CN1414100A - Desmocyte growth factor 13 - Google Patents
Desmocyte growth factor 13 Download PDFInfo
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- CN1414100A CN1414100A CN02126409A CN02126409A CN1414100A CN 1414100 A CN1414100 A CN 1414100A CN 02126409 A CN02126409 A CN 02126409A CN 02126409 A CN02126409 A CN 02126409A CN 1414100 A CN1414100 A CN 1414100A
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- Peptides Or Proteins (AREA)
Abstract
A human fibroblast growth factor-polypeptide 13, the DNA (RNA) for coding it, the process for preparing the said polypeptide by recombination, the method for using the said polypeptide in medical purpose, the antagon of the said polypeptide and its medical application, and the diagnosing method by detecting the mutation in coding sequence and the change in polypeptide concentration are disclosed.
Description
The application be that June 5 nineteen ninety-five, application number are 95197868.3 the applying date, denomination of invention divides an application for the Chinese patent application of " desmocyte growth factor-21 3 ".
Technical field
The purposes of the polynucleotide that the present invention relates to differentiate recently, polypeptide, these polynucleotide and polypeptide and the production method of these polynucleotide and polypeptide by these polynucleotide encodings.More particularly, polypeptide of the present invention has been pushed and has been accredited as fibroblast growth factor/heparin binding growth factor (after this being called " FGF-13 ") qualitatively.The present invention also relates to suppress the method for the effect of these polypeptide.
Background technology
Fibroblast growth factor is to be a family of feature (and being also referred to as heparin binding growth factor (HBGF) thus) with the heparin-binding.In various tissues, find the expression of these proteinic different members, described being expressed under specified time and the spatial control.These protein are effective mitogen of the cell in mesoderm, ectoderm and entoderm source, and described cell comprises inoblast, cornea and vascular endothelial cell, granulocyte, adrenal cortical cell, chondrocyte, sarcoplast, vascular smooth muscle cell, lens epithelial cells, melanophore, keratinocyte, oligodendroglia, stellate cell, scleroblast and hemopoietic cell.
Every kind of member has and other member's eclipsed function, also has unique envelop of function.Except stimulating vascular endothelial cell proliferation, FGF-1 and 2 boths are endotheliocyte chemotaxiss, and FGF-2 has demonstrated and can make endotheliocyte penetrate basilar membrane.FGF-1 and 2 boths have the angiopoietic ability of stimulation.Another key character of these somatomedins is the ability that they promote wound healing.Many other members of FGF family have and FGF-1 and 2 similar activity, for example promote vascularization and wound healing.Several members of FGF family have demonstrated the differentiation of inducing mesoderm to form and regulate neuronal cell, adipocyte and Skeletal Muscle Cell.
Except these biologic activity in healthy tissues, hinted that FGF protein promotes cancer and sarcoma to take place by promoting tumor vessel to form, when their expression was out of control, it was as transforming protein matter.
FGF family is made up of the polypeptide of 8 structurally associateds at present: basic FGF, acid FGF, int 2, hstl/k-FGF, FGF-5, FGF-6, keratinocyte growth factor, AIGF (FGF-8), the neuroglia incitant is ascertained to be new heparin binding growth factor recently, it is culture supernatants purifying (Miyamoto from human glioma cells, M. etc., molecule and cytobiology, 13 (7): 4251-4259 (1993)).The gene of each peptide species is cloned and is checked order.Two member FGF-1 and FGF-2 are characterized with many titles, but modal be to be called as acidity and Prostatropin respectively.Normal gene product influences the general multiplication capacity of the cell in most of mesoderms and neuroderm source.They can form by the interior induction of vascular of body, and play an important role in may growing in early days (Burgess, W.H. and Maciag, T., bioid academic year comments, 58:575-606, (1989)).
The many above member who identifies of FGF family is also coupled on the identical acceptor, and by causing second messenger (message) in conjunction with these acceptors.
The eukaryotic expression system of the FGF-1 of coding secreted form is incorporated in the pig artery by transgenosis.This model has been determined the function of the gene in arterial wall.In transgenosis after 21 days, the induced expression pig endarterium thickening of FGF-1 (Nabel, E.G., etc., nature, 362:844-6 (1993)).Illustrate further, Prostatropin can be regulated the neurospongioma growth, and not relying on its effect in tumor vessel forms carries out, also illustrating Prostatropin release or secretion may be (the Morrison that needs to these effects, R.S., Deng, Neuroscience Research magazine, 34:502-9 (1993)).
Hint in addition fibroblast growth factor (as basic FGF) in the growth of external Kaposi cell, work (Huang, Y.Q., etc., J.Clin.Invest., 91:1191-7 (1993)).The cDNA sequence of human Prostatropin of encoding has been cloned into the transcripting promoter downstream by the identification of phage t7 RNA polymerase.So the Prostatropin that obtains at mitogenesis (mitogenicity), the plasminogen activator is synthetic and vascularization is found out to have the biologic activity (Squires that can't distinguish with human placenta's fibroblast growth factor in measuring, C.H., Deng, journal of biological chemistry, 263:16297-302 (1988)).
United States Patent (USP) 5,155,214 disclose Mammals Prostatropin and its production method of purifying in fact, disclose the aminoacid sequence of ox and human Prostatropin, and the dna sequence dna of coding ox species polypeptide.
Newfound FGF-9 has the sequence similarity with other members of FGF family about 30%.In the FGF-9 sequence, other consensus sequence of two cysteine residues and this family member obtains keeping.Found that FGF-9 does not possess the terminal typical signal sequence of those N-in acid and the basic FGF.Yet, lacking typical signal sequence FGF although find it, FGF-9 is synthesizing the back from emiocytosis (Miyamoto, M. etc., molecule and cytobiology, 13 (7): 4251-4259 (1993).In addition, find that FGF-9 stimulates the cell proliferation of oligodendroglia 2 type stellate cell progenitor cells, BALB/c3T3 and PC-12 cell, but the cell proliferation (Naruo of stimulating human huve cell not, K., Deng, journal of biological chemistry, 268:2857-2864 (1993).
Basic FGF and acid FGF are the strong instrumentalities that cell proliferation, cell move, break up and survive, and act on and derive from ectoderm, on mesoderm and the endoblastic cell type.These two kinds of FGF and KGF and AIGF differentiate through protein purification.Yet other four members separate as oncogene, and its expression is limited in embryo's generation and some types of cancer.Found out that FGF-9 is the mitogen at neurogliocyte.The member who has reported FGF family has the tumorigenesis potentiality.In the time of in transforming the BALB/c3T3 cell, FGF-9 demonstrated transform potentiality (Miyamoto, M., etc., molecule and cytobiology, 13 (7): 4251-4259 (1993).
By being purified into the male hormone inductive somatomedin (AIGF) that also is known as FGF-8 from the conditioned medium of mouse mastocarcinoma cell (SC-3) with the testosterone stimulation.AIGF is tangible class FGF-somatomedin, has the signal peptide sequence of inferring, and has the homology with the known member 30-40% of FGF family.The mammalian cell that transforms with AIGF demonstrates when not having male hormone the tangible hormesis of SC-3 cell.Therefore, the growth of the male hormone inducibility of AIGF mediation SC-3 cell (may also have other cells) is because it is secreted by tumour cell self.
Summary of the invention
Polypeptide of the present invention has been pushed the member who is accredited as FGF family qualitatively, because the result of itself and other member's amino acid sequence homology of FGF family.
According to one aspect of the present invention, the invention provides new mature polypeptide with and bioactive and the diagnosis on or the treatment on useful fragment, analogue and derivative.Polypeptide of the present invention is the people source.
According to another aspect of the present invention, the invention provides the isolated nucleic acid molecule of coding polypeptide of the present invention, comprise mRNA, DNA, cDNA, genomic dna with and antisense analogue and its biologic activity and at useful fragment and derivative in the diagnosis or in the treatment.
According to another aspect of the present invention, the invention provides the method for producing this peptide species through recombinant technology, the reorganization protokaryon and/or the eukaryotic host cell that in said recombinant technology, have adopted recombinant vectors (for example, in recombinant production polypeptide of the present invention as reagent useful clone and expression plasmid) and contained the nucleotide sequence of code book invention polypeptide.
According to another aspect of the present invention, the invention provides the method that polynucleotide with these polypeptide or these polypeptide of encoding are used to screen agonist and antagonist and are used for the treatment of, described methods of treatment for example, the neuronal damage that promotes neuronal damage that wound (as burn and ulceration) healing, prevention are relevant with apoplexy and cause owing to the neurone disorder, promote neure growth, prevent skin aging and epilation, the mesoderm in the stimulation vascularization, stimulation early embryo induces and limb regeneration.
According to another aspect of the present invention, the invention provides the antibody of these polypeptide.
According to another aspect of the present invention, the invention provides the antagonist and the method for using it for the effect that suppresses these polypeptide of said polypeptide, for example, the treatment cell transformation (as, tumour), reduce too much (hyper-vascular) disease of cicatrization and treatment blood vessel.
According to another aspect of the present invention, the invention provides nucleic acid probe, this nucleic acid probe comprises length and is enough to specifically nucleic acid molecule with the multi-nucleotide hybrid of coding polypeptide of the present invention.
According to another aspect of the present invention, the invention provides and detect the disease relevant or the susceptibility of disease and detection diagnostic measurement method by the overexpression of the polypeptide of described sequence encoding with sudden change in the nucleotide sequence of the present invention.
According to another aspect of the present invention, the invention provides these polypeptide, or the method for the relevant purpose of the external preparation that is used for and dna vector synthetic with scientific research, DNA of the polynucleotide of these polypeptide of encoding.
From the instruction of this paper, those skilled in the art can know these and other aspect of the present invention.Description of drawings
Following accompanying drawing only is used for illustrating embodiment of the present invention, and has no intention to be used for limiting the included scope of claim of the present invention.
Fig. 1 has described cDNA sequence and the corresponding deduced amino acid of FGF-13,21 leader sequences that the amino-acid residue representative is inferred.
Specific embodiments
According to one aspect of the present invention, the invention provides a kind of isolated nucleic acid molecule (polynucleotide), this nucleic acid encoding have Fig. 1 deduced amino acid (SEQ ID NO:2) mature polypeptide or by the mature polypeptide of May 12 nineteen ninety-five with the clone's of preserving number ATCC 97148 preservations cDNA coding.
The polynucleotide of FGF-13 polypeptide of the present invention of encoding are at first found in deriving from the cDNA library of human ovarian cancer tissue.The FGF-13 polypeptide is structurally relevant with all members of inoblast family, and it comprises the open reading frame of 212 amino acid whose polypeptide of a coding, 21 leader sequences that amino acid represent one is inferred wherein, so mature polypeptide has 191 amino acid.The situation of coupling is: 1) with mouse AIGF 69% homogeny and 81% similarity are arranged on one section 185 aminoacid sequence; 2) on one section 82 aminoacid sequence, have 30% homogeny and 56% similarity with chicken FGF-4; 3) on one section 78 amino acid whose sequence, 41% homogeny and 64% similarity are arranged with people KGF.
In polypeptide of the present invention, and the mark GXLX of FGF/HBGF family (S, T, A, G) (D, E) (X refers to any amino acid to CXFXE to X6, and (D E) refers to D or E residue, and X6 refers to any 6 amino-acid residues) guards.
Polynucleotide of the present invention can be rna form or dna form, and wherein DNA comprises cDNA, genomic dna and synthetic DNA, and DNA can be two strands or strand.The encoding sequence of this encoding mature polypeptide can be identical with the clone's of encoding sequence (SEQ ID NO:1) shown in Figure 1 or preservation encoding sequence; Because the Feng Yu or the degeneracy of genetic code, this encoding sequence also can be a kind of different encoding sequence, the identical mature polypeptide of cDNA coding of the DNA (SEQ ID NO:1) of its energy and Fig. 1 or said preservation thing.
Mature polypeptide of code pattern 1 (SEQ ID NO:2) or coding can be comprised by the polynucleotide of the mature polypeptide of the cDNA coding of described said preservation thing: the encoding sequence that only is the encoding mature polypeptide; The encoding sequence of encoding mature polypeptide and additional encoding sequence are as leading or secretion sequence or proteinogen (proprotein) sequence; Encoding sequence of encoding mature polypeptide (with dispensable additional encoding sequence) and non-coding sequence are as 5 of intron or mature polypeptide encoded sequence ' and/or 3 ' non-coding sequence.
Like this, " polynucleotide of coded polypeptide " this term comprises polynucleotide that only contain polypeptid coding sequence and the polynucleotide that also contain additional coding and/or non-coding sequence.
The invention still further relates to above-described polynucleotide variant, this variant coding have Fig. 1 deduced amino acid (SEQ ID NO:2) polypeptide or by fragment, analogue and the derivative of the clone's of preservation cDNA encoded polypeptides.The polynucleotide variant that polynucleotide allelic variant that this polynucleotide variant can be natural generation or non-natural produce.
Like this, the present invention includes coding as shown in Figure 1 identical mature polypeptide (SEQ ID NO:2) or by the polynucleotide of the identical mature polypeptide of the clone's of preservation cDNA coding, and the mature polypeptide of coding as Fig. 1 (SEQ ID NO:2) shown in or the polynucleotide variant of fragment, derivative and the analogue of the mature polypeptide of encoding by the clone's of preservation cDNA.These nucleotide variants comprise disappearance variant, replacement variant, interpolation or insert variant.
As above indicated, described polynucleotide can have a kind of encoding sequence, and this sequence is the clone's of encoding sequence shown in Fig. 1 (SEQ ID NO:1) or preservation the allelic variant of natural generation of encoding sequence.Allelic variant known in the art is the another kind of form of polynucleotide sequence, and it can have replacement, disappearance or the interpolation of one or more Nucleotide, and does not change the function of encoded polypeptide in fact.
The present invention also comprises such polynucleotide, wherein the encoding sequence of said mature polypeptide can be in identical reading frame with help to express in the host cell and the polynucleotide sequence of secrete polypeptide merges, described polynucleotide sequence is as controlling the leader sequence that polypeptide works as secretion sequence from cell traffic.Polypeptide with leader sequence is preceding albumen (preprotein), and can have the leader sequence by the polypeptide of host cell cutting formation mature form.This polynucleotide are proteins encoded former (proprotein) also, and this proteinogen is the maturation protein that is added with additional 5 ' amino-acid residue.Maturation protein with former sequence (prosequence) is a proteinogen, is a kind of albumen form of non-activity.In case excise former sequence, what stay is activated maturation protein.
Can encode a kind of maturation protein or coding has the protein of former sequence or the existing former sequence of encoding has the protein of presequence (presequence) (leader sequence) again of polynucleotide like this, for example of the present invention.
Polynucleotide of the present invention also can have the encoding sequence that merges with flag sequence in frame, described flag sequence makes can purifying polynucleotide of the present invention.Under the situation of host bacterium, described flag sequence can be six histidine marks that provided by the pQE-9 carrier, it is used for purifying and merges markd mature polypeptide, perhaps for example when using mammalian cell (as the COS-7 cell), flag sequence can be hemagglutinin (HA) mark.Said HA mark corresponding to derive from the proteinic a kind of epi-position of influenza hemagglutinin (Wilson, I., etc., cell, 37:767 (1984)).
Term " gene " is meant the DNA section relevant with producing polypeptide chain; It comprises coding region region in front and zone subsequently (leader and tailer sequence) and the intervening sequence (intron) between each coding section (exon).
The fragment of total length FGF-13 gene can be as the hybridization probe in cDNA library, be used for separating full-length gene and therewith gene sequence similarity or similar bioactive other gene highly arranged.Such probe preferably has at least 30 bases, and can contain, for example 50 or more base.Said probe also can be used to differentiate the genomic clone of cloning and contain the complete FGF-13 gene that comprises adjusting and promoter region, exon and intron corresponding to the cDNA of total length transcript.The example of screening comprises by utilizing the known dna sequence synthetic oligonucleotide probe to separate the coding region of FGF-13 gene.Labeled oligonucleotide with the sequence that is complementary to gene order of the present invention can be used for screening human cDNA, genomic dna or mRNA library, to determine probe and which library member hybridization.
The invention still further relates to and the polynucleotide (condition is to have at least 70% between two sequences, preferably has at least 90%, more preferably has at least 95% homogeny) of sequence hybridization described above.The present invention be more particularly directed under stringent condition polynucleotide with above-described multi-nucleotide hybrid.As used herein, term " stringent condition " refers to only have at least 95% between sequence, and hybridization just can take place when preferably having at least 97% homogeny.In a preferred embodiment, with the such peptide species of the polynucleotide encoding of above-described multi-nucleotide hybrid, it keeps in fact and identical biological function or the activity of mature polypeptide by cDNA (s) coding of the cDNA (SEQ ID NO:1) of Fig. 1 or preservation.
In addition, described polynucleotide can have at least 20 bases, and preferably at least 30 bases more preferably are at least 50 bases, and itself and multi-nucleotide hybrid of the present invention, and have homogeny as indicated above can keep or retentive activity not.For example, this polynucleotide can be as the probe of SEQ ID NO:1 polynucleotide, for example is used to reclaim polynucleotide or as diagnostic probe or as the PCR primer.
Like this, the present invention relates to and the SEQ ID NO:2 polypeptide of encoding more than Nucleotide have at least 70% homogeny, preferably at least 90% homogeny and the more preferably polynucleotide of at least 95% homogeny and the polypeptide of fragment (this fragment has at least 30 bases, preferably at least 50 bases) and these polynucleotide encodings thereof.
The mentioned preservation thing of this paper will keep according to the regulation of the microbial preservation budapest treaty of the international recognition that is used for patented procedure.These keep thing only is in order to provide convenience to those skilled in the art, is not 112 required preservations of 35 U.S.C.Be included in the described preserved material polynucleotide sequence and by its amino acid sequence coded this paper reference in the lump, and be used to solve any contradiction on this paper sequence description.Any manufacturing, use or sale to preserved material need not give any such permission through permission at this.
The invention still further relates to the FGF polypeptide of deduced amino acid (SEQ ID NO:2) or have polypeptide by the cDNA amino acid sequence coded of preservation with Fig. 1, and the fragment of this peptide species, analogue and derivative.
Term " fragment ", " derivative " and " analogue " during when the polypeptide (SEQ ID NO:2) of relevant Fig. 1 or by the cDNA encoded polypeptides of preservation, refer to the biological function or the active polypeptide that keep identical with such polypeptide basically.Like this, analogue comprises proteinogen, and this analogue can partly excise and then produce active mature polypeptide by proteinogen.
Polypeptide of the present invention can be a recombinant polypeptide, natural polypeptides or synthetic polypeptide, preferably recombinant polypeptide.
The polypeptide of said Fig. 1 (SEQ ID NO:2) or by the fragment of the cDNA encoded polypeptides of preservation, derivative or analogue can be: (i) a kind of like this, wherein one or more amino-acid residues can be also can not be by genetic codon amino acids coding residue by the amino-acid residue that conservative or non-conservative amino acid residues replaces (preferably conservative amino acid residues replacement) and replacement, perhaps (ii) a kind of like this, wherein one or more amino-acid residues comprise substituting group, perhaps (iii) a kind of like this, wherein mature polypeptide and another kind of compound merge, described compound is as increasing the polypeptide compound (for example polyoxyethylene glycol) of half life, perhaps (iv) a kind of like this, wherein additional amino acid and mature polypeptide fusion, for example leading or secretion sequence or be used for the sequence or the former sequence of purifying mature polypeptide.By the elaboration of this paper, can think that such fragment, derivative and analogue is within those skilled in the art's ken.
The present invention preferably provides the polypeptide and the polynucleotide of unpack format, and preferably described polypeptide is become homogeneous with the polynucleotide purifying.
Term " isolating " means described material and has broken away from its primal environment (for example, natural surroundings is if it is natural generation).For example, a kind of polynucleotide or polypeptide that is present in the natural generation in the animal alive is not isolating, but with natural system in the material of some or all coexistence identical polynucleotide or the polypeptide that separate be isolating.Such polynucleotide can be the parts of carrier, and/or such polynucleotide or polypeptide can be the part of composition, and it remains isolating, and this is because this carrier or composition are not the parts of its natural surroundings.
Polypeptide of the present invention comprise SEQ ID NO:2 polypeptide (particularly mature polypeptide) and and the polypeptide of SEQ ID NO:2 have at least 70% similarity (preferably 70% homogeny), it more preferably is 90% similarity (preferably 90% homogeny), it most preferably is the polypeptide of 95% similarity (preferably 95% homogeny), the part that also comprises these polypeptide, the part of this peptide species comprises at least 30 amino acid usually, and at least 50 amino acid preferably.
As known in the art, " similarity " between two polypeptide is by relatively a polypeptide and another amino acid sequence of polypeptide and its conserved amino acid replace to determine.
The fragment of polypeptide of the present invention or part are synthesized by peptide can be used to produce corresponding full-length polypeptide; Therefore, this fragment can be as the intermediate that produces full-length polypeptide.The fragment of polynucleotide of the present invention or part can be used for synthetic total length polynucleotide of the present invention.
The present invention also relates to comprise the carrier of polynucleotide of the present invention and the host cell that produces through genetically engineered with carrier of the present invention, and the method for producing polypeptide of the present invention through recombinant technology.
Host cell produces through genetically engineered operation (transduction, conversion or transfection) with carrier of the present invention, and said carrier can be cloning vector or expression vector.This carrier for example can be, forms such as plasmid, virion and phage.The through engineering approaches host cell can improvement be suitable for activate promotor, select to cultivate in the conventional nutritional medium of transformant or amplification FGF gene.Culture condition, for example temperature and pH value etc. are those of host cell that were used to express selection in the past, are conspicuous to those of ordinary skill.
Polynucleotide of the present invention can be used for producing polypeptide through recombinant technology.Like this, for example, polynucleotide can be included in any of the various expression vectors that are used for express polypeptide.Such carrier comprises karyomit(e), non-chromosome and synthetic DNA sequence, for example SV 40 derivatives; Bacterial plasmid; Phage DNA; Yeast plasmid; Plasmid and phage DNA combination deutero-carrier, viral DNA (as vaccinia virus, adenovirus, fowlpox virus and pseudorabies virus).Yet any other carrier also can use, as long as it is reproducible and stable in the host.
Can suitable dna sequence dna be inserted in the carrier with several different methods.In general, with methods known in the art dna sequence dna is inserted into suitable restriction endonuclease site.Such method and other method are considered in those skilled in the art's ken.Said dna sequence dna in expression vector is to be operably connected to suitable instructing on the mRNA synthetic expression control sequenc (promotor).The representative example of such promotor can should be mentioned that: LTR or SV 40 promotors, colibacillary lac or trp, phage P
LPromotor and known other promotor that controlling gene is expressed in protokaryon or eukaryotic cell or their virus.Said expression vector also comprises the ribosome bind site that is used for translation initiation and Transcription Termination.This carrier also can comprise the suitable sequence of expressing for amplification.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic characteristic of transformed host cells, for example Tetrahydrofolate dehydrogenase of eukaryotic cell culture or neomycin resistance, or tsiklomitsin and the amicillin resistance in the intestinal bacteria for example.
Comprise above-described suitable dna sequence dna and suitable promotor or the carrier of control sequence and can be used to transform suitable host, so that it can marking protein.As the representative example of suitable host, can should be mentioned that here: bacterial cell, as intestinal bacteria, streptomycete, Salmonella typhimurium; The fungal cell is as yeast; Insect cell such as fruit bat S2 and Spodoptera Sf9; Zooblast such as CHO, COS or Bowes melanoma; Adenovirus; Vegetable cell etc.By the elaboration of this paper, within the ken that is chosen in those skilled in the art to suitable host.
More particularly, the present invention also comprises recombinant precursor, and this construct comprises above broadly described one or more sequences.This construct comprises carrier, and as the carrier of plasmid or virus, this carrier has inserted sequence of the present invention forward or backwards.Under the even more ideal situation of this embodiment, construct also comprises the adjusting sequence that can be operationally connected on the described sequence, comprise, for example, promotor.A large amount of carrier and promotors that are fit to are well known by persons skilled in the art, and are obtainable by commercial sources.Provide following carrier by way of example: bacterium: pQE70, pQE60, pQE-9 (Qiagen), pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene), pTRC99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eucaryon: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Parmacia).Yet any other plasmid or carrier can use, as long as they are reproducible and stable in the host.
Can be with CAT (CAT) carrier or other carrier that has selected marker from any required gene Selection promoter region.Two kinds of suitable carriers are pKK232-8 and pCM7.The bacterium promotor of mentioning especially comprises lacI, lacZ, T3, T7, gpt, λ P
RP
L, and trp.Promoter in eukaryote comprises that CMV is early stage immediately, HSV thymidine kinase, early stage and late period SV40, derive from retroviral LTRs and mouse metallothionein(MT)-I.Within the level that is chosen in those of ordinary skills to appropriate carriers and promotor.
In another embodiment, the present invention relates to comprise the host cell of the above construct.Said host cell can be higher eucaryotic cells (as a mammalian cell), or eukaryotic cell (as yeast cell) such as low, and perhaps host cell can be prokaryotic cell prokaryocyte (as a bacterial cell).Can be by the transfection of calcium phosphate transfection, DEAE-dextran mediation, or electroporation is incorporated into construct (Davis, L., Dibner, M., Battey, I., the basic skills in the molecular biology, (1986)) in the host cell effectively.
Construct in the host cell can be used for producing in a usual manner the gene product by the recombination sequence coding.In addition, polypeptide of the present invention can produce by conventional peptide synthesizer is synthetic.
Mature protein can be expressed under suitable promotor control in mammalian cell, yeast, bacterium or other cell.Employing derives from the RNA of DNA construct of the present invention, and cell free translation system also can be used for producing this protein.Sambrook etc., molecular cloning: laboratory manual, second edition, the cold spring port, N.Y., the suitable clone and the expression vector that use with protokaryon and eucaryon host have been described in (1989) (this paper is reference in the lump).
The DNA of polypeptide of the present invention of encoding strengthens in the enhancer sequence that transcribing of higher eucaryote is inserted in the carrier.Enhanser is the cis-acting elements of DNA, and usually about 10 to 300bp, and acting on increases it and transcribe on the promotor.Example comprises polyoma enhanser and the adenovirus enhanser on SV 40 enhansers on the replication orgin side in late period 100 to 270bp, cytomegalovirus early promoter enhanser, the replication orgin side in late period.
In general, recombinant expression vector comprises replication orgin and allows the selected marker (for example, colibacillary ampicillin resistance gene and Saccharomyces cerevisiae TRP1 gene) of host cell conversion and the promotor that instructs the downstream configurations sequence to transcribe that comes from cance high-expression gene.Such promotor can be come the operon of own coding glycolytic ferment (for example glycerol 3-phosphate acid kinase (PGK)), α-factor, acid phosphatase or heat shock protein etc.The allos structure sequence is with suitable manner (phase) and translation initiation and terminator sequence assembling, and preferably, the leader sequence that advances periplasmic space or extracellular substratum with the protein secreting that can instruct translation assembles.Heterologous sequence can be also encoding fusion protein not, this protein comprises the terminal peptide of differentiating of the N-that gives required feature, required feature for example, the stability of the recombinant products of expression or simplify purification step.
Structural DNA sequence by the desired protein of will encoding and suitable translation initiation and termination signal are inserted and are made up the useful expression vector that is used for bacterium with having functional promotor can operate reading method (reading phase).Said carrier comprises one or more Phenotypic Selection marks and replication orgin, to guarantee to keep carrier the host and amplification is provided when needing.The prokaryotic hosts that is fit to transform comprises the various of intestinal bacteria, subtilis, Salmonella typhimurium, Rhodopseudomonas, streptomyces and Staphylococcus, and other bacterium also can be selected to use certainly.
As representational but be not restrictive example, the useful expression vector that is used for bacterium can comprise selected marker and the bacterium replication orgin that comes from commercially available plasmid (genetic elements that comprises well-known cloning vector pBR322 (ATCC37017)).Commercially available carrier like this comprises, for example, and pKK223-3 (Pharmacia Fine chemical company, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, the U.S.).These pBR322 " skeleton " fragment and suitable promotor and structure sequence combination to be expressed.
Transform and after host strain grows to suitable cell density at the appropriate host bacterial strain, induce the promotor of selection with suitable method (for example temperature inversion or chemical induction), other for some time of cell cultures.
Typically,, keep formed crude product to be used for further purifying through physics or chemical process smudge cells through centrifugal cell harvesting.
The microorganism cells that can be used for marking protein through the method fragmentation of any routine, described method comprise freeze-thaw cycle, supersound process, Mechanical Crushing or use the lysis agent.
Various mammalian cell culture systems also can be used for express recombinant protein matter.The example of mammalian expression system comprises by the monkey kidney inoblast COS-7 clone of Gluzman (cell, 23:175 (1981)) description and other can express the clone of compatible carrier, for example, and C127,3T3, CHO, HeLa and bhk cell system.Mammalian expression vector comprises replication orgin, suitable promotor and enhanser, also can comprise any essential ribosome bind site, polyadenylation site, donor splicing site and acceptor site, transcription termination sequence and 5 ' flank non-transcribed sequence.Derive from the required non-transcribed genetic elements of can being used to provide of virus genomic dna sequence dna of SV40 such as SV40 starting point, early promoter, enhanser, spliceosome and polyadenylation site.
Polypeptide of the present invention can reclaim and purifying from the reconstitution cell culture with several different methods, and described method comprises ammonium sulfate or ethanol sedimentation, sour extraction, negatively charged ion or cation-exchange chromatography, phosphorus Mierocrystalline cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and phytohemagglutinin chromatography.In finishing the configuration of mature protein, can use the protein refolding step when needing.At last, can use high performance liquid chromatography (HPLC) as last purification step.
Polypeptide of the present invention can be the product of natural purifying, or the product of chemical synthesis process, or produce from protokaryon or eucaryon host (for example insect of bacterium, yeast, higher plant, cultivation and mammalian cell) through recombinant technology.According to the host who uses in the recombinant method for production, polypeptide of the present invention can be glycosylated maybe can be nonglycosylated.Polypeptide of the present invention also can comprise initial methionine residues.
Owing to have the ability that stimulates vascular endothelial cell growth, polypeptide of the present invention can be used for stimulating the blood vessel of the ischemic tissue that is caused by various diseases (as thrombosis, arteriosclerosis and other cardiovascular disorder) to form the treatment of (revascularization) again.These polypeptide also can be used to stimulate vascularization and limb regeneration.
Described polypeptide also can be used for the treatment of tissue repair and ulcer after damage wound, burn, the operation, because they are mitogenetic to the various cells (as inoblast and Skeletal Muscle Cell) of different sources.And the therefore reparation of promotion damage or illing tissue or alternative.
Described polypeptide also can be used for stimulating neuronal growth, the neuronal damage that is used for the treatment of the neuronal damage relevant with apoplexy with prevention and occurs in some neurone disorder or neurasthenia (Alzheimer's, Parkinson's disease and AIDS-are correlated with syndromes).FGF-13 has the ability that stimulates chondrocyte's growth, and therefore, they can be used to strengthen bone and periodontal regenerative and help tissue transplantation and the bone transplanting.
The skin aging that polypeptide of the present invention also can be caused by sunburn by the growth prevention that stimulates keratinocyte.
The FGF-13 polypeptide also can be used to prevent alopecia, because the FGF family member activates the hair founder cell, and promotes melanocyte growth.Simultaneously, polypeptide of the present invention also can be used to stimulate the growth and the differentiation of hemopoietic cell and medullary cell when using with other combination of cytokines.
The cell culture that the FGF-13 polypeptide also can be used for keeping organ before transplanting or support prior structure.
Polypeptide of the present invention also can be used for the cytodifferentiation that embryo is in early days induced the mesoderm source.
According to another aspect of the present invention, the invention provides for scientific research, DNA syntheticly, the relevant external purpose of dna vector preparation and the diagnostic reagent that human diseases the is provided purpose relevant with therapeutical agent are utilized the method for the polynucleotide of this peptide species or these polypeptide of encoding.
The invention provides the method for the acceptor of differentiating polypeptide of the present invention.Can be with the gene of the said acceptor of many method known to those skilled in the art identifier numbers, described method such as part elutriation and FACS sorting (Coligan etc., immunology scheme in the present age, 1 (2), the 5th chapter, (1991)).Preferably, use cloning by expression, wherein from (for example to the reactive cell of described polypeptide, known NIH3T3 cell and the SC-3 cell that contains some acceptors of FGF family protein) preparation polyadenylic acid RNA, to be divided into a plurality of aggregates from the cDNA library that this RNA produces, be used for rotaring redyeing COS cell or other cell non-reacted described polypeptide.The transfectional cell that is grown on the slide glass is contacted with the polypeptide of the present invention of mark, and described polypeptide can be by several different methods (comprise iodate or introduce site-specific protein kinases recognition site) mark.
Behind fixing and incubation, slide glass is carried out radioautographic analysis.Differentiate positive aggregate, by repeating inferior set and rescreen the choosing method preparation and the inferior aggregate of transfection again, the final mono-clonal that produces the acceptor that coding infers.
The another kind of method that acceptor is differentiated is the polypeptide of mark can be connected with extract light is affine with the cytolemma of expressed receptor molecule, separates cross-linked material through PAGE, and x-ray film is exposed.Can downcut the labeled complex that comprises described polypeptide receptor, be separated into peptide fragment and carry out protein micrometering preface.The aminoacid sequence that obtains from the micrometering preface is used to design the degenerate oligonucleotide probe in a cover screening cDNA library, the gene of the acceptor of inferring with identifier number.
The invention provides the method for SCREENED COMPOUND with the compound of the effect of discriminating adjusting polypeptide of the present invention.An example of this detection is included under the condition of the normal propagation of inoblast Mammals inoblast, polypeptide of the present invention, compound to be screened and
3[H] thymidine mixes.Can treat to carry out under the SCREENED COMPOUND blank determination not existing, and with the amount of its fibroblast proliferation when having described compound relatively, thereby by measuring under every kind of situation
3The absorption of [H] thymidine determines whether described compound stimulates proliferation.By measuring
3The amount of fibroblast proliferation is measured in the liquid scintillation counting(LSC) that [H] thymidine mixes.Agonist and agonist compounds all can be differentiated by this method.
In another approach, mammalian cell or film preparation polypeptide with mark of the present invention in the presence of described compound of expressing the acceptor of polypeptide of the present invention are cultivated.Measure described compound enhancing then or block this interactional ability.Perhaps, be determined at the reaction of the known second messenger system behind compound to be screened and the FGF-13 acceptor interaction, and measure described compound bind receptor and cause the ability that the second messenger reacts, to determine whether described compound is potential agonist or antagonist.This second messenger system includes but not limited to cAMP guanylate cyclase, tyrosine phosphorylation effect, ionic channel or phosphoinositide hydrolysis effect.
The example of agonist compounds comprises antibody, perhaps comprises oligonucleotide in some cases, and it is in conjunction with the acceptor of polypeptide of the present invention, and the second messenger reacts or they are in conjunction with FGF-13 polypeptide itself but do not cause.In addition, the potential antagonist can be the mutant form of described polypeptide, and it is in conjunction with described acceptor but do not cause second messenger reaction, and therefore, the effect of described polypeptide is blocked effectively.
The agonist compounds of another FGF-13 gene and gene product is the antisense constructs that adopts the antisense technology preparation.The expression that antisense technology can come controlling gene by triple helical formation or antisense DNA or RNA, these two kinds of methods are all based on the combination of polynucleotide and DNA or RNA.For example, 5 of the polynucleotide sequence of the mature polypeptide of the present invention of encoding, encoding part can be used for the antisense rna oligonucleotide of about 10 to 40 base pairs of design length.A kind of DNA oligonucleotide is designed to and transcribes related gene regions complementation (triple helical-referring to Lee etc., nucleic acids research, 6:3073 (1979); Cooney etc., science, 241:456, (1988); With Dervan etc., science, 251:1360 (1991)), and then stop transcribing and producing of polypeptide of the present invention.Antisense rna oligonucleotide is hybridized with mRNA in vivo, and the translation becoming of blocking-up mRNA molecule polypeptide of the present invention (antisense-Okano, J.Neurochem., 56:560 (1991); Oligodeoxynucleotide is as the antisense inhibitor (CRC press, Boca Raton, FL (1988)) of genetic expression.Oligonucleotide described above can be sent in the cell, so that can expression in vivo sense-rna and DNA, suppresses the generation of polypeptide of the present invention.
The potential agonist compounds also comprises small molecules, and these small molecules are attached on the binding site of acceptor and occupy this site, make acceptor not stop normal biologic activity thus near its polypeptide.Micromolecular example includes but not limited to little peptide or class peptide molecule.
Agonist compounds also can be used at the cell growth of tumour cell and tissue inhibition polypeptide of the present invention and proliferation function (being the hormesis that tumor vessel takes place), and therefore postpones or stop abnormal cell growth and propagation in for example tumour forms and grows.
Described antagonist also can be used to prevent the too much disease of blood vessel and prevent epithelium lens cell propagation behind the cataract operation outside capsule.Under the situation of the restenosis behind balloon angioplasty for example, the mitogenesis activity of blocking polypeptide of the present invention also may be required.
Described antagonist also can be used for stoping the scar tissue growth during wound healing.
Described antagonist can be used from the composition with pharmaceutically acceptable carrier one as described below.
Polypeptide of the present invention, agonist and antagonist can be used in combination with suitable pharmaceutical carrier, and to constitute the pharmaceutical composition for parenteral administration, such composition comprises said polypeptide and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.Such carrier includes but not limited to salt solution, buffer saline, dextrose, water, glycerine, ethanol and their composition.The mode that its prescription should be suitable for using.
The present invention also provides pharmaceutical pack or test kit, and they comprise one or more containers that are filled with one or more compositions of pharmaceutical composition of the present invention.Can be in this container with the bulletin of government organs' prescribed form of managing medicine and biological products manufacturing, use or sale, this bulletin has reflected manufacturing, use or sold the mankind makes articles for use obtain the agreement of government organs.In addition, polypeptide of the present invention, agonist and antagonist can be treated compound with other and be used in combination.
Described pharmaceutical composition can be used in mode easily, described mode is for example oral, local, in intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the intradermal approach use.Said pharmaceutical composition is used with the significant quantity that treats and/or prevents specified disease.In general, they are used with the amount of about at least 10 micrograms/kg body weight, and in most of the cases, they are used with the amount that is no more than about 8 mg/kg body weight every day.Under most applications, consider factors such as route of administration and illness, dosage from every day about 10 microgram/kilograms to 1 mg/kg body weight.Under the particular case of topical application, preferably with from about 0.1 μ g to 9mg/cm
2Dosage use.
The agonist of polypeptide of the present invention and polypeptide form and antagonist can utilize by the such polypeptide of expression in vivo according to the present invention, and this often is known as " gene therapy ".
Like this, for example, can external pair cell carry out the genetically engineered operation with the polynucleotide (DNA or RNA) of coded polypeptide, the patient who treats to quilt with engineering cell provides said polypeptide.Such method is well-known in the art, and also apparent by the description of this paper.For example, can use the retroviral particle pair cell of the RNA that comprises the polypeptide of the present invention of encoding to carry out the genetically engineered operation with method well known in the art.
Similarly, can carry out the genetically engineered operation by pair cell in the methods known in the art body for example, so that the expression in vivo polypeptide.As known in the art, the production cell that produces the retroviral particle of the RNA comprise the polypeptide of the present invention of encode can be applied to the patient so that body is interior through genetically engineered manipulating cells and the said polypeptide of expression in vivo.Through description of the invention, these or other method of using polypeptide of the present invention in this way is clearly to those skilled in the art.For example, can not retroviral particle through the carrier of genetically engineered manipulating cells, but as adenovirus, it can be used in the body through the genetically engineered manipulating cells after transporting carrier combinations with suitable.
The retrovirus of above-described retrovirus plasmid vector of can deriving includes but not limited to: moloneys mouse leukosis virus, spleen necrosis virus, retrovirus such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis viruses, gibbon orangutan leukosis virus, human immunodeficiency virus, adenovirus, bone marrow proliferation sarcoma virus and mammary tumor virus.In one embodiment, the retrovirus plasmid vector is from the moloneys mouse leukosis virus.
Described carrier comprises one or more promotors.Operable suitable promotor includes but not limited to: retrovirus LTR; SV 40 promotors; And human cytomegalovirus (CMV) promotor (Miller, etc., biotechnology, Vol.7, No.9,980-990 (1989) describes); Perhaps other any promotor (for example eukaryotic cell promotor, as include but not limited to histone, pol III and beta-actin promotor).Other viral promotors that uses includes but not limited to: adenovirus promoter, thymidine kinase (TK) promotor and B19 parvovirus promotor.By the description of this paper, in the ken that is chosen in those skilled in the art of suitable promotor.
The nucleotide sequence of code book invention polypeptide is under suitable promotor control.Operable suitable promotor includes, but are not limited to: adenovirus promoter (as adenovirus major late promoter); Perhaps allogeneic promoter (as cytomegalovirus (CMV) promotor); Respiratory syncytial virus (RSV) promotor; Inducible promoter (as MMT promotor, metallothionein promoter); The heat-shocked promotor; The white protein promotor; The ApoAI promotor; Human globin promoter; Viral thymidine kinase promoter (as herpes simplex thymidine kinase promoter); Retrovirus LTRs (the retrovirus LTRs that comprises above-described modification); The beta-actin promotor; With human growth hormone's promotor.Said promotor also can be the natural promoter of the gene of control coding said polypeptide.
Use retrovirus plasmid vector transduction package cell line so that form production clone.The example of packing cell that can be transfected includes but not limited to: PE501, PA317, Ψ-2, Ψ-AM, PA12, T19-14X, VT-19-17-H2, Ψ CRE, Ψ CRIP, GP+E-86, GP+envAml2 and DAN clone (Miller, the human gene therapy, Vol.1, pgs.5-14 (1990) describes, and its incorporated herein by is reference in the lump).Can be by any known method in this area with described carrier transduction packing cell.These methods include but not limited to: electroporation, use liposome and CaPO
4Precipitation.In addition, the retrovirus plasmid vector can wrap in the liposome, perhaps with the lipid coupling, is administered among the host then.
Production clone produces infective retroviral vector particles, and this particle comprises the nucleotide sequence of coding said polypeptide.Can use in these retroviral vector particles bodies then or external transduction eukaryotic cell.The eukaryotic cell of transduction will be expressed the nucleotide sequence of coding said polypeptide.The eukaryotic cell that can be transduceed includes but not limited to: embryonic stem cells, embryo cancer cells and hemopoietic stem cell, liver cell, inoblast, sarcoplast, keratinocyte, endotheliocyte and bronchial epithelial cell.
The present invention also relates to the purposes of gene of the present invention, in order to detect relevant disease or the disease susceptibility of sudden change in the nucleotide sequence with the polypeptide of the present invention of encoding as the part of diagnostic assay.
Can on dna level, detect individuality with various technology with transgenation of the present invention.Can obtain diagnostic nucleic acid from patient's cell (as blood, urinate, saliva is organized examination of living tissue and necrotomy material).Genomic dna can be directly used in detection, perhaps uses PCR enzymatic amplification (Saiki etc., nature, 324:163-166 (1986)) before analysis.RNA or cDNA also can be used for identical purpose.As an example, can use with the nucleic acid complementary PCR primer of code book invention polypeptide and differentiate and analyze its sudden change.For example, can by with the amplified production size of normal genotype comparison on change detect disappearance or insert.Point mutation can be through DNA and the radiolabeled RNA or the radiolabeled antisense dna sequence hybridization discriminating of amplification.Through ribonuclease A digestion, perhaps distinguish complete paired sequence and mispairing duplex from the difference of melting temperature.
Can finish by detecting when being with or without denaturing agent on the gel change of dna fragmentation electrophoretic mobility based on the genetic test of dna sequence dna difference.Little sequence deletion and insertion can be demonstrated by the high resolving power gel electrophoresis.Not homotactic dna fragmentation can be distinguished on sex change methane amide gradient gel, the migration of wherein different dna fragmentations according to its specific fusing point or part melt temperature and be stuck in gel different positions (referring to, for example, Myers etc., science, 230:1242 (1985)).
Also can protect assay method to disclose sequence variation on the specific position, described assay method such as ribonuclease protecting and S1 protection and chemical cracking method (for example, Cotton etc., PNAS, the U.S., 85:4397-4401 (1985)) by nuclease.
Like this; can detect specific dna sequence by following method; the Southern blotting of for example hybridization of described method, ribonuclease protecting, chemical cracking, direct dna sequencing or use restriction enzyme (for example, restrictive fragment length polymerphism (RFLP)) and genomic dna.
Except that a lot of conventional gel electrophoresises and dna sequencing method, also available original position analyzing and testing sudden change.
Because with respect to the overexpression of the said polypeptide of healthy tissues sample can detect abnormal cell proliferation (for example, tumour) existence, so, the present invention also relates to be used for to detect the diagnostic analysis method of level of the change of various tissue FGF-13 protein.The analytical procedure that is used for detecting host-derived this protein level of sample is known to those skilled in the art, and said method comprises: radioimmunoassay, competition in conjunction with measure, the Western engram analysis, ELISA measures and " sandwich " analyzed.ELISA measures (Coligan, et al., immunology common method, 1 (2), the 6th chapter, (1991)) and comprises the antigenic specific antibody for preparing polypeptide of the present invention, preferably monoclonal antibody at first.In addition, the report antibody of preparation monoclonal antibody.With detectable reagent and report antibodies, said reagent such as radioactivity, fluorescence or in this example, be horseradish peroxidase.Obtain sample by the host, and with its with sample in incubation in the solid support (as the polystyrene ware) of protein bound.By with nonspecific proteins matter (as bovine serum albumin(BSA)) incubation together, will cover any protein binding site freely in the ware.Next with monoclonal antibody incubation in ware, monoclonal antibody and be attached to polypeptide combination any of the present invention on the polystyrene ware during this period.With damping fluid all unconjugated monoclonal antibodies are washed off.At this moment, the report antibody that will be connected with horseradish peroxidase is put into ware, and the result causes reporting antibody and any monoclonal antibody combination that is attached on the proteins of interest.
Then unconjugated monoclonal antibody is washed off.Then add peroxidase substrate and typical curve relatively in ware, the amount of the color that produces in preset time promptly is the proteinic amount that exists in patient's sample of given volume.
Also can use competition assay, wherein will be connected to the antibody of polypeptid specificity of the present invention on the solid support, the sample that makes the FGF-13 of mark and derive from the host can be associated with the amount of polypeptide of the present invention in the sample through for example amount of the mark of liquid scintillation counting(LSC) detection by solid support.
" sandwich " analyzed and measured similar with ELISA, in " sandwich " analyzed, with polypeptide of the present invention by a solid support and with solid support on the antibodies of adhering to, then second antibody is combined with interested polypeptide, afterwards the 3rd antibody mark and that be specific to second antibody is combined by solid support and with second antibody, then quantitatively.
Sequence of the present invention identifies it also is valuable to karyomit(e).This sequence specific ground target is positioned at the specific position on the one human chromosome and can hybridizes with it.In addition, the present specific site that needs to identify on the karyomit(e).At present, only there is the chromosome marking reagent of a few sequence data (repetition polymorphism) can be used for the position of marker chromosomes based on reality.DNA chromosome mapping of the present invention is the important the first step that these sequences and disease related gene are associated.
In brief, just can navigate to sequence on the karyomit(e) by prepare PCR primer (preferred 15-25bp) from cDNA.Adopt the Computer Analysis in 3 ' the untranslated zone can select primer apace, wherein primer should not crossed over above an exon on the genomic dna, otherwise makes amplification method complicated.Adopt these primers to be used for the somatic hybridization body that the PCR screening contains one human chromosome then.Have only those crossbreds that contain the people's gene corresponding just can produce amplified fragments with this primer.
The PCR of somatic hybridization body mapping is that a specific DNA is positioned quick program on the specific karyomit(e).Adopt same Oligonucleolide primers according to the present invention, usefulness comes from one group of fragment of specific karyomit(e) or big genomic clone aggregate, can realize inferior location (sublocalization) in a similar way.Can be used for similarly other mapping strategy of chromosome mapping is comprised in situ hybridization, carry out prescreen and carry out preliminary election with the karyomit(e) through airflow classification of mark, thereby construct the cDNA library of chromosome specific by hybridization.
The cDNA clone can be used for realizing the accurate chromosomal localization of single stage method with the fluorescence in situ hybridization (FISH) of Metaphase Chromosome smear.This technology can adopt the cDNA of 50 or 60 base length.Summary about this technology is consulted Verma etc., human chromosomal: basic fundamental handbook, Pergamon press, New York (1988).
In case sequence has navigated to a chromosome position accurately, then the physical location of this sequence can be associated with the genetic map data on the karyomit(e).These data for example can be at V.McKusick, finds in the human Mendelian inheritance (can by online the obtaining in the Johns Hopkins Welch of university medical science library).Relation between the disease on coming identified gene and navigate to identical chromosomal region by linkage analysis (the common heredity of the adjacent gene of physics) then.
Next need to be determined at the difference in cDNA between the affected and unaffected individuality or the genome sequence.If sudden change be observed in some or all of affected individuals, but in any one normal individual, be not observed again, this sudden change may be the cause of disease of disease so.
According to physical mapping and the present resolving power of genetic mapping technology, the cDNA that quilt accurately navigates to the chromosomal region relevant with disease can be 50-500 potential cause a disease a kind of (wherein supposition the mapping resolving power of 1 megabasse and every 20kb are arranged be a gene) in (causative) gene.
The cell of described polypeptide, its fragment or derivative or analogue or expression above-mentioned substance can be as the immunogen of producing its antibody.These antibody can be for example polyclonal antibody or monoclonal antibody.The present invention also comprises chimeric, strand and humanized antibody, and the product of Fab fragment or Fab expression library.Several different methods known in the art can be used to produce these antibody and fragment.
Can be at the antibody that produces corresponding to polypeptide of sequence of the present invention by this polypeptide being injected directly in the animal body or by this polypeptide is obtained the preferred non-human of animal wherein to animals administer.Then, the antibody that so obtains can be attached on this polypeptide.In this way, even also can be used for producing can be in conjunction with the antibody of whole natural polypeptides for a fragments sequence of this polypeptide of only encoding.Then, this antibody can be used for separating this peptide species from the tissue of expressing this polypeptide.
In order to prepare monoclonal antibody, can adopt any technology of producing antibody of cultivating by successive clone.Example comprises hybridoma technology (Kohler and Milstein, 1975, nature, 256:495-497), trisome hybridoma technology, human B cell hybridoma technology (Kozbor etc., 1983, today immunology, 4:72) and the EBV one hybridoma technology (Cole that produces human monoclonal antibodies, Deng, 1985, monoclonal antibody and cancer therapy, Alan R.Liss, Inc., pp.77-96).
Can will be used for the technology (United States Patent (USP) 4,946,778) of manufacture order chain antibody thus make amendment and produce single-chain antibody at immunogenic polypeptide product of the present invention.Also can use transgenic mice to express humanized antibody at immunogenic polypeptide product of the present invention.
The present invention further is illustrated with reference to the following examples; But, should understand the present invention and be not limited to these embodiment.Unless beyond doing in addition to state, all part or amounts are weight.
To understand following embodiment in order being beneficial to, now to narrate method and/or term that some often occur.
" plasmid " is by a small letter p and/or follow several capitalizations and/or numeral is named the preceding.Initial plasmid herein or can by commercial sources obtain or on unrestricted basis the public can obtain, perhaps can from available plasmid, build according to disclosed method.In addition, be known in the art for plasmid and be conspicuous those of ordinary skills with those described equivalences.
" digestion " of DNA is meant the Restriction Enzyme catalytic pyrolysis DNA that only some sequence on the DNA is worked with a kind of.The multiple Restriction Enzyme that this paper adopted can obtain by commercial sources, and its reaction conditions, cofactor and other service requirements are known to those of ordinary skills.For analysis purposes, usually the plasmid of 1 μ g or the dna fragmentation enzyme with about 2 units that are dissolved in about 20 μ l buffered soln is used.In order to separate the dna fragmentation that is used for plasmid construction, usually in a bigger volume with the DNA of enzymic digestion 5 to the 50 μ g of 20 to 250 units.At concrete Restriction Enzyme, the suitable buffered soln and the amount of substrate are stipulated by the producer.Usually adopt 37 ℃ of following about 1 hour incubation times, but this time can change according to the indication of product supplier.After digestion, reaction mixture directly carries out electrophoresis to isolate required fragment on polyacrylamide gel.
Employing is by Goeddel, D. etc., and nucleic acids research, described 8% polyacrylamide gel of 8:4057 (1980) carries out the size separation of crack fragment.
" oligonucleotide " or refer to a kind of strand poly deoxynucleosides, or refer to can be by two complementary poly deoxynucleosides chains of chemosynthesis.These synthetic oligonucleotide do not have 5, phosphoric acid, and therefore if not in the presence of a kind of kinases during with phosphoric acid of ATP interpolation, this oligonucleotide will can not be connected on another oligonucleotide.The synthetic oligonucleotide will be connected to not by on the dephosphorylized fragment.
" connection " be meant between two double stranded nucleic acid fragments the process that forms phosphodiester bond (Maniatis, T., etc., ibid, p.146).Unless beyond providing separately, adopt dna fragmentation to be connected 10 T4 of the unit dna ligases (" ligase enzyme ") of known damping fluid and condition, the about equimolar amount of per 0.5 μ g to realize being connected.
Except as otherwise noted, press Graham, F. and Van der Eb, A., virusology, the described method of 52:456-457 (1973) transforms.
Embodiment 1 bacterial expression and purifying FGF-13 protein
Utilize the dna sequence dna of the initial amplification coding FGF-13 of PCR Oligonucleolide primers, ATCC#97148, said primer are equivalent to the carrier sequence of proteinic 5 ' sequence of finished FGF-13 (subtraction signal peptide sequence) and gene 3 '.To join respectively in 5 ' and the 3 ' sequence corresponding to the additional nucleotide of gene.Said 5 ' Oligonucleolide primers, 5 ' GCCAGACCATGGAGAATCACCCGTCTCCTAAT 3 ' (SEQ ID NO:3) contains the Nco restriction endonuclease sites.Said 3 ' sequence, 5 ' GATTTAAGATCTCGTGAGGGGCTGGGGCCG3 ' (SEQ ID NO:4) comprises and BglII site complementary sequence, and follows 18 Nucleotide of FGF-13 encoding sequence.
Said restriction endonuclease sites is corresponding to the restriction endonuclease sites of bacterial expression vector pQE-60 (Qiagen, Chatsworth company, CA, 91311).PQE-60 coding antibiotics resistance (Amp
r), bacterium replication orgin (ori), IPTG can regulate promotor operon (P/O), ribosome bind site (RBS), 6-histidine mark and restriction endonuclease sites.Then with NcoI and BglII digestion pQE-60.The sequence of amplification is connected among the pQE-60 and is inserted in the frame that has histidine mark encoding sequence and ribosome bind site (RBS).The method of describing by (molecular clonings: laboratory manual, press of cold spring harbor laboratory, (1989)) such as Sambrook is used to connect mixture transformed into escherichia coli bacterial strain M15/rep4 (Qiagen, company) then.M15/rep4 comprises the multiple copied of plasmid pREP4, and this plasmid expression lacI repressor is given kalamycin resistance (Kan simultaneously
r).Identify transformant by the energy for growth of transformant on the LB flat board, and select penbritin/kalamycin resistance bacterium colony.Separate and the affirmation plasmid DNA by restriction analysis.To contain being cloned in of required construct and replenish (O/N) cultivation of spending the night in the LB liquid nutrient medium of Amp (100 μ g/ml) and Kan (25 μ g/ml).The O/N culture is used to inoculate the large volume culture with 1: 100 to 1: 250 ratio.Cell grows to the optical density(OD) 600 (O.D. between 0.4 and 0.6
600) time, add IPTG (sec.-propyl-B-D-thio-galactose pyran-glucoside) to ultimate density be 1mM.IPTG removes P/O by inactivation lacI repressor, causes the increase of genetic expression.Cell was cultivated 3 to 4 hours again.Pass through centrifugal cell harvesting.Cell precipitation is dissolved in the 6M Guanidinium hydrochloride chaotropic agent.After the clarification, under the condition that the protein that allows to contain the 6-histidine mark is combined closely, in nickel-inner complex post by chromatography purifying dissolved FGF-13 (Hochuli, E. etc., chromatographic science magazine 411:177-184 (1984)) from solution.FGF-13 protein (85% purity) is eluted from post with 6M Guanidinium hydrochloride (pH value 5.0),, transfer to 3M Guanidinium hydrochloride, 100mM sodium phosphate, 10mM gsh (reduced form), 2mM gsh (oxidized form) for renaturation., after 12 hours protein is dialysed to the 10mM sodium phosphate in this solution incubation.
Embodiment 2 utilizes the baculovirus expression system clone and expresses FGF-13
Utilize PCR Oligonucleolide primers amplification coding total length FGF-13 protein DNA sequence (ATCC#97148), said primer is corresponding to 5 ' and 3 ' sequence of this gene: FGF-135 ' primer has sequence 5 ' CTAGTGGATCCCGAGAATCACCCGTCTCCT 3 ' (SEQ ID NO:5), comprise BamHI restriction site (runic), so that the baculovirus signal sequence is placed the frame of 18 Nucleotide of FGF-13 gene in the FGF-13 signal peptide cutting site downstream of inferring the clone in this site.
3 ' primer has sequence 5 ' CGACTTCTAGAACCTCGGGGATCTGGCTCC3 ' (SEQ ID NO:6), comprises the cleavage site and 18 Nucleotide that are complementary to gene 3 '-non-translated sequence of restriction enzyme XbaI.
(" Geneclean " BIO 101 companies, La Jolla Ca.) separate the sequence that increases from 1% sepharose with the commercial reagent box.Then with said fragment with the digestion of corresponding nucleic acids restriction endonuclease, and on 1% sepharose purifying once more.This fragment is appointed as F2.
Carrier pA2gp (is formed by the pVL941 carrier modification, discussion sees below) be used for marking protein, baculovirus expression system is used in this expression, and (summary is referring to Summer, M.D. and Smith, G.E.1987, baculovirus vector and insect cell cultural method handbook, testing station's communique 1555 of Texas's agricultural).This expression vector comprises the strong polyhedrin promotor of the polygonal virus of autographa california multinuclear type (AcMNPV), and its back is the recognition site of restriction endonuclease BamHI and XbaI.The polyadenylation site of monkey disease poison (SV) 40 is used for effective polyadenylation.In order easily to select recombinant virus, with the direction insertion same, be colibacillary beta-galactosidase gene thereafter the polyadenylation signal of polyhedron gene with the polyhedrin promotor.The both sides of polyhedrin sequence are the virus sequences of homologous recombination that is used for the wild-type virus DNA of cell-mediated cotransfection.Can use much other baculovirus vector replacement pA2, and said carrier such as pRG1, pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summer, M.D., virusology, 170:31-39).
Digest said plasmid with restriction enzyme, and utilize the calf intestinal phosphatase enzyme to make the plasmid dephosphorylation with methods known in the art.(La Jolla is Ca.) from 1% sepharose DNA isolation for " Geneclean ", BIO 101 companies to use the commercial reagent box then.This carrier DNA is appointed as V2.
Connect F2 fragment and said dephosphorylated plasmid V2 with the T4 dna ligase.Then transformed into escherichia coli DH5 α cell (the Stratagene cloning system, 11011 North TorreyPines Road La Jolla, Ca.92037) and utilize corresponding restriction enzyme to differentiate the bacterium that contains said plasmid (pBacFGF-13).Confirm the fragments sequence cloned by dna sequencing.
With lipofection (Felgner etc., Proc. Natl. Acad. Sci.USA, 84:7413-7417 (1987)) with 5 μ g plasmid pBacFGF-13 and commercially available the linearized baculovirus (" BaculoGold of 1.0 μ g
TMBaculovirus DNA ", Pharmingen, San Diego, CA.) cotransfection.
In all cases, with 1 μ g BaculoGold
TM(Life Technologies company, Gaithersburg mix in the aseptic hole of droplet plate MD) containing 50 microlitre serum-free Grace ' s substratum for viral DNA and the described plasmid of 5 μ g.Behind the substratum that adds 10 microlitre lipofectin reagents and 90 microlitre Grace ' s, mix to be incorporated under the room temperature and cultivated 15 minutes.Then transfection mixture is dropwise added in the Sf9 insect cell (ATCC CRL1711), said cell inoculation is on the 35mm tissue culture plate with 1ml serum-free Grace ' s substratum.The waggle flat board is to mix the solution of new interpolation.Then flat board was cultivated 5 hours down at 27 ℃.After 5 hours, remove transfection solution, add Grace ' the s insect substratum that 1 microlitre is supplemented with 10% foetal calf serum from flat board.Flat board is put back into incubator, 27 ℃ of following cultured continuously 4 days.
After 4 days, collect supernatant liquor, carry out plaque measurement with similar Summers and the described method of Smith (the same).A bit change sepharose (the LifeTechnologies company that is to use with " Blue Gal ", Gaithersburg), its make be easy to separate dye blue plaque (the detailed description of " plaque measurement " also can the insect cell of Life Technologies company (Gaithersburg) issue cultivate and baculovirus users' guidebook 9-10 page or leaf in find).
After four days, the virus of serial dilution is added in the cell, dye blue plaque with Eppendorf pipette tip picking.The agar that will comprise recombinant virus then is suspended in the Eppendcrf pipe that comprises 200 microlitre Grace ' s substratum.Through the brief centrifugal agar of removing, the supernatant liquor that will comprise recombinant baculovirus is used for infecting the Sf9 cell that is inoculated into 35 millimeters culture dish.After 4 days, gather in the crops the supernatant liquor in these culture dish, in 4 ℃ of storages.
The Sf9 cell is grown in the Grace ' substratum that is supplemented with 10% hot deactivation FBS.Infection multiplicity (MOI) 2 times with recombinant baculovirus V-FGF-13 cells infected.After 6 hours, remove substratum, (LifeTechnologies company Gaithersburg) substitutes with SF900II substratum (deducting methionine(Met) and halfcystine).After 42 hours, add 5 μ Ci
35S methionine(Met) and 5 μ Ci
35S halfcystine (Amersham).Before centrifugal results, cell further to be cultivated 16 hours, labelled protein demonstrates through SDS-PAGE and radioautograph.
The express recombinant FGF-13 of embodiment 3 in the COS cell
Expression plasmid FGF-13-HA derives from carrier pcDNA3/Amp (Invitrogen), it comprises: 1) SV 40 replication orgin, 2) ampicillin resistance gene, 3) colibacillary replication orgin, 4) CMV promotor is thereafter polylinker district, SV 40 introns and polyadenylation site.With the dna fragmentation of the complete FGF-13 precursor of coding with in frame, be integrated into the polylinker district of 3 ' terminal HA marker clone to said carrier, thus, being expressed under the control of CMV promotor of recombinant protein.The HA mark is corresponding to from the proteinic epi-position of influenza hemagglutinin, and this point was described (I.Wilson, H.Niman, R.Heighten, A Cherenson, M.Connolly, and R.Lerner, 1984, cell 37:767, (1984)) in the past.The fusion of HA and target protein makes to be easy to detect recombinant protein with the antibody of discerning the HA epi-position.
Dna fragmentation with corresponding digestion with restriction enzyme pcr amplification also is connected with carrier pcDNA3/Amp.To connect mixture and be transformed among the coli strain SURE (can be, La Jolla, CA 92037 obtains), the culture that transforms will be seeded on the ampicillin medium flat board from the Stratagene cloning system, and the screening resistance clone.Isolated plasmid dna from transformant, and detect whether there is correct fragment with restriction analysis.For express recombinant FGF-13, by DEAE-DEXTRAN method expression vector rotaring redyeing COS cell (J.Sambrook, E.Fritsch, T.Maniatis, molecular cloning: laboratory manual, cold spring harbor laboratory's publication, (1989)).Detect FGF-13-HA protein expression (E.Harlow, D.Lane, antibody: laboratory manual, cold spring harbor laboratory's publication, (1988)) by radio-labeled and immuno-precipitation.With the two day usefulness of cell after transfection
15S-halfcystine mark 8 hours is collected culture then and is used washing agent lysing cell (RIPA damping fluid (150mM NaCl, 1%NP-40,0.1%SDS, 1%NP-40,0.5%DOC, 50mM Tris, pH7.5) (Wilson, I. etc., Id.37:767 (1984)).With HA monoclonal antibody specific sedimentation cell lysate and substratum.Analysing protein precipitation on the 15%SDS-PAGE gel.
Embodiment 5 is via the expression of gene therapy
Inoblast obtains from a research object by Skin biopsy.Be placed on the tissue that obtains on the tissue culture medium (TCM) and be divided into fritter.Small tissue blocks is placed on the wet surface of tissue culture flasks, wherein places about 10 block organizations in every bottle.Bottle is placed upside down, and lid is tight and spend the night under room temperature.At room temperature place counter-rotating bottle after 24 hours, tissue block still is fixed on the bottle bottom, adds fresh culture (for example containing 10%FBS, Ham ' the s F12 substratum of penicillin and Streptomycin sulphate), then in 37 ℃ of about 1 weeks of following incubation.At this moment add fresh culture, changed a subculture subsequently every several days.After cultivating for 2 weeks again, an individual layer inoblast has appearred.With monolayer cell through tryptic digestion and put into bigger bottle.
With EcoRI and Hind III digestion side the long terminal repetition pMV-7 (Kirschmeier, P.T. etc., DNA, 7:219-25 (1988)) of Moloney murine sarcoma virus is arranged, next handle with the calf intestinal phosphatase enzyme.Linear carrier fractional separation and use granulated glass sphere purifying in addition on sepharose.
Adopt the cDNA of corresponding with 5 ' and 3 ' end sequence respectively PCR primer amplification code book invention polypeptide.5 ' primer comprises an EcoRI site, and 3 ' primer contains a HindIII site.In the presence of the T4 dna ligase, the EcoRI of the linearizing skeleton of the Moloney murine sarcoma virus of equivalent and amplification added with Hind III fragment be in the same place, be suitable for keeping resulting mixture under the condition that two fragments connect.Should connect mixture and be used for transform bacteria HB101, in order to confirm this carrier to have the correct gene of interest of insertion bacterium was coated on the agar plate that contains kantlex then.
Amphophilic (amphotropic) pA317 or GP+am12 packing cell are cultivated in the tissue culturing plate of Dulbecco ' the s improvement Eagles substratum (DMEM) that contains 10% calf serum (CS), penicillin and Streptomycin sulphate, be paved with density until reaching.The carrier that will contain described gene then adds in the substratum also with this carrier transduction packing cell.Packing cell is produced immediately and is contained the infective virion of having of this gene (packing cell is known as and produces cell now).
In the production cell of transduction, add fresh substratum, next be paved with the 10cm flat board of producing cell and collect substratum from one.The substratum that contains infectious viral particle filters to remove the production cell of desorption (detached) through millipore filter, utilizes this substratum to go to infect inoblast then.Be paved with from fibroblastic Asia to remove substratum the flat board and promptly to replace and come from the substratum of producing cell.Remove this substratum and replace fresh substratum.If the titre of virus is very high, all so in fact inoblasts are all infected and need not to select.If titre is very low, so need to adopt have as
NeoOr
HisThe retrovirus of alternative mark like this.
Inoblast with through engineering approaches is injected into the host then, it or separately injection or on cytodex 3 microcarrier beads, grown to inject again after being paved with.This moment, inoblast produced protein.
According to above instruction, many improvement of the present invention and variation all are possible, therefore can other mode implement the present invention within the scope of the appended claims.
Sequence table
(1) general information:
(i) applicant: HU etc.
(ii) denomination of invention: fibroblast growth factor-13
(iii) sequence number: 8
(iv) address:
(A) addressee: CARELLA, BYRNE, BAIN, GILFILLAN,
CECCHI, STEWART and OLSTEIN
(B) street: 6 BECKER FARM ROAD
(C) city: ROSELAND
(D) state: New Jersey
(E) country: the U.S.
(F) postcode: 07068
(v) computer-reader form:
(A) media type: 3.5 inches disks
(B) computer: IBM PS/2
(C) operating system: MS-DOS
(D) software: WORD PERFECT 5.1
(vi) current request for data:
(A) application number:
(B) applying date:
(C) classification number:
(vii) request for data formerly
(A) application number: 08/207,412
(B) applying date: on March 8th, 1994
(viii) lawyer/proxy's information:
(A) name: FERRARO, GREGORY D.
(B) registration number: 36,134
(C) case number/number of documents: 325800-364
(ix) telecom information:
(A) phone: 201-994-1700
(B) fax: 201-994-1744
(2) information of SEQ ID NO:1:
(i) sequence signature:
(A) length: 641 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: cDNA
( xi ) ：SEQ ID NO：1CCCGCCTGCT GCCCAACCTC ACTCTGTGCT TACAGCTGCT GATTCTCTGC TGTCAAACTC 60AGGGGGAGAA TCACCCGTCT CCTAATTTTA ACCAGTACGT GAGGGACCAG GGCGCCATGA 120CCGACCAGCT GAGCAGGCGG CAGATCCGCG AGTACCAACT CTACAGCAGG ACCAGTGGCA 180AGCACGTGCA GGTCCCCGGG CGTCGCATCT CCGCCACCGC CGAGGACGGC AACAAGTTTG 240CCAAGCTCAT AGTGGAGACG GACACGTTTG GCAGCCGGGT TCGCATCAAA GGGGCTGAGA 300GTGAGAAGTA CATCTGTATG AACAAGAGGG GCAAGCTCAT CGGGAAGCCC AGCGGGAAGA 360GCAAAGACTG CGTGTTCACG GAGATCGTGC TGGAGAACAA CTATACGGCC TTCCAGAACG 420CCCGGCACGA GGGCTGGTTC ATGGTCTTCA CGCGGCAGGG GCGGCCCCGC CAGGCTTCCC 480GCAGCCGCCA GAACCAGCGC GAGGCCCACT TCATCAAGCG CCTCTACCAA GGCCAGCTGC 540CCTTCCCCAA CCACGCCGAG AAGCAGAAGC AGTTCGAGTT TGTGGGCTCC GCCCCCACCC 600GTCGGACCAA GCGCACACGG CGGCCCCAGC CCCTCACGTA G 641
(2) information of SEQ ID NO:2:
(i) sequence signature:
(A) length: 212 amino acid
(B) type: amino acid
(C) chain:
(D) topological framework: linearity
(ii) molecule type: protein
(xi) sequence description: SEQ ID NO:2Arg Leu Leu Pro Asn Leu Thr Leu Cys Leu Gln Leu Leu Ile Leu
-20 -15 -10Cys?Cys?Gln?Thr?Gln?Gly?Glu?Asn?His?Pro?Ser?Pro?Asn?Phe?Asn
-5 1 5Gln?Tyr?Val?Arg?Asp?Gln?Gly?Ala?Met?Thr?Asp?Gln?Leu?Ser?Arg?10 15 20Arg?Gln?Ile?Arg?Glu?Tyr?Gln?Leu?Tyr?Ser?Arg?Thr?Ser?Gly?Lys?25 30 35His?Val?Gln?Val?Pro?Gly?Arg?Arg?Ile?Ser?Ala?Thr?Ala?Glu?Asp?40 45 50Gly?Asn?Lys?Phe?Ala?Lys?Leu?Ile?Val?Glu?Thr?Asp?Thr?Phe?Gly?55 60 65Ser?Arg?Val?Arg?Ile?Lys?Gly?Ala?Glu?Ser?Glu?Lys?Tyr?Ile?Cys?70 75 80Met?Asn?Lys?Arg?Gly?Lys?Leu?Ile?Gly?Lys?Pro?Ser?Gly?Lys?Ser?85 90 95Lys?Asp?Cys?Val?Phe?Thr?Glu?Ile?Val?Leu?Glu?Asn?Asn?Tyr?Thr100 105 110Ala?Phe?Gln?Asn?Ala?Arg?His?Glu?Gly?Trp?Phe?Met?Val?Phe?Thr115 120 125Arg?Gln?Gly?Arg?Phe?Arg?Gln?Ala?Ser?Arg?Ser?Arg?Gln?Asn?Gln130 135 140Arg?Glu?Ala?His?Phe?Ile?Lys?Arg?Leu?Tyr?Gln?Gly?Gln?Leu?Pro145 150 155Phe?Pro?Asn?His?Ala?Glu?Lys?Gln?Lys?Gln?Phe?Glu?Phe?Val?Gly160 165 170Ser?Ala?Pro?Thr?Arg?Arg?Thr?Lys?Arg?Thr?Arg?Arg?Pro?Gln?Pro175 180 185Leu?Thr190
(2) information of SEQ ID NO:3:
(i) sequence signature:
(A) length: 32 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:3GCCAGACCAT GGAGAATCAC CCGTCTCCTA AT 32
(2) information of SEQ ID NO:4:
(i) sequence signature:
(A) length: 30 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:4GATTTAAGAT CTCGTGAGGG GCTGGGGCCG 30
(2) information of SEQ ID NO:5:
(i) sequence signature:
(A) length: 30 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:5CTAGTGGATC CCGAGAATCA CCCGTCTCCT 30
(2) information of SEQ ID NO:6:
(i) sequence signature:
(A) length: 30 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:6CGACTTCTAG AACCTCGGGG ATCTGGCTCC 30
(2) information of SEQ ID NO:7:
(i) sequence signature:
(A) length: base pair
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:7
(2) information of SEQ ID NO:8:
(i) sequence signature:
(A) length: 30 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:8GATTTACTCG AGCGTGAGGG GCTGGGGCCG 30
Claims (13)
1. isolating polynucleotide, it comprises the member who is selected from as next group:
(a) a kind of polynucleotide, this polynucleotide encoding comprise peptide more than-21 to 191 amino acids shown in the SEQ ID NO:2;
(b) a kind of polynucleotide, this polynucleotide encoding comprise peptide more than 1 to 191 amino acids shown in the SEQ ID NO:2;
(c) a kind of polynucleotide, this polynucleotide can with (a) or multi-nucleotide hybrid (b), and and its have at least 70% homogeny; And
(d) a kind of (a) or (b) or the polynucleotide passage of polynucleotide (c).
2. the polynucleotide of claim 1, wherein said polynucleotide are DNA.
3. the polynucleotide of claim 1, this polynucleotide comprise 65 to 641 Nucleotide shown in the SEQ ID NO:1.
4. isolating polynucleotide, it comprises the member who is selected from as next group:
(a) a kind of polynucleotide, this polynucleotide encoding is a kind of by the mature polypeptide that is included in the dna encoding in No. 97148 preservation things of ATCC;
(b) a kind of polynucleotide, this polynucleotide encoding is by the DNA polypeptide expressed that is included in No. 97148 preservation things of ATCC;
(c) a kind of polynucleotide, this polynucleotide can with (a) or multi-nucleotide hybrid (b), and and they have at least 70% homogeny; And
(d) polynucleotide passage of a kind of (a) and (b) or polynucleotide (c).
5. carrier, this carrier comprises the DNA of claim 2.
6. host cell that obtains through the genetically engineered operation with the carrier of claim 5.
7. method of producing polypeptide, this method comprises the polypeptide of the host cell expression of Accessory Right requirement 6 by said dna encoding.
8. the method for the cell that a production can express polypeptide, this method comprises that the carrier pair cell with claim 5 carries out the genetically engineered operation.
9. a peptide species, this peptide species comprises the member who is selected from as next group:
(i) a kind of polypeptide and its fragment, analogue and derivative with deduced amino acid of SEQ ID NO:2; And (ii) a kind of cDNA encoded polypeptides and its fragment, analogue and derivative by No. 97148 preservation things of ATCC.
10. therapeutic composition that comprises peptide more than the claim 9 for the treatment of significant quantity.
11. therapeutic composition that comprises the DNA of peptide more than the coding claim 9 for the treatment of significant quantity.
12. the nucleic acid of peptide is used for diagnosing application with the diagnostic reagent of not enough relevant disease of described expression of polypeptides or disease susceptibility in preparation more than the coding claim 9.
13. the application of the diagnostic reagent that peptide is used for diagnosing the illness in preparation more than the claim 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN02126409A CN1414100A (en) | 2002-07-12 | 2002-07-12 | Desmocyte growth factor 13 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN02126409A CN1414100A (en) | 2002-07-12 | 2002-07-12 | Desmocyte growth factor 13 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95197868A Division CN1185175A (en) | 1995-06-05 | 1995-06-05 | fibroblast growth factor 13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1414100A true CN1414100A (en) | 2003-04-30 |
Family
ID=4745741
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN02126409A Pending CN1414100A (en) | 2002-07-12 | 2002-07-12 | Desmocyte growth factor 13 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1414100A (en) |
-
2002
- 2002-07-12 CN CN02126409A patent/CN1414100A/en active Pending
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