypeptide refers to both short chains, commonly referred to as peptides, o gopeptides or ohgomers, and to longer chains, generally referred to as proteins Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids
  • Polypeptides include ammo acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known the art Such modifications are well desc ⁇ bed in basic texts and m more detailed monographs, as well as in a voluminous research literature Modifications can occur anywhere m a polypeptide, including the peptide backbone, the ammo acid side-chains and the ammo or carboxyl termini It will be appreciated that the same type of modification may be present in the same or
  • Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods Modifications include acetylation. acylation. ADP- ⁇ bosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide de ⁇ vative, covalent attachment of a hpid or lipid de ⁇ vative, covalent attachment of phosphotidylmositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystme.
  • Va ⁇ ant 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 va ⁇ ant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below
  • a typical va ⁇ ant of a polypeptide differs m ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ m ammo acid sequence by one or
  • Identity is a measure of the identity of nucleotide sequences or ammo acid sequences In general, the sequences are aligned so that the highest order match is obtained "Identity" per se has an art-recognized meaning and can be calculated using published techniques See, e g
  • a polypeptide havmg an ammo acid sequence having at least, for example, 95% "identity" to a reference ammo acid sequence of SEQ ID NO 2 is mtended that the ammo acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five ammo acid alterations per each 100 ammo acids of the reference ammo acid of SEQ ID NO 2
  • the polypeptide sequence may include up to five ammo acid alterations per each 100 ammo acids of the reference ammo acid of SEQ ID NO 2
  • up to 5% of the ammo acid residues in the reference sequence may be deleted or substituted with another ammo acid, or a number of ammo acids up to 5% of the total ammo acid residues in the reference sequence may be inserted into the reference sequence
  • These alterations of the reference sequence may occur at the ammo or carboxy terminal positions of the reference ammo acid sequence or anywhere between those terminal positions, interspersed either individually among residues m the reference
  • the present invention relates to CBFBLH12 polypeptides (or CBFBLH12 protems)
  • the CBFBLH12 polypeptides include the polypeptide of SEQ ID NO 2, as well as polypeptides comprising the ammo acid sequence of SEQ ID NO 2, and polypeptides comp ⁇ smg the ammo acid sequence which have at least 80% identity to that of SEQ ID NO 2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99% are highly preferred Also mcluded within CBFBLH12 polypeptides are polypeptides having the ammo acid sequence which have at least 80% identity to the polypeptide having the ammo acid sequence of SEQ ID NO 2 over its entire length, and still more preferably at least 90% identity, and still more preferably at least 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99% are highly preferred Preferably CBFBLH12 polypeptide
  • the CBFBLH12 polypeptides may be in the form of the "mature" protem or may be a part of a larger protem such as a fusion protein It is often advantageous to include an additional am o acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid m pu ⁇ fication such as multiple histidine residues, or an additional sequence for stability during recombinant production Fragments of the CBFBLH12 polypeptides are also included m the invention A fragment is a polypeptide havmg an ammo acid sequence that entirely is the same as part, but not all, of the ammo acid sequence of the aforementioned CBFBLH12 polypeptides As with CBFBLH12 polypeptides, fragments may be "free-standing,” or comp ⁇ sed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region Representative examples of polypeptide fragments of the mvention, mclude, for example, fragments
  • Preferred fragments m include, for example, truncation polypeptides havmg the ammo acid sequence of CBFBLH12 polypeptides, except for deletion of a continuous senes of residues that mcludes the ammo terminus, or a continuous senes of residues that mcludes the carboxyl terminus or deletion of two continuous senes of residues, one including the ammo terminus and one including the carboxyl terminus
  • fragments characterized by structural or functional attributes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and tum-formmg regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions
  • Other prefened fragments are biologically active fragments Biologically active fragments are those that mediate CBFBLH12 activity, including
  • vanants of the defined sequence and fragments also form part of the present mvention
  • Prefened vanants are those that vary from the referents by conservative ammo acid substitutions - l e , those that substitute a residue with another of like charactenstics Typical such substitutions are among Ala, Val. Leu and lie, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr
  • vanants m which several, 5-10, 1-5, or 1-2 ammo acids are substituted, deleted, or added in any combination
  • the CBFBLH12 polypeptides of the mvention can be prepared m any suitable manner Such polypeptides mclude isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods Means for preparing such polypeptides are well understood m the art Polynucleotides of the Invention
  • CBFBLH12 polynucleotides mclude isolated polynucleotides which encode the CBFBLH12 polypeptides and fragments, and polynucleotides closely related thereto More specifically, CBFBLH12 polynucleotide of the mvention mclude a polynucleotide compnsing the nucleotide sequence contained m SEQ ID NO 1 encodmg a
  • CBFBLH12 polypeptide of SEQ ID NO 2 and polynucleotide havmg the particular sequence of SEQ ID NO 1 CBFBLH12 polynucleotides further mclude a polynucleotide compnsing a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encodmg the CBFBLH12 polypeptide of SEQ ID NO 2, and a polynucleotide comp ⁇ smg a nucleotide sequence that is at least 80% identical to of SEQ ID NO 1 over its entire length
  • polynucleotides at least 90% identical are particularly prefened, and those with at least 95% are especially prefened
  • those with at least 97% are highly prefened and those with at least 98-99% are most highly prefened, with at least 99% being the most prefened
  • Also mcluded under CBFBLH12 polynucleotides are a nucleotide sequence
  • CBFBLH12 of the mvention is structurally related to other proteins of the gene family including NADH-ubiquinone oxireductase, as shown by the results of sequencmg the cDNA of Table 1 (SEQ ID NO 1) encodmg human CBFBLH12
  • the cDNA sequence of SEQ ID NO 1 contains an open reading frame (nucleotide number 9 to 236) encodmg a polypeptide of 76 ammo acids of SEQ ID NO 2
  • the ammo acid sequence of Table 2 (SEQ ID NO 2) has about 78 9% identity (using FASTA) m 76 ammo acid residues with bovine CI-KFYI gene for ubiqumone oxireductase complex (J E Walker, et al , J Mol Biol 226 1051- 1072, 1992)
  • the nucleotide sequence of Table 1 (SEQ ID NO 1) has about 80% identity (usmg FASTA) m 420 nu
  • a nucleotide sequence of a human CBFBLH12 (SEQ ID NO 1)
  • One polynucleotide of the present mvention encodmg CBFBLH12 may be obtained using standard cloning and screening, from a cDNA library denved from mRNA m cells of human cord blood usmg the expressed sequence tag (EST) analysis (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) Polynucleotides of the mvention can also be obtained from natural sources such as genomic DNA hbranes or can be synthesized using well known and commercially available techniques
  • nucleotide sequence encoding CBFBLH12 polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 9 to 236 of SEQ ID NO 2
  • the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself, the coding sequence for the mature polypeptide or fragment m readmg frame with other coding sequences, such as those encodmg a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions
  • a marker sequence which facilitates pu ⁇ fica ⁇ on of the fused polypeptide can be encoded
  • the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed in Gentz et al , Proc NatlAcadSci USA (1989) 86 821-824, or is an HA tag
  • the polynucleot is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed in Gentz e
  • the present mvention further relates to polynucleotides that hyb ⁇ dize to the here above-descnbed sequences
  • the present mvention especially relates to polynucleotides which hybndize under stringent conditions to the herem above-descnbed polynucleotides
  • sh ⁇ ngent conditions means hybndization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97-99% identity between the sequences
  • Polynucleotides of the mvention which are identical or sufficiently identical to a nucleotide sequence contained m SEQ ID NO 1 or a fragment thereof, may be used as hybndization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encodmg CBFBLH12 polypeptide and to isolate cDNA and genomic clones of other genes (including genes encodmg homologs and orthologs from species other than human) that have a high sequence similanty to the CBFBLH12 gene
  • Such hybndization techniques are known to those of skill m the art Typically these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent
  • the probes generally will compnse at least 15 nucleotides Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides Particularly prefened probes will range between 30
  • CBFBLH12 polynucleotides of the present mvention further mclude a nucleotide sequence compnsing a nucleotide sequence that hyb ⁇ dize under st ⁇ ngent condition to a nucleotide sequence havmg SEQ ID NO 1 or a fragment thereof
  • the present mvention also relates to vectors which compnse a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engmeered with vectors of the mvention and to the production of polypeptides of the mvention by recombmant techniques
  • Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs of the present mvention
  • host cells can be genetically engmeered to incorporate expression systems or portions thereof for polynucleotides of the present mvention
  • Introduction of polynucleotides into host cells can be effected by methods desc ⁇ bed m many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press
  • approp ⁇ ate hosts include bactenal cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtilis cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells, and plant cells
  • bactenal cells such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • a great va ⁇ ety of expression systems can be used Such systems mclude, among others, chromosomal, episomal and virus-denved systems, e g , vectors denved from bactenal plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retioviruses, and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids
  • the expression systems may contain control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used The approp ⁇ ate nucle
  • approp ⁇ ate secretion signals may be incorporated mto the desired polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • the cells may be harvested pnor to use m the screening assay If CBFBLH12 polypeptide is secreted mto the medium, the medium can be recovered m order to recover and pu ⁇ fy the polypeptide, if produced intracellularly, the cells must first be lysed before the polypeptide is recovered
  • CBFBLH12 polypeptides can be recovered and punfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography is employed for pu ⁇ fication Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or pu ⁇ fication
  • This mvention also relates to the use of CBFBLH12 polynucleotides for use as diagnostic reagents Detection of a mutated form of CBFBLH12 gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under- expression, over-expression or altered expression of CBFBLH12 Individuals carrying mutations m the CBFBLH12 gene may be detected at the DNA level by a va ⁇ ety of techniques
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, u ⁇ ne, saliva, tissue biopsy or autopsy matenal
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by us g PCR or other amplification techniques pnor to analysis RNA or cDNA may also be used m similar fashion
  • Deletions and insertions can be detected by a change m size of the amplified product m compa ⁇ son to the normal genotype
  • Pomt mutations can be identified by hyb ⁇ dizmg amplified DNA to labeled CBFBLH12 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures
  • DNA sequence differences may also be detected by alterations m electrophoretic mobility of DNA fragments m gels, with or without denatu ⁇ ng agents, or by direct DNA sequencmg See, e g , Myers
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases through detection of mutation in the CBFBLH12 gene by the methods descnbed
  • cardiovascular disease kidney disease, metabolic-associated disorders, and autoimmune diseases
  • methods comp ⁇ smg determining from a sample denved from a subject an abnormally decreased or mcreased level of CBFBLH12 polypeptide or CBFBLH12 mRNA Decreased or mcreased expression can be measured at the RNA level using any of the methods well known m the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods
  • Assay techniques that can be used to determine levels of a protein, such as an CBFBLH12 polypeptide, in a sample denved from a host are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays
  • the present mvention relates to a diagonostic kit for a disease or suspectabi ty to a disease, particularly cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases, which compnses
  • a CBFBLH12 polynucleotide preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof,
  • a CBFBLH12 polypeptide preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or
  • any such kit, (a), (b), (c) or (d) may compnse a substantial component
  • the nucleotide sequences of the present mvention are also valuable for chromosome identification
  • the sequence is specifically targeted to and can hybndize with a particular location on an individual human chromosome
  • the mapping of relevant sequences to chromosomes according to the present mvention is an important first step in conelatmg those sequences with gene associated disease Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be conelated with genetic map data Such data are found, for example, m V McKusick.
  • the differences m the cDNA or genomic sequence between affected and unaffected individuals can also be determined If a mutation is observed m some or all of the affected individuals but not m any normal individuals, then the mutation is likely to be the causative agent of the disease
  • polypeptides of the mvention or their fragments or analogs thereof, or cells expressmg them can also be used as immunogens to produce antibodies lmmunospecific for the CBFBLH12 polypeptides
  • immunospecific means that the antibodies have substantiall greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides m the pnor art
  • Antibodies generated against the CBFBLH12 polypeptides can be obtained by administering the polypeptides or epitope-bea ⁇ ng fragments, analogs or cells to an animal, preferably a nonhuman, usmg routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell lme cultures can be used Examples mclude the hyb ⁇ doma technique (Kohler, G and Milstem, C , Nature (1975) 256 495-497), the t ⁇ oma technique, the human B-cell hyb ⁇ doma technique (Kozbor et al , Immunology Toady (1983) 4 72) and the EBV-hybndoma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985) Techniques for the production of smgle chain antibodies (U S Patent No 4,946,778) can also be adapted to
  • the above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptide or to purify the polypeptides by affinity chromatography
  • Antibodies against CBFBLH12 polypeptides may also be employed to treat cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases, among others
  • Another aspect of the invention relates to a method for mducmg an lmmunological response m a mammal which compnses inoculating the mammal with CBFBLH12 polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases, among others
  • Yet another aspect of the invention relates to a method of inducing lmmunological response m a mammal which comprises, delivering CBFBLH12 polypeptide via a vector directing expression of CBFBLH12 polynucleotide in vivo m order to induce such an lmmunological response to produce antibody to protect said animal from diseases
  • composition which, when mtroduced into a mammalian host, induces an lmmunological response in that mammal to a CBFBLH12 polypeptide wherem the composition comprises a CBFBLH12 polypeptide or CBFBLH12 gene
  • the vaccine formulation may further compnse a suitable earner Smce CBFBLH12 polypeptide may be broken down m the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, mtravenous, intradermal etc injection)
  • parenterally including subcutaneous, intramuscular, mtravenous, intradermal etc injection
  • Formulations suitable for parenteral administration mclude aqueous and non-aqueous stenle injection solutions which may contain anti- oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation mstonic with the blood of the recipient, and aqueous and non-aqueous stenle suspensions which may mclude suspending agents or thickening agents
  • the CBFBLH12 polypeptide of the present mvention may be employed m a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the CBFBLH12 polypeptide of the present mvention
  • polypeptides of the mvention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical hbra ⁇ es, and natural product mixtures
  • agonists or antagonists may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of the polypeptide of the present mvention, or may be structural or functional mimetics of the polypeptide of the present mvention See Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)
  • CBFBLH12 polypeptides are responsible for many biological functions, including many pathologies Accordingly, it is desirous to find compounds and drugs which stimulate CBFBLH12 polypeptide on the one hand and which can inhibit the function of CBFBLH 12 polypeptide on the other hand
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases
  • Antagonists may be employed for a va ⁇ ety of therapeutic and prophylactic purposes for such conditions as cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases
  • screening procedures may involve usmg appropnate cells which express the CBFBLH12 polypeptide or respond to CBFBLH12 polypeptide of the present mvention
  • Such cells mclude cells from mammals, yeast, Drosophila or E cob Cells which express the CBFBLH12 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBFBLH12 polypeptide are then contacted with a test compound to observe binding
  • the assays may simply test binding of a candidate compound wherem adherence to the cells bea ⁇ ng the CBFBLH12 polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor Further, these assays may test whether the candidate compound results m a signal generated by activation of the CBFBLH12 polypeptide, using detection systems approp ⁇ ate to the cells bea ⁇ ng the CBFBLH12 polypeptide Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agomst by the presence of the candidate compound is observed Further, the assays may simply compnse the steps of mixing a candidate compound with a solution containing a CBFBLH12 polypeptide to form a mixture, measuring CBFBLH12 activity m the mixture, and comparing the CBFBLH12 activity of the mixture to a standard
  • the CBFBLH12 cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of CBFBLH12 mRNA and protem in cells
  • an ELISA may be constructed for measunng secreted or cell associated levels of CBFBLH12 protein using monoclonal and polyclonal antibodies by standard methods known m the art, and this can be used to discover agents which may inhibit or enhance the production of CBFBLH12 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the CBFBLH12 protem may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known m the art These mclude, but are not limited to, gand binding and crosslinkmg assays in which the CBFBLH12 is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods mclude biophysical techniques such as surface plasmon resonance and spectroscopy In addition to being used for purification and cloning of the receptor, these bindmg assays can be used to identify agonists and antagonists of CBFBLH12 which compete with the bindmg of CBFBLH12 to its receptors, if any Standard methods for conducting screening assays are well understood in the art Examples of potential CBF
  • the present invention relates to a screenmg kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for CBFBLH12 polypeptides, or compounds which decrease or enhance the production of CBFBLH12 polypeptides, which comprises (a) a CBFBLH12 polypeptide, preferably that of SEQ ID NO 2, (b) a recombmant cell expressmg a CBFBLH12 polypeptide, preferably that of SEQ ID NO 2,
  • a cell membrane expressmg a CBFBLH12 polypeptide, preferably that of SEQ ID NO 2, or
  • This mvention provides methods of treating abnormal conditions such as, cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases, related to both an excess of and insufficient amounts of CBFBLH12 polypeptide activity
  • expression of the gene encoding endogenous CBFBLH12 polypeptide can be inhibited using expression blocking techniques Known such techniques involve the use of antisense sequences, either internally generated or separately administered See, for example, O'Connor, J Neurochem (1991) 56 560 m Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression.
  • Polypeptides and other compounds of the present mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds
  • Polypeptides used m treatment can also be generated endogenously in the subject, in treatment modal-ties often refened to as "gene therapy" as descnbed above.
  • cells from a subject may be engmeered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plas id vector. The cells are then introduced mto the subject.

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Abstract

L'invention concerne des polypeptides et des polynucléotides CBFBLH12 et des procédés d'obtention de ces polypeptides par des techniques recombinantes. Font aussi l'objet de cette invention des procédés d'utilisation des polypeptides et des polynucléotides CBFBLH12 dans la conception de protocoles pour le traitement de maladies cardio-vasculaires, de maladies du rein, de troubles métaboliques et de maladies auto-immunes, entre autres, et des doses diagnostiques pour ces pathologies.
PCT/CN1998/000006 1998-01-19 1998-01-19 Cbfblh12: gene fortement associe au gene ci-kfyi bovin pour complexe d'ubiquinone oxyreductase WO1999036525A1 (fr)

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PCT/CN1998/000006 WO1999036525A1 (fr) 1998-01-19 1998-01-19 Cbfblh12: gene fortement associe au gene ci-kfyi bovin pour complexe d'ubiquinone oxyreductase

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PCT/CN1998/000006 WO1999036525A1 (fr) 1998-01-19 1998-01-19 Cbfblh12: gene fortement associe au gene ci-kfyi bovin pour complexe d'ubiquinone oxyreductase

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WO1999036525A1 true WO1999036525A1 (fr) 1999-07-22

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002079473A3 (fr) * 2001-01-12 2003-10-02 Incyte Genomics Inc Molecules utilisees a des fins diagnostiques et therapeutiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BIOCHEM. BIOPHYS. RES. COMMUN., 241(2), (Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto Hospital, Ontario, Canada), 18 December 1997, pp. 589-594. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002079473A3 (fr) * 2001-01-12 2003-10-02 Incyte Genomics Inc Molecules utilisees a des fins diagnostiques et therapeutiques

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