CN1373217A - Cell multiplication factor Fwap10576 - Google Patents
Cell multiplication factor Fwap10576 Download PDFInfo
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- CN1373217A CN1373217A CN01109261A CN01109261A CN1373217A CN 1373217 A CN1373217 A CN 1373217A CN 01109261 A CN01109261 A CN 01109261A CN 01109261 A CN01109261 A CN 01109261A CN 1373217 A CN1373217 A CN 1373217A
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- polypeptide
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Abstract
A novel polypeptide-cell multiplication factor Fwap10576, the polynucleotide for coding it, the process for preparing said polypeptide by recombination, the antibody and antagon of said polypeptide, the medicinal composition containing said polypeptide and its application in treating cardiovascular disease, and the diagnosis and detection method by detecting the mutation of its sequence and the variation in the content of said polypeptide and the antibody level and disclosed.
Description
The present invention relates to up-to-date identified polynucleotides and by its encoded polypeptides, the purposes of the production method of this polynucleotide and polypeptide and these polynucleotide and polypeptide.Polypeptide of the present invention has been accredited as growth factor, hereinafter is sometimes referred to as " Fwap10576 ".Polynucleotide of the present invention and polypeptide are human origins.
Cardiovascular and cerebrovascular disease are one of major diseases that threaten human health.According to statistics, there are every year 2600000 Chinese to die from this disease approximately, just had a compatriot to be devitalized in average per 12 seconds by this disease.In China, the ratio that the cardiovascular and cerebrovascular disease death toll accounts for total death toll rises to 35.8% of nineteen ninety from 12.1% of nineteen fifty-seven, has raise 2.9 times.Predict according to the World Bank: to the year two thousand twenty, the ratio that whole world cardiovascular and cerebrovascular disease death toll accounts for total death toll will rise to 36.3% from 28.9% of nineteen ninety, and wherein 70% cardiovascular and cerebrovascular disease occur in developing country.The existing hyperpietic people more than 100,000,000 of China, and with annual 3500000 people's speed increase; Cerebral apoplexy disabled person 6,000,000 people, and with annual 1500000 speed increase; Patients with coronary heart disease 1,000,000 people, and with annual 500000 speed increase; In addition, also have myocardosis patient 3,000,000 people.Therefore, the control of cardiovascular and cerebrovascular disease guarantees that to alleviating the human economy burden HUMAN HEALTH has crucial meaning.
Four big stages have been experienced in the treatment of cardiovascular and cerebrovascular disease: in generation nineteen twenty, it is one " pump " that the research of circulation dynamics has disclosed heart, has established treatment basis in heart failure thus; In the 60-70 age,, make the cardiopathic sickness rate of western countries descend nearly 40% to the understanding and the processing of cardiovascular risk factors; 1970,, provide the novel method of anti-arrhythmia, electrophysiologic study and treatment to the electrophysiological research of myocardial cell; To the biological understanding of blood vessel, produced the novel method of heart intervention treating to the beginning of the nineties end of the eighties.
Heart failure, anti-arrhythmia, and interventional therapy to rescuing cardiovascular and cerebrovascular patient's life, improve its quality of life and played active effect.Yet, because these methods are only treated at symptom, fundamentally do not touch the cause of disease, so the treatment specific aim is not strong.Though prolonged patient's life-span, the purpose that does not really reach prevention and cure.Therefore, study, find out the paathogenic factor and the pathogeny of cardiovascular and cerebrovascular disease, be expected to making a breakthrough aspect the treatment of this disease, reach the purpose of fundamentally containing cardiovascular and cerebrovascular disease in molecular biological level.
The cDNA library is one of important research instrument of bioengineering field.Usually from cell, extract mRNA, by the DNA copy (being cDNA, Complementary DNA) of the synthetic mRNA of ThermoScript II.Strand cDNA molecule becomes double chain DNA molecule at the role transformation of archaeal dna polymerase, and then inserts carrier, is transformed in the host bacterium to grow up to the clone.Each clone so only comprises specific mRNA information, and such is called the cDNA library cover clone.
Because cDNA does not contain intron, can directly screen corresponding expressing gene from the cDNA library, so relative gene pool, the cDNA library has advantage simple to operate, easy to use.The difference of the different cell expressing genes of human body has determined the difference of its tissue and organ phenotype, from human aorta cDNA library, separate and the evaluation specific expression gene, particularly separating and identify disease related gene, is one of effective ways of research heredity cardiovascular diseases.
According to one aspect of the present invention, a kind of new mature polypeptide Fwap10576 is provided and has biologic activity and fragment, analogue and the derivative of the Fwap10576 of purposes are arranged in diagnosis or treatment.Polypeptide of the present invention is a human origin.
According to another aspect of the present invention, the isolated nucleic acid molecule of code book invention polypeptide is provided, comprise mRNA, DNA, cDNA, genomic dna, and have biologic activity and fragment, analogue and the derivative of this nucleic acid molecule of purposes are arranged on diagnostics or therapeutics.
According to another aspect of the present invention, the method for producing the Fwap10576 polypeptide with recombinant technology is provided, this method comprises reorganization protokaryon and/or the eukaryotic host cell of cultivating the nucleotide sequence that contains code book invention polypeptide.
According to another aspect of the present invention, the method that provides the polynucleotide with Fwap10576 polypeptide or coding Fwap10576 polypeptide to be used for the treatment of.For example, treat cardiovascular proliferative disease, suppress tumour and form.
According to another aspect of the present invention, provide the antibody of these polypeptide.
According to another aspect of the present invention, the antagonist of described polypeptide is provided, it can be used to suppress the effect of these polypeptide.
According to another aspect of the present invention, provide with nucleotide sequence of the present invention in sudden change and with the diagnostic method of polypeptide unconventionality expression diseases associated of the present invention or disease susceptibility.
According to another aspect of the present invention, provide with the polynucleotide of polypeptide of the present invention or this peptide species of encoding, externally be used for scientific research, DNA is synthetic and the method for artificial constructed dna vector.
According to the instruction of this paper, above-mentioned aspect and related fields are conspicuous to those skilled in the art.
The present invention is achieved through the following technical solutions.Extract mRNA, reverse transcription becomes cDNA, is built into human aorta cDNA library.From the library, obtain the gene fragment of Fwap10576, obtain the full length cDNA sequence of Fwap10576 by the EST splicing.And then research Fwap10576 gene is studied the activity and the function of Fwap10576 polypeptide in the expression and the distribution of different tissues.
According to one aspect of the present invention, the invention provides a kind of isolating nucleic acid (polynucleotide) sequence, its coding have infer, shown in SEQ ID NO:2 the mature polypeptide of aminoacid sequence.Polynucleotide of the present invention are found from the aortal cDNA library of being grown up.It is positioned on No. 16 karyomit(e) of human body cell.It comprises an open reading frame, can encode to have the polypeptide of 156 amino-acid residues.Homology analysis shows that Fwap10576 polypeptide and present known protein do not have sequence homology.
The Fwap10576 polypeptide of inferring is made up of 156 amino acid.Its sequence signature is: (A) 1 to 34 Aa is a signal peptide; (B) 2 to 7 Aa (GSTWGS), 19 to 24 Aa (GLVLSL), 46 to 51 Aa (GTAISC), 96 to 101 Aa (GVSPCC), 118 to 123 Aa (GGLQAL) are N-myristoylation site; (C) 33 to 39 Aa (RARDRDY) are the Tyrosylprotein kinase phosphorylation site; (D) 56 to 58 Aa (SSR) are the protein kinase C phosphorylation site; (E) 77 to 80 Aa (NQSN) are the N-glycosylation site.The molecular weight of analysis revealed Fwap10576 polypeptide is: 16700.5 dalton, iso-electric point is 8.11 (http://www.expasy.ch/tools/protparam.html), and extracellular albumen accounts for 33.3% (http://psort.nibb.a c.jp/form2.html) of Tot Prot.
Polynucleotide of the present invention can be rna form or dna form, and wherein DNA comprises cDNA, genomic dna and synthetic DNA.DNA can be two strands or strand, if strand, then can be coding strand or non-coding (antisense) chain.The encoding sequence of encoding mature polypeptide can be identical with the encoding sequence (213 to 683 Nucleotide) shown in the SIQ ID NO:1 (1083 Nucleotide of total length); Perhaps, because the Feng Yu or the degeneracy of genetic code, encoding sequence also can difference and the encoding sequence shown in the SIQID NO:1.
The polynucleotide of mature polypeptide can comprise shown in the coding SIQ ID NO:2: the encoding sequence of mature polypeptide; The encoding sequence of mature polypeptide and additional encoding sequence are as the polynucleotide of coded polypeptide leader sequence or secretion sequence; Encoding sequence of mature polypeptide (and optional additional code sequence) and non-coding sequence are as the non-coding sequence of intron or mature polypeptide encoded sequence 5 ' and/or 3 ' end.
Therefore, term " polynucleotide of coded polypeptide " comprises polynucleotide that only contain polypeptid coding sequence and the polynucleotide that contain additional coding and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, that its coding is inferred, as to have aminoacid sequence shown in the SIQ ID NO:2 polypeptide fragment, analogue and derivative.Described varient can be the allelic variation body of these polynucleotide of natural generation, or the varient of non-natural generation.As known in the art, the allelic variation body is the another kind of form of one section polynucleotide, and it can have replacement, disappearance or the interpolation of one or more Nucleotide, and does not change the function of coded polypeptide in fact.
Therefore, the present invention includes to encode has the polynucleotide of same acid sequence with mature polypeptide shown in the SIQ ID NO:2, also comprise the polynucleotide varient of can encode fragment, derivative and the analogue of mature polypeptide shown in the SIQ ID NO:2.These varients comprise deletion mutation body, replacement varient, interpolation or insert varient.
The present invention also comprises such polynucleotide, and wherein the encoding sequence of mature polypeptide can be merged by host cell expression and excretory polynucleotide (as producing the polynucleotide of leader sequence) with helping polypeptide in identical reading frame mutually.Leader sequence is controlled polypeptide and is transported from transit cell as secretion sequence.Polypeptide with leader sequence is precursor protein (preprotein), can produce mature polypeptide after cutting its leader sequence by host cell.Polynucleotide of the present invention also can proteins encoded former (proprotein), and proteinogen is the maturation protein with former sequence (prosequence), is the maturation protein that 5 ' end has added amino-acid residue, is a kind of inactive form.After excising former sequence, can produce activated maturation protein.
Therefore, polynucleotide of the present invention a kind of maturation protein of can encoding, also can encode has the albumen of former sequence, and the existing former sequence (prosequence) of can also encoding has the protein of leader sequence (presequence) again.
Polynucleotide of the present invention also are included in the encoding sequence that merges with flag sequence in the same frame, and flag sequence can be used for purifying polypeptide of the present invention.For example, when host cell was bacterium, flag sequence can be provided, be used for six Histidines of purifying fusion product by the pQE-9 carrier.Perhaps, when the host was mammalian cell (as the COS-7 cell), flag sequence can be hemagglutinin (HA), the HA mark corresponding to from a protein derived epi-position of influenza haemagglutination (Wilson, I., etc., cell, 37:767 (1984)).
Term " gene " is meant the dna fragmentation relevant with producing polypeptide, and it comprises before the coding region and zone afterwards, and the intervening sequence (intron) between each coding section (exon).
The fragment of full-length gene of the present invention can be used for separating full-length gene and to this gene high homology or similar bioactive other gene be arranged as the hybridization probe in cDNA library.Said probe preferably has at least 30 bases, and can contain 50 or more a plurality of base.Described probe also can be used for differentiating one or more genomic clones of cloning and contain complete genome corresponding to the cDNA of total length transcript.Wherein, complete genome comprises adjusting sequence, promoter sequence, exon and intron.For example can be according to known dna sequence dna synthetic oligonucleotide probe, and then isolate the encoding part of gene.Can be used for the library member of screening and its hybridization from human cDNA, genomic dna or mRNA library with the oligonucleotide probe of gene order complementary of the present invention, mark.
The invention still further relates to nucleotide sequence with multi-nucleotide hybrid of the present invention.Condition is to have at least 85% between two sequences, preferably at least 90%, and more preferably at least 95% homology.The present invention be more particularly directed under stringent condition polynucleotide with multi-nucleotide hybrid of the present invention.Term used herein " stringent condition " only is meant to have at least 95% between sequence, and hybridization just can take place when preferably having at least 97% homology.Can encode with the polynucleotide of above-mentioned sequence hybridization and mature polypeptide of the present invention has identical biological function or active polypeptide.
In addition, have homology with polynucleotide of the present invention and the polynucleotide that can hybridize can have at least 20 bases, preferred at least 30 bases, more preferably at least 50 bases, it can keep or retentive activity not.Such polynucleotide can be used to reclaim polynucleotide as the probe of SEQ ID NO:1, as diagnostic probe or as the PCR primer.
Therefore, the present invention relates to and the polynucleotide of the polypeptide shown in the SEQ ID NO:2 of encoding have at least 85%, preferably at least 90%, more preferably (described fragment has at least 30 bases for the polynucleotide of at least 95% homology and fragment thereof, preferred at least 50 bases), and by the polypeptide of these polynucleotide encodings.
According to another invention of the present invention, the polypeptide and fragment, analogue and the derivative that the present invention relates to infer, have aminoacid sequence shown in the SIQ ID NO:2.
Term " fragment ", " derivative " and " analogue " when relating to by SIQ ID NO:1 encoded polypeptides or having the polypeptide of aminoacid sequence shown in SIQID NO:2, are meant to have kept described polypeptide biological function or active polypeptide basically.Said analogue can comprise proteinogen, and proteinogen can produce activated mature polypeptide after the part excision.
Polypeptide of the present invention can be a recombinant polypeptide, natural polypeptides or synthetic polypeptide, preferred recombinant polypeptide.
Said polypeptide (SIQ ID NO:2) and fragment thereof, derivative or analogue can be: (i) peptide species, wherein one or more amino-acid residues are replaced by conservative or nonconservative amino-acid residue (preferred conservative amino-acid residue), and substituted amino-acid residue can yes or no by the genetic codon coding, a perhaps (ii) peptide species, wherein one or more amino-acid residues comprise substituting group, a perhaps (iii) peptide species, wherein mature polypeptide and another kind of compound merge, as merging with the compound (for example polyethylene glycol) that increases the polypeptide transformation period, a perhaps (iv) peptide species, wherein mature polypeptide and additional amino-acid residue merge, for example merge with leading or secretion sequence, and for example be used for the sequence of purifying mature polypeptide and merge.By the instruction of this paper, such fragment, derivative and analogue can be considered in those skilled in the art's ken.
Polypeptide of the present invention and polynucleotide preferably provide with unpack format, and preferably are purified into homogeneous (homogeneity) material.
Term " isolating " is meant that described material has broken away from primal environment (for example, if this material is naturally occurring, then referring to natural surroundings).For example, a kind of in living animal naturally occurring polynucleotide or polypeptide be not isolating, but from natural system part or all isolate same polynucleotide in the coexisting substances or polypeptide then is isolating.Such polynucleotide can be the parts of carrier, and such polynucleotide or polypeptide can be the parts of composition, as long as this carrier or composition are not the parts of its natural surroundings.
Polypeptide of the present invention comprises the polypeptide shown in the SEQ ID NO:2 (particularly mature polypeptide) and has at least 85% homology with the polypeptide of SEQ ID NO:2, more preferably 90% homology, the most preferably polypeptide of 95% homology.The present invention also comprises the fragment of aforementioned polypeptides, and polypeptide fragment comprises at least 30 usually, preferably at least 50 amino acid.
As known in the art, " homology " between two polypeptide relatively determined by an amino acid sequence of polypeptide and conserved amino acid thereof are replaced with another polypeptide.
Synthetic by polypeptide, polypeptide fragment of the present invention (or part of polypeptide) can be used for producing full-length polypeptide.This fragment can be used as the intermediate that produces full-length polypeptide.Same, polynucleotide passage of the present invention can be used for synthetic total length polynucleotide of the present invention.
According to another aspect of the present invention, relate to the carrier that contains polynucleotide of the present invention, with the host cell of vector gene through engineering approaches of the present invention, and the method that produces polypeptide of the present invention by recombinant technology.
Host cell produces through genetically engineered (transduction, conversion or transfection) with carrier of the present invention.Said carrier can be clone or expression vector.Carrier can be forms such as plasmid, virion and phage.The engineering host cell can be cultivated in the conventional nutritional medium that is suitable for activating promotor, screening transformant or amplification Fwap10576 gene of the present invention through improvement.Culture condition (as temperature and pH value) is to determine that by different host cells these will be apparent to those skilled in the art.
By recombinant technology, polynucleotide of the present invention can be used to produce polypeptide.Polynucleotide can be included in any carrier that is suitable for express polypeptide.Such carrier comprise karyomit(e) source, the non-chromosome source with the synthetic dna sequence dna.SV40 derivative for example, bacterial plasmid, phage DNA, baculovirus, yeast plasmid, from plasmid and phage DNA in conjunction with the carrier that obtains, viral DNA (as cowpox, adenovirus, fowl avipoxvirus and pseudorabies virus).It in addition, can also use other carrier, as long as can duplicate and survive in the host.
Can suitable dna sequence dna be inserted in the carrier with several different methods.In general, be dna sequence dna to be inserted in the suitable restriction endonuclease site with methods known in the art.
Dna sequence dna in the expression vector can be connected with suitable expression control sequenc (promotor), and is synthetic to instruct mRNA's.The example of promotor has: LTR or SV 40 promotors, colibacillary lac or rtp, phage P
LPromotor, and known other, the promotor of genetic expression in control protokaryon or eukaryotic cell or its virus.Expression vector is also to comprise ribosome bind site and the transcription terminator that instructs translation initiation.Carrier can also comprise the proper sequence that is used to increase and expresses.
In addition, preferred expression vector comprises one or more selectable marker genes, so that the screening of host cell provides phenotypic characteristic in order to transform afterwards.The Tetrahydrofolate dehydrogenase or the neomycin resistance that for example are used for eukaryotic cell culture are perhaps as being used for colibacillary tsiklomitsin and amicillin resistance.
Contain above-mentioned suitable dna sequence dna and suitable promotor or the carrier of regulating and controlling sequence and can be used to transform suitable host, so that its marking protein.
Representative example as suitable host has: bacterial cell, as intestinal bacteria, streptomycete, Salmonella typhimurium; The fungal cell is as yeast; Insect cell such as DrosorpHila S2 and Spodoptera Sf9; Zooblast such as CHO, COS or Bowes melanoma; Adenovirus; Vegetable cell etc.By the instruction of this paper, select appropriate host can regard as in those skilled in the art's ken.
More particularly, the present invention also comprises the recombinant precursor that contains above broadly described one or more sequences.Construct comprises the carrier that has inserted nucleotide sequence of the present invention forward or backwards, as plasmid or virus vector.In even more ideal embodiment, construct has also comprised the adjusting sequence that operationally is connected with described sequence, as promotor.Many suitable carriers and promotor are well known to those skilled in the art, and can obtain by commercial sources.For example, bacteria carrier: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, pHagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS (pHarmaca); Eukaryotic vector: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Parmacia).In addition, can also use other plasmid or carrier, as long as they can duplicate and survive in the host.
Can from gene, select promoter region with the carrier that has CAT (CAT) or other selective marker.Two suitable carriers are PKK232-8 and PCM7.The bacterium promotor of mentioning especially comprises lacI, lacZ, T3, T7, gpt, λ P
R, P
L, and trp.Eukaryotic promoter comprises that CMV is early stage immediately, the SV thymidine kinase, early stage and late period SV40, from retroviral LTRs and mouse metallothionein(MT)-I.Select appropriate carriers and promotor can regard as in those skilled in the art's ken.
In another embodiment, the present invention relates to comprise the host cell of above-mentioned construct.Host cell can be higher eucaryotic cells (as a mammalian cell), or eukaryotic cell (as yeast cell) such as low, or prokaryotic cell prokaryocyte (as bacterial cell).Transfection or electroporation by calcium phosphate transfection, the mediation of DEAE-dextran can be realized the importing (Davis, L., Dibner, M., Battey, I., basic skills in molecular biology, (1986)) of construct to host cell.
Construct in the host cell can produce the product of being encoded by recombination sequence via usual manner.And polypeptide of the present invention can be synthetic with conventional Peptide synthesizer.
Under the control of suitable promotor, maturation protein can be expressed in mammalian cell, yeast cell, bacterial cell or other cell.With the RNA that derives from DNA construct of the present invention, also can produce target protein by cell free translation system.People such as Sambrook (1989, second edition, New York cold spring harbor laboratory) in " molecular cloning laboratory manual " have described and can be used for clone protokaryon and eucaryon host, suitable and expression vector.
By in carrier, inserting enhancer sequence, can improve higher eukaryotic cell transcribing to the DNA of code book invention polypeptide.Enhanser is the cis-acting elements of DNA, and general about 10 to 300bp, and it acts on promotor and transcribes to strengthen it.The example of enhanser has: the SV40 enhanser of replication orgin upstream 100 to 270bp, polyoma enhanser and adenovirus enhanser.
Usually, recombinant expression vector comprises replication orgin and selection markers gene (as the TRP1 gene of colibacillary ampicillin resistance gene and Saccharomyces cerevisiae), and the promotor that obtains, can instruct the downstream configurations genetic transcription from the gene of highly expressing.Such promotor can obtain from the operon of coding glycolytic ferment (for example glycerol 3-phosphate acid kinase (PGK)), α-factor, acid phosphatase or heat shock protein(HSP) etc.Heterologous sequence is with appropriate mode and translation initiation sequence and terminator sequence assembling.Preferably, assemble to cell pericentral siphon or extracellular substratum excretory leader sequence with instructing albumen.Heterologous sequence can be encoded and be contained the fusion rotein of the terminal identification polypeptide of N-, and this identification polypeptide has the ideal feature, as the recombinant products or the simplification purification step of stably express.
With the structure gene of coding target protein, suitable translation initiation and termination signal, and have the promotor of function to insert together, can make up the expression vector that is applicable to bacterium.Said carrier comprises one or more selective markers and a replication orgin, to keep carrier and amplification vector in the host where necessary.The prokaryotic hosts that is fit to transform comprises intestinal bacteria, subtilis, Salmonella typhimurium, Rhodopseudomonas, streptomyces and Staphylococcus a plurality of kinds.
As representative but nonrestrictive example, the expression vector that is used for bacterium can contain selective marker and the replication orgin that is derived from commercially available carrier, and these commercially available carriers comprise the genetic elements of known cloning vector pBR322 (ATCC37017).Commercially available carrier like this comprises, pKK223-3 (pHarmacia Fine chemical company, Uppsala, Sweden) and GEM1 (PromegaBiotec, Madison, WI, the U.S.).These pBR322 " skeleton " part combines with suitable promotor and structure sequence to be expressed.
Transform the appropriate host bacterial strain, treat that it grows to suitable cell density after, induce the promotor of selection and continuation culturing cell for some time with appropriate methods (for example temperature inversion or chemical induction).Centrifuging harvested cell commonly used, with physics or chemical process smudge cells, the crude product that reservation obtains is to be further purified.Can be with any conventional method disruption of microorganisms cell, described method comprises freeze-thaw method, supersound process, Mechanical Crushing or uses the lysis agent that these methods are well known to those skilled in the art.
Various mammalian cell culture systems also can be used for express recombinant protein matter.The example of mammalian expression system has the monkey kidney inoblast COS-7 clone of being described by Gluzman (cell, 23:175 (1981)) and can express other 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, and any essential ribosome bind site, polyadenylation site, donor splicing site and acceptor site, transcription termination sequence and 5 ' flank non-transcribed sequence.The dna sequence dna that obtains from the SV40 viral genome can be used to provide needed non-transcribed genetic elements as SV40 replication orgin, early promoter, enhanser, montage and polyadenylation site.
Can reclaim from the reconstitution cell culture and purifying polypeptide of the present invention with several different methods, described method comprises ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, phytohemagglutinin chromatography and high performance liquid chromatography chromatography (HPLC).
Polypeptide of the present invention can be natural purifying, or chemosynthesis, or with recombinant technology from protokaryon or eucaryon host (for example insect of bacterium, yeast, higher plant, cultivation and mammalian cell) preparation.According to the host who uses in the recombination method, polypeptide of the present invention can be glycosylated or nonglycosylated.Polypeptide of the present invention also can comprise an initial methionine residues.
Polynucleotide of the present invention and polypeptide can be as treatment and the Studies on Diagnosis reagent and the materials of human diseases.Fwap10576 has the effect that promotes cell proliferation, may be used to the treatment of cardiovascular disorder.
According to a further aspect in the invention, provide the method for identifying polypeptide activator of the present invention or antagonist.One of method is under the situation that certain compound exists, the mammalian cell or the film preparation of expressing the Fwap10576 acceptor are cultivated with the Fwap10576 polypeptide, produced second messenger's ability after detecting this compound enhancing or blocking Fwap10576 polypeptide and acceptor interaction.Second messenger system includes but not limited to: protein tyrosine kinase system (PTK), cAMP guanylate cyclase, ionic channel or phosphoinositide hydrolysis effect.The another kind of method of identifying polypeptide antagonist is a competition inhibition method.When there is not certain compound in this method, be made as contrast, when existing, determine the potential antagonist with the variation of the Fwap10576 peptide molecule number of receptors bind by detecting this compound with the Fwap10576 peptide molecule number of receptors bind.
The potential antagonist comprises antibody, perhaps comprises in some cases and polypeptide bonded oligopeptides of the present invention, and they combine and eliminate effectively its function with described polypeptide.
Another potential agonist compounds is the antisense constructs with the antisense technology preparation.Thereby form the expression of antisense DNA or RNA controlling gene by triple helix, aforesaid method is combining based on polynucleotide and DNA or RNA all.For example, can be according to the nucleotide sequence 5 ' end of code book invention mature polypeptide, design is about the sense-rna of 10 to 40 base pairs.And for example design a kind of with transcribe the gene regions complementary DNA that relates to (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)), transcribe and the generation of polypeptide of the present invention thereby stop.Sense-rna is hybridized with mRNA in vivo, and the translation becoming of blocking-up mRNA molecule polypeptide of the present invention (antisense-Okano, J. neurochemistry magazine, 56:560 (1991); Deoxy-oligonucleotide (CRC press, Boca Raton, FL (1988)) as the genetic expression antisense inhibitor.Above-mentioned oligonucleotide can be sent to cell, thereby antisence RNA and DNA are to suppress the generation of polypeptide of the present invention in vivo.
Antagonist also comprises some small molecules, and these small molecules are by combining the interaction that stops polypeptide and its acceptor with polypeptide of the present invention, thereby blocks its normal biological activity.Small molecules includes but not limited to little peptide or class peptide molecule.
Polypeptide of the present invention, its activator and antagonist can combine with suitable carriers and form pharmaceutical composition.Such composition comprises polypeptide and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.Carrier includes but not limited to the combination of salt solution, damping fluid, glucose, water, glycerine, ethanol and above-mentioned substance.Its prescription should be suitable with the mode of administration.
The present invention also provides a kind of drug packages or test kit, and one or more containers are housed in it, and one or more components of pharmaceutical composition of the present invention are housed in the container.What provide simultaneously with it can be through medication management mechanism of government audit, relevant medicine or biological products manufacturing, the information using and sell.Pharmaceutical composition of the present invention can also be used in combination with other treatment compound.
Pharmaceutical composition is administration in the usual way, as in oral, local, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the intradermal approach.To treat and/or prevent the effective dosage drug administration of specified disease composition.Common amount administration with about at least 10 micrograms/kg body weight.In most of the cases, to be no more than the amount administration of about 8 mg/kg body weight every day.Under most applications, consider factors such as route of administration and symptom, dosage from every day about 10 microgram/kilograms to 1 mg/kg body weight.
According to a further aspect in the invention, can use polypeptide of the present invention and activator and antagonist by the mode of expressing in vivo, this mode often is known as " gene therapy ".
Therefore, can carry out genetically engineered processing to patient's cell at the nucleic acid (DNA or RNA) of external use code book invention polypeptide, the cell with through engineering approaches offers the patient who needs treatment again.Aforesaid method is well known in the art.For example, can carry out genetically engineered processing with the retrovirus pair cell of the RNA that contains code book invention polypeptide.
Similarly, can be by methods known in the art genetically engineered cell in vivo, so that express polypeptide in vivo.For example, with the retrovirus transduction packing cell of the RNA that contains code book invention polypeptide, so that it can produce the infectious viral particle that contains goal gene.This production cell is used for the patient, thus through engineering approaches cell and express said polypeptide in vivo.According to instruction of the present invention, use polypeptide of the present invention by aforesaid method or alternate manner and will be apparent to those skilled in the art.
The retrovirus that can obtain retroviral plasmid vector includes but not limited to: Mo Luonishi (Moloney) murine leukemia virus, spleen necrosis virus, retrovirus such as Lloyd's (Rous) sarcoma virus, Harvey sarcoma virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus and mammary tumor virus.
Described carrier comprises one or more promotors.Spendable suitable promotor includes but not limited to: retrovirus LTR; SV 40 promotors; Human cytomegalic inclusion disease virus (CMV) promotor (Miller etc., biotechnology, Vol.7, No.9,980-990 (1989) describes); Or other promotor (for example the eukaryotic cell promotor includes but not limited to histone, pol III and beta-actin promotor).Other adoptable viral promotors includes but not limited to: adenovirus promoter, thymidine kinase (TK) promotor and B19 parvovirus promotor.By the instruction of this paper, suitable promotor will be apparent to those skilled in the art on the selection carrier.
The nucleotide sequence of code book invention polypeptide should have suitable promotor control.Operable suitable promotor includes but 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); Beta-actin promotor and human growth hormone's promotor.Promotor also can be the natural promoter of the gene of coding said polypeptide.
Can produce cell to produce with retrovirus plasmid vector transduction packing cell.Can include but not limited to by transfected packing cell: PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψ CRE, ψ CRIP, GP+E-86, GP+envAml2 and DNA clone (Miller, human gene therapy, Vol.1, pgs.5-14 (1990) describes, and its incorporated herein by is reference in the lump).Carrier can be with any methods known in the art transduction packing cell.These methods include but not limited to: electroporation, use liposome and CaPO
4Precipitation.In addition, retroviral plasmid vector can be embedded in the liposome, perhaps is coupled on the lipid, introduces among the host then.
Production clone produces infectious retroviral vector particle, this particle comprise can coding said polypeptide nucleotide sequence.Can be in vivo or external transduction eukaryotic cell with these retroviral vectors.The eukaryotic cell of being transduceed will be expressed the nucleotide sequence of coding said polypeptide.The eukaryotic cell that can be transduceed includes but not limited to: embryo's stem cell, embryo cells and hematopoiesis stem cell, liver cell, inoblast, sarcoplast, keratinocyte, endotheliocyte and bronchial epithelial cell.
According to a further aspect in the invention, the present invention relates to the Fwap10576 gene in diagnosis or the purposes in detecting, by detecting that sudden change in the Fwap10576 nucleotide sequence can be diagnosed out relative disease or to the susceptibility of disease.
Can on dna level, detect the individuality that carries the Fwap10576 transgenation with multiple technologies.Can be from patient's cell, Tathagata autoblood, urine, saliva, the cell of examination of living tissue and necrotomy material.Genomic dna can be directly used in detection, perhaps can use pcr amplification (Saiki etc., nature, 324:163-166 (1986)) before analysis.RNA or cDNA also can be used for identical purpose.For example, can be with polynucleotide complementary PCR primer of the present invention in differentiating and analyze sudden change.As by comparing, change according to the size of amplified production and to detect disappearance and insert with normal genotype.Can through with can suddenly change with differential point with the nucleic acid array hybridizing after radiolabeled RNA or antisense DNA and the amplification.With RNaseA digestion or the difference by melting temperature(Tm), can distinguish the two strands of perfectly matched sequence and mispairing.
Can directly disclose crt gene and carry sequence difference between the mutator gene by dna sequencing.In addition, clone's dna fragmentation can be used as the special DNA section of probe in detecting.When being used in combination with PCR, the susceptibility of this method improves greatly, for example, uses together with sequencing primer and double-stranded PCR product or by the single-stranded template molecule that the PCR method of improvement produces.Conventional automatic sequencing method is come the definite kernel acid sequence with radio-labeling or fluorescent mark.
Genetic test based on dna sequence dna difference can contain or not contain in the gel of denaturing agent by detecting, and the variation of dna fragmentation electrophoretic mobility realizes.Little sequence deletion and insertion can be shown by the high resolving power gel electrophoresis.Not homotactic dna fragmentation can be distinguished on sex change methane amide gradient gel, and according to its specific fusing point or part melting temperature(Tm), different dna fragmentations will be stuck in the different positions (referring to Myers etc., science, 230:1242 (1985)) of gel.
Also can detect sequence variation on the specific position with the RNase protection analysis method of RNase and S1 protection or chemical cracking method, (as Cotton etc., PNAS, the U.S., 85:4397-4401 (1985)).
Therefore, can detect the difference of dna sequence dna with the Southern blotting of hybridization, ribonuclease protecting, chemical cracking, direct dna sequencing or use restriction enzyme (as restriction fragment length polymorphism (RFLP)) and genomic dna.
Except more conventional gel electrophoresis and dna sequencing, sudden change also can detect with former method of bit analysis.
According to a further aspect in the invention, the present invention relates to a kind of diagnostic analysis method that is undertaken by the change that detects Fwap10576 content of peptides in the different tissues.This is based on normal control tissue and compares, and the overexpression of this polypeptide can detect the existence of disease or disease susceptibility in certain tissue.The analysis on Content method that polypeptide of the present invention in host's the sample is taken from detection is well known to those skilled in the art, method comprises radioimmunoassay, competition measures in conjunction with mensuration, Western engram analysis, enzyme linked immunological absorption (ELISA) and " sandwich " measured, and preferred ELISA detects.ELISA measures and comprises the specific antibody that at first prepares polypeptide of the present invention, preferred monoclonal antibody.The report antibody for preparing this monoclonal antibody then.But will report antibody and combine said reagent such as radioreagent, fluorescent reagent or horseradish peroxidase with a kind of detection reagent.From host sampling, 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, monoclonal antibody and be attached to any polypeptide of the present invention on the polystyrene ware in conjunction with during, with monoclonal antibody incubation in ware.With damping fluid all unconjugated monoclonal antibodies are washed off.At this moment, the receptor antibody that will be connected with horseradish peroxidase is put into ware, and the result causes receptor antibody and any monoclonal antibody combination that is attached on the polypeptide of the present invention.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 detect content of peptides with competition assay.Method comprises that the specific antibody with the Fwap10576 polypeptide is attached on the solid support, mark (as radio-labeling) polypeptide of the present invention then, the sample that to take from the host passes through solid support, then by the certification mark amount, determine the amount of the competitive binding antibody of sample, thereby determine the content of polypeptide of the present invention in the sample.
Polynucleotide sequence of the present invention is also extremely valuable to chromosomal discriminating.This sequence-specific ground target is in the specific position of human chromosome, and hybridization with it.At present, only there is a few karyomit(e) sign reagent to can be used for the painted body position of mark based on actual sequence data (repetition polymorphism).Chromosome mapping based on DNA of the present invention is the primary step that these sequences are related with disease related gene.
In brief, by prepare the chromosomal localization that PCR primer (preferred 15-25bp) can carry out sequence by cDNA.By 3 ' non-translational region of Computer Analysis gene, can select primer fast.Primer should not crossed over first exon of genomic dna, otherwise will make amplification complicated.Then, primer is used for PCR contains one human chromosome with screening the assorted and body of somatocyte.Only contain with the assorted of the corresponding gene of this primer and just can produce amplified fragments with body.
The PCR mapping of somatocyte heterozygote is that specific DNA is positioned specific chromosomal quick method.According to the present invention,, can finish inferior location according to similar approach with identical Oligonucleolide primers with from one group of fragment of specific karyomit(e) or big genomic clone.Other drawing method that can be used for chromosome mapping comprise in situ hybridization, with mark, carry out prescreen and carry out prescreen through the karyomit(e) of airflow classification with hybridization, thereby make up the cDNA library of chromosome specific.
The cDNA clone can realize more accurate chromosome position with the fluorescence in situ hybridization (FISH) of Metaphase Chromosome smear.This technology can adopt the cDNA of 50 or 60 base length.See people's such as Verma summary for details, human chromosomal: basic fundamental handbook, Pergamon press, New York (1988).
In case finished the accurate location of gene on karyomit(e), then physical location and the genetic map data of this gene on karyomit(e) can have been connected.These data can find (can obtain by the internet) at for example V.McKusick in the Welch medical science library of Johns Hopkins university in the human Mendelian inheritance.Then by linkage analysis (the common heredity of the adjacent gene of physics), determine gene and navigated to relation between the disease of karyomit(e) same area.
Need to determine the difference of cDNA between diseased individuals and the normal individual or genome sequence subsequently.If partly or entirely observing sudden change in the diseased individuals, but do not observing in normal individual, then described sudden change may be the cause of disease of disease.
Said polypeptide, its fragment, its derivative or analogue, or the cell of expression above-mentioned substance can be used as immunogen and produces antibody.Antibody can be polyclone or monoclonal antibody.That the present invention also comprises is chimeric, strand and humanized antibody, and the product of Fab fragment or Fab expression library.Several different methods known in the art all can be used for producing these antibody and fragment.
By to animal body (preferred non-human body) direct injection or use polypeptide of the present invention and can obtain corresponding antibody.The antibody of Huo Deing can combine with described polypeptide like this.Like this, even also can produce can be in conjunction with the antibody of whole natural polypeptides for the coded polypeptide fragments sequence.And then with this antibody isolated polypeptide 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.For example hybridoma technology (Kohler and Milstein, 1975, the nature, 256:495-497), trisome hybridoma technology, people B-quadroma technology (Kozbor etc., 1983, today immunology, 4:72) and EBV-hybridoma technology (Cole, Deng, 1985, monoclonal antibody and cancer cancer therapy, Alan R.Liss, Inc., pp.77-96).
The technology (United States Patent (USP) 4,946,778) of described manufacture order chain antibody can be improved, to produce the single-chain antibody of anti-polypeptide of the present invention (tool immunogenicity).Also can express the humanized antibody of anti-polypeptide of the present invention (tool immunogenicity) with transgenic mice.
In order more to be expressly understood essence of the present invention, it is made an explanation referring now to following drawings and Examples.Drawings and Examples are not limit the present invention in any way for explanation.
Under above-described instruction, many improvement of the present invention and variation are possible, therefore, in the scope of appended claims, can implement the present invention in the mode that is different from above specific description.
Fig. 1 has shown the distribution of Fwap10576 in healthy tissues.
Fig. 2 has shown the distribution of Fwap10576 in different tumor cell lines.
Fig. 3 has shown Fwap10576 in normal adult and fetal myocardial tissue, and in the situation of remote myocardial infarction patient ventricular aneurysm tissue expression.
Fig. 4 observes the expression of Fwap10576 at intact animal model cerebral tissue in situ hybridization.
Fig. 5 is the optimization of recombinant protein TRXSX576 expression condition.
Fig. 6 detects the situation that Fwap10576 expresses for the in situ hybridization method in the intact animal cerebral tissue.
Fig. 7 detects the situation that Fwap10576 expresses for the in situ hybridization method in cerebral apoplexy animal brain.
The extraction of structure 1.1 RNA in embodiment one, one-tenth human aorta cDNA library
RNA gents Total RNA Isolation System kit is available from U.S. Promega company (Cat No.Z5110).Operate as follows: take by weighing adult's aortic tissue of preserving in the 0.3g liquid nitrogen, add 10ml sex change liquid (4M guanidinium isothiocyanate, 25mM trisodium citrate) and 1ml 2M sodium acetate (pH4.0) homogenate.Add equal-volume water-saturated phenol and 0.2 times of volume chloroform, thermal agitation 15 seconds was placed 15 minutes on ice.10,000rpm, 4 ℃ are centrifugal 20 minutes.Get supernatant, add the equal-volume Virahol, placed 2 hours for-20 ℃, centrifugal.Suspend with sex change liquid 5ml again and precipitate, repeat above-mentioned steps.With the 75% washing with alcohol precipitation of 1ml ice precooling, evaporation trace ethanol is 5-10 minute under the room temperature, with the deionized water dissolving RNA of baycovin (diethyl pyrocarbonate is hereinafter to be referred as DEPC) processing.1.2mRNA separation
3 of sophisticated mRNA ' end is by a Poly who is made up of 20-250 adenylic acid (AMP) (A).According to this feature, available affinity chromatography separating mRNA and other RNA.Oligo (dT) Mierocrystalline cellulose that this experiment is used contains the poly chain of 12-18 Nucleotide (T).Under many salt condition, the mRNA that has oligo (A) tail combines and stays on the pillar with oligo (dT), and the rRNA and the tRNA that do not have oligo (A) tail are washed off, hangs over mRNA on the pillar with low saline solution wash-out then.
Quick prep micro mRNA purification kit is available from Sweden pHarmacia company.The centrifuge tube 1 that does not have the RNA enzyme at 1.5ml
#Interior 1ml oligo (dT) cellulosic suspensions that adds; Other gets 1.5ml centrifuge tube 2
#, add total RNA 1ml with the sample-loading buffer dilution; Pipe 2
#With pipe 2
#Centrifugal 1 minute of room temperature, 12000rpm; Suction goes to manage 1
#Supernatant will manage 2
#Supernatant add pipe 1
#, jog 5-10 minute; Centrifugal 10 seconds of room temperature, 12000rpm; With 1ml high-salt buffer (10mM Tris-HCl pH7.5,1mM EDTA, 0.5M NaCl) washing 5 times, centrifugal; With 1ml low salt buffer (10mM Tris-HCl pH7.5,1mMEDTA, 0.1M NaCl) washing 5 times, centrifugal; Precipitate and transfer to the microcentrifugation post with 0.3ml low salt buffer suspension oligo (dT), 12,000rpm, centrifugal 5 seconds; With 0.5ml low salt buffer washing 3 times, centrifugal; Collect mRNA with 2 * 0.2ml elutriant; Use the spectrophotometric determination optical density(OD), calculate the ratio of OD260/280; Obtain 300 μ l mRNA, add 7.5 μ l glycogens, 30 μ l 2.5M potassium acetates (pH5.0), 750 μ l dehydrated alcohols are standby-70 ℃ of preservations; Used 75% washing with alcohol, DEPC water dissolution mRNA preceding centrifugal 5 minutes.1.3 cDNA's is synthetic
Synthesis step is with reference to ZAP Express
TmCDNA Synthesis Kit
*And ZAP Express
TmCDNA Gigapack IIGold Cloning Kit
*The specification sheets of (U.S. Stregene company, Catalog No.200403 and 200404).
In the 0.5ml centrifuge tube, add 5 μ l, 10 * the first chain damping fluids successively, the methylated dNTP mixture of 3 μ l, 2 μ lXhoI connexon oligo (dT), 18 primers (1.4 μ g/ μ l), 32.5 the deionized water that μ l handles with DEPC, 1 μ l RNA enzyme inhibitors (40u/ μ l), 5 μ g mRNA put room temperature 10 minutes.Add 1.5 μ l moloneys mouse leukosis virus (MMLV) ThermoScript II (50u/ μ l) again, total reaction volume 50 μ l.Therefrom take out 5 μ l and change another centrifuge tube over to, add 0.5 μ l α-
32P Deoxy-ATP (dATP) is (800ci/mmol) to identify that first chain synthesizes quality.Above-mentioned reaction is all carried out at 37 ℃.
The DNA/RNA hybrid molecule that obtains is synthetic second chain of template with first chain under the effect of dna polymerase i.In ice bath, add 45 μ l, the first chain cDNA successively, 20 μ l, 10 * the second chain damping fluids, 6 μ l dNTP mixtures, 114 μ l deionized waters, 2 μ l[α-
32P] dATP (800ci/mmol), 2 μ l RNA enzyme H (1.5u/ μ l), 1 μ l dna polymerase i (9.0u/ μ l), cumulative volume 200 μ l.Mixing was hatched 2.5 hours for 16 ℃.Reaction is used phenol: chloroform (1: 1) extracting, ethanol sedimentation after finishing.1.4 cDNA is connected with carrier
From test kit, take out 9 μ l EcoRI connexon (sequence is respectively 5 '-AATTCGGCACGAG-3 ' and 3 '-GCCGTGCTC-5 ') dissolution precipitations.Get 1 μ l electrophoresis, identify that first and second chains synthesize quality.In remaining 8 μ l, the second chain cDNA, add 1 μ l, 10 * ligase enzyme damping fluid successively, and 1 μ l 10mmol/L γ-Triphosaden (γ-ATP), 1 μ lT4 dna ligase (4u/ μ l), 8 ℃ of water-baths were hatched 30 minutes for 70 ℃ after 16 hours.With restriction enzyme XhoI digestion 1.5 hours.Sepharose (SepHarose) Cl-2B crosses post and separates, and removes the Nucleotide less than 400 bases, to improve the ratio of full-length cDNA in the cDNA library.1.5 cDNA is cloned into the ZAP phage vector
Multiple clone site on the ZAP phage vector (U.S. Stratagene company) allows to insert the nucleic acid fragment that reaches 10Kb, enter the host after the plasmid part can cut from carrier, form plasmid vector Bluescript.There is T these carrier multiple clone site both sides
7And T
3The promotor of phage, it is synthetic to be used for sequential analysis and probe.Insert the cDNA fragment of carrier and can express tool antigenicity and bioactive fusion rotein.Details are as follows in experiment:
In centrifuge tube, add 100ng cDNA, 0.5 μ l, 10 * ligase enzyme damping fluid, 0.5 μ l 10mmol/L γ-ATP (pH7.5), 1ul ZAP phage vector (1ug/ul), 0.5ul T4 dna ligase (4u/ul) adds deionized water to cumulative volume 5 μ l.12 ℃ connect 16 hours.
Connecting product needs Package casing albumen that the recombinant phage of transfection activity is arranged with generation.From-70 ℃ of taking-up packaging proteins, after the dissolving, in the 25ul packaging protein, add 1ul ligation thing fast, mixing, 22 ℃ were reacted 2 hours, added 500ul SM damping fluid (0.1M NaCl, 0.08M MgSO
4.7H
2O, 0.05M Tris-HCl, 0.01% gelatin), the 20ul chloroform.This mixture is primary cDNA library.1.6 calculate tiring of positive colony
Get 5ul primary cDNA library, with the dilution of 45ul SM damping fluid.With the solution after the 10ul dilution, add in the 200ul competence XL1-Blue MRF ' host bacterium (OD600=0.5).37 ℃ water-bath 20-30 minute, add 3ml top-layer agar sugar, mixing is laid on NZY (0.09M NaCl, 0.08M MgSO
4.7H
2O, 0.5% yeast extract, 1%NZ amine A) on the flat board, to be inverted, 37 ℃ of overnight incubation are calculated the clone's number on the flat board.
Tiring of cDNA library becomes spot number (pfu/ml) expression with plaque.Pfu/ml=(plaque number * extension rate) * 1000/ diluent consumption (ul).Capacity is greater than 1 * 10
6Individual clone's cDNA library, it is 2.4 * 10 that the mRNA. the present invention that can guarantee wherein to contain each single copy becomes the storage capacity in human aorta cDNA library
6Individual independent recombinant clone.1.7 the amplification in cDNA library and preservation
The cDNA library that makes up can be directly used in screening, but unstable.Because the easy inactivation of the phage of non-wild-type, so need amplification with convenient repeatedly screening.Get 5 * 10
4Individual phage transfection XL1-Blue MRF ' host bacterium, bed board was hatched 8-10 hour for 37 ℃, added 8ml SM damping fluid, 4 ℃ of overnight incubation then in the 150mm plate.Centrifugal, collect supernatant, the cDNA library after promptly obtaining increasing.Add 0.3% chloroform, 4 ℃ of preservations.Prolonged preservation need add 7% dimethyl sulfoxide (DMSO) (DMSO), and-70 ℃ frozen.Polymerase chain reaction,PCR (PCR) amplification that embodiment two, new Full Length cDNA Cloning 2.1 are cloned at random
Shop, cDNA library dish, plaque density is 200-500 clone/culture dish (150mm), the limpid single plaque of picking changes in the aseptic centrifuge tube that contains 75ul SM damping fluid and 5ul chloroform, places 4 ℃.Centrifugal with preceding mixing, get supernatant liquor 5ul, carry out pcr amplification with ZAP phage vector 3 ' end primer (5 ' CCAAGCTCGAAATTAACCCTCAC 3 ') and 5 ' end primer (5 ' CAGTCAATTGTAATACGACTCACT 3 '), reaction cumulative volume 50ul.The amplification parameter is 94 ℃ of pre-sex change 3 minutes; Again 94 ℃ 45 seconds, 55 ℃ 30 seconds, 72 ℃ 3 minutes, totally 30 circulations; 72 ℃ were extended 5-10 minute.Estimate the output of PCR product according to the brightness of electrophoresis band in 1% sepharose.2.2 the sequencing of expressed sequence tag (EST)
ABI377 automatic sequencer (ABI PRISM
TM377DNA sequencer) and automatic sequencing test kit (BigDye
TMTerminator Ready Reaction Mix) available from U.S. PE company.Working conditions according to recommending checks order with dideoxy chain termination.
With on-radiation CY5 fluorescein (CY5-fluoroscein) thing of marking, universal sequencing primer thing T
3(5 ' ATTAACCCTCAC TAA AGG GA 3 ') and T
7(5 ' TAA TAC GAC TCA CTA TAG GG3 ') checks order, and the primer consumption is 5pmol/ul in every duplicate samples.Sequencing template is the PCR product, the about 30-100ng of consumption.The pcr amplification parameter be 94 ℃ 3-5 minute; Again 94 ℃ 30 seconds, 50 ℃ 15 seconds, 72 ℃ 1 minute, 20 circulations; 94 ℃ 30 seconds, 72 ℃ 1 minute, 15 circulations; 72 ℃ were extended 5 minutes again.The DNA product is electrophoresis on 8mol/L urea, 6% polyacrylamide gel plate.Voltage 1500V, power 34W, electrophoresis 250-300 minute (ALF Express
TMDNA sequence, Sweden pHarmacia company).2.3 the bioinformatic analysis of EST
BLAST (Basic Local Alignment Search Tool) software with U.S. bioinformation center, each cDNA clone's EST is carried out homology relatively (http://www.ncbi.nlm.nih.gov) with the GenBank/EMBL/DDBJ database. according to the judging criterion (domestic and international most of breadboard universal standards) of BLAST: homologous sequence is less than 100-200 base, and the Score value is new EST less than 100 sequence.The EST of Fwap10576 of the present invention is new EST.2.4 the body intramolecular cyclization of EST primer walking order-checking 2.4.1 ZAP phage
The ZAP phage vector can directly be transferred to plasmid from λShi Juntizaiti with the purpose fragment in the host, cyclisation is PBK-CMV (having a CMV virus early promoter) phagemid.Cyclisation step is with reference to specification sheets (ZAP Express cDNASynthesis kit and ZAP Express cDNA Gigapack
TmGold Doning kit).
Be kept at 4 ℃ of phage and 1ul helper phage (the extremely low bacteriosphage mutation body of a class self-replication ability can provide proteolytic enzyme and coat protein for duplicating with packing of plasmid DNA in the host cell) cotransfection 300ul intestinal bacteria XL1-BlueMRF ' strains (OD600=1.0) of carrying new EST with 3ul.After obtaining single stranded DNA, transfection Escherichia coli XLOLR strain (test kit provides) adds the 5mlLB nutrient solution in vitro again, and the 25ul kantlex was cultivated 12-16 hour for 37 ℃.2.4.2 the extraction of plasmid and evaluation
" QIAPrep Spin Miniprep kit " with German QIAGEN company extracts plasmid.2400rpm, 4 ℃ centrifugal 10 minutes, collect the bacterium of incubated overnight.Abandon supernatant, and adding 25ul damping fluid P1 (100ul/ml RNaseA, 50mM Tris/HCl, 10mM EDTA, pH8.0) suspension bacterium moves in the 1.5ml centrifuge tube of sterilization.(200mMNaOH 1%SDS), puts upside down supernatant several times, fully mixing to add 250ul damping fluid P2.(3.0M KAc pH5.5), shakes pipe 4-6 time at once to add 350ul damping fluid P3.12000rpm, centrifugal 10 minutes (SORVALL
The Mc12V desk centrifuge).Collect supernatant, it is moved to the bottom be with in the miniature purification column (QIA prep Spin Miniprep) of collection tube, 12000rpm, centrifugal 30-60 second.Add 0.75ul damping fluid TE (10mM Tris/HCL, 1mM EDTA, pH8.0), centrifugal 30-60 second.Remove collection tube, the centrifuge tube in a sterilization of the miniature purification column of QLAprep bottom cover adds 50ul damping fluid EB (10mM Tris-HCL pH8.5) or deionized water, keeps in the post bed after 1 minute centrifugal 1 minute, collects the DNA of purifying.
In centrifuge tube, add 3ul enzyme cutting buffering liquid (10 *), 1ul NotI restriction endonuclease (15u/ul), 1ul ECoRI restriction endonuclease (15u/ul), 1ulBSA, 2ulDNA, 22ul deionized water, cumulative volume 30ul.37 ℃ incubation 4-6 hour.Enzyme is cut the back and is identified the plasmid size.2.4.3 the mensuration of new full-length cDNA
ABI377 automatic sequencer and sequencing kit BigDye with U.S. PE company
TMTerminator Ready ReactionMix measures the sequence of PBK-CMV positive colony.
BD4ul is arranged, BOB 2ul (400mM Tris-HCl, pH9.0,10mM MgCl in the reaction solution
2), primer 2 ul (5pmol/ul), dna profiling 30-100ng uses H
2O complements to final volume 20ul.Measure the cDNA full length sequence with walking method (primer method step by step), promptly the sequencing primer of next round by on take turns the order-checking products therefrom terminal bases determine.With oligo 14.0 software design PCR primers.
The result:
Shown in SEQ ID NO:1,1083 base pairs of Fwap10576 cDNA total length.Infer that its initiator codon is 213 to 215 Nucleotide (ATG), terminator codon is 681 to 683 Nucleotide.Open reading frame is 213 to 683 Nucleotide, the polypeptide of being made up of 156 amino-acid residues of encoding.This assignment of genes gene mapping of BLAST analysis revealed and No. 16 karyomit(e)s.
The albumen homology analysis revealed: Fwap10576 albumen does not have the sequence homology with the albumen that suppresses at present.Its protein sequence is characterized as; (A) 1 to 34 Aa is a signal peptide.(B) 2 to 7 Aa (GSTWGS), 19 to 24 Aa (GLVLSL), 46 to 51 Aa (GTAISC), 96 to 101 Aa (GVSPCC), 118 to 123 Aa (GGLQAL) are N-myristoylation site.(C) 33 to 39 Aa (RARDRDY) are network propylhomoserin tyrosine phosphorylation site.(D) 56 to 58 Aa (SSR) are the protein kinase C phosphorylation site.(E) 77 to 80 Aa (NQSN) are the N-glycosylation site.Embodiment three, Fwap10576 are in distribution 3.1 experiment materials of healthy tissues
The many tissue films (MTN film) that contain the mRNA of 12 kinds of tissues detect the distribution of Fwap10576 gene in healthy tissues available from U.S. Clontech company with it.β-actin is a kind of house-keeping gene, expresses homogeneous in multiple tissue, in this experiment with comparing.3.2 Northern hybridization
Method is with reference to " modern molecular biology experimental technique " (Lu Shengdong chief editor, press of China Concord Medical Science University, second edition, 1999) 147-149,202-205,207-213 page or leaf.Details are as follows: the total RNA of 3.2.1 extracts
Take by weighing 0.5g (human adult heart, heart of fetus, ventricular aneurysm tissue) and be organized in the 50ml centrifuge tube, add 10ml GTC, homogenate.Add equal-volume water-saturated phenol and 0.2 volume chloroform, thermal agitation 15 seconds was placed 20 minutes on ice.4 ℃, 12, centrifugal 25 minutes of 000rpm.Get supernatant and place another centrifuge tube, add the equal-volume Virahol ,-20 ℃ precipitate 1 hour.4 ℃ 12, centrifugal 25 minutes of 000rpm abandons supernatant.Behind 5mlGTC dissolution precipitation again, repeat other operation.With 75% washing with alcohol of 1ml precooling, dry, with the dissolving of DEPC treated water.Survey OD (optical density(OD)) 260 and OD280.3.2.2 denaturing formaldehyde gel electrophoresis
Take by weighing the 0.6g sepharose and add in the 52.2ml DEPC treated water, heating for dissolving treats that glue is as cold as 65 ℃, adds 6.0ml 10 * MOPS, 1.8ml formaldehyde, encapsulating.Get the total RNA of 4.5ul (40-60ug), add 2.0ul 10 * MOPS, 3.5ul formaldehyde, 10ul methane amide, mixing, 65 ℃ of sex change 15 minutes, cooled on ice 2 minutes.Add the 2.0ul sample-loading buffer, mixing.50 volts of prerunnings 5 minutes, last sample.After treating that dyestuff all enters glue, voltage is reduced to 40 volts, mixes electrophoretic buffer 1 time in per 10 minutes.3.2.3 commentaries on classics film
Treat that the bromjophenol blue swimming to the gel bottom, stops electrophoresis, take a picture.Wash glue for several times with the DEPC treated water, handled 45 minutes, handled 45 minutes with 20 * SSC with 50mM NaOH.Nylon membrane soaks with deionized water earlier, uses 20 * SSC to handle again 45 minutes.At glue holder upper berth one deck filter paper, soak with 20 * SSC, remove bubble; Glue is inverted in Jiao Tuoshang, puts the plastic tab that hollows out in the middle of above it.Carefully film is placed on the glue, remove bubble,, remove bubble at film upper berth two 20 * SSC wetted filter paper.On filter paper, spread the high thieving paper of 10cm, press the weight of one 500 grams.Changeed film 16 hours, thieving paper is changed 2-3 time in the centre.Carefully take off film, take a picture, with 6 * SSC vacuolar membrane 5 minutes.UV-crosslinked, 80 ℃ of roasting films 1 hour, 4 ℃ of preservations are standby.3.2.4 preparation hybridization template
Upstream primer: 5 '-CC GAATTC
ATGCACCGGCTCATCTTTGTC-3 ', italic is depicted as the protection base, and black matrix is depicted as the EcoRI restriction enzyme site, line part and Fwap10576 nucleotide sequence 107-127 position complementation; Downstream primer: 5 '-GC CTCGAG
TCTTATCGAGGTGGTCTTGAGCTG-3 ', italic is depicted as the protection base, and black matrix is depicted as the XhoI restriction enzyme site, line part and Fwap10576 nucleotide sequence 1198-1221 position complementation.
PCR reaction system: 10 times of damping fluid 5.0ul, thymus nucleic acid 2.0ul, upstream, each 3.0ul of downstream primer, Taq enzyme 1.0ul, Fwap10576 order-checking plasmid (template) 2.0ul, sterilized water 34ul.PCR parameter: 94 ℃ of pre-sex change 3 minutes; 94 ℃ 3 minutes, 94 ℃ 20 seconds, 60 ℃ 30 seconds, 72 ℃ 80 seconds, 30 circulations.72 ℃ 7 minutes.With ammonium acetate/ethanol (1: 5) purified pcr product, be dissolved in 50ulTE, quantitative with agarose, it is diluted to 25ng/ μ l.3.2.5 hybridization
Label probe: in the 0.5ml centrifuge tube, add the PCR product (in 98 ℃ of heating sex change in 4 minutes, cooled on ice 2 minutes) 25ng, 5 * mark damping fluid 10ul, dNTP (no dCTP) 2.0ul, BSA 2.0ul, Klenow enzyme 1.0ul, [α-32P] dCTP 5.0ul mends to cumulative volume 50ul with the water of nuclease free.Room temperature reaction 1-3 hour.
Prehybridization: film was soaked 5 minutes with 6 * SSC, be affixed on the hybridization tube wall, remove bubble.Add 6ml and purchase hybridization solution in Clontech, 68 ℃ 1 hour.
Hybridization: outwell hybridization solution, add the hybridization solution 6ml of preheating, add probe (probe needs in 98 ℃ of heating sex change in 4 minutes, cooled on ice 2 minutes), 68 ℃ 3 hours.
Wash film: (2 * SSC 0.05%SDS) washes film four times in room temperature, each 10 minutes with 200ml washing lotion I earlier.(0.1 * SSC 0.1%SDS) washed 20 minutes at 50 ℃, washed 20 minutes for 56 ℃ to use 200ml washing lotion II again.
Compressing tablet: inhale with filter paper and to remove liquid, wrap, be affixed on the filter paper with the identical size of X-ray sheet compressing tablet with preservative film.-70 ℃ of exposure appropriate times.Develop a film.
The expression level homogeneous (Figure 1A) of result: as shown in Figure 1: β-actin in multiple tissue; Fwap10576 optionally expresses in liver, kidney and heart, and expression level reduces (Figure 1B) successively.Embodiment four, Fwap10576 gene expression 4.1 experiment materials in tumor cell line
The Hybond membrane that contains 8 kinds of tumor cell lines detects the distribution of Fwap10576 gene in different tumor cell lines available from U.S. Clontech company with it.β-actin is as the contrast of this experiment.4.2 Northern hybridization
Referring to embodiment 3.2.
Result: as shown in Figure 2, in 8 kinds of tumor cell lines being checked, following tissue expression Fwap10576 gene: lung cancer A549 cell strain, Burkitt ' s lymphoma, melanoma G-361 cell strain, Lymphocytic leukemia MDL-4 cell strain, chronic myelocytic leukemia K-562 cell strain, its expression level reduces successively.And structure rectal adenocarcinoma SW480 cell strain is not expressed the Fwap10576 gene.Example five, Fwap10576 gene be expression difference 5.1 experiment materials in the decentraction myocyte
Myocardial cell from adult, fetus and outmoded capable myocardial infarction ventricular aneurysm patient.5.2 Northern hybridization
Referring to embodiment 3.2.
The result: the normal adult heart is expressed Fwap10576 hardly, and heart of fetus is expressed Fwap10576, and human adult heart ventricular aneurysm tissue is than 20 times of high expression level Fwap10576 of heart of fetus.Results suggest, Fwap10576 is relevant with heart development.After birth, because the myocardial cell becomes G
0The phase resting cell, fetus period,, a lot of and heart related gene was not expressed, when heart because of ischemic cardiomyocyte apoptosis when downright bad, cardiac repair, the gene that express embryonic stage is expressed once again.The foundation of embodiment six, the Fwap10576 gene expression 6.1 chronic heart failure models in normal, chronic heart failure and subacute heart failure animal hearts
50 male Sprague-Dawley (SD) rat (the 250-300 gram/only) available from the court's Animal House.The calibrated bolck material is provided by Beijing animal center, drinks water to be tap water, and water and feed are arbitrarily absorbed by animal.
Weigh, according to body weight with ketamine and stable intraperitoneal injection general anesthesia rat.Promoting the circulation of qi pipe interpolation pipe and connection small animal respirator.Chest is opened in the left side under the monitoring of ECG monitor, with 6-10 sutures ligation ramus descendens anterior arteriae coronariae sinistrae, closes chest.There to be obvious ST section to raise on the ECG monitor is the sign of ligation success.Treat that its recession of reviving removes respirator.Postoperative raise condition with art before, model promptly builds up after 50 days.6.2 the foundation of subacute heart failure model
10 male Wistar rats (250 grams/only) available from 301 Hospital of PLA, the raising condition is with the chronic heart failure model.
Abdominal injection norepinephrine 30mg/ day/only, injected continuously 4 days, model promptly can use in the 5th day.6.3 RNA-RNA in situ hybridization
Hybridization in situ technique by mRNA specific in probe specificity ground and the cell or DNA hybridization, and shows intracellular gene and expression product based on the principle of base complementrity.This reaction sensitivity is high, and can detect in the tissue differentiation process gene that moment only expresses.Hybridization kit DIG RNA Labeling kit (Cat.No.1175025) is available from German Boehringer Mannheim company.6.3.1 probe preparation
Special primer PCR is cloned into T Vector with the nucleic acid fragment (the 248th to 508 Nucleotide) of 200-300 bp Fwap10576; The recombinant plasmid amplification, purifying; Carry out pcr amplification with T7 and SP6 primer; Special primer and carrier T7 and SP6 primer PCR are decided the gene direction of insertion, order-checking (mRNA and Anti-mRNA).Add the synthetic antisense probe of t7 rna polymerase, SP6 RNA polymerase justice probe (checking the reliability of crossing system).6.3.2 probe mark
The linear DNA that is transcribed with phenol/chloroform extracting uses alcohol precipitation more earlier.The centrifuge tube of no RNA enzyme is put on ice the linear DNA of adding 1 μ l purifying in pipe, 2 μ l NTP mark mixed solutions, 2 μ l 10 * transcribe damping fluid, 1 μ l RNA enzyme inhibitors, 2 μ l (40u) SP6 or T7 TNA polysaccharase, totally 20 μ l.Mixing, centrifugal a little, add the DNA enzyme I that 20u does not contain RNA, 37 ℃ 15 minutes.Add 2 μ l 0.2mol/L EDTA (pH8.0) to stop polyreaction.
In above-mentioned substance, add 2.The abundant mixing of 5 μ l 4mol/L Licl and 75 μ l alcohol (20 ℃) ,-70 ℃ of placements at least 30 minutes (or-20 ℃ placement at least 2 hours).12000rpm is centrifugal, and 70% cold washing with alcohol is after the drying.Add 100 μ l DEPC water and 20u RNase, 37 ℃ leave standstill packing after 30 minutes ,-20 ℃ of preservations.6.3.3 slide is handled and sample preparations
Thoroughly clean slide, autoclave sterilization 30 minutes is dipped slide several times in the solution that contains poly-lysine (0.01%), drying, and 4 ℃ are standby.
Fresh specimens (mouse frozen section) 0.4cm * 0.4cm with 1 * PBS flushing twice, places on the freezing microtome fixed head.8-10 μ m section is equipped with on the slide glass of 1mg/ml poly-lysine and dries 4% Paraformaldehyde 96 fixedly 15-20 minute.1 * PBS was towards Xian 2 times, each 5 minutes.6.3.4 hybridization pre-treatment
Soaked 25 minutes with 0.2N HCl.0.3% Triton X-100 soaked 5 minutes.Wash twice with 1 * PBS, each 5 minutes.With fixing 6 minutes behind 4% the Paraformaldehyde 96.Wash twice with 1 * PBS, each 5 minutes.In 1000ml 0.1mol/L trolamine (pH8.0) solution, add the 5ml diacetyl oxide, treat that it dissolves fully section was placed 10 minutes.More than reaction is all at room temperature carried out.6.3.5 hybridization
Hybridization solution contains the 5ml deionized formamide, 2.5ml 20 * SSC, 500 μ l 100 * Denhardt ' s liquid (10g ficolls, the 10g polyvinylpyrrolidone, 10g bovine serum albumin, the 500ml distilled water of sterilizing), 500 μ l 10%SDS, the salmon sperm dna of 100 μ l 10mg/ml sex change, 400 μ l DEPC water.
To cut into slices and from acetylize solution, pull out, blot sample liquid all around, and be used in sample and draw a ringlet on every side with filter paper.Add prehybridization solution 30ul in ringlet, the interior 42 ℃ of incubations of wet box 2 hours.Get rid of prehybridization solution, cover the Parafilm film, the interior 42 ℃ of incubations of wet box 16 hours, the wet box of sealing with the 25ul hybridization solution.6.3.6 hybridization aftertreatment
Slide is taken out from warm box, remove the Parafilm film.2 * SSC room temperature flushing three times, each 10 minutes.1 * SSC room temperature flushing three times, each 10 minutes.0.1 50 ℃ of flushings twice of * SSC, each 15 minutes.
If background is too high, with slide place the solution that contains 20 μ g/ml RNA enzymes (0.5mol/L Nacl, 10mmol/L Tris-Cl, pH8.0) in, 37 ℃ of digestion 30 minutes.Use the above-mentioned solution that does not contain the RNA enzyme in 37 ℃ of flushings 30 minutes again, wash film.6.3.7 color reaction
Slide is soaked in scavenging solution (1M maleic acid, 0.15M NaCl; 0.3% (V/V) Tween-20).Incubation is 30 minutes in 100ml confining liquid (blocker in the test kit adds maleic acid damping fluid (0.1mol/L maleic acid, 0.15mol/L NaCl) by 10% (W/V), dilution in 1: 10 during use).Add the 20ml antibody-solutions, incubation 30 minutes.Wash twice with the 100ml scavenging solution, each 15 minutes.Add 20ml and detect liquid (0.1M Tris-HCl, 0.1M NaCl, pH9.5) balance 2-5 minute.Incubation in the colour developing liquid (10ml detects in the liquid and adds 200ul NBT/BCIP) of the new preparation of 10ml, lucifuge is not shaken.With sterilization distilled water or TE flushing.
The result: expression is positive in the endochylema of the neurogliocyte of mouse brain, expression especially is positive in the cerebral apoplexy model, this illustrates that this gene not only has conservative property in evolution, and the important organ (liver, kidney and brain) of body is had the important function effect.Point out this gene may be relevant with differentiation, the propagation of the Inflammatory response of cardio-cerebrovascular cell and tumour.Example eight, immunohistochemical analysis 7.1 experiment materials
People's myocardial infarction complication ventricular aneurysm tissue.7.2 immunohistochemical analysis
With correct prokaryotic expression (ptrxfus) plasmid of reorganization, from supernatant liquor, extract expressed fusion protein, through 10% denaturing polyacrylamide electrophoresis, determine the goal gene band, after the Western trace is identified target protein, the serum that immune animal obtained.
5 microns of paraffin sections are attached on the APES slide that is coated with poly-lysine, 75 ℃ of roasting sheets 2 hours.The dimethylbenzene dewaxing is after the entry of cutting into slices behind the gradient alcohol.3%H
2O
2Interior 10 minutes.Distilled water is given a baby a bath on the third day after its birth time, puts into EDTA antigen retrieval liquid (pH8.0), puts in the microwave oven baking (96 ℃-98 ℃) 10 minutes.Room temperature cooling 20-30 minute, distilled water are given a baby a bath on the third day after its birth time.Went into 1 * PBS damping fluid 5 minutes.10% normal rabbit serum 20 minutes.Add the anti-of suitable dilution, overnight incubation in 4 ℃.After 1 * PBS gave a baby a bath on the third day after its birth time, it was anti-to add biotin labeled goat-anti rabbit two, room temperature 10 minutes.1 * PBS gives a baby a bath on the third day after its birth inferior, and enzyme-added Streptavidin three resists room temperature 10 minutes.1 * PBS gives a baby a bath on the third day after its birth inferior, the DAB colour developing.Distilled water three times, Hematorylin understain karyon, dehydration, mounting.
Result: ventricular aneurysm cardiac muscle high expression level Fwap10576 gene.Embodiment eight, the reorganization of Fwap10576 gene in vitro and protein expression 9.1 make up recombinant vectors
Carrier pTrxFus (available from Invitrogen company).Get 20ul pTrxFus carrier, 10 * damping fluid 4.0ul, 1.0ulXhoI, 1.0ul SmaI, the 14ul sterilized water, 37 ℃ of enzymes were cut 16 hours.Purifying enzyme is cut product.
The PCR primer is given birth to worker biotech firm by Shanghai and is synthesized polyacrylamide gel electrophoresis purifying, OD
260=5).Forward primer: 5 ' CGG GAC CGG GAT TAC C 3 ', reverse primer 5 ' ATG TCT AGA TCA TAG AGC ACG AA 3 '.
Get 10ul PCR purified product, 10 * damping fluid 2.0ul, 1.0ul XhoI, the 7ul sterilized water, 37 ℃ of enzymes were cut 16 hours.Purifying enzyme is cut product.
9.1.2 connect
1.0ul the pTrxFus enzyme is cut product, 3.0ul PCR enzyme is cut product, 1.0ul 10X damping fluid, and 1.0ul T4 ligase enzyme, the 4ul sterilized water, 16 ℃ connect 6 hours.
9.1.3 transform
Get above-mentioned connection product 5ul, add 100ul GI724 competent cell.Mixing placed 30 minutes on ice.42 ℃ 60 seconds, 2 minutes on ice.Add 800ul SOC substratum and shook bacterium (less than 225RPM) 45 minutes for 37 ℃.Getting 100ul is applied on the LB flat board that contains Amp (acillin) resistance.Cultivated 16 hours for 30 ℃.Choosing the LB substratum that mono-clonal places 5ml to contain 50ug Amp shook 16 hours for 37 ℃.
9.1.4 plasmid extracts
(rich medium that contains penbritin, component are 6gNa to picking mono-clonal colony inoculation 5ml RM-Amp substratum
2HPO
4(anhydrous), 3g KH
2PO
4, 0.5g NaCl, 1g NH
4Cl, 20g Casamino Acids (acid hydrolysis casein), 0.095gMgCl
2), 30 ℃ are shaken (220-250rpm) and spend the night.
Get bacterium liquid, 2, centrifugal 10 minutes of 000RPM abandons supernatant.Solution I (25mM Tris-HCl, pH8.0,2.0mM EDTA, the 50mM glucose) 140ul that adds precooling in the precipitation, the suspension bacterium.Solution II (0.2M NaOH, 1%SDS) cracking bacterium with the new preparation of 280ul.The solution III (4.0M Potassium acatate) that adds the 450ul precooling again, mixing.Centrifugal 4,000 RPM10 minutes, 12,000 RPM 10 minutes.Get supernatant, add the Virahol of 0.6 times of volume, the same centrifugal.With 100ul TE (10mM Tris-HCl, 0.1mM EDTA, pH8.0) and the RNA enzyme dissolution precipitation of 20ug/ml, 37 ℃ 30 minutes.Add 600ul ammonium acetate/ethanol (1: 5), placed 30 minutes for-70 ℃.Centrifugal back 75% washing with alcohol.With the plasmid that TE 100ul dissolving is extracted, it is quantitative to survey OD260/280.9.1.5 select positive colony
Method with PCR is identified positive colony.9.1.6 sequencing
Restriction enzyme cutting and PCR are accredited as the male clone to check order.Primer is given birth to worker biotech firm by Shanghai and is synthesized polyacrylamide gel electrophoresis purifying, OD
260=5.PTRXFUS forward sequencing primer: 5 '-TTC CTC GAC GCT AACCTG-3 ', pTRXFUS reverse sequencing primer: 5 '-TGT AAA ACG ACG GCC AGT GC-3 '.The final concentration of primer is 5uM.
Order-checking is accredited as male clone (pTrxSX576) is kept in 15% glycerine, deposit for-20 ℃.9.2 the abduction delivering of recombinant protein
Inoculation 50ul bacterium liquid (pTrxSX576) is in 5ml RM-Amp substratum, and 30 ℃ are shaken (220-250rpm) and spend the night.Get 250ul bacterium inoculation 5ml IM inducing culture (the 6g anhydrous Na of spending the night
2HPO
4, 3gKH
2PO
4, 0.5gNaCl, 1gNH
4Cl, 2g acid hydrolysis casein, 0.095gMgCl
2), 30 ℃ were shaken (220-250rpm) 3 hours, took out, and measured OD550 and were about 0.5.Add the 900ml deionized water dissolving, adding water, to make final volume be 1 liter, high pressure 20 minutes (15psi, 121 ℃), and cool to room temperature adds 1ml 100mg/ml ampicillin, and 25ml 20% aseptic glucose is regulated pH=7.0 ± 0.2 ,+4 ℃ of preservations for 20 ℃-25 ℃.Adding tryptophane (Trp) to final concentration is 100ug/ml, 37 ℃ were shaken (220-250rpm) 4 hours, add 10mg/ml tryptophane (Trp), heating 500ml glass distilled water to 80 ℃, add 5g L-tryptophane, be stirred to dissolving fully,, preserved 6 months at most in 4 ℃ of lucifuges with the degerming of 0.45um membrane filtration solution; Take out bacterium liquid, measure OD550; The centrifugal results thalline of 4300g, ultrasonic immediately release albumen or-20 ℃ of preservations.9.3 ultrasonic release fusion rotein
The abduction delivering fusion rotein ,+4 ℃, centrifugal 10 minutes of 4300g, supernatant discarded keeps precipitation.With the resuspended thalline of solution II (20mM Tris-HCl, pH8,2.5mM EDTA), making its OD550 is 100, is positioned on ice.Bacterium liquid is placed on ice, and 3 * 10 pulse per second (PPS)s are put into quick-frozen in the liquid nitrogen immediately, is put in 37 ℃ of water-baths instant then rapidly; Repeat twice; 4 ℃, centrifugal 10 minutes of 4300g, supernatant is transferred in the new pipe, preserves on ice.9.4 polyacrylamide gel electrophoresis and protein immunoblotting 9.4.1 polyacrylamide gel electrophoresis
The fusion rotein solution of getting the ultrasonic release of 20ul adds an amount of albumen load sample damping fluid, boils in the boiling water bath 5 minutes, and centrifugal 5 minutes of 5000rpm gets sample on the 20ul supernatant.The 60V constant voltage is advanced concentrated glue (5%), and the 120V constant voltage is advanced 15% separation gel; Unload glue, Coomassie brilliant blue dyeing 1-2 hour, decolouring analytical results.9.4.2 protein immunoblotting
Half-dried system (Bio-Rad product) changeed film 1 hour, transfering buffering liquid: (referring to " fine works molecular biology experiment guide ").Take off nitrocellulose filter, and the 10ml confining liquid (20mM Tris-Cl, 500mM NaCl, pH=7.5,3%BSA), room temperature was shaken 1 hour.20mlTBST (20mM Tris-Cl, 500mM NaCl, pH=7.5,0.05%Tween-20 (w/v)) washed film 5 minutes, and room temperature is shaken gently.Repeat once.Film is transferred to 1: 20, and in the anti-damping fluid of 000 dilution, room temperature was soaked into 1-2 hour.The 20mlTBST room temperature was washed film 5 minutes.Repeat 1 time; Room temperature was soaked into 1 hour in two anti-damping fluids of 10ml dilution in 1: 5000; 20ml TBST room temperature is washed film 2 times, each 5 minutes; 20ml TBS room temperature is washed film 2 times, each 5 minutes; Prepare fresh substrate solution: 66ul NBT (nitro-blue tetrazolium, nitroblue tetrazolium(NBT), Promega)+10ml alkaline phosphatase damping fluid (100mM Tris-Cl, 100mM NaCl, 5mM MgCl
2, pH=9.5) mixing, (Promega), mixing (using in 1 hour) is standby for 5-bromo-4-chloro-3-indolyl-pHospHate, 5-bromo-4-chloro-3-indolylphosphate to add 33ul BCIP again; 20ml alkaline phosphatase damping fluid room temperature is washed film 2 times, each 5 minutes; 10ml substrate solution, room temperature shake colour developing gently.10-30 minute; Color development stopping: 20ml high pressure water 5 minutes.9.5 the variation of the different soak time recombinant protein of the optimization 9.5.1 of recombinant protein TRXSX576 expression condition TRXSX576 expression amount
30 ℃ of 220-250rpm of TRXSX576 expression strain activate 1 hour respectively, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, adding tryptophane to final concentration then is 100ug/ml, 37 ℃ are shaken (220-250rpm) and induced 4 hours, and polyacrylamide gel electrophoresis detects the variation of expression of recombinant proteins amount, as a result shown in Fig. 5 A.9.5.2 the variation of different induction time recombinant protein TRXSX576 expression amounts
According on choose optimum activating time activation TRXSX576 expression strain, adding tryptophane to final concentration then is 100ug/ml, 37 ℃ of 220-250rpm induced 0 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, polyacrylamide gel electrophoresis detected the variation of recombinant protein TRXSX576 expression amount.The result is shown in Fig. 5 B.9.6 affinitive layer purification fusion rotein TRXSX576
Discharge fusion rotein: under optimum activating time and induction time, expressed fusion protein, ultrasonic release is surveyed OD280, the rough calculation protein concentration.
Balance resin: get 2ml Resin (adding ethanol 4ml altogether) natural sedimentation, discard ethanol; Resuspended with 4-8ml RB (20mM β ME, β mercaptoethanol), leave standstill it is deposited naturally, carefully discard damping fluid; It is resuspended to add 4-8ml RB (20mM β ME), and room temperature was shaken 30-60 minute gently, careful supernatant discarded; Resuspended with 4-8ml RB (not having β ME), leave standstill it is deposited naturally, carefully discard damping fluid; Repeat above-mentioned steps.
Fill out post: Xiang Zhuzhong adds 2ml RB; With the resuspended Resin of RB, carefully be added in the post; Treat that Resin deposits naturally, open control valve and make damping fluid with under about 1/7 seconds velocity flow.
Purifying: application of sample: about 2ml fusion rotein solution, collect with 2ml/12 * 75 test tubes, every pipe is got 5ul and is diluted to 500ul survey OD280, washes post: 8ml RB (1mM β ME), be called for short 1mM β ME, wash-out: respectively with 6ml 5mM β ME, 10mM β ME, 50mM β ME, 100mM β ME, 200mM β ME, 500mM β ME, 1000mM β ME wash-out.In most cases target protein elutes between 10-200mM β ME.
Polyacrylamide gel electrophoresis is identified: every pipe is got the 20ul polyacrylamide gel electrophoresis and is identified (method is the same).9.7 the dialysis of fusion rotein is with concentrated
1 * EK (enterokinase, enteropeptidase) damping fluid ,+4 ℃, the magnetic agitation dialysis was changed dialyzate one time in 4 hours, changed dialyzate at least 4 times; Be concentrated into 1ml with PEG, 4 ℃ of preservations.
The present invention is not limited to above-mentioned specific embodiment, and embodiment only is used to illustrate some aspect of the present invention.The method and the material that are equal on the function comprise within the scope of the invention.In fact, according to description and the accompanying drawing of this paper, be conspicuous to those skilled in the art based on various changes of the present invention.Such change has been included in the scope of claim of the present invention.
Sequence table general information (i) applicant: Cardiovascular Disease Inst. Chinese Academy of Medical Sciences (ii) denomination of invention: growth factor P10576 (iii) sequence number: 2 (iv) address: (A) contact person: Hui Rutai (B) street: No. 167 (C) cities, gift scholar road, north, Xicheng District: Beijing (D) country: China's (E) postcode: 100037 (v) computer-reader form: (A) amboceptor type: 3.5 inches floppy disks (B) computer: information (i) sequence signature of Pentium 166MMX (C) operating system: WINDOWS 95 (D) software: WORD 97 (1) SEQ ID NO:1:
(A) sequence type: Nucleotide
(B) length: 1083 base pairs
(C) chain: strand
(D) topology: linearity is molecule type (ii): cDNA (iii) originates: become the (iv) location in genome, human aorta cDNA library: No. 16 karyomit(e) (v) constitutional features: (A) from 210 to 216bp (ATAATGG) for Kozak sequence (B) from 213 to 683bp be open reading frame (C) from 1044 to 1049bp (AATAAA) for tailing signal (vi) sequence description:
(2) information of SEQ ID NO:2 (i) sequence signature
(A) sequence type: amino acid
(B) length: 156 amino acid
(C) chain: strand
(D) topology: linearity is molecule type (ii): protein is (iii) originated: become (iv) sequence signature of human aorta cDNA library: (A) 1 to 34 Aa is signal peptide (B) 2 to 7 Aa (GSTWGS), 19 to 24 Aa (GLVLSL), 46 to 51 Aa (GTAISC)
96 to 101 Aa (GVSPCC), 118 to 123 Aa (GGLQAL) be N-myristoylation site (C) 33 to 39 Aa (RARDRDY) for network propylhomoserin tyrosine phosphorylation site (D) 56 to 58 Aa (SSR) for protein kinase C phosphorylation site (E) 77 to 80 Aa (NQSN) for the N-glycosylation site (v) sequence description:
5 10 15
| | | 1?Met?Gly?Ser?Thr?Trp Gly?Ser?Pro?Gly?Trp Val?Arg?Leu?Ala?Leu?16?Cys?Leu?Thr?Gly?Leu Val?Leu?Ser?Leu?Tyr Ala?Leu?His?Val?Lys?31?Ala?Ala?Arg?Ala?Arg Asp?Arg?Asp?Tyr?Arg Ala?Leu?Cys?Asp?Val?46?Gly?Thr?Ala?Ile?Ser Cys?Ser?Arg?Val?Phe Ser?Ser?Arg?Trp?Gly?61?Arg?Gly?Phe?Gly?Leu Val?Glu?His?Val?Leu Gly?Gln?Asp?Ser?Ile?76?Leu?Asn?Gln?Ser?Asn Ser?Ile?Phe?Gly?Cys Ile?Phe?Tyr?Thr?Leu?91?Gln?Leu?Leu?Leu?Asp Gly?Val?Ser?Pro?Cys Cys?Pro?Gly?Trp?Ser106?Gln?Ala?Ile?Cys?Leu Pro?Gln?Pro?Pro?Lys Val?Leu?Gly?Gly?Leu121?Gln?Ala?Leu?Pro?Ala Asp?Thr?Leu?Gly?Leu Cys?Pro?Asp?Ala?Ala136?Glu?Leu?Pro?Gly?Val Ser?Arg?Trp?Phe?Cys Leu?Pro?Gly?Leu?Asp151?Pro?Val?Leu?Arg?Ala Leu
Claims (19)
1. isolating polynucleotide, it comprises a member who is selected from following group:
(a) a kind of polynucleotide, the polypeptide of its coding shown in SEQ ID NO:2;
(b) a kind of polynucleotide, it is (a) naturally occurring polynucleotide varient;
(c) a kind of polynucleotide, it can be hybridized with (a), and has at least 85% homology with (a).
2. the polynucleotide of claim 1, wherein said polynucleotide are DNA.
3. the polynucleotide of claim 1, wherein said polynucleotide are RNA.
4. the polynucleotide of claim 1, wherein said polynucleotide are genomic dnas.
5. the polynucleotide of claim 1, said polynucleotide have the sequence shown in SEQ ID NO:1.
6. the polynucleotide of claim 1, said polynucleotide comprise from 213 to 683 Nucleotide shown in SEQ ID NO:1.
7. the polynucleotide of claim 2, its coding polypeptide shown in SEQ ID NO:2.
8. carrier, said carrier comprises the DNA of claim 2.
9. host cell, said host cell is transformed or transfection by the carrier of claim 8.
10. method that produces polypeptide, said method are included in the polypeptide of expressing in the host cell of claim 9 by described dna encoding.
11. a peptide species, it comprises a member who is selected from following group:
(a) peptide species, it has the aminoacid sequence shown in SEQ ID NO:2 of inferring;
(b) peptide species, it is active fragments, analogue or the derivative of (a);
(c) aminoacid sequence shown in the peptide species, itself and SEQ ID NO:2 has at least 85% homology.
12. the antibody of peptide more than the claim 11.
13. the activation of peptide more than a compound, said compound inhibition claim 11.
14. a pharmaceutical composition, said pharmaceutical composition comprise polypeptide or its active fragments of the claim 11 of significant quantity, and one or more pharmaceutically acceptable carrier or vehicle.
15. the purposes of the polypeptide of claim 11 in the pharmaceutical composition of preparation treatment cardiovascular and cerebrovascular diseases.
16. a treatment needs the patient's of Fwap10576 method, said method comprises: to the polypeptide of the claim 11 of patient's administering therapeutic significant quantity.
17. the method for claim 16, said method comprises: provide the DNA of coding said polypeptide to the patient, and at the described polypeptide of patient's expression in vivo.
18. one kind diagnoses the illness or to the method for the susceptibility of disease, said method comprises: measure the sudden change in the polynucleotide of claim 1.
19. a diagnostic method, said method comprises: analyze the polypeptide that whether has claim 11 in the sample that derives from host cell.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN01109261A CN1373217A (en) | 2001-02-28 | 2001-02-28 | Cell multiplication factor Fwap10576 |
| PCT/CN2002/000124 WO2002068639A1 (en) | 2001-02-28 | 2002-02-28 | CELL PROLIFERATION FACTOR Fwap10576 |
| CNB02805508XA CN1231584C (en) | 2001-02-28 | 2002-02-28 | Cell proliferation factor Fwap 10576 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN01109261A CN1373217A (en) | 2001-02-28 | 2001-02-28 | Cell multiplication factor Fwap10576 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1373217A true CN1373217A (en) | 2002-10-09 |
Family
ID=4657825
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN01109261A Pending CN1373217A (en) | 2001-02-28 | 2001-02-28 | Cell multiplication factor Fwap10576 |
| CNB02805508XA Expired - Fee Related CN1231584C (en) | 2001-02-28 | 2002-02-28 | Cell proliferation factor Fwap 10576 |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB02805508XA Expired - Fee Related CN1231584C (en) | 2001-02-28 | 2002-02-28 | Cell proliferation factor Fwap 10576 |
Country Status (2)
| Country | Link |
|---|---|
| CN (2) | CN1373217A (en) |
| WO (1) | WO2002068639A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018119295A1 (en) * | 2016-12-22 | 2018-06-28 | Ventana Medical Systems, Inc. | Fully automated nucleic acid extraction method for tissue samples |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ZA947825B (en) * | 1993-10-06 | 1995-08-22 | Univ Florida | Stem cell proliferation factor. |
| CN1326973A (en) * | 2000-06-07 | 2001-12-19 | 上海博德基因开发有限公司 | New polypeptide-human layer adherent protein gamma 3 link (LAMC3) 69.85 and polynucleotide for encoding such polypeptide |
-
2001
- 2001-02-28 CN CN01109261A patent/CN1373217A/en active Pending
-
2002
- 2002-02-28 CN CNB02805508XA patent/CN1231584C/en not_active Expired - Fee Related
- 2002-02-28 WO PCT/CN2002/000124 patent/WO2002068639A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO2002068639A1 (en) | 2002-09-06 |
| CN1231584C (en) | 2005-12-14 |
| CN1492928A (en) | 2004-04-28 |
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