CN1388132A - Cell death inducing protein and its coding sequence and use - Google Patents
Cell death inducing protein and its coding sequence and use Download PDFInfo
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Abstract
The present invention provides one new kind of cell death-inducing DFF45-like effector C (CIDE-C) protein and polynucleotide for encoding CIDE-C protein. CIDE-C protein is one new member of CIDE family. The present invention also discloses the preparation and application of CIDE-C protein and its polynucleotide. The present invention also discloses the method of using CIDE-C protein for treating various diseases. The present invention also provides medicine composition containing CIDE-C protein.
Description
The invention belongs to molecular biology and medical field, specifically, the present invention relates to the polynucleotide of new coding cell death inducing protein CIDE-C (cell death-inducing DFF45-like effector C), 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 newcomer of CIDE family.
Apoptosis is one of essential characteristic of cell life.It is stable in fetal development, organismic internal environment, play an important role in the scavenging process of bacterium and virus infected cell.The generation of numerous disease is out of control relevant with apoptosis.Reduce closely related as tumour, autoimmunization syndromes and the virus infection that continues and apoptosis; And neurodegenerative disorders (as Parkinson's disease, senile dementia etc.) and ischemia myocardial damage are relevant with the cell transition apoptosis.
Apoptosis can be brought out by multiple factor, and apoptotic cell all shows common morphology, biochemical characteristics, comprises that cell space shrinkage, volume-diminished, nucleus concentrate, rhexis or the like (Tomislav, D.etal., Oncogene, 17:3207-3213,1998).
Many protein factors that the control process of bringing out, wither with apoptosis is relevant are found.Article one, approach is by the mediation of plastosome approach, and cytochrome C discharges from plastosome, forms polymer with Apaf-1, thereby recruits and activate Caspase-9; Caspase-8 is recruited and activated to another approach by the mediation of cell surface death receptor by FADD and FLASH.Article two, approach finally all activates Caspase-3, and Caspase-3 activates the dna segment factor (DFF), causes rhexis (Budihardjo, I., et al., Annual review of cell anddevelopmental biology, 15:269-290,1999).
DFF has two subunits, nuclease (DFF40/CAD) and its inhibition (DFF45/ICAD).By screening with the N end structure territory homology of DFF45 and DFF40, a new apoptosis inducing factor protein family CIDE (cell inducing death DFF45-like effector) (Inohara, N.et al., EMBOJ. have been found, 17:2526-2533,1998).Overexpression CIDE can cell death inducing.The apoptosis inducing factor protein family has two members, CIDE-A and CIDE-B at present.
In view of the closely related property of apoptosis and numerous diseases, this area presses for understands gene and the albumen relevant with apoptosis.
The purpose of this invention is to provide a kind of new cell death inducing protein CIDE-C 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.CIDE-C gene of the present invention is a new gene that does not at home and abroad appear in the newspapers, and will help deepening understanding to the molecular mechanism of apoptosis generation approach to its research of the mechanism of action.
In a first aspect of the present invention, novel isolated CIDE-C polypeptide is provided, this peptide source is from human liver tissue, and it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 75% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned CIDE-C polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 11-724 position among the SEQ ID NO:1; (b) has the sequence of 1-1191 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 CIDE-C protein-active, this method comprises: (a) be fit to cultivate the above-mentioned host cell that is transformed or transduce under the proteic condition of expression CIDE-C; (b) from culture, isolate polypeptide with CIDE-C protein-active.
In a fifth aspect of the present invention, provide and above-mentioned CIDE-C polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 15-1190 Nucleotide in the above-mentioned polynucleotide.
In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism CIDE-C polypeptide active is provided, and the compound that suppresses the CIDE-C polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is the antisense sequences of the encoding sequence (or its fragment) of CIDE-C polypeptide.
In a seventh aspect of the present invention, provide and whether had the proteic method of CIDE-C in the test sample, it comprises: sample is contacted with the proteic specific antibody of CIDE-C, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CIDE-C albumen.
In a eighth aspect of the present invention, a kind of disease relevant with CIDE-C 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.
In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains CIDE-C polypeptide of the present invention or its encoding sequence of safe and effective amount or contains the carrier of this encoding sequence, and pharmaceutically acceptable vehicle, thinner and carrier.These pharmaceutical compositions can be applicable to treat the apoptosis associated conditions.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
The inventor has cloned a kind of new human cell death inducing gene C IDE-C by the homology screening method.This cDNA total length of complete sequence determination is 1191bp (seeing SEQ ID NO:1), the CIDE-C protein of being made up of 238 amino acid (seeing SEQ ID NO:2) of having encoded.Homology analysis shows that CIDE-C belongs to the CIDE gene family.It can cell death inducing.
The present invention has carried out the tissue distribution analysis of CIDE-C gene.By with the Northern hybrid experiment of 12 kinds of mRNA of health adult tissue, show that a large amount that is expressed in colon, heart, small intestine, the stomach of CIDE-C is expressed, trace expression in brain, spleen, liver, the expression product size is about 1.3Kb.In placenta, express the product of 1.1kb.Therefore, the CIDE-C gene can be applied to multiple tissue, has wider ubiquity.
The present invention has made up the carrier for expression of eukaryon of CIDE-C gene, and expresses in people's tire kidney 293T cell, and detects with antibody.Experimental result shows, has expressed the CIDE-C gene in the 293T cell.
The present invention has also carried out the cell in vitro experiment, uses several different methods and detects the gene induced apoptosis of CIDE-C.The 293T cell of at first having used the CIDE-C gene transfection after 24 hours, is collected its genomic dna of cell and extracting.Electrophoresis is identified and is shown that CIDE-C has induced apoptosis.
The inventor has also used the fluorescent microscope 293T cell of foreign gene that detected transfection.In negative control, transfection carrier pCMV-Tag2, its nucleus presents the green fluorescence of structure sample; In positive control, transfection CIDE-B, its nucleus presents the hyperfunction round bead shape corpusculum of fluorescence; In transfection in the cell of CIDE-C, its nucleus also presents the hyperfunction round bead shape corpusculum of fluorescence, illustrates that CIDE-C has also induced apoptosis.
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1, Northern hybridization detects the expression of mRNA in each tissue of people of CIDE-C.
Among the figure: 1, brain; 2, colon; 3, heart; 4, kidney; 5, liver; 6, lung; 7, skeletal muscle; 8, placenta; 9, small intestine; 10, spleen; 11, stomach; 12, testis.
Fig. 2, the immune marking detect the proteic expression of CIDE-C.
Among the figure: 1, pCMV-Tag2; 2, pCMV-Tag2-CIDE-B; 3, pCMV-Tag2-CIDE-C
Fig. 3, extraction genomic dna detect the CIDE-C cell death inducing.
Among the figure: 1, molecular weight standard; 2, pCMV-Tag2; 3, pCMV-Tag2-CIDE-B; 4, pCMV-Tag2-CIDE-C
Fig. 4, fluorescent microscope detect the CIDE-C cell death inducing.
Among the figure: 1, pCMV-Tag2; 2, pCMV-Tag2-CIDE-B; 3, pCMV-Tag2-CIDE-C
The cell of arrow indication is an apoptotic cell.
In the present invention, term " CIDE-C albumen ", " CIDE-C polypeptide " or " cell death inducing protein CIDE-C " be used interchangeably, all refer to have cell death inducing protein CIDE-C amino acid sequence (SEQ ID NO:2) Albumen or polypeptide. They comprise the cell death inducing protein CIDE-C that contains or do not contain initial methionine.
As used herein, " separation " refers to that material separates (if natural from its original environment Material, original environment namely are natural environment). Such as the polynucleotide under the natural state in the active somatic cell and many Peptide does not have separation and purification, but same polynucleotide or polypeptide are such as same other that exist from natural state In the material separately, then for separation and purification.
As used herein, it is natural that " CIDE-C albumen or the polypeptide of separation " refers to that the CIDE-C polypeptide is substantially free of Relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can use the egg of standard White matter purification technique purifying CIDE-C albumen.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptide, synthetic polypeptide, preferred recombinant polypeptide. This The polypeptide of invention can be the product of natural purifying, or the product of chemical synthesis, or use recombinant technique from Produce in protokaryon or the 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 the sugar baseization, or can right and wrong sugar Baseization.
The present invention also comprises fragment, derivative and the similar thing of CIDE-C albumen. As used herein, term " sheet Section ", " derivative " refer to basically keep natural CIDE-C albumen of the present invention identical with " similar thing " Biology function or active polypeptide. Polypeptide fragment of the present invention, derivative or similar thing can be that (i) has one Individual or a plurality of 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 be by the genetic code coding, or (ii) one or The polypeptide that has substituted radical in a plurality of amino acid residues, or (iii) mature polypeptide and another compound (such as Prolong the compound of polypeptide half-life, for example polyethylene glycol) merge formed polypeptide, or (iv) additional ammonia The base acid sequence be fused to this peptide sequence and the polypeptide that forms (as leading sequence or secretion sequence or be used for purifying this The sequence of polypeptide or proteinogen sequence, or with the fusion albumen of the formation of antigen I gG fragment). Religion according to this paper Lead, these fragments, derivative and similar thing belong to the known scope of those skilled in the art.
The special similar thing of CIDE-C of one class is other mammals (such as ox, sheep, rabbit, dog, monkey, mouse etc.) The same source protein of middle CIDE-C. The coded sequence of the same source protein of these other species can disclose according to the present invention Sequence, the method by hybridization or amplification obtains, and then obtains these homology eggs by conventional recombination method In vain.
In the present invention, term " CIDE-C polypeptide " refers to have the SEQ ID NO.2 order of CIDE-C protein active The polypeptide of row. This term also comprises having and change CIDE-C albumen identical function, SEQ ID NO.2 sequence The abnormity formula. These variant forms comprise (but being not limited to): some (are generally 1-50, better ground 1-30 Individual, 1-20 more preferably, 1-10 best) amino acid whose disappearance, insertion and/or replacement, and at the C end (be generally in 20, be in 10 goodly, more preferably is 5 in one or several of the terminal interpolation of end and/or N In individual) amino acid. For example, in the art, when replacing with the close or similar amino acid of performance, Usually can not change the function of protein. Again such as, terminal to add one or several amino at C end and/or N Acid also can not change the function of protein usually. This term comprises that also active fragment and the activity of CIDE-C albumen spread out Biological.
The variant form of this polypeptide comprises: same source sequence, conservative variant, allelic variant, natural sudden change Body, induce mutant, can be coded with the DNA of CIDE-C DNA hybridization under high or low stringency condition Albumen and the polypeptide or the albumen that utilize the antiserum acquisition of anti-CIDE-C polypeptide. The present invention also provides other Polypeptide, as comprise the fusion albumen of CIDE-C polypeptide or its fragment. Except the polypeptide of total length almost, the present invention The soluble fragments that has also comprised the CIDE-C polypeptide. Usually, this fragment has at least about of CIDE-C peptide sequence 10 continuous amino acids, common at least about 30 continuous amino acids, at least about 50 continuous amino acids in better ground, More preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the similar thing of CIDE-C albumen or polypeptide. These similar things and natural CIDE-C polypeptide poor Can not be the difference on the amino acid sequence, also can be the difference that does not affect on the modified forms of sequence, perhaps Have both at the same time. These polypeptide comprise hereditary variant natural or that induce. Induce the variant can be by various skills Art obtains, as by radiation or be exposed to mutagens and produce at random mutagenesis, and also can be by direct mutagenesis method or its The biological technology of his known molecular. Similar thing also comprises having and is different from the amino acid whose residue of natural L-(such as D-Amino acid) similar thing, and have that non-natural exists or synthetic amino acid (such as β, gamma-amino acid) Similar thing. Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(usually the not changing a level structure) form of modifying comprises: in the body or the chemically derived form of external polypeptide as Acetyl or carboxylated. Modify and also to comprise sugar baseization, as those polypeptide synthesize and process in or further add Carry out glycosylation modified in worker's step and polypeptide that produce. This kind modification can be carried out sugar by polypeptide is exposed to The enzyme of baseization (such as mammiferous sugar baseization enzyme or deglycosylating enzyme) and finishing. Modified forms also comprises having phosphorus The sequence of acidifying amino acid residue (such as phosphoric acid tyrosine, phosphoserine, phosphoric acid threonine). Also comprise and being repaiied Thereby decorations have improved its anti-proteolysis performance or have optimized the polypeptide of solubility property.
In the present invention, " CIDE-C albumen conservative variation polypeptide " refers to the amino acid order with SEQ ID NO:2 Row are compared, and have 10 at the most, 8 at the most on better ground, more preferably at the most 5,3 amino acid at the most best Replaced by similar performance or close amino acid and form polypeptide. These conservative variation polypeptide are preferably according to table 1 carries out amino acid substitution and produces.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Polynucleotides of the present invention can be dna form or rna form. Dna form comprises cDNA, genome DNA Or artificial synthetic DNA. 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 or simple with the coding region sequence shown in the SEQ ID NO:1 And variant. As used herein, " letter variant also " refers to that in the present invention coding has SEQ ID NO:2 Protein, but with the differentiated nucleic acid sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotides of the mature polypeptide of coding SEQ ID NO:2 comprise: the coded sequence of an encoding mature polypeptide; The coded sequence of mature polypeptide+various additional code sequences; The coded sequence of mature polypeptide is (with optional additional volume The code sequence)+non-coding sequence.
Term " polynucleotides of coded polypeptide " can be the polynucleotides that comprise this polypeptide of encoding, and also can be The polynucleotides that also comprise additional code and/or non-coding sequence.
The invention still further relates to the variant of above-mentioned polynucleotides, its coding has identical amino acid sequence with the present invention Polypeptide or the fragment of polypeptide, similar thing and derivative. The variant of these polynucleotides can be natural generation The variant that allelic variant or non-natural take place. These nucleotides variants comprise that replacement variant, disappearance become Allosome and insertion variant. As known in the art, allelic variant is the replacement form of polynucleotides, It may be replacement, disappearance or the insertion of one or more nucleotides, but can be from not changing in fact its coding The function of polypeptide.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 60%, better ground is at least 75%, the polynucleotides of at least 80% phase same sex more preferably. The present invention be more particularly directed under strict condition and the present invention The interfertile polynucleotides of described polynucleotides. In the present invention, " strict condition " refers to: (1) is lower Hybridization and wash-out under ion intensity and the higher temperatures degree, such as 0.2 * SSC, 0.1%SDS, 60 ℃; Or (2) hybridization The Shi Jiayou denaturant is such as 50% (v/v) formyl amine, 0.1% little cow's serum/0.1% Ficoll, 42 ℃ etc.; Or (3) Only in the phase same sex 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 biology merit with the mature polypeptide shown in the SEQ ID NO:2 Energy and active.
The invention still further relates to the nucleic acid fragment with above-mentioned sequence hybridization. As used herein, " nucleic acid fragment " Length contains 15 nucleotides at least, better is at least 30 nucleotides, is more preferably at least 50 nucleotides, and is best More than at least 100 nucleotides. The amplification technique (such as PCR) that nucleic acid fragment can be used for nucleic acid with determine and/or The polynucleotide that separates coding CIDE-C albumen.
CIDE-C nucleotides full length sequence of the present invention or its fragment usually can use pcr amplification method, recombination method or Artificial synthetic method obtains. For the pcr amplification method, can be disclosed according to the present invention about the nucleotides sequence, Especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by those skilled in the art known The prepared cDNA storehouse of conventional method as template, amplification and must relevant sequence. When sequence is longer, usually Need to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplified by correct order splicing one Rise.
In case obtained relevant sequence, just can obtain in large quantity relevant sequence with recombination method. This is common Be that it is cloned into carrier, change again cell over to, separate from the host's cell after the propagation by conventional method then Obtain relevant sequence.
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.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.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 CIDE-C 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 CIDE-C polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention CIDE-C 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, the CIDE-C 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: the expression vector based on T7 of expressing in bacterium; The pMSXND expression vector of in mammalian cell, expressing 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 CIDE-C DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P
LPromotor; Eukaryotic promoter comprises CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; Zooblasts such as CHO, COS-1,293 cells.
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 CIDE-C albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease (for example being used for inducing apoptosis of tumour cell) due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism CIDE-C protein function as pharmacological agent CIDE-C protein function.The peptide molecule that can suppress or stimulate the CIDE-C protein function that can be used for seeking therapeutic value with the recombinant C IDE-C protein screening peptide library of expressing.
On the other hand, the present invention also comprises CIDE-C 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 CIDE-C gene product or fragment.Preferably, refer to that those can combine with CIDE-C 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 CIDE-C, comprise that also those do not influence the antibody of CIDE-C protein function.The present invention also comprise those can with modify or without the CIDE-C 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; Or chimeric antibody.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the CIDE-C gene product of purifying or its have antigenic fragment, can be applied to animal (as rabbit, mouse etc.) to induce the generation of polyclonal antibody.Similarly, expressing CIDE-C albumen or its has antigenic segmental cell and can be used to immune animal and produce antibody.Can use the reaction of multiple adjuvant enhancing immunity, comprising (but being not limited to) freund's adjuvant etc.
Monoclonal antibody of the present invention can utilize hybridoma technology to prepare.Each antibody-like of the present invention can utilize the fragment or the functional zone of CIDE-C 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 CIDE-C 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-CIDE-C can be used in the immunohistochemistry technology, detects the CIDE-C albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of CIDE-C, 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.
Utilize albumen of the present invention,, can filter out with CIDE-C albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.
Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
Polypeptide of the present invention can be directly used in disease treatment, for example, is used for the treatment of hepar damnification reparation aspect.When using CIDE-C albumen of the present invention, also can use the other treatment agent simultaneously.
The present invention also provides a kind of pharmaceutical composition, and it contains CIDE-C 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 CIDE-C 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 CIDE-C also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because the proteic no expression of CIDE-C, non-activity or active cell proliferation, growth or metabolic disturbance due to low, also can be used for increasing expression and the activity (for example being used to promote liver regeneration) of CIDE-C.The gene therapy vector (as virus vector) of reorganization can be designed to express CIDE-C albumen, to increase endogenic CIDE-C protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the CIDE-C transgenosis to cell.The method that structure carries the recombinant viral vector of foreign gene is found in existing document (Sambrook, et al.).Recombinant C IDE-C 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 CIDE-C mRNA and ribozyme also within the scope of the invention.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 CIDE-C obtains.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization CIDE-C protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The CIDE-C protein level that is detected in the test can be with laying down a definition the importance of CIDE-C albumen in various diseases and be used to the disease of diagnosing CIDE-C albumen to work.
Whether having the proteic method of CIDE-C in a kind of detection test sample is to utilize the proteic specific antibody of CIDE-C to detect, and it comprises: sample is contacted with the CIDE-C protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CIDE-C albumen.
The proteic polynucleotide of CIDE-C can be used for the diagnosis and the treatment of CIDE-C protein related diseases.Aspect diagnosis, the proteic polynucleotide of CIDE-C can be used for detecting the proteic expression of CIDE-C CIDE-C abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of CIDE-C as the CIDE-C dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of CIDE-C albumen and also can detect the proteic transcription product of CIDE-C.
The sudden change that detects the CIDE-C gene also can be used for the disease of diagnosing CIDE-C albumen relevant.The form of CIDE-C protein mutation comprises that the point mutation compared with normal wild type CIDE-C 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.
The invention has the advantages that:
1, provides a brand-new human apoptosis-related genes CIDE-C.It is a functional gene with cell death inducing.This invention has great importance for the understanding of deepening pair cell apoptosis molecular mechanism.
2, the present invention finds that CIDE-C can cell death inducing, shows that CIDE-C participates in the negative control of withering of cell growth, can be used as medicine or as a series of clinical applications such as target sequence of medicine, comprises early diagnosis, the gene therapy of apoptosis-associated diseases.
3, the expression of CIDE-C in health adult tissue has specificity, and a large amount is expressed in colon, heart, small intestine, stomach, in other trace expression or do not express, shows that this gene has important function in these four kinds of tissues.Can be applicable to diagnostic preparation as colon, heart, small intestine and stomach relative disease.
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
CIDE-C full-length gene clone
Sequence according to CIDE family, carry out homologous sequence relatively with the nucleotide sequence of human genome, polymerase chain reaction (Polymerase Chain Reaction has been synthesized in design, PCR) primer P1:5-＇ gctgacaaggatggaatacg-3 ＇ (SEQ ID NO:3), primer P2:5 ＇-cttgtgggca ctaccagtta a-3 ＇ (SEQ IDNO:4).CDNA library plasmid (GIBCO BRL company product) with liver is a template, and pcr amplification obtains the CIDE-B gene cDNA, and the PCR reaction conditions is 94 ℃ of pre-sex change in 2 minutes, circulating reaction be 94 ℃ 30 seconds, 55 ℃ 30 seconds, 68 ℃ 90 seconds, carry out 36 circulations altogether.The PCR product of about 800bp is reclaimed purifying with the low melting-point agarose gel, be cloned in the T carrier (TaKaRa company product), the method reference " molecular cloning. experiment guide " (Sambrook, J., Fritsh, E.F., and Maniais, T., Molecular Cloning, Cold Spring HarborLaboratory Press, 1989).Carry out complete sequence order-checking (measuring) then by Bo Ya company.
Carry out the PCR reaction with T7 primer (5 ＇-TAATACGACTCACTATAGGGAGA-3 ＇) with primer P1 again, product directly checks order and obtains the 3 ＇ terminal sequences of cDNA.This cDNA total length is 1191bp (SEQ ID NO:1), has comprised a complete protein-coding region (11-724 position among the SEQ ID NO:1), the protein of being made up of 238 amino acid of having encoded (SEQ ID NO:2).
Embodiment 2
The expression analysis of CIDE-C in people's healthy tissues
The poly (A) of 12 kinds of different tissues of normal people will be fixed with
+RNA Hybond membrane (Nanjing University provides) is put into hybrid pipe, add 5mL quick hybridization liquid (Amersham Pharmacia biotech company product), 65 ℃ of prehybridizations 30 minutes, the segmental probe of the random labeled CIDE-C of 32P (the Random Primer dna marker test kit that adds sex change, TaKaRa company), hybridized 1.5 hours for 65 ℃.Hybond membrane with lavation buffer solution I (0.3M NaCl, 0.03M sodium-acetate (PH7.0), 0.1% sodium laurylsulfonate) in room temperature 20 minutes; Use lavation buffer solution II (37.5mM NaCl, 3.75mM sodium-acetate (pH7.0), 0.1% sodium laurylsulfonate (SDS)) to wash twice, each 15 minutes then in 65 ℃.Then, press the X-ray sheet ,-70 ℃ of radioautograph (Fig. 1).
Experimental result shows a large amount expression in colon, heart, small intestine, stomach of CIDE-C gene, trace expression in brain, spleen, liver, and the expression product size is 1.3Kb.In placenta, express the product of 1.1kb.
Embodiment 3
The expression of CIDE-C Construction of eukaryotic and protein product and detection.
According to the cDNA sequences Design primer P3:5 ＇-gctctagaat ggaatacgcc atga-3 ＇ (SEQ IDNO:5) and primer P4:5 ＇-cggaattcac tgcagtatcttc-3 ＇ (SEQ ID NO:6) of CIDE-C, the both sides of primer have the restriction enzyme site of XbaI and EcoRI respectively.With the CIDE-C sequence through sequence verification is template, carries out the PCR reaction.The PCR product reclaims purifying through the low melting-point agarose gel, adds restriction enzyme XbaI and EcoRI (TaKaRa company product).37 ℃ of digestion were reclaimed purifying after one hour.Simultaneously carrier pCMV-Tag2 (Stratagene company product) was cut one hour with identical restriction enzyme XbaI and EcoRI enzyme, the low melting-point agarose gel reclaims purifying.Reclaim product with two kinds and spend the night, be converted in the DH5 α bacterial strain with 16 ℃ of connections of T4 dna ligase (TaKaRa company product).By screening, the CIDE-C plasmid that obtains recombinating, called after pCMV-Tag2-CIDE-C.
Normal tire nephrocyte 293T adds 10% calf serum (GIBCOL BRL company product), 37 ℃, 5%CO with DMEM nutrient solution (GIBCOL BRL company product)
2Cultivate.Cell is inoculated Tissue Culture Dish into 60mm, 37 ℃ of cultivations with 90,000 inoculum size.After 24 hours with 10 μ l Lipofectamine (GIBCOL BRL company product) transfection 293T cell.PCMV-Tag2, the pCMV-Tag2-CIDE-B, the pCMV-Tag2-CIDE-C plasmid that add 5 μ g respectively.After the transfection 24 hours, collect the 293T cell.300 μ l Triton X-100 lysates (10mMTris (pH7.4), 150mM NaCl, 10mM EDTA, 10mM EGTA, 1%Triton X-100,1mM PMSF, 10mM DTT, 5 μ g/ml aprotinin) cracking.Take out 50 μ l lysates and add equivalent 2 * SDS sample-loading buffer, boiling water boiled 10 minutes, and the fine needle head is sheared DNA, the centrifugal precipitation of going.Get 10 μ l and be splined on 15%SDS polyacrylamide (PAGE) gel, carry out protein electrophoresis.Electrophoresis is transferred to poly(vinylidene fluoride) (PVDF) film (NEN company product) with albumen after finishing.Monoclonal antibody (Sigma) with the anti-Flag label protein of mouse is carried out immunoblotting, uses two crosslinked anti-(Santa Cruz company products) of horseradish peroxidase (HRP) to hybridize again.Detect with chemical illuminating reagent at last, press X-ray sheet 1 minute (Fig. 2).
The result shows, has the CIDE-C/Tag fusion rotein of expressing at the 30kDa place.
Embodiment 4
CIDE-C can cell death inducing
The normal tire nephrocyte 293T DMEM nutrient solution that adds 10% calf serum, 37 ℃, 5%CO
2Cultivate.Cell is with 9 * 10
5Individual inoculum size is inoculated the Tissue Culture Dish into 60mm, 37 ℃ of cultivations.After 24 hours with 10 μ lLipofectamine transfection 293T cells.Divide pCMV-Tag2, the pCMV-Tag2-CIDE-B, the pCMV-Tag2-CIDE-C plasmid that add 5 μ g in addition.After the transfection 24 hours, collect the 293T cell, extract the genomic dna of cell.Electrophoresis is identified on 1.8% sepharose.
The result as shown in Figure 3, visible transfection the cell genomic dna of CIDE-B, CIDE-C gene present ladder type.This explanation CIDE-C can cell death inducing.
Embodiment 5
Fluorescent microscope detects CIDE-C inductive apoptosis
The 293T cell is divided to six orifice plates, and 30,000 cells are divided in every hole.The plasmid DNA of transfection 1 μ g after 24 hours, each hole is respectively pCMV-Tag2, pCMV-Tag2-CIDE-B, pCMV-Tag2-CIDE-C.After the transfection 24 hours, acridine orange (AO) and ethidium bromide (EB) dyeing.Fluorescent microscope detects down, and (Fig. 3) takes pictures.Experimental result shows that CIDE-C has induced the apoptosis of 293T cell.
Embodiment 6
The generation of anti-CIDE-C protein antibodies
Get the purified fusion protein that obtains among the 200 μ g embodiment 3, be dissolved in 0.5 ml deionized water, add isopyknic Freund's complete adjuvant and mixing, multi-point injection is in the male new zealand white rabbit intracutaneous of body weight 2.5Kg.Same protein content and the Freund's incomplete adjuvant of 4 weeks back use carries out booster shots.After 2 weeks, strengthen once more.Strengthen the back and get blood, use immune double diffusion method to detect antibody titer at arteria auricularis.When antibody titer reached requirement, the carotid artery bloodletting was collected blood and is prepared antiserum(antisera), added NaN
3To 0.1%, place-20 ℃ of preservations.
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〉Shanghai Inst. of Biochemistry, Chinese Academy of Sciences＜120〉cell death inducing protein, its encoding sequence and purposes＜130〉012165＜160〉7＜170〉PatentIn version 3.0＜210〉1＜211〉1191＜212〉DNA＜213〉Homo sapiens＜220〉＜221〉CDS＜222〉(11) .. (724)＜400〉1gctgacaagg atg gaa tac gcc atg aag tcc ctt agc ctt ctc tac ccc 49
Met?Glu?Tyr?Ala?Met?Lys?Ser?Leu?Ser?Leu?Leu?Tyr?Pro
1 5 10aag?tcc?ctc?tcc?agg?cat?gtg?tca?gtg?cgt?acc?tct?gtg?gtg?acc?cag 97Lys?Ser?Leu?Ser?Arg?His?Val?Ser?Val?Arg?Thr?Ser?Val?Val?Thr?Gln
15 20 25cag?ctg?ctg?tcg?gag?ccc?agc?ccc?aag?gcc?ccc?agg?gcc?cgg?ccc?tgc 145Gln?Leu?Leu?Ser?Glu?Pro?Ser?Pro?Lys?Ala?Pro?Arg?Ala?Arg?Pro?Cys30 35 40 45cgc?gta?agc?acg?gcg?gat?cga?agc?gtg?agg?aag?ggc?atc?atg?gct?tac 193Arg?Val?Ser?Thr?Ala?Asp?Arg?Ser?Val?Arg?Lys?Gly?Ile?Met?Ala?Tyr
50 55 60agt?ctt?gag?gac?ctc?ctc?ctc?aag?gtc?cgg?gac?act?ctg?atg?ctg?gca 241Ser?Leu?Glu?Asp?Leu?Leu?Leu?Lys?Val?Arg?Asp?Thr?Leu?Met?Leu?Ala
65 70 75gac?aag?ccc?ttc?ttc?ctg?gtg?ctg?gag?gaa?gat?ggc?aca?act?gta?gag 289Asp?Lys?Pro?Phe?Phe?Leu?Val?Leu?Glu?Glu?Asp?Gly?Thr?Thr?Val?Glu
80 85 90aca?gaa?gag?tac?ttc?caa?gcc?ctg?gca?ggg?gat?aca?gtg?ttc?atg?gtc 337Thr?Glu?Glu?Tyr?Phe?Gln?Ala?Leu?Ala?Gly?Asp?Thr?Val?Phe?Met?Val
95 100 105ctc?cag?aag?ggg?cag?aaa?tgg?cag?ccc?cca?tca?gaa?cag?ggg?aca?agg 385Leu?Gln?Lys?Gly?Gln?Lys?Trp?Gln?Pro?Pro?Ser?Glu?Gln?Gly?Thr?Arg110 115 120 125cac?cca?ctg?tcc?ctc?tcc?cat?aag?cct?gcc?aag?aag?att?gat?gtg?gcc 433His?Pro?Leu?Ser?Leu?Ser?His?Lys?Pro?Ala?Lys?Lys?Ile?Asp?Val?Ala
130 135 140cgt?gta?acg?ttt?gat?ctg?tac?aag?ctg?aac?cca?cag?gac?ttc?att?ggc 481Arg?Val?Thr?Phe?Asp?Leu?Tyr?Lys?Leu?Asn?Pro?Gln?Asp?Phe?Ile?Gly
145 150 155tgc?ctg?aac?gtg?aag?gcg?act?ttt?tat?gat?aca?tac?tcc?ctt?tcc?tat 529Cys?Leu?Asn?Val?Lys?Ala?Thr?Phe?Tyr?Asp?Thr?Tyr?Ser?Leu?Ser?Tyr
160 165 170gat?ctg?cac?tgc?tgt?ggg?gcc?aag?cgc?atc?atg?aag?gaa?gct?ttc?cgc 577Asp?Leu?His?Cys?Cys?Gly?Ala?Lys?Arg?Ile?Met?Lys?Glu?Ala?Phe?Arg
175 180 185tgg?gcc?ctc?ttc?agc?atg?cag?gcc?aca?ggc?cac?gta?ctg?ctt?ggc?acc 625Trp?Ala?Leu?Phe?Ser?Met?Gln?Ala?Thr?Gly?His?Val?Leu?Leu?Gly?Thr190 195 200 205tcc?tgt?tac?ctg?cag?cag?ctc?ctc?gat?gct?acg?gag?gaa?ggg?cag?ccc 673Ser?Cys?Tyr?Leu?Gln?Gln?Leu?Leu?Asp?Ala?Thr?Glu?Glu?Gly?Gln?Pro
210 215 220ccc?aag?ggc?aag?gcc?tca?tcc?ctt?atc?ccg?acc?tgt?ctg?aag?ata?ctg 721Pro?Lys?Gly?Lys?Ala?Ser?Ser?Leu?Ile?Pro?Thr?Cys?Leu?Lys?Ile?Leu
225 230 235cag?tgaaagccca?agtccttgga?agctttcccc?agtgaaggac?tgactggggg 774Glncctcacgctt?aactggtagt?gcccacaagt?ggtagtggcc?acaagcctgg?cagctgtaga 834gccgcgaacc?tccccacacc?tccctcaccg?cgcaggaccc?tgagtgagga?ggaggagctg 894gaaacctggg?gtgggttggc?caaaggagaa?cctcaagctc?ctggcctgat?ccagctcctt 954cctgcccaag?gcagcttagc?ccatccagac?tggtcctgaa?gtctgtccct?ccattggcat 1014gaagtctgcc?ccttagcaat?ccggcctcgc?aggctgtact?ttcatggtgc?tctctacctt 1074ctggccccca?tcccggaaca?ttcctgagtg?aattcgcaag?cgcactagca?tgtgatatta 1134gggagtttgc?aataaattat?tgaggctgaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaa 1191<210>2<211>238<212>PRT<213>Homo?sapiens<400>2Met?Glu?Tyr?Ala?Met?Lys?Ser?Leu?Ser?Leu?Leu?Tyr?Pro?Lys?Ser?Leu1 5 10 15Ser?Arg?His?Val?Ser?Val?Arg?Thr?Ser?Val?Val?Thr?Gln?Gln?Leu?Leu
20 25 30Ser?Glu?Pro?Ser?Pro?Lys?Ala?Pro?Arg?Ala?Arg?Pro?Cys?Arg?Val?Ser
35 40 45Thr?Ala?Asp?Arg?Ser?Val?Arg?Lys?Gly?Ile?Met?Ala?Tyr?Ser?Leu?Glu
50 55 60Asp?Leu?Leu?Leu?Lys?Val?Arg?Asp?Thr?Leu?Met?Leu?Ala?Asp?Lys?Pro65 70 75 80Phe?Phe?Leu?Val?Leu?Glu?Glu?Asp?Gly?Thr?Thr?Val?Glu?Thr?Glu?Glu
85 90 95Tyr?Phe?Gln?Ala?Leu?Ala?Gly?Asp?Thr?Val?Phe?Met?Val?Leu?Gln?Lys
100 105 110Gly?Gln?Lys?Trp?Gln?Pro?Pro?Ser?Glu?Gln?Gly?Thr?Arg?His?Pro?Leu
115 120 125Ser?Leu?Ser?His?Lys?Pro?Ala?Lys?Lys?Ile?Asp?Val?Ala?Arg?Val?Thr
130 135 140Phe?Asp?Leu?Tyr?Lys?Leu?Asn?Pro?Gln?Asp?Phe?Ile?Gly?Cys?Leu?Asn145 150 155 160Val?Lys?Ala?Thr?Phe?Tyr?Asp?Thr?Tyr?Ser?Leu?Ser?Tyr?Asp?Leu?His
165 170 175Cys?Cys?Gly?Ala?Lys?Arg?Ile?Met?Lys?Glu?Ala?Phe?Arg?Trp?Ala?Leu
180 185 190Phe?Ser?Met?Gln?Ala?Thr?Gly?His?Val?Leu?Leu?Gly?Thr?Ser?Cys?Tyr
195 200 205Leu?Gln?Gln?Leu?Leu?Asp?Ala?Thr?Glu?Glu?Gly?Gln?Pro?Pro?Lys?Gly
210 215 220Lys Ala Ser Ser Leu Ile Pro Thr Cys Leu Lys Ile Leu Gln225 230 235＜210〉3＜211〉20＜212〉DNA＜213〉synthetic primer＜400〉3gctgacaagg atggaatacg 20＜210〉4＜211〉21＜212〉DNA＜213〉synthetic primer＜400〉4cttgtgggca ctaccagtta a 21＜210〉5＜211〉24＜212〉DNA＜213〉synthetic primer＜400〉5gctctagaat ggaatacgcc atga 24＜210〉6＜211〉22＜212〉DNA＜213〉synthetic primer＜400〉6cggaattcac tgcagtatct tc 22＜210〉7＜211〉23＜212〉DNA＜213〉synthetic primer＜400〉7taatacgact cactataggg aga 23
Claims (10)
1. an isolating CIDE-C polypeptide is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 75% 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 ID NO: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 11-724 position among the SEQ ID NO:1;
(b) has the sequence of 1-1191 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. the preparation method with polypeptide of CIDE-C protein-active is characterized in that, this method comprises:
(a) being fit to express under the proteic condition of CIDE-C, cultivate the described host cell of claim 7;
(b) from culture, isolate polypeptide with CIDE-C protein-active.
9. energy and the described CIDE-C protein-specific of claim 1 bonded antibody.
10. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 or the described polynucleotide of claim 3 or the described carrier of claim 6 of safe and effective amount, and pharmaceutically acceptable vehicle, diluent or carrier.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104311654A (en) * | 2014-08-11 | 2015-01-28 | 中国科学院广州生物医药与健康研究院 | CIDE3 polypeptide, preparation of antibody thereof and applications of the antibody |
| US11426445B2 (en) * | 2017-09-19 | 2022-08-30 | Ohio University | Methods for treating cancers using fat specific protein 27 (FSP27) compositions |
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2001
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104311654A (en) * | 2014-08-11 | 2015-01-28 | 中国科学院广州生物医药与健康研究院 | CIDE3 polypeptide, preparation of antibody thereof and applications of the antibody |
| US11426445B2 (en) * | 2017-09-19 | 2022-08-30 | Ohio University | Methods for treating cancers using fat specific protein 27 (FSP27) compositions |
| US20230059413A1 (en) * | 2017-09-19 | 2023-02-23 | Ohio University | Methods for treating cancers using fat specific protein 27 (fsp27) compositions |
| US11975044B2 (en) * | 2017-09-19 | 2024-05-07 | Ohio University | Methods for treating cancers using fat specific protein 27 (FSP27) compositions |
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