CN1408870A

CN1408870A - Human transcription factor, its coding sequence preparation and use

Info

Publication number: CN1408870A
Application number: CN 02137039
Authority: CN
Inventors: 余龙; 唐丽莎; 郭金虎
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2002-09-18
Filing date: 2002-09-18
Publication date: 2003-04-09

Abstract

本发明涉及基因工程技术领域，具体地说，本发明提供了人转录因子(human transcriptionfactor，hTF)，其编码序列、制法及用途。本发明的hTF可形成锌指结构，具有DNA结合区特征，可作为一种转录因子与核酸结合并相互作用以调控基因表达，从而对细胞乃至生物体的生理活动起到调控作用。将本发明的hTF核酸、其反义链及片段制成探针，可检测hTF基因异常；将hTF蛋白序列制成试剂盒或药物，可起到调节细胞生长增殖分化，促进烫伤烧伤、组织切除和器官摘除后的伤口愈合，防止伤口感染以及辅助临床上肿瘤和癌症的诊断、预防和治疗等作用。The invention relates to the technical field of genetic engineering, specifically, the invention provides human transcription factor (human transcription factor, hTF), its coding sequence, preparation method and application. The hTF of the present invention can form a zinc finger structure, has the characteristics of a DNA binding region, and can be used as a transcription factor to bind and interact with nucleic acid to regulate gene expression, thereby regulating the physiological activities of cells and even organisms. The hTF nucleic acid of the present invention, its antisense strand and fragments are made into probes to detect hTF gene abnormalities; the hTF protein sequences are made into kits or medicines, which can regulate cell growth, proliferation and differentiation, and promote burns and tissue resection and wound healing after organ removal, preventing wound infection and assisting clinical diagnosis, prevention and treatment of tumors and cancers.

Description

A kind of human transcription factor, its encoding sequence, method for making and purposes

Technical field

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the present invention relates to the cDNA sequence of a kind of human transcription factor (hTF), the invention still further relates to by this nucleotide sequence coded polypeptide the application of these polynucleotide and polypeptide, and the production method of described polynucleotide and described polypeptide.

Background technology

Transcription factor is to participate in the protein molecule of genetic information from DNA to mRNA in the cell, and great majority are present in the nucleus, so be called the nucleus factor again.They change the conception of DNA, thereby influences the gene transcription level by combining with DNA, and then influence genetic information from RNA to proteinic transmission and even the physiological situation of change organism.

Transcription factor can be divided into positivity transcription factor and negativity transcription factor according to its effect: the positivity transcription factor promotes gene transcription; The negativity transcription factor is transcribing of suppressor gene then.Transcription factor is according to its effect and characteristic distributions can be divided into non-specific and two kinds of idiosyncratic transcription factors.Non-specific transcription factor is present in the various kinds of cell, can act on multiple different promotor and enhanser, can be induced by various factors, by proteinic covalent modification or allosteric adjusting are activated.Idiosyncratic transcription factor only is present in certain specific cell, only combines the tissue specificity and the etap property of decision genetic transcription with certain specific DNA zone of promotor and enhanser.

About transcription factor and DNA bonded feature, it is found that, when transcription factor forms space structure, leucic periodicity occurs with DNA bonded position and repeat.Landschulz proposes a kind of " leucine zipper " model, and promptly the corresponding position of the leucine side chain of a molecule and another molecule is interlaced, forms a kind of zipper, so just makes two molecules couple together (Science 1989 Mar 31; 243 (4899): 1681-8).And more general theory is " Zn finger print type " (Nucleic Acids Res 1989 Nov 25; 17 (22): 9185-92).Zinc refer to be in Xenopus transcription factor TFIIIA, find first can with nucleic acid bonded protein structure domain, so far, this structural domain is found in various nucleic acid binding proteins.A Zinc finger domain is made up of 23-30 amino-acid residue, and is available by two conservative halfcystines and two C-2-C-12-H-3-H expressions simple in structure that conservative Histidine is formed.Wherein, C-2-C represents two amino-acid residues in interval between two adjacent halfcystines.12 amino acid between second halfcystine and first Histidine mainly are polarity and alkalescence, and this shows the obvious and nucleic acid bonded of this structural domain.Normally small-sized self the folded form functional domain of Zinc finger domain, and this wherein zinc atom its tertiary structure has been played critical effect.Zinc atom and halfcystine and Histidine are connected to form the three-dimensional structure (being shown below) of a similar zinc finger-like, and interact with the major groove of nucleic acid.

Discover that zinc fingers combines with five pairs of base pairs that include guanine.They both can also can combine with DNA with RNA.There are some researches show that zinc atom is that the structure at center also can work in proteic interaction.

Zinc fingers will cause many cytodifferentiation disease relevant with fetal development unusually, as: neuropathy refers to toe deformity and Wilms' tumor etc. more.The disease that is caused by the fork head abnormal protein has: angle iris heteroplasia, Li Geshi heteroplasia, glaucoma and aplasia of iris, Bamforth-Lazarus syndrome, T cellular immunity deficiency, congenital alopecia and hypoplastic nails etc.

Summary of the invention

An object of the present invention is to provide a kind of polynucleotide sequence, a kind of human transcription factor of this polynucleotide sequence coding ( hUman cYto kIne rEceptor, hTF).

Another object of the present invention provides a kind of albumen, and this albumen is named as human transcription factor (hTF).

A further object of the present invention provides a kind of recombinant technology that utilizes and produces the proteic method of described hTF.

The present invention also provides this hTF nucleotide sequence and proteic application.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people hTF protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 370-2592 position among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ IDNO.1 in from the nucleotide sequence hybridization of Nucleotide 370-2592 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.2.More preferably, this sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 370-2592 position.

In another aspect of this invention, provide a kind of isolating hTF protein polypeptide, it comprises: have polypeptide or its active fragments of SEQ ID NO.2 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.2 polypeptide of sequence.

In another aspect of this invention, provide a kind of carrier, it contains above-mentioned isolated DNA.

In another aspect of this invention, provide a kind of described carrier transformed host cells.

In another aspect of this invention, the method that provides a kind of generation to have the polypeptide of hTF protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of hTF protein-active operationally is connected in expression regulation sequence, form the hTF protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 370-2592 position among described nucleotide sequence and the SEQ ID NO.1;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of hTF;

(c) be fit to express under the condition of hTF protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with hTF protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 2594 Nucleotide, and its detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 370-2592 position Nucleotide.

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 " hTF albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with HTF protein-active is as 370-2592 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 370-2592 position Nucleotide of SEQ ID NO.1 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.1 in 370-2592 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition with SEQ ID NO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 370-2592 position.In addition, this term also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 370-2592 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form with proteic, the SEQ ID NO.1 sequence of people HTF 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 " hTF protein polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of hTF protein-active.This term also comprises having and variant form people hTF identical function, SEQ ID NO.2 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 hTF and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, induced mutation body, under high or low rigorous degree condition can with the coded albumen of the DNA of hTF DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-hTF polypeptide to obtain.The present invention also provides other polypeptide, as comprises hTF polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of hTF polypeptide.Usually, this fragment have the hTF 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.

Invention also provides the analogue of hTF albumen or polypeptide.The difference of these analogues and natural hTF 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.

The present invention also comprises the antisense sequences of hTF polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of hTF in the cell.

The present invention also comprises a kind of probe molecule, and this molecule has 8-100 of hTF polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the hTF that encodes.

The present invention also comprises the method that detects the hTF nucleotide sequence, 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 the encoding sequence of hTF polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 20-50 Nucleotide.

In the present invention, can select various carrier known in the art for use, as commercially available carrier.

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.

On the other hand, the present invention also comprises hTF DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into hTF gene product or fragment.Preferably, refer to that those can combine with hTF gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of hTF, comprise that also those do not influence the antibody of hTF protein function.The present invention also comprise those can with modify or without the hTF gene product bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the hTF gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing hTF or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology prepare (see people such as Kohler, Nature256; 495,1975; People such as Kohler, Eur.J.Immuno1.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the hTF function and the antibody that does not influence the hTF function.Each antibody-like of the present invention can utilize the fragment or the functional zone of hTF gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of hTF gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

In the present invention, the cDNA nucleotide sequence of people hTF is so to obtain, and is template with people's testis λ gt10cDNA library (available from Clontech company), is primer with two pairs of oligonucleotide: A1 gcttgaacct tgtcacccct c; B1 tcgactacctcctcc aacact is a forward primer; A2:TCACA GCCTGGCCAT TTGCAA; B2 TCTCATTCCAGATC TTCAGAT is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains, size are respectively the fragment and the impurity of about 1,500 and 1,200 base pairs.Remove impurity, with NcoI respectively enzyme cut above-mentioned two fragments, connect then, obtain the purpose fragment.With the nucleotide sequence carrier of packing into, import host cell.Host cell just can give expression to corresponding protein, just will obtain hTF albumen of the present invention through separation and purification.

HTF albumen of the present invention carries out nucleic acid and albumen homology retrieval with BLAST software in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database, found that it has zinc finger protein and the proteic characteristic sequence of fork head.

HTF of the present invention can form zinc fingers, has DNA land feature, can be used as a kind of transcription factor and combine also interaction with nucleic acid with regulate gene expression, thereby the physiological activity of pair cell and even organism plays regulating and controlling effect.These physiological actions comprise the promotion tissue growth, the growth of regulating cell and propagation, and inducing cell is to certain specific trend development or the like.

HTF nucleic acid of the present invention, its antisense strand and fragment are made probe, can detect the hTF gene unconventionality; With hTF protein sequence generate a reagent box or medicine, adjustable ganglion cell's growing multiplication differentiation promotes the wound healing after scalds and burns, cutting tissue and even organ are extractd, and prevents wound infection.With hTF nucleic acid antisense strand of the present invention hTF antibody generate a reagent box can be used for alleviating or assisting therapy owing to the too high disease that causes of hTF expression amount, also can assist the diagnosis of tumour and cancer clinically, prevention and treatment.In addition, hTF of the present invention can also merge with other members of this family or exchange fragment, to produce new albumen, for example the N end of hTF of the present invention and the N end of mouse TF can be exchanged, to produce the albumen that new activity is higher or have new features.At the antibody of hTF of the present invention, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

Embodiment

Embodiment 1

The clone and the mensuration of the cDNA sequence of hTF

1. primer amplification

With people's testis λ gt10cDNA library (available from Clontech company) is template, is primer with two pairs of oligonucleotide: A1GCTTGAACCT TGTCACCCCT C; B1 TCGAC TACCTCCTCC AACACT is a forward primer; A2:TCACA GCCTGGCCAT TTGCAA; B2 TCTC ATTCCAGATC TTCAGAT is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains, size are respectively the fragment and the impurity of about 1,500 and 1,200 base pairs.Remove impurity, with NcoI respectively enzyme cut above-mentioned two fragments, connect then, obtain the purpose fragment.

2.PCR the order-checking of product

With above-mentioned purpose fragment and pGEM-T ^TMCarrier (Promega) connects, transformed into escherichia coli JM103, extract plasmid with QIAprepPlasmid test kit (QIAGEN), carry out the mistake of orientations Lieque to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then with double-stranded nested type disappearance test kit (Pharmacia).Use SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, obtains full length cDNA sequence at last, is total to 2594bp, and detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 370-2592 position Nucleotide.

Derive the aminoacid sequence of hTF according to the full length cDNA sequence that obtains, totally 740 amino-acid residues, its aminoacid sequence sees SEQ ID NO.2 for details.

Embodiment 2

The expression of hTF in intestinal bacteria

In this embodiment,, use PCR Oligonucleolide primers to increase, obtain hTF cDNA as inserting fragment corresponding to 5 of this dna sequence dna ' and 3 ' end with the cDNA sequence of coding hTF.

5 ' Oligonucleolide primers sequence of using in the PCR reaction is:

5 '-G TATGGATCCA TGATGCAGGA ATCTGCGA, this primer contains the restriction enzyme site of BamHI restriction enzyme, is the part encoding sequence of the hTF that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5 '-TTCCAGATC TTCAGATAAAAAGCTTATTACG, this primer contain the part encoding sequence of restriction enzyme site, translation termination and the hTF of HindIII restriction enzyme.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, the CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Amp ^r), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.

With BamHI and HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid cuts with the NcoI enzyme and to identify and insert clip size and direction, and the cDNA fragment of sequence verification hTF has correctly been inserted carrier.

Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved hTF from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out hTF from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps use the dialysis step to remove Guanidinium hydrochloride, perhaps isolate purifying protein from nickel-chelate column, the albumen behind the purifying can be incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed to containing PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.2 with ordinary method.

Embodiment 3

The expression of hTF in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, the following Oligonucleolide primers of cDNA sequence of coding hTF is increased, obtain hTF cDNA as inserting fragment.

5 ' Oligonucleolide primers sequence of using in the PCR reaction is:

5’-G?TATAAGCTTA?TGATGCAGGA?ATCTGCGA

This primer contains the restriction enzyme site of HindIII restriction enzyme, is the part encoding sequence of the hTF that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-TTCCAGATC?TTCAGATAAAAAGCTTATTACG

This primer contains the part encoding sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and hTF.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (fl ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With HindIII and BamHI digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid cuts evaluation with the NcoI enzyme and insert clip size and direction, and the cDNA fragment of sequence verification hTF has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to use lipofection, carries out with Lipofectin (GiBco Life), and transfection is after 48 hours, and through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the SuperdexG-75 of pre-equilibration post and collects above-mentioned proteic active peak.Using 50mMTrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.

Embodiment 4

Homology relatively

HTF albumen (SEQ ID NO.2) the hTF albumen of the present invention that obtains with embodiment 2 and embodiment 3 carries out nucleic acid and albumen homology retrieval with BLAST software in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database, found that it has zinc finger protein and the proteic characteristic sequence of fork head.

Zinc refer to be in Xenopus transcription factor TFIIIA, find first can with nucleic acid bonded protein structure domain, so far, this structural domain is found in various nucleic acid binding proteins.The characteristic sequence of zinc fingers can be represented with following form: C-x (2,4)-C-x (3)-[LIVMFYWC]-x (8)-H-x (3,5)-H.Wherein, x represents arbitrary amino acid, and the quantity of the numeral amino acid deformity in the bracket is chosen one wantonly in the amino acid of [LIVMFYWC] expression from square brackets.The type sequence that the protein sequence of hTF of the present invention has the zinc finger protein structural domain is: VCKWPGCESICEDFGQFLKHLNNEH (371-396 among the SEQ ID NO 2).Zinc fingers will cause many cytodifferentiation disease relevant with fetal development unusually, as: neuropathy refers to toe deformity and Wilms' tumor etc. more.(Science?1988?Sep?16；241(4872)：1489-92；Parraga?G，Horvath?SJ，Eisen?A，Taylor?WE，HoodL，Young?ET，Klevit?RE.；Klug?A.，Rhodes?D.Trends?Biochem.Sci.12：464-469(1987).；Evans?R.M.，Hollenberg?S.M.Cell?52：1-3(1988).)。Zinc fingers is a most general DNA binding domains in the transcription factor of being found at present.

HTF albumen of the present invention also has the fork head protein structure domain.Fork head albumen, functional domain or zinc that it does not contain with other transcription factor homolog refer to feature, also do not have similarity with the DNA land of previous evaluation.It comprises a DNA land of being made up of 100 amino-acid residues, is formed with the spirane structure of flank, and this structural domain is named is " fork head " (Weigel D., Jurgens G., Kuttner F., Seifert E., Jackle H.Cell 57:645-658 (1989)).The fork head functional domain combines with B-form DNA, and this all found (to comprise fruit bat FD1-5, Mammals HNF-3 in many transcription factors, people HTLF, yeast HCM1, or the like) (Clark K.L., Halay E.D., Lai E., Burley S.K.Nature 364:412-420 (1993) .).Although having the transcription factor type of this structural domain has nothing in common with each other, but it is all to participate in the embryo to determine cell fate (Hacker U. in early days that these factors have a common characteristic, Grossniklaus U., Gehring W.J., Jackle H.Proc.Natl.Acad.Sci.U.S.A.89:8754-8758 (1992).The protein sequence of hTF of the present invention contains the proteic characteristic sequence of fork head: VRPPFTYATLIRQAIMESSDRQLTLNEIYSWFTRTFAYFRRNAATWKNAVRHNLSL HKCFVRVENVKGAVWTVDEVEYQKR (527-608 among the SEQ ID NO 2).The disease that is caused by the fork head abnormal protein has: angle iris heteroplasia, Li Geshi heteroplasia, glaucoma and aplasia of iris, Bamforth-Lazarus syndrome, T cellular immunity deficiency, congenital alopecia and hypoplastic nails etc.This further specifies hTF of the present invention and can interact with DNA, participates in transcriptional control,

In sum, hTF of the present invention can form zinc fingers, has DNA land feature, can be used as a kind of transcription factor and combine also interaction with nucleic acid with regulate gene expression, thereby the physiological activity of pair cell and even organism plays regulating and controlling effect.These physiological actions comprise the promotion tissue growth, the growth of regulating cell and propagation, and inducing cell is to certain specific trend development or the like.

HTF 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, hTF of the present invention can also merge with other members of this family or exchange fragment, to produce new albumen, for example the N end of hTF of the present invention and the N end of mouse CKR can be exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of hTF of the present invention, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

Embodiment 5

Preparation antibody

Embodiment 2 or 3 recombinant proteins that obtain are used for immune animal to produce antibody, specific as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation hTF gene translation product with it.

Example 6

Substrate as micromatrix

Micromatrix is called the DNA chip again.(, select by using some famous biosoftwares hTF full-length cDNA, EST or gene fragment substrate samples as micromatrix as LASERGENESOFTWARE (DNASTAR).By using ink-jet technology and a series of chemical process such as ray, chemistry, thermodynamics, mechanical means etc. that sample is fixed on the carrier,, obtain chip (Schena, M.et al. (1995) Science 270:467-470 then as on glass; AndShalon, D.et al. (1996) Genome Res.6:639-645.), the element of formation has point-like, bar shaped or the like.A typical chip contains the element of some amount usually.Behind the hybridization, the unconjugated probe of flush away detects the element of generation hybridization and the degree of reaction with scanner then.The complementarity of probe and each chip component and binding capacity all can be judged by the analysis to the image that scans.

Results of hybridization can be used for detecting hTF genovariation, sudden change and polymorphism, understands because hTF expresses the molecular basis of the undesired disease that causes, diagnoses the illness, and can improve and monitor the activity of related agents.

Embodiment 7:

The preparation of test kit

Test kit 1:

The primer that it contains (1) specific amplification hTF to and operation instruction.This test kit also can contain or not contain the required reagent that the mRNA reverse transcription is become cDNA.Wherein, the sequence of this Auele Specific Primer is derived from the sequence of SEQ ID NO:1.The cDNA that reverse transcription is become carries out specific amplification, whether to contain the nucleotide sequence of hDGF7 in the test sample.This test kit also can contain and carries out other required reagent of PCR reaction, for example damping fluid etc.

In addition, preferably, at least one used primer has been crossed over two exons, because will not produce amplified production corresponding to the genome sequence of hTF this moment.

Test kit 2:

This test kit contains the specific antibody (for example polyclonal antibody of preparation among the embodiment 5, the perhaps monoclonal antibody of the anti-hTF of the hybridoma technology generation of usefulness standard) at hTF, and operation instruction.Whether this test kit is used for direct test sample and exists or lack hTF albumen.Form immunocomplex by specific immune response earlier, detect immunocomplex with routine techniques then.

Test kit 3:

This test kit contains can be specifically and the probe of the mRNA hybridization of hTF.It also can contain or not contain hybridization buffer.This test kit comes whether to exist in the test sample nucleic acid molecule of hTF by the hybridization between nucleic acid molecule and the hTF specific probe.

Test kit also can be used for detecting genovariation, sudden change and polymorphism, and detects the relevant gene of hTF a series of and of the present invention, thereby diagnosis, the treatment of relative disease helped out.

Example 8:

Make medicine

HTF albumen of the present invention and antibody, inhibitor, antagonist or acceptor etc. can be used as medicine, can promote the wound healing after scalds and burns, cutting tissue and even organ are extractd, and prevent wound infection.With hTF nucleic acid antisense strand of the present invention hTF antibody generate a reagent box can be used for alleviating or assisting therapy owing to the too high disease that causes of hTF expression amount, also can assist the diagnosis of tumour and cancer clinically, prevention and treatment.

Albumen of the present invention 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.

In addition, hTF nucleic acid of the present invention (encoding sequence or antisense sequences) can directly be introduced cell, with expression level that improves hTF or the overexpression that suppresses hTF.HTF albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of hTF 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.

When hTF protein polypeptide of the present invention is used as medicine, the treatment effective dose is 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.

＜210〉1＜211〉2594＜212〉＜213〉＜220〉＜221〉＜222〉 ( 370 ) .. ( 2592 )＜223〉＜400〉1gcttgaacct tgtcacccct cacgtgcaca ccaaagacat accctagtga ttaaatgctg 60atttgtgtac gatgtccacg gacgccaaaa caatcacaga gctgcttgat tgttttaatt 120atccagcaca aaatgccatc agtctgggac gtgatcgggc agaggtgtac tcacagtagt 180gtaaatactg ctgtaaatag tgtctgatgg tggcttgaca gtgagctagc ttctgagttt 240tcccttcttt ttatactgtt ttctgtgctg gcttttttga atcttcctaa tttttcatct 300ctttaacaaa ctcctatgaa gttgaaaccg ggaagtttgc tctaacattt ccagagaagg 360tattaagtc atg atg cag gaa tct gcg aca gag aca ata agc aac agt tca 411

Met?Met?Gln?Glu?Ser?Ala?Thr?Glu?Thr?Ile?Ser?Asn?Ser?Ser

1 5 10atg?aat?caa?aat?gga?atg?agc?act?cta?agc?agc?caa?tta?gat?gct?ggc 459Met?Asn?Gln?Asn?Gly?Met?Ser?Thr?Leu?Ser?Ser?Gln?Leu?Asp?Ala?Gly15 20 25 30agc?aga?gat?gga?aga?tca?agt?ggt?gac?acc?agc?tct?gaa?gta?agc?aca 507Ser?Arg?Asp?Gly?Arg?Ser?Ser?Gly?Asp?Thr?Ser?Ser?Glu?Val?Ser?Thr

35 40 45gta?gaa?ctg?cta?cat?ctg?caa?caa?cag?cag?gct?ctc?cag?gca?gca?aga 555Val?Glu?Leu?Leu?His?Leu?Gln?Gln?Gln?Gln?Ala?Leu?Gln?Ala?Ala?Arg

50 55 60caa?ctt?ctt?tta?cag?cag?caa?aca?agt?gga?ttg?aaa?tct?cct?aag?agc 603Gln?Leu?Leu?Leu?Gln?Gln?Gln?Thr?Ser?Gly?Leu?Lys?Ser?Pro?Lys?Ser

65 70 75agt?gat?aaa?cag?aga?cca?ctg?cag?gaa?ttg?ctt?cca?gaa?aca?aaa?tta 651Ser?Asp?Lys?Gln?Arg?Pro?Leu?Gln?Glu?Leu?Leu?Pro?Glu?Thr?Lys?Leu

80 85 90tgt?atc?tgt?ggc?cac?tct?tct?ggt?gat?ggg?cat?cct?cac?aac?aca?ttt 699Cys?Ile?Cys?Gly?His?Ser?Ser?Gly?Asp?Gly?His?Pro?His?Asn?Thr?Phe95 100 105 110gca?gtg?cct?gtg?tca?gtg?gcc?atg?atg?act?ccc?cag?gtg?atc?acc?cct 747Ala?Val?Pro?Val?Ser?Val?Ala?Met?Met?Thr?Pro?Gln?Val?Ile?Thr?Pro

115 120 125cag?caa?atg?cag?cag?atc?ctt?cag?caa?caa?gtc?ctg?tct?cct?cag?cag 795Gln?Gln?Met?Gln?Gln?Ile?Leu?Gln?Gln?Gln?Val?Leu?Ser?Pro?Gln?Gln

130 135 140cta?caa?gcc?ctt?ctc?caa?caa?cag?cag?gct?gtc?atg?ctg?cag?cag?caa 843Leu?Gln?Ala?Leu?Leu?Gln?Gln?Gln?Gln?Ala?Val?Met?Leu?Gln?Gln?Gln

145 150 155caa?cta?caa?gag?ttt?tac?aag?aaa?cag?caa?gag?cag?tta?cat?ctt?cag 891Gln?Leu?Gln?Glu?Phe?Tyr?Lys?Lys?Gln?Gln?Glu?Gln?Leu?His?Leu?Gln

160 165 170ctt?ttg?cag?cag?cag?cag?caa?cag?cag?cag?cag?caa?caa?cag?cag?caa 939Leu?Leu?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln175 180 185 190caa?cag?cag?cag?caa?caa?caa?caa?caa?cag?cag?caa?caa?cag?cag?cag 987Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln

195 200 205cag?cag?caa?cag?cag?cag?cag?cag?caa?cag?cat?cct?gga?aag?caa?gcg 1035Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?His?Pro?Gly?Lys?Gln?Ala

210 215 220aaa?gag?cag?cag?cag?cag?cag?cag?cag?caa?cag?caa?ttg?gca?gcc?cag 1083Lys?Glu?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Leu?Ala?Ala?Gln

225 230 235cag?ctt?gtc?ttc?cag?cag?cag?ctt?ctc?cag?atg?caa?caa?ctc?cag?cag 1131Gln?Leu?Val?Phe?Gln?Gln?Gln?Leu?Leu?Gln?Met?Gln?Gln?Leu?Gln?Gln

240 245 250cag?cag?cat?ctg?ctc?agc?ctt?cag?cgt?cag?gga?ctc?atc?tcc?att?cca 1179Gln?Gln?His?Leu?Leu?Ser?Leu?Gln?Arg?Gln?Gly?Leu?Ile?Ser?Ile?Pro255 260 265 270cct?ggc?cag?gca?gca?ctt?cct?gtc?caa?tcg?ctg?cct?caa?gct?ggc?tta 1227Pro?Gly?Gln?Ala?Ala?Leu?Pro?Val?Gln?Ser?Leu?Pro?Gln?Ala?Gly?Leu

275 280 285agt?cct?gct?gag?att?cag?cag?tta?tgg?aaa?gaa?gtg?act?gga?gtt?cac 1275Ser?Pro?Ala?Glu?llc?Gln?Gln?Leu?Trp?Lys?Glu?Val?Thr?Gly?Val?His

290 295 300agt?atg?gaa?gac?aat?ggc?att?aaa?cat?gga?ggg?cta?gac?ctc?act?act 1323Ser?Met?Glu?Asp?Asn?Gly?Ile?Lys?His?Gly?Gly?Leu?Asp?Leu?Thr?Thr

305 310 315aac?aat?tcc?tcc?tcg?act?acc?tcc?tcc?aac?act?tcc?aaa?gca?tca?cca 1371Asn?Asn?Ser?Ser?Ser?Thr?Thr?Ser?Ser?Asn?Thr?Ser?Lys?Ala?Ser?Pro

320 325 330cca?ata?act?cat?cat?tcc?ata?gtg?aat?gga?cag?tct?tca?gtt?cta?agt 1419Pro?Ile?Thr?His?His?Ser?Ile?Val?Asn?Gly?Gln?Ser?Ser?Val?Leu?Ser335 340 345 350gca?aga?cga?gac?agc?tcg?tca?cat?gag?gag?act?ggg?gcc?tct?cac?act 1467Ala?Arg?Arg?Asp?Ser?Ser?Ser?His?Glu?Glu?Thr?Gly?Ala?Ser?His?Thr

355 360 365ctc?tat?ggc?cat?gga?gtt?tgc?aaa?tgg?cca?ggc?tgt?gaa?agc?att?tgt 1515Leu?Tyr?Gly?His?Gly?Val?Cys?Lys?Trp?Pro?Gly?Cys?Glu?Ser?Ile?Cys

370 375 380gaa?gat?ttt?gga?cag?ttt?tta?aag?cac?ctt?aac?aat?gaa?cac?gca?ttg 1563Glu?Asp?Phe?Gly?Gln?Phe?Leu?Lys?His?Leu?Asn?Asn?Glu?His?Ala?Leu

385 390 395gat?gac?cga?agc?act?gct?cag?tgt?cga?gtg?caa?atg?cag?gtg?gtg?caa 1611Asp?Asp?Arg?Ser?Thr?Ala?Gln?Cys?Arg?Val?Gln?Met?Gln?Val?Val?Gln

400 405 410cag?tta?gaa?ata?cag?ctt?tct?aaa?gaa?cgc?gaa?cgt?ctt?caa?gca?atg 1659Gln?Leu?Glu?Ile?Gln?Leu?Ser?Lys?Glu?Arg?Glu?Arg?Leu?Gln?Ala?Met415 420 425 430atg?acc?cac?ttg?cac?atg?cga?ccc?tca?gag?ccc?aaa?cca?tct?ccc?aaa 1707Met?Thr?His?Leu?His?Met?Arg?Pro?Ser?Glu?Pro?Lys?Pro?Ser?Pro?Lys

435 440 445cct?cta?aat?ctg?gtg?tct?agt?gtc?acc?atg?tcg?aag?aat?atg?ttg?gag 1755Pro?Leu?Asn?Leu?Val?Ser?Ser?Val?Thr?Met?Ser?Lys?Asn?Met?Leu?Glu

450 455 460aca?tcc?cca?cag?agc?tta?cct?caa?acc?cct?acc?aca?cca?acg?gcc?cca 1803Thr?Ser?Pro?Gln?Ser?Leu?Pro?Gln?Thr?Pro?Thr?Thr?Pro?Thr?Ala?Pro

465 470 475gtc?acc?ccg?att?acc?cag?gga?ccc?tca?gta?atc?acc?cca?gcc?agt?gtg 1851Val?Thr?Pro?Ile?Thr?Gln?Gly?Pro?Ser?Val?Ile?Thr?Pro?Ala?Ser?Val

480 485 490ccc?aat?gtg?gga?gcc?ata?cga?agg?cga?cat?tca?gac?aaa?tac?aac?att 1899Pro?Asn?Val?Gly?Ala?Ile?Arg?Arg?Arg?His?Ser?Asp?Lys?Tyr?Asn?Ile495 500 505 510ccc?atg?tca?tca?gaa?att?gcc?cca?aac?tat?gaa?ttt?tat?aaa?aat?gca 1947Pro?Met?Ser?Ser?Glu?Ile?Ala?Pro?Asn?Tyr?Glu?Phe?Tyr?Lys?Asn?Ala

515 520 525gat?gtc?aga?cct?cca?ttt?act?tat?gca?act?ctc?ata?agg?cag?gct?atc 1995Asp?Val?Arg?Pro?Pro?Phe?Thr?Tyr?Ala?Thr?Leu?Ile?Arg?Gln?Ala?Ile

530 535 540atg?gag?tca?tct?gac?agg?cag?tta?aca?ctt?aat?gaa?att?tac?agc?tgg 2043Met?Glu?Ser?Ser?Asp?Arg?Gln?Leu?Thr?Leu?Asn?Glu?Ile?Tyr?Ser?Trp

545 550 555ttt?aca?cgg?aca?ttt?gct?tac?ttc?agg?cgt?aat?gca?gca?act?tgg?aag 2091Phe?Thr?Arg?Thr?Phe?Ala?Tyr?Phe?Arg?Arg?Asn?Ala?Ala?Thr?Trp?Lys

560 565 570aat?gca?gta?cgt?cat?aat?ctt?agc?ctg?cac?aag?tgt?ttt?gtt?cga?gta 2139Asn?Ala?Val?Arg?His?Asn?Leu?Ser?Leu?His?Lys?Cys?Phe?Val?Arg?Val575 580 585 590gaa?aat?gtt?aaa?gga?gca?gta?tgg?act?gtg?gat?gaa?gta?gaa?tac?cag 2187Glu?Asn?Val?Lys?Gly?Ala?Val?Trp?Thr?Val?Asp?Glu?Val?Glu?Tyr?Gln

595 600 605aag?cga?agg?tca?caa?aag?ata?aca?gga?agt?cca?acc?tta?gta?aaa?aat 2235Lys?Arg?Arg?Ser?Gln?Lys?Ile?Thr?Gly?Ser?Pro?Thr?Leu?Val?Lys?Asn

610 615 620ata?cct?acc?agt?tta?ggc?tat?gga?gca?gct?ctt?aat?gcc?agt?ttg?cag 2283Ile?Pro?Thr?Ser?Leu?Gly?Tyr?Gly?Ala?Ala?Leu?Asn?Ala?Ser?Leu?Gln

625 630 635gct?gcc?ttg?gca?gag?agc?agt?tta?cct?ttg?cta?agt?aat?cct?gga?ctg 2331Ala?Ala?Leu?Ala?Glu?Ser?Ser?Leu?Pro?Leu?Leu?Ser?Asn?Pro?Gly?Leu

640 645 650ata?aat?aat?gca?tcc?agt?ggc?cta?ctg?cag?gcc?gtc?cac?gaa?gac?ctc 2379Ile?Asn?Asn?Ala?Ser?Ser?Gly?Leu?Leu?Gln?Ala?Val?His?Glu?Asp?Leu655 660 665 670aat?ggt?tct?ctg?gat?cac?att?gac?agc?aat?gga?aac?agt?agt?ccg?ggc 2427Asn?Gly?Ser?Leu?Asp?His?Ile?Asp?Ser?Asn?Gly?Asn?Ser?Ser?Pro?Gly

675 680 685tgc?tca?cct?cag?ccg?cac?ata?cat?tca?atc?cac?gtc?aag?gaa?gag?cca 2475Cys?Ser?Pro?Gln?Pro?His?Ile?His?Ser?Ile?His?Val?Lys?Glu?Glu?Pro

690 695 700gtg?att?gca?gag?gat?gaa?gac?tgc?cca?atg?tcc?tta?gtg?aca?aca?gct 2523Val?Ile?Ala?Glu?Asp?Glu?Asp?Cys?Pro?Met?Ser?Leu?Val?Thr?Thr?Ala

705 710 715aat?cac?agt?cca?gaa?tta?gaa?gac?gac?aga?gag?att?gaa?gaa?gag?cct 2571Asn?His?Ser?Pro?Glu?Leu?Glu?Asp?Asp?Arg?Glu?Ile?Glu?Glu?Glu?Pro

720 725 730tta tct gaa gat ctg gaa tga ga 2594Leu Ser Glu Asp Leu Glu735 740＜210〉2＜211〉740＜212〉amino acid＜213〉mankind＜400〉2Met Met Gln Glu Ser Ala Thr Glu Thr Ile Ser Asn Ser Ser Met Asn1 5 10 15Gln Asn Gly Met Ser Thr Leu Ser Ser Gln Leu Asp Ala Gly Ser Arg

20 25 30Asp?Gly?Arg?Ser?Ser?Gly?Asp?Thr?Ser?Ser?Glu?Val?Ser?Thr?Val?Glu

35 40 45Leu?Leu?His?Leu?Gln?Gln?Gln?Gln?Ala?Leu?Gln?Ala?Ala?Arg?Gln?Leu

50 55 60Leu?Leu?Gln?Gln?Gln?Thr?Ser?Gly?Leu?Lys?Ser?Pro?Lys?Ser?Ser?Asp65 70 75 80Lys?Gln?Arg?Pro?Leu?Gln?Glu?Leu?Leu?Pro?Glu?Thr?Lys?Leu?Cys?Ile

85 90 95Cys?Gly?His?Ser?Ser?Gly?Asp?Gly?His?Pro?His?Asn?Thr?Phe?Ala?Val

100 105 110Pro?Val?Ser?Val?Ala?Met?Met?Thr?Pro?Gln?Val?Ile?Thr?Pro?Gln?Gln

115 120 125Met?Gln?Gln?Ile?Leu?Gln?Gln?Gln?Val?Leu?Ser?Pro?Gln?Gln?Leu?Gln

130 135 140Ala?Leu?Leu?Gln?Gln?Gln?Gln?Ala?Val?Met?Leu?Gln?Gln?Gln?Gln?Leu145 150 155 160Gln?Glu?Phe?Tyr?Lys?Lys?Gln?Gln?Glu?Gln?Leu?His?Leu?Gln?Leu?Leu

165 170 175Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln

180 185 190Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln

195 200 205Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?His?Pro?Gly?Lys?Gln?Ala?Lys?Glu

210 215 220Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Leu?Ala?Ala?Gln?Gln?Leu225 230 235 240Val?Phe?Gln?Gln?Gln?Leu?Leu?Gln?Met?Gln?Gln?Leu?Gln?Gln?Gln?Gln

245 250 255His?Leu?Leu?Ser?Leu?Gln?Arg?Gln?Gly?Leu?Ile?Ser?Ile?Pro?Pro?Gly

260 265 270Gln?Ala?Ala?Leu?Pro?Val?Gln?Ser?Leu?Pro?Gln?Ala?Gly?Leu?Ser?Pro

275 280 285Ala?Glu?Ile?Gln?Gln?Leu?Trp?Lys?Glu?Val?Thr?Gly?Val?His?Ser?Met

290 295 300Glu?Asp?Asn?Gly?Ile?Lys?His?Gly?Gly?Leu?Asp?Leu?Thr?Thr?Asn?Asn305 310 315 320Ser?Ser?Ser?Thr?Thr?Ser?Ser?Asn?Thr?Ser?Lys?Ala?Ser?Pro?Pro?Ile

325 330 335Thr?His?His?Ser?Ile?Val?Asn?Gly?Gln?Ser?Ser?Val?Leu?Ser?Ala?Arg

340 345 350Arg?Asp?Ser?Ser?Ser?His?Glu?Glu?Thr?Gly?Ala?Ser?His?Thr?Leu?Tyr

355 360 365Gly?His?Gly?Val?Cys?Lys?Trp?Pro?Gly?Cys?Glu?Ser?Ile?Cys?Glu?Asp

370 375 380Phe?Gly?Gln?Phe?Leu?Lys?His?Leu?Asn?Asn?Glu?His?Ala?Leu?Asp?Asp385 390 395 400Arg?Ser?Thr?Ala?Gln?Cys?Arg?Val?Gln?Met?Gln?Val?Val?Gln?Gln?Leu

405 410 415Glu?Ile?Gln?Leu?Ser?Lys?Glu?Arg?Glu?Arg?Leu?Gln?Ala?Met?Met?Thr

420 425 430His?Leu?His?Met?Arg?Pro?Ser?Glu?Pro?Lys?Pro?Ser?Pro?Lys?Pro?Leu

435 440 445Asn?Leu?Val?Ser?Ser?Val?Thr?Met?Ser?Lys?Asn?Met?Leu?Glu?Thr?Ser

450 455 460Pro?Gln?Ser?Leu?Pro?Gln?Thr?Pro?Thr?Thr?Pro?Thr?Ala?Pro?Val?Thr465 470 475 480Pro?Ile?Thr?Gln?Gly?Pro?Ser?Val?Ile?Thr?Pro?Ala?Ser?Val?Pro?Asn

485 490 495Val?Gly?Ala?Ile?Arg?Arg?Arg?His?Ser?Asp?Lys?Tyr?Asn?Ile?Pro?Met

500 505 510Ser?Ser?Glu?Ile?Ala?Pro?Asn?Tyr?Glu?Phe?Tyr?Lys?Asn?Ala?Asp?Val

515 520 525Arg?Pro?Pro?Phe?Thr?Tyr?Ala?Thr?Leu?Ile?Arg?Gln?Ala?Ile?Met?Glu

530 535 540Ser?Ser?Asp?Arg?Gln?Leu?Thr?Leu?Asn?Glu?Ile?Tyr?Ser?Trp?Phe?Thr545 550 555 560Arg?Thr?Phe?Ala?Tyr?Phe?Arg?Arg?Asn?Ala?Ala?Thr?Trp?Lys?Asn?Ala

565 570 575Val?Arg?His?Asn?Leu?Ser?Leu?His?Lys?Cys?Phe?Val?Arg?Val?Glu?Asn

580 585 590Val?Lys?Gly?Ala?Val?Trp?Thr?Val?Asp?Glu?Val?Glu?Tyr?Gln?Lys?Arg

595 600 605Arg?Ser?Gln?Lys?Ile?Thr?Gly?Ser?Pro?Thr?Leu?Val?Lys?Asn?Ile?Pro

610 615 620Thr?Ser?Leu?Gly?Tyr?Gly?Ala?Ala?Leu?Asn?Ala?Ser?Leu?Gln?Ala?Ala625 630 635 640Leu?Ala?Glu?Ser?Ser?Leu?Pro?Leu?Leu?Ser?Asn?Pro?Gly?Leu?Ile?Asn

645 650 655Asn?Ala?Ser?Ser?Gly?Leu?Leu?Gln?Ala?Val?His?Glu?Asp?Leu?Asn?Gly

660 665 670Ser?Leu?Asp?His?Ile?Asp?Ser?Asn?Gly?Asn?Ser?Ser?Pro?Gly?Cys?Ser

675 680 685Pro?Gln?Pro?His?Ile?His?Ser?Ile?His?Val?Lys?Glu?Glu?Pro?Val?Ile

690 695 700Ala?Glu?Asp?Glu?Asp?Cys?Pro?Met?Ser?Leu?Val?Thr?Thr?Ala?Asn?His705 710 715 720Ser?Pro?Glu?Leu?Glu?Asp?Asp?Arg?Glu?Ile?Glu?Glu?Glu?Pro?Leu?Ser

725 730 735Glu?Asp?Leu?Glu

740

Claims

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of people hTF protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 370-2592 position among described nucleotide sequence and the SEQ ID NO.1.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQID NO.2.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence is the sequence of Nucleotide 370-2592 position among the SEQ ID NO.1.

4. isolating hTF protein polypeptide is characterized in that it comprises: have polypeptide or its active fragments of SEQ ID NO.2 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.2 polypeptide of sequence.

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

7. one kind with the described carrier transformed host cells of claim 6.

8. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.

9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.

10. a generation has the method for the polypeptide of hTF protein-active, it is characterized in that this method comprises:

(d) isolate polypeptide with hTF protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 370-2592 position among the SEQ ID NO.1.

12. energy and the described hTF protein polypeptide of claim 4 specificity bonded antibody.

13. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.

14. a probe molecule is characterized in that, it contains 8-100 continuous nucleotide in the described dna molecular of claim 1

15. a test kit is characterized in that, it contains the described dna molecular of claim 1.

16. a test kit is characterized in that, it contains the described antibody of claim 12.

17. the application of the described dna molecular of claim 1 is characterized in that, it is used for nucleic acid amplification reaction as primer or with making gene chip; Perhaps be used to prepare the disease that medicine causes owing to the human transcription factor encoding gene unusually with prevention, diagnosis or treatment.

18. the application of the described protein polypeptide of claim 4 is characterized in that, it uses agonist or inhibitor with the screening human transcription factor; Perhaps be used to prepare the disease that medicine causes owing to the human cell factor expression of receptor unusually with prevention, diagnosis or treatment.