CN1181109A - Transforming growth factor 'alpha'-HII - Google Patents

Abstract

The present invention discloses transforming growth factor alpha HII polypeptides and polynucleotides encoding such polypeptides. Also provided is a procudure for producing such polypeptides by recombinant techniques and therapeutic uses of the polypeptides which include stimulating wound healing, treating neurological disorders, treating ocular disorders, treating kidney and liver disorders and stimulating embryogenesis and angiogenesis. Also disclosed are antagonists against such polypeptide and their use as a therapeutic to treat neoplasia. and diagnostic assage for detecting altered levels of the polypeptide of the present invention and mutations in the nucleic acid sequences which encode the polypeptides of the present invention.

Description

Transforming growth factor alpha-HII

The present invention relates to the purposes of polynucleotide, polypeptide, these polynucleotide and polypeptide of up-to-date discriminating and the production method of this polynucleotide and polypeptide by these polynucleotide encodings.Polypeptide of the present invention has been pushed and has been accredited as the transforming growth factor-alpha homologue qualitatively.More particularly, polypeptide of the present invention has been pushed and has been accredited as transforming growth factor alpha-HII qualitatively, hereinafter is sometimes referred to as " TGF α-HII ".The present invention also relates to suppress the method for the effect of these polypeptide.

Cell growth and differentiation be seemingly initial by multiple stimulation, inhibition and cofactor and hormone, promote, keep and regulate.The variation of cell automatic adjustment mechanism and/or the obstacle relevant disease basic reason of (comprising tumorigenesis) of seemingly growing.Growth pattern (modular) factor is relevant with physiological process with various pathology, and these processes comprise: the survival of signal transduction, cell communication, g and D, embryo's generation, immunne response, hematopoietic cell and differentiation, inflammation, tissue repair and transformation, atherosclerosis and canceration.Albumen is regulated in growth and differentiation that all to be various cells produce under normal physiological conditions or situation that exogenous stimulation is replied such as Urogastron (EGF), transforming growth factor-alpha (TGF α), β-tunicin, amphiregulin and cowpox somatomedin, and these factors also are the members of EGF family.

These peptide growth factors influence the wound cell by autocrine and paracrine mechanism.They also play an important role to the normal wound healing of a lot of tissues (as skin, cornea and gi tract), and all these factors homology of consensus amino acid sequences in fact, and this homology comprises the conservative replacement of three intrachain disulfide bonds.In addition, all factors of this family all are 170,000 transmembrane glycoprotein receptors bind with molecular weight, and the tyrosine kinase activity in activated receptor tenuigenin district (Buhrow, S.A. etc., journal of biological chemistry 258:7824-7826 (1983)).

This acceptor is by many typical cell expressings, and these cells comprise the epithelial cell in skin keratin cell, vascular endothelial cell and GI road.These peptide growth factors are synthetic by several cells relevant with wound healing, and these cells comprise thrombocyte, keratinocyte and activated scavenger cell.These somatomedins were both relevant, also relevant with the restraining effect of other cell type with the hormesis (for example, tumorigenesis) of the growth and the differentiation of some cell.

β-tunicin is a 32-kDa glycoprotein, and this glycoprotein may be to be processed through the proteolysis fracture by a bigger film precursor of striding.The C-terminal district of β-tunicin and the C-terminal district of rat growth factor ' alpha ' have 50% sequence similarity.β-tunicin is a mitogen strong in retinal pigment epithelium and the vascular smooth muscle cell.

Amphiregulin is a kind of cell growth regulator of two-way function, and it demonstrates strong inhibition activity to DNA of tumorigenic cell is synthetic, but promotes some Normocellular growth.Pointed out that amphiregulin has the various extensive uses that comprise treatment wound and cancer.For example, amphiregulin has strong antiproliferative effect external human cancer cell line to several epithelial origins.As U.S. Patent application 5,115, shown in 096, amphiregulin is also induced human foreskin fibroblasts proliferation.

TGF α has the polytropism biological action.Some member's of TGF α generation is to change into fibrocyte synthetic (Ciardiallo etc. by many generation knurls, cellular biochemistry magazine 42:45-57 (1990)), and by the synthetic (Derynck of kinds of tumors (comprising kidney, mammary cancer and squamous cell carcinoma, melanoma and glioblastoma), R. etc., cancer research 47:707-712 (1987)).By analyzing transgenic mice (wherein, the high-caliber TGF α of tumor cells expression), there is direct evidence to show that the expression of TGF α may be the promotion factor that normal cell changes into its tumorigenesis homologue.The transgenic animal of TGF α show the damage of various tumorigenicity, and the tumorigenicity damage is depended on mouse strain system and regulated the selection of the promotor of TGF alpha expression (Sandgren, etc., cell, 61:1121-1135 (1990)).

TGF α also has effect (Derynck, R. cancer progress, 58:27-5 (1992)) in normal fetal development and adult physiological process.TGF α expresses in many tissues (comprising skin, brain, gastrointestinal mucosa and activated scavenger cell).Therefore, TGF α controls the important factor of epithelial cell growth and wound healing is had effect (Schreiber etc., science, 232:1250-1253 (1986)).

Polypeptide of the present invention has been pushed and has been accredited as transforming growth factor TGF α-HII qualitatively.Because aminoacid sequence and human TGF α homology are so made this evaluation.

According to one aspect of the present invention, the invention provides new mature polypeptide with and biologic activity and the diagnosis on or the treatment on useful fragment, analogue and derivative.Polypeptide of the present invention is the people source.

According to another aspect of the present invention, the invention provides the isolated nucleic acid molecule of coding polypeptide of the present invention, comprise mRNA, DNA, cDNA, genomic dna with and analogue and its biologic activity and at useful fragment and derivative in the diagnosis or in the treatment.

According to another aspect of the present invention, the invention provides the method for producing this peptide species by recombinant technology, 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 invention provides the method that the polynucleotide with these polypeptide or these polypeptide of encoding are used for the treatment of, described methods of treatment for example, it is disorderly and promote the hair hair follicle to grow, stimulate vascularization so that treatment burn, ulcer and corneal incision and stimulate the embryo to take place so that recover normal neural function, treatment visual disorder, some cell of target, treatment kidney and liver after wound and AIDS dementia to stimulate wound healing.

According to another aspect of the present invention, the invention provides nucleic acid probe, this nucleic acid probe comprises length and is enough to specifically nucleic acid molecule with nucleic acid array hybridizing of the present invention.

According to another aspect of the present invention, the invention provides the antibody of these polypeptide.

According to another aspect of the present invention, provide the stimulant of polypeptide of the present invention.

According to another aspect of the present invention, the invention provides the antagonist of said polypeptide, it can be used for suppressing the effect of these polypeptide, for example, treatment cornea inflammation, tumorigenesis (for example, tumour, cancer and psoriasis).

According to another aspect of the present invention, the invention provides the diagnostic measurement method of the sudden change diseases associated in the nucleotide sequence that detects with polypeptide overexpression of the present invention and this peptide species of encoding.

According to another aspect of the present invention, the invention provides these polypeptide, or the method for the relevant purpose of the external synthetic that is used for and dna vector synthetic with scientific research, DNA of the polynucleotide of these polypeptide of encoding.

From the instruction of this paper, those skilled in the art can know these and other aspect of the present invention.

Following accompanying drawing is used for illustrating embodiment of the present invention, and has no intention to be used for limiting the included scope of claim of the present invention.

Fig. 1 has described the cDNA sequence corresponding to TGF α-HII deduced amino acid.Used the abbreviation of amino acid standard single-letter.The signal sequence of inferring has underscore.

Fig. 2 is the diagram of the comparing amino acid sequence homology between people's β-tunicin, people TGF α and the people TGF α-HII (the 3rd row). ^*Be illustrated in the EGF motif that demonstrates conservative property in the polypeptide of the present invention.Underscore is represented the mature sequence of people TGF α.

According to one aspect of the present invention, the invention provides a kind of nucleic acid (multinuclear glycosides of separation Acid), this kind nucleic acid coding has the one-tenth of the amino acid sequence (SEQ ID NO:2) of the derivation of Fig. 1 Ripe polypeptide or 15 days Mays nineteen ninety-five of coding are with preserving number ATCC No.__ preservation The mature polypeptide of cDNA clones coding.

Can from human brain or early stage brain tissue, obtain the polynucleotides of code book invention polypeptide. This The polynucleotides of invention are found in the cDNA library of two all instar embryos. Its structurally and EGF family is relevant. It comprises the opening of the protein of 374 amino acid residues of a coding Frame, wherein about 45 amino acid residues are the leading sequences of inferring. Described protein and People TGF α demonstrates the homology of high level, has at one section 263 amino acid sequence 26% the phase same sex and 46% similitude. TGF α-HII contains in all EGF families Whole 6 conservative cysteine residues of finding among the member.

Full-length polypeptide of the present invention shown in SEQ ID NO:2 has the signal order of inferring Row, the sort signal sequence comprises 1 amino acids to 45 amino acids of SEQ ID NO:2, Help this peptide to secrete from cell. This peptide is further processed, SEQ ID NO:2 in the processing The cracking from the described peptide of 46 amino acids to 214 amino acids is got off, because this section amino Acid sequence is the precursor sequence of inferring. And, the representative of 264 amino acids to 344 amino acids The transferring film part of inferring, it is considered to instructing described peptide to specific target site with demonstration hereinafter The biology function of describing is essential. The transferring film part also can be excised from peptide, like this, and this The soluble portions branch that the invention polypeptide is inferred comprises 214 amino acids to 264 of SEQ ID NO:2 Amino acids.

Polynucleotides of the present invention can be rna form or dna form, wherein DNA Comprise cDNA, genomic DNA and synthetic DNA. This kind DNA can be double-stranded or single Chain, if strand, it can be coding strand or non-coding (antisense) chain. This kind encoding mature The coded sequence of polypeptide can and Fig. 1 (SEQ ID NO:1) shown in coded sequence or preservation Clone's coded sequence is identical; Since Feng Yu or the degeneracy of genetic code, this kind code sequence Row also can be a kind of different coded sequences, the DNA (SEQ ID NO:1) of its energy and Fig. 1 Or the identical mature polypeptide of the cDNA of preservation coding.

The cDNA coding of the mature polypeptide of code pattern 1 (SEQ ID NO:2) or coding preservation The polynucleotides of mature polypeptide can comprise: the coded sequence that only is the encoding mature polypeptide; The coded sequence of encoding mature polypeptide and additional coded sequence are such as leading or secretion sequence or egg Former (proprotein) sequence in vain; The coded sequence of encoding mature polypeptide is (with optional additional volume The code sequence) and non-coding sequence, as include 5 of son or mature polypeptide encoded sequence ' and/or 3 ' non-Coded sequence.

Like this, " polynucleotides of coded polypeptide " this term comprises and only contains the peptide coding order Row polynucleotides and also contain additional coding and/or the polynucleotides of non-coding sequence.

The invention still further relates to above-described polynucleotides variant, this kind variant coding has Fig. 1 The amino acid sequence of inferring (SEQ ID NO:2) polypeptide or compiled by the cDNA of preservation clone Fragment, similar thing and the derivative of the polypeptide of code. This kind polynucleotides variant can be natural product The polynucleotides variant that the polynucleotides allelic variant of giving birth to or non-natural produce.

Like this, the present invention includes coding identical mature polypeptide (SEQ ID NO:2) as shown in Figure 1 Or by the polynucleotides of the identical mature polypeptide of the cDNA clones coding of preservation, and coding as Mature polypeptide shown in Fig. 1 (SEQ ID NO:2) or by the one-tenth of the cDNA clones coding of preservation The polynucleotides variant of the fragment of ripe polypeptide, derivative and similar thing. These nucleotide variants bags Draw together disappearance variant, replacement variant, interpolation or insert variant.

As above indicated, described polynucleotides can have a kind of coded sequence, this order Row are coded sequences of the clone of the coded sequence shown in Fig. 1 (SEQ ID NO:1) or preservation The allelic variant of natural generation. Allelic variant known in the art is another of polynucleotide sequence The form of kind, it can have replacement, disappearance or the interpolation of one or more nucleotides, and essence On do not change the function of coded polypeptide.

The present invention also comprises such polynucleotides, wherein the coded sequence of said mature polypeptide Can be in identical reading framework and the multinuclear glycosides that helps host cell expression and secrete polypeptide Acid sequence merges, and described polynucleotide sequence is as controlling polypeptide from cell as secretion sequence The leading sequence that works in the transhipment. Polypeptide with leading sequence is front albumen (preprotein), and can have by host's cell cutting form mature form polypeptide before Lead sequence. This kind polynucleotides are encoding proteins former (proprotein) also, and this kind proteinogen is to add The maturation protein that 5 ' additional amino acid residue is arranged. Maturation with former sequence (prosequence) Albumen is proteinogen, is a kind of albumen form of non-activity. In case excise former sequence, stay That active maturation protein is arranged.

Like this, for example can encode a kind of maturation protein or coding of polynucleotides of the present invention has The protein of former sequence or the existing former sequence of encoding have again front sequence (presequence) (leading order Row) protein.

Polynucleotides of the present invention also can have the coding that merges with flag sequence in frame Sequence, described flag sequence is so that can purifying polynucleotides of the present invention. The bacterium host Situation under, described flag sequence can be the six histidine marks that provided by the pQE-9 carrier Note, it is used for purifying and merges underlined mature polypeptide, perhaps for example when using mammal thin During born of the same parents' (such as COS-7 cell), flag sequence can be haemocyte agglutinin (HA) mark. Said The HA mark corresponding to a kind of epi-position that derives from influenza haemocyte agglutinant protein matter (Wilson, I., etc., cell, 37:767 (1984)).

Term " gene " refers to the DNA section relevant with producing polypeptide chain; It comprises coding The zone of front, district and zone subsequently (leading district and trail sequence) and in each code area Intervening sequence (including son) between the section (extron).

The fragment of total length TGF α-HII gene can be as the hybridization probe in cDNA library, be used for separating full-length gene and therewith gene sequence similarity or similar bioactive other gene highly arranged.Such probe preferably has at least 30 bases, and can contain, for example 50 or more base.Said probe also can be used to differentiate corresponding to the cDNA clone and the genomic clone of total length transcript or contain the clone of the complete TGF α-HII gene that comprises adjusting and promoter region, exon and intron.The example of screening comprises by utilizing the known dna sequence synthetic oligonucleotide probe to come the coding region of isolated genes.Mark with the sequence that is complementary to gene order of the present invention can be used for screening human cDNA, genomic dna or mRNA library, to determine probe and which library member hybridization.

The invention still further relates to and the polynucleotide (condition is to have at least 70% between two sequences, preferably has at least 90%, more preferably has at least 95% homogeny) of sequence hybridization described above.The present invention be more particularly directed under stringent condition polynucleotide with above-described multi-nucleotide hybrid.As used herein, term " stringent condition " refers to only have at least 95% between sequence, and hybridization just can take place when preferably having at least 97% homogeny.In a preferred embodiment, with the such peptide species of the polynucleotide encoding of above-described multi-nucleotide hybrid, it keeps in fact and identical biological function or the activity of mature polypeptide by cDNA (s) coding of the cDNA (SEQ ID NO:1) of Fig. 1 or preservation.

In addition, described polynucleotide can have at least 20 bases, and preferably at least 30 bases more preferably are at least 50 bases, and itself and multi-nucleotide hybrid of the present invention, and have homogeny as indicated above can keep or retentive activity not.For example, this polynucleotide can be as the probe of SEQ ID NO:1 polynucleotide, for example is used to reclaim polynucleotide or as diagnostic probe or as the PCR primer.

Like this, the present invention relates to and the SEQ ID NO:2 polypeptide of encoding more than Nucleotide have at least 70% homogeny, preferably at least 90% homogeny and the more preferably polynucleotide of at least 95% homogeny and the polypeptide of fragment (this fragment has at least 30 bases, preferably at least 50 bases) and these polynucleotide encodings thereof.

The mentioned preservation thing of this paper will keep according to the regulation of the microbial preservation budapest treaty of the international recognition that is used for patented procedure.These keep thing only is in order to provide convenience to those skilled in the art, is not 112 required preservations of 35 U.S.C.Be included in the described preserved material polynucleotide sequence and by its amino acid sequence coded this paper reference in the lump, and be used to solve any contradiction on this paper sequence description.Any manufacturing, use or sale to preserved material need not give any such permission through permission at this.

The invention still further relates to the polypeptide of deduced amino acid (SEQ ID NO:2) or have polypeptide by the cDNA amino acid sequence coded of preservation with Fig. 1, and the fragment of this peptide species, analogue and derivative.

Term " fragment ", " derivative " and " analogue " during when the polypeptide (SEQ ID NO:2) of relevant Fig. 1 or by the cDNA encoded polypeptides of preservation, refer to the biological function or the active polypeptide that keep identical with such polypeptide basically.Like this, analogue comprises proteinogen, and this analogue can partly excise and then produce active mature polypeptide by proteinogen.

Polypeptide of the present invention can be a recombinant polypeptide, natural polypeptides or synthetic polypeptide, preferably recombinant polypeptide.

The polypeptide of said Fig. 1 (SEQ ID NO:2) or by the fragment of the cDNA encoded polypeptides of preservation, derivative or analogue can be: (i) a kind of like this, wherein one or more amino-acid residues can be also can not be by genetic codon amino acids coding residue by the amino-acid residue that conservative or non-conservative amino acid residues replaces (preferably conservative amino acid residues replacement) and replacement, perhaps (ii) a kind of like this, wherein one or more amino-acid residues comprise substituting group, perhaps (iii) a kind of like this, wherein mature polypeptide and another kind of compound merge, described compound is as increasing the polypeptide compound (for example polyoxyethylene glycol) of half life, perhaps (iv) a kind of like this, wherein additional amino acid and mature polypeptide fusion, for example leading or secretion sequence or be used for the sequence or the former sequence of purifying mature polypeptide.By the elaboration of this paper, can think that such fragment, derivative and analogue is within those skilled in the art's ken.

The present invention preferably provides the polypeptide and the polynucleotide of unpack format, and preferably described polypeptide is become homogeneous with the polynucleotide purifying.

Term " isolating " means described material and has broken away from its primal environment (for example, natural surroundings is if it is natural generation).For example, a kind of polynucleotide or polypeptide that is present in the natural generation in the animal alive is not isolating, but with natural system in the material of some or all coexistence identical polynucleotide or the polypeptide that separate be isolating.Such polynucleotide can be the parts of carrier, and/or such polynucleotide or polypeptide can be the part of composition, and it remains isolating, and this is because this carrier or composition are not the parts of its natural surroundings.

Polypeptide of the present invention comprise SEQ ID NO:2 polypeptide (particularly mature polypeptide) and and the polypeptide of SEQ ID NO:2 have at least 70% similarity (preferably 70% homogeny), it more preferably is 90% similarity (preferably 90% homogeny), it most preferably is the polypeptide of 95% similarity (preferably 90% homogeny), the part that also comprises these polypeptide, the part of this peptide species comprises at least 30 amino acid usually, and at least 50 amino acid preferably.

As known in the art, " similarity " between two polypeptide is by relatively a polypeptide and another amino acid sequence of polypeptide and its conserved amino acid replace to determine.

The fragment of polypeptide of the present invention or part are synthesized by peptide can be used to produce corresponding full-length polypeptide; Therefore, this fragment can be as the intermediate that produces full-length polypeptide.The fragment of polynucleotide of the present invention or part can be used for synthetic total length polynucleotide of the present invention.

The present invention also relates to comprise the carrier of polynucleotide of the present invention and the host cell that produces through genetically engineered with carrier of the present invention, and the method for producing polypeptide of the present invention through recombinant technology.

Host cell produces through genetically engineered operation (transduction, conversion or transfection) with carrier of the present invention, and said carrier can be cloning vector or expression vector.This carrier for example can be, forms such as plasmid, virion and phage.The through engineering approaches host cell can the improvement be suitable for activate promotor, select transformant or the conventional nutritional medium of the gene of the present invention that increases in cultivate.Culture condition, for example temperature and pH value etc. are those of host cell that were used to express selection in the past, are conspicuous to those of ordinary skill.

Polynucleotide of the present invention can be used for producing polypeptide through recombinant technology.Like this, for example, polynucleotide can be included in any of the various expression vectors that are used for express polypeptide.Such carrier comprises karyomit(e), non-chromosome and synthetic DNA sequence, for example SV40 derivative; Bacterial plasmid; Phage DNA; Baculovirus; Yeast plasmid; Make up the deutero-carrier from plasmid and phage DNA, viral DNA (as cowpox, adenovirus, fowl avipoxvirus and pseudorabies virus).Yet any other carrier also can use, as long as it is reproducible and stable in the host.

Can suitable dna sequence dna be inserted in the carrier with several different methods.In general, with methods known in the art dna sequence dna is inserted into suitable restriction endonuclease site.Such method and other method are considered in those skilled in the art's ken.

Said dna sequence dna in expression vector is to be operably connected to the suitable mRNA that instructs to synthesize on the expression control sequenc (promotor).The representative example of such promotor can should be mentioned that: LTR or SV40 promotor, colibacillary lac or trp, phage PL promotor and known other promotor that controlling gene is expressed in protokaryon or eukaryotic cell or their virus.Said expression vector also comprises the ribosome bind site that is used for translation initiation and Transcription Termination.This carrier also can comprise the suitable sequence of expressing for amplification.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic characteristic of transformed host cells, for example Tetrahydrofolate dehydrogenase of eukaryotic cell culture or neomycin resistance, or tsiklomitsin and the amicillin resistance in the intestinal bacteria for example.

Comprise above-described suitable dna sequence dna and suitable promotor or the carrier of control sequence and can be used to transform suitable host, so that it can marking protein.

As the representative example of suitable host, can should be mentioned that here: bacterial cell, as intestinal bacteria, streptomycete, Salmonella typhimurium; The fungal cell is as yeast; Insect cell such as fruit bat S2 and Spodoptera Sf9; Zooblast such as CHO, COS or Bowes melanoma; Adenovirus; Vegetable cell etc.By the elaboration of this paper, within the ken that is chosen in those skilled in the art to suitable host.

More particularly, the present invention also comprises recombinant precursor, and this construct comprises above broadly described one or more sequences.This construct comprises carrier, and as the carrier of plasmid or virus, this carrier has inserted sequence of the present invention forward or backwards.Under the even more ideal situation of this embodiment, construct also comprises the adjusting sequence that can be operationally connected on the described sequence, comprise, for example, promotor.A large amount of carrier and promotors that are fit to are well known by persons skilled in the art, and are obtainable by commercial sources.Provide following carrier by way of example: bacterium: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmaca); Eucaryon: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Parmacia).Yet any other plasmid or carrier can use, as long as they are reproducible and stable in the host.

Can be with CAT (CAT) carrier or other carrier that has selected marker from any required gene Selection promoter region.Two kinds of suitable carriers are pKK232-8 and pCM7.The bacterium promotor of mentioning especially comprises lacI, lacZ, T3, T7, gpt, λ P _R, P _L, and trp.Promoter in eukaryote comprises that CMV is early stage immediately, HSV thymidine kinase, early stage and late period SV40, derive from retroviral LTRs and mouse metallothionein(MT)-I.Within the level that is chosen in those of ordinary skills to appropriate carriers and promotor.

In another embodiment, the present invention relates to comprise the host cell of the above construct.Said host cell can be higher eucaryotic cells (as a mammalian cell), or eukaryotic cell (as yeast cell) such as low, and perhaps host cell can be prokaryotic cell prokaryocyte (as a bacterial cell).Can be by the transfection of calcium phosphate transfection, DEAE-dextran mediation, or electroporation is incorporated into construct (Davis, L., Dibner, M., Battey, I., the basic skills in the molecular biology, (1986)) in the host cell effectively.

Construct in the host cell can be used for producing in a usual manner the gene product by the recombination sequence coding.In addition, polypeptide of the present invention can produce by conventional peptide synthesizer is synthetic.

Mature protein can be expressed under suitable promotor control in mammalian cell, yeast, bacterium or other cell.Employing derives from the RNA of DNA construct of the present invention, and cell free translation system also can be used for producing this protein.Sambrook etc., molecular cloning: laboratory manual, second edition, the cold spring port, N.Y., the suitable clone and the expression vector that use with protokaryon and eucaryon host have been described in (1989) (this paper is reference in the lump).

The DNA of polypeptide of the present invention of encoding strengthens in the enhancer sequence that transcribing of higher eucaryote is inserted in the carrier.Enhanser is the cis-acting elements of DNA, and usually about 10 to 300bp, and acting on increases it and transcribe on the promotor.Example comprises polyoma enhanser and the adenovirus enhanser on SV40 enhanser on the replication orgin side in late period 100 to 270bp, cytomegalovirus early promoter enhanser, the replication orgin side in late period.

In general, recombinant expression vector comprises replication orgin and allows the selected marker (for example, colibacillary ampicillin resistance gene and Saccharomyces cerevisiae TRP1 gene) of host cell conversion and the promotor that instructs the downstream configurations sequence to transcribe that comes from cance high-expression gene.Such promotor can be come the operon of own coding glycolytic ferment (for example glycerol 3-phosphate acid kinase (PGK)), α-factor, acid phosphatase or heat shock protein etc.The allos structure sequence is with suitable manner (phase) and translation initiation and terminator sequence assembling, and preferably, the leader sequence that advances periplasmic space or extracellular substratum with the protein secreting that can instruct translation assembles.Heterologous sequence can be also encoding fusion protein not, this protein comprises the terminal peptide of differentiating of the N-that gives required feature, required feature for example, the recombinant products of expression is stable or simplify purification step.

Structural DNA sequence by the desired protein of will encoding and suitable translation initiation and termination signal are inserted and are made up the useful expression vector that is used for bacterium with having functional promotor can operate reading method (reading phase).Said carrier comprises one or more Phenotypic Selection marks and replication orgin, to guarantee to keep carrier the host and amplification is provided when needing.The prokaryotic hosts that be fit to transform comprises various (though other also can select use) of intestinal bacteria, subtilis, Salmonella typhimurium, Rhodopseudomonas, streptomyces and Staphylococcus.

As one representational but be not restrictive example, the useful expression vector that is used for bacterium can comprise selected marker and the bacterium replication orgin that comes from commercially available plasmid (genetic elements that comprises well-known cloning vector pBR322 (ATCC37017)).Commercially available carrier like this comprises, for example, and pKK223-3 (Pharmacia Fine chemical company, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, the U.S.).These pBR322 " skeleton " fragment and suitable promotor and structure sequence combination to be expressed.

Transform and after host strain grows to suitable cell density at the appropriate host bacterial strain, induce the promotor of selection with suitable method (for example temperature inversion or chemical induction), other for some time of cell cultures.

Typically,, keep formed crude product to be used for further purifying through physics or chemical process smudge cells through centrifugal cell harvesting.

The microorganism cells that can be used for marking protein through the method fragmentation of any routine, described method comprise freeze-thaw cycle, supersound process, Mechanical Crushing or use the lysis agent 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 comprises by the monkey kidney inoblast COS-7 clone of Gluzman (cell, 23:175 (1981)) description and other can express the clone of compatible carrier, for example, and C127,3T3, CHO, HeLa and bhk cell system.Mammalian expression vector comprises replication orgin, suitable promotor and enhanser, also can comprise any essential ribosome bind site, polyadenylation site, donor splicing site and acceptor site, transcription termination sequence and 5 ' flank non-transcribed sequence.The dna sequence dna that derives from SV40 montage and polyadenylation site can be used to provide required non-transcribed genetic elements.

Said polypeptide can reclaim and purifying from the reconstitution cell culture with several different methods, and described method comprises ammonium sulfate or ethanol sedimentation, sour extraction, negatively charged ion or cation-exchange chromatography, phosphorus Mierocrystalline cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and phytohemagglutinin chromatography.In finishing the configuration of mature protein, can use the protein refolding step when needing.At last, can use high performance liquid chromatography (HPLC) as last purification step.

Polypeptide of the present invention can be the product of natural purifying, or the product of chemical synthesis process, or produce from protokaryon or eucaryon host (for example insect of bacterium, yeast, higher plant, cultivation and mammalian cell) through recombinant technology.According to the host that reorganization is used in the production method, polypeptide of the present invention can be glycosylated maybe can be nonglycosylated.Polypeptide of the present invention also can comprise 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.

Polypeptide of the present invention can be used for determining the feature of acceptor.At present, the acceptor of EGF family comprises 4 EGF acceptors, is called EGFR1, EGFR2, EGFR3, EGFR4.EGFR2 also can be called ERB-2, and this molecule is being useful (Prigent, S.A., and Lemoine, N.R., somatomedin progress, 4: 1-24 (1992)) aspect various diagnosis and the treatment indication.TGF α-HII polypeptide may be the part of the new EGF type receptors of one or more these acceptors and evaluation.The use of TGF α-HII helps the evaluation of these acceptors, qualitative and clone.For example, the EGF acceptor gene is represented the cell homologue of the v-erb-B oncogene of fowl erythroblastosis virus.The overexpression of EGF acceptor or this protein kinase are regulated the tumorigenesis conversion (Manjusri, D. etc., human cell's factor, 364 and 381 (1991)) that segmental disappearance can cause cell.

Polypeptide of the present invention also can be used for the recovery or the raising of the neural function that reduces because of wound or other destructive pathology (as AIDS dementia, old and feeble dementia or the like).Have been found that TGF α and its homologue are the abundantest parts (Kaser etc., brain research and the research of molecule brain: 16:316-322, (1992)) of the EGF/TGF α acceptor in most of brain.Compare with EGF (it is present in the less dispersive zone), as if TGF α has distribution widely in the various zones of brain, show that TGF α may have physiological action in cerebral tissue.Numerous acceptor site explanation TGF of these TGF α are promoting normal brain activity cytodifferentiation and performance function aspects to have vital role in the brain.Therefore, under the situation that neural function reduces, using polypeptide of the present invention can stimulate brain and improve suitable physiological function.

TGF α-HII and its soluble form also can be used for the treatment of visual disorder, for example, and the cornea inflammation.Various experiments show that the member of TGF α-HII gene family is relevant with this pathology.Nearest paper has been summarized some data relevant with these somatomedins role in ophthalmic (Mann etc., cell 73:249-261 (1993)).Nearest experiment shows the mouse that lacks TGF α gene because white corpuscle and other cell demonstrate the cornea inflammation to the infiltration of the intrinsic matter of eye.

In addition, TGF α somatomedin can be as the mechanism of destroying target cell to the specificity of its target cell.For example, can be by the whole bag of tricks with TGF α-HII or soluble form and lps molecule (for example radiopharmaceuticals of deactivation target cell) coupling.These somatomedins-toxin fusions kills target cell (with killing the adjacent area cell by various " onlooker " effect in some cases).Mesri etc. (journal of biological chemistry 268:4853-62 (1993)) have delivered the up-to-date example of this toxin fusion gene.TGF α-HII and relevant molecule can be formed capsule and with itself and identification and in conjunction with tumour or the antigenic antibodies of cell-specific in liposome, thereby the method for " guiding " cell is provided.

Because the EGF family member demonstrates antiproliferative effect in cell transformed, so TGF α-HII can be used as the compound of anti-tumorigenesis in the same way.For using in the body, polypeptide of the present invention can be used in every way, and these modes include but not limited to: injection, infusion, partly, parenteral or the like.Can use with any physiologically acceptable carrier, these carriers comprise: phosphate-buffered saline, salt solution, sterilized water or the like.

Because have been found that the expression that these somatomedins are arranged in kidney, so TGF α-HII also can be used for the treatment of some renal tubal dysfunction.Like this, keeping of this organ normal physiological of these factor pairs is essential.

Because TGF α and its homologue and pHGF are after partially hepatectomized and acute hepatic necrosis, trigger hepatocellular regeneration, so said treatment also with the regeneration or the liver dysfunction relevant (Masuhara, M. etc., hepatology 16:1241-1249 (1992)) of liver.

The significant treatment relevant with TGF α-HII relates to wound healing.Composition of the present invention can be used for the treatment of the various wounds that comprise all skin wounds, corneal wound and the organ injury of health epithelium arrangement cave in fact.The wound that is suitable for treating comprises the wound that is caused by wound (as burn, scratch, knife wound) and surgical operation (as surgical incision and dermatoplasty).Be suitable for comprising chronic disease (as chronic ulcer, diabetic ulcer) and other disunion (nutrition) illness with other illness of polypeptide treatment of the present invention.

TGF α-HII or its solvable fragment can be incorporated in the physiologically acceptable carrier for use in the zone of infecting.The characteristic variations of said carrier is very big, and this variation depends on the site that expection is used.For using on skin, missible oil and ointment base are normally preferred; Suitable matrix comprises: lanolin, Silvadene (Marion) (particularly for the treatment burn), Aquaphor (the Duke laboratory, South Norwalk, Conn.) or the like.If desired, the composition that will comprise TGF α-HII mixes in bandage and other the wound binder so that wound is exposed under the said peptide constantly.Purposes has also been found in aerocolloidal application.

The concentration of TGF α-HII in therapeutic composition is not crucial but it should be enough to induce epithelial cell proliferation.Can use said composition to the infected area partly, generally be administered to eye or be administered on the skin as missible oil, ointment or lotion as eye drops.For eye, need often treatment, use with 4 hours or still less interval usually.On skin, in the process of healing, need continue the maintenance therapeutic composition in the infected area, every day administering therapeutic composition 2 to 4 times or more times.

The usage quantity of polypeptide of the present invention is with method of application, and the usage quantity of other active compound etc. and changing is usually in the scope of 1 μ g-100 μ g.Polypeptide of the present invention can use together with physiologically acceptable carrier (as salt solution, phosphate-buffered saline or the like).The usage quantity of described compound according to the reaction of cell in vitro and laboratory animal to the experiment polypeptide or to contain the reaction of the preparation of test polypeptide definite by experience.

TGF α-HII or its solvable fragment can be used for the adjusting of vascularization, bone resorption, immunne response and cynapse and neurone effector functions.TGF α-HII also can be used for the adjusting of arachidonic acid cascade.

TGF α-HII or its solvable fragment also can be used for and the relevant application of end differentiation eventually.Many TGF alpha factor and their homologue are induced end differentiation eventually in their target cell.Can be by using the said factor and inducing target cell death to utilize this specific character in vivo.This application method is to consider and the medically relevant disorder of hyper-proliferative of undesirable cell type (as cancer and other proliferative disorders (as inflammation, psoriasis or the like)).Except using in the body, the external situation about using of many permissions is arranged also.For example, can by with said somatomedin and/or the said cell of its derivative external treatment with removing unwanted cells group from marrow.

Said application also relates to depilation, trichomadesis and other influences the skin disorder that the hair hair follicle is grown.Some evidences show that these situations are relevant with TGF α somatomedin." rejecting (the knockout) " mouse that contains null mutation in the TGF α gene through genetically engineered operation demonstrates relevant with hair synthetic quantity and quality unusual.In addition, the research of the genetic map in the mouse has shown that some sudden changes that influence hair growth are positioned at (Mann etc., cell 73:249-261 (1993)) on the TGF α gene locus.The part of TGF α-HII or derivatives thereof or whole body are used the illness that can be used for the treatment of alopecia and trichomadesis, and these claims within the scope of the present invention.

Use TGF α-HII somatomedin by whole body clinical, the pathologic condition of some disease is partially or even wholly improved.Said using can be carried out with the form of gene therapy, or finishes (Woo etc., protein engineering 3:29-37 (1989)) by using by TGF α-HII DNA recombinant precursor or chemistry of peptides synthesis method synthetic peptide or protein.

The invention provides SCREENED COMPOUND with the stimulant of evaluation polypeptide of the present invention or the method for agonist compounds.Example is to cultivate together expressing the mammalian cell of TGF α-HII acceptor or film preparation and potential compound, detects said compound and is produced the ability of second signal by this receptor to determine whether it is effective stimulant.Second messenger system includes but not limited to: cAMP guanylate cyclase, ionic channel or phosphoinositide hydrolysis effect.Effectively antagonist is determined by above-described method, wherein detected agonist compounds and said receptors bind but can not cause that the second messenger reacts, and then blocking-up TGF α-HII and receptors bind.

Another kind of evaluation is a competition assay to the method for the potential antagonist of the receptor-specific of polypeptide of the present invention, and this method comprises the plasmalemma of the acceptor of excessive separation expression polypeptide of the present invention, for example, and human A431 cancer cells.To in medium (volume approximately is 10 μ l), join in the 5 microgram plasmalemmas that contain the potential agonist compounds by the laboratory sample that contains 10nM125I-TGF α-HII of serial dilution, and cultivate 4 hours down at 4 ℃.With the reaction mixture dilution and immediately by millipore filter, rapid then washing nozzle.And in gamma counter, measure the bonded radioactivity.Measure the amount of bonded TGF α-HII then.Under the situation that does not have said compound, carry out check analysis to determine whether antagonist can reduce the amount of bonded TGF α-HII.

The potential agonist compounds comprises under antibody or certain situation and said polypeptide bonded oligopeptides.In addition, the potential antagonist can be a kind of and closely-related protein receptors bind, and this protein is the inactivation form of said polypeptide, and then has stoped the activity of polypeptide of the present invention.

Another kind of potential agonist compounds is the antisense constructs that adopts the antisense technology preparation.The expression that antisense technology can come controlling gene by triple helical formation or antisense DNA or RNA, these two kinds of methods are all based on the combination of polynucleotide and DNA or RNA.For example, the encode 5 ' encoding part of polynucleotide sequence of mature polypeptide of the present invention can be used for the antisense rna oligonucleotide of about 10 to 40 base pairs of design length.A kind of DNA oligonucleotide is designed to and transcribes related gene regions complementation (triple helical-referring to Lee etc., nucleic acids research, 6:3073 (1979); Cooney etc., science, 241:456, (1988); With Dervan etc., science, 251:1360 (1991)), and then stop transcribing and producing of polypeptide of the present invention.Antisense rna oligonucleotide is hybridized with mRNA in vivo, and the translation becoming of blocking-up mRNA molecule polypeptide of the present invention (antisense-Okano, J.Neurochem., 56:560 (1991); Oligodeoxynucleotide is as the antisense inhibitor (CRC press, BocaRaton, FL (1988)) of genetic expression.Oligonucleotide described above can be sent in the cell, so that can expression in vivo sense-rna and DNA, suppresses the generation of polypeptide of the present invention.

Agonist compounds comprises small molecules, and these small molecules and polypeptide of the present invention combination are also blocked its effect acceptor site, like this, have just blocked normal biologic activity.Small molecules also can with the receptors bind of said polypeptide, the result has stoped the combination of polypeptide and acceptor.Micromolecular example includes but not limited to little peptide or class peptide molecule.

Antagonist can be used for treating tumorigenesis, for example, and cancer and tumour.Known restraining effect to tumor cell secretion in the mouse or generation EGF family member can make the tumour decline.

The antagonist of polypeptide of the present invention also can be used for the treatment of to therapeutic some skin disorder, for example, and psoriasis.Have been found that the expression level that said growth factor family member improves will improve (Cook, et al., cancer research, 52:3224-3227 (1992)) again in the Skin biopsy of taking from disease (as the psoriasis damage).Said antagonist and pharmaceutically acceptable carrier (as described below) one is used from the composition.

Polypeptide of the present invention and stimulant or antagonist can be used in combination with suitable pharmaceutical carrier.Such composition comprises said polypeptide and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.Such carrier includes but not limited to salt solution, buffer saline, dextrose, water, glycerine, ethanol and their composition.The mode that its prescription should be suitable for using.

The present invention also provides pharmaceutical pack or test kit, and they comprise one or more containers that are filled with one or more compositions of pharmaceutical composition of the present invention.Can be in this container with the bulletin of government organs' prescribed form of managing medicine and biological products manufacturing, use or sale, this bulletin has reflected manufacturing, use or sold the mankind makes articles for use obtain the agreement of government organs.In addition, polypeptide of the present invention or composition can be treated compound with other and be used in combination.

Described pharmaceutical composition can be used in mode easily, described mode is for example oral, local, in intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the intradermal approach use.Said pharmaceutical composition is used with the significant quantity that treats and/or prevents specified disease.In general, they are used with the amount of about at least 10 micrograms/kg body weight, and in most of the cases, they are used with the amount that is no more than about 8 mg/kg body weight every day.Under most applications, consider factors such as route of administration and illness, dosage from every day about 10 microgram/kilograms to 1 mg/kg body weight.

The stimulant of polypeptide and polypeptide form and antagonist can utilize by the such polypeptide of expression in vivo according to the present invention, and this often is known as " gene therapy ".

Like this, for example, can be external patient's cell be carried out the genetically engineered operation with the polynucleotide (DNA or RNA) of coded polypeptide, the patient who treats to quilt with engineering cell provides said polypeptide.Such method is well-known in the art, and also apparent by the description of this paper.For example, can carry out the genetically engineered operation with the retroviral particle pair cell of the RNA that comprises the polypeptide of the present invention of encoding.

Similarly, can carry out the genetically engineered operation by pair cell in the methods known in the art body for example, so that the expression in vivo polypeptide.For example, will pack of the retrovirus plasmid vector transduction of (packaging) cell, so that packing cell can produce the infectious virus particle that contains interest genes with the RNA that contains code book invention polypeptide.This production cell can be administered to the patient so as in the body with cytogene through engineering approaches and the said polypeptide of expression in vivo.Through description of the invention, these or other method of using polypeptide of the present invention in this way is clearly to those skilled in the art.

The retrovirus of above-described retrovirus plasmid vector of can deriving includes but not limited to: moloneys mouse leukosis virus, spleen necrosis virus, retrovirus such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis viruses, gibbon orangutan leukosis virus, human immunodeficiency virus, adenovirus, bone marrow proliferation sarcoma virus and mammary tumor virus.In one embodiment, the retrovirus plasmid vector is from the moloneys mouse leukosis virus.

Described carrier comprises one or more promotors.Operable suitable promotor includes but not limited to: retrovirus LTR; The SV40 promotor; And human cytomegalovirus (CMV) promotor (Miller, etc., biotechnology, Vo1.7, No.9,980-990 (1989) describes); Perhaps other any promotor (for example eukaryotic cell promotor, as include but not limited to histone, pol III and beta-actin promotor).Other viral promotors that uses includes but not limited to: adenovirus promoter, thymidine kinase (TK) promotor and B19 parvovirus promotor.By the description of this paper, in the ken that is chosen in those skilled in the art of suitable promotor.

The nucleotide sequence of code book invention polypeptide is under suitable promotor control.Operable suitable promotor includes, but are not limited to: adenovirus promoter (as adenovirus major late promoter); Perhaps allogeneic promoter (as cytomegalovirus (CMV) promotor); Respiratory syncytial virus (RSV) promotor; Inducible promoter (as MMT promotor, metallothionein promoter); The heat-shocked promotor; The white protein promotor; The ApoAI promotor; Human globin promoter; Viral thymidine kinase promoter (as herpes simplex thymidine kinase promoter); Retrovirus LTRs (the retrovirus LTRs that comprises above-described modification); The beta-actin promotor; With human growth hormone's promotor.Said promotor also can be the natural promoter of the gene of control coding said polypeptide.

Use retrovirus plasmid vector transduction package cell line so that form production clone.The example of packing cell that can be transfected includes but not limited to: PE501, PA317, ψ-2, ψ-AM, PAl2, T19-14X, VT-19-17-H2, ψ CRE, ψ CRIP, GP+E-86, GP+envAml2 and DAN clone (Miller, human gene therapy, Vol.1, pgs.5-14 (1990) describes, and its incorporated herein by is reference in the lump).Can be by any known method in this area with described carrier transduction packing cell.These methods include but not limited to: electroporation, use liposome and CaPO ₄Precipitation.In addition, the retrovirus plasmid vector can wrap in the liposome, perhaps with the lipid coupling, is administered among the host then.

Production clone produces infective retroviral vector particles, and this particle comprises the nucleotide sequence of coding said polypeptide.Can use in these retroviral vector particles bodies then or external transduction eukaryotic cell.The eukaryotic cell of transduction will be expressed the nucleotide sequence of coding said polypeptide.The eukaryotic cell that can be transduceed includes but not limited to: embryonic stem cell, embryo cells and hemopoietic stem cell, liver cell, inoblast, sarcoplast, keratinocyte, endotheliocyte and bronchial epithelial cell.

The present invention also relates to the purposes of gene of the present invention as diagnostic reagent.The detection of the mutant form of gene of the present invention makes can diagnose the disease that comes from expression of polypeptides deficiency of the present invention (underexpression) or the susceptibility of disease, for example, the wound healing of inappropriate (improper), unsuitable neural function, visual disorder, kidney and hepatic insufficiency, the growth of hair hair follicle, vascularization and embryo form.

Can on dna level, detect individuality with various technology with people's gene sudden change of the present invention.Can obtain diagnostic nucleic acid from patient's cell (as blood, urinate, saliva is organized examination of living tissue and necrotomy material).Genomic dna can be directly used in detection, perhaps uses PCR enzymatic amplification (Saiki etc., nature, 324:163-166 (1986)) before analysis.RNA or cDNA also can be used for identical purpose.As an example, can use with the nucleic acid complementary PCR primer of code book invention polypeptide and differentiate and analyze its sudden change.For example, can by with the amplified production size of normal genotype comparison on change detect disappearance or insert.Point mutation can be through DNA and the radiolabeled RNA or the radiolabeled antisense dna sequence hybridization discriminating of amplification.Through ribonuclease A digestion, perhaps distinguish complete paired sequence and mispairing duplex from the difference of melting temperature.

Disclose reference gene and intergenic sequence difference by direct dna sequencing method with sudden change.In addition, clone's DNA section (segment) can be as probe with detection specificity DNA section.When with PCR in conjunction with the time, the susceptibility of this method improves greatly.For example, the PCR product of sequencing primer and two strands or the single-stranded template molecule that is produced by the PCR method that improves are used together.Radio-labeled Nucleotide method by routine or have fluorescently-labeled automatic sequencing method and determine sequence.

Can finish by detecting when being with or without denaturing agent on the gel change of dna fragmentation electrophoretic mobility based on the genetic test of dna sequence dna difference.Little sequence deletion and insertion can be demonstrated by the high resolving power gel electrophoresis.Not homotactic dna fragmentation can be distinguished on sex change methane amide gradient gel, wherein the migration of different dna fragmentations according to its specific fusing point or part melt temperature and be stuck in gel different positions (referring to, for example, Myers etc., science, 230:1242 (1985)).

Also can protect assay method to disclose sequence variation on the specific position, described assay method such as ribonuclease protecting and S1 protection and chemical cracking method (for example, Cotton etc., PNAS, the U.S., 85:4397-4401 (1985)) by nuclease.

Like this; can detect specific dna sequence by following method; the Southern blotting of for example hybridization of described method, ribonuclease protecting, chemical cracking, direct dna sequencing or use restriction enzyme (for example, restrictive fragment length polymerphism (RFLP)) and genomic dna.

Except that a lot of conventional gel electrophoresises and dna sequencing method, also available original position analyzing and testing sudden change.

Because the overexpression with respect to the said polypeptide of healthy tissues sample (for example can detect some disease, tumorigenesis, skin dysfunction, visual disorder and inflammation) existence, so, the present invention also relates to be used for to detect the diagnostic analysis method of level of the change of various tissues polypeptide of the present invention.The analytical procedure that is used for detecting host-derived sample polypeptide level of the present invention is known to those skilled in the art, and said method comprises: radioimmunoassay, competition in conjunction with measure, the Western engram analysis, preferably ELISA measures.ELISA measures and comprises the antigenic specific antibody for preparing polypeptide of the present invention, preferably monoclonal antibody at first.In addition, the report antibody of preparation monoclonal antibody.With detectable reagent and report antibodies, said reagent such as radioactivity, fluorescence or in this example, be horseradish peroxidase.Obtain sample by the host, and with its with sample in incubation in the solid support (as the polystyrene ware) of protein bound.By with nonspecific proteins matter (as bovine serum albumin(BSA)) incubation together, will cover any protein binding site freely in the ware.Next with monoclonal antibody incubation in ware, monoclonal antibody and be attached to any polypeptide combination of the present invention on the polystyrene ware during this period.With damping fluid all unconjugated monoclonal antibodies are washed off.At this moment, the report antibody that will be connected with horseradish peroxidase is put into ware, and the result causes reporting antibody and any monoclonal antibody combination that is attached on the 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 use competition assay to determine the level of polypeptide of the present invention in host's sample.This measuring method comprises the plasmalemma of excessive separation expression polypeptide receptor of the present invention.To contain then that the laboratory sample of the polypeptide of the present invention of mark joins in the plasmalemma, and with its incubation for some time.Host's sample that may contain polypeptide of the present invention joins in the reaction mixture.Make reaction mixture pass through the filter that is washed rapidly then, and measure the competition amount of bonded radioactivity, thereby determine the amount of polypeptide of the present invention in the sample with definite said acceptor.

Because the cancer cells of a lot of types is just regulated the various members of (upregulate) TGF α family in tumorigenesis or outgrowth process, so the specific antibody of TGF α-HII can be used for the diagnosis and the treatment of cancer.These antibody and TGF α-HII combination and inactivation TGF α-HII.The monoclonal antibody of TGF α-HII (and/or its family member) is used for the diagnosis and the treatment of some dysfunction clinically, and said dysfunction includes, but is not limited to outgrowth and growth failure tumorigenicity.The tumorigenicity tissue has formed the basis of detecting the various determination of serum that somatomedin increases in the infected patient blood to just the transferring of expression of somatomedin.Generally, this mensuration not only is used for diagnostic and makes a definite diagnosis (setting), also is used for prognostic and makes a definite diagnosis (so that detecting the existence of the tumour cell that hides after surgical operation and chemotherapy).

In addition, in receptors bind is measured, can utilize the TGF α-HII of mark or utilize the malignant cell of antibody test expression TGF α-HII acceptor of TGF α-HII acceptor itself.According to the existence and the density discriminate between cells of TGF α-HII acceptor, and then provide the method for the said cell of prediction to the susceptibility of the biologic activity of TGF α-HII.

Sequence of the present invention identifies it also is valuable to karyomit(e).This sequence specific ground target is positioned at the specific position on the one human chromosome and can hybridizes with it.In addition, the present specific site that needs to identify on the karyomit(e).At present, only there is the chromosome marking reagent of a few sequence data (repetition polymorphism) can be used for the position of marker chromosomes based on reality.DNA chromosome mapping of the present invention is the important the first step that these sequences and disease related gene are associated.

In brief, just can navigate to sequence on the karyomit(e) by prepare PCR primer (preferred 10-25bp) from cDNA.Adopt the Computer Analysis in 3 ' the untranslated zone can select primer apace, wherein primer should not crossed over above an exon on the genomic dna, otherwise makes amplification method complicated.Adopt these primers to be used for the somatic hybridization body that the PCR screening contains one human chromosome then.Have only those crossbreds that contain the people's gene corresponding just can produce amplified fragments with this primer.

The PCR of somatic hybridization body mapping is that a specific DNA is positioned quick program on the specific karyomit(e).Adopt same Oligonucleolide primers according to the present invention, usefulness comes from one group of fragment of specific karyomit(e) or big genomic clone aggregate, can realize inferior location (sublocalization) in a similar way.Can be used for similarly other mapping strategy of chromosome mapping is comprised in situ hybridization, carry out prescreen and carry out preliminary election with the karyomit(e) through airflow classification of mark, thereby construct the cDNA library of chromosome specific by hybridization.

The cDNA clone can be used for realizing the accurate chromosomal localization of single stage method with the fluorescence in situ hybridization (FISH) of Metaphase Chromosome smear.This technology can adopt the cDNA of 50 or 60 base length.Summary about this technology is consulted Verma etc., human chromosomal: basic fundamental handbook, Pergamon press, New York (1988).

In case sequence has navigated to a chromosome position accurately, then the physical location of this sequence can be associated with the genetic map data on the karyomit(e).These data for example can be at V.McKusick, finds in the human Mendelian inheritance (can by online the obtaining in the Johns Hopkins Welch of university medical science library).Relation between the disease on coming identified gene and navigate to identical chromosomal region by linkage analysis (the common heredity of the adjacent gene of physics) then.

Next need to be determined at the difference in cDNA between the affected and unaffected individuality or the genome sequence.If sudden change be observed in some or all of affected individuals, but in any one normal individual, be not observed again, this sudden change may be the cause of disease of disease so.

According to physical mapping and the present resolving power of genetic mapping technology, the cDNA that quilt accurately navigates to the chromosomal region relevant with disease can be 50-500 potential cause a disease a kind of (wherein supposition the mapping resolving power of 1 megabasse and every 20kb are arranged be a gene) in (causative) gene.

The cell of described polypeptide, its fragment or derivative or analogue or expression above-mentioned substance can be as the immunogen of producing its antibody.These antibody can be for example polyclonal antibody or monoclonal antibody.The present invention also comprises chimeric, strand and humanized antibody, and the product of Fab fragment or Fab expression library.Several different methods known in the art can be used to produce these antibody and fragment.

Can be at the antibody that produces corresponding to polypeptide of sequence of the present invention by this polypeptide being injected directly in the animal body or by this polypeptide is obtained the preferred non-human of animal wherein to animals administer.Then, the antibody that so obtains can be attached on this polypeptide.In this way, even also can be used for producing can be in conjunction with the antibody of whole natural polypeptides for a fragments sequence of this polypeptide of only encoding.Then, this antibody can be used for separating this peptide species from the tissue of expressing this polypeptide.

In order to prepare monoclonal antibody, can adopt any technology of producing antibody of cultivating by successive clone.Example comprises hybridoma technology (Kohler and Milstein, 1975, nature, 256:495-497), trisome hybridoma technology, human B cell hybridoma technology (Kozbor etc., 1983, today immunology, 4:72) and the EBV-hybridoma technology (Cole that produces human monoclonal antibodies, Deng, 1985, monoclonal antibody and cancer therapy, Alan R.Liss, Inc., pp.77-96).

Can will be used for the technology (United States Patent (USP) 4,946,778) of manufacture order chain antibody thus make amendment and produce single-chain antibody at immunogenic polypeptide product of the present invention.Also can use transgenic mice to express humanized antibody at immunogenic polypeptide product of the present invention.

The present invention further is illustrated with reference to the following examples; But, should understand the present invention and be not limited to these embodiment.Unless beyond doing in addition to state, all part or amounts are weight.

To understand following embodiment in order being beneficial to, now to narrate method and/or term that some often occur.

" plasmid " is by a small letter p and/or follow several capitalizations and/or numeral is named the preceding.Initial plasmid herein or can by commercial sources obtain or on unrestricted basis the public can obtain, perhaps can from available plasmid, build according to disclosed method.In addition, be known in the art for plasmid and be conspicuous those of ordinary skills with those described equivalences.

" digestion " of DNA is meant the Restriction Enzyme catalytic pyrolysis DNA that only some sequence on the DNA is worked with a kind of.The multiple Restriction Enzyme that this paper adopted can obtain by commercial sources, and its reaction conditions, cofactor and other service requirements are known to those of ordinary skills.For analysis purposes, usually the plasmid of 1 μ g or the dna fragmentation enzyme with about 2 units that are dissolved in about 20 μ l buffered soln is used.In order to separate the dna fragmentation that is used for plasmid construction, usually in a bigger volume with the DNA of enzymic digestion 5 to the 50 μ g of 20 to 250 units.At concrete Restriction Enzyme, the suitable buffered soln and the amount of substrate are stipulated by the producer.Usually adopt 37 ℃ of following about 1 hour incubation times, but this time can change according to the indication of product supplier.After digestion, reaction mixture directly carries out electrophoresis to isolate required fragment on polyacrylamide gel.

Employing is by Goeddel, D. etc., and nucleic acids research, described 8% polyacrylamide gel of 8:4057 (1980) carries out the size separation of crack fragment.

" oligonucleotide " or refer to a kind of strand poly deoxynucleosides, or refer to can be by two complementary poly deoxynucleosides chains of chemosynthesis.Therefore these synthetic oligonucleotide do not have 5 ' phosphoric acid, if not in the presence of a kind of kinases during with phosphoric acid of ATP interpolation, this oligonucleotide will can not be connected on another oligonucleotide.The synthetic oligonucleotide will be connected to not by on the dephosphorylized fragment.

" connection " be meant between two double stranded nucleic acid fragments the process that forms phosphodiester bond (Maniatis, T., etc., ibid, p.146).Unless beyond providing separately, adopt dna fragmentation to be connected 10 T4 of the unit dna ligases (" ligase enzyme ") of known damping fluid and condition, the about equimolar amount of per 0.5 μ g to realize being connected.

Except as otherwise noted, press Graham, F. and Van der Eb, A., virusology, the described method of 52:456-457 (1973) transforms.

Embodiment 1

Bacterial expression and purifying TGF α-HII soluble form

Utilize the dna sequence dna (ATCC#__) of the initial amplification coding TGF of PCR Oligonucleolide primers α-HII, said primer is equivalent to the carrier sequence of the proteinic 5 ' sequence of finished TGF α-HII (subtraction signal peptide sequence) and TGF α-HII gene 3 '.To join respectively in 5 ' and the 3 ' sequence corresponding to the additional nucleotide of TGF α-HII.Said 5 ' Oligonucleolide primers has sequence 5 ' CCCGGATCCGCACGAGACATACCTTGTCCG3 ' (SEQ ID NO:3), this primer contains BamHI restriction endonuclease sites (runic), after connect 21 Nucleotide of TGF α-HII encoding sequence that the end amino acid of supposition by protein code of processing begins.Said 3 ' sequence, 5 ' GGGAAGCTTTTAATACTGAAATCGTACAGGAC3 ' (SEQ ID NO:4) comprises and Hind III site complementary sequence, and follows 23 Nucleotide of TGF α-HII.Said restriction endonuclease sites is corresponding to the restriction endonuclease sites (Qiagen, Chatsworth company, CA, 91311) of bacterial expression vector pQE-9.PQE-9 coding antibiotics resistance (Ampr), bacterium replication orgin (ori), IPTG can regulate promotor operon (P/O), ribosome bind site (RBS), 6-histidine mark and restriction endonuclease sites.Then with BamHI and HindIII digestion pQE-9.The sequence of amplification is connected among the pQE-9 and is inserted in the framework of the sequence that has encoding histidine mark and RBS.The method of describing by (molecular clonings: laboratory manual, press of cold spring harbor laboratory, (1989)) such as Sambrook is used to connect mixture transformed into escherichia coli bacterial strain M15/rep 4 (Qiagen, companies) then.M15/rep4 comprises the multiple copied of plasmid pREP4, and this plasmid expression lacI repressor is given kalamycin resistance (Kanr) simultaneously.Identify transformant by the energy for growth of transformant on the LB flat board, and filter out penbritin/kalamycin resistance bacterium colony.Separate and the affirmation plasmid DNA by restriction analysis.The bacterium colony that will contain required construct in the LB liquid nutrient medium that has replenished Amp (100 μ g/ml) and Kan (25 μ g/ml), spend the night (O/N) cultivate.The O/N culture is used to inoculate the large volume culture with 1: 100 to 1: 250 ratio.Cell grows to the optical density(OD) 600 (O.D. between 0.4 and 0.6 ⁶⁰⁰) time, add IPTG (sec.-propyl-B-D-thio-galactose pyran-glucoside) to ultimate density be 1mM.IPTG removes P/O by inactivation lacI repressor, causes the increase of genetic expression.Cell was cultivated 3 to 4 hours again.Pass through centrifugal cell harvesting.Cell precipitation is dissolved in the 6M Guanidinium hydrochloride chaotropic agent.After the clarification, under the condition that the protein that allows to contain the 6-histidine mark is combined closely, in nickel-inner complex post by chromatography purifying dissolved TGF α-HII (Hochuli, E. etc., chromatography magazine 411:177-184 (1984)) from solution.TGF α-HII (85% purity) is eluted from post with 6M guanidine HCl (pH value 5.0),, transfer to 3M Guanidinium hydrochloride, 100mM sodium phosphate, 10M gsh (reduced form), 2M gsh (oxidized form) for renaturation.Incubation was dialysed protein after 12 hours to the 10M sodium phosphate in solution.

Embodiment 2

Utilize the baculovirus expression system clone and express TGF α-HII

Utilize PCR Oligonucleolide primers amplification coding total length TGF α-HII protein DNA sequence (ATCC#__), said primer is corresponding to 5 ' and 3 ' sequence of this gene:

Used three cover primers:

The first cover primer is: 5 ' CGCGGATCCGCCATCATGGTGCTGTGGGAGTCC3 ' (SEQ ID NO:5) and 5 ' GCGTCTAGACTAGTATAGAACACTGTAGTCC3 ' (SEQ ID NO:6), this construct begins at 321 Nucleotide of SEQ ID NO:1, stop at its 1248 Nucleotide, and comprise the leader sequence of inferring;

The second cover primer is: 5 ' CGCGGATCCGCCATCATGCTACTCATCGTAGCC3 ' (SEQ ID NO:7) and 5 ' GCGTCTAGACTAGTATAGAACACTGTAGTCC3 ' (SEQ ID NO:8), this construct begins at 402 Nucleotide of SEQ ID NO:1, stop at its 1248 Nucleotide, and do not comprise the leader sequence of inferring;

The 3rd cover primer is: 5 ' CGCGGATCCAGAACACCACATACCTTGTCCG3 ' (SEQ ID NO:9) and 5 ' GCGTCTAGACTAGTATAGAACACTGTAGTCC3 ' (SEQ ID NO:10), this construct begins at 1100 Nucleotide of SEQ ID NO:1, stopping at its 1248 Nucleotide, is the soluble part that said polypeptide is inferred.All three 5 ' primers all have BamHI restriction endonuclease sites (runic), after meet the Nucleotide (Kozak that represents the effective translation initiation signal of eukaryotic cell, M., molecular biology magazine, 196:947-950 (1987)) (translation initiation codon " ATG " underscore).For this three covers primer, in the pA2GP carrier, built the baculovirus signal peptide sequence.

Said 3 ' primer sequence comprises the cleavage site of restriction endonuclease XbaI, and has 3 ' TGF α district complementary Nucleotide with TGF α-HII gene.(" Geneclean, " biological 101 companies, La Jolla Ca.) separate the sequence that increases from 1% sepharose with the commercial reagent box.With said fragment with endonuclease BamHI and XbaI digestion, and on 1% sepharose purifying once more.This fragment is appointed as F2.

Carrier pA2 is used for preceding two cover primers, and pA2GP is used for the 3rd cover primer.(form with carrier pA2 by the pVL941 carrier modification, discussion sees below) (summary is referring to Summer to express TGF α-HII protein by baculovirus expression system, M.D. and Smith, G.E.1987, the method handbook of baculovirus vector and insect cell culturing process, testing station's communique 1555 of Texas's agricultural).This expression vector comprises the strong polyhedrin promotor of the polygonal virus of autographa california multinuclear type (AcMNPV), and its back is the recognition site of restriction endonuclease.The polyadenylation site of monkey disease poison (SV) 40 is used for effective polyadenylation.In order easily to select recombinant virus, with the direction insertion same, be colibacillary beta-galactosidase gene thereafter the polyadenylation signal of polyhedron gene with the polyhedrin promotor.The both sides of polyhedrin sequence are the virus sequences of homologous recombination that is used for the wild-type virus DNA of cell-mediated cotransfection.Can use much other baculovirus vector replacement pRG1, and said carrier such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summer, M.D., virusology, 170:31-39).

Digest said plasmid with restriction enzyme SmaI and XbaI, utilize the calf intestinal phosphatase enzyme to make the plasmid dephosphorylation by methods known in the art then.(La Jolla is Ca.) from 1% sepharose DNA isolation for " Geneclean ", BIO101 company to use the commercial reagent box then.This carrier DNA is appointed as V2.

Connect F2 fragment and said dephosphorylated plasmid V2 with the T4 dna ligase.Transformed into escherichia coli HB101 cell (Stratagene cloning system then, 11011North TorreyPines Road La Jolla, Ca.92037) and utilize enzyme BamHI and XbaI to differentiate to contain the plasmid (bacterium of pBac TGF α-HII) of said band TGF α-HII gene.Confirm the sequence of cloned sequence by dna sequencing.

With lipofection (Felgner etc., Proc. Natl. Acad. Sci.USA, 84:7413-7417 (1987)) with 5 μ g plasmid pBacTGF α-HII and commercially available the linearized baculovirus (" BaculoGold of 1.0 μ g ^TMBaculovirus DNA ", Pharmingen, San Diego, CA.) cotransfection.

With 1 μ g BaculoGold ^TM(Life Technologies company, Gaithersburg mix in the aseptic hole of droplet plate MD) containing 50 microlitre serum-free Grace ' s substratum for viral DNA and 5 μ g plasmid pBacTGF α-HII.Behind the substratum that adds 10 microlitre lipofectin reagents and 90 microlitre Grace ' s, mix to be incorporated under the room temperature and cultivated 15 minutes.Then transfection mixture is dropwise added in the Sf9 insect cell (ATCC CRL1711), said cell inoculation is on the 35mm tissue culture plate with 1ml serum-free Grace ' s substratum.The waggle flat board is to mix the solution of new interpolation.Then flat board was cultivated 5 hours down at 27 ℃.After 5 hours, remove transfection solution, add Grace ' the s insect substratum that 1 microlitre is supplemented with 10% foetal calf serum from flat board.Flat board is put back into incubator, 27 ℃ of following cultured continuously 4 days.

After 4 days, collect supernatant liquor, carry out plaque measurement with similar Summers and the described method of Smith (the same).Some change is to use the sepharose with " Blue Gal ", and (LifeTechnologies company, Gaithersburg), it makes and is easy to separate the plaque that dyes indigo plant.(the detailed description of " plaque measurement " also can the insect cell of Life Technologies company (Gaithersburg) issue cultivate and baculovirus users' guidebook 9-10 page or leaf in find).

After four days, the virus of serial dilution is added in the cell, dye blue plaque with Eppendorf pipette tip picking.The agar that will comprise recombinant virus then is suspended in the Eppendcrf pipe that comprises 200 microlitre Grace ' s substratum.Through the brief centrifugal agar of removing, the supernatant liquor that will comprise recombinant baculovirus is used for infecting the Sf9 cell that is inoculated into 35 millimeters culture dish.After 4 days, gather in the crops the supernatant liquor in these culture dish, in 4 ℃ of storages.

The Sf9 cell is grown in the Grace ' substratum that is supplemented with 10% hot deactivation FBS.Infection multiplicity (MOI) 2 times with recombinant baculovirus V-TGF α-HII cells infected.After 6 hours, remove substratum, (LifeTechnologies company Gaithersburg) substitutes with SF900II substratum (deducting methionine(Met) and halfcystine).After 42 hours, add 5 μ Ci35S methionine(Met)s and 5 μ Ci ³⁵S halfcystine (Amersham).Before centrifugal results, cell further to be cultivated 16 hours, labelled protein demonstrates through SDS-PAGE and radioautograph.

Embodiment 3

Express recombinant TGF α-HII in the COS cell

Expression plasmid TGF α-HII HA derives from carrier pcDNA3/Amp (Invitrogen), it comprises: 1) SV40 replication orgin, 2) ampicillin resistance gene, 3) colibacillary replication orgin, 4) CMV promotor is thereafter polylinker district, SV40 intron and polyadenylation site.With the dna fragmentation of the complete TGF α-HII precursor of coding with in frame, be integrated into the polylinker district of 3 ' terminal HA marker clone to said carrier, therefore, being expressed under the control of CMV promotor of recombinant protein.The HA mark is corresponding to from the proteinic epi-position of influenza hemagglutinin, and this point was described (I.Wilson, H.Niman, R.Heighten, A Cherenson, M.Connolly, and R.Lerner, 1984, cell 37:767, (1984)) in the past.The fusion of HA and target protein makes to be easy to detect recombinant protein with the antibody of discerning the HA epi-position.

The plasmid construction strategy is described below: the dna sequence dna (ATCC#__) that makes up coding TGF α-HII with two kinds of primers on clone's primary EST by PCR method, said primer is: 5 ' primer, 5 ' CGCGGATCCGCCATCATGGTGCTGTGGGAGTCC3 ' (SEQ ID NO:11), comprising BamHI site (runic), is 18 Nucleotide of TGF α-HII encoding sequence of being begun by initiator codon afterwards; 3 ' primer, 5 ' GCGCTCGAGGTATAGAACACTGTAGTCC3 ' (SEQ ID NO:12) comprises last 19 Nucleotide in XhoI site, TGF α district and the complementary sequence in XhoI site.The pcDNA3/Amp carrier comprises the BamHI/XhoI cloning site, and this site is carried the PCR that is marked in the frame with 3 ' HA and inserted fragment, after connect terminator codon.Therefore, the PCR product comprises the BamHI site, 936 base pair encoding sequences and XhoI site.With BamHI and XhoI digestion with restriction enzyme and dna fragmentation that is connected pcr amplification and carrier pcDNA3/Amp.To connect mixture and be transformed among the coli strain SURE (can be, La Jolla, CA 92037 obtains) from the Stratagene cloning system, with the inoculation that transforms on the ampicillin medium flat board, and screening resistance clone.Isolated plasmid dna from transformant, and detect whether there is correct fragment with restriction analysis.For expression recombinant TGF α-HII, by DEAE-DEXTRAN method expression vector rotaring redyeing COS cell (J.Sambrook, E.Fritsch, T.Maniatis, molecular cloning: laboratory manual, cold spring harbor laboratory's publication, (1989)).Detect TGF α-HII HA protein expression (E.Harlow, D.Lane, antibody: laboratory manual, cold spring harbor laboratory's publication, (1988)) by radio-labeled and immuno-precipitation.With cell after transfection two days with 15S-halfcystine mark 8 hours.Collect culture then and use washing agent lysing cell (RIPA damping fluid (150mM NaCl, 1%NP-40,0.1%SDS, 1%NP-40,0.5%DOC, 50mM Tris, pH7.5) (Wilson, I. etc., Id.37:767 (1984)).With HA monoclonal antibody specific sedimentation cell lysate and culture.Analysing protein precipitation on the 15%SDS-PAGE gel.

Embodiment 4

Expression via gene therapy

Inoblast obtains from a research object by Skin biopsy.Be placed on the tissue that obtains on the tissue culture medium (TCM) and be divided into fritter.Small tissue blocks is placed on the wet surface of tissue culture flasks, wherein places about 10 block organizations in every bottle.Bottle is placed upside down, and lid is tight and spend the night under room temperature.At room temperature place counter-rotating bottle after 24 hours, tissue block still is fixed on the bottle bottom, adds fresh culture (for example containing 10%FBS, Ham ' the s F12 substratum of penicillin and Streptomycin sulphate), then in 37 ℃ of about 1 weeks of following incubation.At this moment add fresh culture, changed a subculture subsequently every several days.After cultivating for 2 weeks again, an individual layer inoblast has appearred.With monolayer cell through tryptic digestion and put into bigger bottle.

With EcoRI and Hind III digestion side the long terminal repetition pMV-7 (Kirschmeier, P.T. etc., DNA, 7:219-25 (1988)) of Moloney murine sarcoma virus is arranged, next handle with the calf intestinal phosphatase enzyme.Linear carrier fractional separation and use granulated glass sphere purifying in addition on sepharose.

Adopt the cDNA of corresponding with 5 ' and 3 ' end sequence respectively PCR primer amplification code book invention polypeptide.5 ' primer comprises an EcoRI site, and 3 ' primer contains a Hind III site.In the presence of the T4 dna ligase, the EcoRI of the linearizing skeleton of the Moloney murine sarcoma virus of equivalent and amplification added with Hind III fragment be in the same place, be suitable for keeping resulting mixture under the condition that two fragments connect.Should connect mixture and be used for transform bacteria HB101, in order to confirm this carrier to have the correct gene of interest of insertion bacterium was coated on the agar plate that contains kantlex then.

Amphophilic (amphotropic) pA317 or GP+am12 packing cell are cultivated in the tissue culturing plate of Dulbecco ' the s improvement Eagles substratum (DMEM) that contains 10% calf serum (CS), penicillin and Streptomycin sulphate, be paved with density until reaching.The carrier that will contain described gene then adds in the substratum also with this carrier transduction packing cell.Packing cell is produced immediately and is contained the infective virion of having of this gene (packing cell is known as and produces cell now).

In the production cell of transduction, add fresh substratum, next be paved with the 10cm flat board of producing cell and collect substratum from one.The substratum that contains infectious viral particle filters to remove the production cell of desorption (detached) through millipore filter, utilizes this substratum to go to infect inoblast then.Be paved with from fibroblastic Asia to remove substratum the flat board and promptly to replace and come from the substratum of producing cell.Remove this substratum and replace fresh substratum.If the titre of virus is very high, all so in fact inoblasts are all infected and need not to select.If titre is very low, so just need to adopt retrovirus with the alternative mark as neo or his.

Inoblast with through engineering approaches is injected into the host then, it or separately injection or on a cytodex3 microcarrier bead, grown to inject again after being paved with.This moment, inoblast produced protein.

According to above instruction, many improvement of the present invention and variation all are possible, therefore can other mode implement the present invention within the scope of the appended claims.

Sequence table (1) general information:

(i) applicant: WEI etc.

(ii) denomination of invention: transforming growth factor alpha-HII

(iii) sequence number: 12

(iv) address:

(A) addressee: CARELLA, BYRNE, BAIN, GILFILLAN,

CECCHI, STEWART and OLSTEIN

(B) street: 6 BECKER FARM ROAD

(C) city: ROSELAND

(D) state: New Jersey

(E) country: the U.S.

(F) postcode: 07068

(v) computer-reader form:

(A) media type: 3.5 inches disks

(B) computer: IBM PS/2

(c) operating system: MS-DOS

(D) software: WORD PERFECT 5.1

(vi) current request for data:

(A) application number:

(B) applying date:

(C) classification number:

(vii) request for data formerly

(A) application number: do not have

(B) applying date: do not have

(viii) lawyer/proxy's information:

(A) name: FERRARO, GREGORYD.

(B) registration number: 36,134

(C) case number/number of documents: 325800-351

(ix) telecom information:

(A) phone: 201-994-1700

(B) fax: the information of 201-994-1744 (2) SEQ ID NO:1:

(i) sequence signature:

(A) length: 1659 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

( xi ) ：SEQ ID NO：1CACTCGTCTG CCCCTGGACT CCCGTCTCCT CCTGTCCTCC GGCTTCCCAG AGCTCCCTCC 60TTATGGCAGC AGCTTCCCGC GTCTCCGGCG CAGTTCTCAG CGGACGACCC TCTCGCTCCG 120GGGCTGAGCC CAGTCCCTGG ATGTTGCTGA AACTCTCGAG ATCATGCGCG GGTTTGGCTG 180CTGCTTCCCC GCCGGGTGCC ACTGCCACCG CCGCCGCCTC TGCTGCCGCC GTCCGCGGGA 240TGCTCAGTAG CCCGCTGCCC GGCCCCCGCG ATCCTGTGTT CCTCGGAAGC CGTTTGCTGC 300TGCAGAGTTG CACGAACTAG TCATGGTGCT GTGGGAGTCC CCGCGGCAGT GCAGCAGCTG 360GACACTTTGC GAGGGCTTTT GCTGGCTGCT GCTGCTGCCC GTCATGCTAC TCATCGTAGC 420CCGCCCGGTG AAGCTCGCTG CTTTCCCTAC CTCCTTAAGT GACTGCCAAA CGCCCACCGG 480CTGGAATTGC TCTGGTTATG ATGACAGAGA AAATGATCTC TTCCTCTGTG ACACCAACAC 540CTGTAAATTT GATGGGGAAT GTTTAAGAAT TGGAGACACT GTGACTTGCG TCTGTCAGTT 600CAAGTGCAAC AATGACTATG TGCCTGTGTG TGGCTCCAAT GGGGAGAGCT ACCAGAATGA 660GTGTTACCTG CGACAGGCTG CATGCAAACA GCAGAGTGAG ATACTTGTGG TGTCAGAAGG 720ATCATGTGCC ACAGATGCAG GATCAGGATC TGGAGATGGA GTCCATGAAG GCTCTGGAGA 780AACTAGTCAA AAGGAGACAT CCACCTGTGA TATTTGCCAG TTTGGTGCAG AATGTGACGA 840AGATGCCGAG GATGTCTGGT GTGTGTGTAA TATTGACTGT TCTCAAACCA ACTTCAATCC 900CCTCTGCGCT TCTGATGGGA AATCTTATGA TAATGCATGC CAAATCAAAG AAGCATCGTG 960TCAGAAACAG GAGAAAATTG AAGTCATGTC TTTGGGTCGA TGTCAAGATA ACACAACTAC 1020AACTACTAAG TCTGAAGATG GGCATTATGC AAGAACAGAT TATGCAGAGA ATGCTAACAA 1080ATTAGAAGAA AGTGCCAGAG AACACCACAT ACCTTGTCCG GAACATTACA ATGGCTTCTG 1140CATGCATGGG AAGTGTGAGC ATTCTATCAA TATGCAGGAG CCATCTTGCA GGTGTGATGC 1200TGGTTATACT GGACAACACT GTGAAAAAAA GGACTACAGT GTTCTATACG TTGTTCCCGG 1260TCCTGTACGA TTTCAGTATG TCTTAATCGC AGCTGTGATT GGAACAATTC AGATTGCTGT 1320CATCTGTGTG GTGGTCCTCT GCATCACAAG GAAATGCCCC AGAAGCAACA GAATTCACAG 1380ACAGAAGCAA AATACAGGGC ACTACAGTTC GGACAATACA ACAAGAGCGT CCACGAGGTT 1440AATCTAAAGG GAGCATGTTT CACAGTGGCT GGACTACCGA GAGCTTGGAC TACACAATAC 1500AGTATTATAG ACAAAAGAAT AAGACAAGAG ATCTACACAT GTTGCCTTGC ATTTGTGGTA 1560ATCTACACCA ATGAAAGCAT GTACTACAGC TATATTTGAT TATGTATGGA TATATTTGAA 1620ATAGTATACA TTGTCTTGAT GTTTTTTCTG TAATGTAAAT AAACTATTTA TATCACACAA 1680AAAAAAAAAA AAAAA 1655 ( 2 ) SEQ ID NO：2：

(i) sequence signature:

(A) length: 374 amino acid

(B) type: amino acid

(C) chain:

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:2Met Val Leu Trp Glu Ser Pro Arg Gln Cys Ser Ser Trp Thr Leu-45-40-35Cys Glu Gly Phe Cys Trp Leu Leu Leu Leu Pro Val Met Leu Leu-30-25-20Ile Val Ala Arg Pro Val Lys Leu Ala Ala Phe Pro Thr Ser Leu-15-10-5Ser Asp Cys Gln Thr Pro Thr Gly Trp Asn Cys Ser Gly Tyr Asp 15 10 15Asp Arg Glu Asn Asp Leu Phe Leu Cys Asp Thr Asn Thr Cys Lys

20 25 30Phe?Asp?Gly?Glu?Cys?Leu?Arg?Ile?Gly?Asp?Thr?Val?Thr?Cys?Val

35 40 45Cys?Gln?Phe?Lys?Cys?Asn?Asn?Asp?Tyr?Val?Pro?Val?Cys?Gly?Ser

50 55 60Asn?Gly?Glu?Ser?Tyr?Gln?Asn?Glu?Cys?Tyr?Leu?Arg?Gln?Ala?Ala

65 70 75Cys?Lys?Gln?Gln?Ser?Glu?Ile?Leu?Val?Val?Ser?Glu?Gly?Ser?Cys

80 85 90Ala?Thr?Asp?Ala?Gly?Ser?Gly?Ser?Gly?Asp?Gly?Val?His?Glu?Gly

95 100 105Ser?Gly?Glu?Thr?Ser?Gln?Lys?Glu?Thr?Ser?Thr?Cys?Asp?Ile?Cys

110 115 120Gln?Phe?Gly?Ala?Glu?Cys?Asp?Glu?Asp?Ala?Glu?Asp?Val?Trp?Cys

125 130 135Val?Cys?Asn?Ile?Asp?Cys?Ser?Gln?Thr?Asn?Phe?Asn?Pro?Leu?Cys

140 145 150Ala?Ser?Asp?Gly?Lys?Ser?Tyr?Asp?Asn?Ala?Cys?Gln?Ile?Lys?Glu

155 160 165Ala?Ser?Cys?Gln?Lys?Gln?Glu?Lys?Ile?Glu?Val?Met?Ser?Leu?Gly

170 175 180Arg?Cys?Gln?Asp?Asn?Thr?Thr?Thr?Thr?Thr?Lys?Ser?Glu?Asp?Gly

185 190 195His?Tyr?Ala?Arg?Thr?Asp?Tyr?Ala?Glu?Asn?Ala?ASn?Lys?Leu?Glu

200 205 210Glu?Ser?Ala?Arg?Glu?His?His?Ile?Pro?Cys?Pro?Glu?His?Tyr?Asn

215 220 225Gly?Phe?Cys?Met?His?Gly?Lys?Cys?Glu?His?Ser?Ile?Asn?Met?Gln

230 235 240Glu?Pro?Ser?Cys?Arg?Cys?Asp?Ala?Gly?Tyr?Thr?Gly?Gln?His?Cys

245 250 255Glu?Lys?Lys?Asp?Tyr?Ser?Val?Leu?Tyr?Val?Val?Pro?Gly?Pro?Val

260 265 270Arg?Phe?Gln?Tyr?Val?Leu?Ile?Ala?Ala?Val?Ile?Gly?Thr?Ile?Gln

275 280 285Ile?Ala?Val?Ile?Cys?Val?Val?Val?Leu?Cys?Ile?Thr?Arg?Lys?Cys

290 295 300Pro?Arg?Ser?Asn?Arg?Ile?His?Arg?Gln?Lys?Gln?Asn?Thr?Gly?His

305 310 315Tyr?Ser?Ser?Asp?Asn?Thr?Thr?Arg?Ala?Ser?Thr?Arg?Leu?Ile

320 325

(2) information of SEQ ID NO:3:

(i) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:3CCCGGATCCG CACGAGACAT ACCTTGTCCG 30

(2) information of SEQ ID NO:4:

(i) sequence signature:

(A) length: 32 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:4GGGAAGCTTT TAATACTGAA ATCGTACAGG AC 32

(2) information of SEQ ID NO:5:

(i) sequence signature:

(A) length: 33 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:5CGCGGATCCG CCATCATGGT GCTGTGGGAG TCC 33

(2) information of SEQ ID NO:6:

(i) sequence signature:

(A) length: 31 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:6GCGTCTAGAC TAGTATAGAA CACTGTAGTC C 31

(2) information of SEQ ID NO:7:

(i) sequence signature:

(A) length: 33 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:7CGCGGATCCG CCATCATGCT ACTCATCGTA GCC 33

(2) information of SEQ ID NO:8:

(i) sequence signature:

(A) length: 31 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:8GCGTCTAGAC TAGTATAGAA CACTGTAGTC C 31

(2) information of SEQ ID NO:9:

(i) sequence signature:

(A) length: 31 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:9CGCGGATCCA GAACACCACA TACCTTGTCC G 31

(2) information of SEQ ID NO:10:

(i) sequence signature:

(A) length: 31 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:10GCGTCTAGAC TAGTATAGAA CACTGTAGTC C 31

(2) information of SEQ ID NO:11:

(i) sequence signature:

(A) length: 33 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:11CGCGGATCCG CCATCATGGT GCTGTGGGAG TCC 33

(2) information of SEQ ID NO:12:

(i) sequence signature:

(A) length: 28 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:12GCGCTCGAGG TATAGAACAC TGTAGTCC 28

Claims (30)

1. isolating polynucleotide, it comprises the member who is selected from as next group:

(a) a kind of polynucleotide, the polypeptide of this polynucleotide encoding shown in SEQ ID NO:2;

(b) a kind of polynucleotide, this polynucleotide encoding comprise peptide more than 1 to 329 amino acids of SEQ ID NO:2;

(c) a kind of polynucleotide, this polynucleotide encoding comprise peptide more than 1 to 264 amino acids of SEQ ID NO:2;

(d) a kind of polynucleotide, this polynucleotide encoding comprise peptide more than 215 to 329 amino acids of SEQ ID NO:2;

(e) a kind of polynucleotide, this polynucleotide encoding comprise peptide more than 215 to 264 amino acids of SEQ ID NO:2;

(f) a kind of polynucleotide, this polynucleotide can with (a), (b), (c), (d) or multi-nucleotide hybrid (e), and and they have at least 70% homogeny.

(h) a kind of (a), (b), (c), (d), (e) or the polynucleotide passage of polynucleotide (f).

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 2, the polypeptide of this polynucleotide encoding shown in SEQ ID NO:2.

6. the polynucleotide of claim 2, this polynucleotide encoding comprises the polypeptide of 1 to 329 amino acids shown in SEQ IDNO:2.

7. the polynucleotide of claim 2, this polynucleotide encoding comprises the polypeptide of 1 to 264 amino acids shown in SEQ IDNO:2.

8. the polynucleotide of claim 2, this polynucleotide encoding comprises the polypeptide of 215 to 329 amino acids shown in SEQ IDNO:2.

9. the polynucleotide of claim 2, this polynucleotide encoding comprises the polypeptide of 215 to 264 amino acids shown in SEQ IDNO:2.

10. isolating polynucleotide, it comprises the member who is selected from as next group:

(a) a kind of polynucleotide, a kind of mature polypeptide of this polynucleotide encoding, this peptide species have by be included in the ATCC preserving number _ _ in the DNA aminoacid sequence of expressing;

(b) a kind of polynucleotide, this polynucleotide can with the multi-nucleotide hybrid of (a), and and its have at least 70% homogeny.

(c) polynucleotide passage of a kind of (a) or polynucleotide (b).

11. the polynucleotide of claim 1, this polynucleotide have the sequence shown in SEQ ID NO:1.

12. the polynucleotide of claim 1, this polynucleotide comprise 321 Nucleotide to 1248 Nucleotide shown in SEQ ID NO:1.

13. the polynucleotide of claim 1, this polynucleotide comprise 402 Nucleotide to 1248 Nucleotide shown in SEQ ID NO:1.

14. the polynucleotide of claim 1, this polynucleotide comprise 1100 Nucleotide to 1248 Nucleotide shown in SEQ ID NO:1.

15. a carrier, this carrier comprises the DNA of claim 2.

16. one kind by the carrier conversion of claim 15 or the host cell of transfection.

17. a method of producing polypeptide, this method are included in the polypeptide of expressing in the host cell of claim 16 by said dna encoding.

18. the method for the cell that a generation can express polypeptide, this method comprises that the carrier pair cell with claim 15 carries out the genetically engineered operation.

19. a peptide species, this peptide species comprises the member who is selected from as next group:

(a) a kind of polypeptide with deduced amino acid of SEQ ID NO:2;

(b) a kind of polypeptide that comprises 1 to 329 amino acids shown in SEQ ID NO:2;

(c) a kind of polypeptide that comprises 215 to 329 amino acids shown in SEQ ID NO:2;

(d) a kind of polypeptide that comprises 215 to 264 amino acids shown in SEQ ID NO:2;

(e) a kind of polypeptide that comprises 1 to 264 amino acids shown in SEQ ID NO:2;

(f) (a), (b), (c), fragment, analogue and the derivative of peptide (d) or (e); With

(g) a kind of by the ATCC preserving number _ _ cDNA encoded polypeptides and its fragment, analogue and derivative.

20. the polypeptide of claim 19, this peptide species comprise 215 amino acids to 264 amino acids of SEQ ID NO:2.

21. the antibody of peptide more than the claim 19.

22. the activation of peptide more than a compound, this compound inhibition claim 19.

23. peptide more than a compound, this compound activation claim 19.

24. a treatment needs the patient's of TGF α-HII method, this method comprises: to the polypeptide of the claim 19 of patient's administering therapeutic significant quantity.

25. a method for the treatment of the patient who need to suppress TGF α-HII, this method comprises: to the compound of the claim 22 of patient's administering therapeutic significant quantity.

26. the method for claim 24, the polypeptide of wherein said treatment significant quantity be by the patient being provided the DNA of the said polypeptide of coding, and express in vivo that said polypeptide uses.

27. an evaluation is as the method for the activated compound of stimulant of the polypeptide of claim 19, this method comprises:

The reaction mixture that will comprise the cell type of expressing TGF α-HII acceptor contacts with compound to be screened; With

Whether determine whether this compound produces signal from said acceptor, be effective stimulant so that identify this compound.

28. an evaluation is as the method for the activated compound of antagonist of the polypeptide of claim 19, this method comprises:

The reaction mixture that will comprise the cell type of expressing TGF α-HII acceptor contacts with compound to be screened; With

With said compound in conjunction with after, determine not have the signal that produces by said acceptor whether be effective antagonist so that identify this compound.

29. one kind diagnoses the illness or to the method for the susceptibility of disease, this method comprises:

Sudden change in the polynucleotide of mensuration claim 1.

30. a diagnostic method, this method comprises:

Analysis derives from the polypeptide that whether has claim 19 in the sample of host cell.

Images