CN1712414A - Method for diagnosing and treating baldness from human Rhbd15 gene and its coding product - Google Patents
Method for diagnosing and treating baldness from human Rhbd15 gene and its coding product Download PDFInfo
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Abstract
Diagnose and treatment for alopecia from human Rhbdl5 gene and its coding product relate to alopecia related protein Rhbd15, polynucleotide of coding Rhbd15 and production of Rhbd15 from recombinant technology.
Description
Technical field
The invention belongs to biotechnology and medical field, specifically, the present invention relates to new people's baldness genes involved Rhbdl5 and proteins encoded thereof.The invention still further relates to the preparation and the application of Rhbdl5 polynucleotide and polypeptide, especially the application aspect diagnosis and treatment baldness.
Background technology
It is comparatively rare that human congenital alopecia, rare are common in the masculinity femininity patient, though do not influence people's life quality, but is that this geneogenous genetic diseases of women has a strong impact on its quality of life for the individual by it, does not regrettably have the method that can fundamentally treat up to now.In addition, collect human congenital alopecia family and have bigger difficulty.
Yet, also do not find or isolate at present the people's gene relevant with baldness.Therefore, this area press for exploitation new with the baldness proteins associated.
Summary of the invention
The purpose of this invention is to provide a kind of new people's baldness associated protein-Rhbdl5 albumen with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of these polypeptide of coding.
Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.
In a first aspect of the present invention, novel isolated Rhbdl5 polypeptide is provided, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQID NO:2 aminoacid sequence.Preferably, this polypeptide is selected from down group: the polypeptide that (i) has SEQ ID NO:2 aminoacid sequence; (ii) have SEQ ID NO:2 aminoacid sequence and the 259th and be the polypeptide of Met; (iii) have SEQ ID NO:2 aminoacid sequence and the 546th and be the polypeptide of Ser.
In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned Rhbdl5 polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is selected from down group: the sequence that (c-1) has 1-3118 position among the SEQ ID NO:1; (c-2) has the sequence of 71-2551 position among the SEQ IDNO:1; (c-3) have among the SEQ ID NO:1 71-2551 position and the 846th and be the sequence of T; (c-4) have among the SEQ ID NO:1 71-2551 position and the 1706th and be the sequence of T.
In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.
In a fourth aspect of the present invention, preparation Rhbdl5 is provided proteic method, this method comprises: (a) expressing under the proteic condition of Rhbdl5, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate Rhbdl5 albumen.
In a fifth aspect of the present invention, provide and above-mentioned Rhbdl5 polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 20-3118 Nucleotide in the above-mentioned polynucleotide.
In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism Rhbdl5 polypeptide active is provided, and the compound that suppresses the Rhbdl5 polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of Rhbdl5 polypeptide.
In a seventh aspect of the present invention, provide and whether had the proteic method of Rhbdl5 in the test sample, it comprises: sample is contacted with the proteic specific antibody of Rhbdl5, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample Rhbdl5 albumen.
In a eighth aspect of the present invention, a kind of disease relevant with Rhbdl5 polypeptide unconventionality expression or method of disease (as baldness) susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.Preferably, described sudden change is the 846th C → T among the SEQ ID NO:1, and the 1706th is C → T.In addition, also provide the reagent corresponding box, it comprises the primer of specific amplification Rhbdl5 gene or transcript.Preferably, described test kit also contains and mutable site bonded probe.
In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes the Rhbdl5 polypeptide active, and perhaps screening suppresses the antagonist of Rhbdl5 polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of Rhbdl5 of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.
In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains Rhbdl5 polypeptide of the present invention or its agonist and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as baldness.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown the cDNA sequence of Rhbdl5, and wherein open reading frame (ORF) is positioned at the 71-2551 position, 827 the amino acid rhbdl5 albumen of encoding.
Fig. 2 has shown the proteic aminoacid sequence of Rhbdl5, and wherein the 205-827 position is a rhomboid protein family high conservative zone (underscore part).
Fig. 3 has shown the sequencing result of a baldness patient Rhbdl5 gene, has the T/C heterozygosis 846 of Rhbdl5 gene, and this sudden change causes the 259th amino acids Thr → Met.
Fig. 4 has shown the sequencing result of a baldness patient Rhbdl5 gene, has the T/C heterozygosis 1706 of Rhbdl5 gene, and this sudden change causes the 546th amino acids Pro → Ser.
Fig. 5 has shown people Rhbdl5 albumen and the proteic aminoacid sequence comparison chart of mouse Rhor.
Embodiment
The inventor is through deeply and extensive studies, separates first and proved a kind of and the closely-related people's gene Rhbdl5 of baldness, the albumen of the similar EGF-R ELISA of this genes encoding.The sudden change of Rhbdl5 or function reduction or disappearance will cause baldness.Finished the present invention on this basis.
In the present invention, term " Rhbdl5 albumen ", " Rhbdl5 polypeptide " or " people's baldness associated protein Rhbdl5 " are used interchangeably, and all refer to have the albumen or the polypeptide of people's baldness associated protein Rhbdl5 aminoacid sequence (SEQ ID NO:2).They comprise the Rhbdl5 albumen that contains or do not contain initial methionine.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating Rhbdl5 albumen or polypeptide " is meant that the Rhbdl5 polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying Rhbdl5 albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.
The present invention also comprises the proteic fragment of Rhbdl5, derivative and analogue.As used herein, term " fragment ", " derivative " are meant biological function or the active polypeptide that keeps natural Rhbdl5 albumen of the present invention identical basically with " analogue ".Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " Rhbdl5 polypeptide " refers to have the SEQ ID NO:2 polypeptide of sequence of Rhbdl5 protein-active.This term also comprises having and variant form Rhbdl5 albumen identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of Rhbdl5 and reactive derivative.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of Rhbdl5 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-Rhbdl5 polypeptide to obtain.The present invention also provides other polypeptide, as comprises Rhbdl5 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of Rhbdl5 polypeptide.Usually, this fragment have the Rhbdl5 peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of Rhbdl5 albumen or polypeptide.The difference of these analogues and natural Rhbdl5 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " Rhbdl5 albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID N0:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of aminoacid sequence shown in the SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of encoding mature polypeptide comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding Rhbdl5.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
Rhbdl5 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The method of using round pcr DNA amplification/RNA is optimized for and obtains gene of the present invention.The primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or Rhbdl5 albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the Rhbdl5 polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention Rhbdl5 polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, the Rhbdl5 polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains Rhbdl5 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, coldSpring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Go into phage PL promotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The Rhbdl5 albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: directly as pharmacological agent Rhbdl5 protein function the disease due to the low or forfeiture (as no sperm, or disease such as low sperm activity) and be used to screen and promote or antibody, polypeptide or other part of antagonism Rhbdl5 protein function.The peptide molecule that can suppress or stimulate the Rhbdl5 protein function that can be used for seeking therapeutic value with the reorganization Rhbdl5 protein screening peptide library of expressing.
On the other hand, the present invention also comprises Rhbdl5 DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into Rhbdl5 gene product or fragment.Preferably, refer to that those can combine with Rhbdl5 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of Rhbdl5, comprise that also those do not influence the antibody of Rhbdl5 protein function.The present invention also comprise those can with modify or without the Rhbdl5 gene product bonded antibody of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the Rhbdl5 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing Rhbdl5 albumen or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.Antibody of the present invention comprises the antibody that can block the Rhbdl5 protein function and the antibody that does not influence the Rhbdl5 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of Rhbdl5 gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of Rhbdl5 gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).
The proteic antibody of anti-Rhbdl5 can be used in the immunohistochemistry technology, detects the Rhbdl5 albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of Rhbdl5, inject in the body and can follow the tracks of its position and distribution.
Available Rhbdl5 albumen of the production of polyclonal antibody or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
Utilize albumen of the present invention,, can filter out with Rhbdl5 albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.
Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): oral, intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
Polypeptide of the present invention can be directly used in disease treatment and prevention, especially prevents and treats baldness.When using Rhbdl5 albumen of the present invention, also can use other to be used for same treatment of conditions agent simultaneously.
The present invention also provides a kind of composition (comprising pharmaceutical composition), and it contains Rhbdl5 polypeptide of the present invention or its agonist, antagonist and (pharmaceutically) acceptable carrier or the vehicle of safe and effective amount (as 0.001-99.9wt%).This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that the Rhbdl5 albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 5 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
Rhbdl5 albumen of the present invention or its agonist also can be added in the hair-care products such as paste shampoo, shampoo, hair conditioner, addition is generally the 0.001-5% (preferably 0.01-1%) of gross weight, thereby just plays the effect of preventing and treating baldness/alopecia when daily use.
The proteic polynucleotide of Rhbdl5 also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because the baldness due to the proteic expression of Rhbdl5 of the proteic nothing expression of Rhbdl5 or unusual/non-activity.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the Rhbdl5 transgenosis to cell.The Rhbdl5 gene of recombinating in addition can be packaged in the liposome, and then is transferred in the cell.
Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.
Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of Rhbdl5 obtains.During screening, must carry out mark to the Rhbdl5 protein molecular.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization Rhbdl5 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The Rhbdl5 protein level that is detected in the test can be with laying down a definition the importance of Rhbdl5 albumen in various diseases and be used to the disease (as baldness) of diagnosing Rhbdl5 albumen to work.
Whether having the proteic method of Rhbdl5 in a kind of detection test sample is to utilize the proteic specific antibody of Rhbdl5 to detect, and it comprises: sample is contacted with the Rhbdl5 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample Rhbdl5 albumen.
The proteic polynucleotide of Rhbdl5 can be used for the diagnosis and the treatment of Rhbdl5 protein related diseases.Aspect diagnosis, the proteic polynucleotide of Rhbdl5 can be used for detecting the proteic expression of Rhbdl5 Rhbdl5 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of Rhbdl5 as the Rhbdl5 dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of Rhbdl5 albumen and also can detect the proteic transcription product of Rhbdl5.
The sudden change that detects the Rhbdl5 gene also can be used for the disease of diagnosing Rhbdl5 albumen relevant.The form of Rhbdl5 protein mutation comprises that the point mutation compared with normal wild type Rhbdl5 dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.Test of the present invention shows that the sudden change of Rhbdl5 is directly related with baldness.
In an example of the present invention, the nucleotide sequence of isolating Rhbdl5 is shown in SEQ ID NO:1, the polynucleotide sequence total length that it comprises is 3118 bases, and its open reading frame is positioned at the 71-2551 position, and the coding total length is 827 amino acid whose Rhbdl5 albumen (SEQ ID NO:2).Studies show that, the sudden change of this gene and human congenital alopecia, rarely send out closely related.Rhbdl5 provides new treatment approach for diseases such as treatment baldnesses, thereby has great application prospect.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The acquisition of Rhbdl5 gene complete open reading frame
According to the total RNA of method extracting human skin cell that the Total RNA purification kit of Qiagen company provides, 1% agarose electrophoresis identifies after the purity that be to use following primer behind the cDNA. with the Reverse Transcript System of Promega company with the RNA reverse transcription:
forward?5’TGTACAGAGCAAGGAGAAGACA3’(SEQ?ID?NO：3)
reverse?5’CTTTCCCACAATGTCCCATC3’(SEQ?ID?NO：4)
Use the TaqGold pyro polymerase of ABI company to carry out pcr amplification, the PCR thermal cycle conditions is:
95 ℃ 10 minutes, 1 circulation; Thermal cycling: 95 ℃ 30 seconds, 56 ℃ 45 seconds, 72 ℃ 3 minutes 30 seconds, totally 40 circulations; 72 ℃ 10 minutes, 1 circulation.
0.8% agarose electrophoresis is separated, and obtains the amplified production of the about 3.2kb of length.The rubber tapping purifying, because of this product longer, so the method for use PRIMER WALKING checks order.Use following 8 pairs of sequencing primers successively:
Hs.rhbdl5?forward?5’-TGTACAGAGCAAGGAGAAGACA-3’(SEQ?ID?NO：3)
Hs.rhbdl5?reverse?5’-GGAGCTCCAGGTCACGCT-3’(SEQ?ID?NO：5)
Hs.rhbdl5?l?forward?5’-CCACAGACTGAGGCTTGACA-3’(SEQ?ID?NO：6)
Hs.rhbdl5?1?reverse?5’-GATGCTCTGGGACAGTGAGG-3’(SEQ?ID?NO：7)
Hs.rhbdl5?2?forward?5’-ACTTGAAGAGCGTCAGCCTC-3’(SEQ?ID?NO：8)
Hs.rhbdl5?2?reverse?5’-ATCGACCACATCCTCCTCC-3’(SEQ?ID?NO：9)
Hs.rhbdl5?3?forward?5’-AGTCCTGTCCCTCACCTCCT-3’(SEQ?ID?NO：10)
Hs.rhbdl5?3?reverse?5’-GGACGAAGGTCAGCCAGTAG-3’(SEQ?ID?NO：11)
Hs.rhbdl5?4?forward?5’-CGGCCCTACTTCACCTACTG-3’(SEQ?ID?NO：12)
Hs.rhbdl5?4?reverse?5’-TTGATCTCGCAGTCCATGTG-3’(SEQ?ID?NO：13)
Hs.rhbdl5?5?forward?5’-GGCCCGATGACATCACTAAG-3’(SEQ?ID?NO：14)
Hs.rhbdl5?5?reverse?5’-TGAACAGGAAGAGCACGATG-3’(SEQ?ID?NO：15)
Hs.rhbdl5?6?forward?5’-CAGTGCCATCTTTCTCCCAT-3’(SEQ?ID?NO：16)
Hs.rhbdl5?6?reverse?5’-CTGTCTTGGGTCTCTGGCTC-3’(SEQ?ID?NO：17)
Hs.rhbdl5?7?forward?5’-TGCGAGAAGTATGAGCTGGA-3’(SEQ?ID?NO：18)
Hs.rhbdl5?7?reverse?5’-CTTTCCCACAATGTCCCATC-3’(SEQ?ID?NO：4)
Order-checking is identified, obtain the full length sequence of Rhbdl5 gene, as SEQ ID NO:1 and shown in Figure 1, wherein, the open reading frame of Rhbdl5 gene (ORF) is positioned at the 71-2551 position, and the coding total length is 827 amino acid whose Rhbdl5 albumen (SEQ ID NO:2 and Fig. 2).
By structural analysis, find that the proteic 205-827 amino acids of Rhbdl5 is a rhomboid protein family high conservative zone (the underscore part of Fig. 2), this shows, Rhbdl5 albumen belongs to Rhomboid protein family member, this family member has report in bacterium and eukaryote, major part is complete membranin, the Histidine of three high conservatives of they predicted transmembrane district existence.
In addition, adopt the method for conventional microsatellite marker that Rhbdl5 is carried out chromosomal localization.The result shows that the Rhbdl5 assignment of genes gene mapping is on human the ten No. five karyomit(e).
Embodiment 2
The authentication method of Rhbdl5 gene cDNA disappearance
200 normal peoples of extracting and 2 baldness patients' scalp cells genomic dna is as template.Use following primer then:
Primer is to 1 Fwd 5-CGTTCAGCTTGCAGAATCTC-3 (SEQ ID NO:20)
Rev?5-CCACATAGGACCATGCCACA-3(SEQ?ID?NO：21)
Primer is to 2 Fwd 5-GACCCACCAGCCTTCTTCT-3 (SEQ ID NO:22)
Rev?5-AAGCCTGTGTGGTTGCTCC-3(SEQ?ID?NO：23)
Use the GoldTaq pyro polymerase of ABI company, carry out pcr amplification, condition is with embodiment 1.Amplified production is after agarose electrophoresis is identified, with the Cycle-Pure Kit purified product of Omega company, the BigDye terminal that uses ABI company is still with above-mentioned primer order-checking.
Sequencing result shows, there is not the coding mutation that causes that amino acid changes in 200 normal peoples' Rhbdl5 gene, and (C → T), this sudden change causes the 259th amino acids Thr → Met (Fig. 3) and there is the T/C heterozygosis in 846 of a baldness patient Rhbdl5 gene.There is the T/C heterozygosis in 1706 of another baldness patient Rhbdl5 gene, and (C → T), this sudden change causes the 546th amino acids Pro → Ser (Fig. 4).This shows that Rhbdl5 gene and baldness are closely related.
Fig. 5 has shown the aminoacid sequence comparison chart of people Rhbdl5 albumen (SEQ ID NO:2) with mouse Rhor albumen (SEQ ID NO:19).Mouse Rhor albumen is the congenital gene (seeing Chinese patent application 02145253.9) that does not have hair that has caused mouse that the inventor discovered in the past.Rhbdl5 albumen and mouse Rhor albumen have higher homology, and this has pointed out them to have similar function, and present embodiment has more confirmed the dependency with baldness.
Embodiment 3
The expression of Rhbdl5 in prokaryotic cell prokaryocyte
Synthetic following primer:
Forward primer: 5 ' ATAAAGCTTATGGCCTCAGCTGACAAGAATGGCAGCAACCTCCCA 3 ' (SEQ ID NO:24) (introduce HindIII point of contact GGATCC, first ATA is the protection base)
Reverse primer: 5 ' CCGCTCGAGGCTCGATCTGGTCCACGATGTGATT 3 ' (SEQID NO:25) (introduce XhoI point of contact CTCGAG, initial CCG is the protection base)
CDNA with people's scalp cells is a template, the proteic DNA of pcr amplification coding Rhbdl5.
Utilize HindIII and Hind III site, pcr amplification product is cloned among the plasmid pET32a (Novagen company) that same enzyme cuts, transform host E.Coli:BL21 (DE3) then.Transformed host cells is expressed under the inducing of IPTG.
Expression product molecular weight in SDS-PA6E glue is approximately 90Kda, conforms to predictor.
Embodiment 4
The preparation of antibody
With obtaining albumen among the embodiment 3, in rabbit, prepare polyclonal antibody as antigen.Specific procedure is as follows: immune purebred new zealand rabbit for the first time, albumen consumption be 240ug/ only, use Freund's complete adjuvant.After 4 weeks, immunity for the second time, the albumen consumption is 140ug/, full adjuvant toos many or too much for use.Afterwards 2 weeks, immune for the third time, the albumen consumption is 120ug/, and full adjuvant toos many or too much for use.
The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people's Rhbdl5 albumen (embodiment 3) with it.Found that antibody can combine with Rhbdl5 albumen specifically.
Embodiment 5
The detection kit of baldness susceptibility
As described in embodiment 1-3, (this sudden change causes the 259th amino acids Thr → Met) and the sudden change of 1706 C → T to 846 C → T among the SEQ ID NO:1, and is closely related with baldness.Therefore, can be that template increases and detects at genome based on these sudden change design primers with patient, SEQ ID NO:20 and 21 for example, SEQ ID NO:22 and 23.
| Sequence (5 '-3 ') | ??SEQ?ID?NO： | |
| Primer is to 1 | ??Fwd?5-CGTTCAGCTTGCAGAATCTC-3 | ????20 |
| ??Rev?5-CCACATAGGACCATGCCACA-3 | ????21 | |
| Primer is to 2 | ??Fwd?5-GACCCACCAGCCTTCTTCT-3 | ????22 |
| ??Rev?5-AAGCCTGTGTGGTTGCTCC-3 | ????23 |
Preparation one detects the sudden change test kit (100 person-times) of 846 C → T among the SEQ ID NO:1, and it contains:
Title numbering quantity
Forward primer SEQ ID NO:20 100pmol
Reverse primer SEQ ID NO:21 100pmol
The PCR damping fluid is some
Preparation one detects the sudden change test kit (100 person-times) of 1706 C → T among the SEQ ID NO:1, and it contains:
Title numbering quantity
Forward primer SEQ ID NO:22 100pmol
Reverse primer SEQ ID NO:23 100pmol
The PCR damping fluid is some
The mRNA of subject's skin cell to be detected is got in extracting, is transcribed into cDNA.PCR primer in the baldness susceptibility detection kit is diluted to 1 μ mol/ μ l, is that template is carried out the PCR reaction with the primer that is provided with prepared cDNA.Behind the PCR product purification, use ABI-PRISM
TM377 dna sequencing instrument carry out the two-way order-checking of the terminal cessation method of fluorescent mark, and the interpretation and the SNP that carry out sequence with Polyphred software confirm.
Detected result shows, the baldness susceptibility of detected object (age) with 846 C → T and 1706 C → T is apparently higher than normal population.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Research Center of Biotechnology
＜120〉utilize people Rhbdl5 gene and coded product thereof to diagnose and treat the method for baldness
<130>040333
<160>25
<170>PatentIn?version?3.1
<210>1
<211>3118
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>
<221>CDS
<222>(71)..(2551)
<223>
<400>1
gccctttcac?agcaagaccc?ggaagccgcg?ggcaaactca?gactcgaagc?cctcccgcct??60
cctgcccaca?atg?gcc?tct?gct?gac?aag?aat?ggc?ggg?agc?gtg?tcc?tct????109
Met?Ala?Ser?Ala?Asp?Lys?Asn?Gly?Gly?Ser?Val?Ser?Ser
1???????????????5???????????????????10
gtg?tcc?agc?agc?cgc?ctg?cag?agc?cgg?aag?cca?ccc?aac?ctc?tcc?atc???157
Val?Ser?Ser?Ser?Arg?Leu?Gln?Ser?Arg?Lys?Pro?Pro?Asn?Leu?Ser?Ile
15??????????????????20??????????????????25
acc?atc?ccg?cca?ccc?gag?aaa?gag?acc?cag?gcc?cct?ggc?gag?cag?gac???205
Thr?Ile?Pro?Pro?Pro?Glu?Lys?Glu?Thr?Gln?Ala?Pro?Gly?Glu?Gln?Asp
30??????????????????35??????????????????40??????????????????45
agc?atg?ctg?cct?gag?agg?aag?aac?cca?gcc?tac?ttg?aag?agc?gtc?agc???253
Ser?Met?Leu?Pro?Glu?Arg?Lys?Asn?Pro?Ala?Tyr?Leu?Lys?Ser?Val?Ser
50??????????????????55??????????????????60
ctc?cag?gag?cca?cgc?agc?cga?tgg?cag?gag?agt?tca?gag?aag?cgc?cct???301
Leu?Gln?Glu?Pro?Arg?Ser?Arg?Trp?Gln?Glu?Ser?Ser?Glu?Lys?Arg?Pro
65??????????????????70??????????????????75
ggc?ttc?cgc?cgc?cag?gcc?tca?ctg?tcc?cag?agc?atc?cgc?aag?ggc?gca???349
Gly?Phe?Arg?Arg?Gln?Ala?Ser?Leu?Ser?Gln?Ser?Ile?Arg?Lys?Gly?Ala
80??????????????????85??????????????????90
gcc?cag?tgg?ttt?gga?gtc?agc?ggc?gac?tgg?gag?ggg?cag?cgg?cag?cag???397
Ala?Gln?Trp?Phe?Gly?Val?Ser?Gly?Asp?Trp?Glu?Gly?Gln?Arg?Gln?Gln
95??????????????????100?????????????????105
tgg?cag?cgc?cgc?agc?ctg?cac?cac?tgc?agc?atg?cgc?tac?ggc?cgc?ctg???445
Trp?Gln?Arg?Arg?Ser?Leu?His?His?Cys?Ser?Met?Arg?Tyr?Gly?Arg?Leu
110?????????????????115?????????????????120?????????????????125
aag?gcc?tcg?tgc?cag?cgt?gac?ctg?gag?ctc?ccc?agc?cag?gag?gca?ccg???493
Lys?Ala?Ser?Cys?Gln?Arg?Asp?Leu?Glu?Leu?Pro?Ser?Gln?Glu?Ala?Pro
130?????????????????135?????????????????140
tcc?ttc?cag?ggc?act?gag?tcc?cca?aag?ccc?tgc?aag?atg?ccc?aag?att???541
Ser?Phe?Gln?Gly?Thr?Glu?Ser?Pro?Lys?Pro?Cys?Lys?Met?Pro?Lys?Ile
145?????????????????150?????????????????155
gtg?gat?ccg?ctg?gcc?cgg?ggc?cgg?gcc?ttc?cgc?cac?ccg?gag?gag?atg???589
Val?Asp?Pro?Leu?Ala?Arg?G1y?Arg?Ala?Phe?Arg?His?Pro?Glu?Glu?Met
160?????????????????165?????????????????170
gac?agg?ccc?cac?gcc?ctg?cac?cca?ccg?ctg?acc?ccc?gga?gtc?ctg?tcc????637
Asp?Arg?Pro?His?Ala?Leu?His?Pro?Pro?Leu?Thr?Pro?Gly?Val?Leu?Ser
175?????????????????180?????????????????185
ctc?acc?tcc?ttc?acc?agt?gtc?cgt?tct?ggc?tac?tcc?cac?ctg?cca?cgc????685
Leu?Thr?Ser?Phe?Thr?Ser?Val?Arg?Ser?Gly?Tyr?Ser?His?Leu?Pro?Arg
190?????????????????195?????????????????200?????????????????205
cgc?aag?aga?atg?tct?gtg?gcc?cac?atg?agc?ttg?caa?gct?gcc?gct?gcc????733
Arg?Lys?Arg?Met?Ser?Val?Ala?His?Met?Ser?Leu?Gln?Ala?Ala?Ala?Ala
210?????????????????215?????????????????220
ctc?ctc?aag?ggg?cgc?tcg?gtg?ctg?gat?gcc?acc?gga?cag?cgg?tgc?cgg????781
Leu?Leu?Lys?Gly?Arg?Ser?Val?Leu?Asp?Ala?Thr?Gly?Gln?Arg?Cys?Arg
225?????????????????230?????????????????235
gtg?gtc?aag?cgc?agc?ttt?gcc?ttc?ccg?agc?ttc?ctg?gag?gag?gat?gtg????829
Val?Val?Lys?Arg?Ser?Phe?Ala?Phe?Pro?Ser?Phe?Leu?Glu?Glu?Asp?Val
240?????????????????245?????????????????250
gtc?gat?ggg?gca?gac?acg?ttt?gac?tcc?tcc?ttt?ttt?agt?aag?gaa?gaa????877
Val?Asp?Gly?Ala?Asp?Thr?Phe?Asp?Ser?Ser?Phe?Phe?Ser?Lys?Glu?Glu
255?????????????????260?????????????????265
atg?agc?tcc?atg?cct?gat?gat?gtc?ttt?gag?tcc?ccc?cca?ctc?tct?gcc????925
Met?Ser?Ser?Met?Pro?Asp?Asp?Val?Phe?Glu?Ser?Pro?Pro?Leu?Ser?Ala
270?????????????????275?????????????????280?????????????????285
agc?tac?ttc?cga?ggg?atc?cca?cac?tca?gcc?tcc?cct?gtc?tcc?ccc?gat????973
Ser?Tyr?Phe?Arg?Gly?Ile?Pro?His?Ser?Ala?Ser?Pro?Val?Ser?Pro?Asp
290?????????????????295?????????????????300
ggg?gtg?caa?atc?cct?ctg?aag?gag?tat?ggc?cga?gcc?cca?gtc?ccc?ggg????1021
Gly?Val?Gln?Ile?Pro?Leu?Lys?Glu?Tyr?Gly?Arg?Ala?Pro?Val?Pro?Gly
305?????????????????310?????????????????315
ccc?cgg?cgc?ggc?aag?cgc?atc?gcc?tcc?aag?gtg?aag?cac?ttt?gcc?ttt????1069
Pro?Arg?Arg?Gly?Lys?Arg?Ile?Ala?Ser?Lys?Val?Lys?His?Phe?Ala?Phe
320?????????????????325?????????????????330
gat?cgg?aag?aag?cgg?cac?tac?ggc?ctc?ggc?gtg?gtg?ggc?aac?tgg?ctg????1117
Asp?Arg?Lys?Lys?Arg?His?Tyr?Gly?Leu?Gly?Val?Val?Gly?Asn?Trp?Leu
335?????????????????340?????????????????345
aac?cgc?agc?tac?cgc?cgc?agc?atc?agc?agc?act?gtg?cag?cgg?cag?ctg????1165
Asn?Arg?Ser?Tyr?Arg?Arg?Ser?Ile?Ser?Ser?Thr?Val?Gln?Arg?Gln?Leu
350?????????????????355?????????????????360?????????????????365
gag?agc?ttc?gac?agc?cac?cgg?ccc?tac?ttc?acc?tac?tgg?ctg?acc?ttc????1213
Glu?Ser?Phe?Asp?Ser?His?Arg?Pro?Tyr?Phe?Thr?Tyr?Trp?Leu?Thr?Phe
370?????????????????375?????????????????380
gtc?cat?gtc?atc?atc?acg?ctg?ctg?gtg?att?tgc?acg?tat?ggc?atc?gca????1261
Val?His?Val?Ile?Ile?Thr?Leu?Leu?Val?Ile?Cys?Thr?Tyr?Gly?Ile?Ala
385?????????????????390?????????????????395
ccc?gtg?ggc?ttt?gcc?cag?cac?gtc?acc?acc?cag?ctg?gtg?ctg?cgg?aac????1309
Pro?Val?Gly?Phe?Ala?Gln?His?Val?Thr?Thr?Gln?Leu?Val?Leu?Arg?Asn
400?????????????????405?????????????????410
aaa?ggt?gtg?tac?gag?agc?gtg?aag?tac?atc?cag?cag?gag?aac?ttc?tgg????1357
Lys?Gly?Val?Tyr?Glu?Ser?Val?Lys?Tyr?Ile?Gln?Gln?Glu?Asn?Phe?Trp
415?????????????????420?????????????????425
gtt?ggc?ccc?agc?tcg?att?gac?ctg?atc?cac?ctg?ggg?gcc?aag?ttc?tca????1405
Val?Gly?Pro?Ser?Ser?Ile?Asp?Leu?Ile?His?Leu?Gly?Ala?Lys?Phe?Ser
430?????????????????435?????????????????440?????????????????445
ccc?tgc?atc?cgg?aag?gac?ggg?cag?atc?gag?cag?ctg?gtg?ctg?cgc?gag????1453
Pro?Cys?Ile?Arg?Lys?Asp?Gly?Gln?Ile?Glu?Gln?Leu?Val?Leu?Arg?Glu
450?????????????????455?????????????????460
cga?gac?ctg?gag?cgg?gac?tca?ggc?tgc?tgt?gtc?cag?aat?gac?cac?tcc????1501
Arg?Asp?Leu?Glu?Arg?Asp?Ser?Gly?Cys?Cys?Val?Gln?Asn?Asp?His?Ser
465?????????????????470?????????????????475
gga?tgc?atc?cag?acc?cag?cgg?aag?gac?tgc?tcg?gag?act?ttg?gcc?act????1549
Gly?Cys?Ile?Gln?Thr?Gln?Arg?Lys?Asp?Cys?Ser?Glu?Thr?Leu?Ala?Thr
480?????????????????485?????????????????490
ttt?gtc?aag?tgg?cag?gat?gac?act?ggg?ccc?ccc?atg?gac?aag?tct?gat????1597
Phe?Val?Lys?Trp?Gln?Asp?Asp?Thr?Gly?Pro?Pro?Met?Asp?Lys?Ser?Asp
495?????????????????500?????????????????505
ctg?ggc?cag?aag?cgg?act?tcg?ggg?gct?gtc?tgc?cac?cag?gac?ccc?agg????1645
Leu?Gly?Gln?Lys?Arg?Thr?Ser?Gly?Ala?Val?Cys?His?Gln?Asp?Pro?Arg
510?????????????????515?????????????????520?????????????????525
acc?tgc?gag?gag?cca?gcc?tcc?agc?ggt?gcc?cac?atc?tgg?ccc?gat?gac????1693
Thr?Cys?Glu?Glu?Pro?Ala?Ser?Ser?Gly?Ala?His?Ile?Trp?Pro?Asp?Asp
530?????????????????535?????????????????540
atc?act?aag?tgg?ccg?atc?tgc?aca?gag?cag?gcc?agg?agc?aac?cac?aca????1741
Ile?Thr?Lys?Trp?Pro?Ile?Cys?Thr?Glu?Gln?Ala?Arg?Ser?Asn?His?Thr
545?????????????????550?????????????????555
ggc?ttc?ctg?cac?atg?gac?tgc?gag?atc?aag?ggc?cgc?ccc?tgc?tgc?atc????1789
Gly?Phe?Leu?His?Met?Asp?Cys?Glu?Ile?Lys?Gly?Arg?Pro?Cys?Cys?Ile
560?????????????????565?????????????????570
ggc?acc?aag?ggc?agc?tgt?gag?atc?acc?acc?cgg?gaa?tac?tgt?gag?ttc????1837
Gly?Thr?Lys?Gly?Ser?Cys?Glu?Ile?Thr?Thr?Arg?Glu?Tyr?Cys?Glu?Phe
575?????????????????580?????????????????585
atg?cac?ggc?tat?ttc?cat?gag?gaa?gca?aca?ctc?tgc?tcc?cag?gtg?cac????1885
Met?His?Gly?Tyr?Phe?His?Glu?Glu?Ala?Thr?Leu?Cys?Ser?Gln?Val?His
590?????????????????595?????????????????600?????????????????605
tgc?ttg?gac?aag?gtg?tgt?ggg?ctg?ctg?ccc?ttc?ctc?aac?cct?gag?gtc????1933
Cys?Leu?Asp?Lys?Val?Cys?Gly?Leu?Leu?Pro?Phe?Leu?Asn?Pro?Glu?Val
610?????????????????615?????????????????620
cca?gat?cag?ttc?tac?agg?ctc?tgg?ctg?tct?ctc?ttc?cta?cat?gct?ggc????198l
Pro?Asp?Gln?Phe?Tyr?Arg?Leu?Trp?Leu?Ser?Leu?Phe?Leu?His?Ala?Gly
625?????????????????630?????????????????635
gtg?gtg?cac?tgc?ctc?gtg?tct?gtg?gtc?ttt?caa?atg?acc?atc?ctg?agg????2029
Val?Val?His?Cys?Leu?Val?Ser?Val?Val?Phe?Gln?Met?Thr?Ile?Leu?Arg
640?????????????????645?????????????????650
gac?ctg?gag?aag?ctg?gcc?ggc?tgg?cac?cgt?atc?gcc?atc?atc?ttc?atc????2077
Asp?Leu?Glu?Lys?Leu?Ala?Gly?Trp?His?Arg?Ile?Ala?Ile?Ile?Phe?Ile
655?????????????????660?????????????????665
ctc?agt?ggc?atc?aca?ggc?aac?ctc?gcc?agt?gcc?atc?ttt?ctc?cca?tac????2125
Leu?Ser?Gly?Ile?Thr?Gly?Asn?Leu?Ala?Ser?Ala?Ile?Phe?Leu?Pro?Tyr
670?????????????????675?????????????????680?????????????????685
cgg?gca?gag?gtg?ggc?ccg?gcc?ggc?tca?cag?ttc?ggc?ctc?ctc?gcc?tgc????2173
Arg?Ala?Glu?Val?Gly?Pro?Ala?Gly?Ser?Gln?Phe?Gly?Leu?Leu?Ala?Cys
690?????????????????695?????????????????700
ctc?ttc?gtg?gag?ctc?ttc?cag?agc?tgg?ccg?ctg?ctg?gag?agg?ccc?tgg????2221
Leu?Phe?Val?Glu?Leu?Phe?Gln?Ser?Trp?Pro?Leu?Leu?Glu?Arg?Pro?Trp
705?????????????????710?????????????????715
aag?gcc?ttc?ctc?aac?ctc?tcg?gcc?atc?gtg?ctc?ttc?ctg?ttc?atc?tgt????2269
Lys?Ala?Phe?Leu?Asn?Leu?Ser?Ala?Ile?Val?Leu?Phe?Leu?Phe?Ile?Cys
720?????????????????725?????????????????730
ggc?ctc?ctg?ccc?tgg?atc?gac?aac?atc?gcc?cac?atc?ttc?ggc?ttc?ctc????2317
Gly?Leu?Leu?Pro?Trp?Ile?Asp?Asn?Ile?Ala?His?Ile?Phe?Gly?Phe?Leu
735?????????????????740?????????????????745
agt?ggc?ctg?ctg?ctg?gcc?ttc?gcc?ttc?ctg?ccc?tac?atc?acc?ttc?ggc????2365
Ser?Gly?Leu?Leu?Leu?Ala?Phe?Ala?Phe?Leu?Pro?Tyr?Ile?Thr?Phe?Gly
750?????????????????755?????????????????760?????????????????765
acc?agc?gac?aag?tac?cgc?aag?cgg?gca?ctc?atc?ctg?gtg?tca?ctg?ctg????2413
Thr?Ser?Asp?Lys?Tyr?Arg?Lys?Arg?Ala?Leu?Ile?Leu?Val?Ser?Leu?Leu
770?????????????????775?????????????????780
gcc?ttt?gcc?ggc?ctc?ttc?gcc?gcc?ctc?gtg?ctg?tgg?ctg?tac?atc?tac????2461
Ala?Phe?Ala?Gly?Leu?Phe?Ala?Ala?Leu?Val?Leu?Trp?Leu?Tyr?Ile?Tyr
785?????????????????790?????????????????795
ccc?att?aac?tgg?ccc?tgg?atc?gag?cac?ctc?acc?tgc?ttc?ccc?ttc?acc????2509
Pro?Ile?Asn?Trp?Pro?Trp?Ile?Glu?His?Leu?Thr?Cys?Phe?Pro?Phe?Thr
800?????????????????805?????????????????810
agc?cgc?ttc?tgc?gag?aag?tat?gag?ctg?gac?cag?gtg?ctg?cac????????????2551
Ser?Arg?Phe?Cys?Glu?Lys?Tyr?Glu?Leu?Asp?Gln?Val?Leu?His
815?????????????????820?????????????????825
tgaccgctgg?gccacacggc?tgcccctcag?ccctgctgga?acagggtctg?cctgcgaggg??2611
ctgccctctg?cagagcgctc?tctgtgtgcc?agagagccag?agacccaaga?cagggcccgg??2671
gctctggacc?tgggtgcccc?cctgccaggc?gaggctgact?ccgcgtgaga?tggttggtta??2731
aggcggggtt?tttctggggc?gtgaggcctg?tgagatcctg?acccaagctc?aggcacaccc??2791
aaggcacctg?cctctctgag?tcttgggtct?cagttcctaa?tatcccgctc?cttgctgaga??2851
ccatctcctg?gggcagggtc?cttttcttcc?caggtcctca?gcgctgcctc?tgctggtgcc??2911
ttctccccca?ctactactgg?agcgtgccct?tgctggggac?gtggctgtgc?cctcagttgc??2971
ccccagggct?gggtgcccac?catgcccctt?cctctttctc?ctcctacctc?tgccctgtga??3031
gcccatccat?aaggctctca?gatgggacat?tgtgggaaag?gctttggcca?tggtctgggg??3091
gcagagaaca?aggggggaga?cacaagt??????????????????????????????????????3118
<210>2
<211>827
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>2
Met?Ala?Ser?Ala?Asp?Lys?Asn?Gly?Gly?Ser?Val?Ser?Ser?Val?Ser?Ser
1???????????????5???????????????????10??????????????????15
Ser?Arg?Leu?Gln?Ser?Arg?Lys?Pro?Pro?Asn?Leu?Ser?Ile?Thr?Ile?Pro
20??????????????????25??????????????????30
Pro?Pro?Glu?Lys?Glu?Thr?Gln?Ala?Pro?Gly?Glu?Gln?Asp?Ser?Met?Leu
35??????????????????40??????????????????45
Pro?Glu?Arg?Lys?Asn?Pro?Ala?Tyr?Leu?Lys?Ser?Val?Ser?Leu?Gln?Glu
50??????????????????55??????????????????60
Pro?Arg?Ser?Arg?Trp?Gln?Glu?Ser?Ser?Glu?Lys?Arg?Pro?Gly?Phe?Arg
65??????????????????70??????????????????75??????????????????80
Arg?Gln?Ala?Ser?Leu?Ser?Gln?Ser?Ile?Arg?Lys?Gly?Ala?Ala?Gln?Trp
85??????????????????90??????????????????95
Phe?Gly?Val?Ser?Gly?Asp?Trp?Glu?Gly?Gln?Arg?Gln?Gln?Trp?Gln?Arg
100?????????????????105?????????????????110
Arg?Ser?Leu?His?His?Cys?Ser?Met?Arg?Tyr?Gly?Arg?Leu?Lys?Ala?Ser
115?????????????????120?????????????????125
Cys?Gln?Arg?Asp?Leu?Glu?Leu?Pro?Ser?Gln?Glu?Ala?Pro?Ser?Phe?Gln
130?????????????????135?????????????????140
Gly?Thr?Glu?Ser?Pro?Lys?Pro?Cys?Lys?Met?Pro?Lys?Ile?Val?Asp?Pro
145?????????????????150?????????????????155?????????????????160
Leu?Ala?Arg?Gly?Arg?Ala?Phe?Arg?His?Pro?Glu?Glu?Met?Asp?Arg?Pro
165?????????????????170?????????????????175
His?Ala?Leu?His?Pro?Pro?Leu?Thr?Pro?Gly?Val?Leu?Ser?Leu?Thr?Ser
l80?????????????????185?????????????????190
Phe?Thr?Ser?Val?Arg?Ser?Gly?Tyr?Ser?His?Leu?Pro?Arg?Arg?Lys?Arg
195?????????????????200?????????????????205
Met?Ser?Val?Ala?His?Met?Ser?Leu?Gln?Ala?Ala?Ala?Ala?Leu?Leu?Lys
210?????????????????215?????????????????220
Gly?Arg?Ser?Val?Leu?Asp?Ala?Thr?Gly?Gln?Arg?Cys?Arg?Val?Val?Lys
225?????????????????230?????????????????235?????????????????240
Arg?Ser?Phe?Ala?Phe?Pro?Ser?Phe?Leu?Glu?Glu?Asp?Val?Val?Asp?Gly
245?????????????????250?????????????????255
Ala?Asp?Thr?Phe?Asp?Ser?Ser?Phe?Phe?Ser?Lys?Glu?Glu?Met?Ser?Ser
260?????????????????265?????????????????270
Met?Pro?Asp?Asp?Val?Phe?Glu?Ser?Pro?Pro?Leu?Ser?Ala?Ser?Tyr?Phe
275?????????????????280?????????????????285
Arg?Gly?Ile?Pro?His?Ser?Ala?Ser?Pro?Val?Ser?Pro?Asp?Gly?Val?Gln
290?????????????????295?????????????????300
Ile?Pro?Leu?Lys?Glu?Tyr?Gly?Arg?Ala?Pro?Val?Pro?Gly?Pro?Arg?Arg
305?????????????????310?????????????????315?????????????????320
Gly?Lys?Arg?Ile?Ala?Ser?Lys?Val?Lys?His?Phe?Ala?Phe?Asp?Arg?Lys
325?????????????????330?????????????????335
Lys?Arg?His?Tyr?Gly?Leu?Gly?Val?Val?Gly?Asn?Trp?Leu?Asn?Arg?Ser
340?????????????????345?????????????????350
Tyr?Arg?Arg?Ser?Ile?Ser?Ser?Thr?Val?Gln?Arg?Gln?Leu?Glu?Ser?Phe
355?????????????????360?????????????????365
Asp?Ser?His?Arg?Pro?Tyr?Phe?Thr?Tyr?Trp?Leu?Thr?Phe?Val?His?Val
370?????????????????375?????????????????380
Ile?Ile?Thr?Leu?Leu?Val?Ile?Cys?Thr?Tyr?Gly?Ile?Ala?Pro?Val?Gly
385?????????????????390?????????????????395?????????????????400
Phe?Ala?Gln?His?Val?Thr?Thr?Gln?Leu?Val?Leu?Arg?Asn?Lys?Gly?Val
405?????????????????410?????????????????415
Tyr?Glu?Ser?Val?Lys?Tyr?Ile?Gln?Gln?Glu?Asn?Phe?Trp?Val?Gly?Pro
420?????????????????425?????????????????430
Ser?Ser?Ile?Asp?Leu?Ile?His?Leu?Gly?Ala?Lys?Phe?Ser?Pro?Cys?Ile
435?????????????????440?????????????????445
Arg?Lys?Asp?Gly?Gln?Ile?Glu?Gln?Leu?Val?Leu?Arg?Glu?Arg?Asp?Leu
450?????????????????455?????????????????460
Glu?Arg?Asp?Ser?Gly?Cys?Cys?Val?Gln?Asn?Asp?His?Ser?Gly?Cys?Ile
465?????????????????470?????????????????475?????????????????480
Gln?Thr?Gln?Arg?Lys?Asp?Cys?Ser?Glu?Thr?Leu?Ala?Thr?Phe?Val?Lys
485?????????????????490?????????????????495
Trp?Gln?Asp?Asp?Thr?Gly?Pro?Pro?Met?Asp?Lys?Ser?Asp?Leu?Gly?Gln
500?????????????????505?????????????????510
Lys?Arg?Thr?Ser?Gly?Ala?Val?Cys?His?Gln?Asp?Pro?Arg?Thr?Cys?Glu
515?????????????????520?????????????????525
Glu?Pro?Ala?Ser?Ser?Gly?Ala?His?Ile?Trp?Pro?Asp?Asp?Ile?Thr?Lys
530?????????????????535?????????????????540
Trp?Pro?Ile?Cys?Thr?Glu?Gln?Ala?Arg?Ser?Asn?His?Thr?Gly?Phe?Leu
545?????????????????550?????????????????555?????????????????560
His?Met?Asp?Cys?Glu?Ile?Lys?Gly?Arg?Pro?Cys?Cys?Ile?Gly?Thr?Lys
565?????????????????570?????????????????575
Gly?Ser?Cys?Glu?Ile?Thr?Thr?Arg?Glu?Tyr?Cys?Glu?Phe?Met?His?Gly
580?????????????????585?????????????????590
Tyr?Phe?His?Glu?Glu?Ala?Thr?Leu?Cys?Ser?Gln?Val?His?Cys?Leu?Asp
595?????????????????600?????????????????605
Lys?Val?Cys?Gly?Leu?Leu?Pro?Phe?Leu?Asn?Pro?Glu?Val?Pro?Asp?Gln
610?????????????????615?????????????????620
Phe?Tyr?Arg?Leu?Trp?Leu?Ser?Leu?Phe?Leu?His?Ala?Gly?Val?Val?His
625?????????????????630?????????????????635?????????????????640
Cys?Leu?Val?Ser?Val?Val?Phe?Gln?Met?Thr?Ile?Leu?Arg?Asp?Leu?Glu
645?????????????????650?????????????????655
Lys?Leu?Ala?Gly?Trp?His?Arg?Ile?Ala?Ile?Ile?Phe?Ile?Leu?Ser?Gly
660?????????????????665?????????????????670
Ile?Thr?Gly?Asn?Leu?Ala?Ser?Ala?Ile?Phe?Leu?Pro?Tyr?Arg?Ala?Glu
675?????????????????680?????????????????685
Val?Gly?Pro?Ala?Gly?Ser?Gln?Phe?Gly?Leu?Leu?Ala?Cys?Leu?Phe?Val
690?????????????????695?????????????????700
Glu?Leu?Phe?Gln?Ser?Trp?Pro?Leu?Leu?Glu?Arg?Pro?Trp?Lys?Ala?Phe
705?????????????????710?????????????????715?????????????????720
Leu?Asn?Leu?Ser?Ala?Ile?Val?Leu?Phe?Leu?Phe?Ile?Cys?Gly?Leu?Leu
725?????????????????730?????????????????735
Pro?Trp?Ile?Asp?Asn?Ile?Ala?His?Ile?Phe?Gly?Phe?Leu?Ser?Gly?Leu
740?????????????????745?????????????????750
Leu?Leu?Ala?Phe?Ala?Phe?Leu?Pro?Tyr?Ile?Thr?Phe?Gly?Thr?Ser?Asp
755?????????????????760?????????????????765
Lys?Tyr?Arg?Lys?Arg?Ala?Leu?Ile?Leu?Val?Ser?Leu?Leu?Ala?Phe?Ala
770?????????????????775?????????????????780
Gly?Leu?Phe?Ala?Ala?Leu?Val?Leu?Trp?Leu?Tyr?Ile?Tyr?Pro?Ile?Asn
785?????????????????790?????????????????795?????????????????800
Trp?Pro?Trp?Ile?Glu?His?Leu?Thr?Cys?Phe?Pro?Phe?Thr?Ser?Arg?Phe
805?????????????????810?????????????????815
Cys?Glu?Lys?Tyr?Glu?Leu?Asp?Gln?Val?Leu?His
820?????????????????825
<210>3
<211>22
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>3
tgtacagagc?aaggagaaga?ca??????????????????????????????????????????????22
<210>4
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>4
ctttcccaca?atgtcccatc?????????????????????????????????????????????????20
<210>5
<211>18
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>5
ggagctccag?gtcacgct???????????????????????????????????????????????????18
<210>6
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>6
ccacagactg?aggcttgaca?????????????????????????????????????????????????20
<210>7
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>7
gatgctctgg?gacagtgagg?????????????????????????????????????????????????20
<210>8
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>8
acttgaagag?cgtcagcctc?????????????????????????????????????????????????20
<210>9
<211>19
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>9
atcgaccaca?tcctcctcc??????????????????????????????????????????????????19
<210>10
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>10
agtcctgtcc?ctcacctcct?????????????????????????????????????????????????20
<210>11
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>11
ggacgaaggt?cagccagtag?????????????????????????????????????????????????20
<210>12
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>12
cggccctact?tcacctactg?????????????????????????????????????????????????20
<210>13
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>13
ttgatctcgc?agtccatgtg?????????????????????????????????????????????????20
<210>14
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>14
ggcccgatga?catcactaag?????????????????????????????????????????????????20
<210>15
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>15
tgaacaggaa?gagcacgatg?????????????????????????????????????????????????20
<210>16
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>16
cagtgccatc?tttctcccat?????????????????????????????????????????????????20
<210>17
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>17
ctgtcttggg?tctctggctc?????????????????????????????????????????????????20
<210>18
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>18
tgcgagaagt?atgagctgga?????????????????????????????????????????????????20
<210>19
<211>827
<212>PRT
＜213〉mouse (Mus musculus)
<400>19
Met?Ala?Ser?Ala?Asp?Lys?Asn?Gly?Ser?Asn?Leu?Pro?Ser?Val?Ser?Gly
1???????????????5???????????????????10??????????????????15
Ser?Arg?Leu?Gln?Ser?Arg?Lys?Pro?Pro?Asn?Leu?Ser?Ile?Thr?Ile?Pro
20??????????????????25??????????????????30
Pro?Pro?Glu?Ser?Gln?Ala?Pro?Gly?Glu?Gln?Asp?Ser?Met?Leu?Pro?Glu
35??????????????????40??????????????????45
Arg?Arg?Lys?Asn?Pro?Ala?Tyr?Leu?Lys?Ser?Val?Ser?Leu?Gln?Glu?Pro
50??????????????????55??????????????????60
Arg?Gly?Arg?Trp?Gln?Glu?Gly?Ala?Glu?Lys?Arg?Pro?Gly?Phe?Arg?Arg
65??????????????????70??????????????????75??????????????????80
Gln?Ala?Ser?Leu?Ser?Gln?Ser?Ile?Arg?Lys?Ser?Thr?Ala?Gln?Trp?Phe
85??????????????????90??????????????????95
Gly?Val?Ser?Gly?Asp?Trp?Glu?Gly?Lys?Arg?Gln?Asn?Trp?His?Arg?Arg
100?????????????????105?????????????????110
Ser?Leu?His?His?Cys?Ser?Val?His?Tyr?Gly?Arg?Leu?Lys?Ala?Ser?Cys
115?????????????????120?????????????????125
Gln?Arg?Glu?Leu?Glu?Leu?Pro?Ser?Gln?Glu?Val?Pro?Ser?Phe?Gln?Gly
130?????????????????135?????????????????140
Thr?Glu?Ser?Pro?Lys?Pro?Cys?Lys?Met?Pro?Lys?Ile?Val?Asp?Pro?Leu
145?????????????????150?????????????????155?????????????????160
Ala?Arg?Gly?Arg?Ala?Phe?Arg?His?Pro?Asp?Glu?Val?Asp?Arg?Pro?His
165?????????????????170?????????????????175
Ala?Ala?His?Pro?Pro?Leu?Thr?Pro?Gly?Val?Leu?Ser?Leu?Thr?Ser?Phe
180?????????????????185?????????????????190
Thr?Ser?Val?Arg?Ser?Gly?Tyr?Ser?His?Leu?Pro?Arg?Arg?Lys?Arg?Ile
195?????????????????200?????????????????205
Ser?Val?Ala?His?Met?Ser?Phe?Gln?Ala?Ala?Ala?Ala?Leu?Leu?Lys?Gly
2l0?????????????????215?????????????????220
Arg?Ser?Val?Leu?Asp?Ala?Thr?Gly?Gln?Arg?Cys?Arg?His?Val?Lys?Arg
225?????????????????230?????????????????235?????????????????240
Ser?Phe?Ala?Tyr?Pro?Ser?Phe?Leu?Glu?Glu?Asp?Ala?Val?Asp?Gly?Ala
245?????????????????250?????????????????255
Asp?Thr?Phe?Asp?Ser?Ser?Phe?Phe?Ser?Lys?Glu?Glu?Met?Ser?Ser?Met
260?????????????????265?????????????????270
Pro?Asp?Asp?Val?Phe?Glu?Ser?Pro?Pro?Leu?Ser?Ala?Ser?Tyr?Phe?Arg
275?????????????????280?????????????????285
Gly?Val?Pro?His?Ser?Ala?Ser?Pro?Val?Ser?Pro?Asp?Gly?Val?His?Ile
290?????????????????295?????????????????300
Pro?Leu?Lys?Glu?Tyr?Ser?Gly?Gly?Arg?Ala?Leu?Gly?Pro?Gly?Thr?Gln
305?????????????????310?????????????????315?????????????????320
Arg?Gly?Lys?Arg?Ile?Ala?Ser?Lys?Val?Lys?His?Phe?Ala?Phe?Asp?Arg
325?????????????????330?????????????????335
Lys?Lys?Arg?His?Tyr?Gly?Leu?Gly?Val?Val?Gly?Asn?Trp?Leu?Asn?Arg
340?????????????????345?????????????????350
Ser?Tyr?Arg?Arg?Ser?Ile?Ser?Ser?Thr?Val?Gln?Arg?Gln?Leu?Glu?Ser
355?????????????????360?????????????????365
Phe?Asp?Ser?His?Arg?Pro?Tyr?Phe?Thr?Tyr?Trp?Leu?Thr?Phe?Val?His
370?????????????????375?????????????????380
Ile?Ile?Ile?Thr?Leu?Leu?Val?Ile?Cys?Thr?Tyr?Gly?Ile?Ala?Pro?Val
385?????????????????390?????????????????395?????????????????400
Gly?Phe?Ala?Gln?His?Val?Thr?Thr?Gln?Leu?Val?Leu?Lys?Asn?Arg?Gly
405?????????????????410?????????????????415
Val?Tyr?Glu?Ser?Val?Lys?Tyr?Ile?Gln?Gln?Glu?Asn?Phe?Trp?Ile?Gly
420?????????????????425?????????????????430
Pro?Ser?Ser?Ile?Asp?Leu?Ile?His?Leu?Gly?Ala?Lys?Phe?Ser?Pro?Cys
435?????????????????440?????????????????445
Ile?Arg?Lys?Asp?Gln?Gln?Ile?Glu?Gln?Leu?Val?Arg?Arg?Glu?Arg?Asp
450?????????????????455?????????????????460
Ile?Glu?Arg?Thr?Ser?Gly?Cys?Cys?Val?Gln?Asn?Asp?Arg?Ser?Gly?Cys
465?????????????????470?????????????????475?????????????????480
Ile?Gln?Thr?Leu?Lys?Lys?Asp?Cys?Ser?Glu?Thr?Leu?Ala?Thr?Phe?Val
485?????????????????490?????????????????495
Lys?Trp?Gln?Asn?Asp?Thr?Gly?Pro?Ser?Asp?Lys?Ser?Asp?Leu?Ser?Gln
500?????????????????505?????????????????510
Lys?Gln?Pro?Ser?Ala?Val?Val?Cys?His?Gln?Asp?Pro?Arg?Thr?Cys?Glu
515?????????????????520?????????????????525
Glu?Pro?Ala?Ser?Ser?Gly?Ala?His?Ile?Trp?Pro?Asp?Asp?Ile?Thr?Lys
530?????????????????535?????????????????540
Trp?Pro?Ile?Cys?Thr?Glu?Gln?Ala?Gln?Ser?Asn?His?Thr?Gly?Leu?Leu
545?????????????????550?????????????????555?????????????????560
His?Ile?Asp?Cys?Lys?Ile?Lys?Gly?Arg?Pro?Cys?Cys?Ile?Gly?Thr?Lys
565?????????????????570?????????????????575
Gly?Ser?Cys?Glu?Ile?Thr?Thr?Arg?Glu?Tyr?Cys?Glu?Phe?Met?His?Gly
580?????????????????585?????????????????590
Tyr?Phe?His?Glu?Asp?Ala?Thr?Leu?Cys?Ser?Gln?Val?His?Cys?Leu?Asp
595?????????????????600?????????????????605
Lys?Val?Cys?Gly?Leu?Leu?Pro?Phe?Leu?Asn?Pro?Glu?Val?Pro?Asp?Gln
610?????????????????615?????????????????620
Phe?Tyr?Arg?Ile?Trp?Leu?Ser?Leu?Phe?Leu?His?Ala?Gly?Ile?Val?His
625?????????????????630?????????????????635?????????????????640
Cys?Leu?Val?Ser?Val?Val?Phe?Gln?Met?Thr?Ile?Leu?Arg?Asp?Leu?Glu
645?????????????????650?????????????????655
Lys?Leu?Ala?Gly?Trp?His?Arg?Ile?Ser?Ile?Ile?Phe?Ile?Leu?Ser?Gly
660?????????????????665?????????????????670
Ile?Thr?Gly?Asn?Leu?Ala?Ser?Ala?Ile?Phe?Leu?Pro?Tyr?Arg?Ala?Glu
675?????????????????680?????????????????685
Val?Gly?Pro?Ala?Gly?Ser?Gln?Phe?Gly?Leu?Leu?Ala?Cys?Leu?Phe?Val
690?????????????????695?????????????????700
Glu?Leu?Phe?Gln?Ser?Trp?Gln?Leu?Leu?Glu?Arg?Pro?Trp?Lys?Ala?Phe
705?????????????????710?????????????????715?????????????????720
Phe?Asn?Leu?Ser?Ala?Ile?Val?Leu?Phe?Leu?Phe?Ile?Cys?Gly?Leu?Leu
725?????????????????730?????????????????735
Pro?Trp?Ile?Asp?Asn?Ile?Ala?His?Ile?Phe?Gly?Phe?Leu?Ser?Gly?Met
740?????????????????745?????????????????750
Leu?Leu?Ala?Phe?Ala?Phe?Leu?Pro?Tyr?Ile?Thr?Phe?Gly?Thr?Ser?Asp
755?????????????????760?????????????????765
Lys?Tyr?Arg?Lys?Arg?Ala?Leu?Ile?Leu?Val?Ser?Leu?Leu?Val?Phe?Ala
770?????????????????775?????????????????780
Gly?Leu?Phe?Ala?Ser?Leu?Val?Leu?Trp?Leu?Tyr?Ile?Tyr?Pro?Ile?Asn
785?????????????????790?????????????????795?????????????????800
Trp?Pro?Trp?Ile?Glu?Tyr?Leu?Thr?Cys?Phe?Pro?Phe?Thr?Set?Arg?Phe
805?????????????????810?????????????????815
Cys?Glu?Lys?Tyr?Glu?Leu?Asp?Gln?Val?Leu?His
820?????????????????825
<210>20
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>20
cgttcagctt?gcagaatctc?????????????????????????????????????????????????20
<210>21
<211>20
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>21
ccacatagga?ccatgccaca?????????????????????????????????????????????????20
<210>22
<211>19
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>22
gacccaccag?ccttcttct??????????????????????????????????????????????????19
<210>23
<211>19
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>23
aagcctgtgt?ggttgctcc??????????????????????????????????????????????????19
<210>24
<211>45
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>24
ataaagctta?tggcctcagc?tgacaagaat?ggcagcaacc?tccca?????????????????????45
<210>25
<211>34
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>25
ccgctcgagg?ctcgatctgg?tccacgatgt?gatt?????????????????????????????????34
Claims (10)
1. an isolating Rhbdl5 polypeptide is characterized in that this polypeptide is selected from down group: polypeptide, its conservative property variation polypeptide, its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is selected from down group:
(i) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(ii) have SEQ ID NO:2 aminoacid sequence and the 259th and be the polypeptide of Met;
(iii) have SEQ ID NO:2 aminoacid sequence and the 546th and be the polypeptide of Ser.
3. isolating polynucleotide is characterized in that, it is selected from down group:
(a) polynucleotide of polypeptide according to claim 1 of encoding;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ IDNO:2.
5. polynucleotide as claimed in claim 3 is characterized in that, these polynucleotide are selected from down group:
(c-1) has the sequence of 1-3118 position among the SEQ ID NO:1;
(c-2) has the sequence of 71-2551 position among the SEQ ID NO:1;
(c-3) have among the SEQ ID NO:1 71-2551 position and the 846th and be the sequence of T;
(c-4) have among the SEQ ID NO:1 71-2551 position and the 1706th and be the sequence of T.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.
8. proteic preparation method of Rhbdl5 is characterized in that this method comprises:
(a) expressing under the proteic condition of Rhbdl5, cultivate the described host cell of claim 7;
(b) from culture, isolate Rhbdl5 albumen.
9. a test kit that detects the baldness susceptibility is characterized in that, it comprises the primer of specific amplification Rhbdl5 gene or transcript.
10. a composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410025361 CN1712414A (en) | 2004-06-23 | 2004-06-23 | Method for diagnosing and treating baldness from human Rhbd15 gene and its coding product |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410025361 CN1712414A (en) | 2004-06-23 | 2004-06-23 | Method for diagnosing and treating baldness from human Rhbd15 gene and its coding product |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1712414A true CN1712414A (en) | 2005-12-28 |
Family
ID=35718229
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410025361 Pending CN1712414A (en) | 2004-06-23 | 2004-06-23 | Method for diagnosing and treating baldness from human Rhbd15 gene and its coding product |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1712414A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480408A (en) * | 2022-02-17 | 2022-05-13 | 南开大学 | RHX6 gene and application thereof in preparation of anti-breast cancer drugs |
-
2004
- 2004-06-23 CN CN 200410025361 patent/CN1712414A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480408A (en) * | 2022-02-17 | 2022-05-13 | 南开大学 | RHX6 gene and application thereof in preparation of anti-breast cancer drugs |
| CN114480408B (en) * | 2022-02-17 | 2023-12-19 | 南开大学 | RHX6 gene and application thereof in preparation of breast cancer resistant medicines |
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