CN1511847A - Ubiquitin-like molecule coming from human dendritic cell and its codnig sequence and use - Google Patents

Abstract

The present invention discloses a kind of human dentritic cell derived ubiquitin-like protein molecule (DC-UbP). The present invention provides the DC-UbP molecule encoding sequence and the recombinant technological process of producing the protein molecule. DC-UbP has lowered expression amount during leukaemia cell differentiation and cell death.

Description

The ubiquitin sample molecule of originated from human dendritic cell and encoding sequence and purposes

Technical field

The invention belongs to biology field, specifically, the present invention relates to the polynucleotide of new coding people ubiquitin sample molecule DC-UbP polypeptide, 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 ubiquitin sample molecule.

Background technology

Specific immune response is by the antigen presenting cell capture antigen, bring out generation after through processing, after handling antigenic information being passed to T, bone-marrow-derived lymphocyte, therefore, antigenic processing treatment is the primary link of organism immune response with offering, and is directly connected to inducing of immuno-stimulating or immunological tolerance.Dendritic cell (dendritic cell, DC) be the strongest sole duty antigen presenting cell of function in the present known body, research to this type of cell, especially the research of its differentiation and development, function controlling is not only helped to understand deeply the Regulation Mechanism of immune response, and the immunne response that can regulate body by the function of regulating DC, formulation to the understanding of the genesis mechanism of tumour, transplant rejection, infection, autoimmune disorder and prophylactico-therapeutic measures is significant, is the focus of current immunology research.

The degraded of intracellular protein is the key link of DC performance function, ubiquitin (Ubiquitin, Ub)-and proteasome (Proteasome) system is the intracellular protein found at present and the main path of foreign protein degraded, the cycle progression of pair cell, transcriptional control, antigen processing are offered and physiological process such as apoptosis has important regulation.Therefore, uiquitin-protease enzyme body system is being brought into play crucial effect in the immunne response process.

Ub is a kind of 76 amino acid whose micromolecule polypeptides, and the lysine residue of the ε amino acid group in the glycine residue by its C-terminal and the protein molecule combines.Ub can be used as monomer, combines with substrate protein white matter molecule, also can combine with other Ub by its inner lysine residue, forms the protein molecule that carries many Ub chain.Ub on the protein molecule can be targeted to the proteolytic enzyme vivo degradation as recognition signal, and many Ub chain can improve target efficient greatly.

Ub is attached to the catalytic process that needs 2-3 step enzyme on the substrate molecule: at first, close by the thioester bond between Ub and the Ub activating enzymes (E1), make the Ub activation; Ub transfers on the Ub conjugate enzyme (Ubcs or E2s) by transacylation then, and the latter is attached to the Ub molecule on the substrate protein molecule, and the effect of perhaps passing through Ub ligase enzyme (E3s) again combines with substrate molecule.In eukaryote, E1s does not have specificity, has only the several of minority; Yet the kind of E2s and E3s is a lot, has specificity.The variation of E2s and E3s shows that the ubiquitin process of protein molecule has specificity, and different substrates needs different E2s and E3s.

Present research also shows, except classical ubiquitin system described above (being the ubiquitin process that Ub participates in), also exists the catalyst system of other proteolytic degradations in the eukaryotic cell, and other combine energy catalysis with target molecule with the similar molecule of ubiquitin.Claim at present this and the similar molecule of ubiquitin be ubiquitin sample molecule (Ubiquitin-like molecules, Ub1).At present the foreign scholar has had been found that multiple new Ub1, and these Ub1 molecules are not only structurally similar to Ub, and with target protein bonded process in, also need on the function similar enzyme system with E2s and E3s.

At present, about Ub and Ub1 system mainly concentrate on following three aspects in the research aspect the immunoregulation: the one, the effect aspect antigen processing of Ub and Ub1 system, studies show that the processing of the restricted antigen peptide of MHC needs to rely on the proteolytic degradation process that the Ub-proteasome mediates; The 2nd, participated in the activation of NF-κ B, be that Ub and Ub1 (mainly being two kinds of new Ub1 molecule Nedd8 and SUMO that the foreign scholar finds) system has participated in the translation post-treatment of NF-κ B precursor and the nuclear translocation of NF-κ B, and, regulated and control the activation of NF-κ B by ubiquitin sample effect to the mutual antagonism of I κ B molecule.The 3rd, also participated in the differentiation and development and the regulation of apoptosis process of immunocyte, just depend on the ubiquitinization of P53 as the performance of P53 function, and control two apoptosis pathway, Ub and Ub1s are also by molecule-cyclin (Cyclin) and CDK supressor to the regulation and control cell cycle progression in addition, NF-κ B molecule, the ubiquitinization of the specific molecular of Bcl-2 family affects the apoptosis of cell.

Therefore, find that new ubiquitin sample molecule has important value for the mechanism of action of understanding immune cell etc.Ubiquitin sample molecule is the effect of performance important regulating and controlling in the generation of tumour, process of growth, and may be worth having important development and application aspect the immunodiagnosis in a plurality of fields such as antitumor, anti-inflammatory response, anti-infective and immunologic function adjusting and the immunotherapy.Therefore, it is significant that this area presses for the ubiquitin sample molecule DC-UbP that develops new originated from human dendritic cell.

Summary of the invention

The purpose of this invention is to provide a kind of new people's ubiquitin sample 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 DC-UbP 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 selected from down group: the polypeptide that (a) has SEQ ID NO:2 aminoacid sequence; (b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have keep the white corpuscle differentiation function by (a) polypeptides derived.

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 80% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people DC-UbP 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 33-350 position among the SEQ ID NO:1; (b) has the sequence of 1-565 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, preparation people DC-UbP is provided proteic method, this method comprises: (a) under expression condition, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate people DC-UbP albumen.

In a fifth aspect of the present invention, provide and above-mentioned people DC-UbP polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 15-565 Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people DC-UbP polypeptide active is provided, and the compound that suppresses people DC-UbP 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 DC-UbP polypeptide.

In a seventh aspect of the present invention, provide and whether had the proteic method of DC-UbP in the test sample, it comprises: sample is contacted with the proteic specific antibody of DC-UbP, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample DC-UbP albumen.

In a eighth aspect of the present invention, a kind of disease relevant with people DC-UbP polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: detect the described DC-UbP expression of polypeptides amount of coding in the cell sample, expression amount is higher than normal control and just represents have cancered possibility to be higher than normal population.

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 DC-UbP polypeptide active, and perhaps screening suppresses the antagonist of people DC-UbP polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people DC-UbP 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 DC-UbP polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.

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

Description of drawings

Fig. 1 has shown people DC-UbP and the proteic amino acid sequence homology comparison diagram of other ubiquitin samples.The top sequence is people DC-UbP, and the below sequence is other ubiquitin sample albumen.Identical amino acid marks with black matrix between a plurality of sequences, and similar amino acid marks with grey body.

Fig. 2 has shown that RT-PCR detects the expression analysis of people DC-UbP in different tumour cells, and prompting DC-UbP is expressed in some tumour cell.

Fig. 3 A-3D has shown interior location of the cell of DC-UbP and subcellular organelle location.Prompting DC-UbP is distributed in the human mitochondrion.

Fig. 4 has shown the apparent molecular weight of people DC-UbP, and the apparent molecular weight of prompting DC-UbP is 20KD.

Fig. 5 has shown that apoptosis-induced or ATRA induces expression analysis in the atomization to DC-UbP through TRAIL at the HL60 cell.

Embodiment

The inventor is through extensive and deep research, separated first novel ubiquitin sample molecule (dendriticcell-derived ubiquitin-like protein, DC-UbP).RT-PCR analysis revealed DC-UbP has expression in some tumour cell, simultaneously be subjected to the apoptosis-induced or ARTA of TRAIL to induce to express in the process of differentiation at people's acute leukemia cells HL60 and descend, this shows that DC-UbP is keeping some tumour cell (as the leukemia cell's) differentiation.Therefore, DC-UbP albumen or its relevant antagonist, agonist etc. can be diseases such as treating tumour provides new immunodiagnosis and targeted therapy approach, thereby has great application prospect.

In the present invention, term " DC-UbP albumen ", " DC-UbP polypeptide " or " ubiquitin sample molecule DC-UbP " are used interchangeably, and all refer to have the albumen or the polypeptide of people's ubiquitin sample molecule DC-UbP aminoacid sequence (SEQ ID NO:2).

As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " isolating DC-UbP albumen or polypeptide " is meant that the DC-UbP polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying DC-UbP albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

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

In the present invention, term " people DC-UbP polypeptide " refers to have the SEQ IDNO.2 polypeptide of sequence of people DC-UbP protein-active.This term also comprises having and variant form people DC-UbP albumen 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 people DC-UbP and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people DC-UbP DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people DC-UbP polypeptide to obtain.The present invention also provides other polypeptide, as comprises people DC-UbP polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people DC-UbP polypeptide.Usually, this fragment have people DC-UbP 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 people DC-UbP albumen or polypeptide.The difference of these analogues and natural human DC-UbP polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

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

In the present invention, " people DC-UbP albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

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

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

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

Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide 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 C-UbP.

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

People DC-UbP 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 DC-UbP 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 DC-UbP polypeptide of reorganization.In general following steps are arranged:

(1). with many nucleic acids (or varient) of coding of the present invention people DC-UbP 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 DC-UbP 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 DC-UbP DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a LaboratoryManual, 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 comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site 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 DC-UbP 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 DC-UbP protein function as pharmacological agent DC-UbP protein function.The peptide molecule that can suppress or stimulate people DC-UbP protein function that can be used for seeking therapeutic value with the recombinant human DC-UbP protein screening peptide library of expressing.

On the other hand, the present invention also comprises people DC-UbP 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 DC-UbP gene product or fragment.Preferably, refer to that those can combine with people DC-UbP 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 DC-UbP, comprise that also those do not influence the antibody of people DC-UbP protein function.The present invention also comprise those can with modify or without the people DC-UbP 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; 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 DC-UbP gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human DC-UbP 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 prepare (see people such as Kohler, Nature256; 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 Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people DC-UbP protein function and the antibody that does not influence people DC-UbP protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people DC-UbP 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 DC-UbP 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 DC-UbP can be used in the immunohistochemistry technology, detects the people DC-UbP albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people DC-UbP, 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 DC-UbP albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people DC-UbP 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 DC-UbP 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, and the exchange by disulfide linkage is incorporated into toxin on the antibody.

The production of polyclonal antibody can choose DC-UbP 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 DC-UbP albumen interactional material takes place, as part, 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 or its agonist and antagonist can be directly used in disease treatment, for example are used for the treatment of tumour aspect.In use, also can use the other treatment agent simultaneously.

The present invention also provides a kind of pharmaceutical composition, and it contains DC-UbP 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 DC-UbP 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 DC-UbP also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of DC-UbP of the proteic nothing expression of DC-UbP or unusual/non-activity.The DC-UbP albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic DC-UbP protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the DC-UbP transgenosis to cell.The method that structure carries the recombinant viral vector of DC-UbP gene is found in existing document (Sambrook, et al.).Recombinant human DC-UbP 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 DC-UbPmRNA 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 DC-UbP obtains.During screening, must carry out mark to people DC-UbP protein molecular.

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

Whether having the proteic method of DC-UbP in a kind of detection test sample is to utilize the proteic specific antibody of DC-UbP to detect, and it comprises: sample is contacted with the DC-UbP protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample DC-UbP albumen.

The proteic polynucleotide of DC-UbP can be used for the diagnosis and the treatment of DC-UbP protein related diseases.Aspect diagnosis, the proteic polynucleotide of DC-UbP can be used for detecting the proteic expression of DC-UbP DC-UbP abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of DC-UbP as the DC-UbPDNA sequence.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 DC-UbP albumen and also can detect the proteic transcription product of DC-UbP.

The sudden change that detects the DC-UbP gene also can be used for the disease of diagnosing DC-UbP albumen relevant.The form of DC-UbP protein mutation comprises that the point mutation compared with normal wild type DC-UbP 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 DC-UbP 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, Mendelian Inheritancein Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.

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 565 bases, and its open reading frame is positioned at the 33-353 position, and the coding total length is 106 amino acid whose people DC-UbP albumen (SEQ ID NO:2).This DC-UbP albumen belongs to the ubiquitin family molecule, with ubiquitin, and sentrin, UCRP, aminoacid sequences such as Nedd8 have certain homology, and consistence can be up to more than 25% in the ubiquitin structural domain, and similarity then can reach 50%.RT-PCR analysis revealed DC-UbP has expression in some tumour cell, simultaneously be subjected to the apoptosis-induced or ARTA of TRAIL to induce the expression that exists in the process of differentiation in various degree at people's acute leukemia cells HL60.Therefore, DC-UbP albumen or its relevant antagonist, agonist etc. can be diseases such as treating tumour provides new immunodiagnosis and targeted therapy approach, thereby has great application prospect.

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:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1: the clone of people DC-UbP cDNA

Extract the total RNA of human dendritic cell with Trizol reagent (Life Technologies 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, and transformed into escherichia coli DH5 α forms the cDNA plasmid library.Measure the sequence of random choose clone's 5 ' end 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 (shown in SEQ ID NO:2) of encoding.This protein is named as the ubiquitin sample molecule DC-UbP of originated from human dendritic cell,

Its encoding gene called after originated from human dendritic cell ubiquitin sample molecule DC-UbP gene.

Sequence SEQ ID NO:1 total length is 565bp, comprises 5 ' the end non-coding region of 32bp and 3 ' the end non-coding region of 212bp, and coding contains 106 amino acid whose polypeptide.Calculating not in theory, the molecular weight of glycosylated ripe molecule is about 12.2kD.22-97 is the ubiquitin functional domain among the SEQ ID NO:2.

They are different with known for the BLAST analysis revealed, have certain homology with the aminoacid sequence of ubiquitin family molecule, and consistence reaches 28%, and similarity reaches 50% (Fig. 1), belongs to the ubiquitin family molecule

Embodiment 2: the cell expressing spectrum analysis that carries out people DC-UbP with the RT-PCR method

Extract the total RNA that is in the corresponding clone of logarithmic phase with Trizol reagent, get 5 μ g cell total rnas and 1 μ g Oligo-dT _12-18Mix, carry out reverse transcription.The reverse transcription system is 20 μ l, and reaction adds 80 μ lddH after finishing ₂O dilutes.The used primer of pcr amplification DC-UbP is as follows: adopted primer 5 '-CCA CGC GTCCGT GCT TGG-3 ' (SEQ ID NO:3) is arranged, antisense primer 5 ' CCG GTA CCG AGT TCT CCA CTGGTG TTG GGT TC (SEQ ID NO:4), simultaneously with β-actin as positive control.The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.5mM primer, 0.2mM dNTP and 1U rTaq archaeal dna polymerase (Takara Inc.), the amplification parameter be 95 ℃ 15 seconds, 57 ℃ 30 seconds, 72 ℃ 30 seconds, capable 1.5% agarose gel electrophoresis of PCR product is tentatively confirmed after 28 circulations.The dna sequence analysis result shows that the DNA sequences encoding of this PCR product and the 1-351 position shown in the SEQ ID NO:1 are identical.

In addition, the expression (Fig. 2) of DC-UbP in multiple different tumour cells, prompting DC-UbP can be used as tumour cell and diagnoses one of genetic marker of these tumours.

Embodiment 3: people DC-UbP is recombinant expressed

In this embodiment, be template with the total length plasmid DNA among the embodiment 1,5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence increases, and obtains people DC-UbP DNA as inserting fragment.

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

5’-GAA?GAT?CTA?TAG?AGG?AAA?AGA?GCG?ACA?TAG?AGA-3’(SEQ?ID?NO：5)

This primer contains the restriction enzyme site of Bgl II restriction enzyme, is the part encoding sequence of people DC-UbP after this restriction enzyme site;

3 ' end primer sequence is:

5’-GGA?ATT?CGG?CTC?AGT?TCA?GTT?CTC?CAC-3’(SEQ?ID?NO：6)

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people DC-UbP of EcoRI restriction enzyme.

With the PCR product purification that obtains after EcoRI and Bgl II enzyme cut and recombinate according to a conventional method with plasmid pRsetB (Invitrogen company) again 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).Plasmid transformation escherichia coli BL21 with the people DC-UbPcDNA of correct sequence.Positive colony is cut evaluation with EcoRI and Bgl II enzyme, the capable 1.0% agarose gel electrophoresis analysis of product.Confirm through order-checking, inserted designed DC-UbP encoding sequence.

Choosing the positive BL21 clone who expresses DC-UbP is inoculated in the 100mlLB substratum, 37 ℃ of 300rpm shaking culture 12-15hr, the LB substratum that is diluted in preheating at 1: 10 continues shaking culture 1.5hr, add behind the 1M IPTG to 1mM 37 ℃ and induce 2-6hr, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml1 * 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 the 2mlHis-Sepharose chromatography column, behind 1 * PBS thorough washing, adding 500ul elution buffer room temperature left standstill after 30 minutes collects elutriant, repeat wash-out 2-3 time, obtain people DC-UbP albumen.

Embodiment 4: anti-people DC-UbP production of antibodies

The recombinant protein people DC-UbP that obtains among the embodiment 4 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 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 people DC-UbP gene translation product with it.Found that antibody can combine with albumen of the present invention specifically.

The structure of embodiment 5:DC-UbP eucaryon fusion expression vector

In this embodiment, be template with the total length plasmid DNA among the embodiment 1,5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence increases, and obtains people DC-UbP DNA as inserting fragment.The PCR reaction parameter be 95 ℃ 15 seconds, 58 ℃ 30 seconds, 72 ℃ 45 seconds, 20 circulations were extended 10 minutes for back 72 ℃,

Total length DC-UbP upstream primer is 5 '-CCA CGC GTC CGT GCT TGG (SEQ ID NO:3), downstream primer is 5 '-CCG GTA CCG AGT TCT CCA CTG GTG TTG GGT TC-3 ' (SEQ ID NO:4), the proteic dna sequence dna of product coding total length DC-UbP.

The PCR product purification and is recombinated according to a conventional method with pcDNA3.1 carrier (Invitrogen company) and is converted into competence bacterium DH5 α after Kpn I enzyme is cut.Picking white clone identifies, also order-checking (sequencing primer is T7 and DC-UbP gene-specific primer CCG GTA CCG AGT TCT CCA CTG GTGTTG GGT TC (SEQ ID NO:4)) of purifying.Confirm through order-checking, inserted the DC-UbP encoding sequence.

Localized immunofluorescence analysis in the embodiment 6:DC-UbP albuminous cell

Utilize LipofectAMINE reagent (Invitrogen) to carry out the gene transfection of eukaryotic cell 293T with DC-UbP total length fusion expression vector plasmid DNA constructed among the embodiment 5, contrast as irrelevant with the pcDNA3.1 plasmid vector.The transient transfection cell after transfection 48 hours with trysinization, be laid on the sterility cover slide, after adherent 8 hours,, under Laser Scanning Confocal Microscope, observe subsequently with anti-FITC-His antibody staining.Organoid is detection and localization altogether, treat cell attachment after, dye with plastosome, endoplasmic reticulum and Golgi's organs, lysosome specificity dyestuff respectively earlier, concentration is 1 μ M, 37 ℃, 15 minutes.After washing with PBS, use anti-FITC-His antibody staining again, under Laser Scanning Confocal Microscope, observe subsequently.

The result shows: the fluorescent signal of DC-UbP transfectional cell all is dispersed in and is distributed in the endochylema; Organoid is the positioning experiment results suggest altogether, and DC-UbP is distributed in the plastosome, and be not present in endoplasmic reticulum, Golgi's organs, lysosome (Fig. 3 A-3D).

The proteic apparent molecular weight of embodiment 7:DC-UbP detects

Utilize LipofectAMINE reagent (Invitrogen) to carry out the gene transfection of eukaryotic cell 293T with DC-UbP total length fusion expression vector plasmid DNA constructed among the embodiment 5, contrast as irrelevant with the pcDNA3.1 plasmid vector.Transient transfection cell after transfection 48 hours, harvested cell.Behind protein extraction damping fluid (PIERCE reagent) lysing cell, obtain the endochylema lysate, measure protein concentration through the BCA method.Split product equivalent is carried out the 12%SDS-PAGE protein electrophoresis, after 100mV is transferred to nitrocellulose filter, detects with anti-His antibody and the HRP coupling two anti-Western blot that carry out.

The result shows that the specific band of a 20KD is arranged in the 293T cell lysate of DC-UbP fusion expression vector transfection, and promptly structure is the fusion rotein (Fig. 4) of " DC-UbP-MYC epi-position-6His ".

Embodiment 8: people DC-UbP is at the expression analysis of HL60 cell in different factor stimulating courses

Extract the HL60 people's acute leukemia cells be in logarithmic phase with Trizol reagent, or induce 8 hours HL60 cell total rna through TRAIL or ARTA.Get 5 μ g cell total rnas and 1 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 DC-UbP is as follows: have adopted primer 5 '-CCA CGC GTC CGT GCT TGG-3 ' (SEQ ID NO:3), antisense primer 5 ' CCG GTA CCG AGT TCT CCA CTG GTG TTGGGT TC (SEQ ID NO:4), simultaneously with beta-actin as positive control.The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.5mM primer, 0.2mM dNTP and 1U rTaq archaeal dna polymerase (TakaraInc.), the amplification parameter be 95 ℃ 15 seconds, 57 ℃ 30 seconds, 72 ℃ 30 seconds, capable 1.5% agarose gel electrophoresis of PCR product is tentatively confirmed after 28 circulations.The dna sequence analysis result shows that the DNA sequences encoding of this PCR product and the 1-351 shown in the SEQID NO:1 are identical.

The result as shown in Figure 5.People DC-UbP is induced by the apoptosis-induced or ATRA of TRAIL to express in the atomization to reduce at people's acute leukemia cells HL60, and prompter DC-UbP plays a role in leukemia cell's differentiation and development or apoptosis, plays the function of keeping leukemia cell's differentiation.

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〉the ubiquitin sample molecule of originated from human dendritic cell and code sequence and purposes

<130>027886

<160>6

<170>PatentIn?version?3.1

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<211>565

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(33)..(350)

<223>

<400>1

ccacgcgtcc?gtgcttggca?ccgccaatca?ac?atg?ata?gag?gaa?aag?agc?gac???????53

Met?Ile?Glu?Glu?Lys?Ser?Asp

1???????????????5ata?gag?act?ctg?gat?att?cct?gag?cca?cca?ccc?aat?tct?gga?tat?gaa???????101Ile?Glu?Thr?Leu?Asp?Ile?Pro?Glu?Pro?Pro?Pro?Asn?Ser?Gly?Tyr?Glu

10??????????????????15??????????????????20tgt?cag?ctt?cgt?ttg?cgc?ctt?tcc?aca?ggc?aaa?gac?ctc?aag?ctt?gtg???????149Cys?Gln?Leu?Arg?Leu?Arg?Leu?Ser?Thr?Gly?Lys?Asp?Leu?Lys?Leu?Val

25??????????????????30??????????????????35gtt?cgc?agc?aca?gac?aca?gta?ttc?cac?atg?aag?aga?cgg?ttg?cat?gca???????197Val?Arg?Ser?Thr?Asp?Thr?Val?Phe?His?Met?Lys?Arg?Arg?Leu?His?Ala40??????????????????45??????????????????50??????????????????55gca?gag?gga?gtg?gaa?cca?ggt?agt?cag?cgg?tgg?ttt?ttt?tct?ggc?aga???????245Ala?Glu?Gly?Val?Glu?Pro?Gly?Ser?Gln?Arg?Trp?Phe?Phe?Ser?Gly?Arg

60??????????????????65??????????????????70cct?ctc?act?gac?aaa?atg?aag?ttc?gaa?gag?ctg?aag?atc?cca?aag?gac???????293Pro?Leu?Thr?Asp?Lys?Met?Lys?Phe?Glu?Glu?Leu?Lys?Ile?Pro?Lys?Asp

75??????????????????80??????????????????85tat?gtt?gta?cag?gtt?ata?gtg?agc?caa?cct?gtg?cag?aac?cca?aca?cca???????341Tyr?Val?Val?Gln?Val?Ile?Val?Ser?Gln?Pro?Val?Gln?Asn?Pro?Thr?Pro

90???????????????????95??????????????????100gtg?gag?aac?tgaactgagc?cctgttggcc?agctcccaca?tccctctgct??????????????390Val?Glu?Asn

105cctttttatg gttcttgttg tcatttccta ctctgcggcg tgaaatctat ttcactgctc 450taaattccct atgaatggat ttagttctga ggaattacca gtgaaaaatt ccatctgtga 510tggagaccaa caaaaataat aaaacacaaa gagccaggca aaaaaaaaaa aaaaa 565＜210〉2＜211〉106＜212〉PRT＜213〉homo sapiens (Homo sapiens)＜400〉2Met Ile Glu Glu Lys Ser Asp Ile Glu Thr Leu Asp Ile Pro Glu Pro1 5 10 15Pro Pro Asn Ser Gly Tyr Glu Cys Gln Leu Arg Leu Arg Leu Ser Thr

20??????????????????25??????????????????30Gly?Lys?Asp?Leu?Lys?Leu?Val?Val?Arg?Ser?Thr?Asp?Thr?Val?Phe?His

35??????????????????40??????????????????45Met?Lys?Arg?Arg?Leu?His?Ala?Ala?Glu?Gly?Val?Glu?Pro?Gly?Ser?Gln

50??????????????????55??????????????????60Arg?Trp?Phe?Phe?Ser?Gly?Arg?Pro?Leu?Thr?Asp?Lys?Met?Lys?Phe?Glu65??????????????????70??????????????????75??????????????????80Glu?Leu?Lys?Ile?Pro?Lys?Asp?Tyr?Val?Val?Gln?Val?Ile?Val?Ser?Gln

85??????????????????90??????????????????95Pro?Val?Gln?Asn?Pro?Thr?Pro?Val?Glu?Asn

100?????????????????105<210>3<211>18

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(18)

＜223〉primer

<400>3

ccacgcgtcc?gtgcttgg???????????????????????????????????????????????????18

<210>4

<211>32

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(32)

＜223〉primer

<400>4

ccggtaccga?gttctccact?ggtgttgggt?tc???????????????????????????????????32

<210>5

<211>33

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(33)

＜223〉primer

<400>5

gaagatctat?agaggaaaag?agcgacatag?aga??????????????????????????????????33

<210>6

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(27)

＜223〉primer

<400>6

ggaattcggc?tcagttcagt?tctccac?????????????????????????????????????????27

Claims (10)

1. an isolating people DC-UbP 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 selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;

(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have keep the white corpuscle differentiation function by (a) polypeptides derived.

3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 80% homogeny with the nucleotides sequence that is selected from down group:

(a) polynucleotide of polypeptide according to claim 1 of encoding;

(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 33-353 position among the SEQ ID NO:1;

(b) has the sequence of 1-565 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. proteic preparation method of people DC-UbP is characterized in that this method comprises:

(a) under expression condition, cultivate the described host cell of claim 7;

(b) from culture, isolate people DC-UbP albumen.

9. energy and the described people DC-UbP of claim 1 polypeptid specificity bonded antibody.

10. a composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.

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