CN1475568A - New type human death resist protein, its coded sequence and use - Google Patents
New type human death resist protein, its coded sequence and use Download PDFInfo
- Publication number
- CN1475568A CN1475568A CNA021365210A CN02136521A CN1475568A CN 1475568 A CN1475568 A CN 1475568A CN A021365210 A CNA021365210 A CN A021365210A CN 02136521 A CN02136521 A CN 02136521A CN 1475568 A CN1475568 A CN 1475568A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- drp
- sequence
- polynucleotide
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A human death-resistant protein (DRP), its coding polynucleotide, the process for recombining said protein, and the application of said protein and its coding sequence in diagnosing and treating cancer are disclosed.
Description
Technical field
The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of the dead opposing of new encoding novel people protein D RP polypeptide, and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.DRP polypeptide of the present invention is a kind ofly new to generate molecule in the closely-related cell with necrocytosis or tumour, and the high expression level of DRP has the function of opposing TNF-α inducing cell death.
Background technology
Tumor necrosis factor alpha (TNF-α) is by various kinds of cell excretory inflammatory cytokine, can induce multiple malignant cell death, comprises apoptosis and necrosis.But normal cell and some tumor cell line are not subjected to the influence of TNF.This resistant function depends on the expression of some TNF inductive protected protein.The effect of TNF cell death inducing is a network engineering of finishing by many signaling molecule fellowships, and wherein TRAFs (TNF receptor-associated factor) family protein, especially TRAF2 is the key link of this Signal Regulation network.TNFR assembles after combining with TNF; and raise TRAF2 and directly combine with TNFR2 born of the same parents' inner segment; or by linking to each other indirectly with TNFR1 in conjunction with other linkers; TRAF2 mainly by activating independently signal transduction pathway pair cell performance apoptosis regulating effect of two of NIK and JNK/SAPK, has the apoptosis defencive function.
The L929 cell is a l cell, generally is used as to detect the Cytotoxic cell model of TNF.TNF both can induce the L929 cell to necrose, and also can induce it that apoptosis takes place.Discovering in the past, mediated cell necrose has some different with the signal transduction pathway of apoptosis.Caspases family is mainly relevant with the apoptotic signal transduction, and the generation of the activation of Phospholipase D, C, A2 and mitochondria activity oxygen class is mainly with downright bad relevant.Therefore infer that two kinds of different cell death ways may also be to be regulated by different death opposing albumen respectively.
In view of the vital role of necrocytosis associated protein, this area presses for the new necrocytosis associated protein of exploitation.
Summary of the invention
The purpose of this invention is to provide a kind of new new type human death opposing protein D RP 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.DRP albumen of the present invention is a kind of TRAF2 homolgous molecule.
In a first aspect of the present invention, novel isolated DRP 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 opposing TNF-α inducing cell death 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 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people DRP 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 214-1164 position among the SEQ ID NO:1; (b) has the sequence of 1-3128 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 DRP protein-active, this method comprises: (a) under the proteic condition of suitable expressing human DRP, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people DRP protein-active.
In a fifth aspect of the present invention, provide and above-mentioned people DRP polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-3128 Nucleotide in the above-mentioned polynucleotide.
In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people DRP polypeptide active is provided, and the compound that suppresses people DRP 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 DRP polypeptide.
In a seventh aspect of the present invention, provide and whether had the proteic method of DRP in the test sample, it comprises: sample is contacted with the proteic specific antibody of DRP, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample DRP albumen.
In a eighth aspect of the present invention, a kind of disease relevant with people DRP 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 DRP polypeptide active, and perhaps screening suppresses the antagonist of people DRP polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people DRP 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 DRP 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 illnesss such as primary cardiac myopathy, liver failure.
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 human PHD P RT-PCR expression analysis result of the present invention.Each swimming lane is as follows: 1.HUT; 2.Jurkat; 3.Molt-4; 4.Ramos; 5.Raji; 6.Daudi; 7.NB4; 8.HL-60; 9.U937; 10.DC; 11. monocyte; 12.T-lymphocyte; 13.B-lymphocyte.
Fig. 2 has shown the shown distribution of people DRP Northern blot hybridization of the present invention.Left side figure is that the healthy tissues of DRP distributes; Right figure is the distribution of DRP in various kinds of cell system.PBL representative peripheral blood leucocyte.Results suggest DRP is the interior molecule of a kind of cell of extensive distribution.
Fig. 3 has shown the lethal effect of people DRP molecule opposing TNF-α of the present invention to the L929 cell.Fig. 3 A is the cell survival curve of different time; Fig. 3 B is the cell survival curve of different TNF dosage.
Embodiment
The inventor is through extensive and deep research, separated first new type human death opposing albumen (death-resistant protein, DRP).DRP and known TRAF2 molecule have higher homology, with DRP stably express in the L929 cell, can significantly suppress the necrocytosis of TRAF2 inductive, Northern hybridization shows that DRP extensively distributes in normal human tissue, the expression of higher level is arranged in tumor tissues, especially meaningfully, normal human T, bone-marrow-derived lymphocyte and monocyte are not expressed this gene, and at T, the tumour cell of B or cells of monocytic origin, as Jurkat, Molt-4, Raji, Daudi, NB4, HL-60, U937, in higher expression is but arranged, this point out this recruit may normal tissue cell cancerate and tumour has been brought into play vital role in forming.Therefore, can determine that DRP is a molecule in a kind of new cell that plays a significant role in the signal transduction of TNF cell death inducing.DRP may have important development and application in a plurality of fields such as the diagnosis of tumour, treatments and be worth.Research shows that the generation of DRP malignant tumour is closely related.Therefore, significant for the new dead opposing of the people albumen of treatment tumour purpose research and development.
In the present invention, term " DRP albumen ", " DRP polypeptide " or " dead opposing protein D RP " are used interchangeably, and all refer to have the albumen or the polypeptide of people's dead opposing protein D RP 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 DRP albumen or polypeptide " is meant that the DRP 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 DRP 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 DRP, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural human DRP 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 DRP polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of people DRP protein-active.This term also comprises having and variant form people DRP 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 DRP 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 DRP DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people DRP polypeptide to obtain.The present invention also provides other polypeptide, as comprises people DRP polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people DRP polypeptide.Usually, this fragment have people DRP 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 DRP albumen or polypeptide.The difference of these analogues and natural human DRP 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 DRP 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 | ?Lvs |
| ??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 RP.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
People DRP 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 DRP 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 DRP polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people DRP 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 DRP 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 BioChem.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 DRP 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 DRP 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 DRP protein function as pharmacological agent DRP protein function.The peptide molecule that can suppress or stimulate people DRP protein function that can be used for seeking therapeutic value with the recombinant human DRP protein screening peptide library of expressing.
On the other hand, the present invention also comprises people DRP 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 DRP gene product or fragment.Preferably, refer to that those can combine with people DRP 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 DRP, comprise that also those do not influence the antibody of people DRP protein function.The present invention also comprise those can with modify or without the people DRP 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 DRP gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human DRP 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 DRP protein function and the antibody that does not influence people DRP protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people DRP 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 DRP 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 DRP can be used in the immunohistochemistry technology, detects the people DRP albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people DRP, 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 DRP albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people DRP 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 DRP 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 DRP protein positive.
The production of polyclonal antibody can choose DRP 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 DRP 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 tumour, inflammation, the treatment of neural system and cardiovascular diseases aspect.In use, also can use the other treatment agent simultaneously.
The present invention also provides a kind of pharmaceutical composition, and it contains DRP 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 DRP 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 DRP 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 DRP of the proteic nothing expression of DRP or unusual/non-activity.The DRP albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic DRP protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the DRP transgenosis to cell.The method that structure carries the recombinant viral vector of DRP gene is found in existing document (Sambrook, et al.).Recombinant human DRP 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 DRPmRNA 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 DRP obtains.During screening, must carry out mark to people DRP protein molecular.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people DRP protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people DRP protein level that is detected in the test can be with laying down a definition the importance of people DRP albumen in various diseases and be used to the disease of diagnosing DRP albumen to work.
Whether having the proteic method of DRP in a kind of detection test sample is to utilize the proteic specific antibody of DRP to detect, and it comprises: sample is contacted with the DRP protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample DRP albumen.
The proteic polynucleotide of DRP can be used for the diagnosis and the treatment of DRP protein related diseases.Aspect diagnosis, the proteic polynucleotide of DRP can be used for detecting the proteic expression of DRP DRP abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of DRP as the DRPDNA 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 DRP albumen and also can detect the proteic transcription product of DRP.
The sudden change that detects the DRP gene also can be used for the disease of diagnosing DRP albumen relevant.The form of DRP protein mutation comprises that the point mutation compared with normal wild type DRP 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 DRP 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 3128 bases, and its open reading frame is positioned at the 214-1164 position, and the coding total length is 317 amino acid whose people DRP albumen (SEQ ID NO:2).This DRP albumen belongs to molecule in the cell, and with TRAF2 aminoacid sequence portion homologous, consistence can be up to 47%, and similarity then can reach 65%.In addition, DRP albumen also comprises TRAF2 conservative zinc fingers and ring finger.The Northern engram analysis shows wide expression in tissue.The research carried out prompting, DRP may be the relevant joint albumen of signal transduction behind new and the TNF-α acceptor.Therefore, DRP albumen or its relevant antagonist, agonist etc. can be diseases such as treating malignant tumour provides new treatment approach, thereby has great application prospect.
Embodiment further sets forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the clone of people DRP 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 inserted into cDNA fragment orientation on the multiple clone site of carrier with SuperScript II clone's test kit (Life Technologies), transforms DH5 α bacterium and form 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 people DRP, and its encoding gene name is people DRP gene.
Sequence SEQ ID NO:1 total length is 3128bp, comprises 5 ' the end non-coding region of 213bp and 3 ' the end non-coding region of 1961bp, and coding contains 317 amino acid whose polypeptide.Calculating not in theory, the molecular weight of glycosylated ripe molecule is about 35.9kD.
They are different with known for the BLAST analysis revealed, and with people TRAF2 aminoacid sequence height homology, consistence reaches 47%, and similarity reaches 65%.In addition, also comprise 2 proteic conservative zinc fingerses of TRAFs and ring finger in DRP albumen, this also further points out it may be signal transducers behind new humanTNF-'s acceptor.
Embodiment 2: with the encoding sequence of RT-PCR method human cloning DRP
Be in logarithmic phase B lymphoma cell strain Raji cell total rna with Trizol (Gibco company) extraction, get 6mg cell total rna and 0.5cgOligo-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: adopted primer 5 '-ATGGGGTAT GATGTAACCCGT-3 ' (SEQ ID NO:3) is arranged, antisense primer 5 '-TATCTCTTCCACGCCA TGCG-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), 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.The dna sequence analysis result shows that the DNA sequences encoding of this PCR product and the 214-1164 shown in the SEQ ID NO:1 are identical.
Experimental result (Fig. 1) shows: DRP is specific expressed in human dendritic cell (DC), but is not expressed in normal people's monocyte, T cell or the B cell.In hematopoiesis such as Jurkat, Molt-4, Ramous, Raji, Daudi, NB4, HL-60, U937 is the expression that also detects DRP mRNA in the tumour cell.
The Northern engram analysis of embodiment 3:DRP
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 (Fig. 2) shows: in many healthy tissuess such as liver, heart, skeletal muscle, placenta and in the various kinds of cell systems such as Molt-4, Raji expression is arranged all, this shows that DRP albumen is a kind of albumen of wide expression.
Embodiment 4: people DRP protokaryon is recombinant expressed
In this embodiment, be template with the pcr amplification product among the embodiment 2,5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence increases, and obtains people DRP DNA as inserting fragment.
5 ' the end Oligonucleolide primers sequence of using in the PCR reaction is:
5’-GCGGATCCATGGGGTAT?GATGTAACCCGT-3’(SEQ?ID?NO：5)
This primer contains the restriction enzyme site of BamHI restriction enzyme, is the part encoding sequence that is begun by initiator codon after this restriction enzyme site;
3 ' end primer sequence is:
5’-GCGAATTCTTATATCTCTTCCACGCCAT-3’(SEQ?ID?NO：6)
This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people DRP of EcoRI restriction enzyme.
With the PCR product purification that obtains after the BamHI/EcoRI enzyme cut and recombinate according to a conventional method with plasmid pGEM-3ZF 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).The people DRP cDNA SalI/NotI endonuclease bamhi of correct sequence is cloned into expression vector Pgex-2T (Pharmacia), forms carrier pGEX-2T-DRP.Positive colony is cut evaluation with the BamHI/EcoRI enzyme, and enzyme is cut the capable 2% agarose gel electrophoresis analysis of product.Confirm through order-checking, inserted complete DRP encoding sequence.
Choosing the positive BL21 clone who expresses DRP 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, 2.7 mM 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, adding 500ul gsh elution buffer (pH 8.0 for 10mM gsh, 50mM Tris-HCl) room temperature left standstill after 30 minutes collects elutriant, repeat wash-out 2-3 time, obtain people DRP-GST fusion rotein.The molecular weight of fusion rotein conforms to predictor.
Embodiment 5: anti-people DRP production of antibodies
The recombinant protein people DRP 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 DRP gene translation product with it.Found that antibody can combine with albumen of the present invention specifically.
Embodiment 6: people DRP eucaryon is recombinant expressed
In this embodiment, be template with the pcr amplification product among the embodiment 2,5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence increases, and obtains people DRP DNA as inserting fragment.
5 ' the end Oligonucleolide primers sequence of using in the PCR reaction is:
5’-GCGAATTCATGGGGTAT?GATGTAACCCGT-3’(SEQ?ID?NO：7)
This primer contains the restriction enzyme site of EcoR I restriction enzyme, is the part encoding sequence that is begun by initiator codon after this restriction enzyme site;
3 ' end primer sequence is:
5’-GCGGATCCTATCTCTTCCACGCCAT-3’(SEQ?ID?NO：8)
This primer contains the restriction enzyme site of BamHII restriction enzyme and the part encoding sequence that people DRP removes translation termination.
With the PCR product purification that obtains after the EcoRI/BamHI enzyme cut and recombinate according to a conventional method with plasmid pGEM-3ZF 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).The people DRP cDNA SalI/NotI endonuclease bamhi of correct sequence is cloned into pcDNA3.1/Myc-His (-) A carrier for expression of eukaryon, forms carrier pcDNA-DRP.Positive colony is cut evaluation with the EcoRI/BamHI enzyme, and enzyme is cut the capable 2% agarose gel electrophoresis analysis of product.Confirm through order-checking, inserted complete DRP encoding sequence.
The biologic activity of embodiment 7:DRP
The pcDNA-DRP plasmid DNA that contains the DRP complete encoding sequence that obtained among the embodiment 6 to import in the L929 cell with liposome, is screened with 600 μ g/ml G418, obtain stably express and clone.Wherein, the cell of stably express DRP represents that with L929-DRP the empty carrier cells transfected is represented with L929-neo.Anti-myc antibody is done the Western engram analysis and confirmed to have in cell actual molecular weight to be the albumen generation of about 38KDa (containing myc and His), and is consistent with theoretical molecular.
By mtt assay and trypan blue staining, analyzed that TNF-α finds that to through the L929-DRP of screening and the lethal effect of L929-neo cell necrocytosis has significant resistant function (Fig. 3 A) to the L929-DRP cell to TNF-α inductive under various dose and different action times.In addition, also by double-tagging fluorescence dye propidium iodide (PI) and rhodamine (rhodamine123) with flow cytometry analysis the mortality ratio of TNF-α/CHX effect two groups of cells in the time of 4 hours, obtained the result consistent with mtt assay, promptly the L929-DRP cell mortality is starkly lower than L929-neo cell (Fig. 3 B).Observe down by fluorescent microscope behind light microscopic and the Hoechst33258 nuclear specific fluorescence dyeing, find that TNF-α effect back target cell presents 3 kinds of different forms: a kind of normal cell form that is, cell attachment, after birth is complete, size is medium, and fluorescence distribution is even; A kind of apoptotic cell form that is, cell volume diminishes, karyopyknosis, but after birth is still complete, the visible blister projection of cell surface, fluorescent brightness height; The third is the non-viable non-apoptotic cell form, and it is big that cell volume becomes, swelling, and fluorescence is inhomogeneous, because nuclear membrane and membranolysis, DNA is excessive and make fluorescence be the irregular status of bar rope type, fluorescent brightness is low.Apoptotic cell quantity is less in three kinds of cells, and does not have significant difference at two groups of iuntercellulars.The a large amount of cells of L929-neo groups of cells are necrosis, and L929-DRP group non-viable non-apoptotic cell quantity obviously is less than the L929-neo group.
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〉new type human death opposing albumen, its coded sequence and purposes＜130〉024970＜160〉8＜170〉PatentIn version 3.1＜210〉1＜211〉3128＜212〉DNA＜213〉homo sapiens (Homo sapiens)＜220〉＜221〉CDS＜222〉(214) .. (1164)＜223〉＜400〉1gggcgagtac tcctgattgt gacatcacat tcatcccctg ggcgatggag cttgtcactg 60ggaaggaata ctcagtcgga gaatagccaa caagatgggt tactgggaga atctcttcag 120tggcactgag tggaggcatc agggggttgg agccttgtga acagggaacc tgccccccaa 180cacttggaag gacctgggtt tcagtgatga gac atg ggg tat gat gta acc cgt 234
Met?Gly?Tyr?Asp?Val?Thr?Arg
1??????????????5ttc?cag?ggg?gat?gtt?gac?gaa?gat?ctt?atc?tgc?cct?att?tgc?agt?gga??????282Phe?Gln?Gly?Asp?Val?Asp?Glu?Asp?Leu?Ile?Cys?Pro?Ile?Cys?Ser?Gly
10??????????????????15??????????????????20gtc?ttg?gag?gag?cca?gta?cag?gca?cct?cat?tgt?gaa?cat?gct?ttc?tgc??????330Val?Leu?Glu?Glu?Pro?Val?Gln?Ala?Pro?His?Cys?Glu?His?Ala?Phe?Cys
25??????????????????30??????????????????35aac?gcc?tgc?atc?acc?cag?tgg?ttc?tct?cag?caa?cag?aca?tgt?cca?gtg??????378Asn?Ala?Cys?Ile?Thr?Gln?Trp?Phe?Ser?Gln?Gln?Gln?Thr?Cys?Pro?Val40??????????????????45???????????????????50?????????????????55gac?cgt?agt?gtt?gtg?acg?gtc?gcc?cat?ctg?cgc?cca?gta?cct?cgg?atc??????426Asp?Arg?Ser?Val?Val?Thr?Val?Ala?His?Leu?Arg?Pro?Val?Pro?Arg?Ile
60??????????????????65??????????????????70atg?cgg?aac?atg?ttg?tca?aag?ctg?cag?att?gcc?tgt?gac?aac?gct?gtg???????474Met?Arg?Asn?Met?Leu?Ser?Lys?Leu?Gln?Ile?Ala?Cys?Asp?Asn?Ala?Val
75??????????????????80???????????????????85ttc?ggc?tgt?agt?gcc?gtt?gtc?cgg?ctt?gac?aac?ctc?atg?tct?cac?ctc???????522Phe?Gly?Cys?Ser?Ala?Val?Val?Arg?Leu?Asp?Asn?Leu?Met?Ser?His?Leu
90??????????????????95??????????????????100agc?gac?tgt?gag?cac?aac?ccg?aag?cgg?cct?gtg?acc?tgt?gaa?cag?ggc???????570Ser?Asp?Cys?Glu?His?Asn?Pro?Lys?Arg?Pro?Val?Thr?Cys?Glu?Gln?Gly
105?????????????????110?????????????????115tgt?ggc?ctg?gag?atg?ccc?aaa?gat?gag?ctg?ccc?aac?cat?aac?tgc?att???????618Cys?Gly?Leu?Glu?Met?Pro?Lys?Asp?Glu?Leu?Pro?Asn?His?Asn?Cys?Ile120?????????????????125?????????????????130?????????????????135aag?cac?ctg?cgc?tca?gtg?gta?cag?cag?cag?cag?aca?cgc?atc?gca?gag???????666Lys?His?Leu?Arg?Ser?Val?Val?Gln?Gln?Gln?Gln?Thr?Arg?Ile?Ala?Glu
140?????????????????145?????????????????150ctg?gag?aag?acg?tca?gct?gaa?cac?aaa?cac?cag?ctg?gcg?gag?cag?aag???????714Leu?Glu?Lys?Thr?Ser?Ala?Glu?His?Lys?His?Gln?Leu?Ala?Glu?Gln?Lys
155?????????????????160?????????????????165cga?gac?atc?cag?ctg?cta?aag?gca?tac?atg?cgt?gca?atc?cgc?agt?gtc???????762Arg?Asp?Ile?Gln?Leu?Leu?Lys?Ala?Tyr?Met?Arg?Ala?Ile?Arg?Ser?Val
170?????????????175?????????????????180aac?ccc?aac?ctt?cag?aac?ctg?gag?gag?aca?att?gaa?tac?aac?gag?atc???????810Asn?Pro?Asn?Leu?Gin?Asn?Leu?Glu?Glu?Thr?Ile?Glu?Tyr?Asn?Glu?Ile
185?????????????????190?????????????????195cta?gag?tgg?gtg?aac?tcc?ctt?cag?cca?gca?aga?gtg?acc?cgc?tgg?gga???????858Leu?Glu?Trp?Val?Asn?Ser?Leu?Gin?Pro?Ala?Arg?Val?Thr?Arg?Trp?Gly200?????????????????205?????????????????210?????????????????215ggg?atg?atc?tcg?act?cct?gat?gct?gtg?ctc?cag?gct?gta?atc?aag?cgc???????906Gly?Met?Ile?Ser?Thr?Pro?Asp?Ala?Val?Leu?Gln?Ala?Val?Ile?Lys?Arg
220?????????????????225?????????????????230tcc?ctg?gtg?gag?agt?ggc?tgt?cct?gct?tct?att?gtc?aac?gag?ctg?att???????954Ser?Leu?Val?Glu?Ser?Gly?Cys?Pro?Ala?Ser?Ile?Val?Asn?Glu?Leu?Ile
235?????????????????240?????????????????245gaa?aat?gcc?cac?gag?cgt?agc?tgg?ccc?cag?ggt?ctg?gcc?aca?cta?gag??????1002Glu?Asn?Ala?His?Glu?Arg?Ser?Trp?Pro?Gln?Gly?Leu?Ala?Thr?Leu?Glu
250?????????????????255?????????????????260act?aga?cag?atg?aac?cga?cgc?tac?tat?gag?aac?tac?gtg?gcc?aag?cgc??????1050Thr?Arg?Gln?Met?Asn?Arg?Arg?Tyr?Tyr?Glu?Asn?Tyr?Val?Ala?Lys?Arg
265?????????????????270?????????????????275atc?cct?ggc?aag?cag?gct?gtt?gtc?gtg?atg?gcc?tgt?gag?aac?cag?cac?????1098Ile?Pro?Gly?Lys?Gln?Ala?Val?Val?Val?Met?Ala?Cys?Glu?Asn?Gln?His280?????????????????285?????????????????290?????????????????295atg?ggg?gat?gac?atg?gtg?caa?gag?cca?ggc?ctt?gtc?atg?ata?ttt?gcg?????1146Met?Gly?Asp?Asp?Met?Val?Gln?Glu?Pro?Gly?Leu?Val?Met?Ile?Phe?Ala
300?????????????????305?????????????????310cat?ggc?gtg?gaa?gag?ata?taagagaact?cgactggcta?tcaggaagag????????????1194His?Gly?Val?Glu?Glu?Ile
315atggaaatca gaaaatccca tcactccagc agctgggacc tgagtcctac ccaccattct 1254taatactgtg gcttatacct gagccacaca tctccctgcc cttctggcac tgaagggcct 1314tggggtagtt tgctcagcct ttcaggtggg aaacccagat ttcctccctt tgccatattc 1374ccctaaaatg tctataaatt atcagtctgg gtgggaaagc ccccacctcc atccattttc 1434ctgcttaggg tccctggttc cagttatttt cagaaagcac aaagagattc aatttccctg 1494gaggatcagg acagaggaag gaatctctaa tcgtccctct cctccaaaac cagggaatca 1554gagcagtcag gcctgttgac tctaagcagc agacatcctg aagaaatggt aagggtggag 1614ccaaatctct agaaataagt agtgaggccg ttaattggcc atcactgatg gcccttaggg 1674aaagactgga cctctgtgcc aagcagtatc cctgttcagc ccaccttaaa ggtgtaggca 1734cccactgggt ctaccagtat gcaggttggg atactgaaaa tttccagatg agctcttctt 1794tcctacaagt tttcataatt agggaatgcc agggtttagg gtaggggtta atctgttggg 1854ggttgatgtg tttagcaaga agctactcct agcttttgct aaaatatggt tggcactgcc 1914tcttgtggca caggccataa ttgttccata gacccctctc tagccctgtg actgtagtta 1974gttactttga tgattttctt tggccattgt ttgtttatat ttcacaaact ccacctactg 2034cccccccccc tctttttttt aagaatggcc tgatcatggc tatctcagcc acattgttgg 2094caatttaatt tatttacttc cttttttttt tttaagaaag gaaaaaagaa aaaaaaatca 2154aacttgaaac ttttcttttg atgttcctat tgtgggggtt ctggataggg tgggacaggg 2214atgggggtgt gttttatatt ttttcctttt cagcacaacc tttggcttta atataggaag 2274agccaaggga gtcctcggct gaacttacga tatctgcccc aaacctctgt aaccccaact 2334gaaatgagga gcttcctctc ttcctgtgaa ggatatgaca gtccagcatc gatgcctgtg 2394ccctctggaa aaatttcctc ctagcccttc cagggcctta tcataaaact ctggatttag 2454agtattcatt ttgaaggcaa ctcccccttc cccaagtttc cttggagctg tatagctggg 2514ttctaagctt caccatgcaa atcagaaatt ttatctctaa gtacaggctg tgccgtgtct 2574cacccacacc cccctgggga cttcagttcc atttcaggtt acctggggta taccttgatc 2634cctagagtga ctggcagagt aagagaaggg gagagataat aggtgtgatt attttaatat 2694gaaggtggag tgtggttgga gatagaaagg ctcctcccca ccatgtaatg gcttcctctc 2754agaattttat tccaggctag cttgctgcag gtctgggtag ttggatcatg gctccactgg 2814gattggggtg gaaagcttga ggggagtagg gttccagctc tgggacattg tgctcaggaa 2874tttgaaaacg ctgctatact tactctggtt actacatttc ttccactccc ctttccccta 2934cctgccttaa ccaaggctca tactgtcctg tccttaccct cagatggagc caggaagctc 2994agtgaaaggc ttccctaccc tttgcactag tgtctctgca ggttgctggt tgtgttgtat 3054gtgctgttcc atggtgttga ctgcactaat aataaacctt ttactcaact ctctaaaaaa 3114aaaaaaaaaa aaaa 3128＜210〉2＜211〉317＜212〉PRT＜213〉 ( Homo sapiens )＜400〉2Met Gly Tyr Asp Val Thr Arg Phe Gln Gly Asp Val Asp Glu Asp Leu1 5 10 15Ile Cys Pro Ile Cys Ser Gly Val Leu Glu Glu Pro Val Gln Ala Pro
20??????????????????25??????????????????30His?Cys?Glu?His?Ala?Phe?Cys?Asn?Ala?Cys?Ile?Thr?Gln?Trp?Phe?Ser
35??????????????????40??????????????????45Gln?Gln?Gln?Thr?Cys?Pro?Val?Asp?Arg?Ser?Val?Val?Thr?Val?Ala?His
50??????????????????55??????????????????60Leu?Arg?Pro?Val?Pro?Arg?Ile?Met?Arg?Asn?Met?Leu?Ser?Lys?Leu?Gln65??????????????????70??????????????????75??????????????????80Ile?Ala?Cys?Asp?Asn?Ala?Val?Phe?Gly?Cys?Ser?Ala?Val?Val?Arg?Leu
85??????????????????90??????????????????95Asp?Asn?Leu?Met?Ser?His?Leu?Ser?Asp?Cys?Glu?His?Asn?Pro?Lys?Arg
100?????????????????105?????????????????110Pro?Val?Thr?Cys?Glu?Gln?Gly?Cys?Gly?Leu?Glu?Met?Pro?Lys?Asp?Glu
115?????????????????120?????????????????125Leu?Pro?Asn?His?Asn?Cys?Ile?Lys?His?Leu?Arg?Ser?Val?Val?Gln?Gln
130?????????????????135?????????????????140Gln?Gln?Thr?Arg?Ile?Ala?Glu?Leu?Glu?Lys?Thr?Ser?Ala?Glu?His?Lys145?????????????????150?????????????????155?????????????????160His?Gln?Leu?Ala?Glu?Gln?Lys?Arg?Asp?Ile?Gln?Leu?Leu?Lys?Ala?Tyr
165?????????????????170?????????????????175Met?Arg?Ala?Ile?Arg?Ser?Val?Asn?Pro?Asn?Leu?Gln?Asn?Leu?Glu?Glu
180?????????????????185?????????????????190Thr?Ile?Glu?Tyr?Asn?Glu?Ile?Leu?Glu?Trp?Val?Asn?Ser?Leu?Gln?Pro
195?????????????????200?????????????????205Ala?Arg?Val?Thr?Arg?Trp?Gly?Gly?Met?Ile?Ser?Thr?Pro?Asp?Ala?Val
210?????????????????215?????????????????220Leu?Gln?Ala?Val?Ile?Lys?Arg?Ser?Leu?Val?Glu?Ser?Gly?Cys?Pro?Ala225?????????????????230?????????????????235?????????????????240Ser?Ile?Val?Asn?Glu?Leu?Ile?Glu?Asn?Ala?His?Glu?Arg?Ser?Trp?Pro
245?????????????????250?????????????????255Gln?Gly?Leu?Ala?Thr?Leu?Glu?Thr?Arg?Gln?Met?Asn?Arg?Arg?Tyr?Tyr
260?????????????????265?????????????????270Glu?Asn?Tyr?Val?Ala?Lys?Arg?Ile?Pro?Gly?Lys?Gln?Ala?Val?Val?Val
275?????????????????280?????????????????285Met?Ala?Cys?Glu?Asn?Gln?His?Met?Gly?Asp?Asp?Met?Val?Gln?Glu?Pro
290 295 300Gly Leu Val Met Ile Phe Ala His Gly Val Glu Glu Ile305 310 315＜210〉3＜211〉21＜212〉DNA＜213〉＜400〉3atggggtatg atgtaacccg t 21＜210〉4＜211〉20＜212〉DNA＜213〉＜400〉4tatctcttcc acgccatgcg 20＜210〉5＜211〉29＜212〉DNA＜213〉＜400〉5gcggatccat ggggtatgat gtaacccgt 29＜210〉6＜211〉28＜212〉DNA＜213〉＜400〉6gcgaattctt atatctcttc cacgccat 28＜210〉7＜211〉29＜212〉DNA＜213〉＜400〉7gcgaattcat ggggtatgat gtaacccgt 29＜210〉8＜211〉25＜212〉DNA＜213〉＜400〉8gcggatccta tctcttccac gccat 25。
Claims (10)
1. an isolating human protein kinase DRP 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 opposing TNF-α inducing cell death 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 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 214-1164 position among the SEQ ID NO:1;
(b) has the sequence of 1-3128 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 human protein kinase DRP polypeptide active is characterized in that this method comprises:
(a) under the condition that is fit to expressing human protein kinase DRP polypeptide, cultivate the described host cell of claim 7;
(b) from culture, isolate polypeptide with human protein kinase DRP polypeptide active.
9. energy and the described human protein kinase DRP of claim 1 polypeptid specificity 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 |
|---|---|---|---|
| CNB021365210A CN100400658C (en) | 2002-08-16 | 2002-08-16 | New type human death resist protein, its coded sequence and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021365210A CN100400658C (en) | 2002-08-16 | 2002-08-16 | New type human death resist protein, its coded sequence and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1475568A true CN1475568A (en) | 2004-02-18 |
| CN100400658C CN100400658C (en) | 2008-07-09 |
Family
ID=34146514
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021365210A Expired - Lifetime CN100400658C (en) | 2002-08-16 | 2002-08-16 | New type human death resist protein, its coded sequence and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100400658C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101897950A (en) * | 2009-05-31 | 2010-12-01 | 中国人民解放军第二军医大学 | Antiviral actions, implementation method and use of novel antiviral molecule DRP |
-
2002
- 2002-08-16 CN CNB021365210A patent/CN100400658C/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101897950A (en) * | 2009-05-31 | 2010-12-01 | 中国人民解放军第二军医大学 | Antiviral actions, implementation method and use of novel antiviral molecule DRP |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100400658C (en) | 2008-07-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1308346C (en) | Novel human phosphotidylethanolamine binding protein, its coding sequence and application | |
| CN1371916A (en) | Human siali acid conjugated immunoglobulin-like agglutinant, its coding sequence and use | |
| CN1475568A (en) | New type human death resist protein, its coded sequence and use | |
| CN1300170C (en) | Novel DnaJ/Hsp40 molecule DC-DJIII, its coding sequence and application | |
| CN1223607C (en) | Human calcium ion/calmodulin dependent protein kinase II inhibitory protein and its application | |
| CN1475502A (en) | New withered induced protein, its coded sequence and use | |
| CN100425695C (en) | Novel human Rab GTP enzyme, its coding sequence and application | |
| CN1475500A (en) | New type human marrow differentiation tagged object its coded sequence and use | |
| CN100443501C (en) | Mitochondria traffic protein molecule for human marrow stromal cell and sequence encoding same and use thereof | |
| CN100480263C (en) | Immunosuppressive receptor derived from dendritic cell, coding sequence and uses thereof | |
| CN1299827A (en) | New human CD20 homolgous molecule and its code sequence and use | |
| CN1299823A (en) | New human cell factor and its code sequence and use | |
| CN1475501A (en) | New type human bone marrow substrate cell source 1 type phosphoric acid enzyme inhibition factor, its coded sequence and use | |
| CN1171901C (en) | New interferon-like protein and its code sequence and use | |
| CN1377891A (en) | Zinc finger protein from dendritic cell, its coding sequence and use | |
| CN1273485C (en) | Immunoglobulin sample receptor originated from human dendritic cell, its coding sequence and application | |
| CN1477116A (en) | Novel human secretion-related membrane protion, its coding sequence and its application | |
| CN1385441A (en) | Novel human lymphokine, its coding sequence and use | |
| CN1299828A (en) | New cell factor acceptor and its code sequence and use | |
| CN1325874A (en) | Human C-type lectin-like receptor and its coding sequence and application | |
| CN1477115A (en) | Novel human cyclin, its coding sequence and application | |
| CN1475499A (en) | New type human cell signal related protein, its code sequence and use | |
| CN1477119A (en) | Novel Human cysteine proteinase inhibitor sample molecule, its coding sequence and application | |
| CN1475503A (en) | New type human bone marrow substrate cell source growth inhibiting factor, its coded sequence and use | |
| CN1380413A (en) | Human 6-phosphoglucosamine isomerase, its code sequence and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
Granted publication date: 20080709 |
|
| CX01 | Expiry of patent term |