CN1407102A - Polypeptide-aconitase-10.57 and polynucleotide for encoding it - Google Patents

Abstract

A polypeptide-aconitase-10.57, the polynucleotide for coding it, the process for preparing said polypeptide by DNA recombination, the application of said polypeptide in treating diseases such as cancer, HIV infection, etc, the antagonist of said polypeptide and its medical action, and the application of said polynucleotide are disclosed.

Description

The polynucleotide of one peptide species---aconitase-10.57 and this peptide species of coding

Technical field

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide---aconitase-10.57, 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.

Background technology

Aconitase is a kind of enzyme of studying more thoroughly, and it can be by the reversible isomerization reaction between citric acid in the cis-aconitic acid catalysis tricarboxylic acid cycle and the isocitric acid, and cis-aconitic acid is as a kind of intermediate product in the reflection process.Why aconitase can attract much attention is to have a multi-functional molecular structure because of it, can adapt to iron ion response regulation and control and some reactions similar and citric acid, isopropylmolic acid and single citric acid of catalysis of various functions such as mRNA translation or stability.

In eukaryote, exist the isozyme of breeding aconitase: a kind of being present in the mitochondrial matrix; Another kind is present in the tenuigenin.Aconitase under the active condition has sulphur-iron-based group of a 4Fe-4S, and its ligand is three cysteine residues.The member of cis-aconitic acid enzyme family comprises that also the element of iron ion response is conjugated protein; 3-isopropylmolic acid isopropylmolic acid dehydratase; Single aconitase and intestinal bacteria ybhJ albumen.

The aconitase of all different sourcess structurally all contains two sections conservative consensus sequence fragment: 1.[LIVM as follows]-x (2)-[GSACIVM]-x-[LIV]-[GTIV]-[STP]-C-x (0,1)-T-N-

[GSTANI]-x (4)-[LIVMA] halfcystine wherein combines with sulphur-iron center.2.G-x(2)-[LIVWPQ]-x(3)-[GAC]-C-[GSTAM]-[LIMPTA]-C-[LIMV]-

[GA] two halfcystines wherein combine with sulphur-iron center.The member of cis-aconitic acid enzyme family has discontinuous subunit group in many structures and the system, the structure of all molecules all depend on its several structural domains different arrangement modes [Megan J.G., Peter J.A.; John R.G. (1997) Trends Biochem.Sci.22 (1): 3-6].

Because aconitase-10.57 albumen plays 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 aconitase-10.57 albumen of more these processes of participation in this area always, particularly identify this proteic aminoacid sequence.The separation of new aconitase-10.57 protein coding gene 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.

Summary of the invention

An object of the present invention is to provide isolating new polypeptide---aconitase-10.57 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 coding aconitase-10.57.

Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain coding aconitase-10.57.

Another object of the present invention provides the method for production aconitase-10.57.

Another object of the present invention provides at polypeptide of the present invention---the antibody of aconitase-10.57.

Another object of the present invention has provided at polypeptide of the present invention---the simulated compound of aconitase-10.57, antagonist, agonist, inhibitor.

Another object of the present invention provides the method for the diagnoses and treatment disease relevant unusually with aconitase-10.57.

The present invention relates to a kind of isolated polypeptide, this polypeptide is the people source, and it comprises: have＜polypeptide or its examples of conservative variations, bioactive fragment or the derivative of 210〉2 aminoacid sequences.Preferably, this polypeptide is to have＜polypeptide of 210〉2 aminoacid sequences.

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＜polynucleotide of the polypeptide of 210〉2 aminoacid sequences;

(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: (a) have＜210〉1 in the sequence of 103-393 position; (b) have＜210〉1 in the sequence of 1-1523 position.

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 aconitase-10.57 protein-active, 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 aconitase-10.57 abnormal protein, 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 aconitase-10.57 disease 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 aconitase-10.57, 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 aconitase-10.57.

" antagonist " or " inhibition " be meant when combining with aconitase-10.57, a kind ofly seals or regulate the biologic activity of aconitase-10.57 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 aconitase-10.57.

" adjusting " is meant that the function of aconitase-10.57 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 aconitase-10.57.

" pure basically " 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 aconitase-10.57 of standard.Basically pure aconitase-10.57 can produce single master tape on the irreducibility polyacrylamide gel.The purity available amino end acid sequence of aconitase-10.57 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, sequence " C-T-G-A " can combine with complementary sequence " G-A-C-T ".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) Gene73:237-244) 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:

Also can be by the Cluster method or with the homogeny percentage (Hein J., (1990) Methods in emzumology183:625-645) 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 aconitase-10.57.

" 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 aconitase-10.57 " is meant that aconitase-10.57 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 aconitase-10.57 of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of aconitase-10.57 polypeptide can be used amino acid sequence analysis.

The invention provides a kind of new polypeptide---aconitase-10.57, it is basically by＜210〉aminoacid sequence shown in 2 forms.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 aconitase-10.57.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of aconitase of the present invention-10.57 or active polypeptide basically.The fragment of polypeptide of the present invention, derivative or analogue can be: (I) 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 by coding have＜polynucleotide of the polypeptide of 210〉2 aminoacid sequences form.Polynucleotide sequence of the present invention comprises＜210〉1 nucleotide sequence.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 1523 bases, its open reading frame 103-393 87 amino acid of having encoded.This polypeptide has the characteristic sequence of MOTIF, and deducibility goes out the 26S Proteasome Structure and Function that this aconitase-10.57 has the MOTIF 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＜210〉1 or the varient of degeneracy.As used herein, " varient of degeneracy " are meant that in the present invention coding has＜210〉2 protein or polypeptide, but with the differentiated nucleotide sequence of coding region sequence shown in＜210〉1.

The polynucleotide of the mature polypeptide of coding＜210〉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 and＜210〉and the mature polypeptide shown in 2 has identical biological function and activity.

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 aconitase-10.57.

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

The special polynucleotide sequence of coding aconitase-10.57 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 aconitase-10.57; (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 aconitase-10.57 genetic expression 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 aconitase-10.57 encoding sequence 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 aconitase-10.57 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 coding aconitase-10.57 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 P of lambda particles phage _LPromotor; 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 aconitase-10.57 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 aconitase-10.57 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention people aconitase-10.57, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;

(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.

Aconitase is a kind of enzyme of studying more thoroughly, and it can be by the reversible isomerization reaction between citric acid in the cis-aconitic acid catalysis tricarboxylic acid cycle and the isocitric acid, and cis-aconitic acid is as a kind of intermediate product in the reflection process.Why aconitase can attract much attention is to have a multi-functional molecular structure because of it, can adapt to iron ion response regulation and control and some reactions similar and citric acid, isopropylmolic acid and single citric acid of catalysis of various functions such as mRNA translation or stability.

The distinctive conserved sequence of aconitase is that its active motif of formation is necessary.The abnormal expression of this characteristic sequence will cause organism metabolic disorder, and then cause relevant disease.

The new polypeptide of the present invention has the homology and the similarity of height with aconitase on structure and function, and also contain above-mentioned conservative characteristic sequence template in the aminoacid sequence.The new polypeptide of the present invention has the physiologically active similar with aconitase.The abnormal expression of above-mentioned distinctive conserved sequence will cause the dysfunction that contains the new polypeptide of this motif of the present invention, thereby cause organism metabolic disorder, and then cause relevant disease.

These metabolic diseases include but not limited to: 1. amino acid metabolism defective disease: pku, tyrosine metabolic deficiency disease such as the high junket base acidaemia of newborn infant's one property crossed, subacute and the chronic tyrosinemia of acute tyrosine, albinism, the sulfur-containing amino acid metabolic defect, the tryptophan metabolism defective is sick as the heredity pellagra, the tryptophane mass formed by blood stasis, the branched chain amino acid metabolic defect, glycine metabolic defect such as glycinemia, hypersarcosinemia, the D-glyceric acidemia, front three aminuria disease, glycinuria and Glucoglycinuria disease, primary oxalic acid disease and oxaluria, proline(Pro) and oxyproline metabolic defect, glutamic acid metabolism defective disease, the metabolic defect of ornithine cycle, the Histidine metabolic defect, the Methionin metabolic defect, and sick 2. mucopolysaccharidosises of other amino acid metabolism defective and other marginality diseases: mucopolysaccharidosis I～VII type, mucopolysaccharidosis marginality disease such as similar rheumatism type mucopolysaccharidosis, sticking fat is stored up disease 3. purine and pyrimidine metabolic defective disease: purine metabolic disturbance is thunderous-and the Ni syndromes, xanthinuria, pyrimidine metabolic is unusual as orotic aciduria, 4. lipid metabolisms of adenosine deaminase defective are unusual: hyperlipoproteinemia, the familial hyperalphalipoproteinemia, familial alpha-lipoprotein blood deficiency disease, the familial tangier's disease, the familial Abetalipoproteinemia, familial hypo-beta-lipoproteinemia, familial LACT deficiency disease 5. carbohydrate metabolism defective diseases: congenital carbohydrate is digested and assimilated defective such as congenital lactose intolerance, the heredity fructose intolerance, monose metabolic defect such as galactosemia, the fructose metabolic deficiency, Glycogen Metabolism is sick as glycogen storage disease 6. organic acidemias: propionic acidemia, methylmalonic aciduria, isovaleric acidemia, associating carboxylase defective, glutaric acidemia I type

Comprehensively above-mentioned, the antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in the diagnosis and treatment of multiple disease, pku for example, mucopolysaccharidosis I～VII type, hyperlipoproteinemia, congenital lactose intolerance etc.

The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) aconitase-10.57.Agonist improves aconitase-10.57 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 aconitase-10.57 be cultivated with the aconitase-10.57 of mark.Measure the medicine raising then or check this interactional ability.

The antagonist of aconitase-10.57 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of aconitase-10.57 can combine and eliminate its function with aconitase-10.57, 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, aconitase-10.57 can be added during bioanalysis measures, determine to interactional influence between aconitase-10.57 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 aconitase-10.57 bonded peptide molecule obtains.During screening, generally tackle aconitase-10.57 molecule 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 aconitase-10.57 antigenic determinant.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 of polyclonal antibody available aconitase-10.57 direct injection immune animal (as rabbit, mouse, rat etc.) 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 aconitase-10.57 include but not limited to hybridoma technology (Kohlerand Milstein.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-aconitase-10.57.

The antibody of anti-aconitase-10.57 can be used in the immunohistochemistry technology, detects the aconitase-10.57 in the biopsy specimen.

With the also available labelled with radioisotope of aconitase-10.57 bonded monoclonal antibody, 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 aconitase-10.57 high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing aconitase-10.57 positive cells.

The disease that antibody among the present invention can be used for treating or prevention is relevant with aconitase-10.57.The antibody that gives suitable dosage can stimulate or block the generation or the activity of aconitase-10.57.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization aconitase-10.57 level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The aconitase that is detected in the test-10.57 level can and be used to the disease of diagnosing aconitase-10.57 to work with the importance of the aconitase that lays down a definition-10.57 in various diseases.

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 aconitase-10.57 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 aconitase-10.57 or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the aconitase-10.57 of expressing variation, to suppress endogenic aconitase-10.57 activity.For example, a kind of aconitase of variation-10.57 can be the aconitase-10.57 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 aconitase-10.57 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 aconitase-10.57 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding aconitase-10.57 is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding aconitase-10.57 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 aconitase-10.57 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 aconitase-10.57 can be used for the diagnosis with the relative disease of aconitase-10.57.The unconventionality expression of the expression that the polynucleotide of coding aconitase-10.57 can be used for detecting aconitase-10.57 aconitase-10.57 whether or under morbid state.As the dna sequence dna of the aconitase of encoding-10.57 can be used for biopsy specimen is hybridized to judge the expression situation of aconitase-10.57.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 aconitase-10.57 with the special primer of aconitase-10.57.

The sudden change that detects aconitase-10.57 gene also can be used for the disease of diagnosing aconitase-10.57 relevant.The form of aconitase-10.57 sudden change comprises that the point mutation compared with normal wild type aconitase-10.57 dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

Sequence of the present invention identifies it also is valuable to karyomit(e).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 15-35bp), sequence can be positioned on the karyomit(e) according to cDNA.Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.

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, Mendelian Inheritancein Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.

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.Aconitase-10.57 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 aconitase-10.57 will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

Description of drawings

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 an aconitase of the present invention-10.57 at the 39-71 amino acid sequence homology comparison diagram of totally 33 amino acid and MOTIF structural domain.The top sequence is an aconitase-10.57, and the below sequence is the MOTIF structural domain.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 aconitase-10.57.10.56kDa be proteinic molecular weight.The arrow indication is isolated protein band.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Embodiment 1: the clone of aconitase-10.57

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 ABI377 automatic sequencer (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 1009e12 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 1009e12 clone is 1523bp (as＜210〉1 shown in), from 103bp to 393bp the open reading frame (ORF) of a 264bp arranged, the new protein of encoding (as＜210〉2 shown in).We are with this clone's called after pBS-1009e12, encoded protein matter called after aconitase-10.57.Embodiment 2:cDNA clone's domain analyses

With the sequence and the encoded protein sequence thereof of aconitase of the present invention-10.57, with profilescan program (Basiclocal Alignment search tool) [Altschul, the SF et al.J.Mol.Biol.1990 among the GCG; 215:403-10], carry out domain analyses at databases such as prosite.Aconitase of the present invention-10.57 has homology with the MOTIF structural domain, and homology the results are shown in Fig. 1, and homology is 25%, must be divided into 8.31; Threshold value is 8.13.Embodiment 3: with the gene of RT-PCR method clones coding aconitase-10.57

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’-CATCCTGAGAACTGAAATTGATCGC-3’(<210>3)

Primer2：5’-ATAAAATTTTTGAATTTATGTTCAA-3’(<210>4)

Primer1 for to be positioned at＜the forward sequence that begins of 1bp of 210〉15 ' end;

Primer2 be＜210〉1 in 3 ' end reverse sequence.

The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/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 the dna sequence dna and＜210 of PCR product〉1-1523bp shown in 1 is identical.Embodiment 4:Northern blotting is analyzed aconitase-10.57 expression of gene:

Extract total RNA[Anal.Biochem1987,162,156-159 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 (pH7.0)-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 aconitase-10.57 coding region sequence (103bp to 393bp) 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 aconitase-10.57

According to＜210〉1 and coding region sequence shown in Figure 1, design a pair of specificity amplification primer, sequence is as follows:

Primer3：5’-CCCCATATGATGCTCTGTCACCTTCAAAGGATGG-3’(<210>5)

Primer4：5’-CCCAAGCTTCTTCAACATGCCGCTTCTGTTCTTC-3’(<210>6)

5 ' end of these two sections primers contains NheI and BamHI restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, NheI and BamHI 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-1009e12 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-1009e12 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 2min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with NheI and BamHI, 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-1009e12) 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-1009e12) 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 aconitase-10.57 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 10.56kDa 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 are identical with 15 amino-acid residues of N-end shown in＜210〉2 as a result.Embodiment 6 anti-aconitase-10.57 production of antibodies

Synthesize the specific polypeptide of following aconitase-10.57 with Peptide synthesizer (PE company product):

NH2-Met-Leu-Cys-His-Leu-Gln-Arg-Met-Val-Ser-Glu-Gln-Cys-His-Leu-COOH(<210>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 aconitase-10.57 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 from polynucleotide of the present invention＜210〉pick out suitable oligonucleotide fragment 1 as hybridization probe, and identify with the filter hybridization method whether some contain polynucleotide sequence of the present invention or its homologous polynucleotide sequence in organizing.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 polynucleotide of the present invention＜210 1 identical or complementary oligonucleotide fragment; The second class probe is part and polynucleotide of the present invention＜210〉1 identical or complementary oligonucleotide fragment.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 of the present invention＜210〉select 1 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 (promptly＜210〉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, with＜210〉1 complete homology of gene fragment or complementation (41Nt):

5’-TGCTTAGTTTTTCAATTATTTCTCCTTTCCCAGGAATTTTT-3’(<210>8)

Probe 2 (probe2) belongs to the second class probe, is equivalent to＜210〉1 gene fragment or its complementary segmental replacement mutant nucleotide sequence (41Nt):

5’-TGCTTAGTTTTTCAATTATTGCTCCTTTCCCAGGAATTTTT-3’(<210>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 phosphate buffered saline buffer (PBS).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 100ul lysis buffer 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 200ug/ml.5) 50 ℃ of insulation reaction 1 hour or 37 ℃ 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 500ul washing precipitation, centrifugal 5 minutes.5) careful sucking-off or pour out ethanol.With the cold washing with alcohol precipitation of 500ul, 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 1ul.

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 100ug/ml, and 37 ℃ are incubated 30 minutes.9) add SDS and Proteinase K, final concentration is respectively 0.5% and 100ug/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 lKina8e damping fluids, 8-10uCi 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.

Sequence table＜110〉Fudan University

Bode Gene Development Co., Ltd., Shanghai＜120〉peptide species---aconitase-10.57＜130〉1009e12＜160〉9＜170〉PatentIn version 3.1＜210〉1＜211〉1523＜212〉DNA＜213〉human＜220〉＜221〉CDS＜222, (103) .., (393)＜223〉＜400〉1caattagatt ttttttcccc acagtctcca tttcccactt actatctctt tcatctcact 60cctcccaatt aaatttattt tgcatactta gagtaacttt aa atg ctt agt ttt 114

Met?Leu?Ser?Phe

1tca?att?att?tct?cct?ttc?cca?gga?att?ttt?atg?atg?ttt?ttt?aat?atc 162Ser?Ile?Ile?Ser?Pro?Phe?Pro?Gly?Ile?Phe?Met?Met?Phe?Phe?Asn?Ile5 10 15 20act?aga?aga?caa?tct?aga?gca?cca?caa?atc?cca?gat?tgg?atc?aac?ctt 210Thr?Arg?Arg?Gln?Ser?Arg?Ala?Pro?6ln?Ile?Pro?Asp?Trp?Ile?Asn?Leu

25 30 35ggg?cct?tcc?tct?gtt?gtt?tta?ctg?gca?tta?ata?gaa?agc?agc?tct?aat 258Gly?Pro?Ser?Ser?Val?Val?Leu?Leu?Ala?Leu?Ile?Glu?Ser?Ser?Ser?Asn

40 45 50agt?caa?gtg?caa?agt?cag?ttt?gta?aat?gct?gaa?caa?ata?aca?gat?aag 306Ser?Gln?Val?Gln?Ser?Gln?Phe?Val?Asn?Ala?Glu?Gln?Ile?Thr?Asp?Lys

55 60 65act?att?gac?tgg?ctt?ctt?ggg?aaa?act?gtt?tgg?tca?gga?gag?aga?aaa 354Thr?Ile?Asp?Trp?Leu?Leu?Gly?Lys?Thr?Val?Trp?Ser?Gly?Glu?Arg?Lys

70 75 80ctg?ttg?aaa?aac?tgt?tgt?act?gat?gtc?acc?ggt?gat?tga?aggggtatct 403Leu?Leu?Lys?Asn?Cys?Cys?Thr?Asp?Val?Thr?Gly?Asp85 90 95ttaattggct?aatttgaaag?aaagtcacaa?aagaaaggca?tgaataacca?aaatcctggg 463atatttctga?aactcagtcg?aggtcagtag?atctgtctgg?gactacattt?tccatcccag 523ttcctaacaa?agtttcattt?tcttttcttt?attctctgat?gtaagagtta?acagtgaaat 583gaccaaaatc?ctgaaagcca?atggagcaac?aataaacata?ctcagataga?ttgcctcata 643aattctttca?agttagtttt?taaaagtaac?acatttttta?aaagtccact?ttggcaaaat 703tgataattta?atatctggtt?atcagcctct?ccaaaggatt?cctggaaaag?ttattttctt 763tttatcattt?aaaaatattc?catttacaaa?gtcactttac?atgaagcttt?tcagtgacat 823aactttctca?taaaatgtgg?tcttctgctt?taaaatccat?ttgatgtttt?ggagaaagta 883aattctccag?cagaattgtc?tcttgtgttt?attggtttta?aataacttgg?cttttctttg 943ctccacctcc?taaaacttgt?atttcacggt?ttttcttttg?gggataggtt?cagaattttc 1003acagctgtat?tcaggtaact?gaagattttg?tgtctccaga?acatcttgta?gagtcatttc 1063atttaacaca?ggaactgaga?cttttgaagg?aagaaatcaa?ttatgatgat?aaactacagg 1123ttaaaaatat?tttgtatcat?gcagtcaaag?aaatggtgag?agccttgaag?atacacgagg 1183atgaagtaga?ggatatggaa?gaaaattaag?tgtgatccag?tttgatattt?ttaggttgtt 1243gaactgggat?tacttaacct?tgaatgatga?tatgtatgca?cactgacttt?aagcttcata 1303aaaccatcag?tgccaagaaa?ttctctttgt?agtaattact?tgttactgac?accacagcag 1363tatagcatat?gtcacagctc?ctgtgattca?atgttataaa?acaagcagaa?ttttaaaagc 1423agcactatat?agctgttttg?tattatagtg?tatatgatgt?ttgtgaaaat?gccagattta 1483aaatgatgta?tttatttttggtaaaaaata?aaaaattcta 1523<210>2<211>96<212>PRT<213>human<400>2Met?Leu?Ser?Phe?Ser?Ile?Ile?Ser?Pro?Phe?Pro?Gly?Ile?Phe?Met?Met1 5 10 15Phe?Phe?Asn?Ile?Thr?Arg?Arg?Gln?Ser?Arg?Ala?Pro?Gln?Ile?Pro?Asp

20 25 30Trp?Ile?Asn?Leu?Gly?Pro?Ser?Ser?Val?Val?Leu?Leu?Ala?Leu?Ile?Glu

35 40 45Ser?Ser?Ser?Asn?Ser?Gln?Val?Gln?Ser?Gln?Phe?Val?Asn?Ala?Glu?Gln

50 55 60Ile?Thr?Asp?Lys?Thr?Ile?Asp?Trp?Leu?Leu?Gly?Lys?Thr?Val?Trp?Ser65 70 75 80Gly?Glu?Arg?Lys?Leu?Leu?Lys?Asn?Cys?Cys?Thr?Asp?Val?Thr?Gly?Asp

85 90 95<210>3<211>24<212>DNA<213>human<400>3CAATTAGATT?TTTTTTCCCC?ACAG 24<210>4<211>24<212>DNA<213>human<400>4TAGAATTTTT?TATTTTTTAC?CAAA 24<210>5<211>33<212>DNA<213>human<400>5CATGCTAGCA?TGCTTAGTTT?TTCAATTATT?TCT 33<210>6<211>33<212>DNA<213>human<400>6CATGGATCCT?CAATCACCGG?TGACATCAGT?ACA 33<210>7<211>15<212>PRT<213>human<400>7Met?Leu?Ser?Phe?Ser?Ile?Ile?Ser?Pro?Phe?Pro?Gly?Ile?Phe?Met1 5 10 15<210>8<211>41<212>DNA<213>human<400>8TGCTTAGTTT?TTCAATTATT?TCTCCTTTCC?CAGGAATTTT?T 41<210>9<211>41<212>DNA<213>human<400>9TGCTTAGTTT?TTCAATTATT?GCTCCTTTCC?CAGGAATTTT?T 41

Claims (18)

1, a kind of isolated polypeptide-aconitase-10.57 is characterized in that it includes: the polypeptide of the aminoacid sequence shown in＜210〉2 or the active fragments of its polypeptide, analogue or derivative.

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＜210〉2.

3, polypeptide as claimed in claim 2 is characterized in that it comprises and has＜polypeptide of the aminoacid sequence shown in 210〉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 have＜210〉2 shown in the multinuclear glycosides of the polypeptide of aminoacid sequence or its fragment, analogue, derivative

Acid;

(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, it is characterized in that described polynucleotide comprise coding and have＜210〉2 shown in the polynucleotide of aminoacid sequence.

6, polynucleotide as claimed in claim 4, it is characterized in that the sequence of described polynucleotide includes＜210〉1 in the 103-393 position sequence or＜210〉1 in the sequence of 1-1523 position.

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 the active polypeptide of aconitase-10.57 is characterized in that described method comprises:

(a) expressing under aconitase-10.57 condition, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate and have the active polypeptide of aconitase-10.57.

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with aconitase-10.57 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 aconitase-10.57.

12, compound as claimed in claim 11 is characterized in that it is＜polynucleotide sequence or its segmental antisense sequences shown in 210〉1.

13, the described application of compound of a kind of claim 11, it is characterized in that described compound be used to regulate aconitase-10.57 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 aconitase-10.57, 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 diagnosis or the treatment disease relevant unusually with aconitase-10.57 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.