CN1275131A - Human lysophospholipase gene (CBFBLH05) - Google Patents
Human lysophospholipase gene (CBFBLH05) Download PDFInfo
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- CN1275131A CN1275131A CN98810060.6A CN98810060A CN1275131A CN 1275131 A CN1275131 A CN 1275131A CN 98810060 A CN98810060 A CN 98810060A CN 1275131 A CN1275131 A CN 1275131A
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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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- 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
Abstract
The CBFBLH0 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilising CBFBLH05 polypeptides and polynucleotides in therapy, and diagnositc assays for such.
Description
Invention field
The present invention relates to new identified polynucleotides, by its encoded polypeptides and these polynucleotide and polypeptide in the evaluation of treatment and its agonist, antagonist and/or the inhibitor compound that may be able to be used for the treatment of purposes and the production of these polypeptide and polynucleotide.Background of invention
The method of seeking medicine is just experiencing the variation of essence, comprises in the new method " functional genomics ", promptly based on the high throughput biology of genome or gene.This method is replacing originally the method based on " position clone " rapidly.At first identify the phenotype show as biological function or inherited disease, then according to its genetic map inverse position to seeking related gene.
Functional genomics mainly relies on various information biology instruments, by identifying possible target gene sequences in existing a plurality of molecular biology databases.This just need constantly identify and characterize other genes and related polypeptide/protein thereof, as the target of seeking medicine.Summary of the invention
The present invention relates to CBFBLH05, particularly CBFBLH05 polypeptide and polynucleotide and recombined material and their production method.On the other hand, the present invention relates to utilize the method for these polypeptide and polynucleotide, comprise treatment hepatopathy, cancer, autoimmune disease and ephrosis diseases such as (limiting disease) hereinafter referred to as the present invention.Again on the one hand, the present invention relates to utilize the method for material evaluation agonist provided by the invention and antagonist/inhibitor and utilize the symptom that compounds identified is treated and CBFBLH05 is uneven relevant.At last, the present invention relates to check the diagnostic test of the disease relevant with CBFBLH05 activity or horizontal abnormality.Detailed Description Of The Invention
On the one hand, the present invention relates to the CBFBLH05 polypeptide.These peptides comprise the isolated polypeptide that contains following aminoacid sequence, the identity of the whole aminoacid sequence of described aminoacid sequence and sequence 2 is at least 70%, preferred identity is at least 80%, more preferably identity is at least 90%, be more preferably identity and be at least 95%, most preferably identity is 97-99% at least.These polypeptide also comprise the polypeptide of the aminoacid sequence that contains sequence 2.
Peptide of the present invention also comprises following isolated polypeptide, the identity of the whole aminoacid sequence of its aminoacid sequence and sequence 2 is at least 70%, preferred identity is at least 80%, more preferably identity is at least 90%, be more preferably identity and be at least 95%, most preferably identity is 97-99% at least.These polypeptide also comprise the polypeptide of sequence 2.
Polypeptide of the present invention also comprises the isolated polypeptide by the polynucleotide encoding that contains sequence 1.
Polypeptide of the present invention cause people's attention be because people's lysophospholipid enzyme clone from umbilical cord blood hematopoietic progenitor cell CD34+ cell, it has two kinds of expression-forms: short chain type and long chain type.Mainly the short chain type in the CD34+ cell.These characteristics are hereinafter referred to as " CBFBLH05 activity " or " CBFBLH05 polypeptide active " or " CBFBLH05 biological activity ".These activity also comprise the antigen and the immunogen activity of described CBFBLH05 polypeptide, the particularly antigen of the polypeptide of sequence 2 and immunogen activity.Preferably, polypeptide of the present invention has the biological activity of at least a CBFBLH05.
Polypeptide of the present invention can be " maturation " proteic form, also can be the part than large protein such as fusion rotein.It is comparatively favourable to generally include additional aminoacid sequence, and additional aminoacid sequence comprises secretion or leader sequence, and former sequence helps the sequence such as the polyhistidyl residue of purifying, or helps stable appended sequence in the recombinant production process.
The present invention also comprises the variant of aforementioned polypeptides, i.e. conserved amino acid generation metathetical polypeptide in the object of reference, and conservative substitution is meant that a residue is had other radical amino acid replacements of similar characteristics.Typical this displacement comprises Ala, Val, the displacement between Leu and the Ile; Displacement between Ser and the Thr; Displacement between acidic residues Asp and the Glu; Displacement between Asn and the Gln; Displacement between alkaline residue Lys and the Arg; Or the displacement between aromatic residue Phe and the Tyr.Particularly preferably be wherein replace, lack or increase several, 5-10, a 1-5 1-3,1-2 is individual or the variant of the combination of 1 amino acid or these modes.
Polypeptide of the present invention can pass through prepared in any suitable way.These polypeptide comprise isolating naturally occurring polypeptide, the polypeptide of recombinant production, the synthetic polypeptide of producing, or the polypeptide of these method combinations produce.The method for preparing these polypeptide is widely known by the people in this area.
The present invention relates to the CBFBLH05 polynucleotide on the other hand.These polynucleotide comprise the separation polynucleotide that contain following nucleotide sequence, the identity of the whole aminoacid sequence of described nucleotide sequence coded polypeptide and sequence 2 is at least 70%, preferred identity is at least 80%, more preferably identity is at least 90%, is more preferably identity and is at least 95%.Polypeptide identity is highly preferred at least 97%, and polypeptide identity is for 98-99% is more highly preferred at least, and polypeptide identity is most preferred at least 99%.These polynucleotide comprise the polynucleotide of the nucleotide sequence in the sequence 1 of the polypeptide that contains encoding sequence 2.
Polynucleotide of the present invention also comprise the isolating polynucleotide that contain following nucleotide sequence, the identity of the whole coding region of nucleotide sequence of above-mentioned nucleotide sequence and encoding sequence 2 aminoacid sequences is at least 70%, preferred identity is at least 80%, more preferably identity is at least 90%, is more preferably identity and is at least 95%.Identity is highly preferred at least 97% polynucleotide, and identity is that the polynucleotide of 98-99% at least are more highly preferred, and identity is most preferred at least 99% polynucleotide.
Polynucleotide of the present invention also comprise the isolating polynucleotide that contain following nucleotide sequence, the identity of the whole nucleotide sequence of above-mentioned nucleotide sequence and sequence 1 is at least 70%, preferred identity is at least 80%, more preferably identity is at least 90%, is more preferably identity and is at least 95%.Identity is highly preferred at least 97% polynucleotide, and identity is that the polynucleotide of 98-99% at least are more highly preferred, and identity is most preferred at least 99% polynucleotide.These polynucleotide comprise the polynucleotide of the polynucleotide that contain sequence 1 and the polynucleotide of sequence 1.
The present invention also provides the complementary polynucleotide of above-mentioned polynucleotide.
The nucleotide sequence of sequence 1 and rat lysophospholipase dna homolog (H.Sugimoto, J.Biol.Chem., 1996:271 (13): 7705-7711).The nucleotides sequence of sequence 1 is classified the cDNA sequence as, contains a polypeptid coding sequence (Nucleotide 6-647), 214 amino acid whose polypeptide of encoding sequence 2.The nucleotide sequence of encoding sequence 2 polypeptide can be identical with the polypeptid coding sequence in the sequence 1, perhaps different, but because the Feng Yu (degeneracy) of genetic code forms but the also polypeptide of encoding sequence 2.Other albumen with this family on the polypeptide structure of sequence 2 are relevant, with rat Phospholipid hydrolase homology and/or structural similitude (H.Sugimoto, J.Biol.Chem., 1996:271 (13): 7705-7711).
Expection preferred polypeptide of the present invention and polynucleotide have its homeopeptide and the similar biological function/characteristic of polynucleotide.And preferred polypeptide of the present invention and polynucleotide have at least a CBFBLH05 activity.
Polynucleotide of the present invention can obtain from the cDNA library by standard clone and triage techniques, this library uses expressed sequence mark (EST) to analyze (Adams that derives by the mRNA from the human cord blood cell, M.K., 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).Polynucleotide of the present invention also can maybe can use well-known commercially available technology synthetic from natural origin such as genome dna library.
When polynucleotide of the present invention were used for recombinant production polypeptide of the present invention, these polynucleotide can comprise the encoding sequence of mature polypeptide itself; The sequence of mature polypeptide encoded sequence and other encoding sequence frame endomixis, for example leading or secretion sequence, preceding former or preceding former protein sequence or other fusion polypeptide encoding sequence partly of other encoding sequences.For example can encode and help the flag sequence of fusion polypeptide purifying.In this certain preferred embodiments on the one hand of the present invention, flag sequence is 6-Histidine peptide or HA mark, the former with the pQE carrier provide (Qiagen, Inc.), relevant discussion is referring to Gentz et al., Proc Natl Acad Sci USA (1989) 86:821-824.Polynucleotide also can contain noncoding 5 ＇ or 3 ＇ sequences, as transcribe, non-translated sequence, shear and polyadenylation signal the sequence of ribosome bind site and stable mRNA.
Further preferred embodiment of the present invention comprises the polynucleotide of coded polypeptide variant, described variant comprises the aminoacid sequence (sequence 2) of sequence 2, wherein replaces, lacks or increase several, 5-10,1-5,1-3,1-2 or 1 amino-acid residue or the combination of these modes.
The nucleotide sequence of polynucleotide of the present invention and sequence 1 is equal to or enough is equal to, therefore can be as the hybridization probe of cDNA and genomic dna or the primer of nucleic acid amplification (PCR) reaction, to separate the full-length cDNA and the genomic clone of code book invention polypeptide, also can separate the cDNA and the genomic clone that have other genes (homologue and the straight gene that comprise the species of coding except that the people) of high sequence similarity with sequence 1 to homologue.The identity of general these nucleotide sequences and object of reference sequence is 70%, preferably identity is 80%, and more preferably identity is 90%, and most preferably identity is 95%.Probe or primer generally comprise at least 15 Nucleotide, and preferably at least 30 Nucleotide also can comprise at least 50 Nucleotide.Particularly preferred probe is 30 to 50 Nucleotide.
The polynucleotide that obtain code book invention polypeptide comprise that the homologue of the species except that the people or straight method to homologue may further comprise the steps: apparatus has sequence 1 or its segmental label probe to screen suitable library under tight hybridization conditions; Separate the full-length cDNA and the genomic clone that contain described polynucleotide sequence.These hybridization techniques are well known to those skilled in the art.Preferred tight hybridization conditions is included in the following solution 42 ℃ and is incubated overnight, then about 65 ℃ with 0.1 * SSC washing filter membrane, described solution comprises 50% methane amide, 5 * SSC (150 mM NaCl, the 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 * Denhardt solution, the salmon sperm DNA that 10% dextran sulfate and 20 μ g/ml sex change are sheared.Therefore, the present invention also comprises by apparatus has the label probe of sequence 1 or its fragments sequence to screen the polynucleotide that suitable library obtains under tight hybridization conditions.
The technician can recognize that under multiple situation, isolating cDNA sequence is incomplete, and wherein the zone of coded polypeptide is prescinded at the 5 ＇ ends of cDNA.This is the effect owing to reversed transcriptive enzyme, and this enzyme itself has lower processivity (the enzyme maintenance is attached to the tolerance of the ability of template in the polymerization process), can not obtain the global DNA copy of mRNA template in the 1st cDNA chain building-up process.
Obtain full-length cDNA or prolong the method for short cDNA existing several be well known to those skilled in the art, for example based on the method (referring to for example Frohman et al., PNAS USA 85,8998-9002,1988) of terminal rapid amplifying (RACE) method of cDNA.Up-to-date technological improvement such as Marathon
TMTechnology (Clontech Laboratories Inc.) has been simplified the search of long-chain cDNA greatly.At Marathon
TMIn the technology, organize extractive mRNA to prepare cDNA, connect " adapter " sequence at its two ends by selected certainly.Use the specific Oligonucleolide primers combination carrying out of gene specific and adapter nucleic acid amplification (PCR) then, the 5 ＇ end of amplification cDNA " disappearance ".Use " nested " primer to repeat the PCR reaction, so-called nested primers is meant and is designed to annealed primer in amplified production (being generally at adapter sequence 3 ＇ downstream annealed adapter Auele Specific Primers with at known sequence 5 ＇ upstream annealed gene-specific primers).Analyze this reaction product by dna sequencing, this product directly is connected the acquisition complete sequence with existing cDNA, or uses new sequence information to design 5 ＇ primers and carry out total length PCR in addition, thereby make up full-length cDNA.
Recombinant polypeptide of the present invention can be by methods known in the art by the genetically engineered host cell preparation that contains expression system.Therefore, the present invention relates to the expression system that contains one or more polynucleotide of the present invention on the other hand, contains the host cell of expression system of the present invention and produces polypeptide of the present invention by recombinant technology through genetically engineered.Utilization is derived from the RNA of DNA construct of the present invention, and cell free translation system can be used to produce these albumen.
For carrying out recombinant production, host cell can be through genetically engineered expression system or its part that contains polynucleotide of the present invention.Polynucleotide are imported host cell can be realized by the method described in the multiple standards laboratory manual, described laboratory manual such as Davis et al., " molecular biology basic skills " (1986) and Sambrook et al., " molecular cloning: laboratory manual " the 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).Preferred these methods comprise transfection, transposition, the microinjection of for example calcium phosphate transfection, the mediation of DEAE-dextran, transfection, electroporation, transduction, friction conversion, particle bombardment or the infection of cationic lipid mediation.
The representative example of suitable host comprises bacterial cell, as suis, staphylococcus, intestinal bacteria, streptomycete and bacillus subtilis cell; The fungal cell is as yeast cell and aspergillus cell; Insect cell is as fruit bat S2 and fall army worm Sf9 cell; Zooblast is as CHO, COS, HeLa, C127,3T3, BHK, HEK 293 and Bowes melanoma cell; And vegetable cell.
Can use multiple expression system, these expression systems comprise karyomit(e), episome and viral deutero-system etc., for example derived from the carrier of bacterial plasmid, bacteriophage, transposon, yeast episome, insertion element, yeast chromosomal element, virus, with carrier derived from the said elements combination, described virus comprises baculovirus, papovavirus such as SV40, vaccinia virus, adenovirus, fowlpox virus, Pseudorabies virus and retrovirus, and the combination carrier is as the carrier derived from plasmid and bacteriophage genetic elements, clay and phagemid.Expression system can contain the control region of regulating and starting expression.In general, any system or carrier that is suitable for keeping in the host, breed or expresses polynucleotide all can use.Suitable nucleotide sequence can be inserted in the expression system by multiple known routine techniques, and described technology can be referring to for example Sambrook et al., " molecular cloning: laboratory manual " the 2nd edition (the same).For the albumen of translating can be secreted in the endoplasmic, periplasmic space or born of the same parents' external environment, can on required polypeptide, add suitable secretion signal.These signals can be that polypeptide is endogenous, also can be the external source secretion signals.
Be used for shaker test if polypeptide of the present invention will be expressed, general preferred polypeptide generates at cell surface.In this case, cell can be gathered in the crops before being used for shaker test.If this polypeptide is secreted in the substratum, can reclaim substratum to reclaim and purified polypeptide; If generate in cell, cell is at first wanted cracking, reclaims polypeptide then.
Polypeptide of the present invention can reclaim and purifying from the reconstitution cell culture by currently known methods, described method comprises ammonium sulfate or ethanol sedimentation, the acid extracting, negatively charged ion or cation-exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatograph, affinity chromatography, hydroxylapatite chromatography and lectin chromatogram.Most preferably high performance liquid chromatography is used for purifying.If polypeptide sex change in the isolated or purified process, the proteic method of known refolding can be used for recovering activity conformation.
The invention still further relates to the application of polypeptide of the present invention as diagnostic reagent.Detected characteristics is that the mutant form of the gene of the sequence relevant with dysfunction 1 polynucleotide can provide a kind of diagnostic tool, the disease that this can increase or limit a kind of because this expression of gene deficiency, it is excessive to express or abnormal expression produces or the diagnosis of disease susceptibility.The individuality that carries sudden change in this gene can detect on dna level by multiple technologies.
Diagnosis nucleic acid can be available from experimenter's cell, as blood, urine, saliva, biopsy sample or postmortem material.Genomic dna can directly be used for detecting, or uses PCR or other amplification technique enzymatic amplifications before analyzing.RNA or cDNA can use in a similar manner.Disappearance can detect by the size variation of comparing amplified production with normal genotype with inserting.Point mutation can be by differentiating the CBFBLH05 nucleotide sequence hybridization of DNA amplification and mark.Pi Pei sequence can be by difference on RNA enzymic digestion or the melting temp and the double-stranded differentiation of mispairing fully.Dna sequence dna difference also can add or not detect (can be referring to for example Myers et al., Science (1985) 230:1242) with electrophoretic mobility on the gel of denaturing agent or direct dna sequencing by dna fragmentation.Also can show (can be referring to Cotton et al., PNASUSA (1985) 85:4397-4401) in the sequence variation of specific position by nuclease protection test such as RNA enzyme and S1 protection method or chemical chop method.In another embodiment, can make up and contain CBFBLH05 nucleotide sequence or its segmental oligonucleotide probe array (array), so that carry out effective screening of for example transgenation.The array technique method is widely known by the people, and suitability is extensive, can be used to solve the various problems in the molecular genetics, comprises genetic expression, genetic linkage and hereditary variability.(referring to for example M.Chee et al., Science, Vol.274, pp610-613 (1996).
Diagnostic test detects the sudden change of CBFBLH05 gene by utilizing aforesaid method, and diagnosis or definite method that the present invention is limited the susceptibility of disease are provided.In addition, these diseases can be diagnosed by the following method: determine to reduce or increase from the horizontal abnormality of polypeptide in experimenter's sample or mRNA.Expressing to reduce or increase to use the known any method in the quantitative field of Nucleotide to determine that on rna level described method is nucleic acid amplification for example, comprises PCR, RT-PCR, RNA enzyme protection, RNA trace and other hybridizing methods.Can be used for determining being well known to those skilled in the art from the experimental technique of the level of albumen in host's the sample such as polypeptide of the present invention.These test methods comprise that radioimmunoassay, competition are in conjunction with test, western blot analysis and ELISA test.
Therefore, on the other hand, the present invention relates to comprise the Sickledex of following composition: (a) polynucleotide of the present invention, the nucleotide sequence of preferred sequence 1, or its fragment; (b) with (a) in Nucleotide complementary nucleotide sequence; (c) polypeptide of the present invention, the polypeptide of preferred sequence 2, or its fragment; (d) antibody of polypeptide of the present invention, the antibody of the polypeptide of preferred sequence 2.
Should be appreciated that in this medicine box, (a), (b), (c) or (d) can comprise a kind of main component (substantial component).This medicine box can be used for diagnosing the illness or to the susceptibility of disease, described disease is hepatopathy, cancer, autoimmune disease and ephrosis etc. particularly.
Nucleotide sequence of the present invention is identified also valuable to karyomit(e).This sequence can special target one by one the specific position on the Autosome and can with its hybridization.The mapping of karyomit(e) correlated series is the important the first step that the gene with these sequences and disease-related connects according to the present invention.In case sequence has been positioned in concrete chromosome position, the physical location of this sequence on karyomit(e) can connect with the genetic map data.These data can be referring to for example V.McKusick, " human Mendelian inheritances) " (by the online acquisition of Johns Hopkins University Welch Medical Library).Being positioned at the gene of same chromosomal region and the relation between the disease can pass through linkage analysis (the common heredity of the adjacent gene of physics) and determine.
The difference of cDNA or genome sequence also can be determined in the influenced and normal individual.If observe a certain sudden change in some or all affected individuals, and do not observe this sudden change in normal individual, then this sudden change may be the cause of disease of disease.
Polypeptide of the present invention or its fragment or analogue or the cell of expressing them also can be used as immunogen, generate the specific antibody of polypeptide immune of the present invention." immunologic opsonin " represents that in the art avidity to polypeptide of the present invention is obviously greater than the avidity to other related polypeptides of this area.
Can be at the antibody of polypeptide of the present invention by ordinary method to the preferred inhuman animals administer polypeptide of animal or contain epi-position fragment, analogue or cell and obtain.In order to prepare monoclonal antibody, can use any technology of the antibody that the generation of continuous cell line culture is provided.Example comprises hybridoma technology (Kohler, G.and Milstein, C., Nature (1975) 256:495-497), three knurls (trioma) technology, human B cell hybridoma technology (Kozbor et al., ImmunologyToday (1983) 4:72) and EBV hybridoma technology (Cole et al., " monoclonal antibody and cancer therapy ", pp77-96, Alan R.Liss, Inc., 1985).
After also can improving, the technology of manufacture order chain antibody such as United States Patent (USP) 4,946,778 described technology are used for the single-chain antibody of production polypeptide of the present invention.Transgenic mice or other organisms comprise that other Mammalss also can be used for expressing humanized antibody.
Above-mentioned antibody also can be used for separating or identify the clone of the described polypeptide of expression or the polypeptide of purifying affinity chromatography technology preparation.
The antibody of anti-polypeptide of the present invention also can be used for treating the present invention and limit disease etc.
On the other hand, the present invention relates to the genetically engineered soluble fusion protein that contains polypeptide of the present invention or its fragment and each subclass (IgG, IgM, IgA, IgE) various piece of heavy chain immunoglobulin or constant region of light chain.Preferred immunoglobulins is the particularly CH part of IgG1 of human IgG, wherein merges to occur in hinge area.In a specific embodiments, can Fc partly be removed by the cutting sequence that blood clotting factor Xa cuts by adding.In addition, the present invention relates to prepare method and its application in drug screening, diagnosis and treatment of these fusion roteins by genetic engineering.Another aspect of the present invention also relates to the polynucleotide of these fusion roteins of encoding.The example of fusion protein technology can be referring to International Patent Application WO 94/29458 and WO94/22914.
Other aspects of the present invention relate to the method for induce immune response in Mammals, comprise with polypeptide immune seeded with mammalian of the present invention, produce antibody and/or t cell response, limit disease etc. thereby protect this animal to avoid the present invention.Other aspects of the present invention also relate to the method for induce immune response in Mammals, and the carrier of coded polypeptide comes administration polypeptide of the present invention by instructing polynucleotide to express also in vivo, so that induce immune response, generation antibody watches for animals and avoids disease.
Other aspects of the present invention relate to immunogen/vaccine preparation (composition), when being introduced into mammalian hosts, can induce the immunne response at polypeptide of the present invention, and wherein said composition contains polypeptide of the present invention or polynucleotide.This vaccine preparation can also contain suitable carriers.Because this polypeptide can be degraded under one's belt, preferably by parenteral route administration (comprising injections such as subcutaneous, muscle, intravenously, intracutaneous).The preparation that is fit to parenteral admin comprises moisture and water-free aseptic parenteral solution, wherein can contain antioxidant, buffer reagent, fungistat and make preparation and the isoosmotic solute of receptor's blood; Moisture and water-free sterile suspensions wherein can contain suspension agent or thickening material.Said preparation can be single agent or multi-agent vessel form, and for example sealed ampoule and bottle also can be kept under the lyophilisation condition, only need add sterile liquid carrier before use and get final product.Vaccine preparation also can contain the immunogenic adjuvant system of enhancing preparation, as oil-in-water system or other system known in the art.Dosage depends on the specific activity of vaccine, can determine easily by routine test.
Polypeptide of the present invention is relevant with multiple biological function, comprises various disease states, and particularly the above the present invention limits disease.Therefore, be necessary to design the screening method of identifying stimulation or suppressing the compound of polypeptide function.Thereby the present invention provides the method for SCREENED COMPOUND on the other hand, to identify the compound that stimulates or suppress the polypeptide function.In general, agonist or antagonist can be used for the above the present invention limit treatment of diseases and the prevention purpose.Compound can be from the evaluation of multiple source, for example cell, acellular prepared product, chemical library and natural product mixture.These agonists, antagonist or the inhibitor of identifying can be the natural of polypeptide of the present invention or the substrate of modifying, part, acceptor, enzyme etc.; Or the structure of polypeptide of the present invention or functional analogue thing.Can be referring to Coligan et al., Current Protocol in Immunology 1 (2): Chapter 5 (1991).
Shaker test can be tested combining of candidate compound and polypeptide, the cell with this polypeptide or cytolemma or its fusion rotein simply, utilizes directly or indirectly and candidate compound bonded marker detection.Perhaps screening method comprises the competition of competing thing with mark.And whether these screening methods can be tested candidate compound can produce the signal that polypeptide activates or suppress to produce, and uses the detection system of the cell that is suitable for having polypeptide.Activation inhibitor test when known agonist exists usually, observing the candidate compound existence influences the agonist activated.The structure-activity polypeptide can be used for screening the method for inverse agonist or inhibitor, measures the activation whether candidate compound suppresses polypeptide when not having agonist or inhibitor.In addition, shaker test can may further comprise the steps simply: mix candidate compound and the solution that contains polypeptide of the present invention, form mixture, measure CBFBLH05 activity in the mixture, relatively the CBFBLH05 activity of mixture and standard substance.Also can be used for the high throughput shaker test as the above-mentioned fusion roteins such as fusion rotein that form by Fc part and CBFBLH05 polypeptide, with the antagonist of evaluation polypeptide of the present invention (referring to D.Bennett et al., JMol Recognition, 8:52-58 (1995); And K.Johanson et al., J Biol Chem, 270 (16): 9459-9471 (1995)).
The antibody of polynucleotide of the present invention, polypeptide and polypeptide also can be used for designing the screening method that detects the influence that mRNA and polypeptide generate in the compound pair cell that adds.For example, can design ELISA, use mono-clonal and polyclonal antibody to measure secretion or cell bonded polypeptide level by standard method known in the art.This can be used for seeking from suitable engineered cell or tissue and can suppress or increase the material (also can be called antagonist or agonist) that polypeptide generates.
This polypeptide can be used for identifying the film combination or the solvable acceptor of existence, uses standard receptors bind technology known in the art.These include but not limited to part combination and crosslinked test, wherein polypeptide with radio isotope (as
125I) mark, chemically modified (as biotinylation), or be fit to detect or the peptide sequence of purifying merges, with infer be subjected to body source (cell, cytolemma, cell conditioned medium, tissue extract, body fluid) incubation.Additive method comprises the biophysics technology, as surperficial kytoplasm resonance and spectroscopy.These screening methods also can be used for identifying the agonist and the antagonist of polypeptide, they and the combine competition of polypeptide to the acceptor that exists.The standard method of carrying out these tests is well known in the art.
The example of possible polypeptide antagonist comprises the antibody of polypeptide, perhaps is in some cases and closely-related oligonucleotide or albumen such as the part of polypeptide, substrate, acceptor, enzyme, for example fragment of part, substrate, acceptor, enzyme etc.; Perhaps be to combine with polypeptide of the present invention but the small molecules of initiating response not, thereby suppress the activity of polypeptide.
Therefore, the present invention relates to a kind of screening medicine box on the other hand, is used for identifying agonist, antagonist, part, acceptor, substrate, enzyme of polypeptide of the present invention etc.; Or reducing or increase the compound that these polypeptide generate, this medicine box comprises: (a) polypeptide of the present invention; (b) reconstitution cell of expression polypeptide of the present invention; (c) cytolemma of expression polypeptide of the present invention; (d) antibody of polypeptide of the present invention; Wherein, polypeptide is preferably the polypeptide of sequence 2.
Should be appreciated that in this medicine box, (a), (b), (c) or (d) can comprise a kind of main component.
Those skilled in the art are easy to recognize that polypeptide of the present invention also can be used for the method based on agonist, antagonist or the inhibitor of structure design polypeptide, comprising: the three-dimensional structure of (a) at first determining polypeptide; (b) infer the reaction possible in agonist, antagonist or the inhibitor or the three-dimensional structure of combining site; (c) synthetic expection and combining of inferring or reactive site in conjunction with or the candidate compound that reacts; (d) whether the test candidate compound is antagonist, antagonist or inhibitor really.Should be appreciated that normally interactive process of this process.
On the other hand, the invention provides and the too high or too low relevant abnormal symptom of CBFBLH05 polypeptide active such as the methods of treatment of hepatopathy, cancer, autoimmune disease and ephrosis.
If polypeptide active is too high, several method can be arranged.A kind of method comprises to the above-mentioned inhibitor compound of experimenter's administration (antagonist), optional and pharmaceutically acceptable carrier makes up, and its consumption can suppress the function of polypeptide, for example passes through the combination of block ligand, substrate, acceptor, enzyme etc., or by the inhibition second signal, thereby alleviate abnormal symptom.Administration can be competed the polypeptide of the soluble form of binding partner, substrate, enzyme, acceptor etc. with endogenous polypeptide in the another kind method.The representative instance of this competition thing comprises the fragment of CBFBLH05 polypeptide.
In another approach, can use the expression of gene of expressing the endogenous CBFBLH05 polypeptide of interrupter technique inhibition coding.Known this technology comprises the use antisense sequences, can be inner that produce or individually dosed (referring to for example O ＇ Connor, J Neurochem (1991) 56:560, " oligodeoxynucleotide of genetic expression antisense inhibitor " CRC Press, Boca Raton, FL (1988)).Perhaps can use with gene form triple helical oligonucleotide (referring to for example Lee, et al., Nucleic Acid Res (1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al., Scicence (1991) 251:1360).Can these oligomer of administration or express relevant oligomer in vivo.
In order to treat CBFBLH05 and the insufficient abnormal symptom of its activity expression, there is several method to use.A kind of method comprises the compound to the activation polypeptide of the present invention of experimenter's drug treatment significant quantity, and promptly above-mentioned agonist in conjunction with the pharmaceutically useful carrier of administration, thereby is alleviated abnormal symptom.Perhaps, can use gene therapy to realize experimenter's relevant cell endogenous production CBFBLH05.For example, polynucleotide of the present invention can be expressed with the replication defect type retroviral vector through engineered as above-mentioned.Separate then in retrovirus expression construct and the packing cell of importing, contain the RNA of code book invention polypeptide in the carrier, thereby packing cell can produce the infectious virus particle that contains goal gene with the retroviral plasmid vector transduction.Can these produce cells to experimenter's administration, so that engineered cells and express polypeptide in vivo in the body.The relevant discussion of gene therapy can be referring to the 20th chapter, " gene therapy and other methods of treatment (and reference wherein); " human molecular genetics ", T Strachan and A P Read, BIOSScientific Publishers Ltd (1996) based on molecular genetics.Another kind method is the polypeptide of the present invention of drug treatment significant quantity, in conjunction with the suitable pharmaceutical carrier of administration.
On the other hand, the invention provides and contain the polypeptide for the treatment of significant quantity and the pharmaceutical composition of pharmaceutically acceptable carrier or vehicle, the soluble form of described polypeptide such as polypeptide of the present invention, agonist/antagonist peptide or micromolecular compound.These carriers include but not limited to salt solution, buffer saline, glucose, water, glycerine, ethanol and its combination.The invention still further relates to cartridge bag and medicine box,, one or more above-mentioned composition compositions of the present invention are housed comprising one or more containers.Polypeptide of the present invention and other compounds can use separately, or unite use with other compounds as the treatment compound.
Composition should be suitable for route of administration, for example whole body or oral route.Preferred whole body administering mode comprises injection, generally is intravenous injection.Also can use other injecting pathways such as subcutaneous, muscle or intraperitoneal approach.The additive method of whole body administration comprises that use cholate or permeate agents such as fusidinic acid or other tensio-active agents are through mucous membrane and percutaneous dosing.In addition, if polypeptide of the present invention or other compounds can be mixed with enteron aisle or capsule, also can be taken orally.These compounds also can be with form surface and/or topicals such as ointment, paste, gels.
The required dosage scope depends on selection, route of administration, preparation nature, the character of experimenter's symptom and attending doctor's the judgement of peptide of the present invention or other compounds.Suitable dosage ranges is 0.1 to 100 μ g/kg.In view of the compound that can obtain has multiple, different route of administration different effects is arranged, the variation range of required dosage is very big to be predictable.For example, the expection oral administration is big than the intravenous injection required dosage.The variation of these dosage levels can be used the standard test method adjustment that is used for optimizing, and this is well known in the art.
Treatment also can endogenous generation in subject with polypeptide, and this form of therapy is commonly referred to " gene therapy ".Therefore, for example can use DNA or the RNA of polynucleotide, utilize retroviral plasmid vector engineered as the polypeptide of coding ex vivo from experimenter's cell.Then these cells are introduced in the subject.
Polynucleotide and peptide sequence have constituted valuable information source, can be used for identifying other sequences with similar homology.By sequence is stored on the computer-readable medium, the data of utilizing storage make evaluation greatly convenient by well-known gopher such as GCC retrieve sequence database then.Therefore, the present invention provides a kind of computer-readable medium on the other hand, stores the polynucleotide that contain sequence 1 on it and/or by its encoded polypeptides sequence.
For ease of understanding term commonly used in this specification sheets, provide to give a definition.
" antibody " comprises mono-clonal and polyclonal antibody, chimeric, strand and humanized antibody and Fab fragment in this article, comprises the product in Fab or other immunoglobulin expression libraries.
" isolating " expression " by artificial " changes its native state.If " isolating " composition or material exist at occurring in nature, then it changes or takes out or taken out and change from its primal environment.For example, when using this term in this article, polynucleotide or the polypeptide that exists in moving object is not " isolating ", but with native state under the material that coexists the as much Nucleotide or the polypeptide that separate be exactly " isolating ".
" polynucleotide " refer generally to poly-ribonucleotide or poly-deoxyribonucleotide, can be the RNA or the DNA of unmodified or modification." polynucleotide " include but not limited to strand and double-stranded DNA, strand and double stranded region blended DNA, strand and double-stranded RNA, strand and double stranded region blended RNA, the hybrid molecule that contains DNA and RNA can be strand or more common mixture for two strands or strand and double stranded region.In addition, " polynucleotide " also refer to contain three sequences of RNA or DNA or RNA and DNA.Term " polynucleotide " also comprises DNA or the RNA that contains one or more modified bases, and is DNA or RNA that stability or other reasons have been modified skeleton." modification " base comprises for example rare base such as tritylation base and inosine.Can carry out multiple modification to DNA and RNA, therefore, " polynucleotide " comprise chemistry, enzymatic or the metabolism modified forms of the polynucleotide that occurring in nature is common, and the DNA and the RNA chemical species of virus and cells characteristic." polynucleotide " also comprise short, that be commonly called oligonucleotide relatively polynucleotide.
" polypeptide " refers to that the peptide bond that contains by peptide bond or modification is interconnected two or more amino acid whose any peptides of peptide isostere (peptideisosteres) or albumen." polypeptide " both comprised the oligopeptides or the oligomer of short chain (being commonly referred to peptide), also comprised long-chain (being commonly referred to albumen).Polypeptide may contain the amino acid outside the amino acid of 20 kinds of genes encodings." polypeptide " comprises the aminoacid sequence of modifying by translation natural method such as post-treatment or chemical modification technology known in the art.It is detailed that these are modified in basic textbook and monograph and a large amount of research paper narration.Modification can occur in any position of polypeptide, comprises peptide backbone, amino acid side chain and amino or C-terminal.Can exist with identical or different degree in the similar modification of the different loci of given polypeptide.And given polypeptide can contain polytype modification.Polypeptide can be owing to ubiquitination branch, also can be branch or unbranched ring-type.Ring-type, branch and branch's ring type polypeptide may be because natural process after the translation or synthetic method generations.Modification comprises acetylize, acidylate, the ADP-ribosylation, amidation, the covalency of flavine stops and closes, the covalency of protoheme stops and closes, the covalency of Nucleotide or nucleotide derivative stops and closes, the covalency of fat or fat derivative stops and closes, the covalency of phosphatidylinositols stops and closes, crosslinked, cyclisation, disulfide linkage forms, demethylation, the formation of covalent cross-linking, Gelucystine forms, Pyrrolidonecarboxylic acid forms, formylation, the γ carboxylation, glycosylation, the GPI deadman forms, hydroxylation, iodate, methylate, the Semen Myristicae acidylate, oxidation, proteolysis processing, phosphorylation, isoamyl two acidylates, racemization, selenium acidylate (selenoylation), sulfation, the amino acid of transfer RNA mediation adds protein, (can be as arginylization and ubiquitination referring to for example " protein: structure and molecular characterization " the 2nd edition, T.E. Creighton, W.H.Freeman andCompany, New York, 1993 and Wold, F., " posttranslational protein is modified: prospect and prospect ", the 1-12 page or leaf, " proteic translation back covalent modification " B.C.Johnson, Ed., AcademicPress, New York, 1983; Seifter et al., " analysis of protein modified and non-albumen cofactor ", Meth Enzymol (1990) 182:626-646 and Rattan et al., " protein synthesis: posttranslational modification and aging ", Ann NY Acad Sci (1992) 663:48-62).
" variant " refers to be different from reference to polynucleotide or polypeptide but kept the polynucleotide or the polypeptide of fundamental characteristics.The nucleotide sequence of general polynucleotide variant is with different with reference to polynucleotide.The variation of variant nucleotide sequence may change or not change the amino acid sequence of polypeptide by the reference polynucleotide encoding.As described below, Nucleotide changes may cause amino acid whose displacement, interpolation, disappearance, fusion and brachymemma in the canonical sequence encoded polypeptides.The aminoacid sequence of general polypeptide variants is with different with reference to polypeptide.Usually, difference is limited, and therefore the sequence with reference to polypeptide and variant is extremely proximate on the whole, is identical in many zones.Variant and one or more displacements, the interpolation of any array configuration, the difference of disappearance can be arranged on aminoacid sequence with reference to polypeptide.Displacement or the amino-acid residue that inserts can yes or no genetic code amino acids coding.Polynucleotide or variant polypeptides can be naturally occurring, as allelic variant, or the non-natural existence.Polynucleotide that non-natural exists and polypeptide variants can be by induced-mutation technique or directly synthetic preparations.
" identity " is known in the art is the two or more peptide sequences determined by comparative sequences or the relation between the polynucleotide sequence.In the art, " identity " also represents the polypeptide determined by the coupling between these sequence chains or the sequence degree of correlation between the polynucleotide sequence." identity " can be calculated easily by currently known methods, includes but not limited to method described in the following document: " calculating molecular biology ", Lesk, A.M., ed.Oxfbrd UniversityPress, New York, 1988; " calculation biology: brief introduction and genome project ", Smith D.W., ed., Academic Press, New York, 1993; " Computer Analysis of sequence data " part 1, Griffin A.M., and Griffin H.G., eds., Humana Press, New Jersey, 1994; " sequential analysis in the molecular biology ", von Heinje, G., Academic Press, 1987; " sequence analysis primer ", Gribskov, M.and Devereux, J., eds., M StocktonPress, New York, 1991; Carillo, H., and Lipman, D., SIAM J.AppliedMath., 48:1073 (1988).The method of determining identity is designed to make between the sequence of test coupling maximum.And, determine that the method for identity has been compiled to the computer program that can openly obtain.The computer program means of determining two identity between the sequence includes but not limited to GCG routine package (Devereux, J., et al., Nucleic Acids Research (1984) 12 (1): 387), BLASTP, BLASTN, FASTA (Atschul, S.F.et al., J Molec Biol (1990) 215:403).BLAST X program can obtain from NCBI or other source (the BLAST handbook, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., etal., J.Mol.Biol.215:403-410 (1990).Well-known Smith Waterman algorithm also can be used for determining identity.
The parameter of peptide sequence comparison comprises: 1) algorithm: Needleman and Wunsch, J.Mol Biol.48:443-453 (1970) comparator matrix (Comparison matrix): BLOSSUM62, from Hentikoff andHentikoff, the compensation of Proc.Natl.Acad.Sci.USA.89:10915-10919 (1992) breach: the compensation of 12 notch length: 4 programs can using these parameters are Genetics Computers Group, the gap program that MadisonWI provides.Above-mentioned parameter is a peptide default parameter (terminal breach uncompensation) relatively.
The parameter of polynucleotide comparison comprises: 1) algorithm: Needleman and Wunsch, J.Mol Biol.48:443-453 (1970) comparator matrix (Comparison matrix): coupling=+ 10, mispairing=0 breach compensation: 50 notch length compensation: 3 programs that can obtain: Genetics Computers Group, the gap program that Madison WI provides.These are peptide default parameters relatively.
The preferred meaning of polynucleotide and polypeptide " identity " provides in following (1) and (2).
(1) the polynucleotide embodiment also comprises isolating polynucleotide, the identity of the canonical sequence of polynucleotide sequence that it is contained and sequence 1 is at least 50,60,70,80,85,90,95,97 or 100%, wherein said polynucleotide sequence can be identical with the canonical sequence of sequence 1, or comprise relatively that with canonical sequence the Nucleotide of some amount changes, wherein said change is selected from least one nucleotide deletion, displacement (comprises conversion, transversion) or insert, described change can occur in any position between terminal or two ends with reference to 5 ＇ of nucleotide sequence or 3 ＇, be dispersed in respectively between the Nucleotide that is distributed in canonical sequence, perhaps be dispersed in and be distributed in the canonical sequence with one or more contigs, the quantity that wherein said Nucleotide changes is following to be determined: the total nucleotide number in the sequence 1 multiply by identity percentage ratio, then its result is reduced from the total nucleotide number of sequence 1, perhaps:
n
n≤x
n-(x
n·y)
N wherein
nFor Nucleotide changes quantity, x
nTotal nucleotide number for sequence 1, the y value was 0.50 at 50% o'clock, was 0.60 at 60% o'clock, was 0.70 at 70% o'clock, at 80% o'clock was 0.80, at 85% o'clock was 0.85, was 0.90 at 90% o'clock, was 0.95 at 95% o'clock, at 97% o'clock was 0.97, if at 100% o'clock was 1.00, was the symbol of multiplying, wherein x
nWith the result of y be not integer, then it is rounded up and gets nearest integer, again from x
nMiddle subduction.The change of the polynucleotide sequence of encoding sequence 2 polypeptide can produce nonsense, missense or phase shift mutation in encoding sequence, therefore change the polypeptide by the polynucleotide encoding after changing.
For example, polynucleotide sequence of the present invention can be identical with the canonical sequence of sequence 2, can be 100% identical, perhaps compare the amino acid change that comprises some amount with canonical sequence, so identity is less than 100%.This change can be selected from least one nucleic acid disappearance, displacement (comprising conversion and transversion) or insert, wherein said change can occur in any position between terminal or two ends with reference to 5 ＇ of polynucleotide sequence or 3 ＇, be dispersed in respectively between the nucleic acid that is distributed in canonical sequence, perhaps be dispersed in and be distributed in the canonical sequence with one or more contigs.The quantity that identity percentage ratio one timing nucleic acid changes is following to be determined: the total amino acid number in the sequence 2 multiply by identity percentage ratio, then its result is reduced from the total amino acid number of sequence 2, perhaps:
n
n≤x
n-(x
n·y)
N wherein
nBe amino acid change quantity, x
nIf be the total amino acid number of sequence 2, the y value was 0.70 at 70% o'clock, was 0.80 at 80% o'clock, was 0.85 at 85% o'clock, and the rest may be inferred, is the symbol of multiplying, wherein x
nWith the result of y be not integer, then it is rounded up and gets nearest integer, again from x
nMiddle subduction.
(2) the polypeptide embodiment also comprises isolated polypeptide, the identity with reference to peptide sequence of peptide sequence that it is contained and sequence 2 is at least 50,60,70,80,85,90,95,97 or 100%, wherein said peptide sequence can be identical with the canonical sequence of sequence 2, or relatively comprise the amino acid change of some amount with canonical sequence, wherein said change is selected from least one aminoacid deletion, displacement (comprising conservative and non-conservative substitution) or insertion, described change can occur in reference to any position between the amino of peptide sequence or C-terminal or two ends, be dispersed in respectively between the amino acid that is distributed in canonical sequence, perhaps be dispersed in and be distributed in the canonical sequence with one or more contigs, the quantity of wherein said amino acid change is following to be determined: the total amino acid number in the sequence 2 multiply by identity percentage ratio, then its result is reduced from the total amino acid number of sequence 2, perhaps:
n
a≤x
a-(x
a·Y)
N wherein
aBe amino acid change quantity, x
aTotal amino acid number for sequence 2, the y value was 0.50 at 50% o'clock, was 0.60 at 60% o'clock, was 0.70 at 70% o'clock, at 80% o'clock was 0.80, at 85% o'clock was 0.85, was 0.90 at 90% o'clock, was 0.95 at 95% o'clock, at 97% o'clock was 0.97, if at 100% o'clock was 1.00, was the symbol of multiplying, wherein x
aWith the result of y be not integer, then it is rounded up and gets nearest integer, again from x
aMiddle subduction.
For example, peptide sequence of the present invention can be identical with the canonical sequence of sequence 2, can be 100% identical, perhaps compare the amino acid change that comprises some amount with canonical sequence, so identity is less than 100%.This change can be selected from least one aminoacid deletion, displacement (comprising conservative and non-conservative substitution) or insert, wherein said change can occur in reference to any position between the amino of peptide sequence or C-terminal or two ends, be dispersed in respectively between the amino acid that is distributed in canonical sequence, perhaps be dispersed in and be distributed in the canonical sequence with one or more contigs.The quantity of identity percentage ratio one timing amino acid change is following to be determined: the total amino acid number in the sequence 2 multiply by identity percentage ratio, then its result is reduced from the total amino acid number of sequence 2, perhaps:
n
a≤x
a-(x
a·y)
N wherein
aBe amino acid change quantity, x
aIf be the total amino acid number of sequence 2, the y value was 0.70 at 70% o'clock, was 0.80 at 80% o'clock, was 0.85 at 85% o'clock, and the rest may be inferred, is the symbol of multiplying, wherein x
aWith the result of y be not integer, then it is rounded up and gets nearest integer, again from x
aMiddle subduction.
" fusion rotein " refers to the albumen by two common incoherent fusion genes or its fragment coding.For example, EP-A-0 464 discloses the fusion rotein that contains immunoglobulin molecules constant region each several part and other people albumen or its part.Under many circumstances, in treatment and diagnosis, use immunoglobulin fc region comparatively favourable, can cause improved pharmacokinetic properties (referring to for example EP-A 0,232 262) as the part of fusion rotein.On the other hand, in certain some application, behind expressing fusion protein, detection and purifying, can excise comparatively ideal of Fc part.
All publications of being quoted in this specification sheets include but not limited to patent and patent application, and are all incorporated by reference, indicate incorporated by reference separately and individually as each publication.Sequence information
1： 1 GGTGTATGTG CGGCAATAAC ATGTCAACCC CGCTGCCCGC CATCGTGCCC 51 GCCGCCCGGA AGGCCACCGC TGCGGTGATT TTCCTGCATG GATTGGGAGA101 TACTGGGCAC GGATGGGCAG AAGCCTTTGC AGGTATCAGA AGTTCACATA151 TCAAATATAT CTGCCCGCAT GCGTTTGATA TTATTGGGCT TTCACCAGAT201 TCACAGGAGG ATGAATCTGG GATTAAACAG GCAGCAGAAA ATATAAAAGC251 TTTGATTGAT CAAGAAGTGA AGAATGGCAT TCCTTCTAAC AGAATTATTT301 TGGGAGGGTT TTCTCAGGGA GGAGCTTTAT CTTTATATAC TGCCCTTACC351 ACACAGCAGA AACTGGCAGG TGTCACTGCA CTCAGTTGCT GGCTTCCACT401 TCGGGCTTCC TTTCCACAGG GTCCTATCGG TGGTGCTAAT AGAGATATTT451 CTATTCTCCA GTGCCACGGG GATTGTGACC CTTTGGTTCC CCTGATGTTT501 GGTTCTCTTA CGGTGGAAAA ACTAAAAACA TTGGTGAATC CAGCCAATGT551 GACCTTTAAA ACCTATGAAG GTATGATGCA CAGTTCGTGT CAACAGGAAA601 TGATGGATGT CAAGCAATTC ATTGATAAAC TCCTACCTCC AATTGATTGA651 CGTCACTAAG AGGCCTTGTG TAGAAGTACA CCAGCATCAT TGTAGTAGAG701 TGTAAACCTT TTCCCATGCC CAGTCTTCAA ATTTCTAATG TTTGCAGTGT751 TTAAATGTTT TGCAAATACA TGCCATTAAC ACAGATCAAT AATATCTCCT801 CTGAGAATTT ATGATCTTAA GTCTATACAT GTATTCTTAT AAGACGACCC851 AGGATCTACT ATATTAGAAT AGATGAAGCA GGTAGCTTCT TTTTTCTCAA 901 ATGTAATTCA GCAAAATAAT ACAGTACTGC CACCAGATTT TTTATTACAT 951 CATTTGAAAA TTAGCAGTAT GCTTAATGAA AATTTGTTCA GGTATAAATG1001 AGCAGTTAAG ATATAAACAA TTTATGCATG CTGTGACTTA GTCTATGGAT1051 TTATTCCAAA ATTGCTTAGT CACCATGCAG TGTCTGTATT TTTATATATG1101 TGTTCATATA TACATAATGA TTATAATACA TAATAAGAAT GAGGTGGTAT1151 TACATTATTC CTAATAATAG GGATAATGCT GTTTATTGTC AAGAAAAAGT1201 AAAATCGTTC TCTTCAATTA ATGGCCCTTT TATTTTGGGA CCAGGCTTTT1251 ATTTTCCCTG ATATTATTTC TATTTAATAC TCTTTTCTCT CAAGAAAAAA2 1 MCGNNMSTPL PAIVPAARKA TAAVIFLHGL GDTGHGWAEA FAGIRSSHIK 51 YICPHAFDII GLSPDSQEDE SGIKQAAENI KALIDQEVKN GIPSNRIILG101 GFSQGGALSL YTALTTQQKL AGVTALSCWL PLRASFPQGP IGGANRDISI151 LQCHGDCDPL VPLMFGSLTV EKLKTLVNPA NVTFKTYEGM MHSSCQQEMM201 DVKQFIDKLL PPID
Sequence table (1) general information (i) applicant:Shanghai Second Emdical University is denomination of invention (ii): people's lysophospholipase gene (CBFBLH05) is sequence number (iii): 2 (iv) addresses:(A) contact person:Ratner ﹠ amp; Prestia (B) street:P.O.Box 980 (C) city:Valley Forge (D) state:PA (E) country:USA (F) postcode:19482 (v) computer-reader form:(A) media type:compatible (C) operating system:DOS (D) software of disk (B) computer:IBM:the current request for data of FastSEQ for Windows Version 2.0 (vi): (A) application number:(B) applying date:(C) classification:(vii) request for data formerly:(A) application number:(B) applying date:(viii) lawyer/agent's information:(A) name:Prestia; Paul F ( B ) ：23,031 ( C ) /：GP-70497 ( ix ) ： ( A ) ：610/407-0700 ( B ) ：610/407-0701 ( C ) ：846169 ( 2 ) 1： ( i ) ： ( A ) ：1300 ( B ) ： ( C ) ： ( D ) ： ( ii ) ：cDNA ( xi ) ：1：GGTGTATGTG CGGCAATAAC ATGTCAACCC CGCTGCCCGC CATCGTGCCC GCCGCCCGGA 60AGGCCACCGC TGCGGTGATT TTCCTGCATG GATTGGGAGA TACTGGGCAC GGATGGGCAG 120AAGCCTTTGC AGGTATCAGA AGTTCACATA TCAAATATAT CTGCCCGCAT GCGTTTGATA 180TTATTGGGCT TTCACCAGAT TCACAGGAGG ATGAATCTGG GATTAAACAG GCAGCAGAAA 240ATATAAAAGC TTTGATTGAT CAAGAAGTGA AGAATGGCAT TCCTTCTAAC AGAATTATTT 300TGGGAGGGTT TTCTCAGGGA GGAGCTTTAT CTTTATATAC TGCCCTTACC ACACAGCAGA 360AACTGGCAGG TGTCACTGCA CTCAGTTGCT GGCTTCCACT TCGGGCTTCC TTTCCACAGG 420GTCCTATCGG TGGTGCTAAT AGAGATATTT CTATTCTCCA GTGCCACGGG GATTGTGACC 480CTTTGGTTCC CCTGATGTTT GGTTCTCTTA CGGTGGAAAA ACTAAAAACA TTGGTGAATC 540CAGCCAATGT GACCTTTAAA ACCTATGAAG GTATGATGCA CAGTTCGTGT CAACAGGAAA 600TGATGGATGT CAAGCAATTC ATTGATAAAC TCCTACCTCC AATTGATTGA CGTCACTAAG 660AGGCCTTGTG TAGAAGTACA CCAGCATCAT TGTAGTAGAG TGTAAACCTT TTCCCATGCC 720CAGTCTTCAA ATTTCTAATG TTTGCAGTGT TTAAATGTTT TGCAAATACA TGCCATTAAC 780ACAGATCAAT AATATCTCCT CTGAGAATTT ATGATCTTAA GTCTATACAT GTATTCTTAT 840AAGACGACCC AGGATCTACT ATATTAGAAT AGATGAAGCA GGTAGCTTCT TTTTTCTCAA 900ATGTAATTCA GCAAAATAAT ACAGTACTGC CACCAGATTT TTTATTACAT CATTTGAAAA 960TTAGCAGTAT GCTTAATGAA AATTTGTTCA GGTATAAATG AGCAGTTAAG ATATAAACAA 1020TTTATGCATG CTGTGACTTA TCTATGGAT TTATTCCAAA ATTGCTTAGT CACCATGCAG 1080TGTCTGTATT TTTATATATG TGTTCATATA TACATAATGA TTATAATACA TAATAAGAAT 1140GAGGTGGTAT TACATTATTC CTAATAATAG GGATAATGCT GTTTATTGTC AAGAAAAAGT 1200AAAATCGTTC TCTTCAATTA ATGGCCCTTT TATTTTGGGA CCAGGCTTTT ATTTTCCCTG 1260ATATTATTTC TATTTAATAC TCTTTTCTCT CAAGAAAAAA 1300 ( 2 ) 2： ( i ) ： ( A ) ：214 ( B ) ： ( C ) ： ( D ) ： ( ii ) ： ( xi ) ：2：
195 200 205Leu?Leu?Pro?Pro?Ile?Asp l?Pro?Ala
210 15Ala?Arg?Lys?Ala?Thr?Ala?Ala?Val?Ile?Phe?Leu?His?Gly?Leu?Gly?Asp
20 25 30Thr?Gly?His?Gly?Trp?Ala?Glu?Ala?Phe?Ala?Gly?Ile?Arg?Ser?Ser?His
35 40 45Ile?Lys?Tyr?Ile?Cys?Pro?His?Ala?Phe?Asp?Ile?Ile?Gly?Leu?Ser?Pro
50 55 60Asp?Ser?Gln?Glu?Asp?Glu?Ser?Gly?Ile?Lys?Gln?Ala?Ala?Glu?Asn?Ile65 70 75 80Lys?Ala?Leu?Ile?Asp?Gln?Slu?Val?Lys?Asn?Gly?Ile?Pro?Ser?Asn?Arg
85 90 95Ile?Ile?Leu?Gly?Gly?Phe?Ser?Gln?Gly?Gly?Ala?Leu?Ser?Leu?Tyr?Thr
100 105 110Ala?Leu?Thr?Thr?Gln?Gln?Lys?Leu?Ala?Gly?Val?Thr?Ala?Leu?Ser?Cys
115 120 125Trp?Leu?Pro?Leu?Arg?Ala?Ser?Phe?Pro?Gln?Gly?Pro?Ile?Gly?Gly?Ala
130 135 140Asn?Arg?Asp?Ile?Ser?Ile?Leu?Gln?Cys?His?Gly?Asp?Cys?Asp?Pro?Leu145 150 155 160Val?Pro?Leu?Met?Phe?Gly?Ser?Leu?Thr?Val?Glu?Lys?Leu?Lys?Thr?Leu
165 170 175Val?Asn?Pro?Ala?Asn?Val?Thr?Phe?Lys?Thr?Tyr?Glu?Gly?Met?Met?His
180 185 190Ser?Ser?Cys?Gln?Gln?Glu?Met?Met?Asp?Val?Lys?Gln?Phe?Ile?Asp?Lys
195 200 205
Leu?Leu?Pro?Pro?Ile?Asp
210
Claims (12)
1. isolated polypeptide that is selected from following polypeptide:
(i) contain with the identity of the whole aminoacid sequence of sequence 2 at least:
(a)70％
(b)80％
(c) 90%; Or
(a)95％
The isolated polypeptide of aminoacid sequence;
The isolated polypeptide that (ii) contains sequence 2 aminoacid sequences; Or
(iii) its aminoacid sequence is the isolated polypeptide of sequence 2.
2. isolating polynucleotide that are selected from following polynucleotide:
(i) isolating polynucleotide, the identity of the whole aminoacid sequence of the nucleotide sequence coded and sequence 2 that it contains are at least:
(a)70％
(b)80％
(c) 90%; Or
(a)95％
The polypeptide of aminoacid sequence;
(ii) isolating polynucleotide, its contain with the identity of the whole coding nucleotide sequence of sequence 2 peptide sequences at least:
(a)70％
(b)80％
(c) 90%; Or
(a)95％
Nucleotide sequence;
(iii) isolating polynucleotide, its contain with the identity of the whole nucleotide sequence of sequence 1 at least:
(a)70％
(b)80％
(c) 90%; Or
(a)95％
Nucleotide sequence;
(iv) isolating polynucleotide, it contains the nucleotide sequence of encoding sequence 2 polypeptide;
(v) isolating polynucleotide, it is the polynucleotide of sequence 1; Or
(vi) isolating polynucleotide, its available mark with sequence 1 or its fragments sequence
The note probe screens suitable library and obtains under tight hybridization conditions;
Or with the described polynucleotide sequence complementary nucleotide sequence that separates.
3. the immunologic opsonin antibody of the polypeptide of claim 1.
4. methods of treatment is used for the treatment of:
(i) need the polypeptide active of claim 1 or the experimenter who expresses raising, comprising:
(a) to the agonist of the described polypeptide of experimenter's drug treatment significant quantity; With/
Or
(b) provide isolating polynucleotide to the experimenter, it contains coding said polypeptide
Nucleotide sequence, its form can realize producing in the body of described polypeptide active
Give birth to; Or
(ii) need to suppress polypeptide active of claim 1 or the experimenter who expresses, bag
Draw together:
(a) to the antagonist of the described polypeptide of experimenter's drug treatment significant quantity; With/
Or
(b) provide the nucleotide sequence of inhibition coding said polypeptide to express to the experimenter
Nucleic acid molecule; And/or
(c) to experimenter's drug treatment significant quantity with described polypeptide compete its part,
The polypeptide of substrate or acceptor.
5. diagnose the method for the susceptibility of disease relevant among the experimenter or disease, comprising with claim 1 polypeptide expression or activity:
(a) whether the coding nucleotide sequence of determining polypeptide described in experimenter's genome exists sudden change; And/or
(b) analyze from whether described expression of polypeptides or its expression amount are arranged in described experimenter's the sample.
6. identify to stimulate or suppress the screening method of compound of the polypeptide function of claim 1, comprise the following a kind of method that is selected from:
(a) utilize directly or indirectly and the combining of candidate compound bonded marker determination candidate compound and the polypeptide cell or the cytolemma of this polypeptide (or have) or its fusion rotein;
(b) when existing, mark competition thing measures combining of candidate compound and the polypeptide cell or the cytolemma of this polypeptide (or have) or its fusion rotein;
(c) cell or the cytolemma suitable detection system of utilization to having polypeptide determines whether candidate compound produces the signal that polypeptide activates or suppresses to produce;
(d) solution of candidate compound with the polypeptide that contains claim 1 is mixed, form mixture, measure the polypeptide active in the mixture, relatively the activity of mixture and standard substance; Or
(e) utilize the ELISA test to wait the influence of the generation of the mRNA that measures polypeptide described in the candidate compound pair cell and coding said polypeptide.
7. the agonist of the polypeptide of claim 1 or antagonist.
8. the expression system that contains a kind of polynucleotide, these polynucleotide can produce the polypeptide of claim 1 when described expression system is present in the compatible cells.
9. the method for preparing recombinant host cell, comprise that the expression system with claim 8 transforms or transfectional cell, make host cell can generate a peptide species under suitable culture condition, it contains with the identity of the whole aminoacid sequence of sequence 2 and is at least 70% aminoacid sequence.
10. the recombinant host cell of the method for claim 9 preparation.
11. the film of the recombinant host cell of claim 10, it expresses a peptide species, and described polypeptide comprises with the identity of the whole aminoacid sequence of sequence 2 and is at least 70% aminoacid sequence.
12. produce the method for polypeptide, be included in the host cell of cultivating claim 10 under the condition that is enough to produce described polypeptide, by reclaiming polypeptide in the culture.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN1998/000164 WO2000009556A1 (en) | 1998-08-11 | 1998-08-11 | Human lysophospholipase gene (cbfblh05) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1275131A true CN1275131A (en) | 2000-11-29 |
Family
ID=4575080
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98810060.6A Pending CN1275131A (en) | 1998-08-11 | 1998-08-11 | Human lysophospholipase gene (CBFBLH05) |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1275131A (en) |
| WO (1) | WO2000009556A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106970220A (en) * | 2006-08-03 | 2017-07-21 | 国立大学法人东京大学 | The special antibody of native form people's autocrine motility factor, its screening technique and detection reagent |
-
1998
- 1998-08-11 CN CN98810060.6A patent/CN1275131A/en active Pending
- 1998-08-11 WO PCT/CN1998/000164 patent/WO2000009556A1/en active Application Filing
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106970220A (en) * | 2006-08-03 | 2017-07-21 | 国立大学法人东京大学 | The special antibody of native form people's autocrine motility factor, its screening technique and detection reagent |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2000009556A1 (en) | 2000-02-24 |
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