CN1380337A - A polypeptide-Down syndrome cell adhesion molecule homoplastic protein 1 and polynucleotide for coding this polypeptide - Google Patents

Abstract

The present invention discloses a polypeptide-Down syndrome cell adhesion molecuale similar protein 1, polynucleotide for coding said polypeptide and method for producing this polypeptide by using DNA recombination technology. Said invention also discloses the method for curing several diseases, such as development disturbance diseases of embryo and nervous system, immune system diseases and tumours and cancers of related tissue by using said polypeptide. Said invention also discloses the antagonist for resisting this polypeptide and its therapeutic action, and also discloses the application of the polynucleotide coding this novel Down syndrome cell adhesion molecule similar protein 1.

Description

The polynucleotide of one peptide species---Down syndrome cell adhesion molecule homoplastic protein 1 and this peptide species of coding

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide---Down syndrome cell adhesion molecule homoplastic protein 1, 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.

Cell adhesion molecule with many bioprocesss in bringing into play very important effect, comprise hematopoiesis, immune response, fetal development, neurological tissue development etc.According to the similarity on the cell adhesion molecule 26S Proteasome Structure and Function, can be divided into three different types: caherin family, comprise N, P, R, B, E-caherin, calcium ion is dependent often for they; Integrin family, often by two subunits, α and β form; Immunoglobulin superfamily, this is a maximum subfamily, often has one or more typical structural domain----immunoglobulin like protein structural domain (Ig-like), form by 70-100 amino acid, and have two conserved cysteine residue, form stable disulfide linkage, they play a role does not need the participation of calcium ion.

Immunoglobulin superfamily comprises classical nerve cell adhesion molecule, NCAM, L1CAM, DCC etc.According to the quantity of FibronectinIII (FNIII) structural domain and the mode and the homology of protein molecular grappling cytolemma, immunoglobulin superfamily can be divided into a lot of subfamilies again.Contain 5 Ig-like structural domains and 2 FNIII structural domains as NCAM, closely related with schizoid generation; L1CAM has 6 Ig-like structural domains and 5 FNIII structural domains, and is relevant with the formation of memory.

Down's syndrome is the sacred disease on a kind of the growth.It becomes cause of disease because of mainly being No. 21 karyomit(e) triplings, promptly forms trisomy 21, causes some genetic expression distortion, have influence on nervous system development, and final one-tenth is sick.Studies show that 21q22.2-22.3 is that Down's syndrome reaches critical area.DSCAM (Down Syndrome CellAdhesion Molecule) is positioned at 21q22.2-22.3, contactin.In situ hybridization shows that the homogenic expression of its mouse mainly concentrates on the tire mouse central nervous system neural ridge of unifying.In view of the above, DSCAM may be closely related with neural growth differentiation, the defective that its overexpression may directly cause Down's syndrome maincenter and peripheral nervous system to be grown.According to another discovering, the homologous gene dscam in the fruit bat has interaction with neural location acceptor Dock, and the disappearance of dscam can cause fruit bat neuronal development location that obstacle takes place.Dscam also is a kind of location acceptor of neuronal development, and by its downstream effects element, Dock, Rho and cytoskeleton play a role, make neurocyte can be in time to external world the change of environment react.They all have 10 IgC2 and 6 FNIII structural domains, and the 10th IgC2 is positioned between the 4th, 5 the FNIII structural domain.

New DSCAML1 gene of the present invention and the DSCAM gene of known mouse and fruit bat do not have tangible homology on nucleotide level, but proteic protein sequence that it is coded and the DSCAM albumen of known mouse have 59% homogeny and 73% similarity, and have identical structural domain array configuration with it.Also contain 10 IgC2 structural domains and 6 Fibronectin III structural domains.Northern is hybridized demonstration, and it only has a tangible band in Adult Human Brain, and size is 7.5kb.And the abundance detection of using PCR to carry out 16 kinds of tissues shows that DSCAML1 only has faint expression in Adult Human Brain, and its expression amount exceeds hundreds of times than Adult Human Brain in the tire brain, and this explanation DSCAML1 is the relevant adhesion molecule of a nervous system development.This proteic sudden change or abnormal expression will cause the organism nervous system development unusual, and the generation of the tumour of embryo and nervous system development disorder disease, disease of immune system and related tissue and cancer etc. is closely related in itself and the organism.

Because Down syndrome cell adhesion molecule homoplastic protein 1 albumen plays an important role in body critical functions such as immune response, fetal development, neurological tissue development as mentioned above, and believe and relate to a large amount of albumen in these regulate processes, thereby need to identify the Down syndrome cell adhesion molecule homoplastic protein 1 albumen of more these processes of participation in this area always, particularly identify this proteic aminoacid sequence.The separation of new Down syndrome cell adhesion molecule homoplastic protein 1 protein coding gene also provides the foundation for determining the effect of this albumen under healthy and morbid state.This albumen may constitute the basis of exploitation medical diagnosis on disease and/or curative, and it is very important therefore separating its coding DNA.

An object of the present invention is to provide isolating new polypeptide---Down syndrome cell adhesion molecule homoplastic protein 1 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 the recombinant vectors of the polynucleotide that contain the Down syndrome cell adhesion molecule homoplastic protein 1 of encoding.

Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain the Down syndrome cell adhesion molecule homoplastic protein 1 of encoding.

Another object of the present invention provides the method for producing Down syndrome cell adhesion molecule homoplastic protein 1.

Another object of the present invention provides at polypeptide of the present invention---the antibody of Down syndrome cell adhesion molecule homoplastic protein 1.

Another object of the present invention has provided at polypeptide of the present invention---the simulated compound of Down syndrome cell adhesion molecule homoplastic protein 1, antagonist, agonist, inhibitor.

Another object of the present invention provides the method for the diagnoses and treatment disease relevant unusually with Down syndrome cell adhesion molecule homoplastic protein 1.

The present invention relates to a kind of isolated polypeptide, this polypeptide is the people source, and it comprises: polypeptide or its examples of conservative variations, bioactive fragment or derivative with SEQ ID No.2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

The invention still further relates to a kind of isolating polynucleotide, it comprises a kind of nucleotide sequence or its variant that is selected from down group:

(a) coding has the polynucleotide of the polypeptide of SEQ ID No.2 aminoacid sequence;

(b) with polynucleotide (a) complementary polynucleotide;

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

More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 13-6174 position among the SEQ ID NO:1; (b) has the sequence of 1-6729 position among the SEQ ID NO:1.

The present invention relates to a kind of carrier that contains polynucleotide of the present invention, particularly expression vector in addition; The host cell that this carrier of a kind of usefulness is genetically engineered comprises the host cell of conversion, transduction or transfection; A kind of method for preparing polypeptide of the present invention of cultivating described host cell and reclaiming expression product that comprises.

The invention still further relates to a kind of can with polypeptid specificity bonded antibody of the present invention.

The invention still further relates to a kind of simulation, activation, antagonism of screening or suppress the method for the compound of Down syndrome cell adhesion molecule homoplastic protein 1 protein-active, it comprises and utilizes polypeptide of the present invention.The invention still further relates to the compound that obtains with this method.

The invention still further relates to a kind of vitro detection and express the relevant disease or the method for disease susceptibility with the Down syndrome cell adhesion molecule homoplastic protein 1 abnormal protein, comprise the sudden change in polypeptide described in the detection of biological sample or its coded polynucleotide sequence, perhaps the amount or the biological activity of polypeptide of the present invention in the detection of biological sample.

The present invention also relates to a kind of pharmaceutical composition, it contains polypeptide of the present invention or its stand-in, activator, antagonist or inhibitor and pharmaceutically acceptable carrier.

The invention still further relates to polypeptide of the present invention and/or polynucleotide and be used for the treatment of cancer, nervous system development disorder disease or immunological disease or other purposes owing to the medicine of Down syndrome cell adhesion molecule homoplastic protein 1 disease that abnormal expression causes in preparation.

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

The following term of using in this specification sheets and claims has following implication unless stated otherwise:

" nucleotide sequence " is meant oligonucleotide, Nucleotide or polynucleotide and fragment or part, also can refer to genome or synthetic DNA or RNA, and they can be strand or two strands, represent sense strand or antisense strand.Similarly, term " aminoacid sequence " is meant oligopeptides, peptide, polypeptide or protein sequence and fragment or part.When " aminoacid sequence " among the present invention related to a kind of aminoacid sequence of naturally occurring protein molecule, this " polypeptide " or " protein " did not mean that aminoacid sequence are restricted to the complete natural amino acid relevant with described protein molecule.

Protein or polynucleotide " variant " are meant a kind of polynucleotide sequence that has the aminoacid sequence of one or more amino acid or Nucleotide change or encode it.Described change can comprise disappearance, insertion or the replacement of amino acid in aminoacid sequence or the nucleotide sequence or Nucleotide.Variant can have " conservative property " and change, and wherein the amino acid of Ti Huaning has structure or the chemical property similar with original acid, as replacing Isoleucine with leucine.Variant also can have non-conservation and change, as replacing glycine with tryptophane.

" disappearance " is meant the disappearance of in aminoacid sequence or nucleotide sequence one or more amino acid or Nucleotide.

" insertion " or " interpolation " is meant that the change in aminoacid sequence or nucleotide sequence causes comparing the increase of one or more amino acid or Nucleotide with naturally occurring molecule." replacement " is meant by different amino acid or Nucleotide and replaces one or more amino acid or Nucleotide.

" biological activity " is meant the protein of structure, regulation and control or biochemical function with natural molecule.Similarly, term " immunologic competence " be meant natural, reorganization or synthetic protein and fragment thereof in suitable animal or cell, induce specific immune response and with specific antibody bonded ability.

" agonist " is meant when combining with Down syndrome cell adhesion molecule homoplastic protein 1, thereby a kind of this protein that causes changes the molecule of regulating this protein active.Agonist can comprise protein, nucleic acid, carbohydrate or any other can be in conjunction with the molecule of Down syndrome cell adhesion molecule homoplastic protein 1.

" antagonist " or " inhibition " be meant when combining with Down syndrome cell adhesion molecule homoplastic protein 1, a kind ofly seals or regulate the biologic activity of Down syndrome cell adhesion molecule homoplastic protein 1 or the molecule of immunologic competence.Antagonist and inhibition can comprise protein, nucleic acid, carbohydrate or any other can be in conjunction with the molecule of Down syndrome cell adhesion molecule homoplastic protein 1.

" adjusting " is meant that the function of Down syndrome cell adhesion molecule homoplastic protein 1 changes, and comprises the change of any other biological property, function or the immune property of the change of the rising of protein active or reduction, binding characteristic and Down syndrome cell adhesion molecule homoplastic protein 1.

" pure basically " is meant and 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 Down syndrome cell adhesion molecule homoplastic protein 1 of standard.Basically pure Down syndrome cell adhesion molecule homoplastic protein 1 can produce single master tape on the irreducibility polyacrylamide gel.The purity available amino end acid sequence of Down syndrome cell adhesion molecule homoplastic protein 1 polypeptide is analyzed.

" complementary " or " complementation " is meant under salt concn that allows and temperature condition by the natural combination of the polynucleotide of base pairing.For example, sequence " C-T-G-A " can combine with complementary sequence " G-A-C-T ".Complementation between two single chain molecules can be part or whole.Complementary degree between the nucleic acid chains has a significant effect for efficient of hybridizing between the nucleic acid chains and intensity.

" homology " is meant the complementary degree, can be portion homologous or complete homology." portion homologous " is meant a kind of part complementary sequence, and it can partly suppress the hybridization of complete complementary sequence and target nucleic acid at least.The inhibition of this hybridization can detect by hybridizing (Southern trace or Northern trace etc.) under the condition that reduces in the severity degree.Basically homologous sequence or hybridization probe can compete and suppress complete homologous sequence and target sequence the condition that reduces of severity degree under combine.This does not mean the conditions permit non-specific binding that the severity degree reduces because the conditional request two sequences that the severity degree reduces mutual be combined into specificity or selectivity interacts.

" homogeny percentage " be meant two or more amino acid or nucleotide sequence relatively in the same or analogous percentage of sequence.The available electron method is measured the homogeny percentage, as passing through MEGALIGN program (Lasergenesoftware package, DNASTAR, Inc., Madison Wis.).The MEGALIGN program can compare two or more sequences (Higgins, D.G. and P.M.Sharp (1988) Gene 73:237-244) according to diverse ways such as Cluster method.The Cluster method is organized the series arrangement cluster by checking the distance between all pairings with each.Then with each bunch with in pairs or become set of dispense.Homogeny percentage between two aminoacid sequences such as sequence A and the sequence B calculates by following formula:

The residue number of mating between sequence A and the sequence B

×100

Interval residue number in the residue number-sequence B of interval in the residue number-sequence A of sequence A

Also can be by the Cluster method or with the homogeny percentage (Hein J., (1990) Methods in emzumology 183:625-645) between known method in this area such as the Jotun Hein mensuration nucleotide sequence.

" similarity " is meant the degree that the identical or conservative property of corresponding position amino-acid residue when arranging contrast between the aminoacid sequence replaces.For example be used for amino acid that conservative property replaces, electronegative amino acid can comprise aspartic acid and L-glutamic acid; Positively charged amino acid can comprise Methionin and arginine; Having uncharged head group has similar hydrophilic amino acid can comprise leucine, Isoleucine and Xie Ansuan; Glycine and L-Ala; L-asparagine and glutamine; Serine and Threonine; Phenylalanine and tyrosine.

" antisense " is meant and specific DNA or RNA sequence complementary nucleotide sequence." antisense strand " is meant and " sense strand " complementary nucleic acid chains.

" derivative " is meant HFP or encodes its chemical modification object of nucleic acid.This chemical modification object can be with alkyl, acyl group or the amino hydrogen atom of replacing.The nucleic acid derivative codified keeps the polypeptide of the main biological characteristics of natural molecule.

" antibody " is meant complete antibody molecule and fragment thereof, as Fa, F (ab ') ₂And Fv, its energy specificity is in conjunction with the antigenic determinant of Down syndrome cell adhesion molecule homoplastic protein 1.

" humanized antibody " is meant that the aminoacid sequence in non-antigen binding domain territory is replaced and becomes more similar to people's antibody, but still keep original in active antibody.

" isolating " speech refers to material is shifted out among its original environment (for example, if spontaneous its natural surroundings that just refers to).Such as it is exactly not to be separated that spontaneous polynucleotide or polypeptide are present in the Live Animals, but same polynucleotide or polypeptide with some or all in natural system with it the material of coexistence separately be exactly isolating.Such polynucleotide may be the parts of a certain carrier, the part that also possible such polynucleotide or polypeptide are a certain composition.Since carrier or composition are not the compositions of its natural surroundings, they remain isolating.

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 Down syndrome cell adhesion molecule homoplastic protein 1 " is meant that Down syndrome cell adhesion molecule homoplastic protein 1 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 Down syndrome cell adhesion molecule homoplastic protein 1 of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of Down syndrome cell adhesion molecule homoplastic protein 1 polypeptide can be used amino acid sequence analysis.

The invention provides a kind of new polypeptide---Down syndrome cell adhesion molecule homoplastic protein 1, it is made up of the aminoacid sequence shown in the SEQ ID NO:2 basically.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 also comprises fragment, derivative and the analogue of Down syndrome cell adhesion molecule homoplastic protein 1.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of Down syndrome cell adhesion molecule homoplastic protein 1 of the present invention 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 the polynucleotide that have a polypeptide of SEQ ID NO:2 aminoacid sequence by coding are formed.Polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO:1.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 6729 bases, its open reading frame 13-6174 2053 amino acid of having encoded.Find relatively that according to amino acid sequence homologous the DSCAM albumen of this polypeptide and mouse has 75% homology, deducibility goes out the 26S Proteasome Structure and Function that this Down syndrome cell adhesion molecule homoplastic protein 1 has the DSCAM protein similar of mouse.

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 " are meant that in the present invention coding has protein or the polypeptide of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The polynucleotide of the mature polypeptide of coding SEQ ID NO: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 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 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.Nucleic acid fragment also can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding Down syndrome cell adhesion molecule homoplastic protein 1.

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

The special polynucleotide sequence of coding Down syndrome cell adhesion molecule homoplastic protein 1 of the present invention can 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 (Qiagene) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., MolecularCloning, 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 Clontech 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) level of the transcript of mensuration Down syndrome cell adhesion molecule homoplastic protein 1; (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 protein product of Down syndrome cell adhesion molecule homoplastic protein 1 genetic expression and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

Use method (Saiki, the et al.Science 1985 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 (Sanger 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 with the host cell of Down syndrome cell adhesion molecule homoplastic protein 1 encoding sequence, and the method that produces polypeptide of the present invention through recombinant technology through the genetically engineered generation.

Among the present invention, the polynucleotide sequence of coding Down syndrome cell adhesion molecule homoplastic protein 1 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 (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 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 the dna sequence dna that contains the Down syndrome cell adhesion molecule homoplastic protein 1 of encoding and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.MolecularCloning, a Laboratory Manual, 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 polynucleotide of coding Down syndrome cell adhesion molecule homoplastic protein 1 or the recombinant vectors that contains these polynucleotide can transform or transduce into host cell, contain 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 Bowes 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 produce Down syndrome cell adhesion molecule homoplastic protein 1 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention people Down syndrome cell adhesion molecule homoplastic protein 1, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;

(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 the tumour of embryo and nervous system development disorder disease, disease of immune system and related tissue and cancer etc.

Cell adhesion molecule is being brought into play very important effect in many bioprocesss, comprise hematopoiesis, immune response, fetal development, neurological tissue development etc.According to the similarity on the cell adhesion molecule 26S Proteasome Structure and Function, can be divided into three different types: caherin family; Integrin family; Immunoglobulin superfamily, this is a maximum subfamily, often has one or more typical structural domain----immunoglobulin like protein structural domain (Ig-like).

Down's syndrome is the sacred disease on a kind of the growth, studies show that 21q22.2-22.3 is that Down's syndrome is expressed critical area.DSCAM is positioned at 21q22.2-22.3, contactin.There are some researches show that DSCAM may be closely related with neural growth differentiation, in vivo, the defective that its overexpression may directly cause Down's syndrome maincenter and peripheral nervous system to be grown.

The DSCAM albumen homology of polypeptide of the present invention and mouse and fruit bat contains the characteristic sequence of this protein family, and both have similar biological function, is the relevant adhesion molecule of nervous system development.This polypeptide unconventionality expression in vivo can cause that the organism nervous system development reaches the tumour and the cancer of related tissue unusually, and then causes relevant disease, and these diseases include but not limited to:

Neural growth disorder disease: dysrhaphia such as spina bifida, anencephalia, brain (meninx) bulging, cranium fissure, neurocele tumour; The brain development deformity is as hole deformity of brain, full forebrain, hydranencephaly; Neurone barrier to migration such as gyrus form unusually; Other deformity is as aqueduct deformity, cerebellar hypoplasia, Down ＇ s syndromes, spinal cord deformity, congenital hydrocephalus, congenital nuclei of cranial nerves underdevelopment syndromes.

Nervous system degenerative disease: senile dementia, Parkinson's disease, multiple sclerosis, chorea, dysthymia disorders, amnesia, Heng Yandun disease, epilepsy, migraine, dementia.

Neuromuscular disease: myasthenia gravis, spinal muscular atrophy, the false hypertrophy of flesh, Duchenne muscular dystrophy, myotonic dystrophy, musculartone disease, slow dyskinesia, myodystonia.

Psychotic disorder: schizophrenia, dysthymia disorders, paranoia, anxiety disorder, obsession, phobia, neural decline.

Peripheral nerve disease: trigeminal neuralgia, facioplegia, bulbar paralysis, sciatica, Green-barre syndrome.

Intracranial spaceoccupying lesion: neurogliocytoma, meningioma, neurofibroma, pituitary adenoma, intracranial granuloma.

Comprehensively above-mentioned, the antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in the diagnosis and treatment of multiple disease, for example cerebellar hypoplasia, Down ' s syndromes, chorea, schizophrenia, Green-barre syndrome and cerebral tumor etc.

The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) Down syndrome cell adhesion molecule homoplastic protein 1.Agonist improves Down syndrome cell adhesion molecule homoplastic protein 1 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 in the presence of medicine, the film preparation of mammalian cell or expression Down syndrome cell adhesion molecule homoplastic protein 1 be cultivated with the Down syndrome cell adhesion molecule homoplastic protein 1 of mark.Measure the medicine raising then or check this interactional ability.

The antagonist of Down syndrome cell adhesion molecule homoplastic protein 1 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of Down syndrome cell adhesion molecule homoplastic protein 1 can combine and eliminate its function with Down syndrome cell adhesion molecule homoplastic protein 1, 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, Down syndrome cell adhesion molecule homoplastic protein 1 can be added in the bioanalysis mensuration, interactional influence between Down syndrome cell adhesion molecule homoplastic protein 1 and its acceptor be determined whether compound is antagonist by measuring compound.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with Down syndrome cell adhesion molecule homoplastic protein 1 bonded peptide molecule obtains.During screening, generally tackle the Down syndrome cell adhesion molecule homoplastic protein 1 molecule and 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 at the Down syndrome cell adhesion molecule homoplastic protein 1 antigenic determinant.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 of the available Down syndrome cell adhesion molecule homoplastic protein 1 direct injection of the production of polyclonal antibody immune animal (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 technology of the monoclonal antibody of preparation Down syndrome cell adhesion molecule homoplastic protein 1 includes but not limited to hybridoma technology (Kohler and Milstein.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 etal, 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 of anti-Down syndrome cell adhesion molecule homoplastic protein 1.

The antibody of anti-Down syndrome cell adhesion molecule homoplastic protein 1 can be used in the immunohistochemistry technology, detects the Down syndrome cell adhesion molecule homoplastic protein 1 in the biopsy specimen.

With the also available labelled with radioisotope of Down syndrome cell adhesion molecule homoplastic protein 1 bonded monoclonal antibody, 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 monoclonal antibody of Down syndrome cell adhesion molecule homoplastic protein 1 high-affinity 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 Down syndrome cell adhesion molecule homoplastic protein 1 positive cells.

The disease that antibody among the present invention can be used for treating or prevention is relevant with Down syndrome cell adhesion molecule homoplastic protein 1.The antibody that gives suitable dosage can stimulate or block the generation or the activity of Down syndrome cell adhesion molecule homoplastic protein 1.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization Down syndrome cell adhesion molecule homoplastic protein 1 level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The Down syndrome cell adhesion molecule homoplastic protein 1 level that is detected in the test can be with laying down a definition the importance of Down syndrome cell adhesion molecule homoplastic protein 1 in various diseases and be used for the disease that the diagnosis of down syndrome cell adhesion molecule homoplastic protein 1 works.

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 polynucleotide of coding Down syndrome cell adhesion molecule homoplastic protein 1 also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the nothing expression of Down syndrome cell adhesion molecule homoplastic protein 1 or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the Down syndrome cell adhesion molecule homoplastic protein 1 of expressing variation, to suppress endogenic Down syndrome cell adhesion molecule homoplastic protein 1 activity.For example, a kind of Down syndrome cell adhesion molecule homoplastic protein 1 of variation can be the Down syndrome cell adhesion molecule homoplastic protein 1 that shortens, lacked signal conduction function territory, 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 of Down syndrome cell adhesion molecule homoplastic protein 1 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 the polynucleotide of coding Down syndrome cell adhesion molecule homoplastic protein 1 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding Down syndrome cell adhesion molecule homoplastic protein 1 is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding Down syndrome cell adhesion molecule homoplastic protein 1 can be packaged in the liposome and 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.

Suppress the oligonucleotide (comprising sense-rna and DNA) of Down syndrome cell adhesion molecule homoplastic protein 1 mRNA 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 polynucleotide of coding Down syndrome cell adhesion molecule homoplastic protein 1 can be used for the diagnosis with the relative disease of Down syndrome cell adhesion molecule homoplastic protein 1.The unconventionality expression of the expression that the polynucleotide of coding Down syndrome cell adhesion molecule homoplastic protein 1 can be used for detecting Down syndrome cell adhesion molecule homoplastic protein 1 Down syndrome cell adhesion molecule homoplastic protein 1 whether or under morbid state.As the dna sequence dna of the Down syndrome cell adhesion molecule homoplastic protein 1 of encoding can be used for biopsy specimen is hybridized to judge the expression situation of Down syndrome cell adhesion molecule homoplastic protein 1.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 the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect Down syndrome cell adhesion molecule homoplastic protein 1 with the special primer of Down syndrome cell adhesion molecule homoplastic protein 1.

The sudden change that detects the Down syndrome cell adhesion molecule homoplastic protein 1 gene also can be used for the relevant disease of diagnosis of down syndrome cell adhesion molecule homoplastic protein 1.The form of Down syndrome cell adhesion molecule homoplastic protein 1 sudden change comprises that the point mutation compared with normal wild type Down syndrome cell adhesion molecule homoplastic protein 1 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.

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, MendelianInheritance in Man (can by with the online acquisition of Johns Hopkins University 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.Down syndrome cell adhesion molecule homoplastic protein 1 comes administration with the amount that treats and/or prevents concrete indication effectively.The amount and the dosage range that are applied to patient's Down syndrome cell adhesion molecule homoplastic protein 1 will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

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 the amino acid sequence homology comparison diagram of the DSCAM of Down syndrome cell adhesion molecule homoplastic protein 1 of the present invention and mouse.The top sequence is a Down syndrome cell adhesion molecule homoplastic protein 1, and the below sequence is the DSCAM of mouse.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+".

Fig. 2 is the polyacrylamide gel electrophoresis figure (SDS-PAGE) of isolating Down syndrome cell adhesion molecule homoplastic protein 1.236.83kDa be proteinic molecular weight.The arrow indication is isolated protein band.

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:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Embodiment 1: the clone of Down syndrome cell adhesion molecule homoplastic protein 1

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 the sequence of all clones' 5 ＇ and 3 ＇ ends 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 DSCAML1 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 DSCAML1 clone is 6729bp (shown in Seq ID NO:1), from 13bp to 6174bp the open reading frame (ORF) of a 6162bp, the new protein (shown in Seq ID NO:2) of encoding arranged.We are with this clone's called after pBS-DSCAML1, encoded protein matter called after Down syndrome cell adhesion molecule homoplastic protein 1.Embodiment 2:cDNA clone's homology retrieval

With the sequence and the encoded protein sequence thereof of Down syndrome cell adhesion molecule homoplastic protein 1 of the present invention, 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.The gene the highest with Down syndrome cell adhesion molecule homoplastic protein 1 homology of the present invention is the DSCAM albumen of a kind of known mouse, and its encoded protein number is AF315558 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 59%; Similarity is 73%.Embodiment 3: with the gene of RT-PCR method clones coding Down syndrome cell adhesion molecule homoplastic protein 1

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’-CCTCTTTATGGCATGTGGCTGCTA-3’(SEQ?ID?NO：3)

Primer2：??5’-AGAAACATAACCTTTATTGCACAC-3’(SEQ?ID?NO：4)

Primer1 is the forward sequence that begins of 1bp that is positioned at the 5 ' end of SEQ ID NO:1;

Primer2 be SEQ ID NO:1 in 3 ' end reverse sequence.

The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/L Tris-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 that the dna sequence dna of PCR product and the 1-6729bp shown in the SEQ ID NO:1 are identical.Embodiment 4:Northern blotting is analyzed the Down syndrome cell adhesion molecule homoplastic protein 1 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 the Down syndrome cell adhesion molecule homoplastic protein 1 coding region sequence (13bp to 6174bp) of pcr amplification shown in Figure 1.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 Phosphor Imager.Embodiment 5: vivoexpression, separation and the purifying of reorganization Down syndrome cell adhesion molecule homoplastic protein 1

According to SEQ ID NO:1 and coding region sequence shown in Figure 1, design a pair of specificity amplification primer, sequence is as follows:

Primer3：5’-GGATCCATGATGTGGCTGCTAACTTTCCTCCTG-3’(Seq?ID?No：5)

Primer4：5’-CATGTCGACCTACACCAGGGTGTAGGATTTGGA-3’(Seq?ID?No：6)

5 ' end of these two sections primers contains BamHI and SalI restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, BamHI and SalI restriction enzyme site are corresponding to expression vector plasmid pQE31 (Qiagen company product, Cat.No.32915) the selectivity restriction enzyme site on.With the pQE-DSCAML1 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pQE-DSCAML1 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantage polymerase 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 pQE31 are carried out double digestion with BamHI and SalI, 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 penbritin (final concentration 100 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 (pQE-DSCAML1) with Calcium Chloride Method with recombinant plasmid transformed coli strain M15.In the LB liquid nutrient medium that contains penbritin (final concentration 100 μ g/ml) and kantlex (final concentration 25 μ g/ml), host bacterium M15 (pQE-DSCAML1) is cultured to logarithmic phase at 37 ℃, add IPTG to final concentration 1mmol/L, continue to cultivate 5 hours.Centrifugal collection thalline, through the broken bacterium of ultrasonic wave, centrifugal collection supernatant, with carrying out chromatography, obtained the target protein Down syndrome cell adhesion molecule homoplastic protein 1 of purifying with 6 Histidines (6His-Tag) bonded affinity column HisTrapAffinity Columns (Amersham-Pharmacia company product).Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 236.83kDa 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 hold 15 amino-acid residues identical with the N-shown in the SEQ ID NO:2 as a result.Embodiment 6 anti-Down syndrome cell adhesion molecule homoplastic protein 1 production of antibodies

Synthesize the specific polypeptide of following Down syndrome cell adhesion molecule homoplastic protein 1 with Peptide synthesizer (PE company product):

NH2-Met-Trp-Leu-Leu-Thr-Phe-Leu-Leu-Leu-Leu-Asp-Ser-Leu-His-Lys-OH(SEQ?ID?NO：7)。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 combine with Down syndrome cell adhesion molecule homoplastic protein 1 specifically.Embodiment 7: polynucleotide passage of the present invention is as the application of hybridization probe

Picking out suitable oligonucleotide fragment from polynucleotide of the present invention is of use in many ways as hybridization probe, as can whether containing polynucleotide sequence of the present invention and detect the homologous polynucleotide sequence to identify it with the healthy tissues of different sources or the genome or the hybridization of cDNA library of pathological tissue with this probe, whether further also available this probe in detecting polynucleotide sequence of the present invention or the expression of its homologous polynucleotide sequence in healthy tissues or pathological tissue cell be unusual.

The purpose of present embodiment is to pick out suitable oligonucleotide fragment as hybridization probe from polynucleotide SEQ ID NO:1 of the present invention, and identifies in some tissues whether contain polynucleotide sequence of the present invention or its homologous polynucleotide sequence with the filter hybridization method.The filter hybridization method comprises dot blotting, Southern blotting, Northern blotting and copy method etc., and they all are that polynucleotide sample to be measured is fixed on use essentially identical step crossover in back on the filter membrane.These identical steps are: the filter membrane of having fixed sample at first carries out prehybridization with the hybridization buffer that does not contain probe, so that nonspecific combining site suppressed by vector of sample and synthetic polymer institute are saturated on the filter membrane.Prehybridization solution is contained the hybridization buffer replacement of label probe then, and insulation makes probe and target nucleic acid hybridization.After the hybridization step, the probe in the hybridization is not removed by a series of film steps of washing.Present embodiment utilizes the film condition of washing (as than low salt concn and higher temperature) of higher-strength, so that hybrid context reduces and only keep the signal of high specificity.The probe that present embodiment is selected for use comprises two classes: first kind probe be fully with the identical or complementary oligonucleotide fragment of polynucleotide SEQ ID NO:1 of the present invention; The second class probe is part and the identical or complementary oligonucleotide fragment of polynucleotide SEQ ID NO:1 of the present invention.Present embodiment selects for use dot blotting that sample is fixed on the filter membrane, higher-strength wash under the film condition, the hybridization specificity of first kind probe and sample is the strongest and kept.One, probe selects for use

From polynucleotide SEQ ID NO:1 of the present invention, select oligonucleotide fragment as hybridization probe, the several aspects that should follow following principle and need to consider: 1, the probe size preferable range is a 18-50 Nucleotide; 2, GC content is 30%-70%, and surpassing then, non-specific hybridization increases; 3, probe interior should not have complementary region; 4, what meet above condition can be used as the primary election probe, further do the computer sequential analysis then, comprise this primary election probe is carried out homology relatively with its source sequence area (being SEQ ID NO:1) and other known genome sequence and complementary district thereof respectively, if with the homology in non-target molecule zone greater than 85% or there are 15 continuous bases of surpassing identical, then this primary election probe is general just should not use; 5, whether the primary election probe finally is chosen to be the probe of actual application value also should further be determined by experiment.

Select and synthetic following two probes after finishing the analysis of above each side:

Probe 1 (probe1) belongs to first kind probe, and complete homology of gene fragment or the complementation (41Nt) of SEQ ID NO:1:

5’-TGTGGCTGCTAACTTTCCTCCTGCTCCTGGACTCTTTACAC-3’(SEQ?ID?NO：8)

Probe 2 (probe2) belongs to the second class probe, is equivalent to gene fragment or its complementary segmental replacement mutant nucleotide sequence (41Nt) of SEQ ID NO:1:

5’-TGTGGCTGCTAACTTTCCTCTTGCTCCTGGACTCTTTACAC-3’(SEQ?ID?NO：9)

Other unlisted common agents and the compound method thereof relevant with following concrete experimental procedure please refer to document: DNA PROBES G.H.Keller; M.M.Manak; Stockton Press, 1989 (USA) and molecular cloning laboratory manual books more commonly used are as works such as " molecular cloning experiment guide " (second edition in 1998) [U.S.] Sa nurse Brooker, Science Press.

Specimen preparation: 1, from fresh or frozen tissue, extract DNA

Step: 1) the fresh or fresh normal liver tissue that thaws is put into the plate that is immersed on ice and fills phosphate buffered saline buffer (PBS).With scissors or scalpel tissue is cut into small pieces.Should keep organizing moistening in the operation.2) with 1000g centrifugal chopper tissue 10 minutes.3) with cooled homogenate damping fluid (0.25mol/L sucrose; 25mmol/LTris-HCl, pH7.5; 25mmol/LnaCl; 25mmol/L MgCl ₂) precipitation that suspends (approximately 10ml/g).4) 4 ℃ with electric homogenizer homogenate tissue suspension at full speed, until tissue by broken fully.5) 1000g is centrifugal 10 minutes.6) with re-suspended cell precipitation (the initial tissue sample of every 0.1g adds 1-5ml), centrifugal 10 minutes again with 1000g.7) with the resuspended precipitation of lysis buffer (the initial tissue sample of every 0.1g adds 1ml), connect following phenol extraction process then.2, the phenol extraction process of DNA

Step: 1) wash cell, centrifugal 10 minutes of 1000g with the cold PBS of 1-10ml.2) with the sedimentary cell (1 * 10 of cold cell pyrolysis liquid resuspension ⁸The minimum of applications 100ul lysis buffer of cell/ml).3) adding SDS is 1% to final concentration, if before re-suspended cell SDS is directly joined in the cell precipitation, cell may form big agglomerate and be difficult to fragmentation, and the overall yield that reduces.This point is especially severe when extracting＞107 cells.4) add Proteinase K to final concentration 200ug/ml.5) 50 ℃ of insulation reaction 1 hour or 37 ℃ gently jolting spend the night.6) use equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, in little centrifuge tube centrifugal 10 minutes.Two corresponding clear separation, otherwise carry out centrifugal again.7) water is transferred to new pipe.8) use the equal-volume chloroform: primary isoamyl alcohol (24: 1) extracting, centrifugal 10 minutes.9) water that will contain DNA is transferred to new pipe.Carry out purifying and the ethanol sedimentation of DNA then.3, the purifying of DNA and ethanol sedimentation

Step: 1) 1/10 volume 2mol/L sodium-acetate and 2 times of cold 100% ethanol of volume are added in the dna solution mixing.-20 ℃ of placements 1 hour or to spending the night.2) centrifugal 10 minutes.3) careful sucking-off or pour out ethanol.4) with 70% cold ethanol 500ul washing precipitation, centrifugal 5 minutes.5) careful sucking-off or pour out ethanol.With the cold washing with alcohol precipitation of 500ul, centrifugal 5 minutes.6) careful sucking-off or pour out ethanol is inverted on thieving paper then residual ethanol is flow to end.Dry air 10-15 minute, so that the volatilization of surperficial ethanol.Note not making the precipitation complete drying, otherwise dissolve again than difficulty.7) with small volume TE or the resuspended DNA precipitation of water.The low speed vortex vibrates or uses the dropper pressure-vaccum, increases TE gradually simultaneously, is mixed to DNA and fully dissolves, every 1-5 * 10 ⁶Cell extracted approximately adds 1ul.

Below the 8-13 step only be used for must removing when depolluting, otherwise can directly carry out the 14th step.8) RNA enzyme A is added in the dna solution, final concentration is 100ug/ml, and 37 ℃ are incubated 30 minutes.9) add SDS and Proteinase K, final concentration is respectively 0.5% and 100ug/ml.37 ℃ are incubated 30 minutes.10) use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extractive reaction liquid, centrifugal 10 minutes.11) carefully shift out water, use isopyknic chloroform: primary isoamyl alcohol (24: 1) extracting again, centrifugal 10 minutes.1 2) carefully shift out water, add 1/10 volume 2mol/L sodium-acetate and the cold ethanol of 2.5 volumes, mixing put-20 ℃ 1 hour.13) with 70% ethanol and 100% washing with alcohol precipitation, dry air, resuspended nucleic acid, process is with the 3-6 step.14) measure A ₂₆₀And A ₂₈₀To detect purity and the productive rate of DNA.15) deposit in-20 ℃ after the packing.The preparation of sample film:

1) get 4 * 2 suitably nitrocellulose filters (NC film) of size, mark point sample position and sample number thereon gently with pencil, each probe needs two NC films, so that wash film with high strength condition and strength condition respectively in the experimental procedure of back.

2) draw and contrast respectively 15 microlitres, put on the sample film, dry at room temperature.

3) place infiltration that 0.1mol/LNaOH is arranged, last 5 minute of filter paper (twice) of 1.5mol/LNaCl, drying to place to soak into has 0.5mol/L Tris-HCl (pH7.0), last 5 minute of filter paper (twice) of 3mol/LNaCl, dries.

4) be sandwiched in the clean filter paper, wrap, 60-80 ℃ of vacuum-drying 2 hours with aluminium foil.

The mark 1 of probe) 3 μ lProbe (0.1OD/10 μ l) add 2 μ lKinase damping fluids, 8-10uCi γ- ³²P-dATP+2UKinase is to add to final volume 20 μ l.2) 37 ℃ are incubated 2 hours.3) add the tetrabromophenol sulfonphthalein indicator (BPB) of 1/5 volume.4) cross Sephadex G-50 post.5) to having ³²Before washing out, P-Probe begins to collect first peak (available Monitor monitoring).6) 5/pipe, collect the 10-15 pipe.7) with liquid glimmer instrument monitoring isotopic weight 8) merge and be required preparation behind the collection liquid of first peak ³²P-Probe (second peak for free γ- ³²P-dATP).

Prehybridization

The sample film is placed plastics bag, and adding 3-10mg prehybridization solution (10 * Denhardt ' s; 6 * SSC, 0.1mg/mlCT DNA (calf thymus DNA).), seal sack after, 68 ℃ of water-baths were shaken 2 hours.

Hybridization

Plastics bag is cut off one jiao, adds the probe prepare, seal sack after, 42 ℃ of water-baths are shaken and are spent the night.

Wash film: high strength is washed film:

1) good sample film has been hybridized in taking-up.

2) 2 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃.

3) 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃.

4) 0.1 * SSC among the 0.1%SDS, washes 30 minutes (2 times) for 55 ℃, and room temperature is dried.Low strength is washed film:

1) good sample film has been hybridized in taking-up.

2) 2 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 37 ℃.

3) 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 37 ℃.

4) 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃, and room temperature is dried.

X-light autography:

-70 ℃, X-light autography (the compressing tablet time decides according to hybridization spot radioactivity is strong and weak).

Experimental result:

Adopt low strength to wash the hybrid experiment that the film condition is carried out, more than two strong and weak not obviously differences of probe hybridization spot radioactivity; And adopting high strength to wash the hybrid experiment that the film condition is carried out, the hybridization spot radioactive intensity of probe 1 obviously is better than the radioactive intensity of another probe hybridization spot.Thereby available probe 1 is qualitative and analyze existence and the differential expression of polynucleotide of the present invention in different tissues quantitatively.

Sequence table (1) general information: (ii) denomination of invention: Down syndrome cell adhesion molecule homoplastic protein 1 and encoding sequence thereof be the sequence number (iii): the information of 9 (2) SEQ ID NO:1: (i) sequence signature:

(A) length: 6729bp

(B) type: nucleic acid

(C) chain: two strands

(D) TOPOLOGY: linear (Ii) MOLECULE TYPE: cDNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 1 CCTCTTTATGGCATGTGGCTGCTAACTTTCCTCCTGCTCCTGGACTCTTTACACAAAGCC 61 CGCCCTGAAG ATGTTGGCACCAGCCTCTACTTTGTAAATGACTCCTTGCAGCAGGTGACC 121 TTTTCCAGCT CCGTGGGGGTGGTGGTGCCCTGCCCGGCCGCGGGCTCCCCCAGCGCGGCC 181 CTTCGATGGT ACCTGGCCACAGGGGACGACATCTACGACGTGCCGCACATCCGGCACGTC 241 CACGCCAACG GGACGCTGCAGCTCTACCCCTTCTCCCCCTCCGCCTTCAATAGCTTTATC 301 CACGACAATG ACTACTTCTGCACCGCGGAGAACGCTGCCGGCAAGATCCGGAGCCCCAAC 361 ATCCGCGTCA AAGCAGTTTTCAGGGAACCCTACACCGTCCGGGTGGAGGATCAAAGGTCA 421 ATGCGTGGCA ACGTGGCCGTCTTCAAGTGCCTCATCCCCTTTTTAGGGCAGGAATATGTT 481 AGCGTTGTAT CTTGGGAGAAAGACACAGTCTCCATCATCCCAGAACACAGGTTTTTTATT 541 ACCTACCACG GCGGGCTGTACATCTCTGACGTACAGAAGGAGGACGCCCTCTCCACCTAT 601 CGCTGCATCA CCAAGCACAAGTATAGCGGGGAGACCCGGCAGAGCAATGGGGCACGCCTC 661 TCTGTGACAG ACCCTGCTGAGTCGATCCCCACCATCCTGGATGGCTTCCACTCCCAGGAA 721 GTGTGGGCCG GCCACACCGTGGAGCTGCCCTGCACCGCCTCGGGCTACCCTATCCCCGCC 781 ATCCGCTGGC TCAAGGATGGCCGGCCCCTCCCGGCTGACAGCCGCTGGACCAAGCGCATC 841 ACAGGGCTGA CCATCAGCGACTTGCGGACCGAGGACAGCGGCACCTACATTTGTGAGGTC 901 ACCAACACCT TCGGTTCGGCAGAGGCCACAGGCATCCTCATGGTCATTGATCCCCTTCAT 961 GTGACCCTGA CACCAAAGAAGCTGAAGACCGGCATTGGCAGCACGGTCATCCTCTCCTGT 1021 GCCCTGACGGGCTCCCCAGAGTTCACCATCCGCTGGTATCGCAACACGGAGCTGGTGCTG 1081 CCTGACGAGGCCATCTCCATCCGCGGGCTCAGCAACGAGACGCTGCTCATCACCTCGGCC 1141 CAGAAGAGCCATTCCGGGGCCTACCAGTGCTTCGCTACCCGCAAGGCCCAGACCGCCCAG 1201 GACTTTGCCATCATTGCACTTGAGGATGGCACGCCCCGCATCGTCTCGTCCTTCAGCGAG 1261 AAGGTGGTCAACCCCGGGGAGCAGTTCTCACTGATGTGTGCGGCCAAGGGCGCCCCGCCC 1321 CCCACGGTCACCTGGGCCCTCGACGATGAGCCCATCGTGCGGGATGGCAGCCACCGCACC 1381 AACCAGTACACCATGTCGGACGGCACCACCATCAGCTACATGAACGTCACAGGCCCCCAG 1441 ATCCGCGACGGGGGCGTGTACCGGTGCACAGCGCGGAACTTGGTGGGCAGTGCTGAATAT 1501 CAGGCGCGAATAAACGTAAGAGGCCCACCCAGCATCCGGGCTATGCGGAACATCACAGCA 1561 GTCGCCGGGCGGGACACCCTTATCAACTGCAGGGTCATCGGCTATCCCTACTACTCCATC 1621 AAGTGGTACAAGGATGCCCTGCTGCTGCCAGACAACCACCGCCAGGTGGTGTTTGAGAAT 1681 GGGACCCTCAAGCTGACTGACGTGCAGAAGGGCATGGATGAGGGGGAGTACCTGTGCAGT 1741 GTCCTCATCCAGCCCCAGCTCTCCATCAGCCAGAGCGTTCACGTAGCCGTCAAAGTGCCC 1801 CCTCTGATCCAGCCCTTCGAATTCCCACCCGCCTCCATCGGCCAGCTGCTCTACATTCCC 1861 TGTGTGGTGTCCTCGGGGGACATGCCCATCCGTATCACCTGGAGGAAGGACGGACAGGTG 1921 ATCATCTCAGGCTCGGGCGTGACTATCGAGAGCAAGGAATTCATGAGCTCCCTGCAGATC 1981 TCTAGCGTCTCCCTCAAGCACAACGGCAACGATACATGCATCGCCAGCAACGCAGCCGCC 2041 ACCGTGAGCCGGGAGCGCCAGCTCATCGTGCGTGTGCCCCCTCGATTTGTGGTGCAACCC 2101 AACAACCAGGATGGCATCTACGGCAAAGCTGGTGTGCTCAACTGCTCGGTGGACGGCTAC 2161 CCCCCACCCAAGGTCATGTGGAAGCATGCCAAGGGGAGCGGGAACCCCCAGCAGTACCAC 2221 CCTGTGCCCCTCACTGGCCGCATCCAGATCCTGCCCAACAGCTCGCTGCTGATCCGCCAC 2281 GTCCTAGAAGAGGACATCGGCTACTACCTCTGCCAGGCCAGCAACGGCGTAGGCACCGAC 2341 ATCAGCAAGTCCATGTTCCTCACAGTCAAGATCCCGGCCATGATCACTTCCCACCCCAAC 2401 ACCACCATCGCCATCAAGGGCCATGCGAAGGAGCTAAACTGCACGGCACGGGGTGAGCGG 2461 CCCATCATCATCCGCTGGGAGAAGGGGGACACAGTCATCGACCCTGACCGCGTCATGCGG 2521 TATGCCATCGCCACCAAGGACAACGGCGACGAGGTCGTCTCCACACTGAAGCTCAAGCCC 2581 GCTGACCGTGGGGACTCTGTGTTCTTCAGCTGCCATGCCATCAACTCGTATGGGGAGGAC 2641 CGGGGCTTGATCCAACTCACTGTGCAAGAGCCCCCCGACCCCCCAGAGCTGGAGATCCGG 2701 GAGGTGAAGGCCCGGAGCATGAACCTGCGCTGGACCCAGCGATTCGACGGGAACAGCATC 2761 ATCACGGGCTTCGACATTGAATACAAGAACAAATCAGATTCCTGGGACTTCAAGCAGTCC 2821 ACACGCAACATCTCCCCCACCATCAACCAGGCCAACATTGTGGACTTGCACCCGGCATCT 2881 GTGTACAGCATCCGCATGTACTCTTTCAACAAGATTGGCCGCAGTGAACCAAGCAAGGAG 2941 CTCACCATCAGCACTGAGGAGGCCGCTCCCGATGGGCCCCCCATGGATGTTACCTTGCAG 3001 CCAGTGACCTCACAGAGCATCCAGGTGACCTGGAAGGCACCCAAGAAGGAGCTGCAGAAC 3061 GGTGTCATCCGGGGCTACCAGATTGGCTACAGAGAGAACAGCCCCGGCAGCAACGGGCAG 3121 TACAGCATCGTGGAGATGAAGGCCACGGGGGACAGCGAGGTCTACACCCTGGACAACCTC 3181 AAGAAGTTCGCCCAGTATGGGGTGGTGGTCCAAGCCTTCAATCGGGCTGGCACGGGGCCC 3241 TCTTCCAGCGAGATCAATGCCACCACTCTGGAGGATGTGCCCAGCCAGCCCCCTGAGAAC 3301 GTCCGGGCCCTGTCCATCACTTCTGACGTGGCCGTCATCTCCTGGTCAGAGCCCCCGCGC 3361 AGCACCCTCAATGGCGTCCTCAAAGGCTATCGGGTCATCTTCTGGTCCCTCTATGTTGAT 3421 GGGGAGTGGGGCGAGATGCAGAACATCACCACCACGCGGGAGCGGGTGGAGCTGCGGGGC 3481 ATGGAGAAGTTCACCAACTACAGCGTCCAGGTGCTGGCCTACACCCAGGCTGGGGACGGC 3541 GTACGCAGCAGTGTGCTCTACATCCAGACCAAGGAGGACGTTCCAGGTCCCCCTGCTGGC 3601 ATCAAAGCTGTCCCTTCATCAGCTAGCAGTGTGGTTGTGTCTTGGCTCCCCCCTACCAAG 3661 CCCAACGGGGTGATCCGCAAGTACACCATCTTCTGTTCCAGCCCCGGGTCTGGCCAGCCG 3721 GCTCCCAGCGAGTACGAGACGAGTCCAGAGCAGCTCTTCTACCGGATCGCCCACCTAAAC 3781 CGCGGTCAGCAGTATCTGCTGTGGGTGGCCGCCGTCACCTCTGCCGGCCGGGGCAACAGC 3841 AGCGAGAAGGTGACCATCGAGCCTGCTGGCAAGGCCCCAGCAAAGATCATCTCCTTTGGG 3901 GGCACCGTGACAACACCTTGGATGAAAGATGTTCGGCTGCCTTGCAATTCAGTGGGAGAT 3961 CCAGCCCCTGCTGTGAAGTGGACCAAGGACAGTGAAGACTCGGCCATTCCAGTGTCCATG 4021 GATGGGCACCGGCTCATCCACACCAATGGCACACTGCTGCTGCGTGCAGTGAAGGCTGAG 4081 GACTCTGGCTACTACACGTGCACGGCCACCAACACTGGTGGCTTTGACACCATCATCGTC 4141 AACCTTCTGGTGCAAGTTCCCCCGGACCAGCCCCGCCTCACTGTCTCCAAAACCTCAGCT 4201 TCGTCCATCACCCTGACCTGGATTCCAGGTGACAATGGGGGCAGCTCCATCCGAGGCTTC 4261 GTGCTACAGTACTCGGTGGACAACAGCGAGGAGTGGAAGGATGTGTTCATCAGCTCCAGC 4321 GAGCGCTCCTTCAAGCTGGACAGCCTCAAGTGTGGCACGTGGTACAAGGTGAAGCTGGCA 4381 GCCAAGAACAGCGTGGGCTCTGGGCGCATCAGCGAGATCATCGAGGCCAAGACCCACGGG 4441 CGGGAGCCCTCCTTCAGCAAAGACCAACACCTCTTCACCCACATCAACTCCACGCATGCT 4501 CGGCTTAACCTGCAGGGCTGGAACAATGGGGGCTGCCCTATCACAGCCATCGTTCTGGAG 4561 TACCGGCCCAAGGGGACCTGGGCCTGGCAGGGCCTCCGGGCCAACAGCTCCGGGGAGGTG 4621 TTTCTGACGGAACTGCGAGAGGCCACGTGGTACGAGCTGCGCATGAGGGCTTGCAACAGT 4681 GCGGGCTGCGGCAATGAAACAGCCCAGTTCGCCACCCTGGACTACGATGGCAGCACCATT 4741 CCACCCATCAAGTCTGCTCAAGGTGAAGGGGATGATGTGAAGAAGCTGTTCACCATCGGC 4801 TGCCCTGTCATCCTGGCCACACTGGGGGTGGCACTGCTCTTCATCGTACGCAAGAAGAGG 4861 AAGGAGAAACGGCTGAAGCGACTCCGAGATGCAAAGAGTTTGGCAGAAATGTTGATAAGC 4921 AAGAACAATAGAAGCTTTGACACCCCTGTGAAAGGGCCACCCCAGGGCCCACGGCTACAC 4981 ATTGACATCCCCAGGGTCCAGCTGCTCATCGAGGACAAAGAAGGCATCAAGCAACTGGGA 5041 GATGACAAGGCCACCATCCCTGTGACAGATGCTGAGTTCAGCCAAGCTGTCAACCCACAG 5101 AGCTTCTGTACTGGCGTCTCCTTGCACCACCCAACCCTCATCCAGAGCACAGGACCCCTC 5161 ATCGACATGTCTGACATCCGGCCAGGAACCAATCCAGTGTCCAGGAAGAATGTGAAGTCA 5221 GCCCACAGCACCCGGAACCGGTACTCAAGCCAGTGGACCCTGACCAAGTGCCAGGCCTCC 5281 ACACCTGCCCGCACCCTCACCTCCGACTGGCGCACCGTGGGCTCCCAGCATGGTGTCACG 5341 GTCACTGAGAGTGACAGCTACAGTGCCAGCCTGTCCCAGGACACAGACAAAGGAAGGAAC 5401 AGCATGGTGTCCACTGAGAGTGCCTCTTCCACCTACGAGGAGCTGGCCCGGGCCTATGAG 5461 CATGCCAAGCTGGAGGAGCAGCTGCAGCACGCCAAGTTTGAGATCACCGAGTGCTTCATC 5521 TCTGACAGTTCCTCTGACCAGATGACCACAGGCACCAACGAGAACGCCGACAGCATGACA 5581 TCCATGAGCACACCCTCAGAGCCTGGCATCTGCCGCTTTACCGCCTCACCACCCAAGCCC 5641 CAGGATGCGGACCGGGGCAAAAACGTGGCTGTGCCCATCCCTCACCGGGCCAACAAGAGT 5701 GACTACTGCAACCTGCCCCTGTATGCCAAGTCAGAGGCCTTCTTTCGAAAGGCAGATGGA 5761 CGTGAGCCCTGCCCCGTGGTCCCACCCCGTGAGGCCTCCATCCGGAACCTGGCTCGAACC 5821 TACCACACCCAGGCTCGCCACCTGACCCTGGACCCTGCCAGCAAGTCCTTGGGCCTTCCC 5881 CACCCAGGGGCCCCCGCTGCCGCCTCCACAGCCACCTTACCTCAGAGGACTCTGGCCATG 5941 CCAGCCCCCCCAGCCGGCACAGCCCCCCCAGCCCCCGGCCCCACCCCTGCTGAGCCACCC 6001 ACCGCCCCCAGCGCTGCCCCTCCGGCCCCCAGCACCGAGCCTCCACGAGCCGGGGGCCCA 6061 CACACCAAAATGGGGGGCTCCAGGGACTCGCTTCTCGAGATGAGCACATCGGGGGTAGGG 6121 AGGTCTCAGAAGCAGGGGGCCGGGGCCTACTCCAAATCCTACACCCTGGTGTAGGGCCGG 6181 CAGGAAGAGCAGCCACGCCTGGGCCGCGCCGCGCCGCAGCCCCACACGCCAGCTCGGCTG 6241 TTTTTCTGCATTATTTATATTCAACTGACAGACAAAAACCAACCAACGACAAAACAAAAA 6301 CCCCCAATCATGAACGCCTGTACATAGAACTCTTTTGTACAAATGAAACTATTTTCTTCT 6361 TCTCCATGAAGCCAGGGCACAAAGAATTTGACAGTACAAGTCAAATCCCCCACCCCACAA 6421 AATATGTGTGGAGATATATATACATATATAGACAGACAGGAACGCGTCCACGAGCTATAT 6481 ATCTATATATTTCTCTCACCCTATTTTGAGACAGAGGCACAAAGACTCAGCAATTTTTTT 6541 CCCTCCTCCTCACCTTCCCCCCAGTCTAGGTGGTTTTGACAAAGACCAAAATCCCAACTC 6601 AGAGACACTGCATGCGATTTTACTGTTCCAAGAAAACCAGGAGTTGCTTCAATTTGCAGA 6661 TGCTTATGTGTTAATACCTTTTTCTATGAAAAAAGACCCAGCGCCGTGTGCAATAAAGGT 6721 TATGTTTCT (3) SEQ ID N0: 2 Information: ...

(i) sequence signature:

(A) length: 2053 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 1 Met Trp Leu Leu Thr Phe Leu Leu Leu Leu Asp Ser Leu His Lys 16 Ala Arg Pro Glu Asp Val Gly Thr Ser Leu Tyr Phe Val Asn Asp 31 Ser Leu Gln Gln Val Thr Phe Ser Ser Ser Val Gly Val Val Val 46 Pro Cys Pro Ala Ala Gly Ser Pro Ser Ala Ala Leu Arg Trp Tyr 61 Leu Ala Thr Gly Asp Asp Ile Tyr Asp Val Pro His Ile Arg His 76 Val His Ala Asn Gly Thr Leu Gln Leu Tyr Pro Phe Ser Pro Ser 91 Ala Phe Asn Ser Phe Ile His Asp Asn Asp Tyr Phe Cys Thr Ala 106 Glu Asn Ala Ala Gly Lys Ile Arg Ser Pro Asn Ile Arg Val Lys 121 Ala Val Phe Arg Glu Pro Tyr Thr Val Arg Val Glu Asp Gln Arg 136 Ser Met Arg Gly Asn Val Ala Val Phe Lys Cys Leu Ile Pro Phe 151 Leu Gly Gln Glu Tyr Val Ser Val Val Ser Trp Glu Lys Asp Thr 166 Val Ser Ile Ile Pro Glu His Arg Phe Phe Ile Thr Tyr His Gly 181 Gly Leu Tyr Ile Ser Asp Val Gln Lys Glu Asp Ala Leu Ser Thr 196 Tyr Arg Cys Ile Thr Lys His Lys Tyr Ser Gly Glu Thr Arg Gln 211 Ser Asn Gly Ala Arg Leu Ser Val Thr Asp Pro Ala Glu Ser Ile 226 Pro Thr Ile Leu Asp Gly Phe His Ser Gln Glu Val Trp Ala Gly 241 His Thr Val Glu Leu Pro Cys Thr Ala Ser Gly Tyr Pro Ile Pro 256 Ala Ile Arg Trp Leu Lys Asp Gly Arg Pro Leu Pro Ala Asp Ser 271 Arg Trp Thr Lys Arg Ile Thr Gly Leu Thr Ile Ser Asp Leu Arg 286 Thr Glu Asp Ser Gly Thr Tyr Ile Cys Glu Val Thr Asn Thr Phe 301 Gly Ser Ala Glu Ala Thr Gly Ile Leu Met Val Ile Asp Pro Leu 316 His Val Thr Leu Thr Pro Lys Lys Leu Lys Thr Gly Ile Gly Ser 331 Thr Val Ile Leu Ser Cys Ala Leu Thr Gly Ser Pro Glu Phe Thr 346 Ile Arg Trp Tyr Arg Asn Thr Glu Leu Val Leu Pro Asp Glu Ala 361 Ile Ser Ile Arg Gly Leu Ser Asn Glu Thr Leu Leu Ile Thr Ser 376 Ala Gln Lys Ser His Ser Gly Ala Tyr Gln Cys Phe Ala Thr Arg 391 Lys Ala Gln Thr Ala Gln Asp Phe Ala Ile Ile Ala Leu Glu Asp 406 Gly Thr Pro Arg Ile Val Ser Ser Phe Ser Glu Lys Val Val Asn 421 Pro Gly Glu Gln Phe Ser Leu Met Cys Ala Ala Lys Gly Ala Pro 436 Pro Pro Thr Val Thr Trp Ala Leu Asp Asp Glu Pro Ile Val Arg 451 Asp Gly Ser His Arg Thr Asn Gln Tyr Thr Met Ser Asp Gly Thr 466 Thr Ile Ser Tyr Met Asn Val Thr Gly Pro Gln Ile Arg Asp Gly 481 Gly Val Tyr Arg Cys Thr Ala Arg Asn Leu Val Gly Ser Ala Glu 496 Tyr Gln Ala Arg Ile Asn Val Arg Gly Pro Pro Ser Ile Arg Ala 511 Met Arg Asn Ile Thr Ala Val Ala Gly Arg Asp Thr Leu Ile Asn 526 Cys Arg Val Ile Gly Tyr Pro Tyr Tyr Ser Ile Lys Trp Tyr Lys 541 Asp Ala Leu Leu Leu Pro Asp Asn His Arg Gln Val Val Phe Glu 556 Asn Gly Thr Leu Lys Leu Thr Asp Val Gln Lys Gly Met Asp Glu 571 Gly Glu Tyr Leu Cys Ser Val Leu Ile Gln Pro Gln Leu Ser Ile 586 Ser Gln Ser Val His Val Ala Val Lys Val Pro Pro Leu Ile Gln 601 Pro Phe Glu Phe Pro Pro Ala Ser Ile Gly Gln Leu Leu Tyr Ile 616 Pro Cys Val Val Ser Ser Gly Asp Met Pro Ile Arg Ile Thr Trp 631 Arg Lys Asp Gly Gln Val Ile Ile Ser Gly Ser Gly Val Thr Ile 646 Glu Ser Lys Glu Phe Met Ser Ser Leu Gln Ile Ser Ser Val Ser 661 Leu Lys His Asn Gly Asn Asp Thr Cys Ile Ala Ser Asn Ala Ala 676 Ala Thr Val Ser Arg Glu Arg Gln Leu Ile Val Arg Val Pro Pro 691 Arg Phe Val Val Gln Pro Asn Asn Gln Asp Gly Ile Tyr Gly Lys 706 Ala Gly Val Leu Asn Cys Ser Val Asp Gly Tyr Pro Pro Pro Lys 721 Val Met Trp Lys His Ala Lys Gly Ser Gly Asn Pro Gln Gln Tyr 736 His Pro Val Pro Leu Thr Gly Arg Ile Gln Ile Leu Pro Asn Ser 751 Ser Leu Leu Ile Arg His Val Leu Glu Glu Asp Ile Gly Tyr Tyr 766 Leu Cys Gln Ala Ser Asn Gly Val Gly Thr Asp Ile Ser Lys Ser 781 Met Phe Leu Thr Val Lys Ile Pro Ala Met Ile Thr Ser His Pro 796 Asn Thr Thr Ile Ala Ile Lys Gly His Ala Lys Glu Leu Asn Cys 811 Thr Ala Arg Gly Glu Arg Pro Ile Ile Ile Arg Trp Glu Lys Gly 826 Asp Thr Val Ile Asp Pro Asp Arg Val Met Arg Tyr Ala Ile Ala 841 Thr Lys Asp Asn Gly Asp Glu Val Val Ser Thr Leu Lys Leu Lys 856 Pro Ala Asp Arg Gly Asp Ser Val Phe Phe Ser Cys His Ala Ile 871 Asn Ser Tyr Gly Glu Asp Arg Gly Leu Ile Gln Leu Thr Val Gln 886 Glu Pro Pro Asp Pro Pro Glu Leu Glu Ile Arg Glu Val Lys Ala 901 Arg Ser Met Asn Leu Arg Trp Thr Gln Arg Phe Asp Gly Asn Ser 916 Ile Ile Thr Gly Phe Asp Ile Glu Tyr Lys Asn Lys Ser Asp Ser 931 Trp Asp Phe Lys Gln Ser Thr Arg Asn Ile Ser Pro Thr Ile Asn 946 Gln Ala Asn Ile Val Asp Leu His Pro Ala Ser Val Tyr Ser Ile 961 Arg Met Tyr Ser Phe Asn Lys Ile Gly Arg Ser Glu Pro Ser Lys 976 Glu Leu Thr Ile Ser Thr Glu Glu Ala Ala Pro Asp Gly Pro Pro 991 Met Asp Val Thr Leu Gln Pro Val Thr Ser Gln Ser Ile Gln Val 1006 Thr Trp Lys Ala Pro Lys Lys Glu Leu Gln Asn Gly Val Ile Arg 1021 Gly Tyr Gln Ile Gly Tyr Arg Glu Asn Ser Pro Gly Ser Asn Gly 1036 Gln Tyr Ser Ile Val Glu Met Lys Ala Thr Gly Asp Ser Glu Val 1051 Tyr Thr Leu Asp Asn Leu Lys Lys Phe Ala Gln Tyr Gly Val Val 1066 Val Gln Ala Phe Asn Arg Ala Gly Thr Gly Pro Ser Ser Ser Glu 1081 Ile Asn Ala Thr Thr Leu Glu Asp Val Pro Ser Gln Pro Pro Glu 1096 Asn Val Arg Ala Leu Ser Ile Thr Ser Asp Val Ala Val Ile Ser 1111 Trp Ser Glu Pro Pro Arg Ser Thr Leu Asn Gly Val Leu Lys Gly 1126 Tyr Arg Val Ile Phe Trp Ser Leu Tyr Val Asp Gly Glu Trp Gly 1141 Glu Met Gln Asn Ile Thr Thr Thr Arg Glu Arg Val Glu Leu Arg 1156 Gly Met Glu Lys Phe Thr Asn Tyr Ser Val Gln Val Leu Ala Tyr 1171 Thr Gln Ala Gly Asp Gly Val Arg Ser Ser Val Leu Tyr Ile Gln 1186 Thr Lys Glu Asp Val Pro Gly Pro Pro Ala Gly Ile Lys Ala Val 1201 Pro Ser Ser Ala Ser Ser Val Val Val Ser Trp Leu Pro Pro Thr 1216 Lys Pro Asn Gly Val Ile Arg Lys Tyr Thr Ile Phe Cys Ser Ser 1231 Pro Gly Ser Gly Gln Pro Ala Pro Ser Glu Tyr Glu Thr Ser Pro 1246 Glu Gln Leu Phe Tyr Arg Ile Ala His Leu Asn Arg Gly Gln Gln 1261 Tyr Leu Leu Trp Val Ala Ala Val Thr Ser Ala Gly Arg Gly Asn 1276 Ser Ser Glu Lys Val Thr Ile Glu Pro Ala Gly Lys Ala Pro Ala 1291 Lys Ile Ile Ser Phe Gly Gly Thr Val Thr Thr Pro Trp Met Lys 1306 Asp Val Arg Leu Pro Cys Asn Ser Val Gly Asp Pro Ala Pro Ala 1321 Val Lys Trp Thr Lys Asp Ser Glu Asp Ser Ala Ile Pro Val Ser 1336 Met Asp Gly His Arg Leu Ile His Thr Asn Gly Thr Leu Leu Leu 1351 Arg Ala Val Lys Ala Glu Asp Ser Gly Tyr Tyr Thr Cys Thr Ala 1366 Thr Asn Thr Gly Gly Phe Asp Thr Ile Ile Val Asn Leu Leu Val 1381 Gln Val Pro Pro Asp Gln Pro Arg Leu Thr Val Ser Lys Thr Ser 1396 Ala Ser Ser Ile Thr Leu Thr Trp Ile Pro Gly Asp Asn Gly Gly 1411 Ser Ser Ile Arg Gly Phe Val Leu Gln Tyr Ser Val Asp Asn Ser 1426 Glu Glu Trp Lys Asp Val Phe Ile Ser Ser Ser Glu Arg Ser Phe 1441 Lys Leu Asp Ser Leu Lys Cys Gly Thr Trp Tyr Lys Val Lys Leu 1456 Ala Ala Lys Asn Ser Val Gly Ser Gly Arg Ile Ser Glu Ile Ile 1471 Glu Ala Lys Thr His Gly Arg Glu Pro Ser Phe Ser Lys Asp Gln 1486 His Leu Phe Thr His Ile Asn Ser Thr His Ala Arg Leu Asn Leu 1501 Gln Gly Trp Asn Asn Gly Gly Cys Pro Ile Thr Ala Ile Val Leu 1516 Glu Tyr Arg Pro Lys Gly Thr Trp Ala Trp Gln Gly Leu Arg Ala 1531 Asn Ser Ser Gly Glu Val Phe Leu Thr Glu Leu Arg Glu Ala Thr 1546 Trp Tyr Glu Leu Arg Met Arg Ala Cys Asn Ser Ala Gly Cys Gly 1561 Asn Glu Thr Ala Gln Phe Ala Thr Leu Asp Tyr Asp Gly Ser Thr 1576 Ile Pro Pro Ile Lys Ser Ala Gln Gly Glu Gly Asp Asp Val Lys 1591 Lys Leu Phe Thr Ile Gly Cys Pro Val Ile Leu Ala Thr Leu Gly 1606 Val Ala Leu Leu Phe Ile Val Arg Lys Lys Arg Lys Glu Lys Arg 1621 Leu Lys Arg Leu Arg Asp Ala Lys Ser Leu Ala Glu Met Leu Ile 1636 Ser Lys Asn Asn Arg Ser Phe Asp Thr Pro Val Lys Gly Pro Pro 1651 Gln Gly Pro Arg Leu His Ile Asp Ile Pro Arg Val Gln Leu Leu 1666 Ile Glu Asp Lys Glu Gly Ile Lys Gln Leu Gly Asp Asp Lys Ala 1681 Thr Ile Pro Val Thr Asp Ala Glu Phe Ser Gln Ala Val Asn Pro 1696 Gln Ser Phe Cys Thr Gly Val Ser Leu His His Pro Thr Leu Ile 1711 Gln Ser Thr Gly Pro Leu Ile Asp Met Ser Asp Ile Arg Pro Gly 1726 Thr Asn Pro Val Ser Arg Lys Asn Val Lys Ser Ala His Ser Thr 1741 Arg Asn Arg Tyr Ser Ser Gln Trp Thr Leu Thr Lys Cys Gln Ala 1756 Ser Thr Pro Ala Arg Thr Leu Thr Ser Asp Trp Arg Thr Val Gly 1771 Ser Gln His Gly Val Thr Val Thr Glu Ser Asp Ser Tyr Ser Ala 1786 Ser Leu Ser Gln Asp Thr Asp Lys Gly Arg Asn Ser Met Val Ser 1801 Thr Glu Ser Ala Ser Ser Thr Tyr Glu Glu Leu Ala Arg Ala Tyr 1816 Glu His Ala Lys Leu Glu Glu Gln Leu Gln His Ala Lys Phe Glu 1831 Ile Thr Glu Cys Phe Ile Ser Asp Ser Ser Ser Asp Gln Met Thr 1846 Thr Gly Thr Asn Glu Asn Ala Asp Ser Met Thr Ser Met Ser Thr 1861 Pro Ser Glu Pro Gly Ile Cys Arg Phe Thr Ala Ser Pro Pro Lys 1876 ​​Pro Gln Asp Ala Asp Arg Gly Lys Asn Val Ala Val Pro Ile Pro 1891 His Arg Ala Asn Lys Ser Asp Tyr Cys Asn Leu Pro Leu Tyr Ala 1906 Lys Ser Glu Ala Phe Phe Arg Lys Ala Asp Gly Arg Glu Pro Cys 1921 Pro Val Val Pro Pro Arg Glu Ala Ser Ile Arg Asn Leu Ala Arg 1936 Thr Tyr His Thr Gln Ala Arg His Leu Thr Leu Asp Pro Ala Ser 1951 Lys Ser Leu Gly Leu Pro His Pro Gly Ala Pro Ala Ala Ala Ser 1966 Thr Ala Thr Leu Pro Gln Arg Thr Leu Ala Met Pro Ala Pro Pro 1981 Ala Gly Thr Ala Pro Pro Ala Pro Gly Pro Thr Pro Ala Glu Pro 1996 Pro Thr Ala Pro Ser Ala Ala Pro Pro Ala Pro Ser Thr Glu Pro 2011 Pro Arg Ala Gly Gly Pro His Thr Lys Met Gly Gly Ser Arg Asp 2026 Ser Leu Leu Glu Met Ser Thr Ser Gly Val Gly Arg Ser Gln Lys 2041 Gln Gly Ala Gly Ala Tyr Ser Lys Ser Tyr Thr Leu Val (4) SEQ ID NO: 3 Information ...

(i) sequence signature

(A) length: 24 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:3:CCTCTTTATGGCATGTGGCTGCTA 24

(5) information of SEQ ID NO:4

(i) sequence signature

(A) length: 24 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:4:AGAAACATAACCTTTATTGCACAC 24

(6) information of SEQ ID NO:5

(i) sequence signature

(A) length: 33 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide (xi) sequence description: the information of SEQ ID NO:5:GGATCCATGATGTGGCTGCTAACTTTCCTCCTG 33 (7) SEQ ID NO:6

(i) sequence signature

(A) length: 33 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide (xi) sequence description: the information of SEQ ID NO:6:CATCCCGGGCTACACCAGGGTGTAGGATTTGGA 33 (8) SEQ ID NO:7:

(i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide (xi) sequence description: the information of SEQ ID NO:7:Met-Trp-Leu-Leu-Thr-Phe-Leu-Leu-Leu-Leu-Asp-Ser-Leu-His-Lys 15 (9) SEQ ID NO:8

(i) sequence signature

(A) length: 41 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide (xi) sequence description: the information of SEQ ID NO:8:TGTGGCTGCTAACTTTCCTCCTGCTCCTGGACTCTTTACAC 41 (10) SEQ ID NO:9

(i) sequence signature

(A) length: 41 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide (xi) sequence description: SEQ ID NO:9:TGTGGCTGCTAACTTTCCTCTTGCTCCTGGACTCTTTACAC 41

Claims (18)

1, a kind of isolated polypeptide-Down syndrome cell adhesion molecule homoplastic protein 1 is characterized in that it includes: the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2 or active fragments, analogue or the derivative of its polypeptide.

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 the SEQ ID NO:2.

3, polypeptide as claimed in claim 2 is characterized in that it comprises the polypeptide with the aminoacid sequence shown in the SEQ ID NO: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 aminoacid sequence shown in the SEQ ID NO:2 polypeptide or its fragment, analogue, derive

The polynucleotide of thing;

(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 is characterized in that described polynucleotide comprise the polynucleotide that coding has aminoacid sequence shown in the SEQID NO:2.

6, polynucleotide as claimed in claim 4, the sequence that it is characterized in that described polynucleotide include the sequence of 1-6729 position among the sequence of 13-6174 position among the SEQ IDNO:1 or the SEQ ID NO:1.

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 preparation method with the active polypeptide of Down syndrome cell adhesion molecule homoplastic protein 1 is characterized in that described method comprises:

(a) expressing under the Down syndrome cell adhesion molecule homoplastic protein 1 condition, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate and have the active polypeptide of Down syndrome cell adhesion molecule homoplastic protein 1.

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with Down syndrome cell adhesion molecule homoplastic protein 1 specificity bonded antibody.

11, the compound of an analoglike or adjusting polypeptide active or expression is characterized in that they are simulation, promotion, antagonism or the active compound that suppresses Down syndrome cell adhesion molecule homoplastic protein 1.

12, compound as claimed in claim 11 is characterized in that it is the polynucleotide sequence shown in the SEQ ID NO:1 or its segmental antisense sequences.

13, the described application of compound of a kind of claim 11, it is characterized in that described compound be used to regulate Down syndrome cell adhesion molecule homoplastic protein 1 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 the stand-in of Down syndrome cell adhesion molecule homoplastic protein 1, agonist, 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 pharmaceutical composition with safe and effective dosage and pharmaceutically acceptable carrier as diagnosis or the treatment disease relevant unusually with Down syndrome cell adhesion molecule homoplastic protein 1 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 medicine as the tumour of embryo and nervous system development disorder disease, disease of immune system and related tissue and cancer etc. with described polypeptide, polynucleotide or compound.

Images