CN1510049A - Polypeptide-human DBH and PAM20, polynucleotide for encoding said polypeptide - Google Patents

Abstract

A novel polypeptide-human DBH and PAM 20, the polynucleotide for coding it, the process for preparing said polypeptide by DNA recombination, the application of said polypeptide in treating diseases, such as cancer, HIV infection, immunopathy, etc, the antagonist of said polypeptide and its medical action, and the application of said polynucleotide are disclosed.

Description

The polynucleotide of a kind of polypeptide-human DBH and PAM20 and this peptide species of coding

Technical field

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide---DBH and PAM20, and the polynucleotide sequence of this polypeptide of encoding.The invention still further relates to the preparation method and the application of these polynucleotide and polypeptide.

Background technology

Copper is comparatively rare as cofactor in Mammals, occur over just in five kinds of known enzymes: monooxygenase [DBH, peptidyl-glycine α-Ntn hydrolase and tyrosine oxidase], amine oxide enzyme, superoxide-dismutase, Terminal oxidase and ceruloplasmin.Although it is the appearance of copper is very limited, most important for the organism homeostasis by the catalytic reaction of copper enzyme.II type copper, the monooxygenase that xitix relies on is to need copper to make cofactor, with the class of enzymes of xitix as a kind of electron donor, has now found that two members:

1) Dopamine-beta-monooxygenase (DBH), the catalysis Dopamine HCL is converted into the neurotransmitter norepinephrine.DBH engages at the big dense-core of the vesicle (chromaffin granule) of medulliadrenal catecholamine secretion and sympathetic nervous system that the catalysis Dopamine HCL is converted into neurotransmitter and hormone norepinephrine in the bubble.In the adrenal chromaffin particle, known DBH all can exist with solvable and embrane-associated protein form, and the distribution alive of the enzyme between the two is almost equal.Stimulate through pheochromocyte, Granulosa Cell Membrane and plasma membrane merge, and discharge its soluble inclusion, comprising the soluble form of Dopamine-beta-monooxygenase.The film combining form of this enzyme by endocytosis, and forms chromaffin granule by gorky's zone circulation of inserting secretory protein in membrana granulosa again from cell surface again.Enzyme in the protoplasma of not knowing as yet owing to functional meaning from the stimulation coupling secretion of the solubility DBH of chromaffin granule.Require dioxygen and external source electron donor (xitix in the body) by the catalytic reaction of Dopamine-beta-monooxygenase, and DBH is placed the monooxygenase family of enzyme.Dopamine-beta-monooxygenase be few several need xitix do cofactor by the enzyme of desirable features mark.Different with two kinds of unit price oxide compound enzymes (Phenylalanine hydroxylase and tyrosine hydroxylase) that contain pterin in the catecholamine biosynthetic pathway, Dopamine-beta-monooxygenase only contains copper as cofactor.

2) peptidyl-glycine α-acid amides monooxygenase (PAM), the carboxyl terminal glycine of many bioactive peptides of catalysis and hormone is converted into amide group, and is attended by secretion, and very important to some incretogenous all biological potential.

The similarity following points of DBH and PAM: the two all is the monooxygenase that contains II type copper, from secretory granules, all needs xitix and molecular oxygen.Xitix the two on all by mitochondrial half DHAR and cytochrome b5 61 recirculation.They have identical dynamic mechanism, can both use catechol or yellow prussiate as the single electron reductive agent.The most important thing is that the two has identical catalysis region.Dopamine-beta-monooxygenase (DBH) and peptidyl-glycine α-acid amides monooxygenase (PAM) has some regional sequences similar between the two, conservative histidine residues bunch is contained in two zones wherein: H-H-M-x (2)-F-x-C and H-x-F-x (4)-H-T-H-x (2)-G, they may be with relevant in conjunction with copper, and we select these two zones as signal mode.Each subunit contains two copper that are reduced, each Cu in the catalytic activity form of DBH and PAM ²⁺The coordination sphere that 3-4 histidyl-imidazoles arranged, the Cu that is reduced of Cu-S and DBH or PAM ⁺The form coordination is close to sequence as part with the methionine(Met) in H-H-M-x (2)-F-x-C template.

In sum, DBH and PAM have similar biochemical property and similar structure, thereby both are classified as same family: II type copper, the monooxygenase family that xitix relies on.

Because DBH and PAM20 albumen play an important role in the body critical function as mentioned above, and believe and relate to a large amount of albumen in these regulate processes, thereby need to identify the DBH and the PAM20 albumen of more these processes of participation in this area always, particularly identify this proteic aminoacid sequence.Separating also of new DBH and PAM20 protein coding gene 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.

Summary of the invention

An object of the present invention is to provide isolating new polypeptide-human DBH and PAM20 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 coding people DBH and PAM20.

Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain coding people DBH and PAM20.

Another object of the present invention provides the method for producing people DBH and PAM20.

Another object of the present invention provides the antibody at polypeptide-human DBH of the present invention and PAM20.

The present invention relates to a kind of isolated polypeptide, this polypeptide is the people source, and it comprises: have＜polypeptide or its examples of conservative variations, bioactive fragment or the derivative of 210〉2 aminoacid sequences.Preferably, this polypeptide is to have＜polypeptide of 210〉2 aminoacid sequences.

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 people DBH and PAM20 protein-active, it comprises and utilizes polypeptide of the present invention.The invention still further relates to the compound that obtains with this method.

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

The invention provides a kind of polypeptide-human DBH and PAM20, it is basically by＜210〉aminoacid sequence shown in 2 forms.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analogue of people DBH and PAM20.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep people DBH of the present invention biological function identical with PAM20 or active polypeptide basically.The fragment of polypeptide of the present invention, derivative or analogue can be: (I) a kind of like this, wherein one or more amino-acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the amino acid that replaces can be also can not encoded by genetic codon; Perhaps (II) is a kind of like this, and certain group on wherein one or more amino-acid residues is replaced by other group and comprises substituting group; Perhaps (III) is a kind of like this, and wherein mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; Perhaps (IV) is a kind of like this, wherein additional aminoacid sequence is integrated into mature polypeptide and the peptide sequence that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying) by the elaboration of this paper, such fragment, derivative and analogue are considered within those skilled in the art's ken.

The invention provides isolating nucleic acid (polynucleotide), substantially by coding have＜polynucleotide of the polypeptide of 210〉2 aminoacid sequences form.Polynucleotide sequence of the present invention comprises＜210〉1 nucleotide sequence.Polynucleotide of the present invention are to find from the cDNA library of people's fetal brain tissue.

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

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

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

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

The invention still further relates to and the polynucleotide (have at least 50% between two sequences, preferably have 70% homogeny) of sequence hybridization described above.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time adds and to use denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And, the polypeptide of interfertile polynucleotide encoding and＜210〉and the mature polypeptide shown in 2 has identical biological function and activity.

The invention still further relates to nucleic acid fragment with sequence hybridization described above.As used herein, " nucleic acid fragment " length contain 10 Nucleotide at least, better be 20-30 Nucleotide at least, be more preferably 50-60 Nucleotide at least, preferably more than at least 100 Nucleotide.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 people DBH and PAM20.

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

The special polynucleotide sequence of coding people DBH of the present invention and PAM20 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., Molecular Cloning, A LaboratoryManual, 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 people DBH and PAM20; (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 people DBH and PAM20 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 the host cell that produces through genetically engineered with carrier of the present invention or directly choose DBH and PAM20 encoding sequence, and the method that produces polypeptide of the present invention through recombinant technology.

Among the present invention, the polynucleotide sequence of coding people DBH and PAM20 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 Na thans, 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 and contains coding people DBH and the dna sequence dna of PAM20 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 PL promotor of lambda particles phage; 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 people DBH and PAM20 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 people DBH and PAM20 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:

(1). with the polynucleotide (or varient) of everybody DBH of coding of the present invention and PAM20, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

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

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

In step (2), according to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

In step (3), recombinant polypeptide can wrap and be expressed or be secreted into the extracellular in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.These methods include, but are not limited to: conventional renaturation is handled, protein precipitant is handled (salt analysis method), centrifugal, the broken bacterium of infiltration, the combination of ultrasonication, super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in disease treatment, for example, can treat malignant tumour, adrenal gland defect, tetter, all kinds of inflammation, HIV infection and immunological disease etc.

Dopamine-beta-monooxygenase (DBH), peptidyl-glycine α-acid amides monooxygenase (PAM) all belong to II type copper, the monooxygenase family that xitix relies on.The two has identical special catalysis region, can both use catechol or yellow prussiate as the single electron reductive agent.But Dopamine-beta-monooxygenase catalysis Dopamine HCL is converted into the neurotransmitter norepinephrine.Engage in the bubble at the big dense-core of the vesicle (chromaffin granule) of medulliadrenal catecholamine secretion and sympathetic nervous system and to find that all DBH catalysis Dopamine HCL is converted into neurotransmitter and hormone norepinephrine.But the carboxyl terminal glycine of many bioactive peptides of PAM catalysis and hormone is converted into amide group, and is attended by secretion, and very important to some incretogenous all biological potential.

Because Dopamine HCL and metabolite are as neurotransmitter; in neural system with various psychotic disorders such as dysthymia disorders; closely related with nervous system degenerative disease such as Parkinson's disease; so contain II type copper, the polypeptide expression of the special catalysis region of monooxygenase family that xitix relies on can cause various nervous system disorderss unusually.Owing to the metabolism and the cardiovascular disorder of Catecholamine matter, especially hypertension is relevant, and is relevant with water-electrolyte metabolism, so polypeptide expression will produce cardiovascular disorder, water-electrolyte metabolism disease unusually again.The polypeptide of DBH of containing of the present invention and the special motif of PAM has above function.

This shows that the abnormal expression of DBH of the present invention and PAM20 will produce various diseases especially nervous system disorders, cardiovascular disorder, water-electrolyte metabolism disease.

The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) people DBH and PAM20.Agonist improves people DBH and PAM20 biological function such as stimulate cellular proliferation, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can be in the presence of medicine, people DBH and the PAM20 with mark cultivates with the film preparation of mammalian cell or expressing human DBH and PAM20.Measure the medicine raising then or check this interactional ability.

The antagonist of people DBH and PAM20 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of people DBH and PAM20 can combine and eliminate its function with people DBH and PAM20, 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, people DBH and PAM20 can be added during bioanalysis measures, determine to interactional influence between people DBH and PAM20 and its acceptor 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 people DBH and PAM20 bonded peptide molecule obtains.During screening, generally tackle people DBH and the PAM20 molecule carries 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 people DBH and PAM20 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.

Can the choose method of DBH and PAM20 direct injection immune animal (as rabbit, mouse, rat etc.) of the production of polyclonal antibody 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 people DBH and PAM20 include 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-people DBH and PAM20.

The antibody of anti-people DBH and PAM20 can be used in the immunohistochemistry technology, detects people DBH and PAM20 in the biopsy specimen.

With people DBH and the also available labelled with radioisotope of PAM20 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 people DBH and PAM20 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 people DBH and PAM20 positive cells.

The disease that antibody among the present invention can be used for treating or prevention is relevant with PAM20 with people DBH.The antibody that gives suitable dosage can stimulate or block generation or the activity of people DBH and PAM20.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people DBH and PAM20 level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.People DBH that is detected in the test and PAM20 level can be with laying down a definition people DBH and the PAM20 importance in various diseases and be used to the disease of diagnosing people DBH and PAM20 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.

The polynucleotide of coding people DBH and PAM20 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 people DBH and PAM20 or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for people DBH and the PAM20 that expresses variation, to suppress endogenic people DBH and PAM20 activity.For example, a kind of people DBH of variation and PAM20 can be people DBH and the PAM20 that shortens, lacked signal conduction function territory, though can combine with the substrate in downstream, lack signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the disease of people DBH and PAM20 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 people DBH and PAM20 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding people DBH and PAM20 is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding people DBH and PAM20 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 people DBH and PAM20mRNA 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 people DBH and PAM20 can be used for the diagnosis with the relative disease of people DBH and PAM20.The unconventionality expression of the expression that the polynucleotide of coding people DBH and PAM20 can be used for detecting people DBH and PAM20 people DBH and PAM20 whether or under morbid state.As the dna sequence dna of encode people DBH and PAM20 can be used for biopsy specimen is hybridized to judge the expression situation of people DBH and PAM20.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.Personnel selection DBH and the special primer of PAM20 carry out the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect people DBH and PAM20.

The sudden change that detects people DBH and PAM20 gene also can be used for the disease of diagnosing people DBH relevant with PAM20.The form of people DBH and PAM20 sudden change comprises that the point mutation compared with the PAM20 dna sequence dna with normal wild type people DBH, 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 Manual ofBasic 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 Inheritance inMan (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.People DBH and PAM20 come administration with the amount that treats and/or prevents concrete indication effectively.Be applied to patient's people DBH and amount and the dosage range of PAM20 and 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 DBH of the present invention and PAM20 at the 23-131 amino acid sequence homology comparison diagram of the characteristic sequence of totally 109 amino acid and structural domain DBH and PAM.The top sequence is DBH and PAM20, and the below sequence is the characteristic sequence of structural domain DBH and PAM.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 people DBH and PAM20.20kDa 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 (Ncw York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1: the clone of people DBH and PAM20

Extract the total RNA of people's tire brain with guanidinium isothiocyanate/phenol/chloroform single stage method.From total RNA, separate poly (A) mRNA with Quik mRNA Isolation Kit (Qiegene company product).2ug poly (A) mRNA forms cDNA through reverse transcription.CDNA fragment orientation is inserted on the multiple clone site of pBSK (+) carrier (Clontech company product) with Smart cDNA clone's test kit (available from Clontech), transforms DH5 α, bacterium forms the cDNA library.Measure all clones' 5 ' and 3 ' terminal sequence with Dye terminate 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 0060E11 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 0060E11 clone is 1005bp (as＜210〉1 shown in), from 105bp to 662bp the open reading frame (ORF) of a 558bp arranged, the new protein of encoding (as＜210〉2 shown in).We are with this clone's called after pBS-0060E11, and the name of encoded protein matter is people DBH and PAM20.

Embodiment 2:cDNA clone's domain analyses

With sequence and the encoded protein sequence thereof of DBH of the present invention and PAM20, with profile scan program (Basiclocal Alignment search tool) [Altschul, the SF et al.J.Mol.Biol.1990 among the GCG; 215:403-10], carry out domain analyses at databases such as prosite.DBH of the present invention and PAM20 have homology at the characteristic sequence of 23-131 and structural domain DBH and PAM, and homology the results are shown in Fig. 1, and homology is 0.29, must be divided into 14.39; Threshold value is 14.37.

Embodiment 3: with the gene of RT-PCR method clones coding people DBH and PAM20

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’-GGGGCCTCTCTCCGTCGCCATGGA-3’

Primer2：5’-GTGATTAAACTACTGTTGTATCAT-3’

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

Primer2 is＜2l0〉1 in 3 ' end reverse sequence.

The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/LTris-Cl, (pH8.5), 1.5mmol/L MgCl ₂, 200 μ mol/L dNTP, 10pmol primer, the Taq archaeal dna polymerase of 1U (Clontech company product).Go up by 25 cycles of following conditioned response at PE9600 type DNA thermal cycler (Perkin-Elmer company): 94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of β-actin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows the dna sequence dna and＜210 of PCR product〉1-1005bp shown in 1 is identical.

Embodiment 4:Northern blotting analyst DBH and PAM20 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)-SmM 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 people DBH and the PAM20 coding region sequence 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: the vivoexpression of recombinant human DBH and PAM20, separate and purifying

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’-CATGCTAGCATGCTGAAAGCCAAGATCCTCTTC-3’

Primer4：5’-CCCAAGCTTCTAGGTCATAATTGACATCTCCTC-3’

5 ' end of these two sections primers contains NheI and BamHI restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, NheI and BamHI restriction enzyme site are corresponding to expression vector plasmid pET-28b (+) (Novagen company product, Cat.No.69865.3) the selectivity restriction enzyme site on.With the pBS-0060E11 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-006OE11 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 pET-28 (+) are carried out double digestion with NheI and BamHI, reclaim big fragment respectively, and connect with the T4 ligase enzyme.Connect product and transform, after the dull and stereotyped overnight incubation of the LB that contains kantlex (final concentration 30 μ g/ml), use the colony polymerase chain reaction (PCR) method screening positive clone, and check order with the big enterobacterial DH5 of Calcium Chloride Method α.Select the correct positive colony of sequence (pET-0060E11) 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-0060E11) 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 people DBH and the PAM20 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 20kDa place.This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end are identical with 15 amino-acid residues of N-end shown in＜210〉2 as a result.

Embodiment 6 anti-people DBH and PAM20 production of antibodies

With synthetic following people DBH of Peptide synthesizer (PE company product) and the specific polypeptide of PAM20: NH ₂-Met-Leu-Lys-Ala-Lys-Ile-Leu-Phe-Val-Gly-Pro-Cys-Glu-Ser-Gly-COOH

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 people DBH and PAM20 specifically.

Sequence table

＜110〉Bode Gene Development Co., Ltd., Shanghai

＜120〉a kind of new polypeptide--the polynucleotide of people DBH and PAM20 and this peptide species of coding

<130>0060e11

<160>2

<170>PatentIn?version?3.1

<210>1

<211>1005

<212>DNA

<213>Homo?sapiens

<220>

<221>CDS

<222>(105)..(662)

<223>

<400>1

ggggcctctc?tccgtcgcca?tggaaacgaa?agcggccaag?tagagctccg?tcctgacgcg 60

ccgcctcccg?tgggctccgg?ccggctaagc?cgcggcggac?aact?atg?ctg?aaa?gcc 116

Met?Leu?Lys?Ala

1

aag?atc?ctc?ttc?gtg?ggg?cct?tgc?gag?agt?gga?aaa?act?gtt?ttg?gcc 164

Lys?Ile?Leu?Phe?Val?Gly?Pro?Cys?Glu?Ser?Gly?Lys?Thr?Val?Leu?Ala

5 10 15 20

aac?ttt?ctg?aca?gaa?tct?tct?gac?atc?act?gaa?tac?agc?cea?acc?caa 212

Asn?Phe?Leu?Thr?Glu?Ser?Ser?Asp?Ile?Thr?Glu?Tyr?Ser?Pro?Thr?Gln

25 30 35

gga?gtg?agg?atc?cta?gaa?ttt?gag?aac?ccg?cat?gtt?acc?agc?aac?aac 260

Gly?Val?Arg?Ile?Leu?Glu?Phe?Glu?Asn?Pro?His?Val?Thr?Ser?Asn?Asn

40 45 50

aaa?ggc?acg?ggc?tgt?gaa?ttc?gag?cta?tgg?gac?tgt?ggt?ggc?gat?gct 308

Lys?Gly?Thr?Gly?Cys?Glu?Phe?Glu?Leu?Trp?Asp?Cys?Gly?Gly?Asp?Ala

55 60 65

aag?ttt?gag?tcc?tgc?tgg?ccg?gcc?ctg?atg?aag?gat?gct?cat?gga?gtg 356

Lys?Phe?Glu?Ser?Cys?Trp?Pro?Ala?Leu?Met?Lys?Asp?Ala?His?Gly?Val

70 75 80

gtg?atc?gtc?ttc?aat?gct?gac?atc?cca?agc?cac?cgg?aag?gaa?atg?gag 404

Val?Ile?Val?Phe?Asn?Ala?Asp?Iie?Pro?Ser?His?Arg?Lys?Glu?Met?Glu

85 90 95 100

atg?tgg?tat?tcc?tgc?ttt?gtc?caa?cag?ccg?tcc?tta?cag?gac?aca?cag 452

Met?Trp?Tyr?Ser?Cys?Phe?Val?Gln?Gln?Pro?Ser?Leu?Gln?Asp?Thr?Gln

105 110 115

tgt?atg?cta?att?gca?cac?cac?aaa?cca?ggc?tct?gga?gat?gat?aaa?gga 500

Cys?Met?Leu?Ile?Ala?His?His?Lys?Pro?Gly?Ser?Gly?Asp?Asp?Lys?Gly

120 125 130

agc?ctg?tct?ttg?tcg?cca?ccc?ttg?aac?aag?ctg?aag?ctg?gtg?cac?tca 548

Ser?Leu?Ser?Leu?Ser?Pro?Pro?Leu?Asn?Lys?Leu?Lys?Leu?Val?His?Ser

135 140 145

aac?ctg?gaa?gat?gac?cct?gag?gag?atc?cgg?atg?gaa?ttc?ata?aag?tat 596

Asn?Leu?Glu?Asp?Asp?Pro?Glu?Glu?Ile?Arg?Met?Glu?Phe?Ile?Lys?Tyr

150 155 160

tta?aaa?agc?ata?atc?aac?tcc?atg?tct?gag?agc?aga?gac?agg?gag?gag 644

Leu?Lys?Ser?Ile?Ile?Asn?Ser?Met?Ser?Glu?Ser?Arg?Asp?Arg?Glu?Glu

165 170 175 180

atg?tca?att?atg?acc?tag?ccagccttca?cctgggactg?ccacatcccc 692

Met?Ser?Ile?Met?Thr

185

agtgaaatca?gcatgtttct?cggtgcagat?ctgaaatcac?atccagctcc?tgatgttttc 752

ttctccctct?gactgcagag?gaagtgttcc?tacctgcagg?aaggcacctg?tcacacaggg 812

cgttcactca?gaccatctgt?gctctgccct?gagttcagtt?gagaaaatcc?tattatcaaa 872

tttggatttc?ctggccccag?aacttcccaa?agacctgtaa?aatggaggga?tttaccacct 932

cacatatgtc?cagttaaaca?gtttgtggac?ttgtaaccgt?cgcagcccaa?tgatacaaca 992

gtagtttaat?cac 1005

<210>2

<211>185

<212>PRT

<213>?Homo?sapiens

<400>?2

Met?Leu?Lys?Ala?Lys?Ile?Leu?Phe?Val?Gly?Pro?Cys?Glu?Ser?Gly?Lys

1 5 10 15

Thr?Val?Leu?Ala?Asn?Phe?Leu?Thr?Glu?Ser?Ser?Asp?Ile?Thr?Glu?Tyr

20 25 30

Ser?Pro?Thr?Gln?Gly?Val?Arg?Ile?Leu?Glu?Phe?Glu?Asn?Pro?His?Val

35 40 45

Thr?Ser?Asn?Asn?Lys?Gly?Thr?Gly?Cys?Glu?Phe?Glu?Leu?Trp?Asp?Cys

50 55 60

Gly?Gly?Asp?Ala?Lys?Phe?Glu?Ser?Cys?Trp?Pro?Ala?Leu?Met?Lys?Asp

65 70 75 80

Ala?His?Gly?Val?Val?Ile?Val?Phe?Asn?Ala?Asp?Ile?Pro?Ser?His?Arg

85 90 95

Lys?Glu?Met?Glu?Met?Trp?Tyr?Ser?Cys?Phe?Val?Gln?Gln?Pro?Ser?Leu

100 105 110

Gln?Asp?Thr?Gln?Cys?Met?Leu?Ile?Ala?His?His?Lys?Pro?Gly?Ser?Gly

115 120 125

Asp?Asp?Lys?Gly?Ser?Leu?Ser?Leu?Ser?Pro?Pro?Leu?Asn?Lys?Leu?Lys

130 135 140

Leu?Val?His?Ser?Asn?Leu?Glu?Asp?Asp?Pro?Glu?Glu?Ile?Arg?Met?Glu

145 150 155 160

Phe?Ile?Lys?Tyr?Leu?Lys?Ser?Ile?Ile?Asn?Ser?Met?Ser?Glu?Ser?Arg

165 170 175

Asp?Arg?Glu?Glu?Met?Ser?Ile?Met?Thr

180 185

Claims (10)

1, a kind of isolated polypeptide-people DBH and PAM20 is characterized in that it includes: the polypeptide of the aminoacid sequence shown in＜210〉2 or the active fragments of its polypeptide, analogue or derivative.

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

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

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

(a) coding have＜210〉2 shown in the 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 105-662 position sequence or＜210〉1 in the sequence of 1-1005 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 people DBH and the active polypeptide of PAM20 is characterized in that described method comprises:

(a) under expressing human DBH and PAM20 condition, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate and have people DBH and the active polypeptide of PAM20.

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with people DBH and PAM20 specificity bonded antibody.