CN1269415A - New human thioredoxin related protein and its code sequence - Google Patents

Abstract

The present invention provides a new human hTrp-m protein expressed in hypothalamus of human body and its code sequence. It also provides preparation method of said protein and nucleic acid sequence and method for detecting human hTrp-m nucleic acid and polypeptide in sample.

Description

A kind of new human thioredoxin related protein and encoding sequence thereof

The present invention relates to fields such as molecular biology, zymetology, physiology and genetically engineered.Particularly, the present invention relates to a kind of hTrp-m albumen and nucleotide sequence thereof of in human hypothalamus, expressing.The invention still further relates to the preparation method and the purposes of this albumen and nucleotide sequence.

Thioredoxin (Thioredoxin) is the small molecular protein that extensively exists in a kind of cell, usually different in the redox reaction process, play a part different.(J?Biol?Chem?1997?Dec?5；272(49)：30841-7)。Discover that thioredoxin can cut off the disulfide linkage between the peptide chain.(J?Biol?Chem1998?Jul?24；273(30)：19160-6)。(thioredoxin-relatedprotein mainly is the redox state that auxiliary thioredoxin is regulated cell in intracellular effect Trp) to thioredoxin related protein.

Among the present invention, we are cloned into the homologous gene hTrp-m of home mouse Trp-m gene in human hypothalamus.

Before the present invention comes forth, any human hTrp-m protein sequence and nucleotide sequence of mentioning in the present patent application thereof that disclose or reported do not arranged as yet.

First purpose of the present invention just provides a kind of new people's gene hTrp-m (Genbank AccessionNo.AF221595), and this gene is a people hTrp-m protein gene.

Second purpose of the present invention provides a kind of new people's albumen hTrp-m.

The 3rd purpose of the present invention provides a kind of recombinant technology that utilizes and produces the above-mentioned new people hTrp-m albumen and the method for nucleotide sequence.

The present invention also provides the application of this people hTrp-m protein polypeptide and encoding sequence.

In one aspect of the invention, a kind of isolated dna molecular is provided, this molecule comprises: coding has the nucleotide sequence of the polypeptide of people hTrp-m protein active, shows at least 70% homology from the nucleotides sequence of Nucleotide 101-733 position dna molecular among described nucleotide sequence and the SEQ ID NO.6; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.6 in from the nucleotide sequence hybridization of Nucleotide 101-733 position.Preferably, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.7.More preferably, described sequence has among the SEQ ID NO.6 nucleotide sequence from Nucleotide 101-733 position.

In another aspect of this invention, provide a kind of isolated people hTrp-m protein and peptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.7 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.7 polypeptide of sequence.

In another aspect of this invention, also provide a kind of carrier, it comprises above-mentioned dna molecular.

In another aspect of this invention, also provide a kind of usefulness above-mentioned carrier transformed host cells.This host cell is intestinal bacteria in an example; In another example, this host cell is an eukaryotic cell.

In another aspect of this invention, also provide a kind of generation to have the method for the polypeptide of people hTrp-m protein active, its step is as follows:

(1) nucleotide sequence that coding is had a polypeptide of people hTrp-m protein-active operationally is connected in expression regulation sequence, form people hTrp-m protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 101-733 position among described nucleotide sequence and the SEQ ID NO.6;

(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of people hTrp-m;

(3) under the condition that is fit to expressing human hTrp-m protein polypeptide, the reconstitution cell in the culturing step (2);

(4) isolate polypeptide with people hTrp-m protein-active.

Preferably, the nucleotide sequence that uses in the method has the sequence of 101-733 position among the SEQ ID NO.6.

The present invention also provides and hTrp-m protein polypeptide specificity bonded antibody.

In the present invention, " isolating ", " purifying " or " pure substantially " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

In the present invention, term " people hTrp-m albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with people hTrp-m protein-active is as 101-733 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.6.This degenerate sequence is meant, is arranged in the encoder block 101-733 position Nucleotide of SEQ ID NO.6 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.6 in 101-733 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.7 of also encoding out.This term also comprises can be under the moderate stringent condition, better under the height stringent condition with SEQ ID NO.6 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 101-733 position.This term also comprise with SEQ ID NO.6 in from the homology of nucleotide sequence at least 70% of Nucleotide 101-733 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.

This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.6 with natural people hTrp-m identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.

In the present invention, term " people hTrp-m albumen or polypeptide " refers to have the SEQ IDNO.7 polypeptide of sequence of people hTrp-m protein-active.This term also comprises having and variant form natural human hTrp-m identical function, SEQ ID NO.7 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people hTrp-m and reactive derivative.

The variant form of people hTrp-m polypeptide of the present invention comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low stringent condition can with the coded albumen of the DNA of people hTrp-m DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people hTrp-m polypeptide to obtain.The present invention also provides other polypeptide, as comprises people hTrp-m polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprises the soluble fragments of people hTrp-m polypeptide.Usually, this fragment have people hTrp-m peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

In the present invention, " people hTrp-m conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.7, has 10 at the most, and preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and formed polypeptide at the most.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn

Glu(E)	Asp	Asp
			Gly(G)	Pro；Ala	Ala
His(H)	Asn；Gln；Lys；Arg	Arg
			Ile(I)	Leu；Val；Met；Ala；Phe	Leu
Leu(L)	Ile；Val；Met；Ala；Phe	Ile
			Lys(K)	Arg；Gln；Asn	Arg
Met(M)	Leu；Phe；Ile	Leu
			Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
Pro(P)	Ala	Ala
			Ser(S)	Thr	Thr
Thy(T)	Ser	Ser
			Trp(W)	Tyr；Phe	Tyr
Tyr(Y)	Trp；Phe；Thr；Ser	Phe
			Val(V)	Ile；Leu；Met；Phe；Ala	Leu

Invention also comprises the analogue of people hTrp-m albumen or polypeptide.The difference of these analogues and natural human hTrp-m polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.When producing people hTrp-m polypeptide of the present invention, people hTrp-m encoding sequence operationally can be connected in expression regulation sequence, thereby form people hTrp-m protein expression vector.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.

In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.

The present invention also provides people hTrp-m specificity bonded antibody, comprises polyclonal antibody and monoclonal antibody.

In the present invention, can use a series of methods known in the art to prepare special antibody at people hTrp-m.For example, the people hTrp-m gene product or its antigen fragment of purifying is injected in the animal body to produce polyclonal antibody.Equally, the cell of expressing human hTrp-m or its antigen fragment also can be used for animal is caused immunity and produces antibody.Antibody prepared in accordance with the present invention also can be monoclonal antibody, and these monoclonal antibodies can prepare (for example, Kohler et al., Nature 256:495,1975 with hybridoma technology; Kohler et al., Eur.J.Immunol.6:511,1976; Kohler et al., Eur.J.Immunol.6:292,1976).Antibody of the present invention comprises the antibody that can prevent people hTrp-m function, also can be the antibody that does not influence people hTrp-m function.Each antibody-like can produce by the fragment of people hTrp-m gene product or functional domain are caused immunity, and people hTrp-m gene product and fragment thereof can produce or synthesize with Peptide synthesizer with recombination method.With the people hTrp-m gene product bonded antibody of non-modified forms, can come immune animal to obtain by being used in the gene product that prokaryotic cell prokaryocyte for example produces among the E.coli.With posttranslational modification form such as glycosylation or phosphorylated protein or polypeptide bonded antibody, can obtain by the immune animal that comes that is used in the gene product that produces in eukaryotic cell such as yeast or the insect cell.

People hTrp-m antibody of the present invention can be used for identifying the cell of expressing human hTrp-m albumen or polypeptide, as Jurkat T cell.For example, can with a kind of detectable molecule for example fluorescein isothiocyanic acid (FITC) come labelling human hTrp-m specific antibody, allow people hTrp-m specific antibody contact then, detect and people hTrp-m specific antibody bonded cell with fluorescent microscope or flow cytometer again with cell sample.

Except cell surface detects people hTrp-m, can also analyze this protein with the Western engram technology.Cell pyrolysis liquid can from culturing cell or take from patient's tissue sample such as hypothalamus extract, and be dissolved in the lysis buffer that contains stain remover.Use sds polyacrylamide gel electrophoresis isolated cell extract (simultaneously with the people hTrp-m polypeptide of purifying as positive control) then, then it is transferred on the nitrocellulose by electrophoresis hybridization.In order to survey people hTrp-m polypeptide, can use typical antibodies detection method, for example radioautograph or alkaline phosphatase enzyme assay method with the immunity of Western trace.And can use the contrast of immunization serum or incoherent monoclonal antibody as non-specific responding.

Whether and quantity the expression of also available Nothern blotting technical Analysis people hTrp-m gene product, the i.e. existence of rna transcription thing in cell of analyst hTrp-m.

The Western engram analysis of the Nothern engram analysis of people hTrp-m DNA and people hTrp-m specific antibody can be united use, with the expression of confirmer hTrp-m in biological specimen.People hTrp-m DNA can also be used for Southern engram analysis or in situ hybridization analysis, with this assignment of genes gene mapping on karyomit(e), and can carry out genetic linkage analysis to find out other possible disease related gene.

In addition, the present invention also provides a kind of nucleic acid molecule that can be used as probe, and this molecule has 8-100 continuous nucleotide of people hTrp-m nucleotide coding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people hTrp-m.

The present invention also provides the method that whether has people hTrp-m nucleotide sequence in the test sample, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to people hTrp-m nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.

In addition, according to people hTrp-m nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening people's hTrp-m homologous gene or homologous protein.

In order to obtain and the people cDNAs of people hTrp-m gene-correlation or the dot matrix of genomic dna s, can screen people cDNA or genome dna library with dna probe, these probes are under low stringent condition, use ³²P people hTrp-m all or part of cooked the radioactivity mark and.The cDNA library that most is suitable for screening is from the hypothalamic library of people.Also can be used for screening purpose from the cDNA library that participates in endocrine other tissue or specific human body cell strain.Structure is that biology field is well-known from the method in the cDNA library of interested cell or tissue.In addition, many such cDNA libraries also can buy, for example available from Clontech, and Palo Alto, Cal..This screening method can be discerned the nucleotide sequence of the gene family relevant with people hTrp-m.

Can finish as follows according to Nucleotide similarity screening people hTrp-m homologue.Human hypothalamic cDNA library, for example (Clontech, Palo Alto Cal.) can use one section all or part of random primer dna probe screening that comprises people hTrp-m gene order to Clontech Cat.#7429-1.Finish having clone's the evaluation of the DNA insertion sequence of 70% homology at least with people hTrp-m sequence, can use hybridization temperature is 55 ℃ hybridization solution, uses 0.5 * SSC and 0.1%SDS to clean then.Shi Bie clone's DNA insertion sequence can be further estimated the similarity of it and people hTrp-m gene with DNA restriction endonuclease analysis and dna sequencing in this way.The distribution of tissue expression can be with above-mentioned Northern blotting technical Analysis.

People hTrp-m homologue also can be used at the antibody of people hTrp-m albumen or polypeptide and discern.For example, can be with the method for standard to commercial or make up with currently known methods, from cell or organize for example hypothalamic expression library to screen.Pour the library into plate, on colony lift to a nitrocellulose membrane, the recombinant protein of expression is attached on the film.Just can carry out typical antibodies and detection then with specific people hTrp-m antibody.Identify the DNA insertion sequence among the clone in this way, can be further analyze to estimate the similarity of it and people hTrp-m gene with DNA restriction endonuclease analysis and dna sequencing.The tissue expression of the gene of new identification distributes and can similarly analyze as stated above.

People hTrp-m Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available artificial chemosynthesis is synthesized relevant sequence.Before the application, prior art fully can be by first synthetic a plurality of polynucleotide small segments, and then connect and obtain the proteic nucleotide sequence of code book contriver hTrp-m.Then, can be with in various existing dna moleculars (as carrier) and the cell in this nucleotide sequence introducing this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WH FreemanCo., San Francisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of Applied Biosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.

The proteic encoding sequence of the present invention can be used for the assignment of genes gene mapping.For example,, the cDNA clone is hybridized with the karyomit(e) of metaphase, can carry out chromosomal localization exactly by fluorescence in situ hybridization technique (FISH).This technology can be used the cDNA that is as short as about 500bp; Also can use and grow to about 2000bp or longer cDNA.For this technology, can be referring to people such as Verma, Human Chromosomes:A Manual of Basic Techniques.Pergamon Press, New York (1988).

In case sequence is located in certain exact position on the karyomit(e), the physical location of sequence on karyomit(e) can be associated with the genetic map data.These genetic map data can obtain, for example by Mendelian (Mendelian) people genetic database (can obtain on the net by Johns Hopkins University Welch Medical Library).Then, come identified gene by linkage analysis and be positioned dependency between the disease of same chromosomal region.

Then, be necessary to determine the cDNA between diseased individuals and the healthy individual or the difference of genome sequence aspect.Be not present in normal individual if a certain sudden change is present in part or all of diseased individuals, this sudden change may be exactly the paathogenic factor of this disease so.

Utilize people hTrp-m albumen of the present invention,, can filter out with people hTrp-m interactional material takes place, perhaps acceptor, inhibitor or antagonist etc. by various conventional screening methods.

Inventor hTrp-m albumen and antibody thereof, inhibitor, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is generally about 5-8, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, intracutaneous or topical.

With people hTrp-m albumen of the present invention is example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains protein and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.People hTrp-m albumen of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When people hTrp-m protein polypeptide of the present invention is used as medicine, this polypeptide of treatment effective dose can be applied to Mammals, wherein should treat effective dose usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

Table 2 is that the homology of people hTrp-m albumen of the present invention and the proteic nucleotide sequence of home mouse Trp-m (GenBankAccession No.AF052660) compares (GAP) table.

Table 3 is that the homology of people hTrp-m albumen of the present invention and the proteic aminoacid sequence of home mouse Trp-m (GenPeptAccession No.AAC40183) compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences, and similar amino acid marks with "+".

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 normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone of people hTrp-m gene

1. separate tissue (hypothalamus isolation)

Hypothalamus derives from 5 normal adult male sex donors, takes out hypothalamus in after death four hours, places the freezing preservation of liquid nitrogen immediately.

2.mRNA separation (mRNA isolation)

Take out tissue, grind, add the 50ml pipe that fills lysate, fully after the vibration, move in the glass homogenizer again with mortar.Move to 50ml after the homogenate and newly manage, and extracted total RNA (TRIzol Reagents, Gibco, NY, USA).Identify total RNA quality with the denaturing formaldehyde gel electrophoresis.Cellulose column with band Oligod (T) separates mRNA among total RNA, quantitatively.

3.cDNA the structure in library (Construction of cDNA library)

With mRNA is template, and synthetic double chain cDNA, reverse transcription primer are seen SEQ ID NO.1.After mending flat end, add the joint that contains the EcoRI point of contact, joint sequence is seen SEQ ID NO.2 and 3 respectively.Behind the phosphorylation EcoRI end, use XhoI digestion with restriction enzyme 1.5 hours, carry out fragment again and separate.Cross the fragment of post screening length＞500bp, use the phenol-chloroform extracting, ethanol sedimentation, the sterilized water dissolving, be connected to Uni-ZAP XR carrier (Stratagene, CA9203, USA), with Zap-cDNA Gigapack III Gold Cloning Kit (Stratagene, CA9203 USA) packs, and the host bacterium is used XL 1-Blue MRF-(Stratagene, CA9203, USA) bacterium.Coated plate is also measured titre.

4. order-checking and database are set up (Seqencing and Database Constructing)

Select the clone who has the external source fragment to insert in the library, amplification back extracting plasmid (Qiagen, Germany), hold as 3 ' and the 5 ' universal primer of holding with T3 and T7, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer carries out the EST large scale sequencing on USA) at ABI 377 sequenators.Sequencing result is removed the carrier sequence with FACTURA software, is transferred to the processing of carrying out next step on SUN Ultra 450 Server.All sequence informations are used the GCG software package again, and (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL) are lower than 95% sequence with no homology or homology and are considered as new gene and set up database.

5. the full-length clone of gene (Cloning of Full-length cDNA)

On the new gene fragment order information basis that obtains, carry out the cDNA full-length clone, carry out in two stages:

(1) " electronic cloning " (Electronic Cloning)

Search the dbEST database with new gene fragment order as probe, with overlap＞50bp, homology is at (the Expressed Sequence Tag of the expressed sequence tag more than 98%, being called for short " EST ") sequence thinks same sequence (consensus sequence), take out and splice with AUTOASSEMBLER software, part EST can the extension probes sequence.Whether the sequence that is extended with the STRIDER software analysis has complete open reading frame (Open Reading Frame again, ORF), on Nucleotide and amino acid levels, whether homology is arranged with definite this sequence with BLAST search Genbank or SwissProt, to help how differentiate resulting full length gene integrity with other species.By the method for electronic cloning, can obtain the full length sequence of people hTrp-m gene usually.

(2) the terminal rapid amplifying of cDNA (Rapid Amplification of cDNA Ends, RACE)

If do not obtain complete cDNA total length yet by " electronic cloning " method, then at 5 ' or 3 ' end design primer of existing sequence, (Clontech Lab, Inc carry out the long range PCR reaction in USA) in human hypothalamus Marathon-Ready cDNA library.Then to PCR product cloning, order-checking.The sequence that is extended with AUTOASSEMBLER and STRIDER software analysis has or not complete ORF, as not having, repeats said process until obtaining total length.

(3)RT-PCR

For 5 ' and 3 ' end known sequences,, can consider to adopt the method for RT-PCR if the centre still has an intersegmental crack (gap) to obtain from existing public database or its data storehouse.At sequence 5 ' end design primer, 3 ' end primer adopts Oligo-dT, increases in the total RNA of hypothalamus storehouse.Then product is cloned, checked order.Splice at last and obtain total length.

By being used in combination above-mentioned 3 kinds of methods, obtained candidate's the proteic complete encoding sequence of people hTrp-m.Obtain on the total length basis of (comprising complete open reading frame at least) in splicing, further R1:5 '-CTTAGTTGCCGGAGCTGAAC-3 ' (SEQ ID NO.4) is a forward primer to the design primer, oligonucleotide R2:5 '-AAAGGAGCAAGCAGGAACAA-3 ' (SEQ ID NO.5) is a reverse primer, with hypothalamic total RNA is template, carry out the RT-PCR amplification, the PCR condition of R1/R2 be 94 ℃ 5 minutes, carried out 35 circulations in 1 minute with 94 ℃ 30 seconds, 60 ℃ 30 seconds and 72 ℃ thereupon, extended 5 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, obtaining expanding fragment length is 1213 bp.Clone, check order with pcr amplification product according to a conventional method then, obtain the sequence shown in the SEQ ID NO.6.

Embodiment 2

The sequence information and the homology analysis of people hTrp-m gene:

People hTrp-m full-length cDNA (the GenBank Accession No.AF221595 that the present invention is new.Unexposed mistake before the application) length is 1213 bp, and detailed sequence is seen SEQ ID NO.6, and wherein open reading frame is positioned at 101-733 position Nucleotide.Derive the aminoacid sequence of people hTrp-m according to full-length cDNA, totally 210 amino-acid residues, molecular weight 24120.79, pI are 5.47.Detailed sequence is seen SEQ ID NO.7.

The full length cDNA sequence of hTrp-m and coded protein thereof are carried out Nucleotide and protein homology retrieval with blast program in Non-redundantGenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that there is certain homology in the Trp-m gene of it and home mouse.On nucleotide level, the mRNA whole coding sequence (GenBank Accession No.AF052660) of it and home mouse Trp-m gene has 50.1% homogeny (subordinate list 2), on amino acid levels, the 138-276 amino acids residue of it and home mouse Trp-m albumen (GenPept Accession No.AAC40183) has 33.3% homogeny and 64.5% similarity (subordinate list 3).Therefore all there are higher homology in hTrp-m gene and home mouse Trp-m gene on nucleic acid still is protein level, can think that both also have very high similarity on function.

People hTrp-m of the present invention is used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, people hTrp-m of the present invention can also merge with other members of this family or exchange fragment, to produce new albumen.For example proteic N end of people hTrp-m of the present invention and the proteic N end of home mouse Trp-m are exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor hTrp-m, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

In addition, inventor hTrp-m nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people hTrp-m or the overexpression that suppresses people hTrp-m.People hTrp-m albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people hTrp-m disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

Because people hTrp-m albumen of the present invention has the natural acid sequence that is derived from the people, therefore, compare with the albumen of the same clan that derives from other species (as home mouse), estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour.

Embodiment 3

The distribution expression pattern of people hTrp-m gene

Electronics Northern express spectra.Press people's such as Ton C. method (Ton C et al., Biochem BiophysRes Commun 1997 Dec 18; 241 (2): 589-594; Hwang DM, et al., Circulation 1997Dec 16; 96 (12): 4146-4203), the BLAST retrieval will be done in the humanEST database of people hTrp-m cDNA sequence in the GCG software package, in the human EST that obtains, probable value＜10e-10, the EST of homogeny＞95% has up to a hundred, can be considered the transcriptional expression of this gene in tissue originally, draw the tissue spectrum of expressing this gene thus, find that it is at hypophysis, melanocyte, parathyroidoma, spleen, kidney, placenta, baby's heart, fetus liver, testis, in the tissues such as tire brain expression is arranged, show that it is bringing into play important effect in these histoorgans of human body.

Embodiment 4

The preparation and the purification of people hTrp-m polypeptide

In this embodiment, the people hTrp-m encoding sequence of total length or fragment are built into commercial protein merge among the expression vector, to express and purification of recombinant proteins.

People hTrp-m polypeptide is carried out prokaryotic expression with the form of gst fusion protein in intestinal bacteria.

Construction of prokaryotic expression vector, and transformed into escherichia coli

Complete encoding sequence (SEQ ID NO.6) according to people hTrp-m, design amplifies complete coding and reads the primer of frame (corresponding respectively to about 20 above Nucleotide of encoding sequence 5 ' and 3 ' end), and on positive anti-primer, introduce restriction endonuclease sites (this decides according to the pGEX-2T carrier of selecting for use) respectively, so that construction of expression vector.With the amplified production that obtains among the embodiment 1 is template, behind pcr amplification, with people hTrp-m gene guarantee to read be cloned under the correct prerequisite of frame the pGEX-2T carrier (Pharmacia, Piscataway, NJ).Identify that good expression vector utilizes CaCl ₂Method changes bacillus coli DH 5 alpha over to, and Screening and Identification obtains containing the engineering bacteria DH5 α-pGEX-2T-hTrp-m of pGEX-2T-hTrp-m expression vector.

Express the isolation identification of the engineering bacteria of GST-hTrp-m recombinant protein

DH5 α-pGEX-2T-hTrp-m the engineering bacteria of picking list bacterium colony contains jolting overnight incubation in the LB substratum of 100 μ g/ml penbritins in 3ml, draw nutrient solution by 1: 100 concentration and in new LB substratum (containing 100 μ g/ml penbritins), cultivated about 3 hours, to OD ₆₀₀After reaching 0.5, adding IPTG continues at 37 ℃ to final concentration 1 mmol/L and cultivated respectively 0,1,2,3 hours.It is centrifugal to get the different 1ml bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2 * SDS sample-loading buffer, 50 μ l, distilled water 45 μ l, 3-mercaptoethanol 5 μ l), the suspendible bacterial precipitation, boiled in the boiling water bath 5 minutes, centrifugal 1 minute of 10000rpm, supernatant adds electrophoresis in the 12%SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing the GST-hTrp-m fusion rotein.

The extraction purifying of GST-hTrp-m fusion rotein

The proteic engineering bacteria DH5 of abduction delivering GST-hTrp-m amalgamation and expression α-pGEX-2T-hTrp-m as stated above.Bacterium centrifugation after inducing adds the resuspended bacterium of 20ml PBS, ultrasonication bacterium by every 400ml bacterium.The ultrasonic completely liquid of broken bacterium adds 50% saturated Triptide Sepharose4B of PBS by every milliliter of amount that adds 20 microlitres, 37 ℃ of joltings were in conjunction with 30 minutes, 10000 rpm precipitated the Triptide Sepharose 4B that combines GST-hTrp-m in centrifugal 10 minutes, abandoned supernatant.Clean twice by the amount that every milliliter of ultrasonic liquid gained precipitation adds 100 μ l PBS, then add 10 μ l reduced glutathione elutriants by every milliliter of ultrasonic liquid gained precipitation, room temperature was put 10 minutes, centrifugal 10 minutes of 10000 rpm, and supernatant is the fusion rotein of wash-out.Repeat twice of wash-out.The supernatant of wash-out is stored in-80 ℃, and carries out the SDS-PAGE electrophoresis, detects purification effect.Protein band at the 24kDa place promptly is a people hTrp-m albumen.

Embodiment 5

People hTrp-m albumen or polypeptide carry out eukaryotic cell expression in insect cell

1. the structure of people hTrp-m rhabdovirus expression vector and transfection Sf 9 insect cell strain

According to the complete encoding sequence (SEQ ID NO.6) of people hTrp-m, design amplifies the primer that complete coding is read frame, and introduces restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively on positive anti-primer, so that construction of expression vector.With the amplified production that obtains among the embodiment 1 is template, behind pcr amplification, with people hTrp-mcDNA under the prerequisite that guarantees reading frame, be cloned into the pVL1392 carrier (Invitrogen, Carlsbad, CA).Identify good expression vector 3 μ g, wild-type linearized baculovirus dna (BaculoGold ^TMACMNPV DNA, Pharmingen, San Diego, CA) 1 μ g and Lipofection (Gibco-BRL, NY) 25 μ l add in the insect substratum of 1ml serum-free, the 15 seconds mixings that vibrate, incubated at room 15 minutes is standby.Get 1ml (2 * 10 ⁶) Sf9 insect cell suspension is in 60mm tissue culturing plate, change transfection media after adherent 1 hour, incubated at room was abandoned substratum after 15 minutes, add the dna vector transfection mixture for preparing previously, Parafilm seals culture plate, cultivated 4 hours in 27 ℃ of joltings of room temperature, then change perfect medium and cultivated 3 days, it is standby to collect supernatant.

2. change the Screening and Identification of the insect cell line of recombinant expression vector over to

The insect cell of transfection after 3 days made cell suspension (2 * 10 with fresh culture ⁶/ 1ml), get the 1ml cell suspension and place 60mm tissue culture ware, add the 3ml substratum, the culture supernatant that 100 μ l collect, adherent 1 hour, abandon the 2ml substratum, continue incubated at room temperature 1 hour, and discarded all substratum, add the 3ml semisolid medium that contains 20 μ l4%X-gal of preheating, cultivate after 5-7 days picking white cell clone and in 96 well culture plates, cultivated 3-5 days, then draw supernatant infection Sf9 insect cell.

Collect the cell that infects and carry out the Western evaluation.The SDS-PAGE electrophoresis will be carried out after the lysis, glue behind the electrophoresis prints to protein transduction on the nitrocellulose membrane in the half-dried electrotransfer instrument of the Multiphor of Pharmacia II, nitrocellulose membrane is placed confining liquid sealing 1 hour, then in the antibody-solutions of anti-people hTrp-m, sealed 1 hour, the jolting of TBS liquid is cleaned 5 minutes 2 times totally, then film is placed the anti-second antibody solution jolting of biotin labeled anti-people hTrp-m one 1 hour, TBS cleans, adding avidin-alkaline phosphatase enzyme complex reacted 30 minutes, TBS cleans 2 times, adds freshly prepared colour developing liquid colour developing and observes protein band.

The Sf9 cell clone of picking high expression level people hTrp-m.

3. the proteic extraction purifying of people hTrp-m

Supernatant with the Sf9 cell clone of high expression level people hTrp-m infects the Sf9 cell in a large number, infects collecting cell after 48 hours, the PBS washing.Per 2 * 10 ⁸Cell adds 20ml cell pyrolysis liquid (0.5%Triton X-100,20mM Na ₃PO ₄(sodium phosphate, pH7.8), 500mM NaCl, 1mM Na ₃VO ₄(vanadic acid sodium), 1mM Pefabloc, 1 μ g/ml pepstatin, leupeptin and aprotinin) broken cell, the centrifugal 20min of 12000 * g removes cell debris, and supernatant is by per 2 * 10 ⁸Cell add 2ml NTA-agarose (Qiagen, Germany), 4 ℃ of absorption 1 hour.Then with containing the His damping fluid washed twice of 100nM imidazoles, with containing 20mM N, N '-two piperazine, 500mM NaCl, the buffer solution elution of 300mM imidazoles is to obtain the albumen of purifying.Elutriant is stored in 4 ℃, and carries out the proteic purity of people hTrp-m that the SDS-PAGE electrophoresis detection is extracted.Protein band at the 24kDa place promptly is a people hTrp-m albumen.

Embodiment 6

The preparation of anti-people hTrp-m antibody

1. the preparation of immune mouse and splenocyte: it is standby that the people hTrp-m albumen that obtains among embodiment 5 and the embodiment 6 is separated the back with chromatography, also can separate with the SDS-PAGE gel electrophoresis, electrophoretic band is cut off from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Get the female mouse of 6-8 week Balb/C in age, the albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days, with the same antigen of non-complete Freund ' s adjuvant emulsion to mouse with the dosage of 50-100 μ g/0.2ml again booster immunization once be used for after 3-5 days merging.Wherein, E Zheng chief editor, " tissue culture and molecular cell learn a skill ", Beijing Publishing House, the 210th page are seen in the splenocyte preparation.

2. by " tissue culture and molecular cell learn a skill " (the same), the method in the 371st page, preparation feeder cell.

3. by " tissue culture and molecular cell learn a skill " (the same), the method in the 213rd page is carried out cytogamy.

4. detection of antibodies: after cytogamy 10-15 days, need to check by the hole, in case find vigorous hybrid cell colony growth, the hTrp-m albumen of just should choosing is done the preliminary screening of antibody activity, and method commonly used has: immunofluorescent test, emission immunity test (RIA), enzyme linked immunosorbent assay (ELISA).After checking out the hole of antibody activity, clone cultivation at once, and isolate antibody.

Sequence that the present invention relates to and mark apportion are as follows:

(1) information of SEQ ID NO.1

(i) sequence signature:

(A) length: 50bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.1GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT 50

(2) information of SEQ ID NO.2

(i) sequence signature:

(A) length: 13bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.2

AATTCGGCAC?GA?G?????????????????????????????????????13

(3) information of SEQ ID NO.3

(i) sequence signature:

(A) length: 9bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(iii) sequence description: SEQ ID NO.3

The information (i) of GCCGTGCTC 9 (4) SEQ ID NO.4. sequence signature:

(A) length: 20bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity is (ii). and molecule type: oligonucleotide is (iii). sequence description: the information (i) of SEQ ID NO.4 CTTAGTTGCCGGAGCTGAAC 20 (5) SEQ ID NO.5. sequence signature:

(A) length: 20bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity is (ii). and molecule type: oligonucleotide is (iii). sequence description: information (i) sequence signature of SEQ ID NO.5AAAGGAGCAAGCAGGAACAA 20 (6) SEQ ID NO.6:

(A) length: 1213bp

(B) type: Nucleotide

(C) chain: strand

( D ) ： ( ii ) ： ( iii ) ：SEQ ID NO.6 1 CTTAGTTGCC GGAGCTGAAC GGCGCGAGCT GGTCTGAGGC GAGCCGAGCC 51 GAGCGAGCGC GGCGGTGGGG CCGAGAGGAC GCGCAGGTGG CGGCGTTGCC101 ATGTCGCACG GTCACAGCCA CGGCGGGGGT GGCTGCCGCT GCGCCGCCGA151 ACGGGAGGAG CCGCCCGAGC AGCGCGGCCT GGCCTACGGC CTGTACCTGC201 GCATCGACCT GGAGCGGCTG CAATGCCTTA ACGAGAGCCG CGAGGGCAGC251 GGCCGCGTCT TCAAGCCGTG GGAGGAGCGG ACCGACCGCT CCAAGTTTGT301 TGAAAGTGAT GCAGATGAAG AGCTTCTGTT TAATATTCCA TTTACGGGCA351 ATGTCAAGCT CAAAGGCATC ATTATAATGG GAGAGGATGA TGACTCACAC401 CCCTCTGAGA TGAGACTGTA CAAGAATATT CCACAGATGT CCTTTGATGA451 TACAGAAAGG GAGCCAGATC AGACCTTTAG TCTGAACCGG GATCTTACAG501 GAGAATTAGA GTATGCTACA AAAATTTCTC GTTTTTCAAA TGTCTATCAT551 CTCTCAATTC ATATTTCAAA AAACTTCGGA GCAGATACGA CAAAGGTCTT601 TTATATTGGC CTGAGAGGAG AGTGGACTGA GCTTCGCCGA CACGAGGTGA651 CCATCTGCAA TTACGAAGCA TCTGCCAACC CAGCAGACCA TAGGGTCCAT701 CAGGTTACCC CACAGACACA CTTTATTTCC TAAGGGCTGG CCAAGGCTCC 751 CATAGAGGCG CTGTGTCAGT GAAGATGTAC GACTACCTGT TGGGAAGGAC 801 AAAGGGATGA GGCTCCAGAG AGAGTTGGCT GCCACAGCCT CTGCCAAGCT 851 TTGTCTTTGG GGCTTGCTGC AGAAACCTGG CCTACGGAAG ATACGACACC 901 ACTGGGAGGG TTGTGTAGGT GCCAGGGGAC CATCGTGGTT CTCTAGGGCG 951 CTGTGGAAAT TGGGTCTTGG GCTGGGTGGC ATCTGGCAGT CATGGATAAC1001 ACTTCCTTTT CCAGTTAATG TGGCCATGTG ATTCCAACTG TCATGTTGCT1051 TTGTGGCAAG ATTGTTGTGT GACTTGTTTT TTTGTTTTTG TTTTTGTTTT1101 TTTAAAGGAA ACTATTTGTG GGCTATAGGA AACTTTCTGA TGCCTCCGGA1151 TTGTGTTAGT AGTAGCCATC AGGAGGGTCT CCAACTAAAA CACTTGTTCC1201 TGCTTGCTCC TTT ( 7 ) SEQ ID NO.7 ( i ) ：

(A) length: 210 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity, (ii) molecule type: polypeptide, (iii) sequence description: SEQ ID NO.7:1 MSHGHSHGGG GCRCAAEREE PPEQRGLAYG LYLRIDLERL QCLNESREGS 51 GRVFKPWEER TDRSKFVESD ADEELLFNIP FTGNVKLKGI IIMGEDDDSH101 PSEMRLYKNI PQMSFDDTER EPDQTFSLNR DLTGELEYAT KISRFSNVYH151 LSIHISKNFG ADTTKVFYIG LRGEWTELRR HEVTICNYEA SANPADHRVH201 QVTPQTHFIS

Table 2Percent Identity:50.1%. 1 CTTAGTTGCCgGAgctGAaCGGCGCGAGCTGGTCTGAGGCGAGCCgaGC. 49

|?|||???|?|?|?|?|?||||??|||???|||||??|?1?...........gggct.ctctgtgag.gatggtgggagtgaagccggtcg?37

.?????????.?????????.?????????.?????????.50?CGAGCGAGCGCgGCGgTGGGGCCGAGAGGA..CGCGCAGGTGGCGGCGTT?97

||||||?|??|???????|?|?|||??||???|?||?||???|?|??||38?ggagcgatccagatttccagccggagctgagcggggcgggctccagactt?87

.?????????.?????????.?????????.?????????.?98?GCCATGTCGCACGGTCAC..AGCCACGGCGgGG...GTGGCTGCCG.CTG?141

|||?||???||?|?||||???|??|?||?|?||???|||?||?|?|??||?88?gccgtg.gtcaagttcaccatgagagggtgtggaccgtgtcttcggattg?136

.?????????.?????????.?????????.?????????.142?CGCCGCCG...AACGGGAGGAGCCGCCCGAGCAGCgCgGCCTGGcCTACg?188

|?|||?||???|?|???|?|||??????|??|??|?|?|?|?|?|?|137?ccccggcgttcagctctatgagtaataagtac.ccacaggcagtcttctt?185

.?????????.?????????.?????????.?????????.??????????.189?GcCTGTACCTGCGCATC...GACCTGGAGCGGCTGCAATGcCTTAACGAG?235

|??|||???||?|||||???|?|??|||?||||||?|???||??|???|186?ggaagtagatgtgcatcagtgccagggaacagctgcca..ccaacaacat?233

.?????????.?????????.?????????.?????????.236?AGCCGCGAgGGCAGCGGCCGCGTCTT...CAAGCCgTGGGAGGAGCGGAC?282

|?|?||?|???|??||?????||?||????||??|??|?|||?|?||??|234?atcagcaacacctacgtttttgttttttcgaaacaaagtgagaatcgatc?283

.?????????.?????????.?????????.?????????.283?CG....ACCGCTCCAAGTTTgTTGAAAGTGATGCAGATGAAGAGCTTCTG?328

|????|??|??|??|???|||?|?|???||?|???|?????|||??|284?agtatcaaggagcagatgctgtgggactagaagagaaaatcaagcagcac?333

.?????????.?????????.?????????.?????????.329?TTTAATATTCCATTTACGGGCAATGTCAAGCTCAAAGGCATCATTATAAT?378

||??|?|?|????|?????||||||???||??|??||?|||????|334?ttagaaaatgatcctggaagcaatg...aggacgcagacattccca....?376

.?????????.?????????.?????????.?????????.379?GGGAGAGGATGATGACTCACACCCCTCTGAGATGAGACTGTACAAGAATA?428

||?|????|||??|???|??|||?|?|????????|??||||???|377?...agggctatatggatttaatgcctttta........ttaacaaagctg?415

.?????????.?????????.???????????.???????.429?TTCCACAGATGTCCTTTGATGATACAGAAAGGGA..GCCAGATCAGACCT?476

|????|?|||||??||?||||?|??||???|?|??||?????||????|416?gttgtga.atgtc..ttaatgaaagtgatgagcacggctttgacaactgt?462

.?????????.?????????.?????????.?????????.477?TTAGTCTGAACCGGGATCTTACAGGAGAATTAGAGTATGCTACAAAAATT?526

|||?????|??|??|??|||???||||??|??||?|?||?|????||?|463?ttacgaaaagacatgtccttcttggag..tctgactgtgatgagcaactc?510

.?????????.?????????.?????????.?????????.527?TCTCGTTTTTCAAATGTCTATCA.TCTCTCAATTCATATTTCAAAAAACT?575

|??|??|???????||?||||??||?|?||????||||?||??||??|511?cttattactgtggcattcaatcagcctgttaagctttattccatgaagtt?560

.?????????.?????????.?????????.?????????.576?TC..GGAGCAGAT.ACGACAAAGGTCTTTTATATTGGCCTGAGAGGAGAG?622

||??|||?|||||?|||????|?|||?|??|?||||????|?????|?|561?tcaaggaccagataacggtcagggtcctaaatat........gtaaaaat?602

.?????????.?????????.?????????.?????????.623?TGGACTGAGCTTCGCCGACACGAGGTGACCATCTGCAATTACGAAGCA.T?671

|????|?|?|?|??||????|?|?|?||???|??|?|??????|||?|?|603?ttttatcaacctaccccgatctatg.gattttgaggaggcggaaaggagt?651

.?????????.?????????.?????????.?????????.672?CTGCCAACCCAGCAGACCATAGGGTCCATCAGGTTACCCCACAG.ACACA?720

||||||?||?????|???|?????||??||?|?||???|?||?|?|652?gagccaactca.agctctggaactgacagaagatgacattaaagaagatg?700

.????????.?????????.?????????.?????????.721?CTTTATTTCCTAAGGGCTGGcCAAGGCTCCCATAGAGGCGCTGTGTCAGT?770

|??|||||????|?|?????|???|?|???|??????|?||||?|?|701?gcattgttcctcttcgttatgttaaatttcagaatgtcaacagtgtaact?750

.?????????.?????????.?????????.?????????.

771?GAAGATGTAC.GACTACCTGTTGGGAAGGACAAAGGGATGAGGCTCCAGA?819

|??|||?|?|?|||?????????????||||?|?||?||?|???||

751?ttatttgttcagtcta............aacaaggagaagaagaaacaac?788

.?????????.??????????.?????????.????????.

820?GAGAGTTGGCTGCCACAGcCTCTGCCAAGCTTTGTCTTTGGGGCTTGCTG?869

||||?||???????????????????|??|||???||||??????|?||

789?gagaatt..................tcatattttactttcattggtactc?820

.?????????.?????????.?????????.?????????.

870?CAGAAACCTGGCCTACGGAAGATACGACACCACtGGGaGGGTTGTGTAGG?919

|||???||?|||???????|???|?|||??||???|???|||||???|

821?cag..tccaggcaacaaatatgaatgacttcaaacgagtggttggcaa..866

.?????????.?????????.?????????.?????????.

920?TgCCAGGGGACCATCGTGGTTCTCTAGGGCGCTGTGGAAATTGG..GTCT?967

||||||????|?|?????|||||??|??????||?|?|||??|?||

867?.aaaaggggaaagccat.......tagggtacaaaagacactggaagact?908

.?????????.?????????.?????????.?????????.

968?TGGGCTGGGTGGCATCTGGCAGTCATGGaTAA.CACTTGCTTTTCCAGTT?1016

|???????||???|??||????||?|||||||???||||?||?||

909?ttactgcagtcagacttgcagctcctggataattgcttgattctcttcgg?958

.?????????.?????????.?????????.?????????.???1017?AATGTGGCCATGTGATTCCAAGTGTCATGTTGCTTTGTGGCAAG.ATTGt?1065

||??||???||?|????????????????||??|||||?||?||?|

959?aaactgttaatct................tttatttgttgccagtaccaa?992

.?????????.?????????.?????????.?????????.

1066?TGtGtGACTTGTTTTTTTGTTTTTGTTT...TTGTTTTTTTAAAGGAAAC?1112

|???|?|?|??||||?||????|?|??|???|?||?|||?||??|?||?|

993?taaatcattgcttttgttcagatggcatcactagtgtttgtagtgtaatc?1042

.?????????.?????????.?????????.?????????.???1113?TATTTGTGGGCTATAGGAAACTTTCTGATGCCTCCGGATTGTGTTAGTAG?1162

|????||??|?|?|||?????|?|??????|???||?||??|???1043?gtgatacatgcagttgtaaa.....taaaagtcactacttttgccaagct?1087

.?????????.???????_?.?????????.?????????.???1163?TAGCCATCAGGAGGGTCTCCAACTAAAACACTTGTTCCTGCTTGCTCCTT?1212

|| | | | || || || | 1088 taaaaaaaaaaa.......aaaaaaaaaaa.................... 1110 are above-listed: (GenBank Accession No.AF221595) is following for the proteic nucleotide sequence of people hTrp-m: the proteic nucleotide sequence of home mouse Trp-m (GenBank Accession No.AF052660)

Table 333.3% identity in 138 aa overlap.64.5% similarity in 88 aa overlap

20????????30????????40?????????50????????60???????69h.pep?????????GCRCAAEREEPPEQRGLAYGLYLRIDLERLQCLNESRE-GSGRVFKPWEERTDRSKFVES

+|||||?|?|????+?????|?|?|?|+||m.pep?????????HLENDPGSNEDADIPKGYMDLMPFINKAGCECLNESDEHGFDNCL-----RKDMS-FLES

110???????120??????130????????140???????150

70????????80????????90???????100???????110????????120h.pep?????????DADEELLFNIPFTGNVKLKGIIIMGEDDDSHPSEMRLYKNIPQ-MSFDDTER-EPDQTFS

|?||+||+++?|+??|||?++?++|?|+?+?|+?++++?|+|+?|+|+++||?||?|++m.pep?????????DCDEQLLITVAFNQPVKLYSMKFQGPDNGQGPKYVKIFINLPRSMDFEEAERSEPTQALE

160????????170???????180???????190???????200???????210

130???????140????????150????????160???????170???????180h.pep???????LNRDLTGELEYAT-KISRFSNVYHLSIHI-SKNFGADTTKVFYIGLRGEWTELRRHEVTI

|++|???|???+??+??+|+||??+++?+?||+???+||++?|+?+?|m.pep???????LTEDDIKEDGIVPLRYVKFQNVNSVTLFVQSKQGEEETTRISYFTFIGTPVQATNMNDFK

220???????230???????240???????250???????260???????270

190???????200???????210h.pep???????CNYEASANPADHRVHQVTPQTHFISm.pep???????RVVGKKGESH

280h.pep: people hTrp-m Argine Monohydrochloride sequence m.pep: home mouse Trp-m Argine Monohydrochloride sequence (GenBank Accession No.AAC40183)

Claims (11)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of polypeptide of people hTrp-m protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 101-733 position among described nucleotide sequence and the SEQ ID NO.6: perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.6 from the nucleotide sequence hybridization of Nucleotide 101-733 position.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.7.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.6 nucleotide sequence from Nucleotide 101-733 position.

4. isolated people hTrp-m protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.7 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID N0.7 polypeptide of sequence.

6. a carrier is characterized in that, it comprises the described DNA of claim 1.

7. one kind with the described carrier transformed host cells of claim 6, it is characterized in that it comprises prokaryotic cell prokaryocyte and eukaryotic cell.

8. a generation has the method for the polypeptide of people hTrp-m protein active, is characterised in that its step is as follows:

(1) nucleotide sequence that coding is had a polypeptide of people hTrp-m protein-active operationally is connected in expression regulation sequence, form people hTrp-m protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 101-733 position among described nucleotide sequence and the SEQ ID NO.6;

(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of people hTrp-m;

(3) under the condition that is fit to expressing human hTrp-m protein polypeptide, the reconstitution cell in the culturing step (2);

(4) isolate polypeptide with people hTrp-m protein-active.

9. energy and the described people hTrp-m of claim 7 protein polypeptide specificity bonded antibody is characterized in that it comprises polyclonal antibody and monoclonal antibody.

10. a nucleic acid molecule is characterized in that, it comprises 8-100 continuous nucleotide in the described dna molecular of claim 1.

11. one kind is used for the method whether test sample exists people hTrp-m nucleotide sequence, it is characterized in that it comprises with described probe of claim 10 and sample hybridizes, whether detection probes combination has taken place then, this sample is the product behind the pcr amplification, wherein the pcr amplification primer is corresponding to people hTrp-m nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence, primer length is 15～50 Nucleotide.