CN1315551A - Polypeptide-human endomannosidase 25 and polynucleotide for coding it - Google Patents

Polypeptide-human endomannosidase 25 and polynucleotide for coding it Download PDF

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CN1315551A
CN1315551A CN 00115107 CN00115107A CN1315551A CN 1315551 A CN1315551 A CN 1315551A CN 00115107 CN00115107 CN 00115107 CN 00115107 A CN00115107 A CN 00115107A CN 1315551 A CN1315551 A CN 1315551A
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polypeptide
polynucleotide
mannosidase
mouse
alpha
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毛裕民
谢毅
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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Abstract

A new polypeptide-human endomannosidase 25, the polynucleotide for coding it, the process for preparing this polypeptide by DNA recombination, the application of said polypeptide in treating several diseases such as cancer, hematopathy, HIV infection, etc., the antagonist against the said polypeptide and its therapeutic action, and the application of said polynucleotide for coding this new human endomannosidase 25 are disclosed.

Description

The polynucleotide of a kind of new polypeptide--people's interior mannosidase 25 and this peptide species of coding
The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide--people's interior mannosidase 25, 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.
Have flourishing endomembrane system and various organoid in the eukaryotic cell, formed the organoid that cell matrix and film center on.The cell endomembrane system is meant that in structure organoid or cellularstructure that function or the film that takes place upward to be correlated with center on mainly refer to endoplasmic reticulum, the lysosome of golgi body and formation thereof and secretory vacuole etc.Wherein endoplasmic reticulum is protein and film fat synthetic base, and the glycosylation of polypeptide and folding and assembling thereof also occur in the endoplasmic reticulum.Golgi body is a kind of polar organoid that has, and is made up of the several sections that interknits.Golgi body plays an important role in proteic processing sorted and packaged and transportation and in intracellular film stream.In addition, proteic glycosylation and complicated processing and modification thereof, the synthetic etc. of the enzymolysis processing of polypeptide and polysaccharide also occurs in the golgi body.
In-α-D-mannosidase is a kind of golgi body enzyme, is the enzyme that participates in the oligosaccharides processing of early stage N-connection.The main chemically modified effect that enters the protein generation in the endoplasmic reticulum has glycosylation, hydroxylation etc.Glycosylation is accompanied by that polypeptide is synthetic to carry out simultaneously, is modal protein modification in the endoplasmic reticulum.Glycosylated main effect is that protein is folded into correct conformation and increases proteinic stability in ripening process; because oligonucleotide chain has certain rigid; thereby limited the membranin of other macromole near cell surface; the outer quilt that makes cell have a protectiveness, the not shape of restrictive cell and motion again.Oligonucleotide chain generally is combined on the four seed amino acid residues of peptide chain in the eukaryotic cell, can be divided into different glycosylation modified of two big classes: N-thus and connect (being connected on the amide nitrogen atom of aspartic acid) and be connected (being connected on the hydroxyl of serine threonine or the hydroxylysine in collegen filament work oxyproline) glycosylation with O-.The glycosylation that N-connects occurs in the rough surfaced endoplasmic reticulum, and oligonucleotide chain by 14 saccharide residues is transferred to from donor phosphoric acid dolichol on the asparagicacid residue of specific tripeptide sequence of new polypeptide chain.The oligonucleotide chain that all N-connects all has a common precursor, in rough surfaced endoplasmic reticulum and by golgi body each at interval in transfer process oligonucleotide chain excise and add specific monose through the processing of a series of enzymes, form sophisticated glycoprotein at last.In-α-D-mannosidase is a kind of enzyme of uniqueness in the glucosylation polymanna processing that participates in the new synthetic N-connection of pruning, cutting discharges the disaccharides that Glc α 1 is converted into 3Man thereby it is in inside, rather than excision individual signals end saccharide residue.Because also having, this kind of enzyme in newborn saccharide unit, removes three and didextrose base seminose (Glc 3Man and Glc 2Man) ability, thereby it also provides a kind of alternative processing approach can surround the Polyglucosidase of block because of inhibitor or enzyme disappearance continues synthetic complicated N-connection oligosaccharides and becomes possibility thereby make.
New interior-α-D-mannosidase of the present invention and mouse be interior-and α-D-mannosidase has 87% identity and 94% similarity on protein level, and in also containing-α-common various characteristic sequential fragments of other members of D-mannoside enzyme family, infer in proper order in this albumen and the mouse-α-D-mannosidase has similar biological function, and the called after mouse is interior-α-D-mannosidase 25.This polypeptide and agonist thereof, inhibitor, antagonist can be used for diagnosis and control proliferative disease, virus infection, diseases such as neoplastic growth and composition transfer.
Because as mentioned above in the mouse-alpha-D-mannosidase 25 albumen play an important role in regulating body critical functions such as cell fission and fetal development, and believe and relate to a large amount of albumen in these regulate processes, thereby need in this area to identify in the mouse of more these processes of participation-alpha-D-mannosidase 25 albumen, particularly identify this proteic aminoacid sequence. alwaysIn the new mouse-separation of alpha-D-mannosidase 25 protein coding genes also is to determine the effect of this albumen under healthy and morbid state to provide the foundation.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--in the mouse-alpha-D-mannosidase 25 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 and contains in the coding mouse-recombinant vectors of the polynucleotide of alpha-D-mannosidase 25.
Another object of the present invention provides and contains in the coding mouse-the genetically engineered host cell of the polynucleotide of alpha-D-mannosidase 25.
Another object of the present invention provides and produces in the mouse-method of alpha-D-mannosidase 25.
Another object of the present invention provides at polypeptide of the present invention--in the mouse-and the antibody of alpha-D-mannosidase 25.
Another object of the present invention has provided at polypeptide of the present invention--in the mouse-and the simulated compound of alpha-D-mannosidase 25, antagonist, agonist, inhibitor.
Another object of the present invention provides in diagnoses and treatment and the mouse-method of the unusual relevant disease of alpha-D-mannosidase 25.
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 88% homogeny.
More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 99-788 position among the SEQ ID NO:1; (b) has the sequence of 1-2098 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 in the mouse-method of the compound of alpha-D-mannosidase 25 protein-actives, it comprises and utilizes polypeptide of the present invention.The invention still further relates to the compound that obtains with this method.
The invention still further relates in a kind of vitro detection and the mouse-alpha-D-mannosidase 25 abnormal proteins express the relevant disease or the method for disease susceptibility, 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 because in the mouse in preparation-purposes of the medicine of alpha-D-mannosidase 25 diseases that abnormal expression causes.
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, and 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 " be meant when with mouse in-when alpha-D-mannosidase 25 combines, thereby a kind of this protein of causing changes the molecule of regulating this protein active.Agonist can comprise protein, nucleic acid, carbohydrate or any other can in conjunction with in the mouse-molecule of alpha-D-mannosidase 25.
" antagonist " or " inhibition " be meant when with mouse in-when alpha-D-mannosidase 25 combines, a kind of seal or regulate in the mouse-biologic activity of alpha-D-mannosidase 25 or molecule of immunologic competence.Antagonist and inhibition can comprise protein, nucleic acid, carbohydrate or any other can in conjunction with in the mouse-molecule of alpha-D-mannosidase 25.
" adjusting " be meant in the mouse-function of alpha-D-mannosidase 25 changes, and comprises in the change and mouse of the rising of protein active or reduction, binding characteristic-change of any other biological property, function or the immune property of alpha-D-mannosidase 25.
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-alpha-D-mannosidase 25 interior with the purified technology of protein purifying mouse of standard.Basically in the pure mouse-alpha-D-mannosidase 25 can produce single master tape on the irreducibility polyacrylamide gel.In the mouse-the purity available amino end acid sequence of alpha-D-mannosidase 25 polypeptide analyzes.
" complementary " or " complementation " is meant under salt concn that allows and temperature condition by the natural combination of the polynucleotide of base pairing.For example, sequence " C-T-G-A " can combine with complementary sequence " G-A-C-T ".Complementation between two single chain molecules can be part or whole.Complementary degree between the nucleic acid chains has a significant effect for efficient of hybridizing between the nucleic acid chains and intensity.
" homology " is meant the complementary degree, can be portion homologous or complete homology." portion homologous " is meant a kind of part complementary sequence, and it can partly suppress the hybridization of complete complementary sequence and target nucleic acid at least.The inhibition of this hybridization can detect by hybridizing (Southern trace or Northern trace etc.) under the condition that reduces in the severity degree.Basically homologous sequence or hybridization probe can compete and suppress complete homologous sequence and target sequence the condition that reduces of severity degree under combine.This does not mean the conditions permit non-specific binding that the severity degree reduces because the conditional request two sequences that the severity degree reduces mutual be combined into specificity or selectivity interacts.
" homogeny percentage " be meant two or more amino acid or nucleotide sequence relatively in the same or analogous percentage of sequence.The available electron method is measured the homogeny percentage, as passing through MEGALIGN program (Lasergenesoftware package, 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:
The residue number of mating between sequence A and the sequence B
100
Interval residue number in the residue number-sequence B of interval in the residue number-sequence A of sequence A
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 can specificity in conjunction with in the mouse-antigenic determinant of alpha-D-mannosidase 25.
" 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 mouse in-alpha-D-mannosidase 25 " be meant in the mouse-alpha-D-mannosidase 25 is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can-alpha-D-mannosidase 25 interior with the purified technology of protein purifying mouse of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.In the mouse-purity of alpha-D-mannosidase 25 polypeptide can use amino acid sequence analysis.
The invention provides a kind of new polypeptide--in the mouse-alpha-D-mannosidase 25, it is made up of the aminoacid sequence shown in the SEQ ID NO:2 basically.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises in the mouse-fragment, derivative and the analogue of alpha-D-mannosidase 25.As used herein, term " fragment ", " derivative " and " analogue " are meant and keep basically in the mouse of the present invention-biological function or active polypeptide that alpha-D-mannosidase 25 is identical.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 2098 bases, its open reading frame 99-788 229 amino acid of having encoded.Relatively to find according to amino acid sequence homologous, in this polypeptide and the mouse-the alpha-D-mannosidase has 75% homology, deducibility goes out in this mouse-and alpha-D-mannosidase 25 has in the mouse-the similar 26S Proteasome Structure and Function of alpha-D-mannosidase.
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.The amplification technique (as PCR) that nucleic acid fragment also can be used for nucleic acid is to determine and/or to separate in the coding mouse-polynucleotide of alpha-D-mannosidase 25.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
In the coding of the present invention mouse-the special polynucleotide sequence of alpha-D-mannosidase 25 can obtain with several different methods.For example, separate polynucleotide with hybridization technique well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect homologous polynucleotide sequence and 2) antibody screening of expression library to be to detect the polynucleotide passage of the clone with common structure feature.
Sequence dna fragment of the present invention also can obtain with following method: 1) separate double chain DNA sequence from genomic dna; 2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of described polypeptide.
In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.The direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.The more frequent method of selecting for use is the separation of cDNA sequence.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qiagene) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.
Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods comprise (but be not limited to-): (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) measure in the mouse-level of the transcript of alpha-D-mannosidase 25; (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 in the mouse-protein product of alpha-D-mannosidase 25 genetic expressions 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 in the mouse-host cell that alpha-D-mannosidase 25 encoding sequences produce through genetically engineered, and the method that produces polypeptide of the present invention through recombinant technology.
Among the present invention, in the coding mouse-polynucleotide sequence of alpha-D-mannosidase 25 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 contain the coding mouse in-dna sequence dna of alpha-D-mannosidase 25 and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; 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, in the coding mouse-polynucleotide of alpha-D-mannosidase 25 or the recombinant vectors that contains these polynucleotide can transform or transduce into host cell, contains 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 in the mouse of reorganization-alpha-D-mannosidase 25 (Science, 1984; 224:1431).In general following steps are arranged:
(1). with in the coding of the present invention people mouse-polynucleotide (or varient) of alpha-D-mannosidase 25, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). in suitable medium, cultivate host cell;
(3). separation, protein purification from substratum or cell.
In step (2), according to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
In step (3), recombinant polypeptide can wrap and be expressed or be secreted into the extracellular in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.These methods include, but are not limited to: conventional renaturation is handled, protein precipitant is handled (salt analysis method), centrifugal, the broken bacterium of infiltration, the combination of ultrasonication, super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in disease treatment, for example, can treat malignant tumour, adrenal gland defect, tetter, all kinds of inflammation, HIV infection and immunological disease etc.
Golgi body plays an important role in proteic processing sorted and packaged and transportation and in intracellular film stream.In addition, proteic glycosylation and complicated processing and modification thereof, the synthetic etc. of the enzymolysis processing of polypeptide and polysaccharide also occurs in the golgi body.In-α-D-mannosidase is a kind of golgi body enzyme, is the enzyme that participates in the oligosaccharides processing of early stage N-connection.The main chemically modified effect that enters the protein generation in the endoplasmic reticulum has glycosylation, hydroxylation etc.Glycosylation is accompanied by that polypeptide is synthetic to carry out simultaneously, is modal protein modification in the endoplasmic reticulum.Glycosylated main effect is that protein is folded into correct conformation and increases proteinic stability in ripening process.In-α-D-mannosidase is a kind of enzyme of uniqueness in the glucosylation polymanna processing that participates in the new synthetic N-connection of pruning.It also has removes three and the ability of didextrose base seminose in newborn saccharide unit, thereby it also provides a kind of alternative processing approach can surround the Polyglucosidase of block because of inhibitor or enzyme disappearance, continues synthetic complicated N-connection oligosaccharides and becomes possibility thereby make.
New interior-α-D-mannosidase of the present invention and mouse be interior-and α-D-mannosidase has 87% identity and 94% similarity on protein level, and in also containing-α-common various characteristic sequential fragments of other members of D-mannoside enzyme family, infer in proper order in this albumen and the mouse-α-D-mannosidase has similar biological function, and the called after mouse is interior-α-D-mannosidase 25.This polypeptide and agonist thereof, inhibitor, antagonist can be used for diagnosis and control proliferative disease, virus infection, diseases such as neoplastic growth and composition transfer.
In polypeptide of the present invention and the mouse-α-D-mannosidase height homology, in containing-characteristic sequence of α-D-mannoside enzyme family, both have similar biological function.It is very important with modification for proteic glycosylation processing, makes albumen have normal biological function.The generation of diseases such as the tumour of substance metabolism disorder disease, Proteometabolism disorder disease and related tissue that its abnormal expression is usually relevant with some and cancer is closely related, and the generation disease of being correlated with.
This shows, in the mouse of the present invention-abnormal expression of alpha-D-mannosidase 25 will produce the especially various tumours of various diseases, fetal development disorders, dysplasia disease, inflammation, immunological disease, and these diseases include but not limited to:
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, bladder cancer, uterus carcinoma, carcinoma of endometrium, colorectal carcinoma, thymus neoplasms, nasopharyngeal carcinoma, laryngocarcinoma, tracheal neoplasm, fibroma, fibrosarcoma, lipoma, liposarcoma
Fetal development disorders: congenital miscarriage, cleft palate, deficiency of skeletal limb, limbs dysdifferentiation, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness
The dysplasia disease: mental retardation, cerebral dysgenesis, skin, fat and amyoplasia disease, B﹠J dysplasia disease, various metabolic defects, cretinism, nanism, emerging comprehensive this in storehouse levied the slow disease of sexual development
Inflammation: chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, multiple sclerosis, glomerulonephritis, myocarditis, myocardosis, atherosclerosis, stomach ulcer, trachelitis, various infective inflammations
Immunological disease: systemic lupus erythematous, rheumatoid arthritis, bronchial asthma, urticaria, atopic dermatitis, infect back myocarditis, scleroderma, myasthenia gravis, Green-barre syndrome, common variable immunodeficient disease, primary bone-marrow-derived lymphocyte immunodeficient disease, acquired immune deficiency syndrome (AIDS)
In the mouse of the present invention-abnormal expression of alpha-D-mannosidase 25 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 the especially various tumours of various diseases, fetal development disorders, dysplasia disease, inflammation, immunological disease, some heredity, blood disease etc.
The present invention also provides SCREENED COMPOUND to improve (agonist) or check in (antagonist) mouse to identify-method of the medicament of alpha-D-mannosidase 25.Agonist improves in the mouse-alpha-D-mannosidase 25 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 be in the presence of medicine, with mammalian cell or express in the mouse-film preparation of alpha-D-mannosidase 25 in the mouse of mark-alpha-D-mannosidase 25 cultivates.Measure the medicine raising then or check this interactional ability.
In the mouse-antagonist of alpha-D-mannosidase 25 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.In the mouse-antagonist of alpha-D-mannosidase 25 can with mouse in-alpha-D-mannosidase 25 combines and eliminates its function, or suppresses the generation of this polypeptide, or combine 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, can with in the mouse-alpha-D-mannosidase 25 adds during bioanalysiss measure, by measure compound in the mouse-alpha-D-mannosidase 25 and its acceptor between interactional influence determine whether compound is antagonist.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.Can with mouse in-alpha-D-mannosidase 25 bonded peptide molecules can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening and obtain.During screening, generally tackle in the mouse-alpha-D-mannosidase 25 molecules 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 provide in the mouse-antibody of alpha-D-mannosidase 25 antigenic determinants.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.
In the available mouse of the production of polyclonal antibody-and the methods of alpha-D-mannosidase 25 direct injection immune animals (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.In the preparation mouse-technology of the monoclonal antibody of alpha-D-mannosidase 25 includes 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 in the anti-mouse-single-chain antibody of alpha-D-mannosidase 25.
In the anti-mouse-and the antibody of alpha-D-mannosidase 25 can be used in the immunohistochemistry technology, detects in the mouse in the biopsy specimen-alpha-D-mannosidase 25.
With in the mouse-the also available labelled with radioisotope of alpha-D-mannosidase 25 bonded monoclonal antibodies, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.
Antibody also can be used for designing the immunotoxin at a certain privileged sites in the body.As in the mouse-monoclonal antibody of alpha-D-mannosidase 25 high-affinities can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, interior-alpha-D-mannosidase 25 positive cells that this hybrid antibody can be used for killing mouse.
Antibody among the present invention can be used for treating or prevention and mouse in-disease that alpha-D-mannosidase 25 is relevant.The antibody that gives suitable dosage can stimulate or block in the mouse-generation or the activity of alpha-D-mannosidase 25.
The invention still further relates to quantitatively and the detection and localization mouse interior-diagnostic testing process of alpha-D-mannosidase 25 levels.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.In the mouse that is detected in the test-alpha-D-mannosidase 25 levels, can with in the mouse that lays down a definition-disease that the importance of alpha-D-mannosidase 25 in various diseases and be used to diagnose in the mouse-alpha-D-mannosidase 25 works.
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.
In the coding mouse-polynucleotide of alpha-D-mannosidase 25 also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because in the mouse-and the nothing of alpha-D-mannosidase 25 is expressed or cell proliferation, growth or the metabolic disturbance of unusual/non-activity due to expressing.The gene therapy vector (as virus vector) of reorganization can be designed in the mouse of expressing variation-alpha-D-mannosidase 25, to suppress in the endogenic mouse-alpha-D-mannosidase 25 activity.For example, in a kind of mouse of variation-alpha-D-mannosidase 25 can be-alpha-D-mannosidase 25 though can combine with the substrate in downstream, to lack signaling activity in the mouse that shortens, lacked signal conduction function territory.Therefore the gene therapy vector of reorganization can be used for treating in the mouse-alpha-D-mannosidase 25 expresses or the disease of active caused by abnormal.Derive from the expression vector of virus such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for in the coding mouse-polynucleotide of alpha-D-mannosidase 25 are transferred in the cell.Structure carries in the coding mouse-and the method for the recombinant viral vector of the polynucleotide of alpha-D-mannosidase 25 is found in existing document (Sambrook, et al.).In addition in the reorganization coding mouse-polynucleotide of alpha-D-mannosidase 25 can be packaged in the liposome and be transferred in the cell.
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 in the mouse-oligonucleotide (comprising sense-rna and DNA) of alpha-D-mannosidase 25mRNA 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.
In the coding mouse-polynucleotide of alpha-D-mannosidase 25 can be used for mouse in-diagnosis of the relative disease of alpha-D-mannosidase 25.In the coding mouse-polynucleotide of alpha-D-mannosidase 25 can be used for detecting in the mouse-expression of alpha-D-mannosidase 25 whether or under morbid state in the mouse-unconventionality expression of alpha-D-mannosidase 25.As in the mouse of encoding-dna sequence dna of alpha-D-mannosidase 25 can be used for biopsy specimen is hybridized to judge in the mouse-the expression situation of alpha-D-mannosidase 25.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.With in the mouse-alpha-D-mannosidase 25 special primers carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro and also can detect in the mouse-transcription product of alpha-D-mannosidase 25.
Detect in the mouse-sudden change of alpha-D-mannosidase 25 genes also can be used for diagnosing in the mouse-disease that alpha-D-mannosidase 25 is relevant.In the mouse-form of alpha-D-mannosidase 25 sudden changes comprise with the normal wild type mouse in-point mutation that alpha-D-mannosidase 25DNA sequence is compared, 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.In the mouse-alpha-D-mannosidase 25 comes administration with the amount that treats and/or prevents concrete indication effectively.Be applied in patient's the mouse-amount and the dosage range of alpha-D-mannosidase 25 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 in the mouse of the present invention-alpha-D-mannosidase 25 and mouse in-the amino acid sequence homology comparison diagram of alpha-D-mannosidase.The top sequence is in the mouse-alpha-D-mannosidase 25, and the below sequence is in the mouse-the alpha-D-mannosidase.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+".
Fig. 2 is in the isolating mouse-the polyacrylamide gel electrophoresis figure (SDS-PAGE) of alpha-D-mannosidase 25.25KDa 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:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Embodiment 1: in the mouse-and the clone of alpha-D-mannosidase 25
Extract the total RNA of people's tire brain with guanidinium isothiocyanate/phenol/chloroform single stage method.From total RNA, separate poly (A) mRNA with Quik mRNA Isolation Kit (Qiegene company product).2ug poly (A) mRNA forms cDNA through reverse transcription.CDNA fragment orientation is inserted on the multiple clone site of pBSK (+) carrier (Clontech company product) with Smart cDNA clone's test kit (available from Clontech), transforms DH5 α, bacterium forms the cDNA library.Measure all clones' 5 ' and 3 ' terminal sequence with Dyeterminate cycle reaction sequencing kit (Perkin-Elmer company product) and ABI 377 automatic sequencers (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genebank) measured are compared, found that the cDNA sequence of one of them clone 0634a08 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 0634a08 clone is 2098bp (shown in Seq ID NO:1), from 99bp to 788bp the open reading frame (ORF) of a 690bp, the new protein (shown in SeqID NO:2) of encoding arranged.We clone called after pBS-0634a08 with this, in the encoded protein matter called after mouse-and alpha-D-mannosidase 25.Embodiment 2:cDNA clone's homology retrieval
With in the mouse of the present invention-sequence and the encoded protein sequence thereof of alpha-D-mannosidase 25, with Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], carry out the homology retrieval at databases such as Genbank, Swissport.With in the mouse of the present invention-gene that alpha-D-mannosidase 25 homologys are the highest is in a kind of known mouse-the alpha-D-mannosidase, its encoded protein number is AF023657 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 87%; Similarity is 94%.Embodiment 3: with in the RT-PCR method clones coding mouse-gene of alpha-D-mannosidase 25
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’-CTGATAACTTGGTACCCACTATTT-3’(SEQ?ID?NO:3)
Primer2: 5’-TTTTAAGGAGATTTATTTTTCGTA-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 1-2098bp shown in the SEQ ID NO:1 are identical.Interior-alpha-D-mannosidase 25 expression of gene that embodiment 4:Northern blotting is analyzed mouse:
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 in the mouse of pcr amplification shown in Figure 1-alpha-D-mannosidase 25 coding region sequences (99bp to 788bp).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: in the reorganization mouse-and vivoexpression, separation and the purifying of alpha-D-mannosidase 25
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’-CCCCATATGATGTACAAAAATGTCAAGTATATT-3’(Seq?ID?No:5)
Primer4: 5’-CATGGATCCTTAAGAAACAGGCAGCTGGCGATC-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-0634a08 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0634a08 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantage polymerase Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 60 ℃ of 30s, 68 ℃ of 2min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with 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-0634a08) 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-0634a08) is cultured to logarithmic phase at 37 ℃, adds IPTG to final concentration 1mmol/L, continues to cultivate 5 hours.Centrifugal collection thalline, through the broken bacterium of ultrasonic wave, centrifugal collection supernatant with carrying out chromatography with 6 Histidines (6His-Tag) bonded affinity column His.Bind Quick Cartridge (Novagen company product), has obtained in the target protein mouse of purifying-alpha-D-mannosidase 25.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 25KDa 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.In the embodiment 6 anti-mouse-alpha-D-mannosidase 25 production of antibodies
With in the synthetic following mouse of Peptide synthesizer (PE company product)-alpha-D-mannosidase 25 specific polypeptide:
NH2-Met-Tyr-Lys-Asn-Val-Lys-Tyr-Ile-Ile-Asp-Lys-Tyr-Gly-Asn-His-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 be specifically with mouse in-alpha-D-mannosidase 25 combines.Embodiment 7: polynucleotide passage of the present invention is as the application of hybridization probe
Picking out suitable oligonucleotide fragment from polynucleotide of the present invention is of use in many ways as hybridization probe, as can whether containing polynucleotide sequence of the present invention and detect the homologous polynucleotide sequence to identify it with the healthy tissues of different sources or the genome or the hybridization of cDNA library of pathological tissue with this probe, whether further also available this probe in detecting polynucleotide sequence of the present invention or the expression of its homologous polynucleotide sequence in healthy tissues or pathological tissue cell be unusual.
The purpose of present embodiment is to pick out suitable oligonucleotide fragment as hybridization probe from polynucleotide SEQ ID NO:1 of the present invention, and identifies in some tissues whether contain polynucleotide sequence of the present invention or its homologous polynucleotide sequence with the filter hybridization method.The filter hybridization method comprises dot blotting, Southern blotting, Northern blotting and copy method etc., and they all are that polynucleotide sample to be measured is fixed on use essentially identical step crossover in back on the filter membrane.These identical steps are: the filter membrane of having fixed sample at first carries out prehybridization with the hybridization buffer that does not contain probe, so that nonspecific combining site suppressed by vector of sample and synthetic polymer institute are saturated on the filter membrane.Prehybridization solution is contained the hybridization buffer replacement of label probe then, and insulation makes probe and target nucleic acid hybridization.After the hybridization step, the probe in the hybridization is not removed by a series of film steps of washing.Present embodiment utilizes the film condition of washing (as than low salt concn and higher temperature) of higher-strength, so that hybrid context reduces and only keep the signal of high specificity.The probe that present embodiment is selected for use comprises two classes: first kind probe be fully with the identical or complementary oligonucleotide fragment of polynucleotide SEQ ID NO:1 of the present invention; The second class probe is part and the identical or complementary oligonucleotide fragment of polynucleotide SEQ ID NO:1 of the present invention.Present embodiment selects for use dot blotting that sample is fixed on the filter membrane, higher-strength wash under the film condition, the hybridization specificity of first kind probe and sample is the strongest and kept.One, probe selects for use
From polynucleotide SEQ ID NO:1 of the present invention, select oligonucleotide fragment as hybridization probe, the several aspects that should follow following principle and need to consider: 1, the probe size preferable range is a 18-50 Nucleotide; 2, GC content is 30%-70%, and surpassing then, non-specific hybridization increases; 3, probe interior should not have complementary region; 4, what meet above condition can be used as the primary election probe, further do the computer sequential analysis then, comprise this primary election probe is carried out homology relatively with its source sequence area (being SEQ ID NO:1) and other known genome sequence and complementary district thereof respectively, if with the homology in non-target molecule zone greater than 85% or there are 15 continuous bases of surpassing identical, then this primary election probe is general just should not use; 5, whether the primary election probe finally is chosen to be the probe of actual application value also should further be determined by experiment.
Select and synthetic following two probes after finishing the analysis of above each side:
Probe 1 (probe1) belongs to first kind probe, and complete homology of gene fragment or the complementation (41Nt) of SEQ ID NO:1:
5’-TGTACAAAAATGTCAAGTATATTATAGACAAATATGGAAAT-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’-TGTACAAAAATGTCAAGTATCTTATAGACAAATATGGAAAT-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 overflows on ice and fill phosphate buffered saline buffer (PBS).With scissors or scalpel tissue is cut into small pieces.Should keep organizing moistening in the operation.2) with 1000g centrifugal chopper tissue 10 minutes.3) with cooled homogenate damping fluid (0.25mol/L sucrose; 25mmol/LTris-HCl, pH7.5; 25mmol/LNaCl; 25mmol/L MgCl 2) precipitation that suspends (approximately 10ml/g).4) 4 ℃ with electric homogenizer homogenate tissue suspension at full speed, until tissue by broken fully.5) 1000g is centrifugal 10 minutes.6) with re-suspended cell precipitation (the initial tissue sample of every 0.1g adds 1-5ml), centrifugal 10 minutes again with 1000g.7) with the resuspended precipitation of lysis buffer (the initial tissue sample of every 0.1g adds 1ml), connect following phenol extraction process then.2, the phenol extraction process of DNA
Step: 1) wash cell, centrifugal 10 minutes of 1000g with the cold PBS of 1-10ml.2) with the sedimentary cell (1 * 10 of cold cell pyrolysis liquid resuspension 8The minimum of applications 100ul lysis buffer of cell/ml).3) adding SDS is 1% to final concentration, if before re-suspended cell SDS is directly joined in the cell precipitation, cell may form big agglomerate and be difficult to fragmentation, and the overall yield that reduces.This point is in extracting＞10 7Especially severe during cell.4) add Proteinase K to final concentration 200ug/ml.5) 50 ℃ of insulation reaction 1 hour or 37 ℃ gently jolting spend the night.6) use equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, in little centrifuge tube centrifugal 10 minutes.Two corresponding clear separation, otherwise carry out centrifugal again.7) water is transferred to new pipe.8) use the equal-volume chloroform: primary isoamyl alcohol (24: 1) extracting, centrifugal 10 minutes.9) water that will contain DNA is transferred to new pipe.Carry out purifying and the ethanol sedimentation of DNA then.3, the purifying of DNA and ethanol sedimentation
Step: 1) 1/10 volume 2mol/L sodium-acetate and 2 times of cold 100% ethanol of volume are added in the dna solution mixing.-20 ℃ of placements 1 hour or to spending the night.2) centrifugal 10 minutes.3) careful sucking-off or pour out ethanol.4) with 70% cold ethanol 500ul washing precipitation, centrifugal 5 minutes.5) careful sucking-off or pour out ethanol.With the cold washing with alcohol precipitation of 500ul, centrifugal 5 minutes.6) careful sucking-off or pour out ethanol is inverted on thieving paper then residual ethanol is flow to end.Dry air 10-15 minute, so that the volatilization of surperficial ethanol.Note not making the precipitation complete drying, otherwise dissolve again than difficulty.7) with small volume TE or the resuspended DNA precipitation of water.The low speed vortex vibrates or uses the dropper pressure-vaccum, increases TE gradually simultaneously, is mixed to DNA and fully dissolves, every 1-5 * 10 6Cell extracted approximately adds 1ul.
Below the 8-13 step only be used for must removing when depolluting, otherwise can directly carry out the 14th step.8) RNA enzyme A is added in the dna solution, final concentration is 100ug/ml, and 37 ℃ are incubated 30 minutes.9) add SDS and Proteinase K, final concentration is respectively 0.5% and 100ug/ml.37 ℃ are incubated 30 minutes.10) use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extractive reaction liquid, centrifugal 10 minutes.11) carefully shift out water, use isopyknic chloroform: primary isoamyl alcohol (24: 1) extracting again, centrifugal 10 minutes.12) carefully shift out water, add 1/10 volume 2mol/L sodium-acetate and the cold ethanol of 2.5 volumes, mixing put-20 ℃ 1 hour.13) with 70% ethanol and 100% washing with alcohol precipitation, dry air, resuspended nucleic acid, process is with the 3-6 step.14) measure A 260And A 280To detect purity and the productive rate of DNA.15) deposit in-20 ℃ after the packing.The preparation of sample film: 1) get 4 * 2 suitably nitrocellulose filters (NC film) of size, mark point sample position and sample number thereon gently with pencil, each probe needs two NC films, so that wash film with high strength condition and strength condition respectively in the experimental procedure of back.2) draw and contrast respectively 15 microlitres, put on the sample film, dry at room temperature.3) place infiltration that 0.1mol/LNaOH is arranged, last 5 minute of filter paper (twice) of 1.5mol/LNaCl, drying to place to soak into has 0.5mol/L Tris-HCl (pH7.0), last 5 minute of filter paper (twice) of 3mol/LNaCl, dries.4) be sandwiched in the clean filter paper, wrap, 60-80 ℃ of vacuum-drying 2 hours with aluminium foil.
The mark 1 of probe) 3 μ lProbe (0.10D/10 μ l) add 2 μ lKinase damping fluids, 8-10uCi γ- 32P-dATP+2UKinase is to add to final volume 20 μ l.2) 37 ℃ are incubated 2 hours.3) add the tetrabromophenol sulfonphthalein indicator (BPB) of 1/5 volume.4) cross Sephadex G-50 post.5) to having 32Before washing out, P-Probe begins to collect first peak (available Monitor monitoring).6) 5/pipe, collect the 10-15 pipe.7) with liquid glimmer instrument monitoring isotopic weight 8) merge and be required preparation behind the collection liquid of first peak 32P-Probe (second peak for free γ- 32P-dATP).
Prehybridization
The sample film is placed plastics bag, and adding 3-10mg prehybridization solution (10 * Denhardt ' s; 6 * SSC, 0.1mg/mlCT DNA (calf thymus DNA).), seal sack after, 68 ℃ of water-baths were shaken 2 hours.
Hybridization
Plastics bag is cut off one jiao, adds the probe prepare, seal sack after, 42 ℃ of water-baths are shaken and are spent the night.
Wash film: high strength is washed film:
1) good sample film has been hybridized in taking-up.
2) 2 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃.
3) 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃.
4) 0.1 * SSC among the 0.1%SDS, washes 30 minutes (2 times) for 55 ℃, and room temperature is dried.Low strength is washed film:
1) good sample film has been hybridized in taking-up.
2) 2 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 37 ℃.
3) 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 37 ℃.
4) 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃, and room temperature is dried.
X-light autography:
-70 ℃, X-light autography (the compressing tablet time decides according to hybridization spot radioactivity is strong and weak).
Experimental result:
Adopt low strength to wash the hybrid experiment that the film condition is carried out, more than two strong and weak not obviously differences of probe hybridization spot radioactivity; And adopting high strength to wash the hybrid experiment that the film condition is carried out, the hybridization spot radioactive intensity of probe 1 obviously is better than the radioactive intensity of another probe hybridization spot.Thereby available probe 1 is qualitative and analyze existence and the differential expression of polynucleotide of the present invention in different tissues quantitatively.
Sequence table (1) general information:
(ⅱ) denomination of invention: in the mouse-alpha-D-mannosidase 25 and encoding sequence thereof
(ⅲ) sequence number: the information of 9 (2) SEQ ID NO:1:
(ⅰ) sequence signature:
(A) length: 2098bp
(B) type: nucleic acid
(C) chain: two strands
(D) topological framework: linearity
(ⅱ) molecule type: cDNA
( ⅹⅰ ) ：SEQ ID NO:1: 1 CTGATAACTTGGTACCCACTATTTTGGATAAAGCTCATAAATATAACCTAAAGGTTACTT 61 TTCACATAGAACCATATAGCAATCGAGATGATCAAAACATGTACAAAAATGTCAAGTATA121 TTATAGACAAATATGGAAATCATCCGGCCTTTTACAGGTACAAGACGAAGACTGGCAATG181 CTCTTCCTATGTTTTATGTCTATGATTCCTATATTACCAAGCCTGAAAAATGGGCCAATC241 TGTTGACCACCTCAGGGTCTCGGAGTATTCGCAATTCTCCTTATGATGGACTGTTTATTG301 CCCTTCTGGTAGAAGAAAAACATAAGTATGATATTCTTCAAAGTGGTTTTGATGGAATTT361 ACACATATTTTGCCACAAATGGCTTTACTTATGGCTCATCACATCAGAATTGGGCTAGCC421 TAAAATTATTTTGTGATAAATACAACTTAATATTTACCCCAAGTGTGGGCCCAGGATACA481 TAGATACCAGCATCCGTCCATGGAACACGCAAAACACTCGGAACCGAATCAATGGGAAGT541 ATTATGAAATTGGTCTGAGTGCCGCACTTCAGACACGCCCCAGCTTAATTTCTATCACCT601 CTTTTAATGAGTGGCATGAAGGAACTCAGATTGAAAAAGCTGTTCCCAAAAGAACCAGTA661 ATACAGTGTACCTAGATTACCGTCCTCATAAACCAGGTCTTTACCTAGAACTGACTCGCA721 AGTGGTCTGAAAAATACAGTAAGGAAAGAGCAACTTATGCATTAGATCGCCAGCTGCCTG781 TTTCTTAATGCATTGATTAAAGTTTAATAGTTATCAAAATCACCTAATTTTTAAAAATAG841 CTTTCGTTTTGAGTTCTGGAAAGAAAACTGTCAAAATCAGTATATACTATTAGTTATATT901 TAAAAATATTTTTTTAAATTCTTTACAGATAATATTATACTTGTTACCCTTCACAATACC 961 ACATGAGAAAATATCTGAGACAAAATGTATACAAATATATTCCTTATGGCATAATTTATT1021 GCATTTCTGACTGAAATCAAAATTCTGATTTGATGGCAATTGAATTTTCATTTTACAATA1081 GATAAATGCTTGTGCTACCTAAAGCACTTAGCACACAGTTAAATTATATTTACATCCTAG1141 ACCCAAATAAATAGGATTGTGTGTATATTTGGGATATCTATTGAAGAAAAAAAGAAAACC1201 CCTTAAAGATAATGTACATGCTTCATGTCGTGTCTTTAAAATAATTTAATCAACTTTATT1261 GTCTTAGTATTTAGACTCTGGATAACTCTACAATAATGAGGAAATTCTTAAGAATAACAA1321 AATCACTGTACCTTCCTCTCAATTTTGCTGTGAACCTGAAATGGCTTTAAATTAATACTC1381 TTATTTTTTATTTAATTTAATTACATAAATTAAACCTTACCATGACCAAATTGTGTTAGG1441 ACGGCCTGCTATCTACAGCACAGTGTGTCATTTGCAGATTTGTGGTTACCTATACCACGC1501 TAGGTGTTTTGACATGTTTAGTGTTTCTGCTTTACAGTGCTGAATTCCATATTTTAGAAG1561 CTATGAAAGTCCTTTTATGAAAAAGTTACTGATTGCTTCTCAGTTATTAGGAAAACAGTT1621 GTTTCACAATTATTATGTAGATATGATGCCCAAATATCATTTTTAGTATATCTTGTCGAT1681 CTTTAAGTTGTTACTATTGTGTTATTCATGTCTTTAAATCAGATACCAAATATTTTTTAG1741 GAAAGAAAAATGTTATTACTGTCATTAGGTTGTCTTTTAATACTTTAAGTTATTTTGACG1801 AAAAGTAATAGAGAAAATTTACTTAGCATTTTAGATTCTAGAGACATGGAAATGAAAATT1861 ATTTTATGTCTAGAGTAGGTCCTGAAGTTTGGCTTTACATTAAGTTTAGCACTGTATCAG1921 AATGAAGAAACTAATATTTTACATAAAAACTAATACTTTCAATTTTTTATATAGTAATAT1981 CCCCATTTTGTAAATGTTAGACTTTTATCATACCTGTAAGTTAAAATACTTGTTATCAAT2041 AACTTGTCATGTTGTGACAAATTGATCACTTGTGTACGAAAAATAAATCTCCTTAAAA ( 3 ) SEQ ID NO:2：
(ⅰ) sequence signature:
(A) length: 229 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅱ) molecule type: polypeptide
( ⅹⅰ ) ：SEQ ID NO:2: 1 Met Tyr Lys Asn Val Lys Tyr Ile Ile Asp Lys Tyr Gly Asn His16 Pro Ala Phe Tyr Arg Tyr Lys Thr Lys Thr Gly Asn Ala Leu Pro31 Met Phe Tyr Val Tyr Asp Ser Tyr Ile Thr Lys Pro Glu Lys Trp46 Ala Ash Leu Leu Thr Thr Ser Gly Ser Arg Ser Ile Arg Asn Ser61 Pro Tyr Asp Gly Leu Phe Ile Ala Leu Leu Val Glu Glu Lys His 76 Lys Tyr Asp Ile Leu Gln Ser Gly Phe Asp Gly Ile Tyr Thr Tyr 91 Phe Ala Thr Asn Gly Phe Thr Tyr Gly Ser Ser His Gln Asn Trp106 Ala Ser Leu Lys Leu Phe Cys Asp Lys Tyr Asn Leu Ile Phe Thr121 Pro Ser Val Gly Pro Gly Tyr Ile Asp Thr Ser Ile Arg Pro Trp136 Asn Thr Gln Asn Thr Arg Asn Arg Ile Asn Gly Lys Tyr Tyr Glu151 Ile Gly Leu Ser Ala Ala Leu Gln Thr Arg Pro Ser Leu Ile Ser166 Ile Thr Ser Phe Asn Glu Trp His Glu Gly Thr Gln Ile Glu Lys181 Ala Val Pro Lys Arg Thr Ser Asn Thr Val Tyr Leu Asp Tyr Arg196 Pro His Lys Pro Gly Leu Tyr Leu Glu Leu Thr Arg Lys Trp Ser211 Glu Lys Tyr Ser Lys Glu Arg Ala Thr Tyr Ala Leu Asp Arg Gln226 Leu Pro Val Ser ( 4 ) 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: the information of SEQ ID NO:3:CTGATAACTTGGTACCCACTATTT 24 (5) 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: the information of SEQ ID NO:4:TTTTAAGGAGATTTATTTTTCGTA 24 (6) 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: the information of SEQ ID NO:5:CCCCATATGATGTACAAAAATGTCAAGTATATT 33 (7) 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: the information of SEQ ID NO:6:CATGGATCCTTAAGAAACAGGCAGCTGGCGATC 33 (8) 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: information (ⅰ) sequence signature of SEQ ID NO:7:Met-Tyr-Lys-Asn-Val-Lys-Tyr-Ile-Ile-Asp-Lys-Tyr-Gly-Asn-His 15 (9) SEQ ID NO:8
(A) length: 41 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: the information of SEQ ID NO:8:TGTACAAAAATGTCAAGTATATTATAGACAAATATGGAAAT 41 (10) 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:TGTACAAAAATGTCAAGTATCTTATAGACAAATATGGAAAT 41

Claims (18)

1, in a kind of isolated polypeptide-mouse-and alpha-D-mannosidase 25, it is characterized in that it includes: the polypeptide of the aminoacid sequence shown in the SEQ IDNO:2 or the active fragments of its polypeptide, analogue or derivative.
2, polypeptide as claimed in claim 1, the aminoacid sequence that it is characterized in that described polypeptide, analogue or derivative has the homogeny with the aminoacid sequence at least 95% shown in 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 88% 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 SEQID NO:2.
6, polynucleotide as claimed in claim 4, the sequence that it is characterized in that described polynucleotide include the sequence of 1-2098 position among the sequence of 99-788 position among the SEQ IDNO: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 ofly have in the mouse-preparation method of alpha-D-mannosidase 25 active polypeptide, it is characterized in that described method comprises:
(a) in expressing mouse-alpha-D-mannosidase 25 conditions under, cultivate the described through engineering approaches host cell of claim 8;
(b) from culture, isolate and have in the mouse-alpha-D-mannosidase 25 active polypeptide.
10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with mouse in-alpha-D-mannosidase 25 specificity bonded antibody.
11, an analoglike or regulate polypeptide active or the compound of expression, it is characterized in that they be simulation, promotion, antagonism or suppress in the mouse-the active compound of alpha-D-mannosidase 25.
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 in the mouse-alpha-D-mannosidase 25 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,, it is characterized in that it is applied to screen in the mouse-stand-in of alpha-D-mannosidase 25, agonist antagonist or inhibitor as the application of polypeptide as described in the arbitrary claim among the claim 1-3; 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, as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11, it is characterized in that with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor with safe and effective dosage and pharmaceutically acceptable carrier form as in diagnosis or treatment and the mouse-pharmaceutical composition of the disease that alpha-D-mannosidase 25 is relevant unusually.
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 00115107 2000-03-24 2000-03-24 Polypeptide-human endomannosidase 25 and polynucleotide for coding it Pending CN1315551A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102286608A (en) * 2011-06-08 2011-12-21 王则宇 Chlamydia diagnosis method and kit

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102286608A (en) * 2011-06-08 2011-12-21 王则宇 Chlamydia diagnosis method and kit
CN102286608B (en) * 2011-06-08 2014-06-25 郑州安图生物工程股份有限公司 Chlamydia diagnosis method and kit

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