CN1477125A - A polypeptide-human amelification protein 23 and polynucleotide for coding said polypeptide - Google Patents

Abstract

The present invention discloses a polypeptide-human amelification protein 23, polynucleotide for coding said polypeptide and method for producing said polypeptide by means of DNA recombination technology. Said invention also discloses the method for using said polypeptide to cure several oral diseases, such as soreness of teeth, odontoseisis and toothache, etc. It also discloses the agonist for resisting said polypeptide and its therapeutic action, and also discloses the application of the polynucleotide coding said novel human amelification protein 23.

Description

A kind of polypeptide-human becomes the polynucleotide of enamel protein 23 and this peptide species of coding

Technical field

The invention belongs to biological technical field, specifically, the invention describes a kind of polypeptide-human and become enamel protein 23, and the polynucleotide sequence of this polypeptide of encoding.The invention still further relates to the preparation method and the application of these polynucleotide and polypeptide.

Background technology

Mammiferous tooth is a special structure, and it interacts by a series of mutual epithelium mesenchymes, and three different mineralized tissues (enamel, dentine and dental cement) are flocked together to grow to form.The forming process of tooth provides many models for regulation and control, form generation and the mineralization that people understand special gene.When cranial nerve ridge deutero-ectoderm formed dentine and dental cement, oral epithelium was derived and is the inner enamel epithelium of mineralized tissue [Paul H.Krebsbach, Suk Keun Lee, et al., J Biol.Chem., 1996,271:4431-4435].In the forming process of tooth, enamel plays an important role in the morphogenetic mineral deposition process of tooth.Enamel has formed the top layer of vertebrates tooth height mineralising, for animal catches and digest food provides the surface [A.G.Fincham, J.Moradian-Oldak et al., J.Struc.Biol., 1999,126:270-299] of highly corrosion.The enamel stromatin is made up of numerous heterogeneous albumen, these albumen play an important role in location, gathering and the crystallization process of mineral, and a plurality of physiological processs [Robinson C such as cellular metabolism, apposition and mineralising are regulated in participation, Brookes SJ et al., Eur J OralSci, 1998 106:282-91].Become enamel protein in the enamel mineralisation process, to play an important role, in the people, become enamel protein to express by the individual gene on the X chromosome, this albumen has the function [A.G.Fincham of high conservative in enamel biomineralization process, J.Moradian-Oldak et al., J.Struc.Biol., 1999,126:270-299].This albumen is high expression level in the tooth that weathers, and it plays an important role in the forming process of cleaning of teeth layer, and its abnormal expression can reduce tooth usually and resist extraneous erosive ability, thereby cause various dental disorders.

The new gene of people of the present invention becomes enamel protein that higher homology degree is arranged with mouse, both have 52% identity and 78% similarity on protein level.Mouse becomes enamel protein proline rich and glycine, and it is except having the higher homology with known protein Y224, does not all have significant homology with any other albumen.This albumen is a kind of albumen of new tooth specifically expressing, high expression level in the tooth that weathers.Discover, this albumen contains some potential phosphorylation sites, the casein kinase i I phosphorylation site that albumen all contained and Tyrosylprotein kinase site and three protein kinase C site [Paul H.Krebsbach of comprising participation mineralizations such as sialoprotein, bone glycoprotein-75, tooth phosphorylated protein, Suk Keun Lee, et al., J Biol.Chem., 1996,271:4431-4435].This albumen plays an important role in the enamel mineralisation process, and its abnormal expression may cause some relevant dental disorders,, toothache loose as tooth acid, gum etc.New people's gene of the present invention becomes enamel protein similar with mouse, also contains Tyrosylprotein kinase II site, protein tyrosine kinase site and protein kinase C site; And also the position one acidic protein, proline rich and glycine.Thereby this albumen is the albumen of new people's tooth specifically expressing, names to the people to become enamel protein 23.And think that it becomes enamel protein similar with mouse, has similar biological function.

Summary of the invention

An object of the present invention is to provide isolating new polypeptide-human become enamel protein 23 with and fragment, analogue and derivative.

Another object of the present invention provides the polynucleotide of this polypeptide of coding.

Another object of the present invention provides and contains the recombinant vectors that the coding people becomes the polynucleotide of enamel protein 23.

Another object of the present invention provides and contains the genetically engineered host cell that the coding people becomes the polynucleotide of enamel protein 23.

Another object of the present invention provides the method that the people becomes enamel protein 23 of producing.

Another object of the present invention provides the antibody that becomes enamel protein 23 at polypeptide-human of the present invention.

Another object of the present invention has provided simulated compound, antagonist, agonist, the inhibitor that becomes enamel protein 23 at polypeptide-human of the present invention.

Another object of the present invention provides the method that diagnoses and treatment and people become the unusual relevant disease of enamel protein 23.

In a first aspect of the present invention, provide novel isolated people to become enamel protein 23, this polypeptide is the people source, and it comprises: have＜polypeptide of 210〉2 aminoacid sequences or its conservative property variation polypeptide or its active fragments or its reactive derivative, analogue.Preferably, this polypeptide is to have＜polypeptide of 210〉2 aminoacid sequences.

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) the above-mentioned people of coding becomes the polynucleotide of enamel protein 23; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding have＜210〉2 shown in the polypeptide of aminoacid sequence.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: (a) have＜210〉1 in the sequence of 6-629 position; (b) have＜210〉1 in the sequence of 1-834 position.

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.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

As used herein, " isolating " are 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 people becomes enamel protein 23 " is meant that the people becomes enamel protein 23 to be substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can become enamel protein 23 with the purified technology of protein purifying people of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The people becomes the purity of enamel protein 23 polypeptide can use amino acid sequence analysis.

The invention provides a kind of polypeptide-human and become enamel protein 23, it is basically by＜210〉aminoacid sequence shown in 2 forms.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.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention comprises that also the people becomes fragment, derivative and the analogue of enamel protein 23.As used herein, term " fragment ", " derivative " and " analogue " are meant and keep people of the present invention to become enamel protein 23 identical biological function or active polypeptide basically.The fragment of polypeptide of the present invention, derivative or analogue can be: (I) a kind of like this, wherein one or more amino-acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the amino acid that replaces can be also can not encoded by genetic codon; Perhaps (II) is a kind of like this, and certain group on wherein one or more amino-acid residues is replaced by other group and comprises substituting group; Perhaps (III) is a kind of like this, and wherein mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; Perhaps (IV) is a kind of like this, wherein additional aminoacid sequence is integrated into mature polypeptide and the peptide sequence that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying) by the elaboration of this paper, such fragment, derivative and analogue are considered within those skilled in the art's ken.

The invention provides isolating nucleic acid (polynucleotide), substantially by coding have＜polynucleotide of the polypeptide of 210〉2 aminoacid sequences form.Polynucleotide sequence of the present invention comprises＜210〉1 nucleotide sequence.Polynucleotide of the present invention are to find from the cDNA library of people's fetal brain tissue.The polynucleotide sequence total length that it comprises is 834 bases, its open reading frame (6-629) 207 amino acid of having encoded.Find relatively that according to amino acid sequence homologous this polypeptide becomes enamel protein that 78% homology is arranged with mouse, deducibility goes out this people and becomes enamel protein 23 to have the similar 26S Proteasome Structure and Function of mouse one-tenth enamel protein.

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＜210〉1 or the varient of degeneracy.As used herein, " varient of degeneracy " are meant that in the present invention coding has＜210〉2 protein or polypeptide, but with the differentiated nucleotide sequence of coding region sequence shown in＜210〉1.

The polynucleotide of the mature polypeptide of coding＜210〉2 comprise: the encoding sequence that has only 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 " is meant polynucleotide that comprise this polypeptide of encoding and the polynucleotide that comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of foregoing description polynucleotide, its coding has the polypeptide of identical aminoacid sequence or segment, 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 the polynucleotide (have at least 50% between two sequences, preferably have 70% homogeny) of sequence hybridization described above.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 adds and to use 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 95%, be more preferably 97% and just hybridize when above.And, the polypeptide of interfertile polynucleotide encoding and＜210〉and the mature polypeptide shown in 2 has identical biological function and activity.

The invention still further relates to nucleic acid fragment with sequence hybridization described above.As used herein, " nucleic acid fragment " length contain 10 Nucleotide at least, better be 20-30 Nucleotide at least, be more preferably 50-60 Nucleotide at least, preferably more than at least 100 Nucleotide.The amplification technique (as PCR) that nucleic acid fragment also can be used for nucleic acid is to determine and/or to separate the polynucleotide that the coding people becomes enamel protein 23.

Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.

Coding people of the present invention becomes the special polynucleotide sequence of enamel protein 23 to obtain with several different methods.For example, separate polynucleotide with hybridization technique well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect homologous polynucleotide sequence and 2) antibody screening of expression library to be to detect the polynucleotide passage of the clone with common structure feature.

Sequence dna fragment of the present invention also can obtain with following method: 1) separate double chain DNA sequence from genomic dna; 2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of described polypeptide.

In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.The direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.The more frequent method of selecting for use is the separation of cDNA sequence.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qiagcnc) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., MolccularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontcch company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.

Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) measure the level that the people becomes the transcript of enamel protein 23; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.

In (1) kind method, hybridizing used probe is and any a part of homology of polynucleotide of the present invention that at least 10 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, within 2000 Nucleotide, preferable is within 1000 Nucleotide to the length of probe usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene order information of the present invention normally.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

In (4) kind method, detect the people and become the protein product of enamel protein 23 genetic expressions can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

Use method (Saiki, the et al.Science1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to from the library, obtain the cDNA of total length, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to polynucleotide 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 gene of the present invention that obtains as mentioned above, perhaps the polynucleotide sequence of various dna fragmentations etc. can with ordinary method such as dideoxy chain termination (Sangcr et al.PNAS, 1977,74:5463-5467) measure.This class polynucleotide sequence is measured also available commercial sequencing kit etc.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and with carrier of the present invention or directly choose into the host cell that enamel protein 23 encoding sequences produce through genetically engineered, and the method that produces polypeptide of the present invention through recombinant technology.

Among the present invention, the coding people becomes the polynucleotide sequence of enamel protein 23 can be inserted in the carrier, contains the recombinant vectors of polynucleotide of the present invention with formation.Term " carrier " 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 carrier.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on the T7 promotor of in bacterium, expressing (Roscnberg, 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 may be used to make up recombinant expression vector.A key character of expression vector is to contain replication origin, promotor, marker gene and translational control element usually.

Method well-known to those having ordinary skill in the art can be used to make up contain the coding people become the dna sequence dna of enamel protein 23 and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, a LaboratoryManual, cold Spring 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; The P of lambda particles phage _LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte 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 that translation initiation is used and transcription terminator etc.Inserting enhancer sequence in carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis acting factor that DNA expresses, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.

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 etc.

Persons skilled in the art all know how to select appropriate carriers/transcriptional regulatory element (as promotor, enhanser etc.) and selected marker.

Among the present invention, the recombinant vectors that the coding people becomes the polynucleotide of enamel protein 23 or contains these polynucleotide can transform or transduce into host cell, contains the genetically engineered host cell of these polynucleotide or recombinant vectors with formation.Term " host cell " refers to 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; Bacterial cell such as Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; Insect cell such as fruit bat S2 or Sf9; Zooblast such as CHO, COS or Bowcs melanoma cells etc.

Can carry out with routine techniques well known to those skilled in the art with dna sequence dna of the present invention or the recombinant vectors transformed host cell that contains described dna sequence dna.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.Alternative 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, perhaps conventional mechanical method such as microinjection, electroporation, liposome packing etc.

By the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention to can be used to express or the people that produces reorganization becomes enamel protein 23 (Science, 1984; 224:1431).In general following steps are arranged:

(1). everybody becomes the polynucleotide (or varient) of enamel protein 23 with coding of the present invention, or transforms or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

(2). in suitable medium, cultivate host cell;

(3). separation, protein purification from substratum or cell.

In step (2), 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.

In step (3), recombinant polypeptide can wrap and be expressed or be secreted into the extracellular 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.These methods include, but are not limited to: conventional renaturation is handled, protein precipitant is handled (salt analysis method), centrifugal, the broken bacterium of infiltration, the combination of ultrasonication, 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 antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in disease treatment, for example, can treat various dental disorders, become flexible as toothache, decayed tooth, tooth acid, gum etc.

Mammiferous tooth structure is very special, and it is flocked together to grow by mineralized tissues such as enamel, dentine and dental cement and forms.The mineralising of dental tissue has important effect for the resistance to corrosion that strengthens tooth.Becoming enamel protein promptly is a kind of albumen of specifically expressing in the dental tissue, and it plays an important role in the enamel mineralisation process.The enamel tissue is a layer tissue of the strong erosion resistance of vertebrates tooth surface tool, and when tooth was subjected to external erosion, what hurt at first was exactly enamel, therefore, become enamel protein high expression level when tooth suffers erosion usually, to strengthen self resistance to corrosion of tooth, opposing is extraneous corrodes.

Specifically with regard to people of the present invention became enamel protein 23, this albumen and fragment thereof or derivative can be used to treat some because of the caused dental disorder of its abnormal expression.These diseases include but not limited to following these, carious tooth, pulpitis, periapical disease, periodontitis, swelling and aching of gum etc.

The present invention also provides SCREENED COMPOUND to improve (agonist) or check the method that (antagonist) people becomes the medicament of enamel protein 23 to identify.Agonist improves the people and becomes enamel protein 23 biological function such as stimulate cellular proliferation, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can be in the presence of medicine, the film preparation that mammalian cell or expressing human is become enamel protein 23 becomes enamel protein 23 to cultivate with the people of mark.Measure the medicine raising then or check this interactional ability.

Antibody, compound, acceptor that the people becomes the antagonist of enamel protein 23 to comprise and filters out lack thing and analogue etc.The people become the antagonist of enamel protein 23 can become with the people enamel protein 23 in conjunction with and eliminate its function, or suppress the generation of this polypeptide, or combine with the avtive spot of this polypeptide and to make this polypeptide can not bring into play biological function.

In screening during as the compound of antagonist, the people can be become enamel protein 23 to add during bioanalysiss measure, become between enamel protein 23 and its acceptor interactional influence to determine whether compound is antagonist by measuring compound to the people.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.Can become enamel protein 23 bonded peptide molecules to be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the people obtains.During screening, generally tackle the people and become enamel protein 23 molecules to carry out mark.

The invention provides and use polypeptide, and fragment, derivative, analogue or their cell are as the method for antigen with production antibody.These antibody can be polyclonal antibody or monoclonal antibody.The present invention also provides the antibody that becomes enamel protein 23 antigenic determinants at the people.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.

The method that the production of polyclonal antibody can be chosen into enamel protein 23 direct injection immune animals (as rabbit, mouse, rat etc.) obtains, and multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.The preparation people become the technology of the monoclonal antibody of enamel protein 23 include but not limited to hybridoma technology (Kohler andMilstein. Nature, 1975,256:495-497), three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison et al, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.Pat No.4946778) also can be used for producing the single-chain antibody that anti-people becomes enamel protein 23.

Anti-people becomes the antibody of enamel protein 23 to can be used in the immunohistochemistry technology, and the people who detects in the biopsy specimen becomes enamel protein 23.

Become the also available labelled with radioisotope of enamel protein 23 bonded monoclonal antibodies with the people, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.

Antibody also can be used for designing the immunotoxin at a certain privileged sites in the body.As the people become enamel protein 23 high-affinities monoclonal antibody can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the people and becomes enamel protein 23 positive cells.

Antibody among the present invention can be used for treating or prevention and people become the relevant disease of enamel protein 23.The antibody that gives suitable dosage can stimulate or block generation or the activity that the people becomes enamel protein 23.

The invention still further relates to the diagnostic testing process that quantitatively becomes enamel protein 23 levels with the detection and localization people.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people who is detected in the test becomes enamel protein 23 levels, can become the importance of enamel protein 23 in various diseases and the disease that is used to diagnose the people to become enamel protein 23 to work with the people that lays down a definition.

Polypeptide of the present invention also can be used as the peptide spectrum analysis, for example, the polypeptide available physical, chemistry or enzyme carry out the specificity cutting, and carries out the two-dimentional or three-dimensional gel electrophoresis analysis of one dimension, be more preferably and carry out mass spectroscopy.

The coding people becomes the polynucleotide of enamel protein 23 also to can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because the people becomes the nothing expression of enamel protein 23 or cell proliferation, growth or the metabolic disturbance due to unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the people who expresses variation and become enamel protein 23, becomes enamel protein 23 activity to suppress endogenic people.For example, it can be that the people who shortens, lacked signal conduction function territory becomes enamel protein 23 that a kind of people of variation becomes enamel protein 23, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the disease that the people becomes enamel protein 23 expression or active caused by abnormal.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for becoming the polynucleotide of enamel protein 23 to be transferred in the cell coding people.Structure carries the coding people and becomes the method for recombinant viral vector of the polynucleotide of enamel protein 23 to be found in existing document (Sambrook, et al.).Reorganization coding people becomes the polynucleotide of enamel protein 23 to be packaged in the liposome to be transferred in the cell in addition.

Polynucleotide import 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.

The inhibition people becomes the oligonucleotide (comprising sense-rna and DNA) of enamel protein 23mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.

The coding people becomes the polynucleotide of enamel protein 23 to can be used for becoming with the people diagnosis of the relative disease of enamel protein 23.The coding people becomes the polynucleotide of enamel protein 23 to can be used for detecting the unconventionality expression that expression that the people becomes enamel protein 23 people whether or under morbid state becomes enamel protein 23.As the people that encodes becomes the dna sequence dna of enamel protein 23 to can be used for biopsy specimen is hybridized to judge that the people becomes the expression situation of enamel protein 23.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.Choosing into enamel protein 23 special primers carries out RNA-polymerase chain reaction (RT-PCR) amplification in vitro and also can detect the transcription product that the people becomes enamel protein 23.

Detecting the people becomes the sudden change of enamel protein 23 genes also to can be used for the disease of diagnosing the people to become enamel protein 23 to be correlated with.The people becomes the form of enamel protein 23 sudden change to comprise with the normal wild type people to become point mutation that enamel protein 23DNA sequence compares, transposition, disappearance, reorganization and other 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.

Sequence of the present invention identifies it also is valuable to karyomit(e).This sequence can be specifically at certain bar human chromosome particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Now, have only chromosomal marker thing seldom to can be used for the marker chromosomes position based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, its important the first step is positioned these dna sequence dnas on the karyomit(e) exactly.

In brief, prepare PCR primer (preferred 15-35bp), sequence can be positioned on the karyomit(e) according to cDNA.Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.

The PCR localization method of somatocyte hybrid cell is that DNA is navigated to concrete chromosomal quick method.Use Oligonucleolide primers of the present invention,, can utilize one group to realize inferior location from specific chromosomal fragment or a large amount of genomic clone by similar approach.Other the similar strategy that can be used for chromosomal localization comprises in situ hybridization, uses the karyomit(e) prescreen and the hybridization preliminary election of the airflow classification of mark, thereby makes up the special cDNA storehouse of karyomit(e).

The cDNA clone is carried out fluorescence in situ hybridization (FISH) with Metaphase Chromosome, can in a step, accurately carry out chromosomal localization.The summary of this technology is referring to Verma etc., Human Chromosomes:a Manualof Basic Techniques, Pergamon Press, New York (1988).

In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritancein Man (can by with the online acquisition of Johns Hopkins Univcrsity Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.

Then, need to measure ill and not cDNA between diseased individuals or genome sequence difference.If observe certain sudden change in some or all of diseased individuals, and this sudden change is not observed in any normal individual, then this sudden change may be the cause of disease of disease.More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as from the horizontal visible of karyomit(e) or use based on detectable disappearance of the PCR of cDNA sequence or transposition.Resolving power according to present physical mapping and assignment of genes gene mapping technology, being accurately positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (the supposing that 1 megabasse mapping resolving power and every 20kb are corresponding to a gene) between 50 to 500 potential Disease-causing genes.

Polypeptide of the present invention, polynucleotide and stand-in thereof, agonist, antagonist and inhibitor and suitable pharmaceutical carrier combination back can be used.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Composition comprises the polypeptide or the antagonist of safe and effective amount and carrier and the vehicle that does not influence effect of drugs.These compositions can be used as medicine and are used for disease treatment.

The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, can have by the given indicative prompting of government authorities of making, using or selling medicine or biological products, the government authorities that this prompting reflects production, uses or sells permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound.

Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.The people becomes enamel protein 23 to come administration with the amount that treats and/or prevents concrete indication effectively.The people who is applied to the patient becomes the amount of enamel protein 23 and dosage range will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

Description of drawings

Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.

Fig. 1 is that the inventor becomes enamel protein 23 to become the amino acid sequence homology comparison diagram of enamel protein with mouse.The top sequence is that the people becomes enamel protein 23, and the below sequence is that mouse becomes enamel protein.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+".

Fig. 2 becomes the polyacrylamide gel electrophoresis figure (SDS-PAGE) of enamel protein 23 for isolating people.23kDa is proteinic molecular weight.The arrow indication is isolated protein band.

Embodiment

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.Embodiment 1: the people becomes the clone of enamel protein 23

Extract the total RNA of people's tire brain with guanidinium isothiocyanate/phenol/chloroform single stage method.From total RNA, separate poly (A) mRNA with Quik mRNA Isolation Kit (Qiegene company product).2ug poly (A) mRNA forms cDNA through reverse transcription.CDNA fragment orientation is inserted on the multiple clone site of pBSK (+) carrier (Clontech company product) with Smart cDNA clone's test kit (available from Clontech), transforms DH5 α, bacterium forms the cDNA library.Measure all clones' 5 ' and 3 ' terminal sequence with Dyeterminate cycle reaction sequencing kit (Perkin-Elmer company product) and ABI 377 automatic sequencers (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genebank) measured are compared, found that the cDNA sequence of one of them clone 0999e02 is new DNA.By synthetic a series of primers the contained insertion cDNA fragment of this clone is carried out two-way mensuration.The result shows, the contained full-length cDNA of 0999e02 clone is 834bp (as＜210〉1 shown in), from 6bp to 629bp the open reading frame (ORF) of a 624bp arranged, the new protein of encoding (as＜210〉2 shown in).We are with this clone's called after pBS-0999e02, and the name of encoded protein matter becomes enamel protein 23 for the people.

Embodiment 2:cDNA clone's homology retrieval

People of the present invention is become the sequence and the encoded protein sequence thereof of enamel protein 23, with Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], carry out the homology retrieval at databases such as Genbank, Swissport.Becoming the highest gene of enamel protein 23 homologys with people of the present invention is that a kind of known mouse becomes enamel protein, and its encoded protein number is U35097 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 58%; Similarity is 72%.Embodiment 3: the gene that becomes enamel protein 23 with RT-PCR method clones coding people

Total RNA is a template with fetus brain cell, is that primer carries out the synthetic cDNA of reverse transcription reaction with oligo-dT, with behind the test kit purifying of Qiagene, carries out pcr amplification with following primer:

Primer1： 5’-CAGGAATGTTGTACGTGCCTTTT-3’ (<210>3)

Primer2： 5’-CAATTGACACATTTTATTTCAAC-3’ (<210>4)

Primer1 for to be positioned at＜the forward sequence that begins of 1bp of 210〉15 ' end;

Primer2 be＜210〉1 in 3 ' end reverse sequence.

The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/LTris-Cl, (pH8.5), 1.5mmol/L MgCl ₂, 200 μ mol/L dNTP, 10pmol primer, the Taq archaeal dna polymerase of 1U (Clontech company product).Go up by 25 cycles of following conditioned response at PE9600 type DNA thermal cycler (Perkin-Elmer company): 94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of β-actin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows the dna sequence dna and＜210 of PCR product〉1-834bp shown in 1 is identical.Embodiment 4:Northern blotting analyst becomes enamel protein 23 expression of gene:

Extract total RNA[Anal.Biochem 1987,162,156-159 with single stage method].This method comprises acid guanidine thiocyanate phenol-chloroform extracting.Promptly use 4M guanidinium isothiocyanate-25mM Trisodium Citrate, 0.2M sodium acetate (pH4.0) carries out homogenate to tissue, adds the phenol of 1 times of volume and the chloroform-primary isoamyl alcohol (49: 1) of 1/5 volume, and is centrifugal after mixing.The sucking-off aqueous phase layer adds Virahol (0.8 volume) and with the centrifugal RNA precipitation that obtains of mixture.With RNA precipitation 70% washing with alcohol that obtains, dry and soluble in water.With 20 μ g RNA, on 1.2% sepharose that contains 20mM 3-(N-morpholino) propanesulfonic acid (pH7.0)-5mM sodium acetate-1mM EDTA-2.2M formaldehyde, carry out electrophoresis.Be transferred on the nitrocellulose filter then.With α- ³²P dATP prepares by random priming ³²The dna probe of P-mark.Used dna probe is that the people of pcr amplification shown in Figure 1 becomes enamel protein 23 coding region sequences (6bp to 629bp).Probe (about 2 * 10 with the 32P-mark ⁶Cpm/ml) spend the night in 42 ℃ of hybridization in a solution with the nitrocellulose filter that has shifted RNA, this solution comprises 50% methane amide-25mM KH ₂PO ₄(pH7.4)-5 * SSC-5 * Denhardt ' s solution and 200 μ g/ml salmon sperm DNAs.After the hybridization, filter membrane is washed 30min in 55 ℃ in 1 * SSC-0.1%SDS.Then, analyze with quantitative with PhosphorImager.Embodiment 5: recombinant human becomes vivoexpression, separation and the purifying of enamel protein 23

According to＜210〉1 and coding region sequence shown in Figure 1, design a pair of specificity amplification primer, sequence is as follows:

Primer3：5’-CCCCATATGATGTTGTACGTGCCTTTTGGAGCA-3’(<210>5)

Primer4：5’-CCCGGATCCAAGTAGCTAATATCTTCGAGCTGT-3’(<210>6)

5 ' end of these two sections primers contains NdeI and BamHI restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, NdeI and BamHI restriction enzyme site are corresponding to expression vector plasmid pET-28b (+) (Novagen company product, Cat.No.69865.3) the selectivity restriction enzyme site on.With the pBS-0999e02 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0999e02 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantagepolymerase Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 60 ℃ of 30s, 68 ℃ of 2min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with NdeI and BamHI, reclaim big fragment respectively, and connect with the T4 ligase enzyme.Connect product and transform, after the dull and stereotyped overnight incubation of the LB that contains kantlex (final concentration 30 μ g/ml), use the colony polymerase chain reaction (PCR) method screening positive clone, and check order with the big enterobacterial DH5 of Calcium Chloride Method α.Select the correct positive colony of sequence (pET-0999e02) with Calcium Chloride Method with recombinant plasmid transformed e. coli bl21 (DE3) plySs (Novagen company product).In the LB liquid nutrient medium that contains kantlex (final concentration 30 μ g/ml), host bacterium BL21 (pET-0999e02) is cultured to logarithmic phase at 37 ℃, adds IPTG to final concentration 1mmol/L, continues to cultivate 5 hours.Centrifugal collection thalline, through the broken bacterium of ultrasonic wave, centrifugal collection supernatant, with carrying out chromatography with 6 Histidines (6His-Tag) bonded affinity column His.Bind Quick Cartridge (Novagen company product), the target protein people who has obtained purifying becomes enamel protein 23.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 23kDa place.This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end are identical with 15 amino-acid residues of N-end shown in＜210〉2 as a result.Embodiment 6 anti-people become enamel protein 23 production of antibodies

Synthetic following people becomes enamel protein 23 specific polypeptide with Peptide synthesizer (PE company product):

NH ₂-。Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, et al.Immunochemistry, 1969; 6:43.Add the complete Freund's adjuvant immunizing rabbit with the above-mentioned hemocyanin polypeptide complex of 4mg, add the incomplete Freund's adjuvant booster immunization once with the hemocyanin polypeptide complex again after 15 days.Employing is done the titre that ELISA measures antibody in the rabbit anteserum through the titer plate of 15 μ g/ml bovine serum albumin polypeptide complex bag quilts.From the rabbit anteserum of antibody positive, separate total IgG with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.Immuno-precipitation proof antibody purified can become enamel protein 23 combinations specifically with the people.

Sequence table＜110〉Bode Gene Development Co., Ltd., Shanghai＜120〉peptide species--the people becomes polynucleotide＜130 of enamel protein 23 and this peptide species of coding〉0999e02＜160〉6＜170〉PatentIn version 3.1＜210〉1＜211〉834＜212〉DNA＜213〉Homo sapiens＜220〉＜221〉CDS＜222, (6) .., (629)＜223〉＜400〉1cagga atg ttg tac gtg cct ttt gga gca aat caa ttg aat gcc cct gcc 50

Met?Leu?Tyr?Val?Pro?Phe?Gly?Ala?Asn?Gln?Leu?Asn?Ala?Pro?Ala

1 5 10 15aga?ctt?ggc?atc?atg?agt?tca?gaa?gaa?gtg?gca?ggc?ggg?aga?gaa?gac 98Arg?Leu?Gly?Ile?Met?Ser?Ser?Glu?Glu?Val?Ala?Gly?Gly?Arg?Glu?Asp

20 25 30cca?atg?gcc?tat?gga?gcc?atg?ttt?cca?gga?ttt?gga?ggc?atg?agg?ccc 146Pro?Met?Ala?Tyr?Gly?Ala?Met?Phe?Pro?Gly?Phe?Gly?Gly?Met?Arg?Pro

35 40 45ggc?ttt?gag?gga?atg?ccc?cac?aac?cca?gct?atg?ggc?ggt?gac?ttc?act 194Gly?Phe?Glu?Gly?Met?Pro?His?Asn?Pro?Ala?Met?Gly?Gly?Asp?Phe?Thr

50 55 60ctg?gaa?ttt?gac?tcc?cca?gtg?gct?gcc?acc?aaa?ggc?cct?gag?aac?gaa 242Leu?Glu?Phe?Asp?Ser?Pro?Val?Ala?Ala?Thr?Lys?Gly?Pro?Glu?Asn?Glu

65 70 75gaa?gga?ggt?gca?caa?ggc?tcc?cct?atg?ccg?gag?gcc?aac?cca?gac?aat 290Glu?Gly?Gly?Ala?Gln?Gly?Ser?Pro?Met?Pro?Glu?Ala?Asn?Pro?Asp?Asn80 85 90 95cta?gaa?aac?cca?gct?ttc?ctt?aca?gag?cta?gaa?cct?gct?ccc?cac?gca 338Leu?Glu?Asn?Pro?Ala?Phe?Leu?Thr?Glu?Leu?Glu?Pro?Ala?Pro?His?Ala

100 105 110ggg?ctc?ctt?gct?ctc?cct?aag?gat?gac?att?ccc?ggc?ctg?cca?agg?agc 386Gly?Leu?Leu?Ala?Leu?Pro?Lys?Asp?Asp?Ile?Pro?Gly?Leu?Pro?Arg?Ser

115 120 125cct?tca?ggg?aag?atg?aag?gga?ctc?ccc?agc?gtc?acc?cca?gca?gct?gct 434Pro?Ser?Gly?Lys?Met?Lys?Gly?Leu?Pro?Ser?Val?Thr?Pro?Ala?Ala?Ala

130 135 140gac?cca?ctg?atg?acc?cct?gaa?tta?gct?gat?gtt?tat?agg?acc?tac?gat 482Asp?Pro?Leu?Met?Thr?Pro?Glu?Leu?Ala?Asp?Val?Tyr?Arg?Thr?Tyr?Asp

145 150 155gct?gac?atg?acc?aca?tcc?gtg?gat?ttc?cag?gaa?gaa?gca?acc?atg?gat 530Ala?Asp?Met?Thr?Thr?Ser?Val?Asp?Phe?Gln?Glu?Glu?Ala?Thr?Met?Asp160 165 170 175acc?acg?atg?gcc?cca?aac?tct?ctg?caa?aca?tcc?atg?cca?gga?aac?aaa 578Thr?Thr?Met?Ala?Pro?Asn?Ser?Leu?Gln?Thr?Ser?Met?Pro?Gly?Asn?Lys

180 185 190gcc?cag?gag?ccc?gag?atg?atg?cat?gac?gca?tgg?cat?ttc?caa?gag?ccc 626Ala?Gln?Glu?Pro?Glu?Met?Met?His?Asp?Ala?Trp?His?Phe?Gln?Glu?Pro

195 200 205tga?cagctctaag?atattagcta?ctttctgtat?gcacaagctt?cccagctttg 679tccccacagt?gtaccttttt?gctaaaacac?ttattaccct?tctgcagcaa?aggcattaaa 739agcgctaagc?atatattaat?aaatgcaagt?ggctagaaat?agtgtaggtc?cccttcttgc 799tttcaatatc ttgttgaaat aaaatgtgtc aattg834<210> 2<211> 207<212> PRT<213> Homo?sapiens<400> 2Met?Leu?Tyr?Val?Pro?Phe?Gly?Ala?Asn?Gln?Leu?Asn?Ala?Pro?Ala?Arg1 5 10 15Leu?Gly?Ile?Met?Ser?Ser?Glu?Glu?Val?Ala?Gly?Gly?Arg?Glu?Asp?Pro

20 25 30Met?Ala?Tyr?Gly?Ala?Met?Phe?Pro?Gly?Phe?Gly?Gly?Met?Arg?Pro?Gly

35 40 45Phe?Glu?Gly?Met?Pro?His?Asn?Pro?Ala?Met?Gly?Gly?Asp?Phe?Thr?Leu

50 55 60Glu?Phe?Asp?Ser?Pro?Val?Ala?Ala?Thr?Lys?Gly?Pro?Glu?Asn?Glu?Glu65 70 75 80Gly?Gly?Ala?Gln?Gly?Ser?Pro?Met?Pro?Glu?Ala?Asn?Pro?Asp?Asn?Leu

85 90 95Glu?Asn?Pro?Ala?Phe?Leu?Thr?Glu?Leu?Glu?Pro?Ala?Pro?His?Ala?Gly

100 105 110Leu?Leu?Ala?Leu?Pro?Lys?Asp?Asp?Ile?Pro?Gly?Leu?Pro?Arg?Ser?Pro

115 120 125Ser?Gly?Lys?Met?Lys?Gly?Leu?Pro?Ser?Val?Thr?Pro?Ala?Ala?Ala?Asp

130 135 140Pro?Leu?Met?Thr?Pro?Glu?Leu?Ala?Asp?Val?Tyr?Arg?Thr?Tyr?Asp?Ala145 150 155 160Asp?Met?Thr?Thr?Ser?Val?Asp?Phe?Gln?Glu?Glu?Ala?Thr?Met?Asp?Thr

165 170 175Thr?Met?Ala?Pro?Asn?Ser?Leu?Gln?Thr?Ser?Met?Pro?Gly?Asn?Lys?Ala

180 185 190Gln?Glu?Pro?Glu?Met?Met?His?Asp?Ala?Trp?His?Phe?Gln?Glu?Pro

195 200 205<210> 3<211> 23<212> DNA<213> Homo?sapiens<400> 3caggaatgtt gtacgtgcct ttt23<210> 4<211> 23<212> DNA<213> Homo?sapiens<400> 4caattgacac attttatttc aac23<210> 5<211> 33<212> DNA<213> Homo?sapiens<400> 5ccccatatga tgttgtacgt gccttttgga gca33<210> 6<211> 33<212> DNA<213> Homo?sapiens<400> 6cccggatcca agtagctaat atcttcgagc tgt33

Claims (18)

1, a kind of isolated polypeptide-people becomes enamel protein 23, it is characterized in that it includes: the polypeptide of the aminoacid sequence shown in＜210〉2 or the active fragments of its polypeptide, analogue or derivative.

2, polypeptide as claimed in claim 1, the aminoacid sequence that it is characterized in that described polypeptide, analogue or derivative has the homogeny with the aminoacid sequence at least 95% shown in＜210〉2.

3, polypeptide as claimed in claim 2 is characterized in that it comprises and has＜polypeptide of the aminoacid sequence shown in 210〉2.

4, a kind of isolating polynucleotide, it is characterized in that described polynucleotide comprise be selected from down the group in a kind of:

(a) coding have＜210〉2 shown in the polypeptide of aminoacid sequence or its fragment, analogue, derivative many

Nucleotide;

(b) with polynucleotide (a) complementary polynucleotide; Or

(c) with (a) or the polynucleotide of at least 70% homogeny (b) are arranged.

5, polynucleotide as claimed in claim 4, it is characterized in that described polynucleotide comprise coding and have＜210〉2 shown in the polynucleotide of aminoacid sequence.

6, polynucleotide as claimed in claim 4, it is characterized in that the sequence of described polynucleotide includes＜210〉1 in the 6-629 position sequence or＜210〉1 in the sequence of 1-834 position.

7, a kind of recombinant vectors that contains exogenous polynucleotide is characterized in that it is the recombinant vectors that is formed by the described polynucleotide of arbitrary claim among the claim 4-6 and plasmid, virus or vehicle expression vector establishment.

8, a kind of genetically engineered host cell that contains exogenous polynucleotide is characterized in that it is to be selected from following a kind of host cell:

(a) host cell that transforms or transduce with the described recombinant vectors of claim 7; Or

(b) host cell that transforms or transduce with the described polynucleotide of the arbitrary claim among the claim 4-6.

9, a kind of people of having becomes the preparation method of enamel protein 23 active polypeptide, it is characterized in that described method comprises:

(a) become under enamel protein 23 conditions in expressing human, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate and have the people and become enamel protein 23 active polypeptide.

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody is to become enamel protein 23 specificity bonded antibody with the people.

11, the compound of an analoglike or adjusting polypeptide active or expression is characterized in that they are simulation, promotion, antagonism or suppress the active compound that the people becomes enamel protein 23.

12, compound as claimed in claim 11 is characterized in that it is＜polynucleotide sequence or its segmental antisense sequences shown in 210〉1.

13, the described application of compound of a kind of claim 11, it is characterized in that described compound be used for the mediator become enamel protein 23 in vivo, the method for external activity.

14, a kind of disease relevant or method of disease susceptibility of detecting with the described polypeptide of arbitrary claim among the claim 1-3, it is characterized in that it comprises the described polypeptide expression amount that detects, perhaps detect the activity of described polypeptide, perhaps detect and cause described expression of polypeptides amount or active unusual nucleotide diversity in the polynucleotide.

15, as the application of polypeptide as described in the arbitrary claim among the claim 1-3, it is characterized in that it is applied to screen stand-in, the agonist that the people becomes enamel protein 23, antagonist or inhibitor; Perhaps be used for the peptide finger print identification.

16, as the application of the described nucleic acid molecule of arbitrary claim among the claim 4-6, it is characterized in that it is used for nucleic acid amplification reaction as primer, perhaps be used for hybridization, perhaps be used to make gene chip or microarray as probe.

17,, it is characterized in that forming the pharmaceutical composition that becomes the relevant unusually disease of enamel protein 23 as diagnosis or treatment with the people with safe and effective dosage and pharmaceutically acceptable carrier with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11.

18, the described polypeptide of arbitrary claim, polynucleotide or the application of compound among the claim 1-6 and 11 is characterized in that being used for the treatment of with described polypeptide, polynucleotide or compound the medicine of dental disorders such as toothache, tooth acid, gum be loosening.