WO1999062948A1 - Gene homologue de la sous-unite ci-b17 de l'ubiquinone oxydoreductase humaine (cblale02) - Google Patents

Gene homologue de la sous-unite ci-b17 de l'ubiquinone oxydoreductase humaine (cblale02) Download PDF

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Publication number
WO1999062948A1
WO1999062948A1 PCT/CN1998/000085 CN9800085W WO9962948A1 WO 1999062948 A1 WO1999062948 A1 WO 1999062948A1 CN 9800085 W CN9800085 W CN 9800085W WO 9962948 A1 WO9962948 A1 WO 9962948A1
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Prior art keywords
polypeptide
identity
seq
sequence
subject
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PCT/CN1998/000085
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English (en)
Inventor
Juan Zhou
Yaping Yu
Qiuhua Huang
Mao Mao
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Shanghai Second Medical University
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Priority to PCT/CN1998/000085 priority Critical patent/WO1999062948A1/fr
Publication of WO1999062948A1 publication Critical patent/WO1999062948A1/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
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in therapy and m identifying compounds which may be agonists, antagonists and/or inhibitors which are potentially useful in therapy, and to production of such polypeptides and polynucleotides
  • the present invention relates to CBNAFA09, m particular CBNAFA09 polypeptides and CBNAFA09 polynucleotides, recombinant materials and methods for their production
  • the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of AIDS, cancer, autoimmune disease, hepatitis, and diabetes, hereinafter referred to as "the Diseases", amongst others
  • the invention relates to methods for identifymg agonists and antagonists/inhibitors usmg the mate ⁇ als provided by the invention, and treating conditions associated with CBNAFA09 imbalance with the identified compounds
  • the invention relates to diagnostic assays for detecting diseases associated with inappropriate CBNAFA09 activity or levels
  • the present invention relates to CBNAFA09 polypeptides
  • pept ⁇ des include isolated polypeptides comprising an ammo acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2
  • polypeptides mclude those comprising the ammo acid of SEQ ID NO 2
  • peptides of the present mvention include isolated polypeptides m which the ammo acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2
  • polypeptides include the polypeptide of SEQ ID NO 2
  • peptides of the present mvention mclude isolated polypeptides encoded by a polynucleotide comp ⁇ smg the sequence contained in SEQ ID NO 1
  • Polypeptides of the present invention are believed to be members of the ubiquinone oxidoreductase family of polypeptides They are therefore of interest because ubiqumone oxidoreductase is is a mitochondria protem complex encoded by nuclear genome, it is composed of many subumts, and it participates in metabolism These properties are hereinafter referred to as "CBNAFA09 activity” or “CBNAFA09 polypeptide activity” or "biological activity of
  • CBNAFA09 Also included amongst these activities are antigenic and lmmunogemc activities of said CBNAFA09 polypeptides, in particular the antigenic and lmmunogenic activities of the polypeptide of SEQ ID NO 2
  • a polypeptide of the present invention exhibits at least one biological activity of CBNAFA09
  • the polypeptides of the present invention may be m the form of the "mature" protein or may be a part of a larger protem such as a fusion protem It is often advantageous to include an additional ammo acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid m purification such as multiple histidine residues, or an additional sequence for stability du ⁇ ng recombinant production
  • the present mvention also includes mclude variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characteristics Typical such substitution
  • polynucleotides of the present mvention mclude isolated polynucleotides comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to SEQ ID NO 1 over the entire length of SEQ ID NO 1
  • polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identiy are more highly preferred, and those with at least 99% identity are most highly preferred
  • Such polynucleotides include a polynucleotide comp ⁇ smg the polynucleotide of SEQ ID NO 1 as well as the polynucleotide of SEQ ID NO 1
  • the invention also provides polynucleotides which are complementary to all the above described polynucleotides
  • the nucleotide sequence of SEQ ID NO 1 shows homology with X64836, bovine ubiquinone oxidoreductase complex subunit CI-B22, (J E Walker, et al J Mol Biol 1992,226 1051-1072 )
  • the nucleotide sequence of SEQ ID NO 1 is a cDNA sequence and comp ⁇ ses a polypeptide encoding sequence (nucleotide 80 to 616) encoding a polypeptide of 179 ammo acids, the polypeptide of SEQ ID NO 2
  • the nucleotide sequence encodmg the polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contamed m SEQ ID NO 1 or it may be a sequence other than the one contained in SEQ ID NO 1, which, as a result of the redundancy (degeneracy) of the
  • Prefe ⁇ ed polypeptides and polynucleotides of the present mvention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides Furthermore, prefe ⁇ ed polypeptides and polynucleotides of the present mvention have at least one CBNAFA09 activity
  • Polynucleotides of the present mvention may be obtained, usmg standard cloning and screening techniques, from a cDNA library de ⁇ ved from mRNA m cells of human umbilical 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 O , et al , Nature (1995) 377
  • EST expressed sequence tag
  • Polynucleotides of the mvention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques
  • the polynucleotide may include the coding sequence for the mature polypeptide, by itself, or the codmg sequence for the mature polypeptide m reading frame with other codmg 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 purification of the fused polypeptide can be encoded
  • the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and desc ⁇ bed m Gentz et al , Proc Natl Acad Sc USA ( 1989) 86 821 -824, or is an HA tag
  • the polynucleotide may also contain non-co
  • Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contamed in SEQ ID NO 1 may be used as hyb ⁇ dization probes for cDNA and genomic DNA or as p ⁇ mers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present mvention 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 SEQ ID NO 1
  • these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to that of the referent
  • the probes or p ⁇ mers will generally comp ⁇ se at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides Particularly prefe ⁇ ed probes will have between 30 and 50 nu
  • an isolated cDNA sequence will be incomplete, m that the region coding for the polypeptide is cut short at the 5' end of the cDNA This is a consequence of reverse transc ⁇ ptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remain attached to the template durmg the polymerisation reaction), failing to complete a DNA copy of the mRNA template durmg 1st strand cDNA synthesis
  • Recombinant polypeptides of the present mvention may be prepared by processes well known m the art from genetically engineered host cells comprising expression systems Accordingly, m a further aspect, the present mvention relates to expression systems which comp ⁇ se a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs de ⁇ ved from the DNA constructs of the present mvention For recombinant production, 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 in many standard laboratory manuals, such as Davis et al , Basic Methods in Molecular Biology (1986) and Sambrook et al ,
  • approp ⁇ ate hosts include bacte ⁇ al cells, such as streptococci, staphylococci, E colt, Streptomyces and Bacillus subtihs cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosophtla S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells, and plant cells
  • bacte ⁇ al cells such as streptococci, staphylococci, E colt, Streptomyces and Bacillus subtihs cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophtla 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, for instance, chromosomal, episomal and virus-denved systems, e g , vectors de ⁇ ved 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, vaccmia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors de ⁇ ved from combinations thereof, such as those de ⁇ ved from plasmid and bacte ⁇ ophage 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 which is able to maintain, propagate or express a polynucleotide to produce a polypeptide m a host may be used The
  • a polypeptide of the present mvention is to be expressed for use m screening assays, it is generally prefe ⁇ ed that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested p ⁇ or to use m the screening assay If the polypeptide is secreted mto the medium, the medium can be recovered m order to recover and pu ⁇ fy the polypeptide If produced lntracellularly, the cells must first be lysed before the polypeptide is recovered
  • Polypeptides of the present invention can be recovered and pu ⁇ fied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, amon 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 durmg isolation and or pu ⁇ fication
  • This invention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of the gene characte ⁇ sed by the polynucleotide of SEQ ID NO 1 which is 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
  • Nucleic acids for diagnosis may be obtamed from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy matenal
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by usmg PCR or other amplification techniques p ⁇ or to analysis RNA or cDNA may also be used m similar fashion
  • Deletions and insertions can be detected by a change m size ofthe amphfied product m companson to the normal genotype
  • Pomt mutations can be identified by hybndrzing amplified DNA to labeled CBNAFA09 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in meltmg temperatures
  • DNA sequence differences may also be detected by alterations m electrophoretic mobility of DNA fragments m gels, with or without denaturing agents, or by direct DNA sequencmg (ee, e g , Myers
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to the Diseases through detection of mutation m the CBNAFA09 gene by the methods desc ⁇ bed
  • diseases may be diagnosed by methods comp ⁇ smg determining from a sample derived from a subject an abnormally decreased or increased level of polypeptide or mRNA Decreased or mcreased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance 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 a polypeptide of the present mvention, m a sample de ⁇ ved from a host are well-known to those of skill in the art
  • Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assay
  • a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO 1 , or a fragment thereof ,
  • polypeptide of the present mvention preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or
  • kits may comp ⁇ se a substantial component
  • a kit will be of use m diagnosing a disease or suspectability to a disease, particularly ADDS, cancer, autoimmune disease, hepatitis, and diabetes, amongst others
  • 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 m co ⁇ elating 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 co ⁇ elated with genetic map data
  • genetic map data are found m, for example, V McKusick, Mendehan Inhe ⁇ tance m Man (available on-line through lohns Hopkins Umversity Welch Medical Library)
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinhentance of physically adjacent genes)
  • the differences m the cDNA or genomic sequence between affected and unaffected individuals can also be determined If a mutation is observed in 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
  • the polypeptides of the mvention or their fragments or analogs thereof, or cells expressing them, can also be used as lmmunogens to produce antibodies lmmunospecific for polypeptides of the present mvention
  • the term "lmmunospecrfic” means that the antibodies have substantially greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides m the pnor art Antibodies generated against polypeptides of the present mvention may be obtamed by administering the polypeptides or epitope-bea ⁇ ng fragments, analogs or cells to an animal, preferably a non-human animal, usmg routine protocols For preparation of monoclonal antibodies, any techmque which provides antibodies produced by continuous cell lme cultures can be used Examples mclude the hyb ⁇ doma techmque (Kohler, G and Milstem, C , Nature (1975) 256 495-497), the t ⁇ oma techmque, the human B-
  • smgle cham antibodies such as those desc ⁇ bed m U S Patent No 4,946,778, can also be adapted to produce smgle chain antibodies to polypeptides of this mvention
  • transgenic mice, or other organisms, including other mammals may be used to express humanized antibodies
  • the above-desc ⁇ bed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography
  • Antibodies against polypeptides of the present mvention may also be employed to treat the
  • the present mvention relates to genetically engmeered soluble fusion proteins compnsing a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of lmmunoglobuhns of various subclasses (IgG, IgM, IgA, IgE) Preferred as an immunoglobulm is the constant part of the heavy cham of human IgG, particularly IgGl, where fusion takes place at the hmge region
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa
  • this invention relates to processes for the preparation of these fusion protems by genetic engineering, and to the use thereof for drug screemng, diagnosis and therapy
  • a further aspect of the invention also relates to polynucleotides encodmg such fusion protems Examples of fusion protem technology can be found m International Patent
  • Another aspect of the mvention relates to a method for inducing an immunological response m a mammal which compnses moculatmg the mammal with a polypeptide of the present mvention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases herembefore mentioned, amongst others
  • Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering a polypeptide of the present mvention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo m order to prise such an immunological response to produce antibody to protect said animal from diseases
  • a further aspect of the mvention relates to an lmmunological/vaccine formulation (composition) which, when mtroduced mto a mammalian host, induces an immunological response in that mammal to a polypeptide of the present invention wherein the composition compnses a polypeptide or polynucleotide of the present mvention
  • the vaccine formulation may further comprise a suitable carrier Smce a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection)
  • Formulations suitable for parenteral administration include aqueous and non-aqueous stenle injection solutions which may contain anti-oxidants, buffers, bactenostats and solutes which render the formulation mstomc with the blood of the recipient, and aqueous and non- aqueous stenle suspensions which may mclude suspending agents or thickening agents
  • the formulations may be presented in unit
  • Polypeptides of the present mvention are responsible for many biological functions, including many disease states, m particular the Diseases herembefore mentioned It is therefore desirous to devise screemng methods to identify compounds which stimulate or which inhibit the function of the polypeptide Accordingly, m a further aspect, the present mvention provides for a method of screemng compounds to identify those which stimulate or which inhibit the function of the polypeptide
  • agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned
  • Compounds may be identified from a va ⁇ ety of sources, for example, cells, cell-free preparations, chemical hbra ⁇ es, and natural product mixtures
  • Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc , as the case may be, of the polypeptide, or may be structural or functional mimetics thereof (see Coligan et al , Current Protocols in Immunology 1(2) Chapter
  • polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screemng methods for detectmg the effect of added compounds on the production of mRNA and polypeptide m cells
  • an ELISA assay may be constructed for measunng secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art These mclude, but are not limited to, hgand bmdmg and crosshnking assays in which the polypeptide is labeled with a radioactive isotope (for instance, 1-"I), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or pu ⁇ fication, and mcubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screemng methods may also be used to identify agomsts and antagonists of the polypeptide which compete with the bmding of the polypeptide to its receptors, if any Standard methods for conducting such assays are well understood m the art
  • polypeptide antagonists examples include antibodies or, m some cases, oligonucleotides or proteins which are closely related to the gands, substrates, receptors, enzymes, etc , as the case may be, of the polypeptide, e g , a fragment of the gands, substrates, receptors, enzymes, etc , or small molecules which bind to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented
  • the present mvention relates to a screenmg kit for identifying agomsts, antagonists, hgands, receptors, substrates, enzymes, etc for polypeptides of the present invention, or compounds which decrease or enhance the production of such polypeptides, which compnses
  • any such kit, (a), (b), (c) or (d) may comprise a substantial component
  • polypeptide of the present invention may also be used m a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by
  • the present invention provides methods of treating abnormal conditions such as, for instance, AIDS, cancer, autoimmune disease, hepatitis, and diabetes, related to either an excess of, or an under-expression of, CBNAFA09 polypeptide activity
  • an inhibitor compound as hereinabove descnbed, optionally in combination with a pharmaceutically acceptable earner, m an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the binding of hgands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition
  • soluble forms of the polypeptides still capable of bindmg the ligand, substrate, enzymes, receptors, etc m competition with endogenous polypeptide may be administered Typical examples of such competitors include fragments of the CBNAFA09 polypeptide
  • expression of the gene encodmg endogenous CBNAFA09 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 O godeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988))
  • oligonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360)
  • These ohgomers can be administered /?er se or the relevant ohgomers can be expressed in vivo
  • a polynucleotide of the mvention may be engineered for expression m a replication defective retroviral vector, as discussed above
  • the retroviral expression construct may then be isolated and introduced mto a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present mvention such that the packaging cell now produces infectious viral particles containmg the gene of interest
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of
  • the present mvention provides for pharmaceutical compositions compnsing a therapeutically effective amount of a polypeptide, such as the soluble form of a polypeptide of the present mvention, agonist/antagonist peptide or small molecule compound, m combination with a pharmaceutically acceptable earner or excipient
  • a pharmaceutically acceptable earner or excipient Such earners mclude, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof
  • the mvention further relates to pharmaceutical packs and kits compnsing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention Polypeptides and other compounds of the present mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds
  • composition will be adapted to the route of admimstration, for instance by a systemic or an oral route
  • Prefe ⁇ ed forms of systemic administration mclude injection, typically by intravenous injection
  • Other injection routes such as subcutaneous, intramuscular, or lntrapentoneal
  • Alternative means for systemic administration mclude transmucosal and transdermal administration usmg penetrants such as bile salts or fusidtc acids or other detergents
  • oral admmistration may also be possible
  • Admimstration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, and the like
  • the dosage range required depends on the choice of peptide or other compounds of the present mvention, the route of admimstration, the nature of the formulation, the nature of the subject's condition, and
  • Polypeptides used in treatment can also be generated endogenously m the subject, m treatment modalities often refe ⁇ ed to as "gene therapy" as descnbed above
  • m treatment modalities often refe ⁇ ed 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 plasmid vector The cells are then introduced mto the subject
  • polypeptide sequences form a valuable information resource with which to identify further sequences of similar homology This is most easily facilitated by storing the sequence m a computer readable medium and then using the stored data to search a sequence database usmg well known searchmg tools, such as GCC Accordingly, in a further aspect, the present mvention provides for a computer readable medium having stored thereon a polynucleotide compnsmg the sequence of SEQ ID NO 1 and/or a polypeptide sequence encoded thereby
  • Antibodies as used herem mcludes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humamzed antibodies, as well as Fab fragments, mcludmg the products of an Fab or other lmmunoglobuhn 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 o ⁇ gmal 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 herem
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeox ⁇ bonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotides include, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double- stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybnd molecules compnsmg DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of smgle- and double-stranded regions
  • polynucleotide refers to triple-stranded regions compnsmg RNA or DNA or both RNA and DNA
  • polynucleotide also mcludes DNAs or RNAs containing one or more modified bases and DNAs
  • Polypeptide refers to any peptide or protem compnsing two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds, l e , peptide isosteres
  • Polypeptide refers to both short chams, commonly referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as protems Polypeptides may contain amino acids other than the 20 gene-encoded ammo acids
  • Polypeptides mclude ammo acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known m the art Such modifications are well descnbed in basic texts and in more detailed monographs, as well as m a voluminous research literature Modifications may occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chains and the ammo or carboxyl termini It
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences
  • Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A M , ed Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, A M , and G ⁇ ffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Heinje, G , Academic Press, 1987, and Sequence Analysis Primer, G ⁇ bskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Canllo, H , and Lipman, D , S AM J Applied Math , 48 1073 (1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified m publicly available computer programs Computer program methods to determine identity between two sequences mclude,
  • polynucleotides and polypeptides are provided m (1) and (2) below
  • Polynucleotide embodiments further mclude an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1, wherem said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may include up to a certain mteger number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides m the reference sequence or m one or more contiguous groups
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NO 1
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-mteger product of x n and y is rounded down to the nearest integer prior to subtracting it from x n
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations m this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical
  • n n is the number of ammo acid alterations
  • x n is the total number of ammo acids in SEQ ID NO 2
  • y is, for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc
  • • is the symbol for the multiplication operator, and wherem any non-mteger product of x n and y is rounded down to the nearest integer pnor to subtracting it from x n
  • Polypeptide embodiments further mclude an isolated polypeptide compnsmg a polypeptide havmg at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may mclude up to a certain mteger number of amino acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one ammo acid deletion,
  • n a is the number of ammo acid alterations
  • x a is the total number of ammo acids in SEQ ID NO 2
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherem any non-mteger product of x a and y is rounded down to the nearest mteger pnor to subtracting it from x a
  • a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may include up to a certam mteger number of ammo acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity
  • Such alterations are selected from the group consistmg of at least one ammo acid deletion, substitution, mcludmg conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence
  • the number of amino acid alterations for a given % identity is determined by multiplymg the total number of ammo acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtractmg that product from said total number of ammo acids m SEQ ID NO 2, or
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NO 2
  • y is, for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc
  • • is the symbol for the multiplication operator, and wherem any non-mteger product of x a and y is rounded down to the nearest mteger pnor to subtractmg it from x a
  • Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof
  • EP-A-0 464 discloses fusion proteins comprising various portions of constant region of immunoglobulm molecules together with another human protein or part thereof
  • employmg an immunoglobulm Fc region as a part of a fusion protein is advantageous for use m therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e g , EP-A 0232 262]
  • Gly Asp Leu Pro Pro Leu Trp Trp Tyr lie Val Thr Arg Pro Arg Glu

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Abstract

L'invention concerne des polypeptides et des polynucléotides CBLALE02 et des procédés permettant de produire ces polypeptides par des techniques recombinantes. L'invention concerne également des procédés d'utilisation de ces polypeptides et polynucléotides CBLALE02 pour des traitements, ainsi que des systèmes de diagnostic de pathologies associées à ces polypeptides.
PCT/CN1998/000085 1998-06-04 1998-06-04 Gene homologue de la sous-unite ci-b17 de l'ubiquinone oxydoreductase humaine (cblale02) WO1999062948A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000085 WO1999062948A1 (fr) 1998-06-04 1998-06-04 Gene homologue de la sous-unite ci-b17 de l'ubiquinone oxydoreductase humaine (cblale02)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000085 WO1999062948A1 (fr) 1998-06-04 1998-06-04 Gene homologue de la sous-unite ci-b17 de l'ubiquinone oxydoreductase humaine (cblale02)

Publications (1)

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WO1999062948A1 true WO1999062948A1 (fr) 1999-12-09

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Application Number Title Priority Date Filing Date
PCT/CN1998/000085 WO1999062948A1 (fr) 1998-06-04 1998-06-04 Gene homologue de la sous-unite ci-b17 de l'ubiquinone oxydoreductase humaine (cblale02)

Country Status (1)

Country Link
WO (1) WO1999062948A1 (fr)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GENOMICS, 35(1), (1996), GU J.Z. et al., "The Human B22 Subunits of the NADH:Ubiquinone Oxidoreductase Maps to the Region of Chromosome 8 Involved in Branchio-oto-renal Syndrome", pages 6-10. *
J. MOL. BIOL., 226(4), (1992), WALKER J.E. et al., "Sequences of 20 Subunits of NADH:Ubiquinone Oxidoreductase from Bovine Heart Mitochondria, Application of a Novel Strategy for Sequencing Proteins using the Polymerase Chain Reaction", pages 1051-1072. *
PEDIATRIC RESEARCH, 39(3), 1996, LANE R.H. et al., "Altered Hepatic Gene Expression of Enzymes Involved in Energy Metabolism in the Growth-Retarded Fetal Rat", pages 390-394. *

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