CN1470527A - Polypeptide-human calcium ion regulatory secretory action protein 48 and polynucleotide encoding this polypeptide - Google Patents

Abstract

The present invention discloses a polypeptide-human calcium ion regulatory secretory action protein 28, polynucleotide for coding said polypeptide and method for producing said polypeptide by using DNA recombination technique. Said invention also discloses the method for using said polypeptide to cure several diseases, such as malignant tumor, hemopathy, HIV infection, immunological diseases and various inflammations. Said invention also discloses the agonist for resisting said polypeptide and its therapeutic action, also discloses the application of polynucleotide coding said novel human calcium ion regulatory secretory action protein 48.

Description

The polynucleotide of a kind of polypeptide-human calcium ion protein 48 for regulating secretion and this peptide species of coding

Technical field

The invention belongs to biological technical field, specifically, the invention describes a kind of sequence of the proteic gene of people's albumen calcium ion regulatory secretion of encoding new, and the polypeptide of this polynucleotide encoding.The present invention has also comprised the purposes and the preparation of this polypeptide.Specifically, polypeptide of the present invention is a kind of new CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 (HCAPS), and encoding gene is CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's gene.

Background technology

Exocytosis is cell secreting outside institute synthetic albumen, neurohumor, the equimolecular approach of hormone, this process is mainly finished by the secretory vesicle of cell, merge and discharge in the process of secretory product at the secretory vesicle of cell and plasmalemma, ATP and cytosol albumen are absolutely necessary.There are some researches show that now calcium ion regulatory secretion albumen plays an important role in some secretion process as norepinephrine, but mechanism of action is not clear at present.(JBiol?Chem?1997?May?30；272(22)：14447-53)

(calcium-dependent activator protein forsecretion CAPS) is a kind of nerve/internal secretion albumen to calcium ion regulatory secretion albumen, mainly works in the exocytosis of emiocytosis vesicle.

CAPS all has existence in the skin of cytolemma and cytosol.In the exocytosis process, ATP is used to synthesize phosphoric acid acyl inositol 4,5-bisphosphate fat (Ptd Ins (4,5) P2), and Ptd Ins (4,5) P2 can improve combining of CAPS and the liposome that acidic phospholipid is arranged significantly.Therefore, can think that CAPS is that effector as Ptd Ins (4,5) P2 works in the exocytosis process.(J?Biol?Chem?1998?Apr3：273(14)：8337-43)

CAPS has very high avidity and saturability, can be incorporated into by the interaction with phospholipid bilayer on the film, and all experiments show that membrane-bound CAPS is one of key element of cell vesicle exocytosis.(Neuron?1998Jul；21(1)：137-45)

Up-to-date studies show that, CAPS regulates the compact nucleus vesicle, and (dense-core vesicle, DCV) with the fusion of plasmalemma, and decision vesicle inclusion is by merging the release of aperture.(J?Neurosci 1999?Sep?1；19(17)：7375-83)

In addition, CAPS is also relevant with the secretion of the transmission carrier of nerve polypeptide and biogenic amine.(Neuron?1998?Jul；21(1)：137-45)

According to amino acid homology result relatively, polypeptide of the present invention is accredited as CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 (HCAPS48) by deduction.

Summary of the invention

An object of the present invention is to provide isolating new polypeptide-human calcium ion protein 48 for regulating secretion 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 that encodes.

Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 that encodes.

Another object of the present invention provides the method for producing the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48.

Another object of the present invention provides the antibody at polypeptide-human calcium ion protein 48 for regulating secretion of the present invention.

Another object of the present invention has provided simulated compound, antagonist, agonist, the inhibitor at polypeptide-human calcium ion regulatory secretion albumen 48 of the present invention.

Another object of the present invention provides the method for the diagnoses and treatment disease relevant unusually with the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48.

In a first aspect of the present invention, novel isolated CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 is provided, this polypeptide is the people source, and it comprises: have＜polypeptide of 210〉2 aminoacid sequences or its conservative property variation polypeptide or its active fragments or its reactive derivative, analogue.Preferably, this polypeptide is to have＜polypeptide of 210〉2 aminoacid sequences.

In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 99% homogeny with a kind of nucleotides sequence that is selected from down group: (a) the above-mentioned CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's of coding polynucleotide; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding have＜210〉2 shown in the polypeptide of aminoacid sequence.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: (a) have＜210〉1 in the sequence of 106-1404 position; (b) have＜210〉1 in the sequence of 1-1716 position.

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.

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

As used herein, " isolating " are meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " isolating " is meant that the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's polypeptide can be used amino acid sequence analysis.

The invention provides a kind of polypeptide-human calcium ion protein 48 for regulating secretion, it is made up of the aminoacid sequence shown in the SEQID 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's fragment, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 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 by coding have＜polynucleotide of the polypeptide of 210〉2 aminoacid sequences form.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 1716 bases, its open reading frame (106-1404) 432 amino acid of having encoded.Find relatively that according to amino acid sequence homologous the calcium ion regulatory secretion albumen of this polypeptide and house mouse has 99% homology.

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in＜210〉1 or the varient of degeneracy.As used herein, " varient of degeneracy " are meant that in the present invention coding has＜210〉2 protein or polypeptide, but with the differentiated nucleotide sequence of coding region sequence shown in＜210〉1.

The polynucleotide of the mature polypeptide of coding＜210〉2 comprise: the encoding sequence that has only mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

Term " polynucleotide of coded polypeptide " is meant polynucleotide that comprise this polypeptide of encoding and the polynucleotide that comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of foregoing description polynucleotide, its coding has the polypeptide of identical aminoacid sequence or segment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

The invention still further relates to and the polynucleotide (have at least 50% between two sequences, preferably have 70% homogeny) of sequence hybridization described above.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time adds and to use denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQID 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 coding CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's polynucleotide.

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

Coding CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's of the present invention special polynucleotide sequence 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 mensuration CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's transcript; (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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's genetic expression and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

Use method (Saiki, the et al.Science1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to from the library, obtain the cDNA of total length, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to polynucleotide sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The gene of the present invention that obtains as mentioned above, perhaps the polynucleotide sequence of various dna fragmentations etc. can with ordinary method such as dideoxy chain termination (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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's encoding sequence, and the method that produces polypeptide of the present invention through recombinant technology through the genetically engineered generation.

Among the present invention, coding CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's polynucleotide sequence 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 that encodes and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, 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, coding CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's polynucleotide 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:

(1). with coding of the present invention people CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's polynucleotide (or varient), 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.

Calcium ion regulatory secretion albumen (calcium-dependent activator protein forsecretion, CAPS) in the exocytosis of emiocytosis vesicle, play an important role, its pair cell secreting outside institute synthetic albumen, neurohumor, the equimolecular process of hormone play regulating effect, so the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 has direct application in a lot of treatment of diseases and prevention.

For example, norepinephrine (norepinephrine, NE) it is relevant with calcium ion regulatory secretion albumen that secretion is proved to be, under CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's effect, can regulate the expression amount of norepinephrine, thereby reach treatment as disease of hematopoietic system, the purpose of radiation disease and immune lymphocytic generation and adjusting relative disease etc.(J Biol?Chem?1997?May?30；272(22)：14447-53)

Utilize polypeptide of the present invention, can treat malignant disease, comprise leukemia and lymphoma class; The tumour class of epithelial cell origin; Between the tumour of matter origin, as the sarcoma class; Central nerve neuroma class or the like.

CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 of the present invention also is a kind of immunomodulator, has immunological enhancement or immunosuppressive action.The CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 can be used for the treatment of some diseases, and these diseases comprise immunoreactive anergy, or abnormal immune reaction, or the host defend invalid.

Polypeptide of the present invention also can be used for treating nervous system disease, and the present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check (antagonist) CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's medicament because the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 plays an important role in neural neurohumor secretion process.Agonist improves human calcium ion regulatory secretion albumen 48 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 expressing human calcium ion protein 48 for regulating secretion be cultivated with the human calcium ion regulatory secretion albumen 48 of mark.Measure the medicine raising then or check this interactional ability.

CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's antagonist comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's antagonist can combine and eliminate its function with the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48, or suppresses the generation of this polypeptide, or combines with the avtive spot of this polypeptide and to make this polypeptide can not bring into play biological function.

In screening during as the compound of antagonist, the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 can be added in the bioanalysis mensuration, interactional influence between CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's bonded peptide molecule obtains.During screening, generally tackle CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's 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 preparation CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's monoclonal antibody 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 (Morrisonetal, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.Pat No.4946778) also can be used for producing anti-CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's single-chain antibody.

Anti-CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's antibody can be used in the immunohistochemistry technology, detects the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 in the biopsy specimen.

With the also available labelled with radioisotope of CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's positive cells.

The disease that antibody among the present invention can be used for treating or prevention is relevant with the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48.The antibody that gives suitable dosage can stimulate or block CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's generation or activity.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's level that is detected in the test can be with laying down a definition the importance of CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 in various diseases and be used to the disease of diagnosing the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 to work.

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.

Coding CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's polynucleotide 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's nothing expression or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 who expresses variation, to suppress endogenic CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's activity.For example, a kind of CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 of variation can be the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 who 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's 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 coding CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's polynucleotide are transferred in the cell.The method of recombinant viral vector that structure carries coding CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's polynucleotide is found in existing document (Sambrook, et al.).Reorganization coding CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's polynucleotide 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 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.

Coding CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's polynucleotide can be used for the diagnosis with CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's relative disease.The expression that coding CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's polynucleotide can be used for detecting CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's whether or under morbid state unconventionality expression.As the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's that encodes dna sequence dna can be used for biopsy specimen is hybridized to judge CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's expression situation.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 the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 with the special primer of CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48.

The sudden change that detects CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's gene also can be used for the disease of diagnosing the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 to be correlated with.The form of CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 sudden change comprises that the point mutation compared with normal wild type CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 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, Mendelian Inheritancein 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.The CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

Description of drawings

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

Fig. 1 is the proteic amino acid sequence homology comparison diagram of the calcium ion regulatory secretion of inventor's calcium ion protein 48 for regulating secretion and house mouse.The top sequence is the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48, and the below sequence is the calcium ion regulatory secretion albumen of house mouse.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+".

Fig. 2 is the proteic polyacrylamide gel electrophoresis figure of isolating human calcium ion regulatory secretion (SDS-PAGE).48kDa is proteinic molecular weight.The arrow indication is isolated protein band.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1: the proteic clone of human calcium ion regulatory secretion

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﹠amp; #61537; Bacterium forms the cDNA library.Measure all clones' 5 ' and 3 ' terminal sequence with Dye terminate cycle reaction sequencing kit (Perkin-Elmer company product) and ABI377 automatic sequencing view (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 0218C06 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 0218C06 clone is 1716bp (as＜210〉1 shown in), from 106bp to 1404bp an open reading frame (ORF) is arranged, the new protein (shown in Seq ID NO:2) of encoding.We are with this clone's called after pBS-0218C06, encoded protein matter called after human calcium ion regulatory secretion albumen.

Embodiment 2:cDNA clone's homology retrieval

With proteic sequence of human calcium ion regulatory secretion of the present invention and encoded protein sequence thereof, with Blast program (Basic

Local 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 human calcium ion regulatory secretion albumen homology of the present invention is the calcium ion regulatory secretion albumen of a kind of known house mouse, and its encoded protein number is D86214 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 99%; Similarity is 99%.Embodiment 3: with RT-PCR method human cloning calcium ion regulatory secretion protein gene

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’-GGGATCAAGAAGTTCGAACATCAGCTCCT-3’(<210>3)

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

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

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

The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/LTris-Cl, (pH8.5), 1.5mmol/L MgCl2,200 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 ℃, 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of β-actin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows the dna sequence dna and＜210 of PCR product〉the 1-{ full length gene shown in 1 } bp is identical.The expression of embodiment 4:Northern blotting analyst calcium ion regulatory secretion protein 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) advances ground 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 HRBP-3 coding region sequence ({ initiation codon position } bp is to { termination codon position } bp) 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 KH2PO4 (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.Example 5: vivoexpression, separation and the purifying of reorganization HRBP-3

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

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

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

5 ' end of these two sections primers contains NcoI and BamHI restriction enzyme site respectively, is respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, and NcoI and BamHI restriction enzyme site are corresponding to expression vector plasmid pET-28b (+) (Novagen

Company's product, Cat.No.69865.3) the selectivity restriction enzyme site on.With the pBS-0218C06 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0218C06 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmmol, 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 pET-28 (+) are carried out double digestion with NcoI and { restriction endonuclease }, reclaim big fragment respectively, and connect with the T4 ligase enzyme.Connect product and transform, after the dull and stereotyped overnight incubation of the LB that contains kantlex (final concentration 30 μ g/ml), use the colony polymerase chain reaction (PCR) method screening positive clone, and check order with the big enterobacterial Dh5 of Calcium Chloride Method α.Select the correct positive colony of sequence (pET-0490B02) with Calcium Chloride Method with recombinant plasmid transformed e. coli bl21 (DE3) plySs (Novagen company product).In the LB liquid nutrient medium that contains kantlex (final concentration 30 μ g/ml), host bacterium BL21 (pET-0490B02) is cultured to logarithmic phase at 37 ℃, adds IPTG to final concentration 1mmol/L, continues to cultivate 5 hours.Centrifugal collection thalline, through the broken bacterium of ultrasonic wave, centrifugal collection supernatant with carrying out chromatography with 6 Histidines (6His-Tag) bonded affinity column His.Bind Quick Cartridge (Novagen company product), has obtained the target protein HRBP-3 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the kDa place.This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end are identical with 15 amino-acid residues of N-end shown in＜210〉2 as a result.The generation of embodiment 6 anti-human calcium ion regulatory secretion protein antibodies

Synthesize the specific polypeptide of following HRBP-3 with Peptide synthesizer (PE company product):

NH2-Met-Ala-Asp-Gln-Ile-Ala-Arg-Glu-Arg-Lys-Phe-Pro-Lys-Phe-Val-OH。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 be specifically and human calcium ion regulatory secretion protein binding.

Sequence table＜110〉Bode Gene Development Co., Ltd., Shanghai＜120〉peptide species--the polynucleotides of human calcium ion regulatory secretion albumen 48 and this peptide species of coding＜130〉0218c06＜160〉6＜170〉PatentIn version 3.1＜210〉1＜211〉1716＜212〉DNA＜213〉Homo sapiens＜220〉＜221〉CDS＜222〉(106) .. (1404)＜223〉＜400〉1gggatcaaga agttcgaaca tcagctcctt tacaatgcct gccagctgga caatccagat 60gagcaagcag cccagatcag acgagagctg gatggacgtc tacaa atg gca gac caa 117

Met?Ala?Asp?Gln

1ata?gcc?agg?gaa?cgc?aaa?ttt?ccc?aag?ttt?gta?tcc?aaa?gaa?atg?gaa 165Ile?Ala?Arg?Glu?Arg?Lys?Phe?Pro?Lys?Phe?Val?Ser?Lys?Glu?Met?Glu5 10 15 20aac?atg?tac?att?gag?gag?ctg?aag?tca?tct?gtc?aac?ctg?ctc?atg?gcc 213Asn?Met?Tyr?Ile?Glu?Glu?Leu?Lys?Ser?Ser?Val?Asn?Leu?Leu?Met?Ala

25 30 35aac?ttg?gag?agc?atg?ccg?gta?tcc?aaa?ggc?ggg?gag?ttc?aag?ctc?cag 261Asn?Leu?Glu?Ser?Met?Pro?Val?Ser?Lys?Gly?Gly?Glu?Phe?Lys?Leu?Gln

40 45 50aaa?ctc?aaa?cgc?agc?cac?aat?gct?tcc?atc?atc?gac?atg?ggc?gag?gag 309Lys?Leu?Lys?Arg?Ser?His?Asn?Ala?Ser?Ile?Ile?Asp?Met?Gly?Glu?Glu

55 60 65agt?gag?aac?cag?ctc?tcc?aag?tca?gat?gtc?gtg?ctg?tct?ttc?tca?ttg 357Ser?Glu?Asn?Gln?Leu?Ser?Lys?Ser?Asp?Val?Val?Leu?Ser?Phe?Ser?Leu

70 75 80gag?gtg?gta?att?atg?gaa?gtc?caa?ggc?ctc?aaa?tct?ttg?gct?cca?aat 405Glu?Val?Val?Ile?Met?Glu?Val?Gln?Gly?Leu?Lys?Ser?Leu?Ala?Pro?Asn85 90 95 100cgc?atc?gta?tat?tgc?aca?atg?gag?gtg?gaa?gga?gga?gag?aaa?cta?cag 453Arg?Ile?Val?Tyr?Cys?Thr?Met?Glu?Val?Glu?Gly?Gly?Glu?Lys?Leu?Gln

105 110 115act?gat?cag?gcc?gag?gct?tct?aaa?cca?acc?tgg?ggc?acc?cag?ggt?gac 501Thr?Asp?Gln?Ala?Glu?Ala?Ser?Lys?Pro?Thr?Trp?Gly?Thr?Gln?Gly?Asp

120 125 130ttc?tcc?aca?acc?cat?gca?ttg?cca?gct?gtg?aag?gtg?aag?ctg?ttc?aca 549Phe?Ser?Thr?Thr?His?Ala?Leu?Pro?Ala?Val?Lys?Val?Lys?Leu?Phe?Thr

135 140 145gag?agc?aca?ggc?gtc?ctg?gcg?ttg?gag?gac?aag?gag?ctt?ggg?cgg?gtt 597Glu?Ser?Thr?Gly?Val?Leu?Ala?Leu?Glu?Asp?Lys?Glu?Leu?Gly?Arg?Val

150 155 160att?ctc?cat?ccc?acc?ccg?aac?agc?ccc?aaa?cag?tca?gag?tgg?cac?aaa 645Ile?Leu?His?Pro?Thr?Pro?Asn?Ser?Pro?Lys?Gln?Ser?Glu?Trp?His?Lys165 170 175 180atg?aca?gtc?tcc?aaa?aac?tgc?ccc?gac?caa?gat?ctc?aaa?atc?aaa?ctt 693Met?Thr?Val?Ser?Lys?Asn?Cys?Pro?Asp?Gln?Asp?Leu?Lys?Ile?Lys?Leu

185 190 195gct?gtc?cga?atg?gat?aag?cct?caa?aac?atg?aag?cat?tct?ggg?tat?tta 741Ala?Val?Arg?Met?Asp?Lys?Pro?Gln?Asn?Met?Lys?His?Ser?Gly?Tyr?Leu

200 205 210tgg?gcc?atc?ggt?aag?aat?gtc?tgg?aag?aga?tgg?aag?aaa?agg?ttt?ttt 789Trp?Ala?Ile?Gly?Lys?Asn?Val?Trp?Lys?Arg?Trp?Lys?Lys?Arg?Phe?Phe

215 220 225gta?ttg?gtg?cag?gtc?agt?cag?tac?acg?ttt?gcc?atg?tgc?agt?tat?cgg 837Val?Leu?Val?Gln?Val?Ser?Gln?Tyr?Thr?Phe?Ala?Met?Cys?Ser?Tyr?Arg

230 235 240gag?aag?aaa?gcg?gag?cct?cag?gaa?ctt?cta?caa?ttg?gat?ggc?tac?act 885Glu?Lys?Lys?Ala?Glu?Pro?Gln?Glu?Leu?Leu?Gln?Leu?Asp?Gly?Tyr?Thr245 250 255 260gtg?gat?tac?acc?gac?ccc?cag?cca?ggt?ttg?gag?ggt?ggc?cga?gcc?ttc 933Val?Asp?Tyr?Thr?Asp?Pro?Gln?Pro?Gly?Leu?Glu?Gly?Gly?Arg?Ala?Phe

265 270 275ttc?aat?gct?gtc?aag?gag?gga?gac?acc?gtg?ata?ttt?gcc?agt?gac?gat 981Phe?Asn?Ala?Val?Lys?Glu?Gly?Asp?Thr?Val?Ile?Phe?Ala?Ser?Asp?Asp

280 285 290gaa?caa?gac?cgc?atc?ctg?tgg?gtc?cag?gcc?atg?tat?cgg?gcc?acg?ggg 1029Glu?Gln?Asp?Arg?Ile?Leu?Trp?Val?Gln?Ala?Met?Tyr?Arg?Ala?Thr?Gly

295 300 305cag?tca?cac?aag?cct?gtg?ccc?ccg?acc?caa?gtc?cag?aaa?ctc?aac?gcc 1077Gln?Ser?His?Lys?Pro?Val?Pro?Pro?Thr?Gln?Val?Gln?Lys?Leu?Asn?Ala

310 315 320aag?gga?gga?aat?gta?cct?cag?ctg?gat?gcc?cct?atc?tct?caa?ttt?tac 1125Lys?Gly?Gly?Asn?Val?Pro?Gln?Leu?Asp?Ala?Pro?Ile?Ser?Gln?Phe?Tyr325 330 335 340gca?gat?aga?gct?caa?aaa?cat?ggc?atg?gat?gaa?ttt?atc?tct?tcc?aac 1173Ala?Asp?Arg?Ala?Gln?Lys?His?Gly?Met?Asp?Glu?Phe?Ile?Ser?Ser?Asn

345 350 355ccc?tgt?aac?ttt?gac?cac?gct?tcc?ctc?ttt?gag?atg?gta?caa?cgc?ctt 1221Pro?Cys?Asn?Phe?Asp?His?Ala?Ser?Leu?Phe?Glu?Met?Val?Gln?Arg?Leu

360 365 370act?ttg?gat?cac?aga?ctt?aat?gat?tcc?tat?tct?tgc?ctg?gca?agt?ata 1269Thr?Leu?Asp?His?Arg?Leu?Asn?Asp?Ser?Tyr?Ser?Cys?Leu?Ala?Ser?Ile

375 380 385ttc?ttt?ggt?cta?cag?caa?ttt?aaa?cat?aac?att?ttt?ttt?cat?tta?aat 1317Phe?Phe?Gly?Leu?Gln?Gln?Phe?Lys?His?Asn?Ile?Phe?Phe?His?Leu?Asn

390 395 400att?tct?cta?cct?tta?tgt?cag?aag?ctc?ttt?tct?cca?tta?cag?aat?ccc 1365Ile?Ser?Leu?Pro?Leu?Cys?Gln?Lys?Leu?Phe?Ser?Pro?Leu?Gln?Asn?Pro405 410 415 420ttt?aac?aat?tat?aaa?tgt?gtt?ccc?gat?ttc?ctg?ggt?tga?gatagtttta 1414Phe?Asn?Asn?Tyr?Lys?Cys?Val?Pro?Asp?Phe?Leu?Gly

425 430cttaccaaag?aaacaagact?gagcaggagg?aagttatcag?tagaaacaga?tccaattccc?1474cagcctccct?atcttagctt?cccccttttc?agctgctaca?tgagaaatat?tcccaagagg?1534gtaatcgagt?ttggcacctg?cttgtggaga?acttgtaaaa?attgcctcag?tagcttagag?1594tttaagatgc?ccttccctcc?ccatcccaga?tctggtaatc?ctatctcatt?acatgaatgt?1654attctctttg?tgtattttct?taaaggttaa?aatgatttgt?aaataaactt?tatttttcat?1714aa1716<210>2<211>432<212>PRT<213>Homo?sapiens<400>2Met?Ala?Asp?Gln?Ile?Ala?Arg?Glu?Arg?Lys?Phe?Pro?Lys?Phe?Val?Ser1 5 10 15Lys?Glu?Met?Glu?Asn?Met?Tyr?Ile?Glu?Glu?Leu?Lys?Ser?Ser?Val?Asn

20 25 30Leu?Leu?Met?Ala?Asn?Leu?Glu?Ser?Met?Pro?Val?Ser?Lys?Gly?Gly?Glu

35 40 45Phe?Lys?Leu?Gln?Lys?Leu?Lys?Arg?Ser?His?Asn?Ala?Ser?Ile?Ile?Asp

50 55 60Met?Gly?Glu?Glu?Ser?Glu?Asn?Gln?Leu?Ser?Lys?Ser?Asp?Val?Val?Leu65 70 75 80Ser?Phe?Ser?Leu?Glu?Val?Val?Ile?Met?Glu?Val?Gln?Gly?Leu?Lys?Ser

85 90 95Leu?Ala?Pro?Asn?Arg?Ile?Val?Tyr?Cys?Thr?Met?Glu?Val?Glu?Gly?Gly

100 105 110Glu?Lys?Leu?Gln?Thr?Asp?Gln?Ala?Glu?Ala?Ser?Lys?Pro?Thr?Trp?Gly

115 120 125Thr?Gln?Gly?Asp?Phe?Ser?Thr?Thr?His?Ala?Leu?Pro?Ala?Val?Lys?Val

130 135 140Lys?Leu?Phe?Thr?Glu?Ser?Thr?Gly?Val?Leu?Ala?Leu?Glu?Asp?Lys?Glu145 150 155 160Leu?Gly?Arg?Val?Ile?Leu?His?Pro?Thr?Pro?Asn?Ser?Pro?Lys?Gln?Ser

165 170 175Glu?Trp?His?Lys?Met?Thr?Val?Ser?Lys?Asn?Cys?Pro?Asp?Gln?Asp?Leu

180 185 190Lys?Ile?Lys?Leu?Ala?Val?Arg?Met?Asp?Lys?Pro?Gln?Asn?Met?Lys?His

195 200 205Ser?Gly?Tyr?Leu?Trp?Ala?Ile?Gly?Lys?Asn?Val?Trp?Lys?Arg?Trp?Lys

210 215 220Lys?Arg?Phe?Phe?Val?Leu?Val?Gln?Val?Ser?Gln?Tyr?Thr?Phe?Ala?Met225 230 235 240Cys?Ser?Tyr?Arg?Glu?Lys?Lys?Ala?Glu?Pro?Gln?Glu?Leu?Leu?Gln?Leu

245 250 255Asp?Gly?Tyr?Thr?Val?Asp?Tyr?Thr?Asp?Pro?Gln?Pro?Gly?Leu?Glu?Gly

260 265 270Gly?Arg?Ala?Phe?Phe?Asn?Ala?Val?Lys?Glu?Gly?Asp?Thr?Val?Ile?Phe

275 280 285Ala?Ser?Asp?Asp?Glu?Gln?Asp?Arg?Ile?Leu?Trp?Val?Gln?Ala?Met?Tyr

290 295 300Arg?Ala?Thr?Gly?Gln?Ser?His?Lys?Pro?Val?Pro?Pro?Thr?Gln?Val?Gln305 310 315 320Lys?Leu?Asn?Ala?Lys?Gly?Gly?Asn?Val?Pro?Gln?Leu?Asp?Ala?Pro?Ile

325 330 335?Ser?Gln?Phe?Tyr?Ala?Asp?Arg?Ala?Gln?Lys?His?Gly?Met?Asp?Glu?Phe

340 345 350?Ile?Ser?Ser?Asn?Pro?Cys?Asn?Phe?Asp?His?Ala?Ser?Leu?Phe?Glu?Met

355 360 365Val?Gln?Arg?Leu?Thr?Leu?Asp?His?Arg?Leu?Asn?Asp?Ser?Tyr?Ser?Cys

370 375 380Leu?Ala?Ser?Ile?Phe?Phe?Gly?Leu?Gln?Gln?Phe?Lys?His?Asn?Ile?Phe385 390 395 400Phe?His?Leu?Asn?Ile?Ser?Leu?Pro?Leu?Cys?Gln?Lys?Leu?Phe?Ser?Pro

405 410 415Leu?Gln?Asn?Pro?Phe?Asn?Asn?Tyr?Lys?Cys?Val?Pro?Asp?Phe?Leu?Gly

420 425 430<210>3<211>29<212>DNA<213>Homo?sapiens<400>3gggatcaaga agttcgaaca tcagctcct29<210>4<211>29<212>DNA<213>Homo?sapiens<400>4ttatgaaaaa taaagtttat ttacaaatc29<210>5<211>30<212>DNA<213>Homo?sapiens<400>5cccccatgga tggcagacca aatagccagg30<210>6<211>31<212>DNA<213>Homo?sapiens<400>6cccggatcct caacccagga aatcgggaac a31

Claims (18)

1, a kind of isolated polypeptide-CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 is characterized in that it includes:＜210〉2

The polypeptide of shown aminoacid sequence or the active fragments of its polypeptide, analogue or derivative.

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

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

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

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

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

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

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

6, polynucleotide as claimed in claim 4, it is characterized in that the sequence of described polynucleotide includes＜210〉1 in the 179-1147 position sequence or＜210〉1 in the sequence of 1-1823 position.

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

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

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

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

9, a kind of preparation method with the active polypeptide of CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 is characterized in that described method comprises:

(a) under expressing human calcium ion protein 48 for regulating secretion condition, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate and have the active polypeptide of CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48.

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's 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 human calcium ion regulatory secretion albumen 48.

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

13, the described application of compound of a kind of claim 11, it is characterized in that described compound be used for mediator's calcium ion protein 48 for regulating secretion 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 CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48's stand-in, 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 the CALCIUM-DEPENDENT ACTIVATOR PROTEIN FOR SECRETION 48 with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11.

18, the described polypeptide of arbitrary claim, polynucleotide or the application of compound among the claim 1-6 and 11 is characterized in that being used for the treatment of with described polypeptide, polynucleotide or compound the medicine of the disease that existence causes of the outer excessive Actin muscle of cancer, disease of immune system, hypertension, kidney depletion, amyotrophy, malnutrition, neurocyte signal conduction disturbance, virus and infectation of bacteria or born of the same parents.