CN1230993A - Expression vectors, cells, and methods for preparing thrombopoietin polypeptides - Google Patents

Abstract

Thrombopoietin polypeptides and vectors, cells, and methods for making the polypeptides are disclosed. The polypeptides are characterized by an amino acid backbone consisting of C-X-B, wherein C is a human thrombopoietin cytokine domain polypeptide; X is a peptide bond or a linker consisting of one or two amino acid residues, subject to the limitation that X, alone or in combination with C or B, does not provide a dibasic amino acid pair; and B is a polypeptide consisting of residues 1 to y of SEQ ID NO:3, wherein y is an integer from 5 to 18, and wherein up to 35 % of the amino acid residues of B are individually replaced by other amino acid residues. The polypeptides are useful with methods of stimulating proliferation or development of hematopoietic cells in vitro and in vivo.

Description

Expression vector, cell and the method for preparation thrombopoietin polypeptides

Background of invention

Hemopoietic is that multipotential stem cell is grown and broken up hemocytoblastic process in the marrow.This process relates to polypeptide growth factor (cytokine) complex interactions, and wherein polypeptide growth factor works by the membrane-bound receptor on the target cell.The cytokine effect produces the special cells factor that common kind is special and/or the stage is special and replys, and causes cell proliferation and differentiation.The complicated coordinative role of the cytokine that need work with suitable order from the unicellular type of stem cell such as hematoblastic growth.

Known cytokine comprises interleukin-, as IL-1, and IL-2, IL-3, IL-6, IL-8 etc.; And G CFS, as G-CSF, M-CSF, GM-CSF, erythropoietin (EPO) etc.Usually, interleukin-is as the medium of immunity and inflammatory reaction.G CFS stimulates bone marrow-derived cells propagation, activates sophisticated white corpuscle, and forms the indispensable part that the host replys inflammation, infection and immune attack.

Various cytokines have been developed as treatment reagent.For example, stimulate the erythropoietin of red blood cell development to be used for the treatment of the anemia that renal failure causes.Several G CFSs have been used for combining with cancer chemotherapy and have added quick-recovery patient immunity system.Interleukin II, alpha-leukocyte are situated between plain and the C-interleukin-is used for some treatment for cancer.Stimulating megakaryocyte generates and thrombopoietic active substance has obtained identifying and being called " thrombopoietin " in the literature (in the recent period by Mcdonald in thrombopoietic animal body fluid, experimental hematology 16:201-205,1988 and McDonald, U.S. children hemooncology magazine 14:8-21,1992 summaries).The work of purifying and this active substance of evaluation causes and cell Mpl receptors bind and stimulating megakaryocyte generation and thrombopoietic proteic clone.See de Sauvage etc., natural 369:533-538,1994; Lok etc., natural 369:565-568,1994; Kaushansky etc., natural 369:568-571,1994; Wendling etc., natural 369:571-574,1994; Bartley etc., cell 77:1117-1124,1994; With the open WO 95/21920 of WIPO.This Mpl receptors bind cytokine is called thrombopoietin.

Amino acid sequence analysis shows that sophisticated people TPO is that amino-acid residue 1 (Ser) from SEQ ID NO:2 extends to amino-acid residue 332 (Gly).TPO is subjected to proteolysis easily and has obtained separating (de Sauvage etc., natural 369:533-538,1994 with allos or degraded form; Bartley etc., cell 77:1117-1124,1994).The small molecules type of having found 25kD has an activity (Bartley etc. external, ibid), and 153 amino acid (deSauvage etc. that have report to recombinate, ibid) and 174 amino acid (Bartley etc., ibid) people TPO polypeptide has an activity external, encoding, (Bartley etc., ibid) expression product of the total length people cDNA of initial translation product also has an activity external to 353 amino acid.

This area needs the method for a large amount of High-efficient Production thrombopoietins; Also need in eukaryotic microorganisms such as yeast, produce the method for thrombopoietin; Further need the method for the thrombopoietin of effectively production lower molecular weight form, the thrombopoietin of lower molecular weight form is compared with full-length molecule and more is not subject to proteolysis.The present invention satisfies these requirements and has other relevant advantage.

Summary of the invention

On the one hand, the invention provides the expression vector that can in eukaryotic host cell, duplicate.Carrier comprises the following element that is operatively connected: (a) transcripting promoter; (b) the 1st segment DNA fragment of coding secretion leading peptide; (c) coding contains the 2nd segment DNA fragment of thrombopoietin (TPO) polypeptide of C-X-B, and wherein C is a human thrombopoietin cytokine structural domain polypeptide; X is peptide bond or the joint that contains 1 or 2 amino-acid residue, and restricted condition is that X does not provide two basic aminoacidss right when combining separately or with C or B; And B is the polypeptide that comprises the residue 1 to y of SEQ ID NO:3, wherein y be from 5 to 18 integer and the B at the most 35% amino-acid residue can be substituted by other amino-acid residue respectively; Reach (d) transcription terminator.In an embodiment of invention, expression vector can duplicate in yeast.In another embodiment, the secretion leading peptide is the alpha factor secretion leading peptide of yeast saccharomyces cerevisiae.In another embodiment, B does not comprise the Arg-Arg dipeptides.In other embodiments, the 4th of B the residue is Thr or Asp; Y be at least 10 and the 10th residue of B be Arg or Glu; And y be at least 14 and the 14th residue of B be Val or Ala.In another embodiment, y is 14 at least, and the 4th residue of B is Thr or Asp, and the 10th residue of B is Arg or Glu, and the 14th residue of B is Val or ala.In other embodiments, X is peptide bond or single amino acids residue.

Providing on the other hand of invention comprises the as above cultivation eukaryotic cell of disclosed expression vector, wherein the synthetic justacrine TPO polypeptide of cell.Cell is a yeast cell in preferred embodiments.

The third aspect of invention provides to contain the thrombopoietin polypeptides that the C-X-B amino acid backbone is a feature, and wherein C is a human thrombopoietin cytokine structural domain polypeptide; X is peptide bond or the joint that contains 1 or 2 amino-acid residue, and restricted condition is that x does not provide two basic aminoacidss right when combining separately or with C or B; And B is the polypeptide that comprises the residue 1 to y of SEQ ID NO:3, and wherein y is from 5 to 18 integer, and wherein among the B at the most 35% residue can be substituted by other amino-acid residue respectively.

The fourth aspect of invention, the method of producing the TPO polypeptide is provided, comprise and cultivating with the as above eukaryotic host cell of disclosed expression vector transfection or conversion, the 1st section of having connected in the carrier and the 2nd segment DNA fragment by host cell expression with synthetic TPO polypeptide, and recovery TPO polypeptide.

The 5th aspect of invention discloses a kind of method that increases platelet count in the Mammals, comprises to administration and pharmaceutically acceptable carrier-bound as above disclosed TPO polypeptide.

With reference to after the following detailed description, will be appreciated that these or others of the present invention.Detailed Description Of The Invention

Before describing the present invention in detail, define some term used herein earlier:

Term " allelic variation " is used to refer to the gene transformation form that produces by sudden change at this, or by the polypeptide of the variation of mutator gene coding.Transgenation can be (no change in encoded polypeptides) of keeping silent maybe can encode polypeptide of vicissitudinous aminoacid sequence.

" expression vector " is segmental wire or the ring-shaped DNA molecule that comprises the desired polypeptides of encoding, and wherein encode fragment is operably connected with other fragment that is used to transcribe.Such fragment comprises promotor and terminator sequence.Expression vector also can comprise one or more replication orgin, one or more selective markers, enhanser, polyadenylation signal etc.Expression vector is derived from plasmid or viral DNA usually, perhaps may comprise the two element.Term " is operably connected " and is meant for intended purposes makes it coordinative role with arrangement of fragments, for example transcribe from promotor begin and through encode fragment to terminator.Expression vector is can be in host living beings independently duplicated or duplicate by being integrated into host genome.

Term " is characterised in that " it is the restricted condition that is used to refer to feature or element.For example, peptide characteristic is, the amino acid backbone of giving sequence comprises cited aminoacid sequence but also comprises other amino-acid residue.And this polypeptide can also comprise sugar chain or other posttranslational modification.

" secretion leading peptide " is the polypeptide that guidance and help albumen transmits through the secretory host cell approach.The secretion leading peptide is also referred to as presequence sometimes.Secretion leading peptide feature is the N-terminal of hydrophobic amino acid core also common (but not being uniquely) at new synthetic proteins.The secretion leading peptide through be everlasting between the secretory phase for 1 time or repeatedly cutting incident in excise from maturation protein.These secretion leading peptides comprise the processing site, can excise from maturation protein when making the secretion leading peptide through Secretory Pathway.

" promotor " is the Gene Partial of RNA polymerase combination and the synthetic zero position of mRNA.

" thrombopoietin " (or " TPO ") is protein, it is characterized in that, the Mpl acceptor of energy specific combination system mutually of the same race also stimulates thrombocyte generation in the body.In normal experimental animal, in 10 days after beginning medication every day, TPO can improve platelet levels 100% or more.Total length TPO comprises N-terminal cytokine structural domain and C-terminal (" C-end ") structural domain.With reference to SEQ ID NO:2, the cytokine structural domain is positioned between the 7th and the 151st half Guang amino-acid residue.

Term " thrombopoietin polypeptides " comprises and contains total length thrombopoietin molecule and its biologically-active moiety that biologically-active moiety is the fragment of the thrombopoietin of the qualitative biological activity of the complete molecule of performance (receptors bind and body internal stimulus thrombocyte generate).

The representative cDNA of SEQ ID NO:1 presentation code total length human thrombopoietin.Those skilled in the art will recognize that there are allelic variation in the DNA of SEQ ID NO:1 and the single allelotrope and the expection of its amino acid sequence coded (SEQ ID NO:2) representative TPO gene.The allelic variation of SEQ ID NO:1 can be by from the cell by Different Individual preparation, and clone and institute's DCRP checked order obtains in tissue or the nucleic acid.

As used herein, term " thrombopoietin cytokine structural domain polypeptide " is meant this core polypeptide (respective regions of the allelic variation of residue 7-151 or SEQ ID NO:2 and SEQ ID NO:2), may further include short N-end (as the residue 1-6 of SEQ ID NO:2) and/or the C-end (for example, the residue 152 of SEQ ID NO:2) extend, this extension is the essential biological activity of saboteur not.In extending, these allow considerable sequence variations.The C-terminal structural domain of people TPO is that the residue 155 (Ala) from SEQ ID NO:2 extends to residue 332 (Gly).This structural domain comprises the glycosylation site that potential O-is connected with N-.Can delete this structural domain wholly or in part and incomplete loss of biological activity.These two structural domains by Arg-Arg dipeptides (residue 153 to 154 of SEQ ID NO:2) separately.Though inferring this dipeptides is that (de Sauvage etc. for example, ibid), the assignee of the present invention carries out studies show that tangible cutting does not take place in this site for the processing site of TPO ripening period cutting.

As used herein, phrase " part in thrombopoietin C-end structure territory " comprises that 1 amino acid in TPOC-end structure territory is to the amino acid that comprises complete TPO C-end structure territory.Usually, the part in C-end structure territory is the continuous fragment in naturally occurring TPO C-end structure territory, the 1st amino-acid residue that contains corresponding complete TPO C-end structure territory is its 1st (N-terminal) amino-acid residue the amino-acid residue of the residue 155 of SEQ ID NO:2 (for example, corresponding to).The part preferred length in the C-end structure territory that the present invention is used is 5 to 18 amino-acid residues, and more preferably length is at least 9 residues, and most preferably length is 14 to 18 residues.

The invention provides the modification method of preparation thrombopoietin polypeptides, and useful expression vector and cell in the method.The present invention is partly based on following discovery: some amino acid changes the secretion that causes eukaryotic host cell in the TPO polypeptide increases.In the present invention, the secretion that improves the TPO polypeptide by the Arg-Arg dipeptides that in disappearance or the alternative natural molecule cytokine structural domain and C-terminal structural domain is separated.Though as inferring in the document, the TPO polypeptide not this Arg-Arg dipeptides place cutting (as, de Sauvage etc. ibid), find this dipeptides secretion inhibitor.So the invention provides the TPO polypeptide, the two basic aminoacidss that it is characterized in that eliminating the C-terminal that the cytokine structural domain is right after are right.Therefore these TPO peptide characteristics are the C-X-B structure, wherein C is a human thrombopoietin cytokine structural domain polypeptide, X is peptide bond or the joint that contains 1 or 2 amino-acid residue, and B is from the part in thrombopoietin C-end structure territory, and restricted condition is that X does not form a pair of alkaline amino acid residue separately or with C or B.

Therefore in albumen of the present invention, the two alkaline sequence (for example residue 153 and 154 of SEQ ID NO:2) that does not exist the junction in the cytokine of wild-type thrombopoietin and C-end structure territory to exist.Preferably, there is not this two alkaline sequence in other position of molecule, especially in B yet.

TPO polypeptide of the present invention comprises naturally occurring people TPO cytokine structural domain aminoacid sequence the most commonly, and this sequence links to each other by the part of peptide bond with the C-end structure territory of Mammals (preferably people) thrombopoietin.In a preferred embodiment of the invention, B comprises 5 to 18 continuous residues in the C-end structure territory of the n terminal residue that originates in C-end structure territory, and wherein nearly 35% amino-acid residue can be alternative by other amino-acid residue respectively among the B.TPO cytokine structural domain also can comprise 1 to about 15, preferably is no more than 10, more preferably is no more than 7 amino acid replacement.Amino acid replacement occurs in non-key amino-acid residue place, and promptly those residues substitute the not serious residue that influences molecular biological activity.The method of identifying non-key amino-acid residue is known in the art, and comprises alanine scanning mutagenesis (Cunningham and Wells, science 244:1081-1085,1989; Bass etc., institute of NAS reports 88:4498-4502, and 1991), wherein import single alanine mutation, and measure the biological activity of gained mutating molecule at each residue place of molecule.Disclosed as Reidhaar-Olson and Sauer (science 241:53-57,1988) or Bowie and Sauer (institute of NAS reports 86:2152-2156,1989), can assess of the influence of a plurality of amino acid replacements to protein-active.Briefly, these authors disclose following method: 2 of random tests simultaneously or a plurality of site and screening have the polypeptide of function in polypeptide, and the polypeptide to mutagenesis carries out sequencing to determine the admissible alternate range of each site then.Other operable method comprises that phage shows (for example, Lowman etc., biological chemistry 30:10832-10837,1991; Ladner etc., U.S. Patent number 5,223,409; Huse, the open WO 92/06204 of WIPO) and mutagenesis (Derbyshire etc., gene 46:145,1986 of area guidance; Ner etc., DNA 7:127,1988).Except the biological activity of test mutagenesis polypeptide, also the expression vector of coded polypeptide can be transformed into culturing cell influences the polypeptide excretory to detect sudden change.

Can design to keep or to strengthen this part molecule the amino acid replacement with respect to naturally occurring TPO sequence among the B the useful influence of excretory.Preferred substituting comprises with the alternative charged residue of uncharged residue.Other preferably substitutes and comprises with the alternative Xie Ansuan (the 168th residue of SEQ IDNO:2) of L-Ala, with L-glutamic acid place of arginine (the 164th residue of SEQ ID NO:2), and substitute Threonine (the 158th residue of SEQ ID NO:2) with aspartic acid.These substitute and can form separately or with any combining form.Preferably compare to substitute and be no more than residue among 25% the B with corresponding naturally occurring sequence.SEQ ID NO:4 to SEQ IDNO:7 represents typical C end structure territory sequence.

Do not accept opinion and limit, the Thr-Thr dipeptides can provide O to connect the attachment site of sugar chain.Therefore in one embodiment of the invention, B comprises a Thr-Thr dipeptides.In particularly preferred embodiments, 5 amino-acid residues of the N-terminal of B are Ala-Pro-Pro-Thr-Thr (SEQ ID NO:8).

Can further improve secretion level by increasing N connection carbohydrate attachment site (Asn-X-Ser/Thr).Yet the existence of such sequence may cause unwanted high-glycosylation in some host cell (as yeast saccharomyces cerevisiae).

Those skilled in the art will recognize that some amino acid whose substituting may be that host cell is specific.So should preferably detect this alternate influence in the host cell type that is used for synthetic polypeptide.

The expression vector that can duplicate in eukaryotic host cell of the present invention comprises transcripting promoter and transcription terminator, and they are operably connected with first segment DNA fragment of aforesaid coding secretion peptide and the second segment DNA fragment of coding TPO polypeptide.Thereby second the segment DNA fragment coding contain the TPO polypeptide of C-X-B, C wherein, X and B are as defined above.In the preferred embodiment of invention, B is the polypeptide that contains the residue 1 to y of SEQ ID NO:3, and wherein y is from 5 to 18 a integer and wherein the B at the most 35%, and preferably being no more than 25% described residue can be substituted by other amino-acid residue respectively.

" coding contains the dna fragmentation of the TPO polypeptide of C-X-B " is meant that the reading direction of the newborn polypeptide that comprises repeat element is to C-terminal from N-terminal.So term " coding " is used to refer to the direct product that dna fragmentation is transcribed and translated.Those skilled in the art are to be understood that this peptide species can wherein can add annexing ingredient such as sugar chain to it through the translation post-treatment.The precise nature of this posttranslational modification can partly be determined by the host cell type of synthetic polypeptide.

Disclosed as the open WO 95/21920 of WIPO, can clone mouse and people TPODNAs, quote in full this open file as a reference at this.The plasmid pZGmpl-1081 that will contain mouse TPO dna sequence dna is kept at American type culture collection according to the Budapest clause of treaty on February 14th, 1994,12301 Parklaw Drive, and Rockville, MD also obtains preserving number 69566.The plasmid pZGmpl-124 that contains people TPO cDNA is kept at American type culture collection as intestinal bacteria DH10b transformant on May 4th, 1994, and preserving number is 69615.These mouse and people cDNAs can be used as probe to separate the DNAs of other coding TPO, comprise genomic dna s, allelic variation and from the DNAs of other kind.

But being used for suitable host cell of the present invention comprises and anyly can express the allogeneic dna sequence DNA incubation growth and have the eukaryotic cell type of Secretory Pathway through operation.

In order to instruct the TPO polypeptide to enter the Secretory Pathway of host cell, the dna sequence dna of coding being secreted leading peptide is used in combination with the dna sequence dna of coding TPO polypeptide.The secretion leading peptide can be the leading peptide of TPO or the leading peptide of other secretory protein such as tissue type plasminogen activator (t-PA) or yeast saccharomyces cerevisiae joint pheromone α-factor.When using allos secretion leading peptide to combine with the TPO polypeptide, corresponding DNA fragments is connected in the correct reading frame, makes the fragment coding fusion rotein of connection.Typically adding the proteolysis cleavage site in the junction of secretion leading peptide and TPO polypeptide excises from the TPO polypeptide so that will secrete leading peptide between the secretory phase.Yet, one skilled in the art will realize that can reclaim fusion rotein also processes subsequently to discharge the TPO polypeptide.

The cell of yeast cell, especially yeast belong is the preferred host of the present invention.Production and cultivation fee that yeast cell is used for human consumption's product for a long time are not high relatively.Disclose the method for also therefrom producing recombinant protein, for example seen Kawasaki, U.S. Patent number 4,599,311 with the foreign DNA transformed yeast cell; Kawasaki etc., U.S. Patent number 4,931,373; Brake, U.S. Patent number 4,870,008; Welch etc., U.S. Patent number 5,037,743; With Murray etc., U.S. Patent number 4,845,075 is incorporated herein by reference.Cell transformed is screened by the phenotype of being determined by selective marker, and selective marker is resistance or the viability when lacking specific nutrition (as leucine) normally.Being used for zymic preferred vector system is as (U.S. Patent number 4,931,373) disclosed POTI carrier systems such as Kauasaki, and this carrier system is by screening transformant at the growing state that contains on the dextrose culture-medium.Be used for suitable promotor of zymic and terminator comprise from the promotor of glycolytic ferment gene and terminator (see, as Kawasaki, U.S. Patent number 4,599,311; Kingsman etc., U.S. Patent number 4,615,974; And Bitter, U.S. Patent number 4,977,092 is incorporated herein by reference) and the promotor and the terminator of alcohol dehydrogenase gene.Other sees U.S. Patent number 4,990,446; 5,063,154; 5,139,936 and 4,661,454, be incorporated herein by reference.Be known in the art and be used for other zymic conversion system, other yeast comprises multiple-shaped nuohan inferior yeast, schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, pichia pastoris phaff, Pichia guilliermondii and maltose candiyeast.See for example Gleeson etc., microbial genetics magazine 132:3459-3465,1986; Cregg, U.S. Patent number 4,882,279; With Stroman etc., U.S. Patent number 4,879,231.

Should preferably use choosing to come the host strain of high-level secretory TPO polypeptide.By ordinary method such as uviolizing or use as the chemomorphosis of ethylmethane sulfonate or nitrosoguanidine is suitable for fermenting to genotype and albumen synthetic parent plant carries out mutagenesis.Detect the protein excretion level of screening survivaling cell with conventional sense method such as filter membrane bacterium colony method.In filter membrane bacterium colony analytical method, cover cell, in western blot analysis, bacterium colony is surveyed subsequently with antibody with nitrocellulose filter.Also can use other to detect as active the detection.

Other fungal cell also is suitable for doing host cell.For example, according to Mcknight etc., U.S. Patent number 4,935,349 method can be utilized the Aspergillus cell, is incorporated herein by reference.Sumino etc., U.S. Patent number 5,162 discloses the method that transforms Acremoniumchrysogenum in 228, is incorporated herein by reference.Lambowitz, U.S. Patent number 4,486 discloses the method that transforms the Neurospora cell in 533, is incorporated herein by reference.

The method that foreign DNA is imported mammalian host cell comprises transfection (Wigler etc., cell 14:725,1978 that calcium phosphate mediates; Corsaro and Pearson, somatic cell genetics 7:603,1981:Graham and Van der Eb, virusology 52:456,1973), electroporation (Neumann etc., EMBO J.1:841-845,1982), the transfection of DEAE-dextran mediation (editor such as Ausubel, the modern molecular biology working method, JohnWiley and Sons company, NY, 1987) and the transfection (Hawley-Nelson etc. of positively charged ion lipid mediation, focus 15:73-79,1993), be incorporated herein by reference.Disclose synthetic recombinant protein in cultivating mammalian cell, for example seen Levinson etc., U.S. Patent number 4,713,339; Hagen etc., U.S. Patent number 4,784,950; Palmiter etc., U.S. Patent number 4,579,821; Mulvihill etc., U.S. Patent number 5,486,471; Foster etc., U.S. Patent number 5,358,932; With Mulvihill etc., U.S. Patent number 5,385,831 is incorporated herein by reference.The preferred mammalian cell of cultivating comprises COS-1 (ATCC NO.CRL 1650), COS-7 (ATCC No.CRL 1651), BHK (ATCCNo.CRL 1632), BHK570 (ATCC No.CRL 10314), 293 (ATCC No.CRL1573; Graham etc., viral genetics magazine 36:59-72,1977) and (CHO-KI for example of Chinese hamster ovary cell system; ATCC No.CCL61).Other suitable clone is known in the art and can be from public depositary institution such as American type culture collection, Rockville, and Maryland obtains.Usually, preferred strong transcripting promoter is for example from the promotor of SV-40 or cytomegalovirus.See for example U.S. Patent number 4,956,288.Other suitable promotor comprises promotor from metallothionein gene (U.S. Patent number 4,579,821 and 4,601,978, be incorporated herein by reference) and adenovirus major late promoter.

Usually screen the cultivation mammalian cell that inserts foreign DNA with medicament selection.These cells so-called " transfectant ".The cell of having cultivated and goal gene can have been passed to the offspring under the situation that selective reagents exists is called " stable transfectant ".Preferred selective marker is coding has resistance to antibiotic neomycin a gene.When existing, Xin Meisu type medicine such as G-418 etc. screen.Selective system also can be used for improving the destination gene expression level, and this process is called " amplification ".Amplification is by cultivating transfectant under the situation about existing at low-level selective reagents, increases the selective reagents amount then and screens the cell of the synthetic quiding gene product of high level and carry out.Preferred amplification selective marker is to give the Tetrahydrofolate dehydrogenase of ammonia first psychopsid resistance.Also can use other medicines resistant gene (for example, hygromycin resistance, multiple drug resistance, tetracycline Transacetylase).

Also can use other higher eucaryotic cells as the host, comprise insect cell, vegetable cell and birds cell.Guarino etc., U.S. Patent number 5,162,222; Bang etc., U.S. Patent number 4,775,624; Openly disclose the conversion of insect cell and synthesizing of middle foreign protein thereof among the WO 94/06463 with WIPO, be incorporated herein by reference.At Sinkar etc., bio-science magazine (Bangalore) 11:47-58, in 1987 to using Agrobacterium rhizogenes to discuss as carrier expressing gene in vegetable cell.

Grow to cultivate in the substratum of required composition and transform or the host cell of transfection containing nutrition and other selected host cell according to conventional methods.The various appropriate culture medium that comprise defined medium and complex medium are known in the art and comprise carbon source, nitrogenous source, indispensable amino acid, VITAMIN and mineral substance usually.If desired, substratum also comprises the composition such as somatomedin or blood plasma.Growth medium will or lack by medicament selection must nutrition screen and contain the cell that external source imports DNA, and it is additional that wherein essential nutrition has by expression vector or cotransfection is gone into the selective marker of host cell.

With method well known in the art as according to the protein affinity purification of proteinic size, electric charge, solubility and further feature with separate to come selective recovery TPO polypeptide prepared in accordance with the present invention.When polypeptide is synthetic in the mammalian cell of cultivating, should be preferably in serum free medium culturing cell with the proteic quantity of limit pollution.Collect and the fractional separation substratum.Preferred fractionation method comprises affinity chromatography, such as the affinity chromatography on solid phase Mpl receptor protein or its ligand binding moiety or by using the affinity chromatography of affine sign (for example poly Histidine, P material or other antibody or other specific binding agents polypeptide or albumen).Can between target protein and affine sign, provide special cleavage site.Also can use other chromatography method such as cation-exchange chromatography, anion-exchange chromatography and hydrophobic interaction chromatography.

TPO polypeptide prepared in accordance with the present invention can use aspect needs strengthen the treatment of cell proliferation in the marrow, uses during as treatment such as the cytopenia that caused by aplastic anemia, aplastic bone marrow syndrome, chemotherapy or congenital hemocytopenia.It is useful when the TPO polypeptide also generates as the treatment thrombocytopenia to the raising thrombocyte.Thrombopenia is relevant with various disease and clinical setting, and these situations may act on the generation disease alone or synergistically.That the minimizing of platelet count can be synthetic damaged by thrombocyte for example, thrombocyte distributes is unusual, because the dilution that a large amount of blood transfusion causes is lost or blood platelet disorders are destroyed and caused.For example, used chemotherapeutics may suppress thrombocyte cells whose development in early stage in the marrow in the cancer therapy, and the thrombopenia that causes has limited chemotherapy and feasible the needs transfuses blood.In addition, some malignant tumour can be damaged the synthetic and thrombocyte distribution of thrombocyte.The radiation therapy that is used to kill malignant cell also kills thrombocyte cell in early stage.Also can cause thrombopenia by the various thrombocyte autoimmune disorders of medicine, newborn infant's alloimmunization or platelet transfusion alloimmunization inductive.The TPO polypeptide can reduce or eliminate the blood transfusion demand, thereby reduces the autoimmune generation of thrombocyte.Hematoblastic abnormal destruction may be derived from: in vascular transplantation or wounded tissue, platelet consumption increases (1); Or (2) and for example drug-induced thrombopenia, idiopathic thrombocytopenic purpure (ITP), autoimmune disease, blood are disorderly as leukemia and lymphoma or relate to the relevant immune mechanism of metastatic cancer of marrow.Other indication of TPO comprises aplastic anemia and infects the drug-induced bone marrow depression that causes by for example chemotherapy or with AZT treatment HIV.

Thrombopenia shows as amount of bleeding to be increased as muzzle district or gastrointestinal tract mucous hemorrhage, and the sepage of wound, ulcer or injection site.

For pharmaceutical use, it is parenteral to pass through to prepare the TPO polypeptide according to conventional methods, especially vein or subcutaneous route transmission.Intravenous administration will carry out during several hrs 1 by a large amount of injections or infusion.Usually, formula of medicine comprises and bonded TPO polypeptide such as pharmaceutically acceptable carrier such as salt solution, buffer saline, 5% D/W.Prescription can further comprise a kind or multiple vehicle, sanitas, and solubility promoter, buffer reagent, albumin is to prevent that the bottle surface protein from losing etc.In addition, the TPO polypeptide can with other cytokine, the especially early stage function cells factor such as STEM CELL FACTOR, IL-3, IL-6, IL-11 or GM-CSF combination.When using this combined treatment, cytokine can be in single prescription in conjunction with or use with the prescription that separates.Compound method is well-known and at for example Remington pharmaceutical science in this area, and Gennaro edit, Mack publishing company, and Easton PA discloses in 1990, is incorporated herein by reference.The therapeutic dose of TPO is generally every kilogram of patient body weight 0.1 microgram to 100 microgram every day, preferably every day the 0.5-50 microgram.Exact dosage desired is treated the character and the decisions such as seriousness and patient characteristics of disease according to acceptable standard, consideration by the clinicist.In some cases, susceptibility increases or when needing extended treatment, dosage range can be appointed as 0.1-20 microgram/kg body weight every day as showing as subject patient.Dosage fixes within those of ordinary skills' level really.The TPO polypeptide reaches after chemotherapy or bone marrow transplantation usually to be used in period of 28 days or reaches greater than 20,000/ cubic millimeters until platelet count, is preferably more than 50,000/ cubic millimeters.More at large, use the TPO polypeptide about 1 week, common 1 to 3 day.Usually, the treatment significant quantity of TPO polypeptide is to be enough to cause the amount that obviously increases cell proliferation in early stage of lymph or marrow and/or differentiation clinically, and this will show as the increase of mature cell (for example thrombocyte or neutrophilic leukocyte) cyclical level.Therefore the treatment of thrombocyte imbalance will last till that platelet count reaches at least 20,000/ cubic millimeter, preferably 50,000/ cubic millimeters.The TPO polypeptide also can with other cytokine such as IL-3, IL-6 and IL-11; STEM CELL FACTOR; Erythropoietin; G-CSF and GM-CSF are in conjunction with using.In the combined treatment scheme, other cytokine every day dosage normally: EPO ,≤150U/ kg body weight; GM-CSF, 5-15 microgram/kg body weight; IL-3,1-5 microgram/kg body weight; And G-CSF, 1-25 microgram/kg body weight.For example be applicable to the anemia patient that the EPO level is low with the EPO combined treatment.

The TPO polypeptide also is the useful tool of differentiation of in vitro study hematopoietic cell and growth, as is used to illustrate cytodifferentiation mechanism and definite mature cell kind system, and finds also to can be used as the proliferant agent in the cell cultures.

The TPO polypeptide also can in vitro as in autologous bone marrow is cultivated use.In brief, before chemotherapy, from the patient body, take out marrow and use the TPO polypeptide, optionally in conjunction with a kind or the processing of the various kinds of cell factor.After chemotherapy, treated marrow is put back in the patient body so that quicken the recovery of marrow then.In addition, the TPO polypeptide also can be used for marrow or the stripped amplification of peripheral blood original (PBPC) cell.Before chemotherapy, stimulate marrow that early stage cell in earlier stage is released into peripheral circulation with STEM CELL FACTOR (SCF) or G-CSF.Can from peripheral blood, collect and concentrate these cells in earlier stage, use a kind or multiple TPO polypeptide then, optionally in conjunction with a kind or the various kinds of cell factor, include but not limited to SCF, G-CSF, IL-3, GM-CSF, IL-6 or IL-11 handle in cultivation, make it differentiation and are proliferated into high-density megalokaryocyte culture, can put back in the patient body behind high dose chemotherapy then.

Further set forth the present invention by following non-limiting example.EXAMPLE Example 1

Make up that 2 expression vectors exist with the 153-154 position Arg-Arg dipeptides of SEQ ID NO:2 relatively or brachymemma people TPO polypeptide expression in the yeast saccharomyces cerevisiae when lacking.These 2 carriers are from plasmid pDPOT (ATCC#68001; At U.S. Patent number 5,128, open in 321).These two carriers comprise and contain yeast saccharomyces cerevisiae triosephosphate isomerase (TPI1) gene promoter (see U.S. Patent number 4,599,311, be hereby incorporated by), yeast saccharomyces cerevisiae MF α 1 presequence, the TPO expression cassette of TPO sequence and yeast saccharomyces cerevisiae TPI1 terminator.Carrier further comprises schizosaccharomyces pombe triosephosphate isomerase (POT) gene (allowing to screen) in containing the substratum of glucose, be used for the anti-penbritin gene and the Leu2-d selective marker of intestinal bacteria screening.The carrier pD85 coding wild-type people TPO sequence from the amino-acid residue 1 of SEQ ID NO:2 to residue 172 wherein substitutes residue 168 (Val) with Ala.The TPO 1-172 sequence of carrier pD79 coding variation wherein deletes 153 and 154 arginine residues codons, and substitutes Xie Ansuan codon (residue 168) with L-Ala codon (with reference to the amino acid position of SEQID NO:2).

Modify total length people TPO DNA with at 5 codons of the 5 ' terminal MF of adding α 1 presequence and in the 3 ' terminal terminator codon that adds by polymerase chain reaction (PCR).PCR uses primer ZC7623 (SEQ ID NO:9) and ZC 7627 (SEQ ID NO:10) to carry out.With Taq polysaccharase and about 20 nanogram template DNAs in 94 ℃, 1 minute; 53 ℃, 1 minute; 72 ℃, carried out 25 circulations and finish PCR in 1.5 minutes.The TPO sequence of separate modifying is 479bp Hind III-Xba I fragment and connects among the pUC18 with the same enzyme cutting.Gained plasmid called after pHB76.

TPO sequence among the pHB76 is modified to import Bbe I and SaI I site at the sequence 5 ' end and 3 in Codocyte factor structure territory ' end respectively.PCR uses primer ZC7868 (SEQ ID NO:11) and ZC7870 (SEQ ID NO:12) to carry out.With Taq polysaccharase and about 20 nanogram template DNAs, 95 ℃ earlier, 1 minute; 68 ℃ were carried out 1 circulation in 10 minutes, then 95 ℃ 1 minute; 68 ℃ were carried out 9 circulations in 4 minutes and finish PCR.Recovery TPO sequence is 482bp Hind III-EcoR I fragment and is connected among the pUC19 that cuts with same enzyme.Gained plasmid called after pTPOGN2.

Then by combining the TPO sequence that makes up variation with three part mode of connection and the pUC19 that digests with Hind III+Xba I from 457 bp Hind III-Sal I fragment of pTPOGN2 and the synthetic fragment that makes up from oligonucleotide ZC8488 (SEQ ID NO:13) and ZC8489 (SEQ IDNO:14).Gained plasmid called after pTPOGN6.The 539bp Hind III-Xba I fragment of from pTPOGN6, separating the TPO polypeptide of coding for alpha last several residues of factor propetide and brachymemma.With this fragment and pDPOT (through cutting of BamH I and alkaline phosphatase treatment), the 1230bp Bgl II-Hind III fragment that comprises TPI1 promotor and MF α 1 presequence is connected with four part mode of connection with the 680bp Xba I that contains the TPI1 terminator-BamH I fragment.Gained plasmid called after pD79.

By with pDPOT (through BamH I cutting and alkaline phosphatase treatment), 1230bp Bgl II-Hind III TP I 1-MF α 1 fragment, the 540bp fragment of the last several residues of coding for alpha factor propetide and the TPO polypeptide of brachymemma is connected the control plasmid of residue 1 to 172 that makes up coding people TPO (SEQ ID NO:2) with 680bp Xba I-BamH I TP I 1 terminator fragment with four part mode of connection.Gained plasmid called after pD85.

As disclosed in (institute of NAS report 75:1929-1933,1978) such as Hinnen, must be with plasmid pD79 and pD85 transformed saccharomyces cerevisiae bacterial strain JG134 (1[cir ° of MAT α Δ tpil ∷ URA3 ura3-52leu2-Δ 2pep4-Δ]).Screen transformant according to them at the energy for growth that contains on the substratum that glucose is unique carbon source.

Transformant is containing 1% yeast extract, and growth is about 60 hours in the liquid nutrient medium of 1% peptone and 5% glucose.By centrifugal from substratum isolated cell.Media samples (has been replenished 10% foetal calf serum with TPO dilution buffer liquid, 2 mmole glutamine, 1 mmole Sodium.alpha.-ketopropionate, 50 mcg/ml penicillin, 50 mcg/ml Streptomycin sulphates, 100 mcg/ml Xin Meisus, 0.00033% β-thin basic ethanol, the RPMI 1640 of 25 mmole Hepes) with dilution in 1: 100.

The BaF3 cell that uses expression vector with coding people mpl receptor (Vigon etc., institute of NAS report 89:5640-5644,1992) transfection is as target cell, detection TPO biological activity in the mitotic division assay method.BaF3 is from lymphoid cell line (Palacios and Steinmetz, cell 41:727-734,1985 before the interleukin 3 dependent form of mouse marrow; Mathey-Prevot etc., molecular cytobiology 6:4133-4135,1986). ³Under the situation that the H-thymus pyrimidine exists cell and given the test agent one are arised from 37 ℃ of placements 16 to 19 hours.By with the typical curve of people TPO relatively, to mixing cell DNA ³The H-thymus pyrimidine carries out quantitatively.The 10U/ milliliter is defined as the amount that obtains maximal stimulation one half in cell fission detects.Two result of experiment see Table 1.

Table 1

Experiment plasmid TPO (units per ml substratum)

1 pD85 40

pD79 740

Below the 2 pD85 boundaries

PD79 160 embodiment 2

Make up the plasmid of a series of coding TPO polypeptide, wherein the TPO polypeptide comprises the people TPO cytokine structural domain (residue 22 to 152 of SEQ IDNO:2) that is connected with the C-terminal fragment by peptide bond or Arg-Arg dipeptides.The structure of table 2 presentation code polypeptide: Arg-Arg represents that dipeptides exists (+) or disappearance (-); The segmental amino acid sequence number of C-terminal is with reference to SEQ ID NO:3.

Table 2

Plasmid Arg-Arg C-terminal sequence

pD117 - 1-5

pD119 - 1-9

pD121 - 1-13

pD123 - 1-18

pD125 + 1-18

With the pUC19 that is digested to line style through Hind III and Xba I, comprise the 457bpHind III-Sal I fragment (embodiment 1) of pTPOGN2 of sequence of encoding part alpha factor secretion leading peptide and groups of people TPO cytokine structural domain and Sal I-Xba I joint of making up from oligonucleotide ZC8486 (SEQ ID NO:15) and ZC8487 (SEQ ID NO:16) with three part mode of connection structure plasmid pTPOGN8.

Make up plasmid pD83:BamH I-digestion, the pDPOT of alkaline phosphatase treatment with following fragment with four part mode of connection; [pHB1054 comprises the yeast saccharomyces cerevisiae TPI1 promotor that is connected with people TPO cytokine structural domain encoding sequence in the pMVR1 plasmid main chain and alpha factor and secretes leading peptide and (be disclosed in U.S. Patent number 5 to comprise 1230bp Bgl II-Hind III fragment of the pHB105-4 of yeast saccharomyces cerevisiae TPI1 promotor and alpha factor secretion leading peptide, in 155,027)]; The 540bp Hind III of pTPOGN8-Xba I fragment, this fragment comprises the encoding sequence of a part of α-factor secretion leading peptide encoding sequence and TPO variant, and wherein the encoding sequence of TPO variant is included in the terminal cytokine structural domain that is connected with 18 residue polypeptide of SEQ ID NO:17 of its C-; And 680bp Xba I-BamH I yeast saccharomyces cerevisiae TPI1 terminator fragment.

At first the Bgl II of the pD83 by will comprising TPI1 terminator and carrier sequence with a pair of oligonucleotide shown in the table 3-EcoR I fragment is inserted pUC19 (cutting with Sal I and EcoR I) and is made up the plasmid shown in the table 2.As shown in table 3, gained plasmid called after pTPOMI1,2,3,4 and 5.To comprise the TPI1 promotor, MF α 1 secretion leading peptide, 2 Bgl I of 5 ' TPO coding region and carrier sequence-Sal I pD83 fragment is with the EcoR I-Bgl I fragment that comprises the carrier sequence and (comprise 3 ' TPO encoding sequence from pTPOMI1-5, TPI1 terminator and carrier sequence) Sal I-EcoR I fragment be connected to make up pD117 respectively, pD119, pD121, pD123 and pD125.

Table 3

The plasmid oligonucleotide

pTPOMI1 ZC10086(SEQ?ID?NO；18)

ZC10087(SEQ?ID?NO:19)

pTPOMI2 ZC10095(SEQ?ID?NO:20)

ZC10096(SEQ?ID?NO:21)

pTPOMI3 ZC10120(SEQ?ID?NO:22)

ZC10121(SEQ?ID?NO:23)

pTPOMI4 ZC10118(SEQ?ID?NO:24)

ZC10119(SEQ?ID?NO:25)

pTPOMI5 ZC10108(SEQ?ID?NO:26)

ZC10109(SEQ?ID?NO:27)

Plasmid is transformed among yeast saccharomyces cerevisiae JG134 or the M35.M35 is the JG134 deutero-that transforms from pD79 by the ultraviolet mutagenesis overnight culture.Cell is diluted at 1: 1000, and 50 microlitre diluents are coated on the YEPD (1% yeast extract, 2% peptone, 2%D-glucose, 0.004% VITAMIN B4,0.006% leucine).In the darkroom,, be placed in the lighttight box, and cultivated 2 days in 30 ℃ to dull and stereotyped uv irradiating 20 seconds.Then with every flat board replica plate on fresh YEPD plate, with the nitrocellulose filter covering and in 30 ℃ of overnight incubation.Remove nitrocellulose filter then and wash any adherent yeast cell off.Use sealing of 5% milk and mouse-anti-human T PO antibody among 1 * PBS to make the nitrocellulose filter colour developing by the standard protein engram technology.Shown that the bacterium colony of high-level secretory further detects by western blotting and activity detection in the first screening.The mutant of a called after GN35 continues to produce the activity doubly than the high 2-4 of parent strain.Obtain GN35 by transforming pD79 with 2 microns big miniplasmids that comprise prokaryotic cell prokaryocyte kalamycin resistance gene and yeast saccharomyces cerevisiae TPI1 gene.Screening has the transformant of resistance to 2 mg/ml G418 and cultivates in containing the YEPD of G418, then at YEPGGE (0.004% VITAMIN B4,0.006%L-leucine, 1% yeast extract, 0.4%D-semi-lactosi, 2% peptone, 3% glycerine, 1% ethanol) the middle cultivation.Screening can not be the cell of growing on the substratum of carbon source at glucose then, and this shows that the triose-phosphate isomerase gene on the pD79 loses.The cell called after M35 strain system of processing treatment.Transformant is containing 2% peptone, grows 70 hours under the ventilation condition in the liquid nutrient medium of 1% yeast extract and 5% glucose.Comprising plasmid pD79 and pD85 is control group.In addition, detect plasmid pHB109 (the people TPO cytokine of encoding separately structural domain) and pBJ118 (polypeptide that coding is identical with pD85 and pD125).

Disclosed as embodiment 1, collect and detect substratum.Detected result sees Table 4.

Table 4

Host range plasmid TPO (ng/ml)

JG134 pD117 26

pD119 51

pD121 53

pD123 35

pD125 3

pD79 70

pD85 3

Below the pHB109 boundary

pBJ118 8

M35 pD79 180 embodiment 3

Make up the plasmid based on pDPOT of a series of coding TPO polypeptide with conventional Protocols in Molecular Biology.The TPO polypeptide of each coding comprises the people TPO cytokine structural domain that links to each other with the C-terminal fragment that is derived from people TPO C-end structure territory through peptide bond.The C-terminal fragment sequence of these polypeptide is face table 5 as follows.Amino acid is represented with conventional one-letter symbol.

Table 5

Plasmid C-terminal structural domain

pD91 APPDTAVPSRTSLVLTLN

(SEQ?ID?NO:5)

pD93 APPDTAVPSETSLVLTLN

(SEQ?ID?NO:6)

pD95 APPTTAVPSETSLVLTLN

(SEQ?ID?NO:7)

Plasmid is transformed among the bacterial strain JG134, and as embodiment 2 disclosed cultivation transformants.Disclosed as embodiment 1, results and detection substratum.

The results are shown in Table 6

Table 6

Plasmid TPO (units per ml)

pDPOT 0

pD79 700

pD85 100

pD91 200

pD93 300

pD95 600

From above-mentioned aspect,, under the situation of not leaving the spirit and scope of the present invention, multiple modification can be arranged although should be appreciated that and described specific embodiments of the invention at this for illustrative purposes.Therefore, except that additional claim, the present invention is not limited by other.

Sequence table (1) essential information (ⅰ) applicant: ZymoGenetics, Inc.

1201?Eastlake?Avenue?East

Seattle,washington?98102

United State of America (ⅱ) denomination of invention: carrier, cell and method (ⅲ) sequence number of preparation thrombopoietin polypeptides: 27 (ⅳ) contact address

(A) addressee: ZymoGenetics, Inc.

(B) street: 1201 Eastlake Avenue East

(C) city: Seattle

(D) state name: WA

(E) country: the U.S.

(F) postcode: 98102 (ⅴ) computer-reader form

(A) media type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, version #1.25 (ⅵ) is application materials at present:

(A) application number:

(B) Date to Tender Notice of Readiness:

(C) classification: (ⅶ) lawyer/proxy's information

(A) title: Parker.Gary E.

(B) registration number: 31,648

(C) reference/number of recording: 95-34 (ⅶ) telecom information:

(A) phone: 206-442-6673

(B) fax: 206-442-6678 (2) is about information (ⅰ) sequence signature of SEQ ID NO:1

(A) length: 1062 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅱ) molecule type: cDNA (ⅸ) feature:

(A) title/key: mat-peptide

(B) position: 64..1062 (ⅸ) feature:

(A) title/key: sig-peptide

(B) position: 1..63 (ⅸ) feature:

(A) title/key: CDS

(B) position: 1..1062 (ⅹ ⅰ) sequence description: SEQ ID NO:1:ATG GAG CTG ACT GAA TTG CTC CTC GTG GTC ATG CTT CTC CTA ACT GCA48Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala-21-20-15-10AGG CTA ACG CTG TCC AGC CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC96Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val-5 15 10CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC144Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser

15 20 25CAG?TGC?CCA?GAG?GTT?CAC?CCT?TTG?CCT?ACA?CCT?GTC?CTG?CTG?CCT?GCT192Gln?Cys?Pro?Glu?Val?His?Pro?Leu?Pro?Thr?Pro?Val?Leu?Leu?Pro?Ala

30 35 40GTG?GAC?TTT?AGC?TTG?GGA?GAA?TGG?AAA?ACC?GAG?ATG?GAG?GAG?ACC?AAG240Val?Asp?Phe?Ser?Leu?Gly?Glu?Trp?Lys?Thr?Gln?Met?Glu?Glu?Thr?Lys

45 50 55GCA?CAG?GAC?ATT?CTG?GGA?GCA?GTG?ACC?CTT?CTG?CTG?GAG?GGA?GTG?ATG288Ala?Gln?Asp?Ile?Leu?Gly?Ala?Val?Thr?Leu?Leu?Leu?Glu?Gly?Val?Met?60 65 70 75GCA?GCA?CGG?GGA?CAA?CTG?GGA?CCC?ACT?TGC?CTC?TCA?TCC?CTC?CTG?GGG336Ala?Ala?Arg?Gly?Gln?Leu?Gly?Pro?Thr?Cys?Leu?Ser?Ser?Leu?Leu?Gly

80 85 90CAG?CTT?TCT?GGA?CAG?GTC?CGT?CTC?CTC?CTT?GGG?GCC?CTG?CAG?AGC?CTC384Gln?Leu?Ser?Gly?Gln?Val?Arg?Leu?Leu?Leu?Gly?Ala?Leu?Gln?Ser?Leu

95 100 105CTT?GGA?ACC?CAG?CTT?CCT?CCA?CAG?GGC?AGG?ACC?ACA?GCT?CAC?AAG?GAT432Leu?Gly?Thr?Gln?Leu?Pro?Pro?Gln?Gly?Arg?Thr?Thr?Ala?His?Lys?Asp

110 115 120CCC?AAT?GCC?ATC?TTC?CTG?AGC?TTC?CAA?CAC?CTG?CTC?CGA?GGA?AAG?GTG480Pro?Asn?Ala?Ile?Phe?Leu?Ser?Phe?Gln?His?Leu?Leu?Arg?Gly?Lys?Val

125 130 135CGT?TTC?CTG?ATG?CTT?GTA?GGA?GGG?TCC?ACC?CTC?TGC?GTC?AGG?CGG?GCC528Arg?Phe?Leu?Met?Leu?Val?Gly?Gly?Ser?Thr?Leu?Cys?Val?Arg?Arg?Ala140 145 150 155CCA?CCC?ACC?ACA?GCT?GTC?CCC?AGC?AGA?ACC?TCT?CTA?GTC?CTC?ACA?CTG576Pro?Pro?Thr?Thr?Ala?Val?Pro?Ser?Arg?Thr?Ser?Leu?Val?Leu?Thr?Leu

160 165 170AAC?GAG?CTC?CCA?AAC?AGG?ACT?TCT?GGA?TTG?TTG?GAG?ACA?AAC?TTC?ACT624Asn?Glu?Leu?Pro?Asn?Arg?Thr?Ser?Gly?Leu?Leu?Glu?Thr?Asn?Phe?Thr

175 180 185GCC?TCA?GCC?AGA?ACT?ACT?GGC?TCT?GGG?CTT?CTG?AAG?TGG?CAG?CAG?GGA672Ala?Ser?Ala?Arg?Thr?Thr?Gly?Ser?Gly?Leu?Leu?Lys?Trp?Gln?Gln?Gly

190 195 200TTC?AGA?GCC?AAG?ATT?CCT?GGT?CTG?CTG?AAC?CAA?ACC?TCC?AGG?TCC?CTG720Phe?Arg?Ala?Lys?Ile?Pro?Gly?Leu?Leu?Asn?Gln?Thr?Ser?Arg?Ser?Leu

205 210 215GAC?CAA?ATC?CCC?GGA?TAC?CTG?AAC?AGG?ATA?CAC?GAA?CTC?TTG?AAT?GGA768Asp?Gln?Ile?Pro?Gly?Tyr?Leu?Asn?Arg?Ile?His?Glu?Leu?Leu?Asn?Gly220 225 230 235ACT?CGT?GGA?CTC?TTT?CCT?GGA?CCC?TCA?CGC?AGG?ACC?CTA?GGA?GCC?CCG816Thr?Arg?Gly?Leu?Phe?Pro?Gly?Pro?Ser?Arg?Arg?Thr?Leu?Gly?Ala?Pro

240 245 250GAC?ATT?TCC?TCA?GGA?ACA?TCA?GAC?ACA?GGC?TCC?CTG?CCA?CCC?AAC?CTC864Asp?Ile?Ser?Ser?Gly?Thr?Ser?Asp?Thr?Gly?Ser?Leu?Pro?Pro?Asn?Leu

255 260 265CAG?CCT?GGA?TAT?TCT?CCT?TCC?CCA?ACC?CAT?CCT?CCT?ACT?GGA?CAG?TAT912Gln?Pro?Gly?Tyr?Ser?Pro?Ser?Pro?Thr?His?Pro?Pro?Thr?Gly?Gln?Tyr

270 275 280ACG?CTC?TTC?CCT?CTT?CCA?CCC?ACC?TTG?CCC?ACC?CCT?GTG?GTC?CAG?CTC960Thr?Leu?Phe?Pro?Leu?Pro?Pro?Thr?Leu?Pro?Thr?Pro?Val?Val?Gln?Leu

285 290 295CAC?CCC?CTG?CTT?CCT?GAC?CCT?TCT?GCT?CCA?ACG?CCC?ACC?CCT?ACC?AGC1008His?Pro?Leu?Leu?Pro?Asp?Pro?Ser?Ala?Pro?Thr?Pro?Thr?Pro?Thr?Ser300 305 310 315CCT?CTT?CTA?AAC?ACA?TCC?TAC?ACC?CAC?TCC?CAG?AAT?CTG?TCT?CAG?GAA1056Pro?Leu?Leu?Asn?Thr?Ser?Tyr?Thr?His?Ser?Gln?Asn?Leu?Ser?Gln?Glu

320 325 330GGG TAA1062Gly (2) are about information (ⅰ) sequence signature of SEQ ID NO:2

(A) length: 353 amino acid

(B) type: amino acid

(D) topological structure: line style, (ⅱ) molecule type: protein, (ⅹ ⅰ) sequence description: SEQ ID NO:2:Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala-21-20-15-10Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val-5,15 10Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser

15 20 25Gln?Cys?Pro?Glu?Val?His?Pro?Leu?Pro?Thr?Pro?Val?Leu?Leu?Pro?Ala

30 35 40Val?Asp?Phe?Ser?Leu?Gly?Glu?Trp?Lys?Thr?Gln?Met?Glu?Glu?Thr?Lys

45 50 55Ala?Gln?Asp?Ile?Leu?Gly?Ala?Val?Thr?Leu?Leu?Leu?Glu?Gly?Val?Met?60 65 70 75Ala?Ala?Arg?Gly?Gln?Leu?Gly?Pro?Thr?Cys?Leu?Ser?Ser?Leu?Leu?Gly

80 85 90Gln?Leu?Ser?Gly?Gln?Val?Arg?Leu?Leu?Leu?Gly?Ala?Leu?Gln?Ser?Leu

95 100 105Leu?Gly?Thr?Gln?Leu?Pro?Pro?Gln?Gly?Arg?Thr?Thr?Ala?His?Lys?Asp

110 115 120Pro?Asn?Ala?Ile?Phe?Leu?Ser?Phe?Gln?His?Leu?Leu?Arg?Gly?Lys?Val

125 130 135Arg?Phe?Leu?Met?Leu?Val?Gly?Gly?Ser?Thr?Leu?Cys?Val?Arg?Arg?Ala140 145 150 155Pro?Pro?Thr?Thr?Ala?Val?Pro?Ser?Arg?Thr?Ser?Leu?Val?Leu?Thr?Leu

160 165 170Asn?Glu?Leu?Pro?Asn?Arg?Thr?Ser?Gly?Leu?Leu?Glu?Thr?Asn?Phe?Thr

175 180 185Ala?Ser?Ala?Arg?Thr?Thr?Gly?Ser?Gly?Leu?Leu?Lys?Trp?Gln?Gln?Gly

190 195 200Phe?Arg?Ala?Lys?Ile?Pro?Gly?Leu?Leu?Asn?Gln?Thr?Ser?Arg?Ser?Leu

205 210 215Asp?Gln?Ile?Pro?Gly?Tyr?Leu?Asn?Arg?Ile?His?Glu?Leu?Leu?Asn?Gly220 225 230 235Thr?Arg?Gly?Leu?Phe?Pro?Gly?Pro?Ser?Arg?Arg?Thr?Leu?Gly?Ala?Pro

240 245 250Asp?Ile?Ser?Ser?Gly?Thr?Ser?Asp?Thr?Gly?Ser?Leu?Pro?Pro?Asn?Leu

255 260 265Gln?Pro?Gly?Tyr?Ser?Pro?Ser?Pro?Thr?His?Pro?Pro?Thr?Gly?Gln?Tyr

270 275 280Thr?Leu?Phe?Pro?Leu?Pro?Pro?Thr?Leu?Pro?Thr?Pro?Val?Val?Gln?Leu

285 290 295His?Pro?Leu?Leu?Pro?Asp?Pro?Ser?Ala?Pro?Thr?Pro?Thr?Pro?Thr?Ser300 305 310 315Pro?Leu?Leu?Asn?Thr?Ser?Tyr?Thr?His?Ser?Gln?Asn?Leu?Ser?Gln?Glu

320 325 330Gly (2) are about information (ⅰ) sequence signature of SEQ ID NO:3

(A) length: 178 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: line style (ⅱ) molecule type: protein (ⅹ ⅰ) sequence description: SEQ ID NO:3:Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr1 5 10 15Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe

20 25 20Thr?Ala?Ser?Ala?Arg?Thr?Thr?Gly?Ser?Gly?Leu?Leu?Lys?Trp?Gln?Gln

35 40 45Gly?Phe?Arg?Ala?Lys?Ile?Pro?Gly?Leu?Leu?Asn?Gln?Thr?Ser?Arg?Ser

50 55 60Leu?Asp?Gln?Ile?Pro?Gly?Tyr?Leu?Asn?Arg?Ile?His?Glu?Leu?Leu?Asn65 70 75 80Gly?Thr?Arg?Gly?Leu?Phe?Pro?Gly?Pro?Ser?Arg?Arg?Thr?Leu?Gly?Ala

85 90 95Pro?Asp?Ile?Ser?Ser?Gly?Thr?Ser?Asp?Thr?Gly?Ser?Leu?Pro?Pro?Asn

100 105 110Leu?Gln?Pro?Gly?Tyr?Ser?Pro?Ser?Pro?Thr?His?Pro?Pro?Thr?Gly?Gln

115 120 125Tyr?Thr?Leu?Phe?Pro?Leu?Pro?Pro?Thr?Leu?Pro?Thr?Pro?Val?Val?Gln

130 135 140Leu?His?Pro?Leu?Leu?Pro?Asp?Pro?Ser?Ala?Pro?Thr?Pro?Thr?Pro?Thr145 150 155 160Ser?Pro?Leu?Leu?Asn?Thr?Ser?Tyr?Thr?His?Ser?Gln?Asn?Leu?Ser?Gln

165 170 175Glu Gly (2) are about information (ⅰ) sequence signature of SEQ ID NO:4

(A) length: 18 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: line style (ⅱ) molecule type: peptide (ⅹ ⅰ) sequence description: SEQ ID NO:4:Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Ala Leu Thr1 5 10 15Leu Asn (2) are about information (ⅰ) sequence signature of SEQ ID NO:5

(A) length: 18 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: line style (ⅱ) molecule type: peptide (ⅹ ⅰ) sequence description: SEQ ID NO:5:Ala Pro Pro Asp Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr1 5 10 15Leu Asn (2) are about information (ⅰ) sequence signature of SEQ ID NO:6

(A) length: 18 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: line style (ⅱ) molecule type: peptide (ⅹ ⅰ) sequence description: SEQ ID NO:6:Ala Pro Pro Asp Thr Ala Val Pro Ser Glu Thr Ser Leu Val Leu Thr 15 10 15 Leu Asn (2) are about information (ⅰ) sequence signature of SEQ ID NO:7

(A) length: 18 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: line style (ⅱ) molecule type: peptide (ⅹ ⅰ) sequence description: SEQ ID NO:7:Ala Pro Pro Thr Thr Ala Val Pro Ser Glu Thr Ser Leu Val Leu Thr 15 10 15 Leu Asn (2) are about information (ⅰ) sequence signature of SEQ ID NO:8

(A) length: 5 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: line style (ⅱ) molecule type: peptide (ⅹ ⅰ) sequence description: SEQ ID NO:8 Ala Pro Pro Thr Thr 15 (2) information (ⅰ) sequence signature about SEQ ID NO:9

(A) length: 49 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC7623 (ⅹ ⅰ) sequence description: SEQ ID NO:9:GCGCGCAAGC TTGGACAAGA GAAGCCCGGC TCCTCCTGCT TGTGACCTC 49 (2) information (ⅰ) sequence signature about SEQ ID NO:10

(A) length: 51 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC7627 (ⅹ ⅰ) sequence description: SEQ ID NO:10:GCGCGCAAGC TTCTAGACT ATCAGACGCA GAGGGTGGAC CCTCCTACAA G 51 (2) information (ⅰ) sequence signature about SEQ ID NO:11

(A) length: 40 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC7868 (ⅹ ⅰ) sequence description: SEQ ID NO:11:GTGTGTGAAT TCTAGACTAT CAGACGCAGA GGGTCGACCC 40 (2) information (ⅰ) sequence signature about SEQ ID NO:12

(A) length: 35 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC7870 (ⅹ ⅰ) sequence description: SEQ ID NO:12:GTGTGTAAGC TTGGACAAGA GAAGCCCGGC GCCTC 35 (2) information (ⅰ) sequence signature about SEQ ID NO:13

(A) length: 82 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC8488 (ⅹ ⅰ) sequence description: SEQ ID NO:13:TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCAGAACTTC TTTGGCTTTG 60 ACTTTGAACT GATAGAGATC TT, 82 (2) information (ⅰ) sequence signature about SEQ ID NO:14

(A) length: 82 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC8489 (ⅹ ⅰ) sequence description: SEQ ID NO:14:CTAGAAGATC TCTATCAGTT CAAAGTCAAA GCCAAAGAAG TTCTGGATGG AACAGCAGTG 60 GTTGGTGGGG CGACGCAGAG GG, 82 (2) information (ⅰ) sequence signature about SEQ ID NO:15

(A) length: 85 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC8486 (ⅹ ⅰ) sequence description: SEQ ID NO:15:TCGACCCTCT GCGTCGGAGC TCCACCAGAC GAAGCTGTTC CAGACAGAGA CGAATTGGTT 60 TTGGAATTGA ACTGATAGAG ATCTT, 85 (2) information (ⅰ) sequence signature about SEQ ID NO:16

(A) length: 85 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC8487 (ⅹ ⅰ) sequence description: SEQ ID NO:16:CTAGAAGATC TCTATCAGTT CAATTCCAAA ACCAATTCGT CTCTGTCTGG AACAGCTTCG 60 TCTGGTGGAG CTCCGACGCA GAGGG, 85 (2) information (ⅰ) sequence signature about SEQ ID NO:17

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅹ ⅰ) sequence description: SEQ ID NO:17:Ala Pro Pro Asp Glu Ala Val Pro Asp Arg Asp Glu Leu Val Leu Glu 15 10 15 Leu Asn (2) are about information (ⅰ) sequence signature of SEQ ID NO:18

(A) length: 37 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC10086 (ⅹ ⅰ) sequence description: SEQ ID NO:18:TCGACCCTCT GCGTCGCCCC ACCAACCACT TGATAGA 37 (2) information (ⅰ) sequence signature about SEQ ID NO:19

(A) length: 37 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC10087 (ⅹ ⅰ) sequence description: SEQ ID NO:19:GATCTCTATC AAGTGGTTGG TGGGGCGACG CAGAGGG 37 (2) information (ⅰ) sequence signature about SEQ ID NO:20

(A) length: 49 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC10095 (ⅹ ⅰ) sequence description: SEQ ID NO:20:TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCTGATAGA 49 (2) information (ⅰ) sequence signature about SEQ ID NO:21

(A) length: 49 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC10096 (ⅹ ⅰ) sequence description: SEQ ID NO:20:GATCTCTATC AGGATGGAAC AGCAGTGGTT GGTGGGGCGA CGCAGAGGG 49 (2) information (ⅰ) sequence signature about SEQ ID NO:22

(A) length: 61 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC10120 (ⅹ ⅰ) sequence description: SEQ ID NO:22:TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCAGAACTTC TTTGTGATAG 60 A, 61 (2) information (ⅰ) sequence signature about SEQ ID NO:23

(A) length: 61 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC10121 (ⅹ ⅰ) sequence description: SEQ ID NO:23:GATCTCTATC ACAAAGAAGT TCTGGATGGA ACAGCAGTGG TTGGTGGGGC GACGCAGAGG 60 G, 61 (2) information (ⅰ) sequence signature about SEQ ID NO:24

(A) length: 76 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC10118 (ⅹ ⅰ) sequence description: SEQ ID NO:24:TCGACCCTCT GCGTCGCCCC ACCAACCACT GCTGTTCCAT CCAGAACTTC TTTGGTTTTG 60 ACTTTGAACT GATAGA, 76 (2) information (ⅰ) sequence signature about SEQ ID NO:25

(A) length: 76 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC10119 (ⅹ ⅰ) sequence description: SEQ ID NO:25:GATCTCTATC AGTTCAAAGT CAAAACCAAA GAAGTTCTGG ATGGAACAGC AGTGGTTGGT 60 GGGGCGACGC AGAGGG, 76 (2) information (ⅰ) sequence signature about SEQ ID NO:26

(A) length: 82 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC10108 (ⅹ ⅰ) sequence description: SEQ ID NO:26:TCGACCCTCT GCGTCAGGCG GGCCCCACCA ACCACTGCTG TTCCATCCAG AACTTCTTTG 60 GTTTTGACTT TGAACTGATA GA, 82 (2) information (ⅰ) sequence signature about SEQ ID NO:27

(A) length: 82 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC10109 (ⅹ ⅰ) sequence description: SEQ ID NO:27:GATCTCTATC AGTTCAAAGT CAAAACCAAA GAAGTTCTGG ATGGAACAGC AGTGGTTGGT 60 GGGGCCCGCC TGACGCAGAG GG 82

Claims (21)

1. reproducible expression vector in eukaryotic host cell comprises the following element that is operatively connected:

(a) transcripting promoter;

(b) the first segment DNA fragment of coding secretion leading peptide;

(c) coding contains the second segment DNA fragment of thrombopoietin (TPO) polypeptide of C-X-B, and wherein C is a human thrombopoietin cytokine structural domain polypeptide; X is peptide bond or the joint that comprises 1 or 2 amino-acid residue, and restricted condition is that X does not provide two basic aminoacidss right when combining separately or with C or B; And B is the polypeptide that contains the residue 1 to y of SEQ ID NO:3, wherein y be from 5 to 18 integer and wherein the B at the most 35% described residue can be substituted by other amino-acid residue respectively; And

(d) transcription terminator.

2. expression vector according to claim 1, wherein said carrier can duplicate in yeast.

3. expression vector according to claim 2, the secretion leading peptide that wherein said secretion leading peptide is the yeast saccharomyces cerevisiae alpha factor.

4. expression vector according to claim 1, wherein y is 9 at least.

5. expression vector according to claim 1, wherein B comprises the Thr-Thr dipeptides.

6. expression vector according to claim 1, wherein B does not comprise the Arg-Arg dipeptides.

7. expression vector according to claim 1, wherein 25% described residue is alternative by other amino-acid residue respectively at the most among the B.

8. expression vector according to claim 1, wherein the residue 1 to 5 of B is Ala-Pro-Pro-Thr-Thr (SEQ ID NO:8).

9. expression vector according to claim 1, wherein the 4th of B the residue is Thr or Asp.

10. expression vector according to claim 1, wherein y be at least 10 and the 10th residue of B be Arg or Glu.

11. expression vector according to claim 1, wherein y be at least 14 and the 14th residue of B be Val or Ala.

12. expression vector according to claim 11, wherein B is Ala-Pro-Pro-Thr-Thr-Ala-Val-Pro-Ser-Arg-Thr-Ser-Leu-Ala-Leu-Thr-Leu-Asn (SEQ ID NO:4).

13. expression vector according to claim 1, wherein B is selected from SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

14. expression vector according to claim 1, wherein X is a peptide bond.

15. expression vector according to claim 1, wherein X is the single amino acids residue.

16. expression vector according to claim 1, wherein C comprises the residue 1 to 152 of SEQ ID NO:2.

17. the eukaryotic cell that contains the cultivation of expression vector according to claim 1, wherein said cell synthesizes the described TPO polypeptide of justacrine.

18. yeast cell according to claim 17.

19. thrombopoietin polypeptides is characterized in that comprising the C-X-B amino acid backbone, wherein C is a human thrombopoietin cytokine structural domain polypeptide; X is peptide bond or the joint that contains 1 or 2 amino-acid residue, and restricted condition is that X does not provide two basic aminoacidss right when combining separately or with C or B; And B is the polypeptide that comprises the residue 1 to y of SEQID NO:3, and wherein y is from 5 to 18 integer, and 35% described residue can be alternative by other amino-acid residue respectively at the most among the B.

20. produce the method for TPO polypeptide, comprising:

Cultivate with can in host cell, duplicating and comprise following expression vector transfection or the transformed host cells that is operatively connected element:

(a) transcripting promoter;

(b) the first segment DNA fragment of coding secretion leading peptide;

(c) coding comprises the second segment DNA fragment of the thrombopoietin of C-X-B, and wherein C is a human thrombopoietin cytokine structural domain polypeptide; X is peptide bond or the joint that contains 1 or 2 amino-acid residue, and restricted condition is that X does not provide two basic aminoacidss right when combining separately or with C or B; And B is the polypeptide that contains the residue 1 to y of SEQ ID NO:3, and wherein y is from 5 to 18 integer, and 35% described residue can be respectively by other amino acid replacement at the most among the B; And

(d) transcription terminator;

Wherein first section of connecting of host cell expression and the second segment DNA fragment are synthesized the TPO polypeptide; And

Reclaim the TPO polypeptide.

21. increase platelet count purpose method in the Mammals, comprise to described animal and using and pharmaceutically acceptable carrier-bound thrombopoietin polypeptides, this polypeptide is characterised in that the amino acid backbone that comprises C-X-B, and wherein C is a human thrombopoietin cytokine structural domain polypeptide; X is peptide bond or the joint that contains 1 or 2 amino-acid residue, and restricted condition is that X does not provide two basic aminoacidss right when combining separately or with C or B; And B is the polypeptide that contains the residue 1 to y of SEQID NO:3, and wherein y is from 5 to 18 integer, and 35% described residue can be alternative by other amino-acid residue respectively at the most among the B.