CN1306976A - Polypeptide-clathrin 76 and polynucleotide for coding said polypeptide - Google Patents

Polypeptide-clathrin 76 and polynucleotide for coding said polypeptide Download PDF

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CN1306976A
CN1306976A CN00111507A CN00111507A CN1306976A CN 1306976 A CN1306976 A CN 1306976A CN 00111507 A CN00111507 A CN 00111507A CN 00111507 A CN00111507 A CN 00111507A CN 1306976 A CN1306976 A CN 1306976A
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polypeptide
polynucleotide
clathrin
sequence
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毛裕民
谢毅
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Shanghai Bodao Gene Technology Co Ltd
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Shanghai Bodao Gene Technology Co Ltd
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Abstract

The present invention discloses one new kind of polypeptide, clathrin 76, polynucleotides encoding this polypeptide and DNA recombination process to produce the polypeptide. The present invention also discloses the method of applying the polypeptide in treating various diseases, such as malignant tumor, nosohemia, HIV infection, immunological diseases and inflammations. The present invention also discloses the antagonist resisting the polypeptide and its treatment effect. The present invention also discloses the application of the polynucleotides encoding the clathrin 76.

Description

The polynucleotide of a kind of new polypeptide---clathrin 76 and this peptide species of coding
The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide---clathrin 76, and the polynucleotide sequence of this polypeptide of encoding.The invention still further relates to the preparation method and the application of these polynucleotide and polypeptide.
The interior active mechanism of cellular uptake material mainly contains two kinds: phagolysis (i.e. " cell is eaten "), (smaller particles (diameter＜0.2.mu.m) is taken in diameter＞0.5.mu.m), pinosome (" cell is drunk ") in the vesicle to digest larger particles usually.Pinosome is the most normal to occur in the specific site that cytolemma is called as the pothole of clathrin-Bao quilt.Clathrin is called as triskelion because of the profile that it has three pin.The vesicle mediation of clathrin bag quilt transportation of the receptor-selective to another cytolemma from a cytolemma.They help the sorting protein in receptor mediated endocytosis and the anti--Golgi network.Clathrin is that a kind of main film forms albumen, and it wraps the vesicle of similar coating shrinkage pool, and forms the cell surface fragment that participates in integrating film traffic in the biology.The adventitia of clathrin (being called as the triangle protein complexes) constitutes (23 to 27Kd) by three heavy chains (180Kd) and three light chains.The heavy chain of clathrin provides structural framing for grid, forms three structures, and light chain is crossed over the interior segments of each and can be interacted with other cytoplasmic proteins.Bag is required being polymerized to of triskelion to be positioned at the polyhedron network of cytolemma kytoplasm one side by the formation of clathrin vesicle.The assembling of clathrin finally forms spherical bag quilt on every side at the film bubble that sprouts by itself and connector molecularity selectivity decoy receptor.After sprouting, clathrin is separated assembling again, discharges vesicle and goes to merge with target film.On different cell inner membrances, adjust the time and the site of clathrin, in cell, replenish clathrin subunit and control mesh albumen the pond and separate assembling and need the polymolecular signal.
The clathrin tumor-necrosis factor glycoproteins is seven folding tumor-necrosis factor glycoproteinss that are positioned at clathrin heavy chain arm regions, and each tumor-necrosis factor glycoproteins contains 140 amino acid approximately, and structure is the α spiral, is positioned at centre portions in whole clathrin.This tumor-necrosis factor glycoproteins is very conservative in Mammals, and it forms spherical outstanding at the trimerical drift angle of clathrin, keep triangle protein complexes shape, the mediation dimerization.
The Rap Phosphoric acid esterase is a kind of albumen aspartic acid Phosphoric acid esterase (AspPc) family, and this albumen is the sPOoF-P intermediate dephosphorylation of phosphate cpd, thereby prevents sporulation.This family member is assembled by TPRs TPR.They be by with the physiological process institute inductive regulatory factor of sporulation opposition.The RapA phosphatase activity is by a kind of small protein, and PhrA controls.
Polypeptide of the present invention contains the conservative characteristic sequence of conservative clathrin tumor-necrosis factor glycoproteins and class RapA albumen aspartic acid Phosphoric acid esterase (AspPc), this polypeptide and antagonist agonist inhibitor thereof, diagnosable prevention are held concurrently and are treated and the relevant imbalance of unusual vesicle transportation.
Because clathrin 76 albumen play an important role in the critical function in body as mentioned above, and believe and relate to a large amount of albumen in these regulate processes, thereby need to identify clathrin 76 albumen of more these processes of participation in this area always, particularly identify this proteic aminoacid sequence.The separation of new clathrin 76 protein coding genes also provides the foundation for determining the effect of this albumen under healthy and morbid state.This albumen may constitute the basis of exploitation medical diagnosis on disease and/or curative, and it is very important therefore separating its coding DNA.
An object of the present invention is to provide isolating new polypeptide---clathrin 76 with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of this polypeptide of coding.
Another object of the present invention provides the recombinant vectors of the polynucleotide that contain the clathrin 76 of encoding.
Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain the clathrin 76 of encoding.
Another object of the present invention provides the method for producing clathrin 76.
Another object of the present invention provides at polypeptide of the present invention---the antibody of clathrin 76.
Another object of the present invention has provided at polypeptide of the present invention---the simulated compound of clathrin 76, antagonist, agonist, inhibitor.
Another object of the present invention provides the method for the unusual relevant disease of diagnoses and treatment and clathrin 76.
The present invention relates to a kind of isolated polypeptide, this polypeptide is the people source, and it comprises: polypeptide or its examples of conservative variations, bioactive fragment or derivative with SEQ ID No.2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
The invention still further relates to a kind of isolating polynucleotide, it comprises a kind of nucleotide sequence or its variant that is selected from down group:
(a) coding has the polynucleotide of the polypeptide of SEQ ID No.2 aminoacid sequence;
(b) with polynucleotide (a) complementary polynucleotide;
(c) with (a) or polynucleotide sequence (b) have the polynucleotide of at least 70% homogeny.
More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 251-2326 position among the SEQ ID NO:1; (b) has the sequence of 1-2658 position among the SEQ ID NO:1.
The present invention relates to a kind of carrier that contains polynucleotide of the present invention, particularly expression vector in addition; The host cell that this carrier of a kind of usefulness is genetically engineered comprises the host cell of conversion, transduction or transfection; A kind of method for preparing polypeptide of the present invention of cultivating described host cell and reclaiming expression product that comprises.
The invention still further relates to a kind of can with polypeptid specificity bonded antibody of the present invention.
The invention still further relates to a kind of simulation, activation, antagonism of screening or suppress the method for the compound of clathrin 76 protein-actives, it comprises and utilizes polypeptide of the present invention.The invention still further relates to the compound that obtains with this method.
The invention still further relates to a kind of vitro detection and express the relevant disease or the method for disease susceptibility with clathrin 76 abnormal proteins, comprise the sudden change in polypeptide described in the detection of biological sample or its coded polynucleotide sequence, perhaps the amount or the biological activity of polypeptide of the present invention in the detection of biological sample.
The present invention also relates to a kind of pharmaceutical composition, it contains polypeptide of the present invention or its stand-in, activator, antagonist or inhibitor and pharmaceutically acceptable carrier.
The invention still further relates to polypeptide of the present invention and/or polynucleotide and be used for the treatment of cancer, developmental character disease or immunological disease or other purposes owing to the medicine of clathrin 76 diseases that abnormal expression causes in preparation.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
The following term of using in this specification sheets and claims has following implication unless stated otherwise: " nucleotide sequence " is meant oligonucleotide, Nucleotide or polynucleotide and fragment or part, also can refer to genome or synthetic DNA or RNA, they can be strand or two strands, represent sense strand or antisense strand.Similarly, term " aminoacid sequence " is meant oligopeptides, peptide, polypeptide or protein sequence and fragment or part.When " aminoacid sequence " among the present invention related to a kind of aminoacid sequence of naturally occurring protein molecule, this " polypeptide " or " protein " did not mean that aminoacid sequence are restricted to the complete natural amino acid relevant with described protein molecule.
Protein or polynucleotide " variant " are meant a kind of polynucleotide sequence that has the aminoacid sequence of one or more amino acid or Nucleotide change or encode it.Described change can comprise disappearance, insertion or the replacement of amino acid in aminoacid sequence or the nucleotide sequence or Nucleotide.Variant can have " conservative property " and change, and wherein the amino acid of Ti Huaning has structure or the chemical property similar with original acid, as replacing Isoleucine with leucine.Variant also can have non-conservation and change, as replacing glycine with tryptophane.
" disappearance " is meant the disappearance of in aminoacid sequence or nucleotide sequence one or more amino acid or Nucleotide.
" insertion " or " interpolation " is meant that the change in aminoacid sequence or nucleotide sequence causes comparing the increase of one or more amino acid or Nucleotide with naturally occurring molecule." replacement " is meant by different amino acid or Nucleotide and replaces one or more amino acid or Nucleotide.
" biological activity " is meant the protein of structure, regulation and control or biochemical function with natural molecule.Similarly, term " immunologic competence " be meant natural, reorganization or synthetic protein and fragment thereof in suitable animal or cell, induce specific immune response and with specific antibody bonded ability.
" agonist " is meant when combining with clathrin 76, thereby a kind of this protein that causes changes the molecule of regulating this protein active.Agonist can comprise protein, nucleic acid, carbohydrate or any other can be in conjunction with the molecule of clathrin 76.
" antagonist " or " inhibition " be meant when combining with clathrin 76, a kind ofly seals or regulate the biologic activity of clathrin 76 or the molecule of immunologic competence.Antagonist and inhibition can comprise protein, nucleic acid, carbohydrate or any other can be in conjunction with the molecule of clathrin 76.
" adjusting " is meant that the function of clathrin 76 changes, and comprises the change of any other biological property, function or the immune property of the change of the rising of protein active or reduction, binding characteristic and clathrin 76.
The pure basically ＂ of ＂ is meant and 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 clathrin 76 of standard.Basically pure clathrin 76 can produce single master tape on the irreducibility polyacrylamide gel.The purity available amino end acid sequence of clathrin 76 polypeptide is analyzed.
" complementary " or " complementation " is meant under salt concn that allows and temperature condition by the natural combination of the polynucleotide of base pairing.For example, " C-T-G-A ' can with complementary sequence " G-A-C-T ' combines sequence.Complementation between two single chain molecules can be part or whole.Complementary degree between the nucleic acid chains has a significant effect for efficient of hybridizing between the nucleic acid chains and intensity.
" homology " is meant the complementary degree, can be portion homologous or complete homology." portion homologous " is meant a kind of part complementary sequence, and it can partly suppress the hybridization of complete complementary sequence and target nucleic acid at least.The inhibition of this hybridization can detect by hybridizing (Southern trace or Northern trace etc.) under the condition that reduces in the severity degree.Basically homologous sequence or hybridization probe can compete and suppress complete homologous sequence and target sequence the condition that reduces of severity degree under combine.This does not mean the conditions permit non-specific binding that the severity degree reduces because the conditional request two sequences that the severity degree reduces mutual be combined into specificity or selectivity interacts.
" homogeny percentage " be meant two or more amino acid or nucleotide sequence relatively in the same or analogous percentage of sequence.The available electron method is measured the homogeny percentage, as passing through MEGALIGN program (Lasergene softwarepackage, DNASTAR, Inc., Madison Wis.).The MEGALIGN program can compare two or more sequences (Higgins, D.G. and P.M.Sharp (1988) Gene 73:237-244) according to diverse ways such as Cluster method.The Cluster method is organized the series arrangement cluster by checking the distance between all pairings with each.Then with each bunch with in pairs or become set of dispense.Homogeny percentage between two aminoacid sequences such as sequence A and the sequence B calculates by following formula:
Figure A0011150700071
Also can be by the Cluster method or with the homogeny percentage (Hein J., (1990) Methods in emzumology 183:625-645) between known method in this area such as the Jotun Hein mensuration nucleotide sequence.
" similarity " is meant the degree that the identical or conservative property of corresponding position amino-acid residue when arranging contrast between the aminoacid sequence replaces.For example be used for amino acid that conservative property replaces, electronegative amino acid can comprise aspartic acid and L-glutamic acid; Positively charged amino acid can comprise Methionin and arginine; Having uncharged head group has similar hydrophilic amino acid can comprise leucine, Isoleucine and Xie Ansuan; Glycine and L-Ala; L-asparagine and glutamine; Serine and Threonine; Phenylalanine and tyrosine.
" antisense " is meant and specific DNA or RNA sequence complementary nucleotide sequence." antisense strand " is meant and " sense strand " complementary nucleic acid chains.
" derivative " is meant HFP or encodes its chemical modification object of nucleic acid.This chemical modification object can be with alkyl, acyl group or the amino hydrogen atom of replacing.The nucleic acid derivative codified keeps the polypeptide of the main biological characteristics of natural molecule.
" antibody " is meant complete antibody molecule and fragment thereof, as Fa, F (ab ') 2And Fv, its energy specificity is in conjunction with the antigenic determinant of clathrin 76.
" humanized antibody " is meant that the aminoacid sequence in non-antigen binding domain territory is replaced and becomes more similar to people's antibody, but still keep original in active antibody.
" isolating " speech refers to material is shifted out among its original environment (for example, if spontaneous its natural surroundings that just refers to).Such as it is exactly not to be separated that spontaneous polynucleotide or polypeptide are present in the Live Animals, but same polynucleotide or polypeptide with some or all in natural system with it the material of coexistence separately be exactly isolating.Such polynucleotide may be the parts of a certain carrier, the part that also possible such polynucleotide or polypeptide are a certain composition.Since carrier or composition are not the compositions of its natural surroundings, they remain isolating.
As used herein, " isolating " are 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 clathrin 76 " is meant that clathrin 76 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 clathrin 76 of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of clathrin 76 polypeptide can be used amino acid sequence analysis.
The invention provides a kind of new polypeptide---clathrin 76, it is made up of the aminoacid sequence shown in the SEQ ID NO:2 basically.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 fragment, derivative and the analogue of clathrin 76.As used herein, term " fragment ", " derivative " and " analogue " are meant biological function or the active polypeptide that keeps clathrin of the present invention 76 identical basically.The fragment of polypeptide of the present invention, derivative or analogue can be: (I) is a kind of like this, wherein one or more amino-acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the amino acid that replaces can be also can not encoded by genetic codon; Perhaps (II) is a kind of like this, and certain group on wherein one or more amino-acid residues is replaced by other group and comprises substituting group; Perhaps (III) is a kind of like this, and wherein mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; Perhaps (IV) is a kind of like this, wherein additional aminoacid sequence is integrated into mature polypeptide and the peptide sequence that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying) by the elaboration of this paper, such fragment, derivative and analogue are considered within those skilled in the art's ken.
The invention provides isolating nucleic acid (polynucleotide), substantially the polynucleotide that have a polypeptide of SEQ ID NO:2 aminoacid sequence by coding are formed.Polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO:1.Polynucleotide of the present invention are to find from the cDNA library of people's fetal brain tissue.The polynucleotide sequence total length that it comprises is 2658 bases, its open reading frame 251-2326 691 amino acid of having encoded.This polypeptide has the characteristic sequence of clathrin tumor-necrosis factor glycoproteins and class RapA albumen aspartic acid Phosphoric acid esterase characteristic sequence, and deducibility goes out the 26S Proteasome Structure and Function that this clathrin 76 has clathrin tumor-necrosis factor glycoproteins and class RapA albumen aspartic acid Phosphoric acid esterase characteristic sequence representative.
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 " are meant that in the present invention coding has protein or the polypeptide 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 that has only 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 " is meant polynucleotide that comprise this polypeptide of encoding and the polynucleotide that comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of foregoing description polynucleotide, its coding has the polypeptide of identical aminoacid sequence or segment, 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 the polynucleotide (have at least 50% between two sequences, preferably have 70% homogeny) of sequence hybridization described above.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 adds and to use 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 95%, be more preferably 97% 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 sequence hybridization described above.As used herein, " nucleic acid fragment " length contain 10 Nucleotide at least, better be 20-30 Nucleotide at least, be more preferably 50-60 Nucleotide at least, preferably more than at least 100 Nucleotide.Nucleic acid fragment also can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding clathrin 76.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
The special polynucleotide sequence of coding clathrin 76 of the present invention can obtain with several different methods.For example, separate polynucleotide with hybridization technique well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect homologous polynucleotide sequence and 2) antibody screening of expression library to be to detect the polynucleotide passage of the clone with common structure feature.
Sequence dna fragment of the present invention also can obtain with following method: 1) separate double chain DNA sequence from genomic dna; 2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of described polypeptide.
In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.The direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.The more frequent method of selecting for use is the separation of cDNA sequence.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qiagene) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.
Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) level of the transcript of mensuration clathrin 76; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.
In (1) kind method, hybridizing used probe is and any a part of homology of polynucleotide of the present invention that at least 10 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, within 2000 Nucleotide, preferable is within 1000 Nucleotide to the length of probe usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene order information of the present invention normally.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.
In (4) kind method, detect the protein product of clathrin 76 genetic expressions and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.
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 from the library, obtain the cDNA of total length, 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 polynucleotide 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 gene of the present invention that obtains as mentioned above, perhaps the polynucleotide sequence of various dna fragmentations etc. can with ordinary method such as dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467) measure.This class polynucleotide sequence is measured also available commercial sequencing kit etc.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and with carrier of the present invention or directly with the host cell of clathrin 76 encoding sequences through the genetically engineered generation, and the method that produces polypeptide of the present invention through recombinant technology.
Among the present invention, the polynucleotide sequence of coding clathrin 76 can be inserted in the carrier, contains the recombinant vectors of polynucleotide of the present invention with formation.Term " carrier " 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 carrier.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on the T7 promotor of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier may be used to make up recombinant expression vector.A key character of expression vector is to contain replication origin, promotor, marker gene and translational control element usually.
Method well-known to those having ordinary skill in the art can be used to make up the dna sequence dna that contains the clathrin 76 of encoding and the expression vector of suitable transcribing/translational control element.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; The PL promotor of lambda particles phage; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte 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 that translation initiation is used and transcription terminator etc.Inserting enhancer sequence in carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis acting factor that DNA expresses, 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.
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 etc.
Persons skilled in the art all know how to select appropriate carriers/transcriptional regulatory element (as promotor, enhanser etc.) and selected marker.
Among the present invention, the polynucleotide of coding clathrin 76 or the recombinant vectors that contains these polynucleotide can transform or transduce into host cell, contain the genetically engineered host cell of these polynucleotide or recombinant vectors with formation.Term " host cell " refers to 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; Bacterial cell such as Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; Insect cell such as fruit bat S2 or Sf9; Zooblast such as CHO, COS or Bowes melanoma cells etc.
Can carry out with routine techniques well known to those skilled in the art with dna sequence dna of the present invention or the recombinant vectors transformed host cell that contains described dna sequence dna.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.Alternative 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, perhaps conventional mechanical method such as microinjection, electroporation, liposome packing etc.
By the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention to can be used to express or produce clathrin 76 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding human clathrin 76 of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;
(2). in suitable medium, cultivate host cell;
(3). separation, protein purification from substratum or cell.
In step (2), 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.
In step (3), recombinant polypeptide can wrap and be expressed or be secreted into the extracellular 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.These methods include, but are not limited to: conventional renaturation is handled, protein precipitant is handled (salt analysis method), centrifugal, the broken bacterium of infiltration, the combination of ultrasonication, 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 antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in disease treatment, for example, can treat malignant tumour, adrenal gland defect, tetter, all kinds of inflammation, HIV infection and immunological disease etc.
Clathrin is significant in cytophagy, pinosome and blebbing, is regulating and control the transmission of protein signal molecule.Clathrin has distinctive clathrin tumor-necrosis factor glycoproteins and forms its biological activity.
Polypeptide of the present invention contains conservative clathrin tumor-necrosis factor glycoproteins, contains the conservative characteristic sequence of class RapA albumen aspartic acid Phosphoric acid esterase (AspPc) simultaneously.The conservative characteristic sequence of RapA Phosphoric acid esterase relates to the dephosphorylation of substrate.Polypeptide expression of the present invention will cause the unusual of cytophagy, pinosome and blebbing effect unusually, and make the dephosphorylation of substrate unusual, influence the transmission of protein signal molecule, and produce relevant disease.
This shows, the abnormal expression of clathrin 76 of the present invention will produce especially dysplasia disease of various diseases, various tumours, inflammation, immunological disease, these diseases include but not limited to: dysplasia disease: mental retardation, cerebral dysgenesis, dysnoesia, stravismus, skin, fat and amyoplasia disease such as congenital cutis laxa, albinism, senium praecox, congenital dyskeratosis, B﹠J dysplasia disease such as fetal rickets, epiphyseal dysplasia, metabolic osteopathy, various metabolic defects such as various amino acid metabolism defective disease, cretinism, nanism, emerging comprehensive this in storehouse levied the slow disease of sexual development
The tumour of various tissues: cancer of the stomach, liver cancer, lung cancer, the esophageal carcinoma, mammary cancer, leukemia, lymphoma, thyroid tumor, hysteromyoma, neurocytoma, astrocytoma, ependymoma, glioma, neurofibroma, colorectal carcinoma, melanoma, adrenal carcinoma, bladder cancer, osteocarcinoma, osteosarcoma, myelomatosis, bone marrow cancer, uterus carcinoma, carcinoma of endometrium, carcinoma of gallbladder, colorectal carcinoma, thymus neoplasms, nasopharyngeal carcinoma, laryngocarcinoma, tracheal neoplasm, fibroma, fibrosarcoma, lipoma, liposarcoma, leiomyoma
Inflammation: atopic reaction, bronchial asthma, sarcoidosis, osteoarthritis, dermatomyositis, urticaria, atopic dermatitis, intestines are answered acute syndromes, atrophic gastritis, chronic gastritis, myasthenia gravis, Green-barre syndrome, intracranial granuloma, multiple scleroderma, pancreatitis, cholecystitis, glomerulonephritis, myocarditis, myocardosis, atherosclerosis, stomach ulcer, hyperplasia of prostate, trachelitis
Immunological disease: rheumatoid arthritis, chronic active hepatitis infects back myocarditis, systemic lupus erythematous, scleroderma, myasthenia gravis, Green-barre syndrome, autoimmune hemolytic anemia, acquired immune deficiency syndrome (AIDS), bronchial asthma, Asprin asthma, rhinallergosis, diffuse interstitial pulmonary fibrosis, urticaria, atopic dermatitis
The abnormal expression of clathrin 76 of the present invention also will produce some heredity, blood disease etc.
The antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in disease treatment, for example, can treat various diseases especially dysplasia disease, various tumour, inflammation, immunological disease, some heredity, blood disease etc.
The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) clathrin 76.Agonist improves clathrin 76 biological function such as stimulate cellular proliferation, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can in the presence of medicine, the film preparation of mammalian cell or expression clathrin 76 be cultivated with the clathrin 76 of mark.Measure the medicine raising then or check this interactional ability.
The antagonist of clathrin 76 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of clathrin 76 can combine and eliminate its function with clathrin 76, or suppresses the generation of this polypeptide, or combines with the avtive spot of this polypeptide and to make this polypeptide can not bring into play biological function.
In screening during as the compound of antagonist, clathrin 76 can be added during bioanalysiss measure, determine to interactional influence between clathrin 76 and its acceptor whether compound is antagonist by measuring compound.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with clathrin 76 bonded peptide molecules obtains.During screening, generally tackle clathrin 76 molecules and carry out mark.
The invention provides and use polypeptide, and fragment, derivative, analogue or their cell are as the method for antigen with production antibody.These antibody can be polyclonal antibody or monoclonal antibody.The present invention also provides the antibody at clathrin 76 antigenic determinants.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.
The method of the production available grids albumen 76 direct injection immune animals (as rabbit, mouse, rat etc.) of polyclonal antibody obtains, and multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.The technology of monoclonal antibody of preparation clathrin 76 include but not limited to hybridoma technology (Kohler andMilstein.Nature, 1975,256:495-497), three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison et al, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.Pat No.4946778) also can be used for producing the single-chain antibody of anti-clathrin 76.
The antibody of anti-clathrin 76 can be used in the immunohistochemistry technology, detects the clathrin 76 in the biopsy specimen.
With the also available labelled with radioisotope of clathrin 76 bonded monoclonal antibodies, 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 also can be used for designing the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of clathrin 76 high-affinities 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 clathrin 76 positive cells.
The disease that antibody among the present invention can be used for treating or prevention and clathrin 76 are relevant.The antibody that gives suitable dosage can stimulate or block the generation or the activity of clathrin 76.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization clathrin 76 levels.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.Clathrin 76 levels that detected in the test can be with laying down a definition the importance of clathrin 76 in various diseases and be used to the disease of diagnosing clathrin 76 to work.
Polypeptide of the present invention also can be used as the peptide spectrum analysis, for example, the polypeptide available physical, chemistry or enzyme carry out the specificity cutting, and carries out the two-dimentional or three-dimensional gel electrophoresis analysis of one dimension, be more preferably and carry out mass spectroscopy.
The polynucleotide of coding clathrin 76 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 nothing expression of clathrin 76 or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the clathrin 76 of expressing variation, to suppress endogenic clathrin 76 activity.For example, a kind of clathrin 76 of variation can be the clathrin 76 that shortens, lacked signal conduction function territory, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the disease of clathrin 76 expression or active caused by abnormal.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the polynucleotide of coding clathrin 76 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding clathrin 76 is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding clathrin 76 can be packaged in the liposome and be transferred in the cell in addition.
Polynucleotide import 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.
Suppress the oligonucleotide (comprising sense-rna and DNA) of clathrin 76 mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.
The polynucleotide of coding clathrin 76 can be used for the diagnosis with the relative disease of clathrin 76.The unconventionality expression of the expression that the polynucleotide of coding clathrin 76 can be used for detecting clathrin 76 clathrin 76 whether or under morbid state.As the dna sequence dna of the clathrin 76 of encoding can be used for biopsy specimen is hybridized to judge the expression situation of clathrin 76.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 the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect clathrin 76 with clathrin 76 special primers.
The sudden change that detects clathrin 76 genes also can be used for diagnosing the relevant disease of clathrin 76.The form of clathrin 76 sudden change comprises that the point mutation compared with normal wild type clathrin 76 dna sequence dnas, 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).This sequence can be specifically at certain bar human chromosome particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Now, have only chromosomal marker thing seldom to can be used for the marker chromosomes position based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, its important the first step is positioned these dna sequence dnas on the karyomit(e) exactly.
In brief, prepare PCR primer (preferred 1 5-35bp), sequence can be positioned on the karyomit(e) according to cDNA.Then, these primers being used for PCR screening contains each bar and goes into chromosomal somatocyte hybrid cell.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.
The PCR localization method of somatocyte hybrid cell is that DNA is navigated to concrete chromosomal quick method.Use Oligonucleolide primers of the present invention,, can utilize one group to realize inferior location from specific chromosomal fragment or a large amount of genomic clone by similar approach.Other the similar strategy that can be used for chromosomal localization comprises in situ hybridization, uses the karyomit(e) prescreen and the hybridization preliminary election of the airflow classification of mark, thereby makes up the special cDNA storehouse of karyomit(e).
The cDNA clone is carried out fluorescence in situ hybridization (FISH) with Metaphase Chromosome, can in a step, accurately carry out chromosomal localization.The summary of this technology is referring to Verma etc., Human Chromosomes:a Manualof Basic Techniques, Pergamon Press, New York (1988).
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 Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
Then, need to measure ill and not cDNA between diseased individuals or genome sequence difference.If observe certain sudden change in some or all of diseased individuals, and this sudden change is not observed in any normal individual, then this sudden change may be the cause of disease of disease.More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as from the horizontal visible of karyomit(e) or use based on detectable disappearance of the PCR of cDNA sequence or transposition.Resolving power according to present physical mapping and assignment of genes gene mapping technology, being accurately positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (the supposing that 1 megabasse mapping resolving power and every 20kb are corresponding to a gene) between 50 to 500 potential Disease-causing genes.
Polypeptide of the present invention, polynucleotide and stand-in thereof, agonist, antagonist and inhibitor and suitable pharmaceutical carrier combination back can be used.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Composition comprises the polypeptide or the antagonist of safe and effective amount and carrier and the vehicle that does not influence effect of drugs.These compositions can be used as medicine and are used for disease treatment.
The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, can have by the given indicative prompting of government authorities of making, using or selling medicine or biological products, the government authorities that this prompting reflects production, uses or sells permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound.
Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.Clathrin 76 comes administration with the amount that treats and/or prevents concrete indication effectively.The amount and the dosage range that are applied to patient's clathrin 76 will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.
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 is the amino acid sequence homology comparison diagram of clathrin 76 of the present invention and structural domain clathrin tumor-necrosis factor glycoproteins and class RapA albumen aspartic acid Phosphoric acid esterase characteristic sequence.The top sequence is a clathrin 76, and the below sequence is structural domain clathrin tumor-necrosis factor glycoproteins and class RapA albumen aspartic acid Phosphoric acid esterase characteristic sequence.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+".
Fig. 2 is the polyacrylamide gel electrophoresis figure (SDS-PAGE) of isolating clathrin 76.74KDa is proteinic molecular weight.The arrow indication is isolated protein band.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Embodiment 1: the clone of clathrin 76
Extract the total RNA of people's tire brain with guanidinium isothiocyanate/phenol/chloroform single stage method.From total RNA, separate poly (A) mRNA with Quik mRNA Isolation Kit (Qiegene company product).2ug poly (A) mRNA forms cDNA through reverse transcription.CDNA fragment orientation is inserted on the multiple clone site of pBSK (+) carrier (Clontech company product) with Smart cDNA clone's test kit (available from Clontech), transforms DH5 α, bacterium forms the cDNA library.Measure all clones' 5 ' and 3 ' terminal sequence with Dyeterminate cycle reaction sequencing kit (Perkin-Elmer company product) and ABI 377 automatic sequencers (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genebank) measured are compared, found that the cDNA sequence of one of them clone 0367f12 is new DNA.By synthetic a series of primers the contained insertion cDNA fragment of this clone is carried out two-way mensuration.The result shows, the contained full-length cDNA of 0367f12 clone is 2658bp (shown in Seq ID NO:1), from 251bp to 2326bp the open reading frame (ORF) of a 2076bp, the new protein (shown in Seq ID NO:2) of encoding arranged.We are with this clone's called after pBS-0367f12, encoded protein matter called after clathrin 76.Embodiment 2:cDNA clone's domain analyses
With the sequence and the encoded protein sequence thereof of clathrin 76 of the present invention, with profile scan program (Basiclocal Alignment search tool) [Alt schul, the SF et al.J.Mol.Biol.1990 among the GCG; 215:403-10], carry out domain analyses at databases such as prosite.Clathrin 76 of the present invention has homology with structural domain clathrin tumor-necrosis factor glycoproteins and class RapA albumen aspartic acid Phosphoric acid esterase characteristic sequence, and homology the results are shown in Fig. 1.Embodiment 3: with the gene of RT-PCR method clones coding clathrin 76
Total RNA is a template with fetus brain cell, is that primer carries out the synthetic cDNA of reverse transcription reaction with oligo-dT, with behind the test kit purifying of Qiagene, carries out pcr amplification with following primer:
Primer1:5’-GGATAAAGGTGTATTTATTGCTTA-3’(SEQ?ID?NO:3)
Primer2:5’-TCAACAATTTTATTTTAAAGTTTT-3’(SEQ?ID?NO:4)
Primer1 is the forward sequence that begins of 1bp that is positioned at the 5 ' end of SEQ ID NO:1;
Primer2 be SEQ ID NO:1 in 3 ' end reverse sequence.
The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 1 0mmol/LTris-Cl, (pH8.5), 1.5mmol/L MgCl 2, 200 μ mol/L dNTP, 10pmol primer, the Taq archaeal dna polymerase of 1U (Clontech company product).Go up by 25 cycles of following conditioned response at PE9600 type DNA thermal cycler (Perkin-Elmer company): 94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of β-actin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows that the dna sequence dna of PCR product and the 1-2658bp shown in the SEQ ID NO:1 are identical.Embodiment 4:Northern blotting is analyzed clathrin 76 expression of gene:
Extract total RNA[Anal.Biochem 1987,162,156-159 with single stage method].This method comprises acid guanidine thiocyanate phenol-chloroform extracting.Promptly use 4M guanidinium isothiocyanate-25mM Trisodium Citrate, 0.2M sodium acetate (pH4.0) carries out homogenate to tissue, adds the phenol of 1 times of volume and the chloroform-primary isoamyl alcohol (49: 1) of 1/5 volume, and is centrifugal after mixing.The sucking-off aqueous phase layer adds Virahol (0.8 volume) and with the centrifugal RNA precipitation that obtains of mixture.With RNA precipitation 70% washing with alcohol that obtains, dry and soluble in water.With 20 μ g RNA, on 1.2% sepharose that contains 20mM 3-(N-morpholino) propanesulfonic acid (pH7.O)-5mM sodium acetate-1mM EDTA-2.2M formaldehyde, carry out electrophoresis.Be transferred on the nitrocellulose filter then.With α- 32P dATP prepares by random priming 32The dna probe of P-mark.Used dna probe is clathrin 76 coding region sequences (251bp to 2326bp) of pcr amplification shown in Figure 1.Probe (about 2 * 10 with the 32P-mark 6Cpm/ml) spend the night in 42 ℃ of hybridization in a solution with the nitrocellulose filter that has shifted RNA, this solution comprises 50% methane amide-25mM KH 2PO 4(pH7.4)-5 * SSC-5 * Denhardt ' s solution and 200 μ g/ml salmon sperm DNAs.After the hybridization, filter membrane is washed 30min in 55 ℃ in 1 * SSC-0.1%SDS.Then, analyze with quantitative with Phosphor Imager.Embodiment 5: vivoexpression, separation and the purifying of reorganization clathrin 76
According to SEQ ID NO:1 and coding region sequence shown in Figure 1, design a pair of specificity amplification primer, sequence is as follows:
Primer3:5’-CCCCATATGATGCTGGCACAGAATTTAATGCTA-3’(Seq?ID?No:5)
Primer4:5’-CATGGATCCTCACAGTTCCTCCTCCGTTCGCAG-3’(Seq?ID?No:6)
5 ' end of these two sections primers contains Nde I and BamH I restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, Nde I and BamH I restriction enzyme site are corresponding to expression vector plasmid pET 28b (+) (Novagen company product, Cat.No.69865.3) the selectivity restriction enzyme site on.With the pBS-0367f12 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0367f12 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantage polymerase Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 60 ℃ of 30s, 68 ℃ of 2 min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with Nde I and BamH I, reclaim big fragment respectively, and connect with the T4 ligase enzyme.Connect product and transform, after the dull and stereotyped overnight incubation of the LB that contains kantlex (final concentration 30 μ g/ml), use the colony polymerase chain reaction (PCR) method screening positive clone, and check order with the big enterobacterial DH5 of Calcium Chloride Method α.Select the correct positive colony of sequence (pET-0367f12) with Calcium Chloride Method with recombinant plasmid transformed e. coli bl21 (DE3) plySs (Novagen company product).In the LB liquid nutrient medium that contains kantlex (final concentration 30 μ g/ml), host bacterium BL21 (pET-0367f12) is cultured to logarithmic phase at 37 ℃, adds IPTG to final concentration 1mmol/L, continues to cultivate 5 hours.Centrifugal collection thalline, through the broken bacterium of ultrasonic wave, centrifugal collection supernatant with carrying out chromatography with 6 Histidines (6His-Tag) bonded affinity column His.Bind Quick Cartridge (Novagen company product), has obtained the target protein clathrin 76 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 74KDa place.This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end hold 15 amino-acid residues identical with the N-shown in the SEQ ID NO:2 as a result.Embodiment 6 anti-clathrin 76 production of antibodies
Synthesize following clathrin 76 specific polypeptide with Peptide synthesizer (PE company product):
NH2-Met-Leu-Ala-Gln-Asn-Leu-Met-Leu-Lys-Arg-Phe-Ser-Asp-Ala-Trp-COOH(SEQ?ID?NO:7)。Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, et al.Immunochemistry, 1969; 6:43.Add the complete Freund's adjuvant immunizing rabbit with the above-mentioned hemocyanin polypeptide complex of 4mg, add the incomplete Freund's adjuvant booster immunization once with the hemocyanin polypeptide complex again after 15 days.Employing is done the titre that ELISA measures antibody in the rabbit anteserum through the titer plate of 15 μ g/ml bovine serum albumin polypeptide complex bag quilts.From the rabbit anteserum of antibody positive, separate total IgG with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.Immuno-precipitation proof antibody purified can combine with clathrin 76 specifically.Embodiment 7: polynucleotide passage of the present invention is as the application of hybridization probe
Picking out suitable oligonucleotide fragment from polynucleotide of the present invention is of use in many ways as hybridization probe, as can whether containing polynucleotide sequence of the present invention and detect the homologous polynucleotide sequence to identify it with the healthy tissues of different sources or the genome or the hybridization of cDNA library of pathological tissue with this probe, whether further also available this probe in detecting polynucleotide sequence of the present invention or the expression of its homologous polynucleotide sequence in healthy tissues or pathological tissue cell be unusual.
The purpose of present embodiment is to pick out suitable oligonucleotide fragment as hybridization probe from polynucleotide SEQ ID NO:1 of the present invention, and identifies in some tissues whether contain polynucleotide sequence of the present invention or its homologous polynucleotide sequence with the filter hybridization method.The filter hybridization method comprises dot blotting, Southern blotting, Northern blotting and copy method etc., and they all are that polynucleotide sample to be measured is fixed on use essentially identical step crossover in back on the filter membrane.These identical steps are: the filter membrane of having fixed sample at first carries out prehybridization with the hybridization buffer that does not contain probe, so that nonspecific combining site suppressed by vector of sample and synthetic polymer institute are saturated on the filter membrane.Prehybridization solution is contained the hybridization buffer replacement of label probe then, and insulation makes probe and target nucleic acid hybridization.After the hybridization step, the probe in the hybridization is not removed by a series of film steps of washing.Present embodiment utilizes the film condition of washing (as than low salt concn and higher temperature) of higher-strength, so that hybrid context reduces and only keep the signal of high specificity.The probe that present embodiment is selected for use comprises two classes: first kind probe be fully with the identical or complementary oligonucleotide fragment of polynucleotide SEQ ID NO:1 of the present invention; The second class probe is part and the identical or complementary oligonucleotide fragment of polynucleotide SEQ ID NO:1 of the present invention.Present embodiment selects for use dot blotting that sample is fixed on the filter membrane, higher-strength wash under the film condition, the hybridization specificity of first kind probe and sample is the strongest and kept.One, probe selects for use
From polynucleotide SEQ ID NO:1 of the present invention, select oligonucleotide fragment as hybridization probe, the several aspects that should follow following principle and need to consider: 1, the probe size preferable range is a 18-50 Nucleotide; 2, GC content is 30%-70%, and surpassing then, non-specific hybridization increases; 3, probe interior should not have complementary region; 4, what meet above condition can be used as the primary election probe, further do the computer sequential analysis then, comprise this primary election probe is carried out homology relatively with its source sequence area (being SEQ ID NO:1) and other known genome sequence and complementary district thereof respectively, if with the homology in non-target molecule zone greater than 85% or there are 15 continuous bases of surpassing identical, then this primary election probe is general just should not use; 5, whether the primary election probe finally is chosen to be the probe of actual application value also should further be determined by experiment.Select and synthetic following two probes after finishing the analysis of above each side:
Probe 1 (probe1) belongs to first kind probe, and complete homology of gene fragment or the complementation (41Nt) of SEQ ID NO:1:
5’-TGCTGGCACAGAATTTAATGCTAAAGAGGTTTTCTGATGCT-3’(SEQ?ID?NO:8)
Probe 2 (probe2) belongs to the second class probe, is equivalent to gene fragment or its complementary segmental replacement mutant nucleotide sequence (41Nt) of SEQ ID NO:1:
5’-TGCTGGCACAGAATTTAATGCTAAAGAGGTTTTCTGATGCT-3’(SEQ?ID?NO:9)
Other unlisted common agents and the compound method thereof relevant with following concrete experimental procedure please refer to document: DNA PROBES G.H.Keller; M.M.Manak; Stockton Press, 1989 (USA) and molecular cloning laboratory manual books more commonly used are as works such as " molecular cloning experiment guide " (second edition in 1998) [U.S.] Sa nurse Brooker, Science Press.
Specimen preparation: 1, from fresh or frozen tissue, extract DNA
Step: 1) the fresh or fresh normal liver tissue that thaws is put into the plate that is immersed on ice and fills the slow liquid (PBS) of phosphoric acid salt.With scissors or scalpel tissue is cut into small pieces.Should keep organizing moistening in the operation.2) with 1000g centrifugal chopper tissue 10 minutes.3) with cooled homogenate damping fluid (0.25mol/L sucrose; 25mmol/LTris-HCl, pH7.5; 25mmol/LnaCl; 25mmol/L MgCl 2) precipitation that suspends (approximately 10ml/g).4) 4 ℃ with electric homogenizer homogenate tissue suspension at full speed, until tissue by broken fully.5) 1000g is centrifugal 10 minutes.6) with re-suspended cell precipitation (the initial tissue sample of every 0.1g adds 1-5ml), centrifugal 10 minutes again with 1000g.7) with the resuspended precipitation of lysis buffer (the initial tissue sample of every 0.1g adds 1ml), connect following phenol extraction process then.2, the phenol extraction process of DNA
Step: 1) wash cell, centrifugal 10 minutes of 1000g with the cold PBS of 1-10ml.2) with the sedimentary cell (1 * 10 of cold cell pyrolysis liquid resuspension 8The minimum of applications 100 μ l lysis buffers of cell/ml).3) adding SDS is 1% to final concentration, if before re-suspended cell SDS is directly joined in the cell precipitation, cell may form big agglomerate and be difficult to fragmentation, and the overall yield that reduces.This point is in extracting＞10 7Especially severe during cell.4) add Proteinase K to final concentration 200 μ g/ml.5) 50 ℃ of insulation reaction 1 hour or 37C gently jolting spend the night.6) use equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, in little centrifuge tube centrifugal 10 minutes.Two corresponding clear separation, otherwise carry out centrifugal again.7) water is transferred to new pipe.8) use the equal-volume chloroform: primary isoamyl alcohol (24: 1) extracting, centrifugal 10 minutes.9) water that will contain DNA is transferred to new pipe.Carry out purifying and the ethanol sedimentation of DNA then.3, the purifying of DNA and ethanol sedimentation
Step: 1) 1/10 volume 2mol/L sodium-acetate and 2 times of cold 100% ethanol of volume are added in the dna solution mixing.-20 ℃ of placements 1 hour or to spending the night.2) centrifugal 10 minutes.3) careful sucking-off or pour out ethanol.4) with 70% cold ethanol 500 μ l washing precipitations, centrifugal 5 minutes.5) careful sucking-off or pour out ethanol.With the cold washing with alcohol precipitation of 500 μ l, centrifugal 5 minutes.6) careful sucking-off or pour out ethanol is inverted on thieving paper then residual ethanol is flow to end.Dry air 10-15 minute, so that the volatilization of surperficial ethanol.Note not making the precipitation complete drying, otherwise dissolve again than difficulty.7) with small volume TE or the resuspended DNA precipitation of water.The low speed vortex vibrates or uses the dropper pressure-vaccum, increases TE gradually simultaneously, is mixed to DNA and fully dissolves, every 1-5 * 10 6Cell extracted approximately adds 1 μ l.
Below the 8-13 step only be used for must removing when depolluting, otherwise can directly carry out the 14th step.8) RNA enzyme A is added in the dna solution, final concentration is 100 μ g/ml, and 37 ℃ are incubated 30 minutes.9) add SDS and Proteinase K, final concentration is respectively 0.5% and 100 μ g/ml.37 ℃ are incubated 30 minutes.10) use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extractive reaction liquid, centrifugal 10 minutes.11) carefully shift out water, use isopyknic chloroform: primary isoamyl alcohol (24: 1) extracting again, centrifugal 10 minutes.12) carefully shift out water, add 1/10 volume 2mol/L sodium-acetate and the cold ethanol of 2.5 volumes, mixing put-20 ℃ 1 hour.13) with 70% ethanol and 100% washing with alcohol precipitation, dry air, resuspended nucleic acid, process is with the 3-6 step.14) measure A 260And A 280To detect purity and the productive rate of DNA.15) deposit in-20 ℃ after the packing.The preparation of sample film:
1) get 4 * 2 suitably nitrocellulose filters (NC film) of size, mark point sample position and sample number thereon gently with pencil, each probe needs two NC films, so that wash film with high strength condition and strength condition respectively in the experimental procedure of back.
2) draw and contrast respectively 15 microlitres, put on the sample film, dry at room temperature.
3) place infiltration that 0.1mol/LNaOH is arranged, last 5 minute of filter paper (twice) of 1.5mol/LNaCl, drying to place to soak into has 0.5mol/L Tris-HCl (pH7.0), last 5 minute of filter paper (twice) of 3mol/LNaCl, dries.
4) be sandwiched in the clean filter paper, wrap, 60-80 ℃ of vacuum-drying 2 hours with aluminium foil.
The mark of probe
1) 3 μ lProbe (0.1OD/10 μ l) add 2 μ lKinase damping fluids, 8-10 μ Ci γ- 32P-dATP+2UKinase is to add to final volume 20 μ l.
2) 37 ℃ are incubated 2 hours.
3) add the tetrabromophenol sulfonphthalein indicator (BPB) of 1/5 volume.
4) cross Sephadex G-50 post.
5) to having 32Before washing out, P-Probe begins to collect first peak (available Monitor monitoring).
6) 5/pipe, collect the 10-15 pipe.
7) monitor isotopic weight with liquid glimmer instrument
8) merge and to be required preparation behind the collection liquid of first peak 32P-Probe (second peak for free γ- 32P-dATP).
Prehybridization
The sample film is placed plastics bag, and adding 3-10mg prehybridization solution (10 * Denhardt ' s; 6 * SSC, 0.1mg/mlCT DNA (calf thymus DNA).), seal sack after, 68 ℃ of water-baths were shaken 2 hours.
Hybridization
Plastics bag is cut off one jiao, adds the probe prepare, seal sack after, 42 ℃ of water-baths are shaken and are spent the night.
Wash film: high strength is washed film:
1) good sample film has been hybridized in taking-up.
2) 2 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃.
3) 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃.
4) 0.1 * SSC among the 0.1%SDS, washes 30 minutes (2 times) for 55 ℃, and room temperature is dried.Low strength is washed film:
1) good sample film has been hybridized in taking-up.
2) 2 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 37 ℃.
3) 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 37 ℃.
4) 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃, and room temperature is dried.
X-light autography:
-70 ℃, X-light autography (the compressing tablet time decides according to hybridization spot radioactivity is strong and weak).
Experimental result:
Adopt low strength to wash the hybrid experiment that the film condition is carried out, more than two strong and weak not obviously differences of probe hybridization spot radioactivity; And adopting high strength to wash the hybrid experiment that the film condition is carried out, the hybridization spot radioactive intensity of probe 1 obviously is better than the radioactive intensity of another probe hybridization spot.Thereby available probe 1 is qualitative and analyze existence and the differential expression of polynucleotide of the present invention in different tissues quantitatively.Embodiment 8 DNA Microarray
Gene chip or gene micromatrix (DNA Microarray) are that present many National Laboratories and big drugmaker are all in the new technology of setting about developing and developing, it is meant and is arranged in a large amount of target fragments on the carriers such as glass, silicon in an orderly manner, to high-density, carry out the comparison and the analysis of data then with fluoroscopic examination and computer software, to reach purpose quick, efficient, that bioinformation is analyzed on high-throughput ground.Polynucleotide of the present invention can be used as target DNA and are used for biochip technology and are used for high-throughput and study new gene function; Seek and the new gene of the screening tissue specificity new gene of disease-related such as tumour particularly; The diagnosis of disease is as heredopathia.The existing in the literature multiple report of its concrete grammar step is as consulting document DeRisi, J.L., Lyer, V.﹠amp; Brown, P.O. (1997) Science278,680-686. and document Helle, R.A., Schema, M., Chai, A., Shalom, D., (1997) PNAS 94:2150-2155. () point sample
Various full-length cDNA amounts to 4000 polynucleotide sequences as target DNA, comprising polynucleotide of the present invention.They are increased by PCR respectively, behind the purifying gained amplified production its concentration is transferred to about 500ng/ul, (available from U.S. Cartesian company) puts on glass medium with Cartesian 7500 point sample instruments, and distance between points is 280 μ m.Slide behind the point sample is carried out hydration, drying, places UV-crosslinked instrument crosslinked, and the wash-out after drying is fixed on DNA and is prepared into chip on the sheet glass.The existing in the literature multiple report of its concrete grammar step, the point sample post-processing step of present embodiment is:
1. hydration 4 hours in the wet environment;
2.0.2%SDS washed 1 minute;
3.ddH 2The O washed twice, each 1 minute;
4.NaBH 4Sealed 5 minutes;
5.95 in ℃ water 2 minutes;
6.0.2%SDS washed 1 minute;
7.ddH 2Twice of O flushing;
8. airing, 25 ℃ to be stored in the dark place standby.(2) probe mark
With the single stage method total mRNA of extracting from normal hepatocytes and liver cancer respectively, and with Oligotex mRNA Midi Kit (available from QiaGen company) purified mRNA, by reverse transcription respectively with fluorescent reagent Cy3dUTP (5-Amino-propargyl-2 '-deoxyuridine 5 '-triphate coupled to Cy3 fluorescent dye, available from Amersham Phamacia Biotech company) mRNA of mark normal liver tissue, with fluorescent reagent Cy5dUTP (5-Amino-propargyl-2 '-deoxyuridine 5 '-triphate coupled to Cy5fluorescent dye, available from Amersham Phamacia Biotech company) the labelling liver cancer tissue mRNA, prepare probe after purified.Concrete steps reference and method are seen: Schena, M., Shalon, D., Heller, R. (1996) Proc.Natl.Acad.Sci.USA.Vol.93:10614-10619.Schena, M., Shalon, Dari., Davis, R.W. (1995) Science.270. (20): 467-480. (three) hybridization
Respectively will be from the probe of above two kinds of tissues with chip at UniHyb TMHybridized 16 hours in HybridizationSolution (available from the TeleChem company) hybridization solution, room temperature washings (1 * SSC, 0.2%SDS) the washing back is scanned with ScanArray 3000 scanners (available from U.S. General Scanning company), the image of scanning carries out data analysis with Imagene software (U.S. Biodiscovery company) to be handled, calculate the Cy3/Cy5 ratio of each point, this ratio is considered to express discrepant gene less than 0.5 greater than 2 point.Experimental result shows, Cy3signal=11135.64 (getting the mean value of four experiments), Cy5signal=13857.52 (getting the mean value of four experiments), Cy3/Cy5=0.80358, the expression no significant difference of polynucleotide of the present invention in above two kinds of tissues.
Sequence table
(1) general information:
(ⅱ) denomination of invention: clathrin 76 and encoding sequence thereof
(ⅲ) sequence number: 9
(2) information of SEQ ID NO:l:
(ⅰ) sequence signature:
(A) length: 2658bp
(B) type: nucleic acid
(C) chain: two strands
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
( ⅹⅰ ) ：SEQ ID NO:1: 1 GGATAAAGGTGTATTTATTGCTTATGATGATGATAAGGTGTACACTTATGTCTTTCACAA 61 GGACACTATACAAGGAGCCAAGGTTATTTTGGCTGGTAGCACCAAAGTTCCTTTTGCTCA121 TAAACCTTTGCTGCTATATAATGGAGAGCTGACCTGCCAAACACAGAGTGGAAAAGTAAA181 CAACATCTACCTTAGCACCCATGGCTTTCTCAGCAACTTAAAAGATACGGGGCCTGACGA241 ACTGAGACCAATGCTGGCACAGAATTTAATGCTAAAGAGGTTTTCTGATGCTTGGGAAAT301 GTGCAGGATTCTGAATGATGAGGCTGCCTGGAATGAGTTGGCCAGAGCTTGTCTACATCA361 CATGGAAGTGGAGTTTGCAATCCGTGTTTATCGGAGAATTGGAAATGTAGGCATAGTGAT421 GTCCTTGGAACAAATAAAGGGAATAGAGGACTACAATCTTTTGGCAGGACACCTTGCCAT481 GTTTACCAACGATTATAACCTGGCTCAGGACTTGTACCTTGCATCCAGCTGTCCTATTGC541 TGCCCTGGAGATGAGAAGGGATTTACAGCATTGGGACAGTGCTCTACAGCTGGCAAAGCA601 TTTGGCCCCAGACCAGATACCTTTTATATCAAAAGAATATGCTATTCAGCTTGAATTCGC661 GGGTGATTATGTAAATGCTTTGGCTCATTATGAGAAAGGAATAACAGGTGATAATAAGGA721 ACATGATGAAGCTTGTCTGGCTGGAGTGGCCCAGATGTCCATAAGAATGGGAGACATACG781 TCGAGGGGTTAACCAAGCCCTCAAGCATCCCAGCAGGGTCCTTAAAAGAGACTGTGGAGC841 CATATTGGAGAATATGAAGCAATTTTCAGAAGCGGCCCAACTGTATGAAAAAGGTCTCTA901 CTACGATAAAGCAGCATCTGTTTACATCCGCTCTAAGAATTGGGCAAAAGTTGGTGATCT 961 TCTGCCCCACGTTTCTTCTCCTAAGATCCATTTGCAGTATGCCAAAGCCAAGGAAGCAGA1021 TGGAAGATACAAAGAAGCTGTTGTAGCTTATGAAAATGCAAAACAGTGGCAAAGTGTAAT1081 CCGCATCTATCTGGATCACCTCAATAATCCTGAAAAAGCTGTCAATATTGTTAGAGAGAC1141 CCAGTCTCTGGATGGAGCCAAAATGGTAGCCAGGTTTTTTCTACAGCTTGGTGACTATGG1201 GTCTGCCATCCAGTTTCTTGTCATGTCCAAATGCAACAATGAAGCTTTCACACTGGCTCA1261 GCAACACAACAAAATGGAAATCTATGCAGATATTATTGGTTCTGAAGACACTACTAATGA1321 AGACTATCAAAGCATTGCCTTATACTTTGAAGGAGAAAAGAGATATCTTCAGGCTGGAAA1381 ATTCTTCTTGCTGTGTGGCCAATATTCACGAGCACTTAAACACTTCCTGAAATGCCCAAG1441 CTCGGAAGATAATGTGGCAATAGAAATGGCAATTGAAACTGTTGGTCAGGCCAAAGATGA1501 ACTGCTGACCAATCAGCTGATAGACCATCTCCTGGGGGAGAACGATGGCATGCCTAAGGA1561 TGCCAAGTACCTGTTCCGCTTGTACATGGCTCTGAAGCAATACCGAGAAGCTGCCCAGAC1621 TGCCATCATCATTGCCAGAGAAGAGCAGTCTGCAGGCAACTACCGGAATGCACACGATGT1681 TCTCTTCAGTATGTATGCAGAACTGAAATCCCAGAAGATCAAAATTCCCTCCGAGATGGC1741 CACCAACCTCATGATTCTGCACAGCTATATACTAGTAAAGATTCATGTTAAAAATGGAGA1801 TCACATGAAAGGGGCTCGCATGCTCATTCGGGTGGCCAACAACATCAGCAAATTTCCATC1861 ACACATTGTACCCATCCTGACGTCAACTGTGATTGAGTGTCACAGGGCAGGCCTGAAGAA1921 CTCTGCTTTCAGCTTCGCAGCTATGTTGATGAGGCCTGAATACCGCAGCAAAATAGATGC1981 CAAATACAAAAAGAAGATCGAGGGAATGGTCAGGAGACCCGATATATCTGAGATAGAAGA2041 GGCCACGACTCCATGTCCATTCTGCAAATTTCTTCTCCCAGAGTGTGAACTCCTCTGTCC2101 TGGATGTAAAAACAGTATCCCATATTGCATTGCAACAGGTCGACACATGTTGAAAGATGA2161 CTGGACGGTGTGTCCACATTGTGACTTCCCTGCTCTATACTCAGAATTGAAGATCATGCT2221 AAACACTGAAAGCACATGTCCTATGTGTTCAGAAAGATTAAACGCTGCTCAGCTGAAAAA2281 GATTTCAGACTGTACCCAGTACCTGCGAACGGAGGAGGAACTGTGATTGGCACGTGCAGA2341 TACAATGCTCCTGAGAAGACAGCATTTTCCACAGGAGGCTGTTTCCTCCCCTGGTGGATT2401 TAAGAGACGGTCCTTTCTGGATACAGAGAAATGAAACAACGGTGACCTCTCCAGGTCGGC2461 ACTTTCCACTTCTGTACGGTGGCAAAACGATGACATGTAACCTTGCTGTTTATTGTACTT2521 TGTATATTATTTCCTCTTCAAAGTCTTTCTTACACACTCTATCCTCTGCACTGTTAATAG2581 TAACCTATGACATAATTGTAAATATTCAGCTTTTTGCTAACTTTTGTATTTTGAAAAACT2641 TTAAAATAAAATTGTTGA
(3) information of SEQ ID NO:2:
(ⅰ) sequence signature:
(A) length: 691 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: polypeptide
( ⅹⅰ ) ：SEQ ID NO:2: 1 Met Leu Ala Gln Asn Leu Met Leu Lys Arg Phe Ser Asp Ala Trp 16 Glu Met Cys Arg Ile Leu Asn Asp Glu Ala Ala Trp Asn Glu Leu 31 Ala Arg Ala Cys Leu His His Met Glu Val Glu Phe Ala Ile Arg 46 Val Tyr Arg Arg Ile Gly Asn Val Gly Ile Val Met Ser Leu Glu 61 Gln Ile Lys Gly Ile Glu Asp Tyr Asn Leu Leu Ala Gly His Leu 76 Ala Met Phe Thr Asn Asp Tyr Asn keu Ala Gln Asp Leu Tyr Leu 91 Ala Ser Ser Cys Pro Ile Ala Ala Leu Glu Met Arg Arg Asp Leu106 Gln His Trp Asp Ser Ala Leu Gln Leu Ala Lys His Leu Ala Pro121 Asp Gln Ile Pro Phe Ile Ser Lys Glu Tyr Ala Ile Gln Leu Glu136 Phe Ala Gly Asp Tyr Val Asn Ala Leu Ala His Tyr Glu Lys Gly151 Ile Thr Gly Asp Asn Lys Glu His Asp Glu Ala Cys Leu Ala Gly166 Val Ala Gln Met Ser Ile Arg Met Gly Asp Ile Arg Arg Gly Val181 Asn Gln Ala Leu Lys His Pro Ser Arg Val Leu Lys Arg Asp Cys196 Gly Ala Ile Leu Glu Asn Met Lys Gln Phe Ser Glu Ala Ala Gln211 Leu Tyr Glu Lys Gly Leu Tyr Tyr Asp Lys Ala Ala Ser Val Tyr226 Ile Arg Ser Lys Asn Trp Ala Lys Val Gly Asp Leu Leu Pro His241 Val Ser Ser Pro Lys Ile His Leu Gln Tyr Ala Lys Ala Lys Glu256 Ala Asp Gly Arg Tyr Lys Glu Ala Val Val Ala Tyr Glu Asn Ala271 Lys Gln Trp Gln Ser Val Ile Arg Ile Tyr Leu Asp His Leu Asn286 Asn Pro Glu Lys Ala Val Asn Ile Val Arg Glu Thr Gln Ser Leu301 Asp Gly Ala Lys Met Val Ala Arg Phe Phe Leu Gln Leu Gly Asp316 Tyr Gly Ser Ala Ile Gln Phe Leu Val Met Ser Lys Cys Asn Asn331 Glu Ala Phe Thr Leu Ala Gln Gln His Asn Lys Met Glu Ile Tyr346 Ala Asp Ile Ile Gly Ser Glu Asp Thr Thr Asn Glu Asp Tyr Gln361 Ser Ile Ala Leu Tyr Phe Glu Gly Glu Lys Arg Tyr Leu Gln Ala376 Gly Lys Phe Phe Leu Leu Cys Gly Gln Tyr Ser Arg Ala Leu Lys391 His Phe Leu Lys Cys Pro Ser Ser Glu Asp Asn Val Ala Ile Glu406 Met Ala Ile Glu Thr Val Gly Gln Ala Lys Asp Glu Leu Leu Thr421 Asn Gln Leu Ile Asp His Leu Leu Gly Glu Asn Asp Gly Met Pro436 Lys Asp Ala Lys Tyr Leu Phe Arg Leu Tyr Met Ala Leu Lys Gln451 Tyr Arg Glu Ala Ala Gln Thr Ala Ile Ile Ile Ala Arg Glu Glu466 Gln Ser Ala Gly Asn Tyr Arg Asn Ala His Asp Val Leu Phe Ser481 Met Tyr Ala Glu Leu Lys Ser Gln Lys Ile Lys Ile Pro Ser Glu496 Met Ala Thr Asn Leu Met Ile Leu His Ser Tyr Ile Leu Val Lys511 Ile His Val Lys Asn Gly Asp His Met Lys Gly Ala Arg Met Leu526 Ile Arg Val Ala Asn Asn Ile Ser Lys Phe Pro Ser His Ile Val541 Pro Ile Leu Thr Ser Thr Val Ile Glu Cys His Arg Ala Gly Leu556 Lys Asn Ser Ala Phe Ser Phe Ala Ala Met Leu Met Arg Pro Glu571 Tyr Arg Ser Lys Ile Asp Ala Lys Tyr Lys Lys Lys Ile Glu Gly586 Met Val Arg Arg Pro Asp Ile Ser Glu Ile Glu Glu Ala Thr Thr601 Pro Cys Pro Phe Cys Lys Phe Leu Leu Pro Glu Cys Glu Leu Leu616 Cys Pro Gly Cys Lys Asn Ser lle Pro Tyr Cys Ile Ala Thr Gly631 Arg His Met Leu Lys Asp Asp Trp Thr Val Cys Pro His Cys Asp646 Phe Pro Ala Leu Tyr Ser Glu Leu Lys Ile Met Leu Asn Thr Glu661 Ser Thr Cys Pro Met Cys Ser Glu Arg Leu Asn Ala Ala Gln Leu676 Lys Lys Ile Ser Asp Cys Thr Gln Tyr Leu Arg Thr Glu Glu Glu691 Leu
(4) information of SEQ ID NO:3
(ⅰ) sequence signature
(A) length: 24 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:3:
GGATAAAGGTGTATTTATTGCTTA 24
(5) information of SEQ ID NO:4
(ⅰ) sequence signature
(A) length: 24 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:4:
TCAACAATTTTATTTTAAAGTTTT 24
(6) information of SEQ ID NO:5
(ⅰ) sequence signature
(A) length: 33 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:5:CCCCATATGATGCTGGCACAGAATTTAATGCTA 33
(7) information of SEQ ID NO:6
(ⅰ) sequence signature
(A) length: 33 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:6:CATGGATCCTCACAGTTCCTCCTCCGTTCGCAG 33
(8) information of SEQ ID NO:7:
(ⅰ) sequence signature:
(A) length: 15 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: polypeptide
(ⅹ ⅰ) sequence description: SEQ ID NO:7:
Met-Leu-Ala-Gln-Asn-Leu-Met-Leu-Lys-Arg-Phe-Ser-Asp-Ala-Trp 15
(9) information of SEQ ID NO:8
(ⅰ) sequence signature
(A) length: 41 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:8:TGCTGGCACAGAATTTAATGCTAAAGAGGTTTTCTGATGCT 41
(10) information of SEQ ID NO:9
(ⅰ) sequence signature
(A) length: 41 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:9:TGCTGGCACAGAATTTAATGCTAAAGAGGTTTTCTGATGCT 41

Claims (18)

1, a kind of isolated polypeptide-clathrin 76 is characterized in that it includes: the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2 or active fragments, analogue or the derivative of its polypeptide.
2, polypeptide as claimed in claim 1, the aminoacid sequence that it is characterized in that described polypeptide, analogue or derivative has the homogeny with the aminoacid sequence at least 95% shown in the SEQ ID NO:2.
3, polypeptide as claimed in claim 2 is characterized in that it comprises the polypeptide with the aminoacid sequence shown in the SEQ ID NO:2.
4, a kind of isolating polynucleotide, it is characterized in that described polynucleotide comprise be selected from down the group in a kind of:
(a) coding has the polynucleotide of the polypeptide of aminoacid sequence shown in the SEQ ID NO:2 or its fragment, analogue, derivative;
(b) with polynucleotide (a) complementary polynucleotide; Or
(c) with (a) or the polynucleotide of at least 70% homogeny (b) are arranged.
5, polynucleotide as claimed in claim 4 is characterized in that described polynucleotide comprise the polynucleotide that coding has aminoacid sequence shown in the SEQ IDNO:2.
6, polynucleotide as claimed in claim 4, the sequence that it is characterized in that described polynucleotide include the sequence of 1-2658 position among the sequence of 251-2326 position among the SEQ ID NO:1 or the SEQ ID NO:1.
7, a kind of recombinant vectors that contains exogenous polynucleotide is characterized in that it is the recombinant vectors that is formed by the described polynucleotide of arbitrary claim among the claim 4-6 and plasmid, virus or vehicle expression vector establishment.
8, a kind of genetically engineered host cell that contains exogenous polynucleotide is characterized in that it is to be selected from following a kind of host cell:
(a) host cell that transforms or transduce with the described recombinant vectors of claim 7; Or
(b) host cell that transforms or transduce with the described polynucleotide of the arbitrary claim among the claim 4-6.
9, a kind of preparation method with clathrin 76 active polypeptide is characterized in that described method comprises:
(a) expressing under clathrin 76 conditions, cultivate the described through engineering approaches host cell of claim 8;
(b) from culture, isolate and have clathrin 76 active polypeptide.
10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with clathrin 76 specificity bonded antibody.
11, the compound of an analoglike or adjusting polypeptide active or expression is characterized in that they are simulation, promotion, antagonism or the active compound that suppresses clathrin 76.
12, compound as claimed in claim 11 is characterized in that it is the polynucleotide sequence shown in the SEQ ID NO:1 or its segmental antisense sequences.
13, the described application of compound of a kind of claim 11, it is characterized in that described compound be used to regulate clathrin 76 in vivo, the method for external activity.
14, a kind of disease relevant or method of disease susceptibility of detecting with the described polypeptide of arbitrary claim among the claim 1-3, it is characterized in that it comprises the described polypeptide expression amount that detects, perhaps detect the activity of described polypeptide, perhaps detect and cause described expression of polypeptides amount or active unusual nucleotide diversity in the polynucleotide.
15, as the application of polypeptide as described in the arbitrary claim among the claim 1-3, it is characterized in that it is applied to screen the stand-in of clathrin 76, agonist, antagonist or inhibitor; Perhaps be used for the peptide finger print identification.
16, as the application of the described nucleic acid molecule of arbitrary claim among the claim 4-6, it is characterized in that it is used for nucleic acid amplification reaction as primer, perhaps be used for hybridization, perhaps be used to make gene chip or microarray as probe.
17,, it is characterized in that forming pharmaceutical composition with safe and effective dosage and pharmaceutically acceptable carrier as the relevant unusually disease of diagnosis or treatment and clathrin 76 with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11.
18, the described polypeptide of arbitrary claim, polynucleotide or the application of compound among the claim 1-6 and 11, it is characterized in that being used for the treatment of as malignant tumour with described polypeptide, polynucleotide or compound, hemopathy, the medicine of HIV infection and immunological disease and all kinds of inflammation.
CN00111507A 2000-01-26 2000-01-26 Polypeptide-clathrin 76 and polynucleotide for coding said polypeptide Pending CN1306976A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110869043A (en) * 2017-04-07 2020-03-06 奥列格·阿尔卡季耶维奇·科京 Specific peptide clathrin inhibitors

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110869043A (en) * 2017-04-07 2020-03-06 奥列格·阿尔卡季耶维奇·科京 Specific peptide clathrin inhibitors

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