CN1377891A - Zinc finger protein from dendritic cell, its coding sequence and use - Google Patents
Zinc finger protein from dendritic cell, its coding sequence and use Download PDFInfo
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Abstract
The present invnetion relates to one new type of zinc finger protein and provides polynucleotides for coding this protein molecule and the recombination technology method of producing this protein molecule. The present invention also discloses the use of the polynucleotides for coding this cell period regulating protein molecule. The present invention also discloses the antibody to the protein and its application method in diagnosing the treating diseases, especially in diagnosing and treating hemopoietic disorder, immunological response abnormity and tumor.
Description
The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding Novel Human Zinc Finger Protein DPZF, and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.Specifically, polypeptide of the present invention is a kind of new BTB/POZ zinc finger protein.
Gene expression regulation is the key link of the complicated vital movement of cell, and it has determined the specific functional features of cell.At present, found that by the research of transcriptional regulatory many gene family codifieds produce transcription factors, these transcription factors have has cell or promotor specificity, has plenty of the cell base of keeping and transcribes movable necessary.
C2H2 zinc finger protein family is one of family maximum in the transcription factor, is characterized in containing the C2H2 zinc fingers of some repeated arrangement.The concensus sequence of C2H2 zinc fingers is CX
2-4CX
3FX
5LX
2HX
3-4, Cys wherein and His can combine with zine ion, thus make the key amino acid of zinc in referring to be combined into can with DNA bonded structure.
At Kruppel C2H2 zinc finger protein subfamily, H/C catenation sequence (TGEKY/F) is contained in the zone that connects contiguous zinc fingers usually.
Dezincification refers to beyond the district that the N of zinc finger protein end has diversity and conservative property usually, and the interaction between the mediation albumen participates in transcriptional control.The N end structure of C2H2 zinc finger protein comprises FAX, KRAB, BTB/POZ etc.BTB/POZ is the hydrophobic region of high conservative, contains about 120 amino acid, mainly mediates between the BTB/POZ albumen self combination.
The zinc finger protein that contains BTB/POZ and Kruppel zinc fingers claims POK again.Except POK, BTB/POZ also is present in Bach albumen, and Bach albumen belongs to another kind of transcription factor, and its C end does not contain zinc fingers, and contains the leucine zipper structure of alkalescence.In POK albumen, the BTB/POZ structure it has been generally acknowledged that to appraise and decide a feature relevant with zinc finger protein specific, can mediate transcripting suppressioning action.
BCL-6 (B-cell lymphoma-6) and preceding myelogenous leukemia zinc finger protein PLZF are the POK important members, may play a significant role in processes such as hematopoiesis, tumour generation and immunne response, thereby be subjected in recent years paying close attention to widely.The BCL-6 assignment of genes gene mapping mainly is expressed in the B cell of germinal center in 3q27, participates in the adjusting of lymphocyte generation, inflammation and immunne response.The BCL-6 deficient mice shows as germinal center's dyspoiesis and lymphocyte generates impaired.The BCL-6 gene transposition is to the immunoglobulin gene site, or 5 ' non-coding region point mutation of BCl-6 gene, and the imbalance that can cause BCL-6 to express is relevant with the lymphadenomatous generation of B.
Zinc finger protein will cause that unusually gene transcript expression is unusual, and cause for example multiple disease such as tumour, hematopoiesis disorder.Therefore, significant for the Novel Human Zinc Finger Protein of diagnosing and the therapeutic purpose research and development is new.
The purpose of this invention is to provide a kind of new Novel Human Zinc Finger Protein-DPZF albumen with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of these polypeptide of coding.
Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.
In a first aspect of the present invention, novel isolated DPZF polypeptide is provided, this polypeptide is the people source, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people DPZF polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 187-2412 position among the SEQ ID NO:1; (b) has the sequence of 1-2764 position among the SEQ ID NO:1.
In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.
In a fourth aspect of the present invention, provide preparation to have the method for the polypeptide of people DPZF protein-active, this method comprises: (a) under the proteic condition of suitable expressing human DPZF, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people DPZF protein-active.
In a fifth aspect of the present invention, provide and above-mentioned people DPZF polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 187-2412 Nucleotide in the above-mentioned polynucleotide.
In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people DPZF polypeptide active is provided, and the compound that suppresses people DPZF polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of people DPZF polypeptide.
In a seventh aspect of the present invention, provide and whether had the proteic method of DPZF in the test sample, it comprises: sample is contacted with the proteic specific antibody of DPZF, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample DPZF albumen.
In a eighth aspect of the present invention, a kind of disease relevant with people DPZF polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.
In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes people DPZF polypeptide active, and perhaps screening suppresses the antagonist of people DPZF polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people DPZF of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.
In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains people DPZF polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated the multiple disease of hematopoiesis disorder, abnormal immune response, tumour.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that is defined.
Fig. 1 has shown the aminoacid sequence of zinc finger protein DPZF of the present invention.Wherein, the POZ district represented in black matrix, and underscore is represented the C2H2 zinc fingers.
Fig. 2 is that the proteic homology of zinc finger protein DPZF of the present invention and people BCL-6 compares.Wherein Fig. 2 A is that the POZ district compares; Fig. 2 B is that C2H2 zinc refers to the comparison distinguished.Among the figure, conservative halfcystine (Cys) and Histidine (His) among " * " expression C2H2.
Fig. 3 has shown that zinc finger protein DPZF is at distribution expression pattern.
Fig. 4 has shown that zinc finger protein DPZF has high expression level in lymphoma cell strain.
Fig. 5 has shown zinc finger protein DPZF immunohistochemical analysis result in tonsilla.
Fig. 6 has shown zinc finger protein DPZF immunohistochemical analysis result in the B lymphoma cell.
In the present invention, term " DPZF albumen ", " DPZF polypeptide " or " zinc finger protein DPZF " can Alternate, the albumen of (SEQ ID NO:2) or many that all refers to have human zinc-finger protein DPZF amino acid sequence Peptide. They comprise the zinc finger protein DPZF that contains or do not contain initial methionine.
As used herein, " separation " refers to that material separates (if natural from its primal environment Material, primal environment namely is natural surroundings). Such as the polynucleotide under the native state in the active somatic cell Do not have separation and purification with polypeptide, but same polynucleotide or polypeptide are as depositing together from native state Other materials in separately, then for separation and purification.
As used herein, " DPZF albumen or the polypeptide of separation " refers to that the DPZF polypeptide is substantially free of the sky Right relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can be with marking Accurate purified technology of protein purifying DPZF albumen. Basically pure polypeptide is solidifying at non-reduced polyacrylamide Can produce single master tape on the glue.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide. Polypeptide of the present invention can be the product of natural purifying, or the product of chemical synthesis, or uses the restructuring skill Art is from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell) The middle generation. The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, or Can be nonglycosylated. Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises fragment, derivative and the analog of people DPZF albumen. As used herein, term " fragment ", " derivative " and " analog " refer to basically keep natural human DPZF egg of the present invention The biological function of Bai Xiangtong or active polypeptide. Polypeptide fragment of the present invention, derivative or analog can Being that (i) has one or more conservative or non-conservation amino acid residue (preferred conservative amino acid residue) quilts The polypeptide that replaces, and the amino acid residue of such replacement can be also can not be to be encoded by genetic code , or (ii) in one or more amino acid residues, have the polypeptide of substituted radical, or (iii) maturation is many Peptide and another compound (such as the compound that prolongs the polypeptide half-life, for example polyethylene glycol) merge institute's shape The polypeptide that becomes, or (iv) additional amino acid sequence be fused to this peptide sequence and the polypeptide that forms (as leading Sequence or secretion sequence or be used for sequence or the proteinogen sequence of this polypeptide of purifying, or with antigen I gG fragment The fusion of formation). According to the instruction of this paper, these fragments, derivative and analog belong to ability The known scope of territory those of skill in the art.
In the present invention, term " people DPZF polypeptide " refers to have the SEQ ID NO. of people DPZF protein active The polypeptide of 2 sequences. This term also comprise have with people DPZF albumen identical function, SEQ ID NO.2 The variant form of sequence. These variant forms comprise (but being not limited to): several (be generally 1-50, 1-30 preferably, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or Replace, and add one or several and (be generally in 20, preferably in that C end and/or N are terminal Being in 10, more preferably is in 5) amino acid. For example, in the art, close with performance Or similar amino acid can not change the function of protein when replacing usually. Again such as, at the C end One of the terminal interpolation of end and/or N or several amino acid also can not change the function of protein usually. This term The active fragment and the reactive derivative that also comprise people DPZF albumen.
The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural Mutant, induced mutation body, under high or low stringency condition can with the DNA of people DPZF DNA hybridization Coded albumen and the polypeptide or the albumen that utilize the antiserum acquisition of anti-people DPZF polypeptide. The present invention Other polypeptide also are provided, as have comprised the fusion of people DPZF polypeptide or its fragment. Except total length almost Polypeptide outside, the present invention has also comprised the soluble fragments of people DPZF polypeptide. Usually, this fragment has the people The DPZF peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, Goodly 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 analog of people DPZF albumen or polypeptide. These analogs and natural human DPZF are many The difference of peptide can be the difference on the amino acid sequence, also can be not affect on the modified forms of sequence Difference perhaps haves both at the same time. These polypeptide comprise genetic variant natural or that induce. The induce variation body Can obtain by various technology, as by radiation or be exposed to mutagens and produce random mutagenesis, also can By direct mutagenesis method or the biological technology of other known moleculars. Analog also comprises having the sky of being different from The analog of the right amino acid whose residue of L-(such as D-amino acid), and have that non-natural exists or synthetic The analog of amino acid (such as β, gamma-amino acid). Should be understood that polypeptide of the present invention is not limited to above-mentioned example The representational polypeptide of lifting.
(usually the not changing primary structure) form of modification comprises: the chemically derived shape of the polypeptide that body is interior or external Formula such as acetylation or carboxylated. Modify and also to comprise glycosylation, as those in the synthetic and processing of polypeptide or Further carry out glycosylation modified in the procedure of processing and polypeptide that produce. This modification can be passed through polypeptide Be exposed to and carry out glycosylated enzyme (such as mammiferous glycosylase or deglycosylating enzyme) and finish. Modify Form also comprises having the phosphorylated amino acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine) Sequence. Thereby also comprising being modified has improved its anti-proteolysis performance or has optimized the many of solubility property Peptide.
In the present invention, " people DPZF albumen conservative variation polypeptide " refers to the ammonia with SEQ ID NO:2 The base acid sequence is compared, and has 10 at the most, and preferably at the most 8, more preferably at the most 5, best extremely Many 3 amino acid are replaced by similar performance or close amino acid and are formed polypeptide. These conservatives become Different polypeptide preferably carries out amino acid substitution according to table 1 and produces.
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 |
| Thr (T) | Ser | Ser |
| Trp (W) | Tyr；Phe | Tyr |
| Tyr (Y) | Trp；Phe；Thr；Ser | Phe |
| Val (V) | Ile；Leu；Met；Phe；Ala | Leu |
Polynucleotides of the present invention can be dna form or rna form. Dna form comprises cDNA, base Because of group DNA or artificial synthetic DNA. DNA can be strand or double-stranded. DNA can be coding Chain or noncoding strand. The coding region sequence of encoding mature polypeptide can with the coding shown in the SEQ ID NO:1 The variant of the identical or degeneracy of region sequence. As used herein, " variant of degeneracy " is in the present invention In refer to encode and have the protein of SEQ ID NO:2, but with the code area order shown in the SEQ ID NO:1 Be listed as differentiated nucleotide sequence.
The polynucleotides of the mature polypeptide of coding SEQ ID NO:2 comprise: the coding of an encoding mature polypeptide Sequence; The coded sequence of mature polypeptide and various additional code sequence; The coded sequence of mature polypeptide (with appoint The additional code sequence of choosing) and non-coding sequence.
Term " polynucleotides of coded polypeptide " can be the polynucleotides that comprise this polypeptide of encoding, and also can To be the polynucleotides that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation encoding D PZF.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
People DPZF 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 synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
Use method (Saiki, the et al.Science1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or DPZF albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the DPZF polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people DPZF polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, people DPZF polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains people DPZF DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, aLaboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P
LPromotor; Eukaryotic promoter comprise CMV immediate early promoter, HSV thymidine kinase start give, early stage and late period SV40 promotor, retrovirus LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The people DPZF albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism DPZF protein function as pharmacological agent DPZF protein function.The peptide molecule that can suppress or stimulate people DPZF protein function that can be used for seeking therapeutic value with the recombinant human DPZF protein screening peptide library of expressing.
On the other hand, the present invention also comprises people DPZF 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 people DPZF gene product or fragment.Preferably, refer to that those can combine with people DPZF 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 people DPZF, comprise that also those do not influence the antibody of people DPZF protein function.The present invention also comprise those can with modify or without the people DPZF 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 people DPZF gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human DPZF albumen or its have 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 to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodiesand T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people DPZF protein function and the antibody that does not influence people DPZF protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people DPZF 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 people DPZF 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).
The proteic antibody of anti-people DPZF can be used in the immunohistochemistry technology, detects the people DPZF albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people DPZF, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.
Antibody among the present invention can be used for treating or prevention and the relevant disease of people DPZF albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people DPZF or activity.
Antibody also can be used for being designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people DPZF albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell (for example lymph-node cell etc.) of people DPZF protein positive.
The production of polyclonal antibody can choose DPZF albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
Utilize albumen of the present invention,, can filter out with DPZF albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.
Albumen of the present invention and antibody thereof, inhibitor, agonist, 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 about 5-8 usually, 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, intravenously, subcutaneous, intracutaneous or topical.
Polypeptide of the present invention can be directly used in disease treatment, for example, is used for multiple treatment of diseases such as hematopoiesis disorder, abnormal immune response, tumour.When using DPZF albumen of the present invention, also can use the other treatment agent simultaneously, as IFN, EPO etc.
The present invention also provides a kind of pharmaceutical composition, and it contains DPZF polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.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.Pharmaceutical composition 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 making pharmaceutical composition, be that the DPZF albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount 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.
The proteic polynucleotide of people DPZF also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell endocytic, secretion or the metabolic disturbance due to the proteic expression of DPZF of the proteic nothing expression of DPZF or unusual/non-activity.The DPZF albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic DPZF protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the DPZF transgenosis to cell.The method that structure carries the recombinant viral vector of DPZF gene is found in existing document (Sambrook, et al.).Recombinant human DPZF gene can be packaged in the liposome in addition, and then is transferred in the cell.
Suppress the oligonucleotide (comprising sense-rna and DNA) of people DPZF mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.
Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.
Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of people DPZF obtains.During screening, must carry out mark to people DPZF protein molecular.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people DPZF protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people DPZF protein level that is detected in the test can be with laying down a definition the importance of people DPZF albumen in various diseases and be used to the disease of diagnosing DPZF albumen to work.
Whether having the proteic method of DPZF in a kind of detection test sample is to utilize the proteic specific antibody of DPZF to detect, and it comprises: sample is contacted with the DPZF protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample DPZF albumen.
The proteic polynucleotide of DPZF can be used for the diagnosis and the treatment of DPZF protein related diseases.Aspect diagnosis, the proteic polynucleotide of DPZF can be used for detecting the proteic expression of DPZF DPZF abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of DPZF as the DPZF dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of DPZF albumen and also can detect the proteic transcription product of DPZF.
The sudden change that detects the DPZF gene also can be used for the disease of diagnosing DPZF albumen relevant.The form of DPZF protein mutation comprises that the point mutation compared with normal wild type DPZF dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.
Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of DPZF prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.
In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, MendelianInheritance in Man (can by with the online acquisition of Johns Hopkins University Welch MedicalLibrary).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ IDNO:2.Polynucleotide of the present invention are isolated from human dendritic cell cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 2764 bases, and its open reading frame is positioned at the 187-2412 position, and the coding total length is 741 amino acid whose new BTB/POZ zinc finger proteins.This albumen n end contains the BTB/POZ structure, and the C end contains the C2H2 zinc fingers of 4 repeated arrangement, the POZ zinc finger protein (DPZF) in called after dendritic cell source.
POZ district and the BCL-6 homology of DPZF are the highest.DPZF is wide expression in hemopoietic tissue, comprises spleen, lymphoglandula, thymus gland, peripheral blood leucocyte and tire liver.Immunohistochemical methods shows that DPZF mainly is expressed in germinal center in hemopoietic tissue.Identical with BCL-6, DPZF also is positioned 3q27.These show that DPZF is and the zinc finger protein of BCl-6 height correlation, may participate in the adjusting that hematopoiesis generates.Different with BCl-6 is, DPZF has wider express spectra, and DPZF is expressed in DC, peripheral blood lymphocytes, B cell and T cell; The tumour cell of cells of monocytic origin is not expressed DPZF as U937, HL-60, NB4 and THP-1 cell strain etc., and tumor tissues, especially the B lymphoma high expression level DPZF in lymph source.
Except BCl-6 and DPZF, many genes relevant with hematopoiesis are arranged on No. 3 karyomit(e)s, comprise MLF-1 (myeloid leukemia factor-1), IKAROS and thrombopoietin (TPO) etc., these expression of gene imbalances can cause the disorder of body hemopoietic function, even cause the generation of tumour.MLF-1 is relevant with acute leukemia, and IKAROS plays the regulating effect at center in the lymphocyte differentiation, relevant with diffusivity maxicell B lymphoma (DLCL).The expression imbalance of this hint DPZF may cause the unusual of hematopoiesis disorder and immunne response.
Therefore, DPZF albumen or its relevant antagonist, agonist etc. can be the diagnosis and the treatment of treatment hematopoiesis disorder, abnormal immune response, tumour and offer help, and may be, thereby has great application prospect directly for diseases such as hematopoiesis disorder, abnormal immune response, tumour provide new treatment approach.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the clone of people DPZF cDNA
Extract the total RNA of human dendritic cell with Trizol (Gibco company).Then, from total RNA, separate poly (A) mRNA.Poly (A) mRNA after reverse transcription forms cDNA, is cloned test kit (available from Gibco) with SuperScriptII cDNA fragment orientation is inserted on the multiple clone site of carrier, transform DH5 α bacterium and form the cDNA plasmid library.Measure random choose clone's 5 ' terminal sequence with dideoxy method.CDNA sequence and the existing public dna sequence data storehouse measured are compared, found that a cDNA clone's dna sequence dna is new full-length cDNA.By synthetic a series of primers the contained dna sequence dna of new clone is carried out two-way mensuration.Computer Analysis shows that cloning contained full-length cDNA is a new cDNA sequence (shown in SEQ ID NO:1), the new protein (as SEQ ID NO:2 and shown in Figure 1) of encoding.This protein is named as Novel Human Zinc Finger Protein DPZF, its encoding gene called after Novel Human Zinc Finger Protein DPZF gene.
Sequence SEQ ID NO:1 total length is 2764bp, comprises 3 ' end non-coding region of 5 of 186bp ' end non-coding region and 352bp, and the coding region is positioned at the 187-2412 position, 741 the amino acid whose polypeptide of encoding.Calculating not in theory, the molecular weight of glycosylated ripe molecule is about 81kD.
They are different with known for the BLAST analysis revealed, at protein level and people BCL-6 to a certain degree homology are arranged.Especially refer to district and BCL-6 albumen and preceding myelogenous leukemia zinc finger protein PLZF albumen height homology (Fig. 2 A and 2B) in POZ district and C2H2 zinc.This shows that albumen of the present invention is a kind of new zinc finger protein, the POZ zinc finger protein (DPZF) in called after dendritic cell source.
Embodiment 2: with the encoding sequence of RT-PCR method human cloning DPZF
Be in logarithmic phase B lymphoma cell strain Raji cell total rna with Trizol (Gibco company) extraction, get 6mg cell total rna and 0.5 μ g Oligo-dT
12-18Mix, carry out reverse transcription.The reverse transcription system is 20 μ l, and reaction adds 80 μ l ddH after finishing
2O dilutes.The used primer of pcr amplification is as follows: have adopted primer 5 '-gc TCT AGA GA TCT ATG CTA GAA CGG AAG AAA CCC-3 ' (SEQ IDNO:3), antisense primer 5 ' ggaa ttc ACT ACT TAT CCG TCA GAC AC-3 ' (SEQ ID NO:4).The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.4mM primer, 0.2mM dNTP and 1U ExTaq archaeal dna polymerase (Takara Inc.), the amplification parameter be 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds, capable 2% agarose gel electrophoresis of PCR product is tentatively confirmed after 25 circulations.Detect the amplified production of about 2.2kb.
The dna sequence analysis result shows that the DNA sequences encoding of this PCR product and the 187-2412 position shown in the SEQ ID NO:1 are identical.
The Northern engram analysis of embodiment 3 DPZF
Carry out the Northern trace by following ordinary method: filter membrane to be checked places the hybridization solution of 10ml through 68 ℃ of preheatings, in hybrid heater (Bellco) in 68 ℃ of prehybridizations 30 minutes; The cDNA probe that mark is good was in 95～100 ℃ of sex change 2～5 minutes, and (cDNA probe final concentration is 2～10ng/ml or 1～2 * 10 to put rapid cooling back adding hybridization solution on ice
6Cpm/ml), fully mixing was hybridized 2 hours in 68 ℃.After hybridization finishes, filter membrane with 2 * SSC, the drip washing of 0.05%SDS room temperature for several times, the washing lotion several is changed in continuing vibration flushing 30～40 minutes therebetween.Use 0.1 * SSC, 0.1%SDS in 50 ℃ of vibration flushings 20～40 minutes subsequently.Last filter membrane wrapped up with plastic fresh-keeping membrane, in-70 ℃ of exposure X line films 24～48 hours.
Northern blot hybridization result shows: certain expression is arranged in liver, spleen, peripheral blood, thymus gland and lymphoglandula.This shows that DPZF albumen is a kind of expression albumen (Fig. 3) more widely.
The RT-PCR that embodiment 4 DPZF express analyzes
Be in logarithmic phase cell or cell strain Raji cell total rna with Trizol (Gibco company) extraction, get 6mg cell total rna and 0.5 μ g Oligo-dT
12-18Mix, carry out reverse transcription.The reverse transcription system is 20 μ l, and reaction adds 80 μ l ddH after finishing
2O dilutes.The used primer of pcr amplification is as follows: have adopted primer 5 '-TCT CCA GAC CCA GCC CTC AT-3 ' (SEQ ID NO:5), antisense primer 5 ' CGACTG CGA GAC CCG TAG C-3 ' (SEQ ID NO:6).Estimate the about 378bp of amplified production.The amplimer of beta-actin is 5 '-GCATCGTGATGGACTCCG-3 ' (justice is arranged) (SEQ ID NO:9), 5 '-TCGGAAGGTGGACAGCGA-3 ' (antisense) (SEQ ID NO:10), the expectation amplified production is 600bp.The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.4mM primer, 0.2mM dNTP and 1U ExTaqDNA polysaccharase (Takara Inc.), the amplification parameter be 94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 45 seconds, capable 2% agarose gel electrophoresis of PCR product is tentatively confirmed after 30 circulations.
The result as shown in Figure 4, DPZF albumen especially has the high expression level of high level at B lymphoma cell strains such as Raji, Daudi.
In this embodiment, express the N end parts fragment of DPZF and the fusion rotein that GST forms.With the pcr amplification product among the embodiment 2 is template, increases with the PCR Oligonucleolide primers of 5 ' and 3 following ' end of sequence, obtains people DPZF DNA as inserting fragment.
5 ' end Oligonucleolide primers sequence of using in the PCR reaction is:
5′-g?gaa?tt?CTA?GAA?CGG?AAG?AAA?CCC-3′(SEQ?ID?NO：7)
This primer contains the restriction enzyme site of BamHI restriction enzyme, is the part Nucleotide of the encoding sequence that begun by initiator codon after this restriction enzyme site;
3 ' end primer sequence is:
5′-g?gaa?ttc?ggt?acc?TCA?GCC?CGA?CTC?CGT?GTC?GCT-3′(SEQ?ID?NO.8)，
This primer contains restriction enzyme site, translation termination of EcoRI restriction enzyme, and amplified production contains 230 amino acid whose encoding sequences of N end of people DPZF.
DPZF cDNA PCR product purification is after XbaI cuts with the EcoRI enzyme again is connected with plasmid pGEM3, recombinate according to a conventional method and be converted into the competence bacillus coli DH 5 alpha, the picking positive colony is identified back purifying and order-checking (377 type sequenators of ABI company, BigDye Terminator test kit, PE company).The DPZF cDNA BglII/EcoRI endonuclease bamhi of correct sequence is cloned into expression vector pGEX-2T (Pharmacia company), forms carrier pGST-DPZF and be converted into bacillus coli DH 5 alpha.Positive colony is cut the evaluation direction with BamHI/Kpn I enzyme, and enzyme is cut the capable 2% agarose gel electrophoresis analysis of product.Confirm through order-checking, inserted the DPZF encoding sequence, the N end merges correct with GST.
Choosing the positive DH5 α clone who expresses DPZF is inoculated in 100ml 2 * YTA substratum, 37 ℃ of 300rpm shaking culture 12-15hr, 2 * YTA the substratum that is diluted in preheating at 1: 10 continues shaking culture 1.5hr, add behind the 100mM IPTG to 0.1mM 30 ℃ and induce 2-6hr, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml 1 * PBS (0.14M NaCl on ice, 2.7mM KCl, 10.1mM Na
2HPO
4, 1.8mM KH
2PO
4PH7.3) resuspended, add 20%Triton X-100 to 1% jog 30min again after ultrasonic (B.Braun Labsonic U) fragmentation, then 12,4 ℃ of centrifugal 10min of 000g, supernatant with 0.8 μ m membrane filtration after, cross 1ml 50% glutathione S epharose 4B chromatography column, behind 1 * PBS thorough washing, add 500ul gsh elution buffer (10mM gsh, 50mM Tris-HCl, pH 8.0) room temperature leaves standstill after 30 minutes and collects elutriant, repeat wash-out 2-3 time, obtain the people DPZF fusion rotein that the N end contains GST, molecular weight is about 54kD.
Embodiment 6: anti-people DPZF production of antibodies
The DPZF gst fusion protein that obtains among the embodiment 5 is used for immune animal to produce antibody, and concrete grammar is 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 500-600 μ g/1ml emulsification carries out subcutaneous multi-point injection to rabbit.After 60 days,, mouse is carried out subcutaneous multi-point injection with booster immunization with the dosage of 300-400 μ g/1ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carry out booster immunization one time every 28 days, carry out secondary at least.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people DPZF gene translation product with it.Found that antibody can combine with albumen of the present invention specifically.
Embodiment 7: immunohistochemical analysis and chromosomal localization
Get the tonsilla and the B lymphoma biopsy specimen of clinical excision, the row paraffin section carries out immunohistochemical staining according to a conventional method.Spend the night earlier, add the goat anti-rabbit igg (DAKO company) of horseradish peroxidase (HRP) mark after developing a film,, add the DAB substrate after developing a film and develop the color, after mounting, microscopy behind the haematoxylin redyeing in room temperature effect 1 hour with the how anti-incubated at room of anti-DPZF rabbit.
The result as illustrated in Figures 5 and 6.DPZF mainly is expressed in germinal center (Fig. 5) at tonsilla, presents high expression level (Fig. 6) in the B lymphoma cell.
In addition, with ordinary method DPZF has been carried out chromosomal localization, found identically with BCL-6, DPZF also is positioned 3q27.These show that DPZF is and the zinc finger protein of BCl-6 height correlation, may participate in the adjusting that hematopoiesis generates.
The proteic eukaryotic expression of embodiment 8 people DPZF
In this embodiment, express the people DPZF albumen of total length.
Pcr amplification product among the embodiment 2 is cloned into pCMV-tag2 carrier (Stratagen company), is built into the carrier for expression of eukaryon pDPZF-FLAG of DPZF, its N end merges with FLAG sequence (DYKDDDDK).Dna sequence dna and reading frame do not have mistake through sequence verification.
With expression vector pDPZF-FLAG through liposome transfection COS7 cell, lysing cell after 48 hours.After cell pyrolysis liquid carried out electrophoretic separation with 8%SDS-PAGE, electrotransfer detected with anti-FLAG antibody (Stratagen company) to nitrocellulose filter.The result shows that the proteic molecular weight of DPZF is about 94kDa.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table＜110〉Immunology Inst., No.2 Military Medical Univ.＜120〉zinc finger protein, its coded sequence and purposes＜130 of Cells Derived from Dendritic〉011794＜160〉10＜170〉PatentIn version 3.0＜210〉1＜211〉2764＜212〉DNA＜213〉Homo sapiens＜220〉＜221〉CDS＜222〉(187) .. (2412)＜400〉1gggcaggttg ggaaacagcc cagtggtata aggatgagga aactgaagcc cagagaggtg 60aagtgaggtg cccaaggcca cacagcaagt tagaggcaca gctagtacgg tagctcaagt 120ctcctgactc ccagtccagt gctcctccta ttactccacg agtcctgtct ctaagcttcc 180tgacaa atg cta gaa cgg aag aaa ccc aag aca gct gaa aac cag aag 228
Met?Leu?Glu?Arg?Lys?Lys?Pro?Lys?Thr?Ala?Glu?Asn?Gln?Lys
1 5 10gca?tct?gag?gag?aat?gag?att?act?cag?ccg?ggt?gga?tcc?agc?gcc?aag 276Ala?Ser?Glu?Glu?Asn?Glu?Ile?Thr?Gln?Pro?Gly?Gly?Ser?Ser?Ala?Lys15 20 25 30ccg?ggc?ctt?ccc?tgc?ctg?aac?ttt?gaa?gct?gtt?ttg?tct?cca?gac?cca 324Pro?Gly?Leu?Pro?Cys?Leu?Asn?Phe?Glu?Ala?Val?Leu?Ser?Pro?Asp?Pro
35 40 45gcc?ctc?atc?cac?tca?aca?cat?tca?ctg?aca?aac?tct?cac?gct?cac?acc 372Ala?Leu?Ile?His?Ser?Thr?His?Ser?Leu?Thr?Asn?Ser?His?Ala?His?Thr
50 55 60ggg?tca?tct?gat?tgt?gac?atc?agt?tgc?aag?ggg?atg?acc?gag?cgc?att 420Gly?Ser?Ser?Asp?Cys?Asp?Ile?Ser?Cys?Lys?Gly?Met?Thr?Glu?Arg?Ile
65 70 75cac?agc?atc?aac?ctt?cac?aac?ttc?agc?aat?tcc?gtg?ctc?gag?acc?ctc 468His?Ser?Ile?Asn?Leu?His?Asn?Phe?Ser?Asn?Ser?Val?Leu?Glu?Thr?Leu
80 85 90aac?gag?cag?cgc?aac?cgt?ggc?cac?ttc?tgt?gac?gta?acg?gtg?cgc?atc 516Asn?Glu?Gln?Arg?Asn?Arg?Gly?His?Phe?Cys?Asp?Val?Thr?Val?Arg?Ile95 100 105 110cac?ggg?agc?atg?ctg?cgc?gcg?cac?cgc?tgc?gtg?ctg?gca?gcc?ggc?agc 564His?Gly?Ser?Met?Leu?Arg?Ala?His?Arg?Cys?Val?Leu?Ala?Ala?Gly?Ser
115 120 125ccc?ttc?ttc?cag?gac?aaa?ctg?ctg?ctt?ggc?tac?agc?gac?atc?gag?atc 612Pro?Phe?Phe?Gln?Asp?Lys?Leu?Leu?Leu?Gly?Tyr?Ser?Asp?Ile?Glu?Ile
130 135 140ccg?tcg?gtg?gtg?tca?gtg?cag?tca?gtg?caa?aag?ctc?att?gac?ttc?atg 660Pro?Ser?Val?Val?Ser?Val?Gln?Ser?Val?Gln?Lys?Leu?Ile?Asp?Phe?Met
145 150 155tac?agc?ggc?gtg?cta?cgg?gtc?tcg?cag?tcg?gaa?gct?ctg?cag?atc?ctc 708Tyr?Ser?Gly?Val?Leu?Arg?Val?Ser?Gln?Ser?Glu?Ala?Leu?Gln?Ile?Leu
160 165 170acg?gcc?gcc?agc?atc?ctg?cag?atc?aaa?aca?gtc?atc?gac?gag?tgc?acg 756Thr?Ala?Ala?Ser?Ile?Leu?Gln?Ile?Lys?Thr?Val?Ile?Asp?Glu?Cys?Thr175 180 185 190cgc?atc?gtg?tca?cag?aac?gtg?ggc?gat?gtg?ttc?ccg?ggg?atc?cag?gac 804Arg?Ile?Val?Ser?Gln?Asn?Val?Gly?Asp?Val?Phe?Pro?Gly?Ile?Gln?Asp
195 200 205tcg?ggc?cag?gac?acg?ccg?cgg?ggc?act?ccc?gag?tca?ggc?acg?tca?ggc 852Ser?Gly?Gln?Asp?Thr?Pro?Arg?Gly?Thr?Pro?Glu?Ser?Gly?Thr?Ser?Gly
210 215 220cag?ggc?agc?gac?acg?gag?tcg?ggc?tac?ctg?cag?agc?cac?cca?cag?cac 900Gln?Gly?Ser?Asp?Thr?Glu?Ser?Gly?Tyr?Leu?Gln?Ser?His?Pro?Gln?His
225 230 235agc?gtg?gac?agg?atc?tac?tcg?gca?ctc?tac?gcg?tgc?tcc?atg?cag?aat 948Ser?Val?Asp?Arg?Ile?Tyr?Ser?Ala?Leu?Tyr?Ala?Cys?Ser?Met?Gln?Asn
240 245 250ggc?agc?ggc?gag?cgc?tct?ttt?tac?agc?ggc?gca?atg?gtc?agc?cac?cac 996Gly?Ser?Gly?Glu?Arg?Ser?Phe?Tyr?Ser?Gly?Ala?Met?Val?Ser?His?His255 260 265 270gag?act?gcg?ctc?ggc?ctg?ccc?cgc?gac?cac?cac?atg?gaa?gac?ccc?agc 1044Glu?Thr?Ala?Leu?Gly?Leu?Pro?Arg?Asp?His?His?Met?Glu?Asp?Pro?Ser
275 280 285tgg?atc?aca?cgc?atc?cat?gag?cgc?tcg?cag?cag?atg?gag?cgc?tac?ctg 1092Trp?Ile?Thr?Arg?Ile?His?Glu?Arg?Ser?Gln?Gln?Met?Glu?Arg?Tyr?Leu
290 295 300tcc?acc?acc?ccc?gag?acc?acg?cac?tgc?cgc?aag?cag?ccc?cgg?cct?gtg 1140Ser?Thr?Thr?Pro?Glu?Thr?Thr?His?Cys?Arg?Lys?Gln?Pro?Arg?Pro?Val
305 310 315cgc?atc?cag?acc?cta?gtg?ggc?aac?atc?cac?atc?aag?cag?gag?atg?gag 1188Arg?Ile?Gln?Thr?Leu?Val?Gly?Asn?Ile?His?Ile?Lys?Gln?Glu?Met?Glu
320 325 330gac?gat?ttc?gac?tac?tac?ggg?cag?caa?agg?gtg?cag?atc?ctg?gaa?cgc 1236Asp?Asp?Phe?Asp?Tyr?Tyr?Gly?Gln?Gln?Arg?Val?Gln?Ile?Leu?Glu?Arg335 340 345 350aac?gaa?tcc?gag?gag?tgc?acg?gaa?gac?aca?gac?cag?gcc?gag?ggc?acc 1284Asn?Glu?Ser?Glu?Glu?Cys?Thr?Glu?Asp?Thr?Asp?Gln?Ala?Glu?Gly?Thr
355 360 365gag?agt?gag?ccc?aaa?ggt?gaa?agc?ttc?gac?tcg?ggc?gtc?agc?tcc?tcc 1332Glu?Ser?Glu?Pro?Lys?Gly?Glu?Ser?Phe?Asp?Ser?Gly?Val?Ser?Ser?Ser
370 375 380ata?ggc?acc?gag?cct?gac?tcg?gtg?gag?cag?cag?ttt?ggg?cct?ggg?gcg 1380Ile?Gly?Thr?Glu?Pro?Asp?Ser?Val?Glu?Gln?Gln?Phe?Gly?Pro?Gly?Ala
385 390 395gcg?cgg?gac?agc?cag?gct?gaa?ccc?acc?caa?ccc?gag?cag?gct?gca?gaa 1428Ala?Arg?Asp?Ser?Gln?Ala?Glu?Pro?Thr?Gln?Pro?Glu?Gln?Ala?Ala?Glu
400 405 410gcc?ccc?gct?gag?ggt?ggt?ccg?cag?aca?aac?cag?cta?gaa?aca?ggt?gct 1476Ala?Pro?Ala?Glu?Gly?Gly?Pro?Gln?Thr?Asn?Gln?Leu?Glu?Thr?Gly?Ala415 420 425 430tcc?tct?ccg?gag?aga?agc?aat?gaa?gtg?gag?atg?gac?agc?act?gtt?atc 1524Ser?Ser?Pro?Glu?Arg?Ser?Asn?Glu?Val?Glu?Met?Asp?Ser?Thr?Val?Ile
435 440 445act?gtc?agc?aac?agc?tcc?gac?aag?agc?gtc?cta?caa?cag?cct?tcg?gtc 1572Thr?Val?Ser?Asn?Ser?Ser?Asp?Lys?Ser?Val?Leu?Gln?Gln?Pro?Ser?Val
450 455 460aac?acg?ttc?atc?ggg?cag?cca?ttg?cca?agt?acc?cag?ctc?tac?tta?cgc 1620Asn?Thr?Phe?Ile?Gly?Gln?Pro?Leu?Pro?Ser?Thr?Gln?Leu?Tyr?Leu?Arg
465 470 475cag?aca?gaa?acc?ctc?acc?agc?aac?ctg?agg?atg?cct?ctg?acc?ttg?acc 1668Gln?Thr?Glu?Thr?Leu?Thr?Ser?Asn?Leu?Arg?Met?Pro?Leu?Thr?Leu?Thr
480 485 490agc?aac?acg?cag?gtc?att?ggc?aca?gct?ggc?aac?acc?tac?ctg?cca?gcc 1716Ser?Asn?Thr?Gln?Val?Ile?Gly?Thr?Ala?Gly?Asn?Thr?Tyr?Leu?Pro?Ala495 500 505 510ctc?ttc?act?acc?cag?ccc?gtg?ggc?agt?ggc?ccc?aag?cct?ttc?ctc?ttc 1764Leu?Phe?Thr?Thr?Gln?Pro?Val?Gly?Ser?Gly?Pro?Lys?Pro?Phe?Leu?Phe
515 520 525agc?ctg?cca?cag?ccc?ctg?gca?ggc?cag?cag?acc?cag?ttt?gtg?aca?gtg 1812Ser?Leu?Pro?Gln?Pro?Leu?Ala?Gly?Gln?Gln?Thr?Gln?Phe?Val?Thr?Val
530 535 540tcc?cag?ccc?ggt?ctg?tcg?acc?ttt?act?gca?cag?ctg?cca?gcg?cca?cag 1860Ser?Gln?Pro?Gly?Leu?Ser?Thr?Phe?Thr?Ala?Gln?Leu?Pro?Ala?Pro?Gln
545 550 555ccc?ctg?gcc?tca?tcc?gca?ggc?cac?agc?aca?gcc?agt?ggg?caa?ggc?gaa 1908Pro?Leu?Ala?Ser?Ser?Ala?Gly?His?Ser?Thr?Ala?Ser?Gly?Gln?Gly?Glu
560 565 570aaa?aag?cct?tat?gag?tgc?act?ctc?tgc?aac?aag?act?ttc?acc?gcc?aaa 1956Lys?Lys?Pro?Tyr?Glu?Cys?Thr?Leu?Cys?Asn?Lys?Thr?Phe?Thr?Ala?Lys575 580 585 590cag?aac?tac?gtc?aag?cac?atg?ttc?gta?cac?aca?ggt?gag?aag?ccc?cac 2004Gln?Asn?Tyr?Val?Lys?His?Met?Phe?Val?His?Thr?Gly?Glu?Lys?Pro?His
595 600 605caa?tgc?agc?atc?tgt?tgg?cgc?tcc?ttc?tcc?tta?aag?gat?tac?ctt?atc 2052Gln?Cys?Ser?Ile?Cys?Trp?Arg?Ser?Phe?Ser?Leu?Lys?Asp?Tyr?Leu?Ile
610 615 620aag?cac?atg?gtg?aca?cac?aca?gga?gtg?agg?gca?tac?cag?tgt?agt?atc 2100Lys?His?Met?Val?Thr?His?Thr?Gly?Val?Arg?Ala?Tyr?Gln?Cys?Ser?Ile
625 630 635tgc?aac?aag?cgc?ttc?acc?cag?aag?agc?tcc?ctc?aac?gtg?cac?atg?cgc 2148Cys?Asn?Lys?Arg?Phe?Thr?Gln?Lys?Ser?Ser?Leu?Asn?Val?His?Met?Arg
640 645 650ctc?cac?cgg?gga?gag?aag?tcc?tac?gag?tgc?tac?atc?tgc?aaa?aag?aag 2196Leu?His?Arg?Gly?Glu?Lys?Ser?Tyr?Glu?Cys?Tyr?Ile?Cys?Lys?Lys?Lys655 660 665 670ttc?tct?cac?aag?acc?ctc?ctg?gag?cga?cac?gtg?gcc?ctg?cac?agt?gcc 2244Phe?Ser?His?Lys?Thr?Leu?Leu?Glu?Arg?His?Val?Ala?Leu?His?Ser?Ala
675 680 685agc?aat?ggg?acc?ccc?cct?gca?ggc?aca?ccc?cca?ggt?gcc?cgc?gct?ggc 2292Ser?Asn?Gly?Thr?Pro?Pro?Ala?Gly?Thr?Pro?Pro?Gly?Ala?Arg?Ala?Gly
690 695 700ccc?cca?ggc?gtg?gtg?gcc?tgc?acg?gag?ggg?acc?act?tac?gtc?tgc?tcc 2340Pro?Pro?Gly?Val?Val?Ala?Cys?Thr?Glu?Gly?Thr?Thr?Tyr?Val?Cys?Ser
705 710 715gtc?tgc?cca?gca?aag?ttt?gac?caa?atc?gag?cag?ttc?aac?gac?cac?atg 2388Val?Cys?Pro?Ala?Lys?Phe?Asp?Gln?Ile?Glu?Gln?Phe?Asn?Asp?His?Met
720 725 730agg?atg?cat?gtg?tct?gac?gga?taa?gtagtatctt?tctctctttc?ttatgaacaa 2442Arg?Met?His?Val?Ser?Asp?Gly735 740aacaaaacaa?caacaaaaaa?caaacaaaca?aaaaagctat?ggcactagaa?tttaagaaat 2502gttttggttt?catttttact?ttctgttttt?gtttttgttt?cgtttcattt?tgtactacat 2562gaagaactgt?tttttgcctg?ctggtacatt?acatttccgg?aggcttgggt?gaataatagt 2622tttcccagtc?tccctcggat?ggtggcctta?aggcctggta?gtgcttcaag?aggtccactg 2682gttggatctc?tagctactgg?cctctaaata?caacccttct?ttacaaaaaa?atcttttaaa 2742aaaaagtaaa?aaaaaaaaaa?aa 2764<210>2<211>741<212>PRT<213>Homo?sapiens<400>2Met?Leu?Glu?Arg?Lys?Lys?Pro?Lys?Thr?Ala?Glu?Asn?Gln?Lys?Ala?Ser1 5 10 15Glu?Glu?Asn?Glu?Ile?Thr?Gln?Pro?Gly?Gly?Ser?Ser?Ala?Lys?Pro?Gly
20 25 30Leu?Pro?Cys?Leu?Asn?Phe?Glu?Ala?Val?Leu?Ser?Pro?Asp?Pro?Ala?Leu
35 40 45Ile?His?Ser?Thr?His?Ser?Leu?Thr?Asn?Ser?His?Ala?His?Thr?Gly?Ser
50 55 60Ser?Asp?Cys?Asp?Ile?Ser?Cys?Lys?Gly?Met?Thr?Glu?Arg?Ile?His?Ser65 70 75 80Ile?Asn?Leu?His?Asn?Phe?Ser?Asn?Ser?Val?Leu?Glu?Thr?Leu?Asn?Glu
85 90 95Gln?Arg?Asn?Arg?Gly?His?Phe?Cys?Asp?Val?Thr?Val?Arg?Ile?His?Gly
100 105 110Ser?Met?Leu?Arg?Ala?His?Arg?Cys?Val?Leu?Ala?Ala?Gly?Ser?Pro?Phe
115 120 125Phe?Gln?Asp?Lys?Leu?Leu?Leu?Gly?Tyr?Ser?Asp?Ile?Glu?Ile?Pro?Ser
130 135 140Val?Val?Ser?Val?Gln?Ser?Val?Gln?Lys?Leu?Ile?Asp?Phe?Met?Tyr?Ser145 150 155 160Gly?Val?Leu?Arg?Val?Ser?Gln?Ser?Glu?Ala?Leu?Gln?Ile?Leu?Thr?Ala
165 170 175Ala?Ser?Ile?Leu?Gln?Ile?Lys?Thr?Val?Ile?Asp?Glu?Cys?Thr?Arg?Ile
180 185 190Val?Ser?Gln?Asn?Val?Gly?Asp?Val?Phe?Pro?Gly?Ile?Gln?Asp?Ser?Gly
195 200 205Gln?Asp?Thr?Pro?Arg?Gly?Thr?Pro?Glu?Ser?Gly?Thr?Ser?Gly?Gln?Gly
210 215 220Ser?Asp?Thr?Glu?Ser?Gly?Tyr?Leu?Gln?Ser?His?Pro?Gln?His?Ser?Val225 230 235 240Asp?Arg?Ile?Tyr?Ser?Ala?Leu?Tyr?Ala?Cys?Ser?Met?Gln?Asn?Gly?Ser
245 250 255Gly?Glu?Arg?Ser?Phe?Tyr?Ser?Gly?Ala?Met?Val?Ser?His?His?Glu?Thr
260 265 270Ala?Leu?Gly?Leu?Pro?Arg?Asp?His?His?Met?Glu?Asp?Pro?Ser?Trp?Ile
275 280 285Thr?Arg?Ile?His?Glu?Arg?Ser?Gln?Gln?Met?Glu?Arg?Tyr?Leu?Ser?Thr
290 295 300Thr?Pro?Glu?Thr?Thr?His?Cys?Arg?Lys?Gln?Pro?Arg?Pro?Val?Arg?Ile305 310 315 320Gln?Thr?Leu?Val?Gly?Asn?Ile?His?Ile?Lys?Gln?Glu?Met?Glu?Asp?Asp
325 330 335Phe?Asp?Tyr?Tyr?Gly?Gln?Gln?Arg?Val?Gln?Ile?Leu?Glu?Arg?Asn?Glu
340 345 350Ser?Glu?Glu?Cys?Thr?Glu?Asp?Thr?Asp?Gln?Ala?Glu?Gly?Thr?Glu?Ser
355 360 365Glu?Pro?Lys?Gly?Glu?Ser?Phe?Asp?Ser?Gly?Val?Ser?Ser?Ser?Ile?Gly
370 375 380Thr?Glu?Pro?Asp?Ser?Val?Glu?Gln?Gln?Phe?Gly?Pro?Gly?Ala?Ala?Arg385 390 395 400Asp?Ser?Gln?Ala?Glu?Pro?Thr?Gln?Pro?Glu?Gln?Ala?Ala?Glu?Ala?Pro
405 410 415Ala?Glu?Gly?Gly?Pro?Gln?Thr?Asn?Gln?Leu?Glu?Thr?Gly?Ala?Ser?Ser
420 425 430Pro?Glu?Arg?Ser?Asn?Glu?Val?Glu?Met?Asp?Ser?Thr?Val?Ile?Thr?Val
435 440 445Ser?Asn?Ser?Ser?Asp?Lys?Ser?Val?Leu?Gln?Gln?Pro?Ser?Val?Asn?Thr
450 455 460Phe?Ile?Gly?Gln?Pro?Leu?Pro?Ser?Thr?Gln?Leu?Tyr?Leu?Arg?Gln?Thr465 470 475 480Glu?Thr?Leu?Thr?Ser?Asn?Leu?Arg?Met?Pro?Leu?Thr?Leu?Thr?Ser?Asn
485 490 495Thr?Gln?Val?Ile?Gly?Thr?Ala?Gly?Asn?Thr?Tyr?Leu?Pro?Ala?Leu?Phe
500 505 510Thr?Thr?Gln?Pro?Val?Gly?Ser?Gly?Pro?Lys?Pro?Phe?Leu?Phe?Ser?Leu
515 520 525Pro?Gln?Pro?Leu?Ala?Gly?Gln?Gln?Thr?Gln?Phe?Val?Thr?Val?Ser?Gln
530 535 540Pro?Gly?Leu?Ser?Thr?Phe?Thr?Ala?Gln?Leu?Pro?Ala?Pro?Gln?Pro?Leu545 550 555 560Ala?Ser?Ser?Ala?Gly?His?Ser?Thr?Ala?Ser?Gly?Gln?Gly?Glu?Lys?Lys
565 570 575Pro?Tyr?Glu?Cys?Thr?Leu?Cys?Asn?Lys?Thr?Phe?Thr?Ala?Lys?Gln?Asn
580 585 590Tyr?Val?Lys?His?Met?Phe?Val?His?Thr?Gly?Glu?Lys?Pro?His?Gln?Cys
595 600 605Ser?Ile?Cys?Trp?Arg?Ser?Phe?Ser?Leu?Lys?Asp?Tyr?Leu?Ile?Lys?His
610 615 620Met?Val?Thr?His?Thr?Gly?Val?Arg?Ala?Tyr?Gln?Cys?Ser?Ile?Cys?Asn625 630 635 640Lys?Arg?Phe?Thr?Gln?Lys?Ser?Ser?Leu?Asn?Val?His?Met?Arg?Leu?His
645 650 655Arg?Gly?Glu?Lys?Ser?Tyr?Glu?Cys?Tyr?Ile?Cys?Lys?Lys?Lys?Phe?Ser
660 665 670His?Lys?Thr?Leu?Leu?Glu?Arg?His?Val?Ala?Leu?His?Ser?Ala?Ser?Asn
675 680 685Gly?Thr?Pro?Pro?Ala?Gly?Thr?Pro?Pro?Gly?Ala?Arg?Ala?Gly?Pro?Pro
690 695 700Gly?Val?Val?Ala?Cys?Thr?Glu?Gly?Thr?Thr?Tyr?Val?Cys?Ser?Val?Cys705 710 715 720Pro?Ala?Lys?Phe?Asp?Gln?Ile?Glu?Gln?Phe?Asn?Asp?His?Met?Arg?Met
725 730 735His?Val?Ser?Asp?Gly
740＜210〉3＜211〉34＜212〉DNA＜213〉＜400〉3gctctagaga tctatgctag aacggaagaa accc 34＜210〉4＜211〉27＜212〉DNA＜213〉＜400〉4ggaattcact acttatccgt cagacac 27＜210〉5＜211〉20＜212〉DNA＜213〉＜400〉5tctccagacc cagccctcat 20＜210〉6＜211〉19＜212〉DNA＜213〉＜400〉6cgactgcgag acccgtagc 19＜210〉7＜211〉24＜212〉DNA＜213〉＜400〉7ggaattctag aacggaagaa accc 24＜210〉8＜211〉34＜212〉DNA＜213〉＜400〉8ggaattcggt acctcagccc gactccgtgt cgct 34＜210〉9＜211〉18＜212〉DNA＜213〉＜400〉9gcatcgtgat ggactccg 18＜210〉10＜211〉18＜212〉DNA＜213〉＜400〉10tcggaaggtg gacagcga 18
Claims (10)
1. an isolating people DPZF polypeptide is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group:
(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ IDNO:2.
5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:
(a) has the sequence of 187-2412 position among the SEQ ID NO:1;
(b) has the sequence of 1-2764 position among the SEQ ID NO:1.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.
8. preparation method with polypeptide of people DPZF protein-active is characterized in that this method comprises:
(a) under the proteic condition of suitable expressing human DPZF, cultivate the described host cell of claim 7;
(b) from culture, isolate polypeptide with people DPZF protein-active.
9. energy and the described people DPZF of claim 1 protein-specific bonded antibody.
10. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01105817 CN1377891A (en) | 2001-04-02 | 2001-04-02 | Zinc finger protein from dendritic cell, its coding sequence and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01105817 CN1377891A (en) | 2001-04-02 | 2001-04-02 | Zinc finger protein from dendritic cell, its coding sequence and use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1377891A true CN1377891A (en) | 2002-11-06 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01105817 Pending CN1377891A (en) | 2001-04-02 | 2001-04-02 | Zinc finger protein from dendritic cell, its coding sequence and use |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101940780A (en) * | 2010-07-08 | 2011-01-12 | 中国人民解放军第二军医大学 | Zinc finger protein ZBTB20 in chondrocyte-specific knockout mouse model and application thereof |
| WO2022235482A1 (en) * | 2021-05-03 | 2022-11-10 | Rutgers, The State University Of New Jersey | Immunotherapy for inflammatory bowel disease and/or cancer |
-
2001
- 2001-04-02 CN CN 01105817 patent/CN1377891A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101940780A (en) * | 2010-07-08 | 2011-01-12 | 中国人民解放军第二军医大学 | Zinc finger protein ZBTB20 in chondrocyte-specific knockout mouse model and application thereof |
| WO2022235482A1 (en) * | 2021-05-03 | 2022-11-10 | Rutgers, The State University Of New Jersey | Immunotherapy for inflammatory bowel disease and/or cancer |
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