CN1306975A - Polypeptide-leucine multiple repeat sequence 71 and polynucleotide for coding said polypeptide - Google Patents

Polypeptide-leucine multiple repeat sequence 71 and polynucleotide for coding said polypeptide Download PDF

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CN1306975A
CN1306975A CN 00111505 CN00111505A CN1306975A CN 1306975 A CN1306975 A CN 1306975A CN 00111505 CN00111505 CN 00111505 CN 00111505 A CN00111505 A CN 00111505A CN 1306975 A CN1306975 A CN 1306975A
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polypeptide
polynucleotide
sequence
multiple repeat
repeat 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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Priority to CN 00111505 priority Critical patent/CN1306975A/en
Priority to AU29981/01A priority patent/AU2998101A/en
Priority to PCT/CN2001/000045 priority patent/WO2001055374A1/en
Publication of CN1306975A publication Critical patent/CN1306975A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Abstract

The present invention discloses one new kind of polypeptide, leucine repeated sequence 71, 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 leucine repeated sequence 71.

Description

The polynucleotide of a kind of new polypeptide---leucine multiple repeat sequence 71 and this peptide species of coding
The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide---leucine multiple repeat sequence 71, 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.
Leucine multiple repeat sequence is a template that sequence is short and small in some functions and cell position of living in protein all inequality.These tumor-necrosis factor glycoproteinss participate in the reaction between albumen and the albumen usually.Each leucine tumor-necrosis factor glycoproteins is made of a β-α unit.These unit cell arrangement form parallel β lamella, and have one side to be revealed in the solvent, form the non-globosity that extends, because this particular structure is so have stronger albumen and the reaction between the albumen usually.The zone of halfcystine or l-asparagine normally is rich in the both sides of leucine multiple repeat sequence, and near aliphatics and the die aromatischen Aminosaeuren of comparatively guarding that also contain is sweet ammonia and dried meat acid propylhomoserin sometimes.The total residue of the hydrophobicity of this tumor-necrosis factor glycoproteins carboxyl terminal part contains 3,4 or 7 residues usually.
This sequence is an albumen and the territory that combines of cell regulating factor, and albumen combines the activity that suppresses regulatory factor by it with cell regulating factor, and the albumen with this sequence has the signal conduction usually, sometimes with the protein-interacting that causes pathologic reaction.For example just contain this sequence in the RNA enzyme inhibition factor, this factor is a kind of cytoplasmic protein, it is combined closely and suppresses pancreas RNA enzyme superfamily, form the RNA enzyme in conjunction with territory and RNA enzyme surface bonding by parallel β lamella and β α ring, the adjusting that participation RNA upgrades in mammalian cell is also worked in blood vessel takes place.If proteic leucine multiple repeat sequence is undergone mutation in the human body, can cause the Bernard-Soulier disease of various degree, this is a kind of bleeding disorder.Find that in addition the single leucine template that is rich in human I-type immune deficiency virus Rev albumen is this viral trans-activation domain.
The template that contains leucine multiple repeat sequence in the polypeptide of the present invention, this polypeptide and antagonist thereof, agonist and inhibitor can be used for diagnosing and prevent and treat the Bernard-Soulier disease of various degree and RNA more neovascularity relevant disease takes place, and the disease that causes of human I-type immune deficiency virus.
Because leucine multiple repeat sequence 71 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 the leucine multiple repeat sequence 71 albumen of more these processes of participation in this area always, particularly identify this proteic aminoacid sequence.The separation of new leucine multiple repeat sequence 71 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.
An object of the present invention is to provide isolating new polypeptide---leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 of encoding.
Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain the leucine multiple repeat sequence 71 of encoding.
Another object of the present invention provides the method for producing leucine multiple repeat sequence 71.
Another object of the present invention provides at polypeptide of the present invention---the antibody of leucine multiple repeat sequence 71.
Another object of the present invention has provided at polypeptide of the present invention---the simulated compound of leucine multiple repeat sequence 71, antagonist, agonist, inhibitor.
Another object of the present invention provides the method for the diagnoses and treatment disease relevant unusually with leucine multiple repeat sequence 71.
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 20-1963 position among the SEQ ID NO:1; (b) has the sequence of 1-2316 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 leucine multiple repeat sequence 71 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 the leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 7l 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 leucine multiple repeat sequence 71, 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 leucine multiple repeat sequence 71.
" antagonist " or " inhibition " be meant when combining with leucine multiple repeat sequence 71, a kind ofly seals or regulate the biologic activity of leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71.
" adjusting " is meant that the function of leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71.
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 leucine multiple repeat sequence 71 of standard.Basically pure leucine multiple repeat sequence 71 can produce single master tape on the irreducibility polyacrylamide gel.The purity available amino end acid sequence of leucine multiple repeat sequence 71 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 A0011150500071
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 leucine multiple repeat sequence 71.
" 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 leucine multiple repeat sequence 71 " is meant that leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of leucine multiple repeat sequence 71 polypeptide can be used amino acid sequence analysis.
The invention provides a kind of new polypeptide---leucine multiple repeat sequence 71, it is made up of the aminoacid sequence shown in the SEQ IDNO: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 leucine multiple repeat sequence 71.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of leucine multiple repeat sequence 71 of the present invention or active polypeptide 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 2316 bases, its open reading frame (20-1963) 647 amino acid of having encoded.This polypeptide has repeatedly repetitive series of leucine, and deducibility goes out this leucine multiple repeat sequence 71 and has the repeatedly 26S Proteasome Structure and Function of repetitive series representative of leucine.
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 leucine multiple repeat sequence 71.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
The special polynucleotide sequence of coding leucine multiple repeat sequence 71 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 literary composition village, often used in village names.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 leucine multiple repeat sequence 71; (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 leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 of encoding and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, aLaboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; 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 leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:
(1). with coding human leucine of the present invention polynucleotide of tumor-necrosis factor glycoproteins 71 (or varient) repeatedly, 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.
Leucine multiple repeat sequence is as the combine territory of a kind of albumen with cell regulating factor, albumen combines the activity that suppresses regulatory factor by it with cell regulating factor, albumen with this sequence has the signal conduction usually, sometimes with the protein-interacting that causes pathologic reaction.For example just contain this sequence in the RNA enzyme inhibition factor, this factor is a kind of cytoplasmic protein, and it is combined closely and suppresses pancreas RNA enzyme superfamily, and the adjusting that participation RNA upgrades in mammalian cell is also worked in blood vessel takes place.
Discover, if human leucine repeatedly tumor-necrosis factor glycoproteins undergo mutation, can cause the Bernard-Soulier disease of various degree, this is a kind of bleeding disorder.In addition, the single leucine template that is rich in human I-type immune deficiency virus Rev albumen is this viral trans-activation domain.
The polypeptide that contains leucine multiple repeat sequence, its characteristic leucine multiple repeat sequence are that its biological activity institute of formation is essential.
Polypeptide of the present invention is the polypeptide that contains the characteristic leucine multiple repeat sequence, its abnormal expression will make the activity of cell regulating factor suppress unusual, the inhibition of pancreas RNA enzyme superfamily is unusual, and may take place at blood vessel, in the immune response of I type effect unusual, and produce relevant disease.
This shows that the abnormal expression of leucine multiple repeat sequence 71 of the present invention will produce various diseases especially Bernard-Soulier disease, vascular conditions, I type immunological disease, various tumour, these diseases include but not limited to:
Vascular conditions: atherosclerosis, vasculitis, vascular tumor, hemangioendothelial sarcoma I type immunological disease: bronchial asthma, Asprin asthma, rhinallergosis, supersensitivity broncho-pulmonary aspergillosis, hypersensitivity pneumonitis, lung eosinophilia, diffuse interstitial pulmonary fibrosis, sarcoidosis, urticaria, atopic dermatitis
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, nasal cavity and tumor of sinus of nose, nasopharyngeal carcinoma, laryngocarcinoma, tracheal neoplasm, fibroma, fibrosarcoma, lipoma, liposarcoma, leiomyoma
The abnormal expression of leucine multiple repeat sequence 71 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 Bernard-Soulier disease, vascular conditions, I type immunological disease, various tumour, 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) leucine multiple repeat sequence 71.Agonist improves leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 be cultivated with the leucine multiple repeat sequence 71 of mark.Measure the medicine raising then or check this interactional ability.
The antagonist of leucine multiple repeat sequence 71 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of leucine multiple repeat sequence 71 can combine and eliminate its function with leucine multiple repeat sequence 71, 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, leucine multiple repeat sequence 71 can be added in the bioanalysis mensuration, interactional influence between leucine multiple repeat sequence 71 and its acceptor be determined 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 leucine multiple repeat sequence 71 bonded peptide molecule obtains.During screening, generally tackle the leucine multiple repeat sequence 71 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 the leucine multiple repeat sequence 71 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 available leucine multiple repeat sequence 71 direct injection of the production of polyclonal antibody 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 leucine multiple repeat sequence 71 include but not limited to hybridoma technology (Kohler and 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-leucine multiple repeat sequence 71.
The antibody of anti-leucine multiple repeat sequence 71 can be used in the immunohistochemistry technology, detects the leucine multiple repeat sequence 71 in the biopsy specimen.
With the also available labelled with radioisotope of leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the leucine multiple repeat sequence 71 positive cells.
The disease that antibody among the present invention can be used for treating or prevention is relevant with leucine multiple repeat sequence 71.The antibody that gives suitable dosage can stimulate or block the generation or the activity of leucine multiple repeat sequence 71.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization leucine multiple repeat sequence 71 level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The leucine multiple repeat sequence 71 level that is detected in the test can be with laying down a definition the importance of leucine multiple repeat sequence 71 in various diseases and be used to the disease of diagnosing leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the leucine multiple repeat sequence 71 of expressing variation, to suppress endogenic leucine multiple repeat sequence 71 activity.For example, a kind of leucine multiple repeat sequence 71 of variation can be the leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding leucine multiple repeat sequence 71 is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 can be used for the diagnosis with the relative disease of leucine multiple repeat sequence 71.The unconventionality expression of the expression that the polynucleotide of coding leucine multiple repeat sequence 71 can be used for detecting leucine multiple repeat sequence 71 leucine multiple repeat sequence 71 whether or under morbid state.As the dna sequence dna of the leucine multiple repeat sequence 71 of encoding can be used for biopsy specimen is hybridized to judge the expression situation of leucine multiple repeat sequence 71.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 leucine multiple repeat sequence 71 with the special primer of leucine multiple repeat sequence 71.
The sudden change that detects the leucine multiple repeat sequence 71 gene also can be used for the disease of diagnosing leucine multiple repeat sequence 71 relevant.The form of leucine multiple repeat sequence 71 sudden change comprises that the point mutation compared with normal wild type leucine multiple repeat sequence 71 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, 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.Leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71 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 repeatedly amino acid sequence homology comparison diagram of repetitive series of leucine multiple repeat sequence 71 of the present invention and structural domain leucine.The top sequence is a leucine multiple repeat sequence 71, and the below sequence is a repeatedly repetitive series of structural domain leucine.Same amino acid represents with monocase amino acid between two sequences, similar amino acid with "+' expression.
Fig. 2 is the polyacrylamide gel electrophoresis figure (SDS-PAGE) of isolating leucine multiple repeat sequence 71.71KDa 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 leucine multiple repeat sequence 71
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 QuikmRNA 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 0465e10 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 0465e10 clone is 2316bp (shown in Seq ID NO:1), from 20bp to 1963bp the open reading frame (ORF) of a 1944bp, the new protein (shown in Seq ID NO:2) of encoding arranged.We are with this clone's called after pBS-0465e10, encoded protein matter called after leucine multiple repeat sequence 71.Embodiment 2:cDNA clone's domain analyses
With the sequence and the encoded protein sequence thereof of leucine multiple repeat sequence 71 of the present invention, with profile scan 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.Leucine multiple repeat sequence 71 of the present invention and structural domain leucine repeatedly repetitive series have homology, and homology the results are shown in Fig. 1.Embodiment 3: with the gene of RT-PCR method clones coding leucine multiple repeat sequence 71
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’-GGAGGGCTCCGTTTGAAACATGGC-3’(SEQ?ID?NO:3)
Primer2:5’-AGAAAATTATTTTCTTTAATTATC-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, 10mmol/L Tris-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 1-2316bp shown in the SEQ ID NO:1 are identical.Embodiment 4:Northern blotting is analyzed leucine multiple repeat sequence 7l 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 (pH7.0)-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 the leucine multiple repeat sequence 71 coding region sequence (20bp to 1963bp) 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 leucine multiple repeat sequence 71
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’-CCCCATATGATGGCGCGGGCTGGCCCTCGGCTG-3’(Seq?ID?No:5)
Primer4:5’-CATGGATCCTCAGCAGGCCTGGCAGCCTCCATG-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-0465e10 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0465e10 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-0465e10) 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-0465e10) is cultured to logarithmic phase at 37 ℃, adds IPTG to final concentration lmmol/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 leucine multiple repeat sequence 71 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 71KDa 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-leucine multiple repeat sequence 71 production of antibodies
Synthesize the specific polypeptide of following leucine multiple repeat sequence 71 with Peptide synthesizer (PE company product):
NH2-Met-Ala-Arg-Ala-Gly-Pro-Arg-Leu-Val-Leu-Ser-Glu-Glu-Ala-Val-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 leucine multiple repeat sequence 71 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 conform to 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’-TGGCGCGGGCTGGCCCTCGGCTGGTGCTGAGCGAGGAGGCG-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’-TGGCGCGGGCTGGCCCTCGGCTGGTGCTGAGCGAGGAGGCG-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 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 200 μ g/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 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/ml CT 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/ μ l, (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=4013.63 (getting the mean value of four experiments), Cy5signal=4464.43 (getting the mean value of four experiments), Cy3/Cy5=0.89902, 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: leucine multiple repeat sequence 71 and encoding sequence thereof
(ⅲ) sequence number: 9
(2) information of SEQ ID NO:1:
(ⅰ) sequence signature:
(A) length: 2316bp
(B) type: nucleic acid
(C) chain: two strands
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
( ⅹⅰ ) ：SEQ ID NO:1: 1 GGAGGGCTCCGTTTGAAACATGGCGCGGGCTGGCCCTCGGCTGGTGCTGAGCGAGGAGGC 61 GGTTCGGGCGAAGAGCGGCTTAGGGCCTCACCGCGACCTGGCTGAGCTTCAGTCATTGTC121 TATTCCTGGAACTTACCAAGAGAAGATCACCCACCTGGGACATTCTCTGATGAGTTTAAC181 AGGTCTGAAATCTTTGGATCTCTCGCGCAACTCCTTGGTTGGTCTGGAGGGCATTCAGTA241 CCTGACTGCATTGGAGAGTCTCAATCTCTACTACAACTGCATCTCCTCGTTGGCAGAAGT301 GTTTCGGCTCCACGCCTTAACCGAGCTCGTGGATGTGGACTTCCGGCTGAACCCCGTGGT361 GAAGGTTGAGCCTGACTACCGCCTTTTTGTTGTGCACCTGCTCCCCAAGCTCCAGCAGCT421 GGACGATCGCCCCGTGAGAGCAAGCGAGCGGAAGGCTTCCCGACTGCATTTTGCATCAGA481 GGACTCACTCGACTCCAAAGAGAGCGTCCCAGCTTCTTTGAAAGAGGGCAGACCACACCA541 CCCCAGAGCCAAGTGCACCGAGGCCTTGGCCAAGCAGAGCCTGGTCATGGATGCGGATGA601 CGAGGCAGTCCTGAACCTCATTGCAGAGTGCGAGTGGGACCTCGGCAGGCCTCCCGGGAG661 CACGAGCTTCAGCCAGAAGGGGCGTGAGGCCGACTCTCGTGGTTCCCAAGAATCCAGACA721 TCTGTTGAGCCCGCAGTTGGTACAGTACCAGTGTGGGGACTCTGGGAAGCAGGGCCGTGA781 GACGAGGAGGAGCAGCTGCAGAGGGTGCTGTCTGGAGAAGATGCCTTGGAGCCAGCTCTG841 TGGAGAGCTTCCGCCACTGTACGGAGCGGAGCCAGAGGCCTCCCGTGCCCCCAGGCCACA901 CACGTACTTCACCCCACACCCAGACTCCATGGATACCGAGGACTCGGCCTCTTCTCAGAA 961 GTTGGATTTGTCAGGAGAAATGGTGCCTGGTCCCCTGCCAGCCCCCGGAAAGTGCAGGAA1021 GCGAAGAATGCCTGTTGGAAGATTCCAGACGTTTTCGGACCAGGAGGGTTTGGGCTGCCC1081 GGAGAGAACTCATGGGTCCTCCGTGCCCAAGGAGAGCCTGAGCAGACAGGACAGCTCAGA1141 AAGCAGGAACGGGAGGACCTTGTCTCAGCCTGAGGCCTCGGAGACTGAGGAGCAGAGGTC1201 TCGGGGTGTGACCGACACCAGAGAGCCGTCTCCCGGGTCACACTCGGCTCTACCCGGGAA1261 GAAGACGGCCCTGCAGGCGGCGCTCCTGGAGACGCTCTTGGACCTGGTGGACAGGAGCTG1321 GGGCGGCTGCAGGTCCCTGCACAGCAACGAGGCATTCCTTGCTCAGGCAAGACACATCTT1381 GTCATCTGTTGAAGAATTCACAGCAGCTCAGGACAGCTCTGCGATGGTGGGTGAAGATGT1441 CGGCTCCCTGGCTCTGGAGAGTAAGTCCCTGCAAAGCCGCCTTGCTGAGCAGCAGCAGCA1501 GCACGCCCGGGAGATGAGCGAGGTGACGGCGGAGCTGCACCACGCACACAAGGAGCTGGA1561 TGATTTGAGACAACATTTAGATAAATCTTTGGAAGAGAACAGTAGGTTAAAATCGCTTTT1621 GTTGAGTATGAAAAAGGAAGTGAAGAGTGCAGACACTGCAGCCACGTTAAATTTGCAGAT1681 CGCTGGACTTCAAACAAGTGTGAAGAGGCTGTGTGGCGAGATTGTGGAACTGAAGCAGCA1741 CCTGGAGCACTACGACAAGATCCAGGAGCTCACGCAGATGCTGCAGGAGAGCCACAGCTC1801 CCTGGTCAGCACCAATGAACACCTGCTGCAGGAGCTGAGCCAGGTGCGGGCGCAGCACAG1861 AGCCGAGGTGGAGCAGATGCACTGGGGCTACCAGGAGCTCAAGAAGACCATGGCCCTGTT1921 TCCACACAGCAGCGCCAGCCATGGAGGCTGCCAGGCCTGCTGACTCCTGCCGAGAAGCTG1981 GGCCACCCCTTAAGCTTCCTGGTAAAGTTACATTGTCTGCACCTTTGTACTTCTTTACTG2041 AGTGTACTGGCTGGCAAGAGTTCTCTCTTCTGTTGGTAATTATTTAGGATTTTTGGAATG2101 TATTCAGGACCTGTAGCTTGGTTTTCTAAAGCACCTCGTAAAATGATATGATTACTCCAA2161 GCCCCTCTGCATGTTTTCAGACAGAACACATTGACATATTTTGAGACAAACTGACTATTA2221 ATCTTGTATCCAGTATCCTGAGATGAAGTAAATGCAGTGTTCTACTGCCTGATGCGAAAG2281 AGAGCTATGTATGATAATTAAAGAAAATAATTTTCT
(3) information of SEQ ID NO:2:
(ⅰ) sequence signature:
(A) length: 647 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: polypeptide
( ⅹⅰ ) ：SEQ ID NO:2:1 Met Ala Arg Ala Gly Pro Arg Leu Val Leu Ser Glu Glu Ala Val 16 Arg Ala Lys Ser Gly Leu Gly Pro His Arg Asp Leu Ala Glu Leu 31 Gln Ser Leu Ser Ile Pro Gly Thr Tyr Gln Glu Lys Ile Thr His 46 Leu Gly His Ser Leu Met Ser Leu Thr Gly Leu Lys Ser Leu Asp 61 Leu Ser Arg Asn Ser Leu Val Gly Leu Glu Gly Ile Gln Tyr Leu 76 Thr Ala Leu Glu Ser Leu Asn Leu Tyr Tyr Asn Cys Ile Ser Ser 91 Leu Ala Glu Val Phe Arg Leu His Ala Leu Thr Glu Leu Val Asp106 Val Asp Phe Arg Leu Asn Pro Val Val Lys Val Glu Pro Asp Tyr121 Arg Leu Phe Val Val His Leu Leu Pro Lys Leu Gln Gln Leu Asp136 Asp Arg Pro Val Arg Ala Ser Glu Arg Lys Ala Ser Arg Leu His151 Phe Ala Ser Glu Asp Ser Leu Asp Ser Lys Glu Ser Val Pro Ala166 Ser Leu Lys Glu Gly Arg Pro His His Pro Arg Ala Lys Cys Thr181 Glu Ala Leu Ala Lys Gln Ser Leu Val Met Asp Ala Asp Asp Glu196 Ala Val Leu Asn Leu Ile Ala Glu Cys Glu Trp Asp Leu Gly Arg211 Pro Pro Gly Ser Thr Ser Phe Ser Gln Lys Gly Arg Glu Ala Asp226 Ser Arg Gly Ser Gln Glu Ser Arg His Leu Leu Ser Pro Gln Leu241 Val Gln Tyr Gln Cys Gly Asp Ser Gly Lys Gln Gly Arg Glu Thr256 Arg Arg Ser Ser Cys Arg Gly Cys Cys Leu Glu Lys Met Pro Trp271 Ser Gln Leu Cys Gly Glu Leu Pro Pro Leu Tyr Gly Ala Glu Pro286 Glu Ala Ser Arg Ala Pro Arg Pro His Thr Tyr Phe Thr Pro His301 Pro Asp Ser Met Asp Thr Glu Asp Ser Ala Ser Ser Gln Lys Leu316 Asp Leu Ser Gly Glu Met Val Pro Gly Pro Leu Pro Ala Pro Gly331 Lys Cys Arg Lys Arg Arg Met Pro Val Gly Arg Phe Gln Thr Phe346 Ser Asp Gln Glu Gly Leu Gly Cys Pro Glu Arg Thr His Gly Ser361 Ser Val Pro Lys Glu Ser Leu Ser Arg Gln Asp Ser Ser Glu Ser376 Arg Asn Gly Arg Thr Leu Ser Gln Pro Glu Ala Ser Glu Thr Glu391 Glu Gln Arg Ser Arg Gly Val Thr Asp Thr Arg Glu Pro Ser Pro406 Gly Ser His Ser Ala Leu Pro Gly Lys Lys Thr Ala Leu Gln Ala421 Ala Leu Leu Glu Thr Leu Leu Asp Leu Val Asp Arg Ser Trp Gly436 Gly Cys Arg Ser Leu His Ser Asn Glu Ala Phe Leu Ala Gln Ala451 Arg His Ile Leu Ser Ser Val Glu Glu Phe Thr Ala Ala Gln Asp466 Ser Ser Ala Met Val Gly Glu Asp Val Gly Ser Leu Ala Leu Glu481 Ser Lys Ser Leu Gln Ser Arg Leu Ala Glu Gln Gln Gln Gln His496 Ala Arg Glu Met Ser Glu Val Thr Ala Glu Leu His His Ala HisS11 Lys Glu Leu Asp Asp Leu Arg Gln His Leu Asp Lys Ser Leu Glu526 Glu Asn Ser Arg Leu Lys Ser Leu Leu Leu Ser Met Lys Lys Glu541 Val Lys Ser Ala Asp Thr Ala Ala Thr Leu Asn Leu Gln Ile Ala556 Gly Leu Gln Thr Ser Val Lys Arg Leu Cys Gly Glu Ile Val Glu571 Leu Lys Gln His Leu Glu His Tyr Asp Lys Ile Gln Glu Leu Thr586 Gln Met Leu Gln Glu Ser His Ser Ser Leu Val Ser Thr Asn Glu601 His Leu Leu Gln Glu Leu Ser Gln Val Arg Ala Gln His Arg Ala616 Glu Val Glu Gln Met His Trp Gly Tyr Gln Glu Leu Lys Lys Thr631 Met Ala Leu Phe Pro His Ser Ser Ala Ser His Gly Gly Cys Gln646 Ala Cys
(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:
GGAGGGCTCCGTTTGAAACATGGC 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:
AGAAAATTATTTTCTTTAATTATC 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:CCCCATATGATGGCGCGGGCTGGCCCTCGGCTG 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:CATGGATCCTCAGCAGGCCTGGCAGCCTCCATG 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-Ala-Arg-Ala-Gly-Pro-Arg-Leu-Val-Leu-Ser-Glu-Glu-Ala-Val 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:TGGCGCGGGCTGGCCCTCGGCTGGTGCTGAGCGAGGAGGCG 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:TGGCGCGGGCTGGCCCTCGGCTGGTGCTGAGCGAGGAGGCG 41

Claims (18)

1, a kind of isolated polypeptide-leucine multiple repeat sequence 71 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 1-2316 position among the sequence of 20-1963 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 the active polypeptide of leucine multiple repeat sequence 71 is characterized in that described method comprises:
(a) expressing under the leucine multiple repeat sequence 71 condition, cultivate the described through engineering approaches host cell of claim 8;
(b) from culture, isolate and have the active polypeptide of leucine multiple repeat sequence 71.
10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71.
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 leucine multiple repeat sequence 71 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 leucine multiple repeat sequence 71, 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 leucine multiple repeat sequence 71 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.
CN 00111505 2000-01-26 2000-01-26 Polypeptide-leucine multiple repeat sequence 71 and polynucleotide for coding said polypeptide Pending CN1306975A (en)

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CN 00111505 CN1306975A (en) 2000-01-26 2000-01-26 Polypeptide-leucine multiple repeat sequence 71 and polynucleotide for coding said polypeptide
AU29981/01A AU2998101A (en) 2000-01-26 2001-01-15 A novel polypeptide, a leucine multirepeating sequence 71 and the polynucleotideencoding the polypeptide
PCT/CN2001/000045 WO2001055374A1 (en) 2000-01-26 2001-01-15 A novel polypeptide, a leucine multirepeating sequence 71 and the polynucleotide encoding the polypeptide

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108721646A (en) * 2017-04-17 2018-11-02 中山大学 A kind of method and antiviral drugs inhibiting virus infection

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108721646A (en) * 2017-04-17 2018-11-02 中山大学 A kind of method and antiviral drugs inhibiting virus infection
CN108721646B (en) * 2017-04-17 2021-10-01 中山大学 Method for inhibiting virus infection and antiviral drug

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