CN1298882A - Human Na-dependent phosphate cotransporter 35 and its coding sequence - Google Patents

Abstract

The present invention relates to a new polypeptide-haman sodium dependent type phosphate co-transfer protein 35, polynucleotide for encoding said polypeptide, and method of producing said polypeptide using DNA recombination technology. The present invention also discloses the method of treating various diseases using said polypeptide such as hypophasphaturia, hypercalcemia, hypophosphatemic recikets, nephritis etc.. The present invention also discloses antagonistic for resisting the said polypeptide and its therapeutic action.

Description

People's NTP35 and encoding sequence thereof

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide---NTP35 (abbreviating " NTP35 " as), 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.

Phosphorus is one of must element in the organism, and its balance in animal body depends on that regulation and control are eaten, intestinal absorption, bone deposition and heavily absorb and renal excretion and heavily absorb etc.Relating to phosphoric acid salt renal excretion and re-absorbed major protein is the sodium/phosphorus cotransporter (NPT) that is positioned at the kidney proximal tubule.

Present known kidney has four types of Na-dependent inorganic phosphorus cotransporters.Wherein, II type translocator (NPT2) is the main action protein of kidney inorganic phosphorus running balance, and it is subjected to inorganic phosphorus that diet eats and the regulation and control of hormone Parathyroid (people (1999) J.Biol.Chem Vol 274 such as Shinsuke Kido, Issue40,28256-2826, Oct1).I type (NPT1/SLC17A1), III type (NPT3/SLC17A2), IV type (NPT4/SLC17A3) also have transhipment other molecules such as organic anionic effect concurrently, and (Biber J waits people (1996) Kidney Int 1996 Apr; 49 (4): 981-5).

In the small intestinal mucosa tissue, the sequence of people such as Shibui discovery and NPT1 similar structures, called after SLC17A4. (people (1999) Hum.Genet.44:190-192 such as Shibui) in addition, specific Na-dependent inorganic phosphorus translocator (BNPT) is also arranged in the brain, and BNPT is at conservative (people (1994) the Proc Natl Acad sci USA Jun 7 such as Ni B of spinal animal evolutionary process camber; 91 (12): 5607-11).The common constructional feature that has these NPT albumen promptly all contains 6-8 and strides diaphragm area, and this illustrates that this proteinoid is a membranin.

NPT is positioned on the cytolemma, utilizes the inside and outside Na ion concentration difference of cell and the electro-chemical potential gradient that causes, thereby transportation phosphoric acid salt is crossed over cytolemma people (1999) Hum.Genet.44:190-192 such as () Shibui.

Discover, because the unusual and phosphoric acid metabolism confusion of NPT and coding nucleic acid thereof and some relative diseases such as the low-phosphorous acidaemia of familial, low-phosphorous acidemia rickets and the hypercalcemia that causes thereof, low-phosphorous acid bone disease, hemochromatosis etc. are relevant.Therefore, significant for therapeutic purpose research and development people's NPT.

An object of the present invention is to provide isolating new NPT polypeptide---NTP35 (abbreviating " NTP35 " as) 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 NPT35 that encodes.

Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain the NPT35 that encodes.

Another object of the present invention provides the method for producing NPT35.

Another object of the present invention provides at polypeptide of the present invention---the antibody of NPT35.

Another object of the present invention has provided at polypeptide of the present invention---the simulated compound of NPT35, antagonist, agonist, inhibitor.

Another object of the present invention provides the method for the diagnosis disease relevant unusually with NPT35 with treatment.

In a first aspect of the present invention, novel isolated NPT35 is provided, preferably, this polypeptide is the people, and it comprises: have the polypeptide of SEQ ID NO:2 aminoacid sequence or its conservative property variation polypeptide or its active fragments or its reactive derivative, analogue.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

In a second aspect of the present invention, the polynucleotide of these polypeptide of separated coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned NPT35 of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 24-968 position among the SEQ ID NO:1; (b) has the sequence of 1-2670 position among the SEQ IDNO:1.

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.

The polypeptide of novelty of the present invention according to homology relatively can predicate new people's NTP35 (being designated hereinafter simply as NPT35), and the characteristic that this NPT35 has a NPT family strides diaphragm area, thereby has similar biological function.

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 NTP35 or NPT35 " is meant that NTP35 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 NPT35 of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of NPT35 polypeptide can be used amino acid sequence analysis.

The invention provides a kind of new polypeptide---NPT35, it is made up of the aminoacid sequence shown in the SEQ ID NO:2 basically.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analogue of NTP35.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of NPT35 of the present invention or active polypeptide basically.The fragment of polypeptide of the present invention, derivative or analogue can be: one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue) (i) are arranged, and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merge formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying).According to the instruction of this paper, these fragments, derivative and analogue belong within the known scope of those skilled in the art.

In one embodiment of the invention, the isolating nucleic acid (polynucleotide) that is provided substantially is made up of the polynucleotide that coding has a polypeptide of SEQ ID NO:2 aminoacid sequence.More preferably, 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 2670 bases, its open reading frame (24bp to 968bp) 314 amino acid of having encoded.Relatively find according to amino acid sequence homologous, the inorganic phosphorus translocator of this polypeptide and fruit bat [Drosophilaananassae] has 42% homology, and this polypeptide has the membrane structure of striding of NPT family, and deducibility goes out this new people NPT35 and has the similar 26S Proteasome Structure and Function of NPT gene family.

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " are meant that in the present invention coding has protein or the polypeptide of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence that has only mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

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

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is a kind of 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 is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with sequence hybridization described above.As used herein, " nucleic acid fragment " length contain 10 Nucleotide at least, better be at least 30 Nucleotide, be more preferably at least 50 Nucleotide, 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 NTP35.

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

The special polynucleotide sequence of coding NTP35 of the present invention can obtain with several different methods.For example, separate polynucleotide with hybridization technique well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect homologous polynucleotide sequence and 2) antibody screening of expression library to be to detect the polynucleotide passage of the clone with common structure feature.

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

In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.The direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.The more frequent method of selecting for use is the separation of cDNA sequence.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qiagene) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination polymerase chain reaction technique, 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 NPT35 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.Dna probe can carry out mark with radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

In (4) kind method, detect the protein product of NTP35 genetic expression and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

The method (Saiki, et al.Science1985:230:1350-1354) of using round pcr DNA amplification/RNA is optimized for and obtains 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 or the nucleotide sequence of its various dna fragmentations, and available 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.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 NPT35 encoding sequence through the genetically engineered generation, and the method that produces polypeptide of the present invention through recombinant technology.

Among the present invention, the polynucleotide sequence of coding NTP35 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 NPT35 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 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 NPT35 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.Another kind method is to use MgCl ₂Handle.If 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.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), utilize polynucleotide sequence of the present invention to can be used to express or produce the NTP35 of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention people NTP35, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;

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

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

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

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

The antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in disease treatment, for example, can treat the low-phosphorous acidaemia of familial, low-phosphorous acidemia rickets and the hypercalcemia that causes thereof, low-phosphorous acid bone disease, hemochromatosis, ephritis etc.

Comprise that the humans and animals experimenter treats the NPT35 of effective dose, its biologically active derivatives or agonist, can treat, alleviate and/or prevent to be selected from following disease: the low-phosphorous acidaemia of familial, low-phosphorous acidemia rickets and the hypercalcemia that causes thereof, low-phosphorous acid bone disease, ephritis (including but not limited to the ephritis that low-phosphorous acidaemia causes) etc.

Comprise that the humans and animals experimenter treats the NPT35 antagonist or the inhibitor of effective dose, can treat, alleviates and/or prevent to be selected from following disease: hemochromatosis; Ephritis (including but not limited to the ephritis that hyperphosphatemia causes) etc.

The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) NPT35.Agonist improves NPT35 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, mammalian cell be cultivated with the albumen of the present invention of mark.Measure the medicine raising then or check the proteic ability of the present invention.

The antagonist of NPT35 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of NPT35 can combine and eliminate its function with the NPT35 polypeptide, 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, NPT35 can be added during bioanalysis measures, by measuring compound interactional influence between NPT35 and its acceptor is determined whether compound is antagonist.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 NPT35 bonded peptide molecule obtains.During screening, generally tackle the NPT35 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 NPT35 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 NPT35 direct injection of the production of polyclonal antibody immune animal (as rabbit, mouse, rat etc.) obtains.There is multiple adjuvant to 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 NPT35 includes but not limited to: and 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 (Morrisonet al, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.Pat No.4946778) also can be used for producing the single-chain antibody of anti-NPT35.

The antibody of anti-NPT35 can be used in the immunohistochemistry technology, detects the NPT35 in the biopsy specimen.

With the also available labelled with radioisotope of NPT35 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 NPT35 high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the NPT35 positive cells.

The disease that antibody among the present invention can be used for treating or prevention is relevant with NPT35.The antibody that gives suitable dosage can stimulate or block generation or the activity of NPT35.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization NPT35 level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The NPT35 level that is detected in the test can be with laying down a definition the importance of NPT35 in various diseases and be used to the disease of diagnosing NPT35 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 NPT35 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 NPT35 or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the NPT35 that expresses variation, to suppress endogenic NPT35 activity.For example, a kind of NPT35 of variation can be the NPT35 that shortens, lacked signal conduction function territory, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the disease of NPT35 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 NPT35 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding NPT35 is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding NPT35 can be packaged in the liposome and then 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 NPT35mRNA 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 NPT35 can be used for diagnosing the disease relevant with NPT35.The unconventionality expression of the expression that the polynucleotide of coding NPT35 can be used for detecting NPT35 NPT35 whether or under morbid state.As the dna sequence dna of the NPT35 that encodes can be used for biopsy specimen is hybridized to judge the expression situation of NPT35.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 NPT35 with the special primer of NPT35.

The sudden change that detects the NPT35 gene also can be used for the disease of diagnosing NPT35 relevant.The form of NPT35 sudden change comprises that the point mutation compared with normal wild type NPT35DNA sequence, 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).Yet, have only chromosomal marker thing seldom to can be used for the marker chromosomes position now 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.NPT35 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 NPT35 will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

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

Fig. 1 be inventor NPT35 and the amino acid sequence homology comparison diagram.The top sequence is NPT35, the below sequence be fruit bat infer the inorganic phosphorus cotransporter.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 NPT35.35kDa is proteinic molecular weight.Swimming lane 1 is a molecular weight marker, and swimming lane 2 is the albumen before separating with 3; Swimming lane 4 is the NPT35 albumen after separating.The arrow indication is isolated protein band.

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

The clone of embodiment 1:NPT35

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 Quick mRNA separating kit (Qiegene company product).2ug poly (A) mRNA forms cDNA through reverse transcription.CDNA fragment orientation is inserted on the multiple clone site of pBSK (+) carrier (Clontech company product) with Smart cDNA clone's test kit (available from Clontech), transforms DH5 α, bacterium forms the cDNA library.Measure the sequence of all clones' 5 ＇ and 3 ＇ ends with dyestuff termination circulating reaction sequencing kit (Perkin-Elmer company product) and ABI377 automatic sequencer (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genbank) measured are compared, found that the cDMA sequence of one of them clone 0978b08 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 0957b08 clone is 2670bp (shown in SEQ ID NO:1), from 24bp to 968bp the open reading frame (ORF) of a 945bp, the new protein (shown in HSEQ ID NO:2) of encoding arranged.This clone is named as pBS-0957b08, encoded protein matter called after NPT35.

Embodiment 2:cDNA clone's homology retrieval

With sequence and the encoded protein sequence thereof of NPT35 of the present invention, with Blast program (BasiclocalAlignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], carry out the homology retrieval at databases such as Genbank, Swissport.With the highest gene of NPT35 homology of the present invention be a kind of known fruit bat infer the inorganic phosphorus cotransporter, its encoded protein number is AF024691 in the access of Genbank.Protein homology the results are shown in accompanying drawing 1, both height homologies, and its homogeny is 42%; Similarity is 60%.Spscan program and McGeoch Scan according to GCG version10.0 software package infer that NPT35 is a secretory protein, and its signal peptide position is 4-71.

In addition, NPT35 membranin prediction of the present invention has eight sections to stride diaphragm area, and they lay respectively at amino acid 23-43,48-68,99-119,147-167,185-205,209-229,239-259,277-297 position.

Embodiment 3: with the gene of RT-PCR method clones coding NPT35

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:

Primer 1:5 ＇-GAGAGGGTGTTACATTTCCAGCCATGCATGCCATGT-3 ＇ (SEQ ID NO:3)

Primer 2: 5 ＇-AAATCAATTCACAAAGGTTTATTTTGATGATTTAT-3 ＇ (SEQ ID NO:4)

The forward sequence that the 1bp that primer 1 is held for 5 ＇ that are positioned at SEQ ID NO:1 begins;

Primer 2 be SEQ ID NO:1 in 3 ＇ end reverse sequence.

The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/L Tris-Cl, (pH8.5), 1.5mmol/L MgCl ₂, 200 μ mol/L dNTP, 10pmol primer, the Taq archaeal dna polymerase of 1U (Clontech company product).Go up by 25 cycles of following conditioned response at PE9600 type DNA thermal cycler (Perkin-Elmer company): 94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of beta-actin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows that the dna sequence dna of PCR product and the 1-2670bp shown in the SEQ ID NO:1 are identical.

Embodiment 4:Northern blotting is analyzed the NPT35 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 gRNA, 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 NPT35 coding region sequence (24bp to 968bp) of pcr amplification shown in Figure 1.Will ³²The probe of P-mark (about 2 * 10 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.

Example 5: vivoexpression, separation and the purifying of reorganization NPT35

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

Primer 3:5 ＇-GCTAGCATGCATGCCATGTGGTCTTCTTGGGCT-3 ＇ (SEQ ID No:5)

Primer 4:5 ＇-GGATCCGTTCCTTCAGTGTCTGTGTCCATGGTG-3 ＇ (SEQ ID No:6)

The 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-0957b08 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0957b08 plasmid 10pg, primer 3 and primer 4 among the cumulative volume 50 μ l and be respectively l0pmol, Advantage polymerase Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 60 ℃ of 30s, 72 ℃ 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-0957b08) 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-0957b08) 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 NPT35 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 35kDa place.This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end hold 15 amino-acid residues identical with the N-shown in the SEQ ID NO:2 as a result.

Embodiment 6 anti-NPT35 production of antibodies

Synthesize the specific polypeptide of following NPT35 with Peptide synthesizer (PE company product):

NH ₂-Met-His-Ala-Met-Trp-Ser-Ser-Trp-Ala-Pro-Pro-Leu-Glu-Arg-Ser-OH(SEQ?ID?NO：7)。Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, et al.Immunochemistry, 1969; 6:43.Add the complete Freund's adjuvant immunizing rabbit with the above-mentioned hemocyanin polypeptide complex of 4mg, add the incomplete Freund's adjuvant booster immunization once with the hemocyanin polypeptide complex again after 15 days.Employing is done the titre that ELISA measures antibody in the rabbit anteserum through the titer plate of 15 μ g/ml bovine serum albumin polypeptide complex bag quilts.From the rabbit anteserum of antibody positive, separate total IgG with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.Immuno-precipitation proof antibody purified can combine with NPT35 specifically.

Embodiment 7: polynucleotide passage of the present invention is as the application of hybridization probe

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

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

One, probe selects for use

From polynucleotide SEQ ID NO:1 of the present invention, select oligonucleotide fragment as hybridization probe, the several aspects that should follow following principle and need to consider:

1, the probe size preferable range is a 18-50 Nucleotide;

2, GC content is 30%-70%, and surpassing then, non-specific hybridization increases;

3, probe interior should not have complementary region;

4, what meet above condition can be used as the primary election probe, further do the computer sequential analysis then, comprise this primary election probe is carried out homology relatively with its source sequence area (being SEQ ID NO:1) and other known genome sequence and complementary district thereof respectively, if with the homology in non-target molecule zone greater than 85% or there are 15 continuous bases of surpassing identical, then this primary election probe is general just should not use;

5, whether the primary election probe finally is chosen to be the probe of actual application value also should further be determined by experiment.

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

Probe 1 (probe1) belongs to first kind probe, with complete homology of gene fragment or the complementation (41Nt) of SEQ ID NO:1: 51-GAGAGGGTGTTACATTTCCAGCCATGCATGCCATGTGGTCT-3 ＇ (SEQ ID NO:8)

Probe 2 (probe2) belongs to the second class probe, is equivalent to gene fragment or its complementary segmental replacement mutant nucleotide sequence (41Nt) of SEQ ID NO:1: 51-ACGCTTAGATCCAGCCATGCATGCCATGTCTTGCACAACAT-3 ＇ (SEQ ID NO:9)

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

Specimen preparation:

1, from fresh or frozen tissue, extract DNA

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

2, the phenol extraction process of DNA

Step: 1) wash cell, centrifugal 10 minutes of 1000g with the cold PBS of 1-10ml.2) with the sedimentary cell of cold cell pyrolysis liquid resuspension (the minimum of applications 100ul lysis buffer of 1 * 108 cell/ml).3) adding SDS is 1% to final concentration, if before re-suspended cell SDS is directly joined in the cell precipitation, cell may form big agglomerate and be difficult to fragmentation, and the overall yield that reduces.This point is in extracting＞10 ⁷Especially severe during cell.4) add Proteinase K to final concentration 200ug/ml.5) 50 ℃ of insulation reaction 1 hour or 37 ℃ gently jolting spend the night.6) use equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, in little centrifuge tube centrifugal 10 minutes.Two corresponding clear separation, otherwise carry out centrifugal again.7) water is transferred to new pipe.8) use the equal-volume chloroform: primary isoamyl alcohol (24: 1) extracting, centrifugal 10 minutes.9) water that will contain DNA is transferred to new pipe.Carry out purifying and the ethanol sedimentation of DNA then.

3, the purifying of DNA and ethanol sedimentation

Step: 1) 1/10 volume 2mol/L sodium-acetate and 2 times of cold 100% ethanol of volume are added in the dna solution mixing.-20 ℃ of placements 1 hour or to spending the night.2) centrifugal 10 minutes.3) careful sucking-off or pour out ethanol.4) with 70% cold ethanol 500ul washing precipitation, centrifugal 5 minutes.5) careful sucking-off or pour out ethanol.With the cold washing with alcohol precipitation of 500ul, centrifugal 5 minutes.6) careful sucking-off or pour out ethanol is inverted on thieving paper then residual ethanol is flow to end.Dry air 10-15 minute, so that the volatilization of surperficial ethanol.Note not making the precipitation complete drying, otherwise dissolve again than difficulty.7) with small volume TE or the resuspended DNA precipitation of water.Low speed vortex vibration or use the dropper pressure-vaccum, the while increases TE gradually, is mixed to DNA and fully dissolves, and every 1-5 * 106 cells are extracted approximately adds 1ul.

Below the 8-13 step only be used for must removing when depolluting, otherwise can directly carry out the 14th step.

8) RNA enzyme A is added in the dna solution, final concentration is 100ug/ml, and 37 ℃ are incubated 30 minutes.9) add SDS and Proteinase K, final concentration is respectively 0.5% and 100ug/ml.37 ℃ are incubated 30 minutes.10) use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extractive reaction liquid, centrifugal 10 minutes.11) carefully shift out water, use isopyknic chloroform: primary isoamyl alcohol (24: 1) extracting again, centrifugal 10 minutes.12) carefully shift out water, add 1/10 volume 2mol/L sodium-acetate and the cold ethanol of 2.5 volumes, mixing put-20 ℃ 1 hour.13) with 70% ethanol and 100% washing with alcohol precipitation, dry air, resuspended nucleic acid, process is with the 3-6 step.14) measure A260 and A280 to detect purity and the productive rate of DNA.15) deposit in-20 ℃ after the packing.

The preparation of 4 sample films:

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

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

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

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

The mark of 5 probes

1) 3 μ lProbe (0.1OD/10 μ l) add 2 μ lKinase damping fluids, 8-10 uCi γ- ³²The P-dATP+2U kinases is to add to final volume 20 μ l.

2) 37 ℃ are incubated 2 hours.

3) add the tetrabromophenol sulfonphthalein indicator (BPB) of 1/5 volume.

4) cross Sephadex G-50 post.

5) to having ³²Before washing out, P-Probe begins to collect first peak (available watch-dog monitoring).

6) 5/pipe, collect the 10-15 pipe.

7) monitor isotopic weight with liquid glimmer instrument

8) merge and to be required preparation behind the collection liquid of first peak ³²(second peak is free γ to P-Probe ³²P-dATP).

6 prehybridizations

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

7 hybridization

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

8 wash film: the washing of film is with one of following two kinds of conditions:

High strength is washed film:

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

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

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

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

Low strength is washed film:

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

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

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

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

9X-light autography:

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

Experimental result:

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

Sequence table (1) general information: (i) applicant:

(A) name: Shengyuan Gene Development Co., Ltd., Shanghai

(B) street: No. 668 610 Room, East Road, Beijing

(C) city: Shanghai

(D) country: China

(E) postcode: 200001 (ii) denominations of invention: people's NTP35 and encoding sequence thereof be the sequence number (iii): the information of 9 (2) SEQ ID NO:1: (i) sequence signature:

(A) length: 2670bp

(B) type: nucleic acid

(C) chain: two strands

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：1：GAGAGGGTGT TACATTTCCA GCCATGCATG CCATGTGGTC TTCTTGGGCT CCCCCTCTTG 60AAAGAAGCAA ACTTCTTAGC ATTTCATATG CAGGAGCACA GCTTGGGACA GTAATTTCTC 120TTCCTCTTTC TGGAATAATT TGCTACTATA TGAATTGGAC TTATGTCTTC TACTTTTTTG 180GTACTATTGG AATATTTTGG TTTCTTTTGT GGATCTGGTT AGTTAGTGAC ACACCACAAA 240AACACAAGAG AATTTCCCAT TATGAAAAGG AATACATTCT TTCATCATTA AGAAATCAGC 300TTTCTTCACA GAAGTCAGTG CCGTGGGTAC CCATTTTAAA ATCCCTGCCA CTTTGGGCTA 360TCGTAGTTGC ACACTTTTCT TACAACTGGA CTTTTTATAC TTTATTGACA TTATTGCCTA 420CTTATATGAA GGAGATCCTA AGGTTCAATG TTCAAGAGAA TGGGTTTTTA TCTTCATTGC 480CTTATTTAGG CTCTTGGTTA TGTATGATCC TGTCTGGTCA AGCTGCTGAC AATTTAAGGG 540CAAAATGGAA TTTTTCAACT TTATGTGTTC GCAGAATTTT TAGCCTTATA GGAATGATTG 600GACCTGCAGT ATTCCTGGTA GCTGCTGGCT TCATTGGCTG TGATTATTCT TTGGCCGTTG 660CTTTCCTAAC TATATCAACA ACACTGGGAG GCTTTTGCTC TTCTGGATTT AGCATCAACC 720ATCTGGATAT TGCTCCTTCG TATGCTGGTA TCCTCCTGGG CATCACAAAT ACATTTGCCA 780CTATTCCAGG AATGGTTGGG CCCGTCATTG CTAAAAGTCT GACCCCTGAT AACACTGTTG 840GAGAATGGCA AACCGTGTTC TATATTGCTG CTGCTATTAA TGTTTTTGGT GCCATTTTCT 900TTACACTATT CGCCAAAGGT GAAGTACAAA ACTGGGCTCT CAATGATCAC CATGGACACA 960GACACTGAAG GAACCAATAA ATAATCCTGC CTCTATTAAT GTATTTTTAT TTATCATGTA 1020ACCTCAAAGT GCCTTCTGTA TTGTGTAAGC ATTCTATGTC TTTTTTTAAT TATACTTGTA 1080TTAGATTTTT AAGGCCTATA ATCATGAAAT ATCACTAGTT GCCAGAATAA TAAAATGAAC 1140TGTGTTTAAT TATGAATAAT ATGTAAGCTA GGACTTCTAC TTTAGGTTCA CATACCTGCC 1200TGCTAGTCGG GCAACATGAA GTAGGACAGT TCTGTTGATT TTTTAGGGCC ATACTAAAGG 1260GAATGAGCTG AAACAGACCT CCTGATACCT TTGCTTAATT AAACTAGATG ATAATTCTCA 1320GGTACTGATA AACACCTGTT GTTGTTCACT TTCCTCATAA AAATTGTCAG CTCTCTCTGA 1380CACTTAGACC TCAAACTTTA GCATCTCTGT GGAGCTGCCA TCCACTGTAT AATTTCGCCT 1440GGCAACTGGA CTGAGGGGAG TGTGCCCAGG CAGCTGCCAA GCACTCCCTC CCTGGCTTCA 1500GGGTCAGAGT GCCCAGCGTT TATCAGAGGC AGCATCCAAG CCCAGAGCCA GTGTCGACTC 1560TTCGGCTGGT GCCTTTCCTC TGAGGGGCTA TCAATGTGTA GATAAAGCCC TGAGTAGGCA 1620AGAGCAGTGA GATCCACTGC TATGGTCTTG GTACATCCTC AAACTTTCCC TTCCCAGCAC 1680AGAGGAATAT TGGCTGGCAT GCAACCTGCA AAAGAAAAAT GCGAAGCGGC CGGGCACGGT 1740GGCTCATGCC TGTAATCCCA GCACTTTGGG GGGCTGAGGT GGGCGAATCA TGAGATCAGG 1800AGTTCGAGAC CAGCCTGGCC AGCATGGTGA AACCCCATCT CTACTAAAAA TACAAAAAAT 1860TAGCTGGGCG TGGTGACGGG CGCCTGTAAT CCCAGATACT CAGGAGGCTG AGGTAGGAGA 1920ATCACTTGAA CCTGGGAGGT GGAAGTTGCA GTGAACCAAG ATCACGCCAC TGCACTCCAG 1980CCTGGGCGAT GGAGCGAGAC TCCAACTCAA AAAAAAAAAA AAGAATAAAG AAAGAAAAGT 2040GCGATGCCCA GTCAATCACA AATAAGATCA TCCTGGTTTA AATCTACTCT CACATGGATC 2100ACAGTATAAA TTTCTATGTG CTGTGTTTTG TTCGTTTGTA TTTTGTAGAG ATGGGGTCTC 2160GTTTTGTCGC CCAGGCTGGT TTTGAACTCC TGGCTTCAAG CGATCCTCCT GTCTCGGCCT 2220CACAAAGTGT TGAGACTACA GGCATGAGCC ACTGTGCCCA GCCTGTTCTA TGTTTTTAAG 2280CTACACGAGA ATTTTTTTTT TAATTAATTC TCACTGTTTG TTCAGTCTGT CTTCATCTAA 2340GTTTGTGTTG CAGTTTAAAG TTAAAGTGAC TTTTAAAGGC CACATCACCT GAGACTAGGG 2400TAATCATCTT TACTTCTGGT TCCTGAAATC ATATTTTTCC AGTGGACCAC CCTCCAGTGG 2460CTGTGGTTGT TGAGCATGCT TTCAGAACAC CTATGTGGCT TAAAACTTAG TTTATGTTTT 2520GTGTTCAACA CTACGTGTAA TATTTTAAAA CTGTTTAATG TGATGTGAAT ACATTTATGT 2580ACATTTATTT TTAAATTTGT AAATAGCTTT AAATTGCTAT GGCAATGTTT CTTTTATAAA 2640TCATCAAAAT AAACCTTTGT GAATTGATTT 2670 ( 2 ) SEQ ID NO：2： ( i ) ：

(A) length: 314 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO：2：Met His Ala Met Trp Ser Ser Trp Ala Pro Pro Leu Glu Arg Ser 15Lys Leu Leu Ser Ile Ser Tyr Ala Gly Ala Gln Leu Gly Thr Val 30Ile Ser Leu Pro Leu Ser Gly Ile Ile Cys Tyr Tyr Met Asn Trp 45Thr Tyr Val Phe Tyr Phe Phe Gly Thr Ile Gly Ile Phe Trp Phe 60Leu Leu Trp Ile Trp Leu Val Ser Asp Thr Pro Gln Lys His Lys 75Arg Ile Ser His Tyr Glu Lys Glu Tyr Ile Leu Ser Ser Leu Arg 90Asn Gln Leu Ser Ser Gln Lys Ser Val Pro Trp Val Pro Ile Leu 105Lys Ser Leu Pro Leu Trp Ala Ile Val Val Ala His Phe Ser Tyr 120Asn Trp Thr Phe Tyr Thr Leu Leu Thr Leu Leu Pro Thr Tyr Met 135Lys Glu Ile Leu Arg Phe Asn Val Gln Glu Asn Gly Phe Leu Ser 150Ser Leu Pro Tyr Leu Gly Ser Trp Leu Cys Met Ile Leu Ser Gly 165Gln Ala Ala Asp Asn Leu Arg Ala Lys Trp Asn Phe Ser Thr Leu 180Cys Val Arg Arg Ile Phe Ser Leu Ile Gly Met Ile Gly Pro Ala 195Val Phe Leu Val Ala Ala Gly Phe Ile Gly Cys Asp Tyr Ser Leu 210Ala Val Ala Phe Leu Thr Ile Ser Thr Thr Leu Gly Gly Phe Cys 225Ser Ser Gly Phe Ser Ile Asn His Leu Asp Ile Ala Pro Ser Tyr 240Ala Gly Ile Leu Leu Gly Ile Thr Asn Thr Phe Ala Thr Ile Pro 255Gly Met Val Gly Pro Val Ile Ala Lys Ser Leu Thr Pro Asp Asn 270Thr Val Gly Glu Trp Gln Thr Val Phe Tyr Ile Ala Ala Ala Ile 285Asn Val Phe Gly Ala Ile Phe Phe Thr Leu Phe Ala Lys Gly Glu 300Val Gln Asn Trp Ala Leu Asn Asp His His Gly His Arg His 314 ( 2 ) SEQ ID NO：3 ( i )

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:3:GAGAGGGTGT TACATTTCCA GCCATGCATG CCATGT 36 (2) SEQ ID NO:4

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii); Oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:4:AAATCAATTC ACAAAGGTTT ATTTTGATGA TTTA 35 (2) SEQ ID NO:5

(A) length: 31 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:5:GCTAGCATGC ATGCCATGTG GTCTTCTTGG GCT 23 (2) SEQ ID NO:6

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:6:GGATCCGTTC CTTCAGTGTC TGTGTCCATG GTG 23 (2) SEQ ID NO:7: (i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): polypeptide (xi) sequence description: information (i) sequence signature of SEQ ID NO:7:Met His Ala Met Trp Ser Ser Trp Ala Pro Pro Leu Glu Arg Ser 15 (2) SEQ ID NO:8

(A) length: 41 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:8:GAGAGGGTGT TACATTTCCA GCCATGCATG CCATGTGGTC T 41 (2) SEQ ID NO:9

(A) length: 41 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO:9:ACGCTTAGAT CCAGCCATGC ATGCCATGTC TTGCACAACA T 41

Claims (18)

1, a kind of isolating people's NTP35 is characterized in that, it comprises: have the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2 or active fragments, analogue or the derivative of its polypeptide.

2, polypeptide as claimed in claim 1 is characterized in that, the aminoacid sequence of described polypeptide, analogue or derivative has the homogeny with the aminoacid sequence at least 95% shown in the SEQ ID NO:2.

3, polypeptide as claimed in claim 2 is characterized in that, it has the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2.

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

(a) coding has the polynucleotide of the polypeptide of aminoacid sequence shown in the SEQ ID NO:2 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 is characterized in that, described polynucleotide comprise the polynucleotide that coding has aminoacid sequence shown in the SEQ ID NO:2.

6, polynucleotide as claimed in claim 4 is characterized in that, the sequence of described polynucleotide includes the sequence of 1-2670 position among the sequence of 24-968 position among the SEQ ID NO:1 or the SEQ ID NO:1.

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

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

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

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

9, a kind of preparation method with the active polypeptide of people's NTP35 is characterized in that, described method comprises:

(a) under the condition of expressing human NTP35, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate and have the active polypeptide of NTP35.

10, a kind of can with polypeptide bonded antibody, it is characterized in that, described antibody be can with NTP35 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 NTP35.

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

13, the described application of compound of a kind of claim 11 is characterized in that, described compound be used to regulate NTP35 in vivo, the method for external activity.

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

15, as the application of polypeptide as described in the arbitrary claim among the claim 1-3, it is characterized in that it is applied to screen stand-in, the agonist of NTP35, antagonist or inhibitor; Perhaps be used for the peptide finger print identification.

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

17, the described polypeptide of arbitrary claim, polynucleotide or the application of compound among the claim 1-6 and 11, it is characterized in that, form pharmaceutical composition with safe and effective dosage and pharmaceutically acceptable carrier as diagnosis or the treatment disease relevant unusually with NTP35 with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor.

18, the described polypeptide of arbitrary claim, polynucleotide or the application of compound among the claim 1-6 and 11, it is characterized in that, be used for the treatment of the medicine of the low-phosphorous acidaemia of familial, low-phosphorous acidemia rickets and the hypercalcemia that causes thereof, low-phosphorous acid bone disease, hemochromatosis, ephritis with described polypeptide, polynucleotide or compound.

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