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CN1110558C - Novel growth/differentiation factor of TGF-beta family - Google Patents

Novel growth/differentiation factor of TGF-beta family Download PDF

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CN1110558C
CN1110558C CN95193816A CN95193816A CN1110558C CN 1110558 C CN1110558 C CN 1110558C CN 95193816 A CN95193816 A CN 95193816A CN 95193816 A CN95193816 A CN 95193816A CN 1110558 C CN1110558 C CN 1110558C
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albumen
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tgf
leu
protein
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G·霍藤
H·奈德哈特
R·贝克托尔德
J·波尔
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Biopharm Gesellschaft zur Biotechnologischen Entwicklung von Pharmaka mbH
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Abstract

The present invention includes a protein of the TGF-beta family, DNA encoding the same, and a pharmaceutical composition containing the protein.

Description

Growth/the differentiation factor of new TGF-'beta ' family
The present invention relates to a kind of growth/differentiation factor and dna encoding sequence thereof of new TGF-'beta ' family.
The somatomedin that belongs to the TGF-'beta ' family has BMP-, TGF-and activator/statin-relevant albumen (Roberts und Sporn, Handbook of Experimental Pharmacology 95,419-472, (1990)).This type of somatomedin is widely used in clinical treatment and related application.This type of factor is suitable for trauma care and tissue regeneration.And, but the more multiplefactor induced tissue such as the bone growth of TGF-'beta ' family.
Wozney (Progress in Growth Factor Research 1 (1989), 267-280) and people such as Vale (Handbook of Experimental Pharmacology 95 (1990), 211-248) reported various somatomedin as with the BMP-and the factor that activator/the statin group is close.Has tangible structural similarity between this member.This type of proteic precursor contains 110 to 140 amino acid whose C terminal sequences by a N end signal sequence, propetide and one and constitutes.The C end becomes maturation protein after the precursor division.This type of factor is divided according to the homology of its aminoacid sequence.Maturation protein contains the sequence of high conservative, and especially this type of factor has 7 common cysteine residues of this family member.TGF-β proteinoid is multi-functional somatomedin with hormonal activity, and also have other relevant biological activitys such as cell chemotropism, promote the differentiation of cell and organize inducibility.EP 0 222 491 A1 disclose the sequence of statin α and β chain.
In a word, the difference structurally of TGF-β proteinoid makes it have significantly different biological action, and this proteinoid is present in extensively different types of organization and developmental stages.Thereby carry out aspect the concrete function different, as the cell physiological environment that requires, life-span, location, demand, anti-degradation capability to cofactor.Have the inductive of organizing potentiality although described a lot of albumen, in body, especially also need study in great detail in medically effect to it.Also have unknown TGF-'beta ' family member protein probably, they are for the differentiation of different tissues/induce meaningful especially.Owing to its function is understood inadequately, differentiate the proteic biological detecting method of different TGF-β and lack, thereby these new proteic separation of TGF-β is had great difficulty.On the other hand, too little with the proteic nucleic acid sequence homology of known TGF-β, traditional nucleic acid hybridization technique also can not be used to screen unknown TGF-β albumen.However, being badly in need of the new TGF-β albumen of isolation identification goes up and neededly has (tissue) and induce/albumen of Differentiation to satisfy all medical treatment.Such (albumen) factor can be used for treating various tissue injurys and tissue deterioration disease.
The PCT/EP93/00350 patent application provides nucleic acid and the protein sequence of TGF-β albumen MP121, and has indicated most of and the corresponding sequence of mature peptide.The complete sequence of MP121 precursor peptide is also unexposed to be delivered.
Basic task of the present invention is to obtain new TGF-β proteic dna encoding sequence, the especially DNA of TGF-albumen MP121 and the amino acid full length sequence that has short cell fission and/or induce differentiation potential.
The solution of this problem is to obtain a coding TGF-'beta ' family proteic dna molecular, and it comprises: (a) one of nucleotide sequence of being obtained according to the degeneracy of genetic code by sequence (a) of other function section (b) of encoding mature albumen section and the nucleotide sequence randomly indicated among the SEQ ID NO.1 (c) a kind of nucleotide sequence (d) that waits bit derivant corresponding to sequence (a) and one of (b) is derived from other vertebrates based on it and one of sequence different with sequence (a) (e) and (a), (b), (c), or the nucleotide sequence of one of sequence (d) hybridization and prerequisite are the coding regions that will contain the maturation protein of TGF-'beta ' family according to the dna molecular of (e) at least.
Other form of implementation of the present invention relates to the theme of claim 2 to 10, and other advantage of the present invention and feature are illustrated by preferred embodiment.Sequence and collection of illustrative plates now are summarized as follows:
SEQ ID NO.1 represents the complete nucleotide sequence of coding DNA of people's TGF-β albumen MP121.Codon ATG starts from the 128th Nucleotide.Fully matured albumen especially preferably starts from the 836th Nucleotide.
SEQ ID NO.2 is the full length amino acid sequence according to the former precursor protein of the people TGF-β albumen MP121 of SEQ ID NO.1 nucleotide sequence release.The maturation protein starting point is preferably placed at the zone between the 217th to 240 amino acid, and especially the 236th or the 237th and the 237th amino acid most preferably.
SEQ ID NO.3 be mouse TGF-β albumen MP121 coding DNA complete nucleotide sequence.The coding region starts from the ATG initial code at the 131st Nucleotide place and ends at stop code at the 1187th Nucleotide place.The proteic initial code of preferred maturity starts from the 839th Nucleotide, and it is the intron of 5.5kb that a length is arranged between the 446th and 447 position of its genomic dna.
SEQ ID NO.4 is the complete amino acid sequence of the former precursor protein of mouse TGF-β albumen MP121, and this aminoacid sequence is that the nucleotide sequence according to SEQ ID NO.3 obtains.The proteic starting point of people MP121 of this maturation protein starting point and SEQ ID NO.2 is suitable, i.e. zone between the 217th to 240 amino acid.The most preferred starting point of this maturation protein is the 237th amino acid, and the maturation protein part is made up of 116 amino acid as people's MP121 maturation protein like this.Thereby the precursor protein of TGF-'beta ' family cuts after the RXXR site usually and separates maturation protein (seeing zkaynak et al., J.Biol.Chem.267,25220-25227, (1992) and the document that is drawn thereof).Therefore with regard to the MP121 of mouse, maturation protein to the small part starting point also may be the 236th amino acid.
SEQ ID NO.5 shows the nucleotide sequence that the exon of people's MP121 gene is joined.The Nucleotide of two exons is represented by capitalization, and intron sequences is represented by lowercase.
Fig. 1 is the comparison of people MP121 and some TGF-'beta ' family members' (statin α and β chain) aminoacid sequence, and this figure's relatively is to be begun by first of 7 conserved cysteine residue. *Represent that this amino acid all is identical in participating in all albumen relatively; + being illustrated in the albumen and the people MP121 that have at least a participation to compare all contains this amino acid.
Fig. 2 be illustrated in the Oligonucleolide primers of using in the present invention nucleotide sequence and with known TGF-'beta ' family member's sequence relatively.M represents A or C, and S represents C or G, and R represents A or G, and K represents G or T.2a represents the sequence of primer OD, and 2b represents the sequence of primer OID.
Fig. 3 shows is the sketch that utilizes the Western trace that the chicken antibody of anti-people MP121 carries out.
Fig. 4 is illustrated in that MP121 compares with the expression of activator β A and β B in the different tissues of mice.
Fig. 5 represents to utilize partially purified MP121 that dopamine neuron is handled, to the positive effect that this neuronic viability produced.
The concept that relates among the present invention in addition " maturation protein " also comprises the functional area of total protein, it Have almost same BA and preferably will contain at least 7 conservative half of TGF-'beta ' family Those zones of the section at cystine place. Particularly the N of maturation protein end might slightly be repaiied Decorations, so its sequence can be different with the sequence of SEQ ID NO 2 and 4. May in the maturation protein Have additional amino acid to exist, its existence does not affect the function of albumen, may have amino acid deletions yet, As long as these amino acid whose disappearances are the function of limit protein not also. But the albumen of preferred people and mouse has The 237th all ammonia that amino acid is later in SEQ ID NO 2 and SEQ ID NO 4 amino acid sequences Base acid. Have been found that in other member of TGF-'beta ' family, be positioned at the attached of maturation protein N end Adding amino acid does not affect the activity of albumen, namely belongs to this kind situation such as 6 additional histidines of N end.
The present invention includes the coding section of above-mentioned defined maturation protein, randomly also comprise SEQ Other function section of ID NO 1 nucleotide sequence, and the institute that produces according to the degeneracy of genetic code The derivative that sequence and these sequences are arranged. And, the present invention includes from other mammiferous by In the dna sequence dna of the slightly different coding TGF-'beta ' family albumen of source difference and sequence, but this The coded protein sequence of a little DNA is close and have a roughly the same biological action. These sequence phases Has very big common ground mutually, as the more drawn similitude of SEQ ID NO 1 and NO 3 The same.
In addition, the present invention also comprises the sequence with above-mentioned sequence hybridization, prerequisite be these dna sequence dnas extremely Contain less section and the reservation of complete coding TGF-'beta ' family maturation protein (according to above-mentioned definition) Biologically active.
The concept of " functional area " refers to possess MP121 albumen from angle of the present invention The protein fragments of one of the biological action in natural zone is as signal peptide, precursor peptide or ripe egg White part.
The dna encoding section of preferred people MP121 maturation protein part is preferably from the 836th nucleosides It is the 1184th nucleotides of sequence shown in the SEQ ID NO 1 that acid begins to stop code. Randomly should Dna molecular also may comprise other function section of sequence shown in the SEQ ID NO 1, i.e. the coding letter Number peptide is or/and the nucleotide sequence of precursor peptide part. Especially preferred this dna molecular comprises for signal-and the sequence of the part of front peptide moiety and maturation protein, namely the of sequence shown in the SEQ ID NO 1 128 to 1184 nucleotides. Shown in SEQ ID NO.3, the MP121 maturation of preferred mouse The code area of albumen starts from the 839th nucleotides until the stop code of the 1187th nucleotides, some feelings Under the condition, this dna molecular also comprises other function section of sequence shown in the SEQ ID NO.3, i.e. letter Number peptide is or/and the coded sequence of precursor peptide part.
On the other hand, except the mature peptide code area, dna molecular of the present invention also comprises other albumen The function signal peptide and/or the code area of precursor peptide part, these albumen are as having " cysteine knot " The albumen of (Cystine Knot Motif) (Cell, Vol.73 (1993) .S.421-424), especially Other albumen activin/inhibin as mentioned above or the BMP-albumen of TGF-B-family, the spy Not MP52 (seeing PCT/EP94/02630). Related nucleotide sequence derives from above-mentioned institute The document of publishing of row. Importantly, these sequences still keep the correct reading of maturation protein Framework. But owing in different host cells, obtain expressing, thereby different bursts or/ And precursor peptide may have positive influences to its expression. Precursor peptide is substituted by the precursor peptide of other albumen Existing such as following report (Mol.Endocrinol.5 (1991), 149-155; Proc.Natl.Acad. Sci.USA 90 (1993), 2905-2909). Although this patent cover all are moving from other vertebra The degenerate sequence of thing and hybridization sequences, owing to its nucleotides or/and the slight change of amino acid sequence show Reveal structural difference, still, the coded albumen of this class sequence still has roughly the same useful Characteristic makes it still may medically obtain essentially identical application.
According to the present invention, " hybridization " is hybridization conditions commonly used, preferably utilizes the salinity of 6 * SSC, and is assorted Handing over temperature is 62 ℃-66 ℃, and the film condition of washing afterwards is to use 0.6 * SSC, and 0.1%SDS is in 62 ℃-66 ℃ were washed 1 hour.
The preferred embodiment of the present invention is to come from as defined above especially mammal of vertebrate Such as pig, chicken and rodent such as rat or mouse especially derive from primate such as people's dna sequence dna And the corresponding sequence that copies.
Sequence shown in the SEQ ID NO.1 and 3 and that be called people and mouse MP121 is the special preferred implementing form of the present invention.The MP121 gene transcription is carried out in hepatic tissue, and the aminoacid sequence of the maturation protein of its proteins encoded and statin/activator class has very big homology (see figure 1).People's α-Yi Zhisu, statin β A(activator β A), and statin β B(activator β B) protein sequence by report such as Mason (Biochem.Biophys.Res.Comm.135,957-964 (1986)).The peculiar typical sequence homology of known statin sequence also occurs in MP121 precursor peptide part, and the precursor peptide of the other parts of MP121 precursor peptide and statin has obvious difference.
But as far as is known, the expression pattern of MP121 and activator is different.Activator is mainly expressed (activator β in sexual gland AAt ovary, activator β BAt testis and ovary), and MP121 mainly expresses in liver.Used so far experimental technique also is not enough to detect its trace expression.According to the literature, confirmed activator except expressing at the sexual gland of different mouse tissues also at adult animals (Meunier et al., Proc.Natl.Acad.Sci.USA 85,247-251 (1988)) expresses (R0berts et al. and in embryo development procedure, Endocrinology 128,3122-3129 (1991)). so MP121 also may express this further confirmation that awaits in other tissue.
Another content of the present invention is the carrier that contains a copy of the dna molecular among the present invention at least.In examples of such carriers, preferably this dna molecular joins with the sequence that control is expressed.This class carrier is applicable to produces TGF-β proteinoid in the cell of stable or instantaneous conversion.Different animals, plant, fungi and bacterial system can be used for transforming and follow-up cultivation.Carrier among preferred the present invention contains duplicate necessary sequence in host cell, but and self-replicating.But the carrier that preferred in addition utilization contains selectable marker gene makes the conversion energy of host cell access evaluation.
Another content of the present invention is the host cell that DNA in the present invention or carrier transform, and suitable host cell example comprises various eucaryons and prokaryotic cell prokaryocyte, as: intestinal bacteria, insect cell, vegetable cell, mammalian cell and fungi such as yeast.
An albumen of the TGF-'beta ' family that the dna sequence dna that another important content of the present invention is a claim 1 is coded.Preferred this albumen has the aminoacid sequence shown in SEQ ID NO.2 or the SEQ ID NO.4 or its funtion part (as above definition) randomly, and show may be relevant with medical applications biological nature as organizing induced activity.
Because this albumen forms homodimer or heterodimer with the albumen with " cysteine knot " especially TGF-β albumen, thereby this proteic above-mentioned characteristic can change to some extent.These couple structures may be applied equally clinically, so also be the content of present patent application.Preferred heterodimer comprise by the protein monomer among the present invention and α-, β A-or β BThe heterodimer that the monomer of-statin chain forms.More the character with activator and statin is close for the character of preferred heterodimer.For example: if form heterodimer, MP121/ statin (α-chain) or MP121/ activator (β so with statin α-albumen or other statin beta-proteins A-or β B-chain) heterodimer just may suppress or promote the synthetic of prolan a (FSH).MP121/ activator heterodimer also may influence mesoblastic growth.Can further predict, form heterodimer with the member of the proteic BMP class of TGF-β, can strengthen class BMP activity, such as inducing or promote bone to form, cartilage forms and the ability of the formation of reticular tissue.
Therefore another content of the present invention is a heterodimer as described below: by the coded TGF-'beta ' family albumen of the present invention of the dna sequence dna of claim 1, with the albumen with " cysteine knot " especially with the formed heterodimer of the proteic monomer of other TGF-'beta ' family.Similarly heterodimer albumen has among EPO 626451A2 and J.Biol.Chem.265 (1990) .13198-13205 and describes at WO93/09229.
Another content of the present invention is following chimeric protein: promptly have proteic functional deriv or the section of dna sequence encoding albumen of the present invention shown in preferred SEQ ID NO.2 and SEQ ID NO.4, especially have the function section of maturation protein and contain the segmental chimeric protein of other proteic part in addition.Other albumen can be the albumen that contains " cysteine knot " that preferably belongs to the TGF-'beta ' family, as MP52 (PCT/EP94/02630) especially.The regional glairy receptor binding site of other intact proteins can make the proteic specificity of primary MP121 change
The biological nature of the preferred MP121 of albumen among the present invention can be determined (Cell 71.1003-1014 (1992)) such as Wrana according to the activation analysis method in the following document, Ling etc. (Proc.Natl.Acad.of Science.82.7217-7221 (1985)), Takuwa etc. (Am.J.Physiol.257.E797-E803 (1989)), Fann and Patterson (Proc.Natl.Acad.of Science.91.43-47 (1994)), Broxmeyer etc. (Proc.Natl.Acad.of Science.85.9052-9056 (1988)), Green etc. (Cell.71.731-739 (1992)), (EMBO is (1995) J.14.736-742) such as Partridge etc. (Endocrinology.108.213-219 (1981)) or Krieglstein.
Existing report, activator A and TGF-β 1 ,-2 and-3 have the effect that improves the dopamine neuron viability (EMBO such as Krieglstein J.14,736-742 (1995)) external; Neurosciene 63.1189-1196 (1994) such as Krieglstein).Partially purified MP121 has promoter action (comparing with the supernatant liquor of contrast) (Fig. 5) to the viability of the dopamine neuron cultivated through 8 days.
Another content of the present invention is the following method of producing TGF-β-family protein: it is characterized in that, cultivate the host cell that DNA or carrier of the present invention transforms, extract the TGF-beta-protein from cell or/and in the supernatant liquor of nutrient solution.This method is included in the purification of the TGF-beta-protein that transforms the cultivation of host cell in the suitable culture medium and produced.Can produce the target protein of q.s, the cell culture technology that makes it may be applied to medical treatment or need somatomedin by this method.Host cell can be bacterium such as bacillus category or intestinal bacteria, fungi such as yeast, vegetable cell such as tobacco, potato or Arabidopis thaliana, or zooblast especially vertebrate cells system as Mo-, Cos-or CHO-clone, or insect cell line.Utilize rhabdovirus system, can be implemented in the expression in the insect larvae.Utilize bacterium to produce, albumen then of the present invention can the inclusion body form produce.Can carry out renaturation according to known method to these inclusion bodies, to obtain having active albumen (seeing Jaenicke, R. and Rudolph, R.Protein Structure.ed.Creighton, T.E., IRL Press.Chapter 9).If produce and the formed heterodimer of other TGF-'beta ' family member protein, two kinds of proteic monomers can be expressed in same clone or be expressed respectively, are suitable to the same refolding method of inclusion body that forms.The method that is applicable to the coexpression in same clone mainly contains viral system such as rhabdovirus system or acne bacterium virus system.The proteinic production of heterodimer professional in principle is known, described in WO93/09229 and EP 0626 451A2.
Production contains the chimeric protein of other protein part, need on dna level, make corresponding changes, these normally the professional know and by its operation (EMBO is (1991) J.10,2105-2110; Cell 69 (1992), 329-341; J.Neurosci.39 (1994), 195-210).
Another content of the present invention is to contain the pharmaceutical composition of the TGF-β albumen of medicine effective quantity as effective constituent.This composition randomly contains acceptable carrier in the pharmacy, auxiliary, dilution or weighting agent.This pharmaceutical composition can be used for trauma care and tissue regeneration separately, also can be used in combination with other effective constituent, as: other TGF-β albumen or somatomedin such as EGF (Urogastron or PDGF (PDGF).And this kind pharmaceutical composition also can be used for preventing disease.
Content among the present invention also has: contain heterodimer albumen of the present invention or/and the pharmaceutical composition of chimeric protein.
Preferred pharmaceutical composition of the present invention is used for the treatment of or prevents the damage of bone/cartilage/reticular tissue/skin, mucous membrane, endothelial tissue, epithelium, nerve, brain, kidney or tooth; And be used for tooth implantation, wound healing and tissue regeneration process; As promoting phytokinin to be used to induce the hepatic tissue growth, induce the hyperplasia of medullary cell or precursor cell; Be used to keep differentiation state and treatment infertility and be used for contraception.Pharmaceutical composition of the present invention in addition also is used for the treatment of the disease such as the digestive system of exchange of substance aspect or relates to the disease of glucose level.
Proteic another the possible clinical application of TGF-β among the present invention be as immunoreactive supressor avoiding the rejection after the organ transplantation, or be used to promote vasculogenesis.And the albumen among the present invention also can be used for promoting potentia concipiendi or contraception.Medical composition among the present invention also can be used for diseases prevention or esthetic surgery.The use of said composition is not limited to the mankind, also is applicable to animal, especially domestic or economic animal.
In addition, use range and the specificity according to different needs heterodimer albumen and chimeric protein also can change to some extent owing to the section difference of other albumen or protein monomer.
In a word, the albumen among the present invention can be used for treating the expression diseases associated with MP121, and amount that on the one hand can be by improving MP121 or the activity of existing M121 on the other hand, then can realize by the activity that suppresses MP121.Therefore another content of the present invention is synthetic antisense nucleic acid and ribozyme, to be used to suppress translating of MP121.Inhibition is because messenger RNA(mRNA) is cut in conjunction with " hidden " or by ribozyme by antisense nucleic acid.
Existing report (Weintraub, H.M, Scientific American262:40 (1990)) about synthetic antisense nucleic acid.Antisense nucleic acid and corresponding messenger RNA(mRNA) hybridization and form the duplex molecule that to be translated.Marcus-Sekura is for example seen in the utilization of antisense nucleic acid, and the report of C.J (Anal.Biochem.172 (1988), S.289-295).
Ribozyme is to have the RNA molecule that the specificity cutting is similar to other single stranded RNA molecule ability of DNA restriction enzyme.The synthetic of ribozyme appears in the newspapers: Cech, and J.Amer.Med.Assn.260 (1988), S.3030.
According to the present invention, the suitable carrier with dna sequence dna of the present invention is used in external or the interior transfection sick body cell of body, maybe this carrier is used for the cell in vitro transfection earlier, again with in these transfectional cell implant patient bodies.Equally, the MP121 antisense polynucleotides can be changed over to the cell of not expecting that MP121 expresses.
The active compacting of MP121 can not undertaken by will not causing on the contrary with MP121 that molecule that signal continues conduction be incorporated on the MP121 acceptor.
This shows that within the scope of the invention, the MP121 acceptor of cell is also very interesting.For finding the MP121 acceptor, at first by crosslinked experiment, measure different clones to radiolabeled MP121 ( 125J-MP121) binding ability.Then, to cell, utilize expression vector (InVitrogen provides) construction cDNA library in conjunction with MP121.Utilization filters out by acceptor-cDNA cells transfected the binding ability of radiolabeled MP121.This is a method known to those skilled in the art, is used to separate activin receptor (Mathews, L.S.﹠amp as it; Vale, W.W, Cell 65 (1991), 973-982) and TGF-β Type II acceptor (Lin, H.Y, etc., Cell 68 (1992), 775-785).MP121 acceptor and known activin receptor are similar by inference, belong to the acceptor complex body of same family, thereby also can utilize the oligonucleotide of known method degeneracy to carry out PCR, to find the part fragment of allos complex body.This method also is used for activator and TGF-β TYPE I acceptor, and (Tsuchida waits Proc.Natl.Acad.Sci.USA 90 (1993), 11242-11246; Attisano etc., Cell, 75 (1993), 671-680; Franzen etc., Cell 75 (1993), 681-692).
Last content of the present invention is can a proteic antibody of specific combination the present invention or an one antibody fragment (as Fab or Fab ').The method of producing this strain specific antibodies or antibody fragment belongs to general professional general knowledge.Preferred this antibody is a monoclonal antibody.This antibody or antibody fragment also are applicable to diagnostic method.
In addition, the present invention will be illustrated by following examples.Separation 1.1 total RNA of example 1:MP121 are the (Biochemistry that extract from (40 years old) people's hepatic tissue according to the method for Chirgwin etc., 18,5294-5299 (1979)) .Poly (A+)-RNA then separates (method Stratagene Poly (A) Quick-Column that sees the product supplier) by Oligo-(dT) chromatography from total RNA.1.2。Reverse transcription reaction is as follows: with 1-2.5 μ g Poly (A)+-RNA was heated to 65 ℃ in 5 minutes, forwards cooled on ice fast to.Reaction mixture comprises 27u RNA-Guard (Pharmacia), 2.5 μ g Oligo (dT) 12-18 (Pharmacia), 5 * damping fluid (250mmol/l Tris/HClpH8.5,50mmol/l MgCl 2, 50mmol/l DTT, each dNTP of 5mmol/l, 600mmol/lKCl), and the required 20U AMV ThermoScript II (BoehringerMannheim) of every μ g Poly (A+)-RNA.Reaction mixture (25 μ l) is stored in-20 ℃ with gained cDNA after 42 ℃ of incubations 2 hours.1.3 deoxynucleotide primer OD shown in Figure 2 and OID are synthetic by full-automatic dna synthesizer (Biosearch).Earlier by the polyacrylamide gel electrophoresis of sex change, utilize isotachophoresis that the master tape on the gel is separated again, thereby with the primer purifying.Nucleotide sequence by more known TGF-'beta ' family member and select higher conserved regions, with design oligonucleotides, this conserved regions more as shown in Figure 2.For the ease of the clone, two oligonucleotide all have the EcoRI restriction enzyme site, and primer OD also has a NcoI restriction enzyme site at its 5 ' end.1.4.PCR being the reaction volume of cDNA (seeing 1.2) the .50 μ l that is equivalent to 20ng Poly (A+) RNA, the initiator of reaction comprises 1 * PCR damping fluid (16.6mmol/l (NH 4) 2SO 4, 67mmol/l Tris/HCl pH8.8,2mmol/l MgCl 2, 6.7 μ mol/l EDTA, 10mmol/l beta-mercaptoethanol, 170 μ g/ml bovine serum albumins (BSA) (Gibco), 200 each dNTP of μ mol/l (Pharmacia), each oligonucleotide of 30pmol (OD ﹠amp; OID) and 1.5UTaq-polysaccharase (Ampli Taq, Perkin Elmer Cetus).On reaction mixture, add one deck paraffin oil, carry out 40 round-robin PCR reactions then.The PCR reaction product concentrates with ethanol sedimentation through behind phenol/chloroform extracting and purifying.1.5.PCR SphI (Pharmacia) and AlwNI (Biolabs) endonuclease reaction (method that provides according to company) are provided half of reaction product.Second half then is used for AvaI (BRL), AlwNI (Biolabs) and TfiI (Biolabs) endonuclease reaction.The endonuclease reaction volume is 100 μ l (containing the 8U enzyme), carries out in 37 ℃ of incubations 2~12 hours (except the TfiI, being 65 ℃ of incubations).1.6. utilizing agarose gel electrophoresis that enzyme is cut product separates.Behind ethidium bromide staining, not digested amplified fragments is cut out from gel, and after the phenol extracting, separate.The DNA of gained is again through purifying after twice phenol/chloroform extracting.1.7. 1/4 of the DNA of gained or 1/5 again in order to amplification behind ethanol sedimentation, reaction conditions is identical with primary amplification condition, different is that cycle index is reduced to 13 times, again amplified production purified after, be used for endonuclease reaction same as described above, never digested then product is separated on the sepharose as described for amplified production, is used further to repeat amplification protcol.1.8 will cut (reaction conditions: the supplier is described according to enzyme) with 4U EcoRI (Pharmacia) enzyme from last isolated amplified production on the gel.Enzyme is cut 1/4 of mixture and is used for being connected with pBluescript Sk+ (Stratagene) carrier of EcoRI cutting.24 clones that connect each enzyme complex of back do the sequence analysis.AlwNI and SphI endonuclease bamhi only have BMP6 and statin β A sequence and do not contain any new sequence.In the endonuclease bamhi of AvaI, AlwNI and TfiI, found 19 identical new sequences and be named as MP121.These plasmids are named as pSK-MP121 (OD/OID).The master operation of one of them sequence and discovery is listed on two Nucleotide different.Ligation and colibacillary conversion are the methods (MolecularCloning:A Laboratory Manual (1989)) according to Sambrook etc.
Method according to Frohmann (see the Amplifications that Perkin-ELmer Corp publishes, 5,11-15 (1990)) has obtained cDNA clone's 3 ' terminal sequence, thereby has possessed the cDNA clone of total length.Being used to separate the segmental liver mRNA of a MP121 and being connected with reverse transcription under Oligo dT (16mer) situation (AGAATTCGCATGCCATGGTCGACGAAGC-T16) of adapter primer in use as mentioned above. the used adapter primer of amplified reaction is (AGAATTCGCATGCCATGGTCGACG) and according to MP121 sequence institute synthetic one inner primer (GGCTACGCCATGAACTTCTGCATA).Amplified production is used for amplified reaction again, and the primer is: adapter primer and another inside primer (ACATAGCAGGC ATGCCTGGTATTG) according to the MP121 preparation.Again amplified production is cloned in the SphI site of pT7/T3 U19 carrier (Pharmacia) and measures its sequence after the SphI enzyme is cut.The intersection of utilization and known MP121 sequence is identified institute's DCRP.A clone who is named as P121Lt3 ' MP13 is used to separate a NcoI/SphI fragment (the NcoI end is treated to blunt end through the T4 polysaccharase).This fragment is cloned on above mentioned pSK-MP121 (OD/OID) carrier, and the OD-primer sequence is corresponding with the direction of the T7 primer of the polyclone point of pSK.Used carrier is cut through the SphI/SmaI enzyme.Constructed recombination called after pMP121DFus6.It contains the MP121 sequence part of the 922nd to 1360 Nucleotide shown in the SEQ ID NO.1.1.9 the DdeI fragment among the pMP121 DFus6, be the 931st to 1304 nucleotide segment among the SEQ ID NO.1, be used to screen the cDNA library (Clontech of people's liver, #HL3006b, Lot 36223) (method is seen Current Protocols inMolecular Biology such as Ausubel, and Greene Publishing Associates and Wiley-Interscience (1989) publishes).From 8.1 * 10 5Filter out 24 phagocytosis tagmas (Mischplaques) in the phage, and purifying has obtained single phage respectively.Therefrom select through 10 positive clones of PCR reaction detection, and be purified into single phage.The PCR the primer is that LO2 (ACATAGCAGGCATGCCTGGTATTG) reaches from the segmental primer LOI1 of DdeI (CTGCAGCTGTGTTGGCCTTGAGA).With the cDNA fragment in the phage through the cutting of EcoRI enzyme from coming out, and be cloned in carrier with EcoRI cracked pBluescript SK.
Sequencing to one of gained plasmid SK121L9.1 shows.Initiation codon starts from the 128th Nucleotide among the SEQ IDNO.1, and three stop codes that are in same reading frame were arranged before this initiation codon, respectively at the 62nd, 77 and 92 Nucleotide place.Through and the proteic sequence homology of other TGF-β relatively, the MP121 maturation protein starts from the 836th Nucleotide among the SEQ ID NO.1, i.e. the 237th amino acid among the SEQ NO.2, stop code starts from the 1184th Nucleotide of SEQ ID NO.1.
Plasmid SK121L9.1 (preserving number: 9177) be deposited among the DSM on April 26th, 1994.1.10. the separation of MP121 cDNA and genomic dna thereof in the mouse:
With the MP121 sequence that people MP121 sequence information is used to separate mouse, the method that wherein relates to is the known method of professional, sees Current Protocols in MolecularBiology (Ausubel et al; Greene Publishing Associates and Wiley-Interscience, Wiley ﹠amp; Sohs, 1987-1995) or Molecular Cloning (Sambrooket al, 2nd edition, Cold Spring Harbour Laboratory Press 1989).
At first, from synthetic two primer: the ACGAATTCCGACGAGGCATCGACTGC of people MP121 sequence, and GCGTCGACTACCATGTCAGGTATGTC, designed an enzyme point of contact (EcoRI and SalI) at 5 ' of two primers end.After two primers are used for the amplification of mouse gene group DNA, the fragment that the 0.35Kb of gained is long is cloned on Bluescript (Stratagene) carrier again, and be used to prepare radioactive probe, utilize λ library and the cDNA library of this probe according to standard method screening mouse gene group DNA.CDNA is synthetic by the isolated RNA of the liver of mouse, adds that EcoRI/NotI connector sequence rear clone is on λ gt10.
From gene pool and cDNA storehouse, all isolated the MP121 clone.The cDNA subclone that will contain complete encoding sequence is in the EcoRI site of Bluescript SK (Stratagene), and gained plasmid called after mouse SKMP121 also is deposited in (DSM9964) in the DSM database May 10 nineteen ninety-five.Sequential analysis gained complete sequence is shown in SEQ ID NO.3.Initial code starts from the 131st Nucleotide and stop code starts from the 1187th Nucleotide in SEQ ID NO.3 sequence.See SEQ ID NO.4 according to the protein sequence that this sequence is released.
From the subclone analysis revealed that contains the MP121 clone of genomic library, an intron that about 5.5kb is long is contained in the coding region of MP121 precursor peptide.This intron is between the 446th and 447 Nucleotide of sequence shown in the SEQ ID NO.3.The interface point of exon is shown in SEQ IDNO.5.The expression of example 2:MP121
In eucaryon and prokaryotic system, all might express MP121.
Only the maturation protein section with MP121 is used for prokaryotic expression.After purifying,, be foldable to binary at the ripe monomer of the MP121 of expression in escherichia coli.For simplifying the proteic purge process of MP121, can add 6 histidine residues at the N-of maturation protein end, thereby be easy to proteic purifying with combining of Ni inner complex Column chromatography column by 6 histidine residues.
For example: with prokaryotic vector pBP4 expressing human MP121 maturation protein section (the 237-352 amino acid among the SEQ ID No.2), total 13 the additional amino acid (MHHHHHHKLEFAM) that comprise 6 Histidines of its N-end.This carrier is the derivative of pBR322 plasmid, and contains the tetracycline resistance gene and from the T7 promotor of pBluescriptII SK plasmid (Stratagene).In addition, this carrier also has a ribosome binding site and an initial code after its T7 promotor, then be 6 Histidine passwords after the initial code.Terminator (T) is positioned at a plurality of be used to insert intron single endonuclease digestion insertion site such as EcoRI, and XhoI is after the stop code of SmaI and ApaI and three kinds of reading frames.With plasmid SK121L9.1 (DSM preserving number: 9177) be template, amplify the cDNA of MP121 maturation protein through the PCR reaction, the primer is: the end of GAATTCGCCATGGGCATCGACTGCCAAGGAGG and two oligonucleotide of CCGCTCGAGAAGCTTCAACTGCA CCCACAGGC. all has additional restriction enzyme site (to be respectively EcoRI/NcoI, or XhoI/HindIII.The fragment cloning that the 377bp of gained is long is in the EcoRV site of pBluescriptII SK (Stratagene).The fragment of the 0.38kb of gained is also cloned the EcoRI site in the pBP4 carrier after the EcoRI enzyme is cut with one of them clone (MP1215 ' end points to the T7 promotor).Insert segmental correct trend among the gained plasmid pBP4MP121His after restriction analysis and sequential analysis and determined.The pBP4MP121His plasmid is deposited in (preserving number is 9704) in the DSM database January 30 nineteen ninety-five.
MP121 albumen also can obtain expressing by utilizing the T7-RNA polysaccharase.Can utilize the method for different expression T7-RNA polysaccharases, as: utilize the phage of another plasmid that contains the T7-RNA pol gene or coding T7-RNA polysaccharase to infect, or by special bacterial isolates BL21 (DE3) pLysS (Novagen who contains T7-RNA polysaccharase integrator gene, #69451-1), under the situation of IPTG abduction delivering T7-RNA polysaccharase, can obtain having the inclusion body of the MP121 maturation protein (MP121His) of histidine mark (His-Tag).In SDS polyacrylamide (15%) gel electrophoresis, and the nearly 16KD of this proteic apparent molecular weight (theoretical molecular: 14.2KD), shown in the Western blot of Fig. 3.The bacterium that transforms with the pBP4 plasmid is not then found special protein band in contrast.Because this albumen contains histidine mark (His-Tag), so, the method for Ni sequestrant-chromatography column purifying protein can be utilized, as (BIO/Technology Vol.6,1321-1325 (1988)) as described in the Hochuli etc.Can make further purifying to albumen by reversed-phase high pressure liquid chromatography (HPLC).Utilize a reverse-phase chromatographic column (Art:715023), used chromatographic condition is for Nucleosil 300-7C4, Macherey-Nagel: flow velocity 2 ml/min, be dissolved in the acetonitrile of 0.1%TFA, gradient is 0~90%, the time: 100 minutes.With this understanding, MP121His albumen is to begin wash-out at 40% o'clock at acetonitrile concentration.
Utilize the MP121 specific antibody, analyze to confirm the MP121 albumen of above-mentioned albumen behaviour by Western blot.Used MP121 polyclonal antibody is from chicken and rabbit.For obtaining being used for the antigen of immunity,, in intestinal bacteria, obtain expressing with the fusion rotein of a section in the MP121 maturation protein (the 260th to 352 amino acid among the SEQ ID NO.2) with preceding 98 amino acid fragments of MS2-phage polysaccharase.Isolated fusion rotein (MS2-MP121) inclusion body is separated through polyacrylamide gel electrophoresis,, develop and be used for immunity (chicken or rabbit) (Tessmer, U.﹠amp from gel according to after the Tongran color method dyeing; Dernick, R., IBL (1990) 8-13).Can both detect the specifically expressing of MP121 from the antibody of chicken or rabbit acquisition.Western blot shown in Figure 3 analyzes, the MP121 antibody that used is from chicken.This antibody is through PEG precipitation (Thalley B.S and Carroll, S.B.BIO/Technology, Vol.8,934-938 (1990)) and the processing of film conjugated antigen (fusion rotein (MS2-MP121)) and obtain being further purified method and see Sambrook et al., Molecular Cloning, 2nd edition, Cold Spring Harbour Laboratory Press1989.18.17 page or leaf).Used two anti-ly are the anti-chicken immune Lysozyme (IgG) mutually coupled with alkaline phosphatase (Sigma A9171).Utilize Tropix Western-Light Protein Detection Kit (Serva #WL10RC) test kit, further detect according to the method that the supplier provided.
For obtaining the material of biologically active, can will after the MP121 of expression in escherichia coli monomer is purified, be folded into binary.Method therefor is seen Jaenicke, R. and Rudolph, R (ProteinStructure, ed, Creighton, T.E.IRL Press, Chapter9).
Express MP121 in eukaryotic cell, used is the vaccinia virus expression system.This expression system has play-by-play Current Protocols in Molecular Biology (Ausubel etc., Greene Publishing Associates and Wiley-Interscience, Wiley ﹠amp in following document; Sons, Chapter 16, Unit 16.15~16.18) (following the document is called for short CP), those skilled in the art can imitate.The main foundation of this system is to use the specific support foreign DNA to be incorporated on the genome of vaccinia virus by homologous recombination.Based on this principle, used carrier contains TK (thymidine kinase) gene on the vaccinia virus genome.In addition, carrier also carries colibacillary xantheine-guanine-phosphoribosyl transferase gene (gpt) (Falkner, F.G.﹠amp; Moss, B., J.of Virol.62 (1988), 1849-1854)), be intended to recombinant celo virus has been cloned the whole coding region that has MP121 in this carrier cDNA.
For shortening plasmid SK121L9.1 (DSM preserving number: the non-translational region of 5 '-ends and 3 '-ends 9177), and, need carry out a series of PCR and react and the subclone step in interpolation single endonuclease digestion site, two ends.All PCR reactions all are as template with SK121L9.1 plasmid (DSM preserving number 9177).For the used primer of the non-translational region that shortens 5 '-ends is CCCGGATCCGCTAGCACCATGACCTCCTCATTG CTTCTG (two ends has BamHI and NheI restriction enzyme site respectively), and an inner primer (CCCTGTTGTCCTCTAGAAGTG).The PCR fragment cloning of gained on Bluescript SK (Stratagene), is compared by sequential analysis and with the intersection of sequence shown in the SEQ ID NO.1, so that institute's calling sequence is determined.For the used template of non-translational region that shortens 3 ' end is a SphI/EcoRI fragment (0.22kb) among the plasmid pBP4M121His.
Two end fragments of people MP121 are connected by the centre dna sequence dna that lacks among the MP121 among inner restriction enzyme site (XbaI and SphI) and the plasmid SK121L9.1 (DSM preserving number 9177), (Molecular Cloning such as Sambrook are seen in standard method, 2nd editeon, ColdSpring Harbour Laboratory Press, 1989).The cDNA that has been shortened still has complete MP121 reading frame (128-1184 Nucleotide among the SEQ ID NO.1), and as BamHI and EcoRI fragment cloning on the pBP1 carrier of cutting through BamHI and EcoRI enzyme.The plasmid pBP1MP121 that is obtained thus is deposited in (preserving number: 9665) in the DSM database January 12 nineteen ninety-five.
Plasmid pBP1MP121 is used to make up the vaccinia virus of reorganization.For this reason, the wild-type vaccinia virus of 1 milliliter of PBS damping fluid and the 143B cell (HuTK-of 80% symphysis will be suspended in, ATCC CRL 8303) mix in diameter is the culture dish of 35mm, moderate vibration 30 minutes is to realize infect (1 virus/10 cells) of viral pair cell under the room temperature.Supernatant liquor removed and add 2 milliliters of nutrient solutions (MEM, Gibco BRL #041-01095 contain the penicillin and the Streptomycin sulphate that are diluted to 1/500 concentration, Gibco BRL #043-05140), in 37 ℃ of incubations 2 hours.After at last nutrient solution being removed, add 100ng pBP1MP121 and the 2 μ g media DNA (calf thymus DNAs of ultrasonication, Boehringer Mannheim #104175) and 10 μ l Lipofektin (GibcoBRL #18292-011), about 15 hours of incubation (37 ℃) in 1ml MEM, add after the 1ml MEM contain 20%FCS (Sigma #F-7524) in 37 ℃ and continued incubations 24 hours, at last lysing cell is frozen preservation deeply.
Based on the gpt screening of xantheine-guanine-phosphoribosyl transferase, and the separation and the amplification procedure of recombinant virus, roughly as described in Unit 16.17 among the document CP, carry out.Be used cell be RK13 (ATCC CCL37).
Utilize spot hybridization (seeing CP.Unit 16.18) to determine whether MP121 cDNA is incorporated on the viral genome.Through recombinant virus and the wild-type virus that the pBPMP121 transfection is obtained, all be used to analyze the expression of MP121 in clone 143B (people HuTK-, ATCC CRL 8303) and NIH-3T3 (DSM ACC59, Switzerland's mice embryonic).Cell cultures is carried out in explanation according to the clone supplier, carry out (37 ℃ of cellular invasions with the virus of about 3 times of Yu Liansheng cell quantities, 30 minutes), add then and contained 10%FCS and penicillin/streptomycin (1: 500, GibcoBRL #043-05140) nutrient solution, and 37 ℃ of incubations 6 hours.Remove cell behind the nutrient solution with for example HBSS (Gibco, BRL #14180-046) after twice washing, (MEM is used for HuTK-clone, and DMEM+4.5g/l glucose+NEAA (Gibco, BRL #11140-035) is used for NIH-3T3 clone to add the production nutrient solution that does not contain FCS again; Add Aprotinin (Fluka #10820,50U/ml) and penicillin/streptomycin) respectively.After cultivating in 20 to 22 hours, collect the supernatant liquor of culturing cell, utilize Western blots to analyze expression level (CP is seen in standard method, and Unit 10.8).In the supernatant liquor of 1~3 milliliter cell culture fluid, add isopyknic acetone and protein precipitation and carry out centrifugal after the incubation at least one hour on ice.Gained precipitation is suspended among the electrophoresis sample solution, and is used for 15% polyacrylamide gel electrophoresis (sample solution: 7M urea, 1%SDS, 7mM NaH 2PO 4, 0.01% tetrabromophenol sulfonphthalein, and optional 1% beta-mercaptoethanol).Used labelled protein is prestained molecular weight of albumen standard substance (Gibco BRL #6041-020).Albumen change film (pvdf membrane: Immobilon #IPVH 00010) and the envelope membrane process identical with standard method.
From (Fig. 3) Western blot sketch as can be seen, the cell that infects through recombinant virus shows specific MP121 band.The MP121 that expresses in the NIH-3T3 cell is a secretory protein, and this albumen shows that behind electrophoresis under the non-reduced condition molecular weight is about 18KD (theoretical molecular of expection is 25KD).The molecular weight that this albumen shows in the reductibility electrophoresis is 15KD (theoretical molecular of expectation is 12.5KD).This result shows, MP121 is that the binary as maturation protein obtains expressing just as expected.To slow slightly, this is likely because the formation of ball-like structure in the HuTk-cell, finds that also precursor protein is processed into maturation protein than monomer M P121 mobile phase for MP121 albumen binary.The cell (HuTK-or NIH-3T3) that infects through wild-type virus (not containing foreign DNA) does not show band at Western blot.
The cowpox expression system is applicable to and utilizes recombined vaccinia virus to carry out cotransformation, especially is suitable for the proteic formation of the proteic heterodimer of different TGF-β.Utilization contains a kind of affinity post of TGF-β protein monomer specific antibody, heterodimer and homodimer might be separated.Wherein the α of statin-and β A-β B chain is especially interesting.Example 3: the research that MP121 expresses in the different tissues of mice
From 6 the week age mouse different tissues (brain, heart, kidney, liver, lung, spleen, muscle, ovary, testis) and embryonic stem cell the secundum legem method extract total RNA.By produce family's explanation with the total RNA of 10 μ g carry out ribonuclease protecting experiment (RPA) (Ambion RPAII Kit, #1410).For obtaining activator β AAnd β BSpecific probe, utilize Auele Specific Primer, to increasing with the corresponding mouse of maturation protein (129SV) genomic dna.For making amplified fragments be easy to the clone, the end of primer has respectively been introduced EcoRI and/or BamHI or HindIII restriction enzyme site.Amplification activator β APrimer sequence from the mRNA sequence (GenBank Accession #M 37482) of mouse:
GGATCCGAATTCGGCTTGGAGTGTGATGGCAAGG and GGATCCGAATTCCTCTG GGACCTGGCAACTCTAG.
Amplification activator β BUsed degenerated primer is then from people's sequence (Mason et al, Molecular Endocrinology 3,1352-1358 (1989)): GAGAATTCCA (GA) CA (GA) TT (TC) TT (CT) AT and GCAAGCTTT (GA) TA (TC) TC (GA) TC (GA) TC.The PCR fragment that obtains is subcloned on carrier pGEM-4 (Promega) and goes up and it is identified.The activator specific sequence fragment that the RPA test is protected, activator β ABe 369bp activator β BBe 254bp.The protection fragment of MP121 is SEQ ID NO.887-1164 Nucleotide in 3 sequences.The fragment of cloning in pGEM-4 is used for the isolated transcription reaction, with the antisense RNA probes of synthesizing radioactive mark.According to supplier (Promega, the Riboprobe Gemini Systems) reaction conditions that provides, use 100 μ M CTP and α 32P-CTP (800Ci/mmol, Amersham).Equally, wire plasmid pTri-GAPDH (Ambion #7431) the synthetic radioactive mark RNA of institute (CTP concentration is 1mM) who cuts with DdeI is in order in contrast.4 antisense RNA probes are separated from polyacrylamide gel, and be respectively applied for RNA (the total RNA/1 of 10 μ g * 10 with the mouse respective organization 5The cpm probe) mixes, be incubated overnight at 42 ℃.Carry out denaturing gel electrophoresis and radioactive automatic developing (4 days) according to standard method.The partial purification of example 4:MP121 and the activity research of partially purified MP121
MP121 albumen in that cowpox expression system (seeing example 2) is obtained can obtain partial purification by two kinds of chromatography columns.
The MP121 production process is as follows: NIH-3T3 cell (the DSM ACC59 of adhesion, Switzerland's mice embryonic) recombinant virus with same quantity mixes, to finish infection processs, then, add the nutrient solution that contains 10%FCS and penicillin/streptomycin in 37 ℃ of incubations 30 minutes.
After 37 ℃ are cultivated 4 hours, remove nutrient solution with cell washing twice, add the production nutrient solution (seeing example 2) that is provided with FCS.After continuing to cultivate 20-22 hour.Collect supernatant liquor and carry out centrifugal (40000 * g, 30 minutes, 4 ℃), then supernatant liquor is filtered to remove virus removal (filtering net: 0.1 μ m aperture, MillexVV, Millipore #SLVV25LS) again.Utilize the wild-type vaccinia virus to infect the cell of gained, obtain contrast supernatant liquor (Wt) by above method.Analyze according to Western blot, the expression amount of MP121 is estimated as 50~100 μ g/l.
In 1.1 liters of cell conditioned medium liquid that contain MP121, add proteinase inhibitor PMSF (1 μ M) respectively, (NH 4) 2SO 4(final concentration is 1M), Tris-pH8.0 (final concentration is 20mM) is added to this sample through buffer A (1M (NH 4) 2SO 420mM Tris-pH8.0) (volume is 5 milliliters (FastFloW (high sub) Pharmacia #17-0973-05) to equilibrated phenyl sepharose post, utilize 15 times to the buffer A of column volume and 10 times of buffer B (20mM Tris.pH8.0) flushing to column volume and with linear gradient from 100% damping fluid C (20mM Tris pH8.0,80% ethylene glycol) wash-out, flow velocity is 1 ml/min, wash-out 50 minutes (each cut 5ml), Western blot analysis revealed, MP121 albumen are to be come out by wash-out in 50~80% o'clock in glycol concentration.The part of protein ingredient through polyacrylamide gel (concentration 15%) electrophoresis, is determined to contain the component of MP121 and it is put together according to silver staining (SilverStain-II, Daiichi #SE140000).The respective components of purifying contrast supernatant liquor gained after the silver staining analysis, is also put together.
The MP121 protein ingredient of collecting, (HPLC) is further purified by anti-phase high pressure liquid chromatography.Earlier with buffer A (aqueous solution of 0.1% trifluoroacetic acid) balance C8 chromatographic column (Aquapore RP300, Applied Biosystems, particle diameter 7 μ m, aperture: 300A °).With containing after the proteic component of MP121 is added on the chromatographic column of above-mentioned collection, earlier with buffer A thorough washing chromatographic column.The linear gradient of using per minute 1.5% buffer B (90% acetonitrile, 0.1% trifluoroacetic acid) again elutes conjugated protein with 0.2ml/ minute flow velocity.With collected different components (volume: 600 μ l) be used for Western blot and according to gel electrophoresis analysis that silver staining carried out.MP121 albumen is to begin wash-out at 55% o'clock at acetonitrile concentration.To contain the proteic component of MP121 and put together, and the respective components in the purge process of supernatant liquor is also concentrated.Show through silver-colored stained gel electrophoretic analysis, still be mixed with other albumen in the MP121 protein ingredient.For obtaining the MP121 of purifying, need further purification step.
The purification process that available professional was familiar with is further purified, as: use gel sieve post, ion exchange column, affinity column or metal chelating column.Estimate according to Western blot analytical results, separablely from the supernatant liquor of 1 liter of cell cultures go out the partially purified MP121 of about 8 grams.Partially purified albumen is stored in-80 ℃ after lyophilize.
Be to confirm the influence of MP121, distinguish from neurone (method is seen Brain Res.586 such as Shimoda, 319-331 (1992)) from the midbrain of the rat embryo (E14) in 14 day age to dopamine neuron.According to (Neuroscience 63,1189-1196 (1994)) described methods such as Krieglstein, carry out neural single celled separation and cultivation.Scribbling on the slide glass of polyornithine/laminine, the design cell density be 200000 cells/centimetre 2After 24 hours cultivate, every three days 2/3 nutrient solution (500 μ l) removed and add the fresh medium of same amount and other required composition.The MP121 albumen of phenyl sepharose post and anti-phase high pressure liquid chromatography (HPLC) purifying and lyophilize rear section purifying is dissolved in 50% acetonitrile, adds cell culture fluid.The final concentration of MP121 in nutrient solution is 20ng/ml (final concentration of acetonitrile is 0.3%).Contrast supernatant liquor (wt) sample of purifying of amount equally is dissolved in 50% acetonitrile and adds in the contrast culture liquid, and wherein the final concentration of acetonitrile also is 0.3%.After cultivating in 8 days, at room temperature the gained culture is fixed with 4% Paraformaldehyde 96, (10 minutes ,-20 ℃) use PBS (salts solution of phosphoric acid buffer) flushing again after the acetone osmotic treated, through 1%H 2O 2(being dissolved in PBS) handles, and with after horse serum flushing, the sealing treatment, carries out immunohistochemical staining.Tyrosine hydroxylase (TH) is the certain enzyme in Dopamine HCL and other catecholamine biosynthetic process, so TH can be used as the mark (cell that contains norepinephrine is not separated) that above-mentioned neuronal cell is cultivated.Mouse monoclonal antibody with the mouse TH of the Chinese People's Anti-Japanese Military and Political College (diluted 1: 200, and BoehringerMannheim), through 37 ℃, after incubation was handled in 1 hour, used test kit (Vectastain ABC Kit:Vecto Labs) further to detect again.From 0.12cm 2The quantity of statistics TH-positive cell in the area.As ise apparent from FIG. 5, MP121 has favourable influence to the growth of dopamine neuron.
Fig. 5 represents from the isolating neurone of rat embryo (E14) midbrain, the survival quantity of the dopamine neuron that the TH immune response is positive after cultivating in 8 days.The treatment effect of the contrast supernatant liquor product (wt) of partially purified MP121 (concentration 20ng/ml), equivalent and undressed neurone (contrast: contain 0.3% acetonitrile nutrient solution).Institute's indicating value is the mean value ± standard error of mean of three repeated samplings.The detailed description of Fig. 3-5: Fig. 3: the Western blot sketch that chicken antibody carried out that utilizes anti-MP121.1: the Bacillus coli cells that (1% beta-mercaptoethanol) transforms through pBP4MP121His under reductive condition.2: the culture supernatant of the NIH-3T3 cell that (1% beta-mercaptoethanol) infects through recombinant virus (the MP121 cDNA that contains insertion) under reductive condition.3: the culture supernatant of the NIH-3T3 cell that under non-reduced condition, infects through recombinant virus (the MP121 cDNA that contains insertion).M: the molecular weight of albumen mark that dyes in advance (indicating the apparent molecular weight of major protein band) (Gibco BRL#26041-020).Fig. 4: the radioautograph collection of illustrative plates of the gel analysis of ribonuclease protecting experiment.Used specific probe is activator β AA)-, activator β BB)-,, MP121-reached GAPDH-probe in contrast, used total RNA is from different mouse tissue (1: brain, 2: heart, 3: kidney, 4: liver, 5: lung, 6: muscle, 9: ovary, 10: spleen, 11: testis), from embryonic stem cells (12:CJ7) and yeast in contrast (swimming lane 13), sample 14 does not contain RNA with in contrast.Not protected sense-rna sample in the 8 and 15 expression hybridization: the not protected fragment length of having indicated expectation in the bracket on right side; Protected fragment length then indicates in the left side, sample 7 be as molecular weight marker enzyme cuts and the end-labelled pBR322 plasmid of γ-32P-ATP (Amersham) through MsDI (Biolabs #303).Fig. 5: the dopamine neuron number with the immunoreactive rat embryo of TH-(E14) midbrain of expression survival after cultivating in 8 days.The effect of surveying from following different treatment: (contrast: the nutrient solution that contains 0.3% acetonitrile), institute's indicating value is the mean value ± standard error of mean of three repeated samplings for the partially purified contrast supernatant liquor (wt) of partially purified MP121 (concentration is 20ng/ml), equivalent and undressed neurone.
Sequence explanation (1) general information
(i) applicant
(A) title: Biopharm Gesellschaft zur biotechnologischen
Entwicklung?von?Pharmaka?mhH
(B) street number: Czernyring 22
(C) place: Heidelberg
(E) country: Germany
(F) postcode: 69115
(ii) denomination of invention: the growth/differentiation factor of new TGF-'beta ' family
(iii) sequence number: 6
(iv) computer-reader form:
(A) media type: floppy disk
(B) computer: IBM PC compatible
(C) operating system: PC-DOS/MS-DOS
(D) software: PatentIn Release #1.0, Version #1.30 (EPA)
(v) application information:
Application number: WO PCT/EP95/02552 (2) SEQ ID NO:1 explanation
(i) sequence signature:
(A) length: 2272BP
(B) kind: Nucleotide
(C) chain: two strands
(D) topology: wire
(vi) primary source:
(A) organism: Homo sapiens
( xi ) :SEQ ID NO:1:CAAGGAGCCA TGCCAGCTGG ACACACACTT CTTCCAGGGC CTCTGGCAGC CAGGACAGAG 60TTGAGACCAC AGCTGTTGAG ACCCTGAGCC CTGAGTCTGT ATTGCTCAAG AAGGGCCTTC 120CCCAGCAATG ACCTCCTCAT TGCTTCTGGC CTTTCTCCTC CTGGCTCCAA CCACAGTGGC 180CACTCCCAGA GCTGGCGGTC AGTGTCCAGC ATGTGGGGGG CCCACCTTGG AACTGGAGAG 240CCAGCGGGAG CTGCTTCTTG ATCTGGCCAA GAGAAGCATC TTGGACAAGC TGCACCTCAC 300CCAGCGCCCA ACACTGAACC GCCCTGTGTC CAGAGCTGCT TTGAGGACTG CACTGCAGCA 360CCTCCACGGG GTCCCACAGG GGGCACTTCT AGAGGACAAC AGGGAACAGG AATGTGAAAT 420CATCAGCTTT GCTGAGACAG GCCTCTCCAC CATCAACCAG ACTCGTCTTG ATTTTCACTT 480CTCCTCTGAT AGAACTGCTG GTGACAGGGA GGTCCAGCAG GCCAGTCTCA TGTTCTTTGT 540CCAGCTCCCT TCCAATACCA CTTGGACCTT GAAAGTGAGA GTCCTTGTGC TGGGTCCACA 600TAATACCAAC CTCACCTTGG CTACTCAGTA CCTGCTGGAG GTGGATGCCA GTGGCTGGCA 660TCAACTCCCC CTAGGGCCTG AAGCTCAAGC TGCCTGCAGC CAGGGGCACC TGACCCTGGA 720GCTGGTACTT GAAGGCCAGG TAGCCCAGAG CTCAGTCATC CTGGGTGGAG CTGCCCATAG 780GCCTTTTGTG GCAGCCCGGG TGAGAGTTGG GGGCAAACAC CAGATTCACC GACGAGGCAT 840CGACTGCCAA GGAGGGTCCA GGATGTGCTG TCGACAAGAG TTTTTTGTGG ACTTCCGTGA 900GATTGGCTGG CACGACTGGA TCATCCAGCC TGAGGGCTAC GCCATGAACT TCTGCATAGG 960GCAGTGCCCA CTACACATAG CAGGCATGCC TGGTATTGCT GCCTCCTTTC ACACTGCAGT 1020GCTCAATCTT CTCAAGGCCA ACACAGCTGC AGGCACCACT GGAGGGGGCT CATGCTGTGT 1080ACCCACGGCC CGGCGCCCCC TGTCTCTGCT CTATTATGAC AGGGACAGCA ACATTGTCAA 1140GACTGACATA CCTGACATGG TAGTAGAGGC CTGTGGGTGC AGTTAGTCTA TGTGTGGTAT 1200GGGCAGCCCA AGGTTGCATG GGAAAACACG CCCCTACAGA AGTGCACTTC CTTGAGAGGA 1260GGGAATGACC TCATTCTCTG TCCAGAATGT GGACTCCCTC TTCCTGAGCA TCTTATGGAA 1320ATTACCCCAC CTTTGACTTG AAGAAACCTT CATCTAAAGC AAGTCACTGT GCCATCTTCC 1380TGACCACTAC CCTCTTTCCT AGGGCATAGT CCATCCCGCT AGTCCATCCC GCTAGCCCCA 1440CTCCAGGGAC TCAGACCCAT CTCCAACCAT GAGCAATGCC ATCTGGTTCC CAGGCAAAGA 1500CACCCTTAGC TCACCTTTAA TAGACCCCAT AACCCACTAT GCCTTCCTGT CCTTTCTACT 1560CAATGGTCCC CACTCCAAGA TGAGTTGACA CAACCCCTTC CCCCAATTTT TGTGGATCTC 1620CAGAGAGGCC CTTCTTTGGA TTCACCAAAG TTTAGATCAC TGCTGCCCAA AATAGAGGCT 1680TACCTACCCC CCTCTTTGTT GTGAGCCCCT GTCCTTCTTA GTTGTCCAGG TGAACTACTA 1740AAGCTCTCTT TGCATACCTT CATCCATTTT TTGTCCTTCT CTGCCTTTCT CTATGCCCTT 1800AAGGGGTGAT TTGCCTGAGC TCTATCACCT GAGCTCCCCT GCCCTCTGGC TTCGTGCTGA 1860GGTCAGGGCA TTTCTTATCC CTGTTCCCTC TCTGTCTAGG TGTCATGGTT CTGTGTAACT 1920GTGGCTATTC TGTGTCCCTA CACTACCTGG CTACCCCCTT CCATGGCCCC AGCTCTGCCT 1980ACATTCTGAT TTTTTTTTTT TTTTTTTTTT TGAAAAGTTA AAAATTCCTT AATTTTTTAT 2040TCCTGGTACC ACTACCACAA TTTACAGGGC AATATACCTG ATGTAATGAA AAGAAAAAGA 2100AAAAGACAAA GCTACAACAG ATAAAAGACC TCAGGAATGT ACATCTAATT GACACTACAT 2160TGCATTAATC AATAGCTGCA CTTTTTGCAA ACTGTGGCTA TGACAGTCCT GAACAAGAAG 2220GGTTTCCTGT TTAAGCTGCA GTAACTTTTC TGACTATGGA TCATCGTTCC TT 2272 ( 2 ) SEQ ID NO:2:
(i) sequence signature:
(A) length: 352 amino acid
(B) kind: amino acid
(C) chain:
(D) topology: wire
(ii) molecular species: peptide
(vi) primary source:
(A) organism: Homo sapiens
(xi) sequence explanation: SEQ ID NO:2:
Met?Thr?Ser?Ser?Leu?Leu?Leu?Ala?Phe?Leu?Leu?Leu?Ala?Pro?Thr?Thr
1 5 10 15
Val?Ala?Thr?Pro?Arg?Ala?Gly?Gly?Gln?Cys?Pro?Ala?Cys?Gly?Gly?Pro
20 25 30
Thr?Leu?Glu?Leu?Glu?Ser?Gln?Arg?Glu?Leu?Leu?Leu?Asp?Leu?Ala?Lys
35 40 45
Arg?Ser?Ile?Leu?Asp?Lys?Leu?His?Leu?Thr?Gln?Arg?Pro?Thr?Leu?Asn
50 55 60
Arg?Pro?Val?Ser?Arg?Ala?Ala?Leu?Arg?Thr?Ala?Leu?Gln?His?Leu?His
65 70 75 80
Gly?Val?Pro?Gln?Gly?Ala?Leu?Leu?Glu?Asp?Asn?Arg?Glu?Gln?Glu?Cys
85 90 95
Glu?Ile?Ile?Ser?Phe?Ala?Glu?Thr?Gly?Leu?Ser?Thr?Ile?Asn?Gln?Thr
100 105 110
Arg?Leu?Asp?Phe?His?Phe?Ser?Ser?Asp?Arg?Thr?Ala?Gly?Asp?Arg?Glu
115 120 125
Val?Gln?Gln?Ala?Ser?Leu?Met?Phe?Phe?Val?Gln?Leu?Pro?Ser?Asn?Thr
130 135 140
Thr?Trp?Thr?Leu?Lys?Val?Arg?Val?Leu?Val?Leu?Gly?Pro?His?Asn?Thr
145 150 155 160
Asn?Leu?Thr?Leu?Ala?Thr?Gln?Tyr?Leu?Leu?Glu?Val?Asp?Ala?Ser?Gly
165 170 175
Trp?His?Gln?Leu?Pro?Leu?Gly?Pro?Glu?Ala?Gln?Ala?Ala?Cys?Ser?Gln
180 185 190
Gly?His?Leu?Thr?Leu?Glu?Leu?Val?Leu?Glu?Gly?Gln?Val?Ala?Gln?Ser
195 200 205
Ser?Val?Ile?Leu?Gly?Gly?Ala?Ala?His?Arg?Pro?Phe?Val?Ala?Ala?Arg
210 215 220
Val?Arg?Val?Gly?Gly?Lys?His?Gln?Ile?His?Arg?Arg?Gly?Ile?Asp?Cys
225 230 235 240
Gln?Gly?Gly?Ser?Arg?Met?Cys?Cys?Arg?Gln?Glu?Phe?Phe?Val?Asp?Phe
245 250 255
Arg?Glu?Ile?Gly?Trp?His?Asp?Trp?Ile?Ile?Gln?Pro?Glu?Gly?Tyr?Ala
260 265 270
Met?Asn?Phe?Cys?Ile?Gly?Gln?Cys?Pro?Leu?His?Ile?Ala?Gly?Met?Pro
275 280 285
Gly?Ile?Ala?Ala?Ser?Phe?His?Thr?Ala?Val?Leu?Asn?Leu?Leu?Lys?Ala
290 295 300
Asn?Thr?Ala?Ala?Gly?Thr?Thr?Gly?Gly?Gly?Ser?Cys?Cys?Val?Pro?Thr
305 310 315 320
Ala?Arg?Arg?Pro?Leu?Ser?Leu?Leu?Tyr?Tyr?Asp?Arg?Asp?Ser?Asn?Ile
325 330 335
Val?Lys?Thr?Asp?Ile?Pro?Asp?Met?Val?Val?Glu?Ala?Cys?Gly?Cys?Ser
340 345 350 (2) explanations to SEQ ID NO:3
(i) sequence signature:
(A) length: 1558BP
(B) kind: Nucleotide
(C) chain: two strands
(D) topology: wire
(vi) primary source:
(A) organism: mouse
( xi ) :SEQ ID NO:3:AAGGAGTCAT GCCAGTCGGA GGTCAGTCAC ATTCCTCCCA GGGTCCCTGG TGCCCAGGAC 60AGAGTTGAAG CACTCCCGTT GAGACCCTGA ATATAGGCTT TGGGTCCTTT AAGGAGGCTA 120TCCTCCAGCA ATGGCCTCCT CCTTGCTCCT GGCTCTTCTG TTCCTGACTC CAACCACAGT 180AGTGAACCCC AAAACTGAGG GTCCATGCCC AGCATGTTGG GGTGCCATCT TTGACCTGGA 240GAGCCAGCGG GAGCTGCTTC TCGATTTGGC CAAGAAAAGT ATCCTGGACA AGCTGCACCT 300CAGCCAGCGC CCCATACTCA GTCGGCCAGT GTCCAGAGGG GCTCTCAAGA CCGCGCTGCA 360GCGCCTCCGC GGGCCTTGAC GGGAAACCCT GTTGGAGCAT GACCAGAGAC AAGAAGAGTA 420TGAGATCATC AGCTTTGCTG ACACAGACCT CTCCAGCATC AACCAGACCC GGCTCGAGTT 480CCACTTCTCT GGTAGAATGG CCAGTGGCAT GGAGGTCCGG CAGACCCGCT TCATGTTCTT 540CGTGCAGTTC CCCCACAATG CCACCCAGAC CATGAATATA AGAGTTCTTG TGCTAAGACC 600ATATGACACC AACCTCACCT TGACAAGTCA GTACGTGGTG CAGGTGAATG CCAGTGGCTG 660GTACCAGCTT CTCCTGGGAC CTGAAGCTCA AGCTGCTTGC AGCCAGGGAC ACCTTACTCT 720GGAGCTGGTA CCAGAAAGCC AGGTGGCCCA CAGTTCCTTG ATCCTGGGCT GGTTTTCCCA 780CAGGCCTTTT GTGGCAGCCC AGGTAAGGGT TGAGGGCAAG CATCGGGTTC GCCGGCGAGG 840TATCGATTGC CAGGGGGGGT CCAGGATGTG CTGTCGACAA GAGTTTTTTG TAGACTTCCG 900TGAGATTGGC TGGAATGACT GGATCATCCA GCCTGAAGGC TATGCCATGA ACTTCTGCAC 960TGGGCAGTGC CCACTACATG TGGCAGGCAT GCCTGGCATC TCTGCCTCCT TTCACACTGC 1020AGTGCTGAAT CTGCTCAAAG CCAACGCAGC TGCTGGCACC ACTGGCAGGG GCTCGTGCTG 1080CGTGCCTACA TCTCGGCGCC CTCTGTCTTT GCTCTACTAT GACAGGGACA GCAACATTGT 1140CAAGACGGAT ATACCTGACA TGGTGGTCGA GGCCTGCGGG TGTAGTTAGC TTATGGGTGA 1200TACAGGCTGC CTGAGGTAGA ATGGCCTTCC TCAGGAAGGG AAACTCTGTT CCCACTTCTG 1260TCCAGAATGG AAACACCTTT CTAAGCATGC AGACATCCCT CTGTGGACTT CAGGGGATCC 1320ACCTCTAAAG AGAGTCACTA GTGACCAACA GCCTTTCTCT CTCCTGGGAC ATGGTTGACC 1380CAGTACACCC ATCCTCAGCC TTAAGTTAGA GGCTAATCGA CTCCTACATA TATATGTCAT 1440TTTGTCCTAG CAAACACCCC TTAGCTCCCC TTAGTCAACT ATGTAATCTA CTCTGCCTCC 1500CTGACCCTGC CACCGGAAGG TTCCTATTCC ACGATGATAT GCCTTAGTGT CTCCCCTT 1558 ( 2 ) SEQ ID NO:4:
(i) sequence signature:
(A) length: 352 amino acid
(B) kind: amino acid
(C) chain:
(D) topology: wire
(ii) molecular species: peptide
(vi) primary source:
(A) organism: mouse
(xi) sequence explanation: SEQ ID NO:4:
Met?Ala?Ser?Ser?Leu?Leu?Leu?Ala?Leu?Leu?Phe?Leu?Thr?Pro?Thr?Thr
1 5 10 15
Val?Val?Asn?Pro?Lys?Thr?Glu?Gly?Pro?Cys?Pro?Ala?Cys?Trp?Gly?Ala
20 25 30
Ile?Phe?Asp?Leu?Glu?Ser?Gln?Arg?Glu?Leu?Leu?Leu?Asp?Leu?Ala?Lys
35 40 45
Lys?Ser?Ile?Leu?Asp?Lys?Leu?His?Leu?Ser?Gln?Arg?Pro?Ile?Leu?Ser
50 55 60
Arg?Pro?Val?Ser?Arg?Gly?Ala?Leu?Lys?Thr?Ala?Leu?Gln?Arg?Leu?Arg
65 70 75 80
Gly?Pro?Arg?Arg?Glu?Thr?Leu?Leu?Glu?His?Asp?Gln?Arg?Gln?Glu?Glu
85 90 95
Tyr?Glu?Ile?Ile?Ser?Phe?Ala?Asp?Thr?Asp?Leu?Ser?Ser?Ile?Asn?Gln
100 105 110
Thr?Arg?Leu?Glu?Phe?His?Phe?Ser?Gly?Arg?Met?Ala?Ser?Gly?Met?Glu
115 120 125
Val?Arg?Gln?Thr?Arg?Phe?Met?Phe?Phe?Val?Gln?Phe?Pro?His?Asn?Ala
130 135 140
Thr?Gln?Thr?Met?Asn?Ile?Arg?Val?Leu?Val?Leu?Arg?Pro?Tyr?Asp?Thr
145 150 155 160
Asn?Leu?Thr?Leu?Thr?Ser?Gln?Tyr?Val?Val?Gln?Val?Asn?Ala?Ser?Gly
165 170 175
Trp?Tyr?Gln?Leu?Leu?Leu?Gly?Pro?Glu?Ala?Gln?Ala?Ala?Cys?Ser?Gln
180 185 190
Gly?His?Leu?Thr?Leu?Glu?Leu?Val?Pro?Glu?Ser?Gln?Val?Ala?His?Ser
195 200 205
Ser?Leu?Ile?Leu?Gly?Trp?Phe?Ser?His?Arg?Pro?Phe?Val?Ala?Ala?Gln
210 215 220
Val?Arg?Val?Glu?Gly?Lys?His?Arg?Val?Arg?Arg?Arg?Gly?Ile?Asp?Cys
225 230 235 240
Gln?Gly?Gly?Ser?Arg?Met?Cys?Cys?Arg?Gln?Glu?Phe?Phe?Val?Asp?Phe
245 250 255
Arg?Glu?Ile?Gly?Trp?Asn?Asp?Trp?Ile?Ile?Gln?Pro?Glu?Gly?Tyr?Ala
260 265 270
Met?Asn?Phe?Cys?Thr?Gly?Gln?Cys?Pro?Leu?His?Val?Ala?Gly?Met?Pro
275 280 285
Gly?Ile?Ser?Ala?Ser?Phe?His?Thr?Ala?Val?Leu?Asn?Leu?Leu?Lys?Ala
290 295 300
Asn?Ala?Ala?Ala?Gly?Thr?Thr?Gly?Arg?Gly?Ser?Cys?Cys?Val?Pro?Thr
305 310 315 320
Ser?Arg?Arg?Pro?Leu?Ser?Leu?Leu?Tyr?Tyr?Asp?Arg?Asp?Ser?Asn?Ile
325 330 335
Val?Lys?Thr?Asp?Ile?Pro?Asp?Met?Val?Val?Glu?Ala?Cys?Gly?Cys?Ser
340 345 350 (2) explanations to SEQ IDNO:5
(i) sequence signature:
(A) length: 18bp
(B) kind: Nucleotide
(C) chain: two strands
(D) topology: wire
(vi) primary source:
(A) organism: Homo sapiens
(ix) feature:
(A) title/key: exon
(B) position: 1..3
(ix) feature:
(A) title/key: intron
(B) position: 4..18
(xi) sequence explanation: SEQ ID NO:5:CAGGTAGGTC CATGGTCG 18 (2) explanations to SEQ ID NO:6
(i) sequence signature:
(A) length: 18bp
(B) kind: Nucleotide
(C) chain: two strands
(D) topology: wire
(vi) primary source:
(A) organism: Homo sapiens
(ix) feature:
(A) title/key: intron
(B) position: 1..15
(ix) feature:
(A) title/key: exon
(B) position: 16..18
(xi) sequence explanation: SEQ ID NO:6:CTTGATTTTT AACAGACC 18

Claims (14)

1. the proteic dna molecular of TGF-'beta ' family of encoding, it comprises
(a) proteic part of the encoding mature of nucleotide sequence shown in the SEQID NO.1 or encoding mature albumen plus signal peptide be or/and the part of precursor peptide,
(b) one of nucleotide sequence that obtains according to the degeneracy of genetic code by sequence (a),
(c) with (a) or the nucleotide sequence of under following stringent condition, hybridizing of sequence (b):
In 62-66 ℃ of hybridization, use 0.6 * SSC, 0.1%SDS to wash film 1 hour in 62-66 ℃ with 6 * SSC again,
And prerequisite is the coding region with the bioactive maturation protein of MP121 that will contain complete TGF-'beta ' family according to the dna molecular of (c) at least.
2. dna molecular according to claim 1
It is characterized by: a kind of fusion rotein thereby its nucleotide sequence that also contains the coding proteic maturation protein of another TGF-'beta ' family or signal peptide and/or precursor peptide is encoded.
3. carrier
It is characterized by: it is to insert plasmid by the dna molecular with claim 1 or 2 to obtain.
4. the albumen of the coded TGF-'beta ' family of dna sequence dna in the claim 1 or 2.
5. the albumen in the claim 4
It is characterized by: it contains the maturation protein part of aminoacid sequence shown in SEQ ID NO.2 or the SEQ ID NO.4 at least.
6. chimeric protein
It is characterized by:
It contains albumen and a receptor binding domains in claim 4 or 5.
7. heterodimer albumen
It is characterized by: it is made of the protein monomer in the claim 4,5 or 6 and other TGF-'beta ' family protein monomer.
8. pharmaceutical composition
It is characterized by: it contains in the claim 4 to 7 a kind of albumen of one at least as effective constituent.
9. the pharmaceutical composition of claim 8, it also contains pharmacy common carrier, auxiliary agent, thinner or weighting agent.
10. be used to prepare the purposes of the medicine for the treatment of hepatic diseases according to each a kind of albumen among the claim 4-7.
11. be derived from the sense-rna of the dna molecular of claim 1.
12. dna sequence dna in claim 1 or 2 or the carrier in the claim 3 are in the application of the sick body cell being carried out in-vitro transfection.
13. antibody,
It is characterized by: its specificity is in conjunction with each a kind of albumen in the claim 4 to 7.
14. each albumen of claim 4-7 is used for promoting the purposes of the pharmaceutical composition of dopamine neuron survival in preparation.
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DEP4423190.3 1994-07-01
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