CN1134539C - Thrombopoietin - Google Patents

Abstract

Isolated thrombopoietin (TPO), isolated DNA encoding TPO, and recombinant or synthetic methods of preparing and purifying TPO are disclosed. Various forms of TPO are shown to influence the replication, differentiation or maturation of blood cells, especially megakaryocytes and megakaryocyte progenitor cells. Accordingly, these compounds may be used for treatment of thrombocytopenia.

Description

Thrombopoietin

Field of the present invention

The present invention relates to influence hematopoietic cell, particularly thrombocyte remote ancestor cell survival, propagation, differentiation or sophisticated proteinic separation, purification and recombinant chou or chemosynthesis.The present invention is specifically related to encode can be in conjunction with the also clone and the expression of the nucleic acid of the protein ligands of activating cells factor acceptor superfamily member mpl.The invention still further relates to and use these protein separately or comprise the immunity or the hematopoiesis disease of thrombopenia with treatment in conjunction with other cytokines.

Background of the present invention I. hemopoietic system

Hemopoietic system produces the mature blood cell of the essential eggcase of all Mammals existence.These mature cells comprise: the red corpuscle of special delivering oxygen and carbonic acid gas; Undertake cell-mediated and antibody-mediated immunoreactive T and bone-marrow-derived lymphocyte; The special thrombocyte that generates clot; And as scavenger cell or accessory cell granulocyte and the scavenger cell to resist infection.Granulocyte is further divided into the specialized cell with standalone feature: neutrophil cell, eosinophil, basophilic cell and mastocyte.It should be noted that, the mature blood cell of all these specializations sees single common initiating cell type (Dexter etc. in the marrow, that be called multipotency (or all-round) stem cell at first from a kind of, Ann.Rve.Cell Biol., 3:423-441 (1987)).

In mammiferous life whole process, must continue, produce in large quantities the mature blood cell of eggcase.The hemocyte of overwhelming majority specialization can only be kept several hours functionally activies (Cronkite etc., Blood, Cells, 2:263-284 (1976)) to several weeks.Therefore,, need to bring in constant renewal in mature blood cell, primordial stem cell itself in order to keep the normal stable state hemocyte of Mammals demand, and any middle remote ancestor's clone between initiating cell and mature cell or remote ancestor's clone of pedigree orientation.

The core of hemopoietic system is a multipotential stem cell.These cell relative quantities are few, carry out self upgrade producing the filial generation stem cell by propagation, or are converted into through a series of differentiation step and increase and remote ancestor's cell that sophisticated pedigree limits, finally form the mature blood cell of eggcase.

For example, some the multipotency remote ancestor cell that is called CFC-Mix from stem cell produces the colony that comprises whole different medullary cells through propagation (self upgrades) with growing: red corpuscle, neutrophil cell, megalokaryocyte (hematoblastic predecessor), scavenger cell, basophilic cell, eosinophil and newborn maxicell.Remote ancestor's cell proliferation of other lymph pedigree and bud into T cell and B cell.

In addition, between CFC-Mix remote ancestor cell and medullary cell, exist another group to be oriented to middle remote ancestor's cell of filial generation.Remote ancestor's cell that these pedigrees limit is by the filial generation table sort of its generation.Therefore, the immediate predecessor of known bone myelocyte is: produce erythrocytic CFU-E (CFU-E), produce granulocyte/macrophage colony formation cell (GM-CFC) of neutrophil cell and scavenger cell, the eosinophil colony forming cell (Eos-CFC) that produces Megakaryocytic megakaryocyte colony forming cell (Meg-CFC), generation eosinophil and the basophilic cell colony forming cell (Bas-CFC) that produces mastocyte.Other middle precursory cell between multipotential stem cell and mature blood cell also known (stating as follows) maybe can be found the pedigree with various degree and limit and self updating ability.

Limit and maturity because lost versatility and obtained pedigree, so as if the chief cell in the normal hematopoiesis cell system reduce the ability that self upgrades.Therefore, at an end of hematopoietic cell spectrum, be to have the multipotential stem cell that self upgrades and be divided into directed remote ancestor's cell ability of all various pedigree specializations.This ability is the basis of bone marrow transplantation treatment, and primordial stem cell refills in the whole hematopoietic cell system in this treatment.At the other end of hematopoietic cell spectrum, be remote ancestor's cell and the filial generation thereof that the height pedigree limits, they have lost the ability of self upgrading, but have obtained sophisticated functionally active.

Controlling propagation and the growth that stem cell and pedigree limit remote ancestor's cell subtly by various hemopoieticgrowth factors or cytokine.The effect in vivo of these somatomedins is complicated, is not understood fully as yet.Some somatomedins, for example interleukin 3 (IL-3) can irritate remote ancestor's cell of multipotential stem cell and some pedigree orientations, comprises as megalokaryocyte.Originally other factor is considered to its effect as granulocyte/macrophage colony stimulating factor (GM-CSF) and is limited to GM-CFC ' s, but finds afterwards that GM-CSF also influenced particularly Megakaryocytic propagation and growth.Therefore, find that IL-3 and GM-CSF more have overlapping biological activity, although they have different potential.Recently, discovery interleukin-6 (IL-6) and interleukin-11 (IL-11) form megakaryocyte colony does not individually all have obviously influence, then irritate Megakaryocytic maturation (Yonemura etc., Exp.Hematol., 20:1011-1016 (1992)) when acting synergistically with IL-3.

Therefore, hemopoieticgrowth factor can influence the growth and the differentiation of one or more pedigrees, can be overlapping aspect the single remote ancestor's clone of influence with other somatomedin, or can act synergistically with other somatomedin.

And, limiting the cell development different steps of remote ancestor's cell from the pedigree of myeloid-lymphoid stem cell by various orientations to mature blood cell, hemopoieticgrowth factor can demonstrate their effect.For example, as if erythropoietin (epo) only promotes the propagation of remote ancestor's cell of mature erythrocyte.The IL-3 former effect that cell and middle pedigree limit remote ancestor's cell that starts from that as if wields influence earlier.Other somatomedin such as STEM CELL FACTOR (SCF) can influence even primary cell development more.

Can understand in sum, the survival, propagation, differentiation or the sophisticated new hemopoieticgrowth factor that influence any hemocyte or its predecessor will be useful, and the reconstruction of rib in the hemopoietic system of the weakening that is caused by disease or radiotherapy or chemotherapy arranged especially. II. megalokaryocyte generates---and thrombocyte produces

The adjusting that megalokaryocyte generates and thrombocyte produces is referring to summary Mazur, Exp.Hematol., 15:248 (1987) Hoffman, Blood, 74:1196-1212 (1989).In brief, the marrow multipotential stem cell is divided into megalokaryocyte, red corpuscle and myelocytic series.It is believed that directed this one-level of megalokaryocyte remote ancestor cell is arranged between stem cell and megalokaryocyte.At least identify three class megalokaryocyte remote ancestor cells, i.e. burst forming unit megalokaryocyte (BFU-MK), colony forming unit megalokaryocyte (CFU-MK) and low density megalokaryocyte remote ancestor cell (LD-CFU-MK).Megakaryocytic maturation itself is the continuity of growing, and can be divided into some stages according to the form standard of standard.Megalokaryocyte (MK or meg) the discerned member the earliest of family is a megakaryoblast.Diameter was 20-30 μ m when these cells began, and basophilic cell matter and an irregular slightly nuclear are arranged, and that this nuclear has is loose, slightly be netted chromatin and several kernel.After this, megakaryoblast can contain 32 of as many as nuclear polyploid, but that tenuigenin is still kept is sparse and immature.In ripening process, nuclear becomes and more is little lobate and pyknosis, and cytoplasmic amount increases, and acidophilia and be particulate state becomes more.Can there be the hematoblastic outward appearance of release at the edge of most of mature cells of this family.Usually, the megalokaryocyte below 10% is in the embryonic cell stage, is mature cell more than 50%.The authoritative morphological classification that is generally used for megalokaryocyte series is the megakaryoblast of very early time form; Mesomorphic promegakaryocyte or basophilic megakaryocyte; The maturation of later stage form (acidophilia, particulate state or produce hematoblastic) megalokaryocyte.Ripe megalokaryocyte extends into the blood sinus chamber with the tenuigenin fibril, and they are fragmented into single thrombocyte (Williams etc., Hematlolgy, 1972) in the blood sinus chamber.

It is believed that the megalokaryocyte generation relates to some regulatory factors (Williams etc., Br.J.Haematol., 52:173 (1982) and Willams etc., J.Cell Physiol., 110:101 (1982)).The commitment supposition that megalokaryocyte generates is mitotic division, relate to from the cell proliferation of CFU-MK and the initial formation of cell colony, but be not subjected to the influence (Burstein etc. of platelet counts, J.Cell Physiol., 109:333 (1981) and Kimura etc., Exp.Hematol., 13:1048 (1985)).Sophisticated later stage is non-mitotic division, relates to the polyploidization and the tenuigenin maturation of nuclear, may regulate (Odell etc. with Feedback mechanism by the periphery number of platelets, Blood, 48:765 (1976) and Ebbe etc., Blood, 32:787 (1968)).

To existing of megakaryocyte colony stimulating factor uniqueness, specific (MF-CSF) too controversial (Mazur, Exp.Hematol., 15:340﹠amp; 350 (1987)).But most authors believe, produce the adjusting that the extremely important process of this a pair of existence is subjected to cytokine utterly as thrombocyte.Having megalokaryocyte/this hypothesis of blood-platelet specific cytokine is that 30 years of researches provide the foundation, but still do not have so far resemble that the such cytokine of single MK-CSF (TPO) is purified, order-checking and Analysis and Identification.

Though it is reported, MK-CSF ' s is by the thrombopenia (Hill etc. from experimental generation, Exp.Hematol., 14:752 (1986)) and human embryo kidney (HEK) conditioned medium (CM) (McDonald etc., J.Lab.Clin.Med., 85:59 (1975) and from plasticity-anaemia and idiopathic thrombocytopenic purpura urine extract (Kawakita etc., Blood, 6:556 (1983) and blood plasma (Hoffman etc., J.Clin.invest., 75:1174 (1985)) partly purify in, but in most of cases, their physiological function is not understood as yet.

Pokeweed mitogen activated spleen cell-conditioned medium (PWM-SpCM) and mouse myelomonocyte clone WEHI-3 (WEHI-3CM) are used as megalokaryocyte promotor.PWM-SpCM contains the factor (Met-calf etc., Pro.Natl.Acad.Sci., USA, the 72:1744﹠amp of multiple promotion CFU-MK growth; 1748 (1975); Quesenberry etc., Blood, 65:2141985:; And lscove, N.N., inHematopoietic Cell Differentiation, ICN-UCLA Symposia onMolecular and Cellular Biology, Vol.10, Golde etc. edit (NewYork, Academy Press) pp37-52 (1978)), one of them is interleukin 3 (IL-3), this be a kind of multispectral be cell colony stimulating factor (multi-CSF (Burstein, Blood Cells, 11:469 (1986))).Other factor in this substratum is not identified and is separated as yet.Relatively a large amount of IL-3 and the more a spot of GM-CSF of WEHI-3 mouse myelomonocyte system secretion.Have now found that IL-3 promotes large-scale hematopoietic cell growth (lhle etc., J.lmmunol., 13:282 (1983)).Also find, in very early stage multipotency precursor is induced and in the formation of very a large amount of mixing hematopoietic cell colonies, IL-3 and a lot of known hematopoiesis hormone or somatomedin synergy, (Bartelmez etc., J.Cell Physiol., 122:382﹠amp; 369 (1985) and Warren etc., Cell, 46:667﹠amp; 674 (1988)), comprise erythropoietin (EPO) and interleukin 1 (IL-1).

Other source of megalokaryocyte promotor also sees in the conditioned medium of mouse lung, bone, macrophage system, peritoneal effusion cell and HEKC.Although some inconsistent data (Mazur, Exp.Hematol., 15:340﹠amp are arranged; 350 (1987)), but have some evidences (Geissler etc., Br.J.Haematol., 60:233-238 (1985)) to show, activated T lymphocyte but not monocyte play a driving role in megalokaryocyte generates.These find prompting, and activated T lymphocytic emiocytosis thing such as interleukin-can be the developmental regulatory factor of MK (Geissler etc., Exp.Hematol., 15:845-853 (1987)).Erythropoietin (EPO) with purifying generates some researchs (Vainchenker etc., Blood, the 54:940 (1979) that carries out to megalokaryocyte; McLeod etc., Nature, 261:492-4 (1976) and Williams etc., Exp.Hematol., 12:734 (1984)) show that this hormone forms the MK colony has promoter action.This is not containing seroculture and is containing in the seroculture, and has all obtained proof (Williams etc., Exp.Hematol., 12:734 (1984) under the situation of shortage accessory cell.Opposite with the effect of PWM-SpCM, EPO is assumed to be and relates to unicellular and two cell stages that megalokaryocyte generates more, and PWM-SpCM relates to four cell stages that megalokaryocyte is grown.The Megakaryocytic interaction early stage and late period of all these factor pairs remains to be illustrated.

The data prompting that obtains from some laboratories, only have separately the MK-colony stimulating active multispectral be that the factor is GM-CSF and IL-3, and on than low degree, be B cell stimulating factor-1 L-6 (Ikebuchi etc., Proc.Natl.Acad.Sci.USA, 84:9035 (1987).Recently, some authors have reported that IL-11 and leukaemia inhibitory factor (LIF) play synergy with IL-3, increase megalokaryocyte volume and ploidy (Yonemura etc., British Journal of Hematology, 84:16-23 (1993); Burstein etc., J.Cell.Physiol., 153:305-312 (1991); Bruno etc., Blood, 76:50-56 (1990); Metcalf etc., Blood, 77:2150-2153 (1991); Bruno etc., Exp.Hematol., 19:378-381 (1991); With Yonemura etc., Exp.Hematol., 20:1011-1016 (1992).

Other interesting document comprises: Eppstein etc., U.S. Patent No. 4,962,091; Chong, United States Patent (USP) NNo.4,879,111; Fernandes etc., U.S. Patent No. 4,604,377; Wissler etc., U.S. Patent No. 4,512,971; Got-tlieb, U.S. Patent No. 4,468,379; Bennett etc., U.S. Patent No. 5,215,895; Kogan etc., U.S. Patent No. 5,250,732; Kimura etc., Eur.J.lm-munol., 20 (9): 1927-1931 (1990); Secor etc., J.of lmmunol., 144 (4): 1484-1489 (1990); Warren etc., J.of lmmunol., 140 (1): 94-99 (1988); Warren etc., Exp.Hematol., 17 (11): 1095-1099 (1989); Bruno etc., Exp.Hematol., 17 (10): 1038-1043 (1989); Tamikawa etc., Exp.Hematol., 17 (8): 883-888 (1989); Koike etc., Blood, 75 (12): 2286-2291 (1990); Lotem, Blood, 75 (5): 1545-1551 (1989); Rennick etc., Blood, 73 (7): 1828-1835 (1989); With Clutterbuck etc., Blood, 73 (6): 1504-1512 (1989). III. thrombopenia

Thrombocyte is the key element in the blood clotting mechanism.The exhaustion that is called the thrombocyte cyclical level of little blood plate minimizing is present in various clinical conditions and the disease.Thrombopenia is normally defined platelet count and is lower than 150 * 10 ⁹/ liter.According to platelet life span, thrombocytopenic major cause can be divided three classes roughly, promptly the thrombocyte that causes of (1) marrow produces impairedly, and (2) spleen thrombocyte compiles that platelet destruction increases (for example autoimmunity thrombopenia or chemotherapy and radiation) in (splenomegaly) or (3) peripheral circulation.In addition, accept heavy dose of patient who uses the hematoblastic blood product of destruction fast, can thrombopenia take place because of dilution.

Thrombocytopenic clinical bleeding depend on thrombocytopenic seriousness, the cause of disease and may with the blood coagulation defective.Usually, platelet count is in 20-100 * 10 ⁹/ liter the patient too much danger of bleeding is arranged after the wound, and platelet count is lower than 20 * 10 ⁹But/liter patient's hematostaxis.A kind of patient in back is the candidate of thrombocyte transfusion, is attended by immunity and viral danger.When the cause of disease was thrombocyte generation minimizing rather than destruction increase, its bleeding tendency of the thrombopenia of any degree was even more serious.At latter event, the transhipment of the thrombocyte of acceleration causes upgrading, the more effective thrombocyte that more stops blooding circulates.Thrombopenia can be summarized as follows from various diseases.More detailed description can be referring to Schafner, A.L., " Thrombocytopenia and Disorders of Platelet Function; " InternalMedicine, the third edition, John J.Hutton etc., editor, Litte Brown and Co., Boston/Toronto/London (1990).

(a) produce the impaired thrombopenia that causes by thrombocyte

The congenital thrombocytopenic cause of disease comprises CAA (Fanconi syndromes) and congenital no megalokaryocyte aleukia, and these may be relevant with skeleton deformity.Acquired thrombocyte produces disease and is caused by megalokaryocyte underdevelopment or the generation of poor efficiency thrombocyte.There is multiple situation can cause megalokaryocyte underdevelopment, comprising the invasion and attack of hypoplastic bone marrow (comprising the special bone marrow depression of sending out the property form or causing), myelofibrosis, leukemia and metastatic tumour or granuloma to marrow by chemotherapeutics or radiotherapy.In some situation, toxin, infector or medicine can disturb thrombocyte to generate in relative selectivity ground; The example comprises the property sent out the temporarily thrombopenia and the slight thrombopenia relevant with taking thiazide diuretic that is caused by alcohol or some virus infection.At last, megaloblast forming process (folic acid or B ₁₂Shortage) the poor efficiency thrombocyte of secondary generates and also can cause thrombopenia in, follows usually with anaemia and oligoleukocythemia.

Current to depend on evaluation and reverse by thrombocyte generation the weakening thrombocytopenic treatment that causes to the bone marrow disorder cause of disease that exists.Usually only carry out the thrombocyte transfusion to patient that the severe haemorrhage complication is arranged or for the protection in the surgical procedure, because isoimmunization may cause producing refractoriness to continuing the input thrombocyte.The mucosal bleeding that is caused by serious thrombopenia can be eased with anti-molten scleroproein agent by oral or intravenous injection.But, if use anti-molten scleroproein agent that the thrombosis complication is further developed to disseminated intravascular coagulation (DIC) patient.

(b) compile the thrombopenia that causes by spleen (thrombocyte)

The splenomegaly that is caused by any reason all may be with slight relevant to the thrombopenia of moderate.With the spleen in immune-mediated thrombopenia that hereinafter will discuss hematoblastic enthusiasm destruction is compared, this is that a very passive spleen thrombocyte compiles process (hypersplenism).Though the most commonly encountered diseases of hypersplenism is because of being the congested splenomegaly that portal hypertension that alcoholic cirrhosis causes causes, congested, the wetting property or the lymphadenosis splenomegaly of other form are also relevant with thrombopenia.Single thrombopenia that is caused by hypersplenism counting usually is not less than 50 * 10 ⁹/ liter.

(c) thrombopenia that causes by non-immune-mediated platelet destruction

Various non-immunologic processes can cause thrombopenia to hematoblastic accelerating the failure.Such disease comprises disseminated intravascular coagulation, prosthetic appliance in the blood vessel, external circulation of blood and as thrombotic microangiopathies such as thrombotic thrombocyte purpuras.In all these situations, the thrombocyte that is exposed in the circulation of prosthetic surface or abnormal vascular inner membrance can be consumed in these positions or be destroyed by reticuloendothelial system, then before prematurity with its removing.Editor's such as Braunwald Harrison ' s Principles of Internal Medicine the 11st edition, P1478 has narrated the disease condition that may cause disseminated intravascular coagulation (DIC) in more detail among the McGraw Hill (1987).Prosthetic appliance comprises that heart valve and intra-aortic oalloon can cause slightly the destructive thrombopenia to moderate in the blood vessel, and accepts sending out a property thrombopenia temporarily and may be caused by hematoblastic consumption or destruction in the extracorporeal circuit among the patient of cardiopulmonary bypass or hemodialysis.

(d) drug-induced immunity thrombopenia

There is more than 100 kind of medicine relevant with immune-mediated thrombopenia.But, only Quinidine, quinine, gold, sulfonamides, cefoxitin and heparin have been carried out detailed qualitative.Drug-induced thrombopenia takes place in usually very serious and general after patient has taken the sensitization medicine several days immediately.

(e) immunity (autoimmunity) thrombopenic purpura (ITP)

ITP among the adult is a kind of chronic disease, it is characterized in that the autoimmunity platelet destruction.This autoantibody is IgG normally, but other immunoglobulin (Ig) is also had report.Though the ITP autoantibody has been found and platelet membrane GPII _bIII _aRelevant, but the platelet antigen specificity in most of case is not identified out yet.The outer destruction of the blood vessel of sensitized platelet occurs in the reticuloendothelial system of spleen and liver.Though ITP cases more than half are idiopathic, most patient suffers from rheumatic or autoimmune disorder (as systemic lupus erythematous) or lymphoproliferative disease's (as lymphocytic leukemia).

(f) HIV inductive ITP

ITP is that more and more common a kind of HIV infects complication (Morris etc., Ann.Intern.Med., 96:714-717 (1982)), and can appear at any stage of disease progression, infecting the patient of the acquired immune deficiency syndrome (AIDS) of having been made a definite diagnosis (AIDS) patient, AIDS AIDS-related complex AIDS with by HIV but not having among the patient of AIDS symptom all has generation.It is a kind of communicable disease that HIV infects, and finally shows as the degree of depth defective and the generator opportunistic infections of cellular immune function and sb.'s illness took a turn for the worse.Infecting the initial dysimmunity that causes by HIV is to express the lymphocytic expansionary minimizing of T and the functional damage (Lane etc., Ann.Rev.Immunol.3:477 (1985)) of cd4 cell surface glycoprotein.May be CD4 complementary/forfeiture of induced T lymphocyte function caused and caused opportunistic infection and AIDS sb.'s illness took a turn for the worse the cellular immunization of feature and the degree of depth defective of humoral immunization.(H.Lane is the same).

Though also do not know the ITP mechanism that HIV is relevant, it is believed that to be different from and infect incoherent ITP mechanism (walsh etc., N.Eng.J.Med.311:635-639 (1984) with HIV; And Ratner, Am.J.Med., 86:194-198 (1989)).IV. current to thrombocytopenic methods of treatment

The seriousness and the urgency that thrombopenia patient's methods of treatment are depended on clinical symptom.Similar to the HIV dependency with the thrombocytopenic methods of treatment of HIV irrelevance, and though used multiple different methods of treatment, such treatment is still disputable.

Glucocorticoid (as Ultracortene-H) therapy has successfully improved makes a definite diagnosis the platelet count of suffering from the thrombocytopenia patient, but in most patient, this reaction is incomplete, perhaps recurrence can occur when the dosage that reduces glucocorticoid or interruption are taken.Based on the research of carrying out with the relevant ITP patient of HIV, some investigator proposes the glucocorticoid therapy may cause easy infection AIDS.Usually be lower than 20 * 10 at thrombocyte ⁹/ liter below or when hematostaxis occurring, take glucocorticoid.

As for the patient that glucocorticoid is not answered, compound 4-(2-chloro-phenyl-)-9-methyl-2-(3-(4-morpholinyl)-3-acetone-1-yl) 6H-thieno-(3,2, f) (1,2,4) triazole (4,3, a) (1,4) diazepine (WEB 2086) has been successfully used to treat the incoherent ITP grave illness of HIV example.With WEB 2086 treatments one platelet count is 37,000-58, and the patient of 000/ microlitre, after the treatment in 1-2 week, platelet count rises to 140,000-190,000/ microlitre.(EP 361,077 and Lohman etc., Lancet, 1147 (1988)).

Though the optimal treatment method of acquired no megalokaryocyte thrombopenic purpura (AATP) is also uncertain, antithymocyte globulin (ATG) (a kind of horse anteserum at people's thymic tissue) has been shown and can have produced secular, alleviation completely.(Trimble etc., Am.J.Hematol., 37:126-127 (1991)).But nearest report first points out that the hemoposieis of ATG is because of Thiomersalate, and protein is played a part the mercury carrier by hypothesis.(Panella etc., Cancer Research, 50:4429-4435 (1990)).

Have and report that the spleen excision has good result.Spleen is extractd and eliminated most of platelet destructions position and most of autoantibody generation source in many patient.This method can produce alleviation secular, that need not to treat in a large amount of patients.But, the patient who lacks immunizing power should avoid surgical operation usually, only is applicable to severe thrombopenia case (as severe HIV dependency ITP) and patient reactionless to the glucocorticoid treatment in 2 to 3 weeks or that interrupt can't obtaining after glucocorticoid is taken the persistence reaction so spleen is extractd.Based on existing scientific knowledge, also do not remove the spleen excision and whether can make patient tend to suffer from AIDS.

Except Ultracortene-H therapy and spleen are extractd, some cytotoxic agent, (AZT zidovudine) also demonstrates the possibility that is used for the treatment of HIV inductive ITP, but the result is preliminary examination as vincristine(VCR) and Zidovodine.

Hence one can see that, and treating thrombocytopenic a kind of method should be to obtain a kind of megalokaryocyte or the differentiation of its precursor and maturation of quickening to become the medicament that thrombocyte produces form.Identifying that a kind of like this being commonly referred to spent a large amount of effort aspect " thrombopoietin " medicament (TPO).Other title of common TPO also has in the document: thrombocyte produces stimulating factor (TSF), megakaryocyte colony stimulating factor (MK-CSF), megakaryocyte stimulating factor and megalokaryocyte promotor.As far back as nineteen fifty-nine, (Rak etc., Med.Exp. 1:125), carry out qualitative and effort purifying to it and continue so far always just to have had been found that the TPO activity.Though the partially purified report that the TPO-active polypeptide is arranged is (for example referring to Tayrien etc., J.Biol.Chem., 262:3262 (1987) and Hoffman etc., J.Clin.Invest.75:1174 (1985)), but other report infers that then TPO itself is not a kind of separate component, and is multi-functional performance form (IL-3, the Sparrow etc. of certain known hormone, Prog.Clin.Biol.Res., 215:123 (1986)).No matter which kind of form and source, the molecule with thrombopoietic activity all should have important therapeutic value.Definitely be not accredited as TPO though still there is protein, sizable interest is mpl round a nearest discovery then, a kind of cytokine receptor of supposition, and the thrombocyte of may transduceing generates signal.V.Mpl is a kind of megalokaryocyte generative nature cytokine receptor

The propagation and the maturation that it is believed that hematopoietic cell are closely regulated by some factor, the propagation of these factor forwards or negative regulation multipotential stem cell and the differentiation of multispectral system.These effects are to combine mediation with the high affinity of specific cell surface receptor by extracellular protein factor.These cell surface receptors have the homology of height, and are classified as the member of cytokine receptor superfamily usually.The member of this superfamily comprises the acceptor of following material: IL-2 (β and γ chain) (Hatakeyama etc., Science, 244:551-556 (1989); Takeshita etc., Science, 257:379-382 (1991)), IL-3 (itoh etc., Science, 247:324-328 (1990); Gorman etc., Proc.Natl.Acad.Sci.USA, 87:5459-5463 (1990); Kitamura etc., Cell, 66:1165-1174 (1991a); Kitamura etc., Proc.Natl.Acad.Sci.USA, 88:5082-5086 (1991b)), IL-4 (Mosley etc., Cell, 59:335-348 (1989)), IL-5 (Takaki etc., EMBO J., 9:4367-4374 (1990); Tavernier etc., Cell, 66:1175-1184 (1991)), IL-6 (Yamasaki etc., Science, 241:825-828 (1988); Hibi etc., Cell, 63:149-1157 (1990I), IL-7 (Goodwin etc., Cell, 60:941-951 (1990)), IL-9 (Renault etc., Proc.Natl.A-cad.Sci.USA, 89:5690-5694 (1992)), granulocyte-macrophage colony stimutaing factor (GM-CSF) (Gearing etc., EMBO are (1991) J.8:3667-3676); Hayashida etc., Proc.Natl.Acad.Sci.USA, 244:9655-9659 (1990)), granulocyte colony-stimulating factor (G-CSF) (Fukunaga etc., Cell, 61:341-350 (1990a); Fukunaga etc., Proc.Natl.Acad.Sci.USA, 87:8702-8706 (1990b); Larsen etc., J.Exp.Med., 172:1559-1570 (1990)), EPO (D ' Andrea etc., Cell, 57:277-285 (1989); Jones etc., Blood, 76:31-35 (1990)), leukaemia inhibitory factor (LIF) (Gearing etc., EMBO J., 10:2839-2848 (1991)), oncostatin M (OSM) (Rose etc., Proc.Natl.Acad.Sci.USA, 88:8641-8645 (1991)) and prolactin antagonist (Boutin etc., Proc.Natl.Acad.Sci.USA, 88:7744-7748 (1988); Edery etc., Proc.Natl.Acad.Sci.USA, 86:2112-2116 (1989)), (330:536-543 (1987) and ciliary neurotrophic factor (CNTF) be (Davis etc. (GH) for Leung etc., Nature for tethelin, Science, 253:59-63 (1991)) acceptor.

Member in the cytokine receptor superfamily can be divided into three kinds of functional categories (relevant summary can be referring to Nicola etc., Cell, 67:1-4 (1991)).The first kind is made up of the strand acceptor, and as erythropoietin receptor (EPO-R) or granulocyte colony stimulating factor receptor (G-CSF-R), they are by extracellular domain and signal in the part high affinity combines and produce a kind of cell.Receptor of second order claims α-subunit again, comprises the member of interleukin-6 receptor (IL6-R), granulocyte-macrophage colony stimulating factor acceptor (GM-CSF-R), interleukin 3 acceptor (IL3-R α) and other cytokine receptor superfamily.These α-subunits combine with the low affinity of part but can not the interior signals of transducer cell.A kind of high affinity acceptor of energy transduction signal is by α-subunit and is called the 3rd type cytokines member of β-subunit (as β _c, three kinds of α-subunits such as IL3-R α and GM-CSF-R common beta-subunit) between a kind of heterodimer of forming produce.

Mpl is that the evidence of a member in the cytokine superfamily comes from sequence homology (Gear-ing, EMBO be (1988) J.8:3667-3676; Bazan, Proc.Natl.Sci.USA, 87:6834-6938 (1990); Davis etc., Science, 253:59-63 (1991) and Vigon etc., Proc.Natl.Acad.Sci.USA, 89:5640-5644 (1992)) and the ability of its transduction proliferation signal.

The protein sequence of being derived by the molecular cloning of mouse c-mpl discloses this albumen and other cytokine receptor has homology.Its ectodomain contains 465 amino-acid residues, and this structural domain is made of two subdomains, and each subdomain has the halfcystine of 4 high conservatives and one section special motif of N end subdomain and C end subdomain.The ectodomain of estimating binding partner has similar dual beta-Folding bucket geometry.The low affinity binding domains of this multiple ectodomain and IL-3, IL-5 and GM-CSF acceptor common signal transduction chain and LIF (Vigon etc., Oncogene, 8:2607-2615 (1993)) has the homology of height.So mpl may belong to the low affinity ligands association class of cytokine receptor superfamily.

Mouse mpl and people are ripe, and mplp shows that relatively two kinds of protein show 81% sequence identity.Especially, N end and the outer subdomain of C end born of the same parents have 75% and 80% sequence identity respectively.The most conservative mpl district is the cytoplasmic structure territory, demonstrates 91% amino acid consistence, and one section on all four 37 residue sequence in two species is being arranged near the membrane spaning domain.So mpl is reported as one of member the most conservative in the cytokine receptor superfamily (Vigon, the same).

Mpl can transduce the source of evidence of functional receptor of proliferation signal in the structure of Chimerical receptor, and Chimerical receptor contains and the ectodomain of the cytokine receptor of certain known cytokine high affinity and the cytoplasmic structure territory of mpl.Owing to also do not report the part of known mpl, so must make up the mosaic type high affinity ligands in conjunction with ectodomain by first kind cytokine receptor such as IL-4R or G-CSFR.Vigon etc. (the same) merge the ectodomain of G-CS-FR and cytoplasmic structure territory and the membrane spaning domain of c-mpl.Clone BAF/B03 (Ba/F3) with this G-CSFR/c-mpl mosaic and the dependence of total length G-CSFR transfection IL-3 in contrast.With mosaic cells transfected well-grown when cytokine IL-3 or G-CSF exist.Equally, with G-CSFR cells transfected also well-grown in IL-3 or G-CSF.When not having somatomedin, all cells is all dead.(EMBO is (7) J.12: 2645-2653 (1993)) also carried out similar experiment for Skoda etc., in the experiment cytoplasmic structure territory of the ectodomain of people IL-4 acceptor (hIL-4-R) and membrane spaning domain and mouse mpl is merged, and with the IL-3 dependency Ba/F3 clone of this transfection mouse.All can normally breed when specific specificity IL-4 or IL-3 exist with wild-type hIL-4-R cells transfected.Normal with the Ba/F3 cell of hIL-4R/mpl transfection when not having IL-3 (have or) propagation when hIL-4 exists, show thus that in the Ba/F3 cell cytoplasmic structure territory of mpl has comprised the necessary whole key elements of transduction proliferation signal.

These chimeric abilities that experimental results show that mpl cytoplasmic structure territory transduction proliferation signal, but do not mention whether the ectodomain of mpl can binding partner.These results are consistent with at least two kinds of possibilities, and promptly mpl is as EPO-R or G-CSFR, be a kind of strand acceptor (first kind) or be the signal transduction β-subunit (the 3rd class) (Skoda etc., the same) that needs an IL-3 sample α-subunit.The VI.Mpl part is a kind of thrombopoietin (TPO)

As mentioned above, the someone proposes to contain in the serum a kind of unique factor that is called as thrombopoietin (TPO) sometimes, and it assists various other cytokines to promote Megakaryocytic growth with ripe.Also from serum or other source, do not isolate this natural factor, though many research organizations have carried out a large amount of effort for this reason.Even do not know whether mpl can be directly in conjunction with certain megalokaryocyte stimulating factor, nearest experiment has still proved mpl and transduction relevant (Methia etc. from the proliferation signal of one or more factors of finding among the aplastic marrow patients serum, Blood, 82 (5): 1395-1401 (1993)).

Having a kind of serum colony that is different from IL-1 α, IL-3, IL-4, IL-6, IL-11, SCF, EPO, G-CSF and GM-CSF of uniqueness to form the factor comes from the detection of c-mpl expression and distribution and the mpl antisense research of carrying out in a kind of these clones in the original and directed hematopoietic cell system by the transduce evidence of proliferation signal of mpl.

In the human hematopoietic cell of immune purifying, use reversed transcriptive enzyme (RT)-PCR (Methia etc., the same) only to show at CD34 ⁺Found strong mpl mRNA information in the cell of purifying, megalokaryocyte and the thrombocyte.The CD34 that gets by marrow (BM) purifying ⁺Cell accounts for 1% of whole BM cells, and is enriched in (as red corpuscle, grain scavenger cell and megalokaryocyte) in original and directed remote ancestor's cell of all pedigrees.

There is report to show that the Mpl antisense oligodeoxyribonucleotide suppresses from multipotency CD34 ⁺The megakaryocyte colony of cell forms, these multipotencys CD34 ⁺Cell cultures is in the serum of taking from aplastic marrow (the abundant source of a kind of megakaryocyte colony stimulating activity (MK-CSA)) patient.These identical antisense oligodeoxyribonucleotide all do not have effect to red corpuscle or the formation of grain macrophage colony.

Whether mpl directly binding partner and whether serum factor shows can cause the megalokaryocyte generation still unclear by the mpl effect.But once the someone proposed, if the really direct binding partner of mpl, its aminoacid sequence may be a high conservative, and owing to the height sequence identity between people and the mouse mpl ectodomain has cross reactivity (Vigon etc., the same (1993)) between kind.

VII. purpose

In sum, be appreciated that the requirement that exists a kind of now in the art and will continue, promptly separate and identify can promote hematopoietic cell, in particular for treating thrombocytopenic megalokaryocyte or its precursor propagation, differentiation and sophisticated molecule.It is believed that this quasi-molecule is a kind of mpl part, so also further need isolate this part to estimate their effects in cell growth and differentiation.

So one of the object of the invention is to obtain a kind of pharmacy pure energy stimulating megakaryocyte propagation, differentiation and/or maturation to be the molecule of sophisticated thrombocyte generation form.

Two of the object of the invention provides and is used for the treatment of the hematopoiesis disease, the molecule of especially thrombocytopenic form of therapy.

Three of the object of the invention is to separate, purifying, and especially identifying can be in vivo in conjunction with the protein ligands and the proliferation signal of transduceing that are called as certain cytokine superfamily receptors of mpl.

Four of the object of the invention provides the nucleic acid molecule of this proteinoid part of coding and produces in the reconstitution cell culture with these nucleic acid molecule and is used to the mpl binding partner diagnosing and treat.

Five of the object of the invention provides the derivative and the modified forms of protein ligands, comprises their variant amino acid sequence body, variation glycoprotein form and their covalence derivative.

Six of the object of the invention provides fusion polypeptide that mpl part and certain heterologous protein combine and their covalence derivative.

Seven of the object of the invention provides the variation polypeptide that the EPO combined sequence after mpl part and aminoacid addition and the replacement forms, thereby generates a kind of protein that can regulate thrombocyte and red corpuscle remote ancestor cell proliferation and growth.

Another purpose of the present invention is that preparation is used to improve at mpl part or its and merges the immunogen of the antibody of form, and obtain can be in conjunction with the antibody of this class part.

With reference to following explanation, these and other purpose of the present invention will be conspicuous concerning persons skilled in the art.The present invention's general introduction

The present invention seeks to by providing a kind of isolating Mammals megalokaryocyte generative nature propagation and ripe promotion albumen to reach, this albumen be named as " mpl part " (ML) or " thrombopoietin " (TPO), its can stimulating megakaryocyte propagation, ripe and/or be divided into sophisticated thrombocyte generation form.

Can be in order to following method from the protein of this basic homogeneous of certain natural origin purifying: (1) can make mpl ligand molecular to be purified selective adsorption under the condition on the immobilization receptor polypeptides, certain source blood plasma and certain immobilization receptor polypeptides that will contain mpl ligand molecular to be purified, especially be fixed on mpl or the contact of mpl fusion polypeptide on certain carrier, (2) washing immobilization receptor polypeptides and carrier thereof to be removing not adsorbent, and (3) are with elution buffer wash-out mpl ligand molecular from the immobilization receptor polypeptides that is adsorbed with the mpl ligand molecular.Natural origin preferably contains the mammalian plasma or the urine of mpl part.This Mammals can suffer from aplastic anemia, and the immobilization acceptor is the mpl-IgG syzygy.

Optionally, preferably megalokaryocyte generative nature propagation and maturation promote the mpl ligand polypeptide that albumen is isolating and the utilization of basic homogeneous is synthesized or recombination method makes.

" mpl part " polypeptide of the present invention or " TPO " preferably have 70% complete sequence consistence at least with the pig mpl ligand polypeptide aminoacid sequence of highly purified basic homogeneous, and have the sequence identity of 8Q% at least with pig mpl body Quito peptide " EPO structural domain ".Optionally, mpl part of the present invention is the one-tenth acquaintance mpl part (hML) with mature amino acid sequence shown in Figure 1 (SEQ IDNO:1), or its varient or its post transcriptional modificaiton form or have and the protein that becomes about 80% sequence identity of acquaintance mpl part.Mpl ligand variant body can be a fragment, the aminoterminal of especially sophisticated people mpl part (hML) or " EPO structural domain " fragment.Best, n terminal fragment remains with almost whole people ML sequences between first to fourth cysteine residues, but can contain this extra-regional general interpolation, disappearance or replacement.According to this embodiment, the fragment polypeptide can be represented by following structural formula:

X-hML(7-151)-Y

Wherein hML (7-151) represents Cys ⁷To Cys ¹⁵¹Between people TPO (hML) aminoacid sequence (comprise Cys ⁷And Cys ¹⁵¹); X represents Cys ⁷Aminoterminal amino-acid residue or its amino-acid residues amino or one or more ripe hMl extend to as Met, Tyr or to the homing sequence (as Xa factor or zymoplasm) that contains just like the proteolysis position; Y represents Cys ¹⁵¹Carboxyl or the carboxyl terminal amino-acid residue of one or more ripe hMl or its extend amino-acid residue.

Optionally, mpl ligand polypeptide or its fragment can merge (mosaic) with certain heterologous polypeptide.Preferred heterologous polypeptide is certain cytokine, G CFS or interleukin-or its fragment, and especially complete part (kit-ligand, KL), IL-1, IL-3, IL-6, IL-11, EPO, GM-CSF or LIF.A kind of preferred heterologous polypeptide is an immunoglobulin chain, and especially human IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgD, IgM or its fragment especially contain the IgG CH.

On the other hand, the invention provides a kind of composition that contains separative mpl agonist, biologically active and preferably can stimulate mark Nucleotide (as ³The H-thymidine) mixes in that the IL-3 of personnel selection mpl transfection relies on, the Ba/F3 cell DNA.Optionally, this mpl agonist is that bioactive mpl part is arranged, and preferably can measure moderate stimulation in the rebound of mouse thrombocyte ³⁵S mixes in the round-robin thrombocyte.Suitable mpl agonist comprises hML ₁₅₃, hML (R153A, R154A), hML2, hML3, hML4, mML, mML2, mML3, pML and pML2 or their fragment.

In other embodiments, the invention provides a kind of isolating can be in conjunction with the antibody of mpl part.This isolating can the fusion with another kind of polypeptide alternatively in conjunction with the antibody of mpl part, and this antibody or its syzygy can be used for separating from aforementioned source and purifying is used for fixing the mpl part of mpl.In the content on the other hand of this embodiment, the invention provides a kind of be used in the body or in the method for vitro detection mpl part, comprise making antibody and being contacted and detect whether combination take place by doubtful certain sample (especially serum sample) that contains part.

In other embodiments, the invention provides a kind of separated coding mpl part or its segmental nucleic acid molecule, this nucleic acid molecule can detect composition with certain alternatively and carry out mark; And providing a kind of has and the nucleic acid molecule complementation of certain mpl part encoding sequence or the nucleic acid molecule that can hybridize with it to the height stringent condition in moderate.Preferred nucleic acid molecule is the coding nucleic acid of those people, pig and mouse mpl part, comprises RNA and DNA, is genome and cDNA.This embodiment on the other hand in, nucleic acid molecule is the DNA of coding mpl part and contains a replicable vector, wherein DNA with can be operably connected by the regulating and controlling sequence of discerning with this carrier host transformed.Optionally, DNA has among Fig. 15 '-3 ' (SEQ ID NO:2) or cDNA or its fragment of 3 '-5 ' sequence.This one side content also comprises the method that produces the mpl part with this carrier transformed host cells (preferably Chinese hamster ovary celI) and a kind of DNA of use, preferably includes the cDNA that expresses coding mpl part after conversion in the host cell culture and reclaim the mpl part from host cell or host cell culture.Zhi Bei mpl part people mpl part preferably in this way.

The present invention comprises that also a kind of being used for the treatment of suffer from certain hematopoiesis disease, especially thrombocytopenic mammiferous method, and it comprises certain mpl part of taking effective therapeutic dose to Mammals.Optionally, can be with this mpl part and certain cytokine, especially certain G CFS or interleukin-are united and are taken.Preferred G CFS or interleukin-comprise: complete part (KL), ILF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-3, IL-6 and IL-11.

The present invention also comprises and separating from the microorganism that produces TPO and the method for purifying TPO (ML):

(1) cracking or dissolving contain the cell of TPO,

(2) alternatively soluble substance is separated with the insoluble substance that contains TPO,

(3) with the TPO in the dissolving damping fluid dissolving insoluble substance,

(4) dissolved TPO is separated with insoluble substance with other solubility,

(5) at oxygen refolding TPO in the damping fluid also,

(6) TPO that will correctly fold separates with the TPO of false folding.

This method is dissolved the insoluble substance that contains TPO with chaotropic agent, and this chaotropic agent is selected from guanidinesalt, Sodium Thiocyanate 99 or urea.This method also proposes, and dissolved TPO separates with insoluble substance with other solubility with a step or multistep in the reverse-phase chromatography by centrifugal, gel-filtration.The refolding step of this method provides a kind of potential buffer solution that had not only contained oxygenant but also contained reductive agent.Usually, oxygenant is oxygen or the compound that contains at least one disulfide linkage, and reductive agent is the compound that contains at least one free sulfhydryl groups.Best, oxygenant is selected from already oxidised gsh (GSSG) and halfcystine, and reductive agent is selected from as-reduced gsh (GSH) and halfcystine.Most preferred oxygenant is already oxidised gsh (GSSG), and most preferred reductive agent is as-reduced gsh (GSH).And the mol ratio of oxygenant preferably is equal to or greater than reductive agent.Potential buffer solution also contains washing agent in addition, preferably is selected from CHAPS and CHAPSO, and content is at least 1%.It is that good NaCl and concentration are to be good glycerine greater than 15% with about 0.1-0.5M that potential buffer solution also contains concentration.The pH of potential buffer solution is good with pH7.5-pH9.0, and the refolding step was carried out 12-48 hour at 4 ℃.The refolding step produces the TPO of biologically active, wherein at the N-terminal Cys of the most close EPO structural domain with form a disulfide linkage between the Cys of close its carboxyl terminal.

The present invention also comprise a kind of from microorganism the method for purifying biologically active TPO, it comprises:

(1) microbivorous at least epicyte,

(2) handle the lysate that contains TPO with chaotropic agent,

(3) refolding TPO,

(4) TPO of separation decon and false folding from the correct TPO that folds.

The accompanying drawing summary

Fig. 1 shows aminoacid sequence (SEQID NO:1) and the nucleotide sequence coding (SEQ ID NO:2) of people mpl part (hML) cDNA of deduction.Beginning at each row is numbered Nucleotide.5 ' and 3 ' non-translational region is represented with lowercase.Ser1 from ripe mpl part (ML) protein sequence is numbered amino-acid residue above sequence.The border of the exon 3 of supposing marks with arrow, and adds collimation mark and go out potential N-glycosylation site.Stain with the sequence top marks cysteine residues.The line sequence is corresponding to the N terminal sequence that is recorded from the mpl of porcine blood plasma part by purifying.

Fig. 2 shows usefulness ³The experimentation that the H-thymidine mixes.In order to measure the existence of mpl in the various sources, mpl P Ba/F3 cell under the condition that lacks IL-3,37 ℃, 5%CO in the humidity incubator ₂Cultivated 24 hours down with air.After the hungry cultivation of IL-3, cell is paved on 96 well culture plates that contain or do not contain dilute sample, in cell culture incubator, cultivated 24 hours.20 μ l are contained 1 μ Ci ³The serum-free RPMI substratum of H-thymidine adds and carries out last 6-8 hour cultivation in each hole.Harvested cell and washing with water on 96 hole filter plates then.Then filter plate is counted.

Fig. 3 has shown that PRONASE A, DTT and high temperature stimulate the effect effect of Ba/F3-mpl cell proliferation to APP.In order to digest APP, after PRONASE A (Boehringer Mannheim company) or bovine serum albumin and Affi-gel10 (Bio-rad company) coupling, hatched 18 hours at 37 ℃ respectively with APP with PRONASE A.Then, centrifugal removal resin and supernatant liquor is analyzed.APP also be heated to 80 ℃ 4 minutes or make the DTT of 100 μ M after the PBS that dialyses.

Fig. 4 shows from Phenyl-Toyopearl, the mpl ligand activity thing wash-out on Blue-Sepharose and the Ultra-lind-mpl post.Elution fraction 4-8 on the mpl affinity column is the peak activity part.

Fig. 5 shows the SDS-PAGE of Ultralink-mpl elution fraction.Add 1ml-20 ℃ respectively among each elution fraction 2-8 of 200 μ l, contain the acetone of 1mM HCl.After 3 hours, sample carries out centrifugal at-20 ℃, then gained is deposited in-20 ℃ with twice of washing with acetone.Then the precipitation behind the washing with acetone is dissolved in the 30 μ l SDS-dissolving damping fluid, makes 100 μ M DTT and 90 ℃ of heating 5 minutes.Then sample is separated on the 4-20%SDS-polyacrylamide gel, can estimate protein by silver dyeing.

Fig. 6 shows the mpl ligand activity thing on the SDS-PAGE.Under non-reduced condition, the elution fraction 6 of mpl-affinity column separates on the SDS-of 4-20% polyacrylamide gel.Behind the electrophoresis gel is cut into 12 and wait subregion, as described in embodiment, carry out electroelution then.Sample behind the electroelution is dialysed into PBS, analyze with 1/20 diluent.Molecular weight (Mr) standard that is used to calibrate gel is Novex Mark 12 standard substances.

Fig. 7 demonstration has exhausted the influence of the mpl part of APP to the generation of people's megalokaryocyte.1ml is prepared the mpl part that exhausts APP by 1ml mpl-affinity column (700 μ g mpl-IgG/ml NHS-superose, Pharmacia company).People's peripheral stem cell be made into 10%APP or 10% exhausted APP mpl part and cultivated 12 days.Generate as quantitative analysis megalokaryocyte as described in the embodiment.

Fig. 8 shows the influence that people's megalokaryocyte is generated hormesis by APP mpl-IgG.People's peripheral stem cell is made into 10%APP and cultivated 12 days.The 0th, 2, added mpl-IgG (0.5 μ g) or ANP-R-IgG (0.5 μ g) in 4 days.After 12 days, generate as quantitative analysis megalokaryocyte as described in the embodiment.Provide actual repeating data in the bracket and represent the mean value of repeat samples.

Fig. 9 has shown the human gene group DNA's of coding mpl part 390bp fragment two strands.The derivation aminoacid sequence (SEQ ID NO:3) that has also shown " exon 3 ", encoding sequence (SEQ IS NO:4) and its complementary sequence (SEQ ID NO:5).

Figure 10 has shown the derivation aminoacid sequence of sophisticated people mpl part (hML) (SEQ ID NO:6) and sophisticated erythropoietin (hEPO) (SEQ ID NO:7).The supposition aminoacid sequence of people mpl part and the sequence of erythropoietin are come into line.Add collimation mark and go out identical amino acid, the neutral gear of introducing for alignment dots.Mark potential N-glycosylation position with underscore, to the hML solid line, to the hEPO with dashed lines.To erythropoietin activity is that critical two halfcystines mark with big stain.

Figure 11 shows derivation aminoacid sequence hML (SEQ ID NO:6), hML2 (SEQ ID NO:8), hML3 (SEQ ID NO:9) and the hML4 (SEQ ID NO:10) of sophisticated people mpl ligand isoforms.Add collimation mark and go out identical amino acid, the neutral gear of introducing for alignment dots.

Figure 12 A, 12B and 12C have shown the influence (A) of people mpl part to the Ba/F3-mpl cel l proliferation, with radiolabeled specificity at megalokaryocyte glycoprotein GPII _bThe mouse IgG monoclonal antibody quantitative analysis vitro human megalokaryocyte of IIIa generates (B) and detect the mouse thrombocyte in the thrombocyte rebound is measured and generates (C).

293 cells utilize CaPO ₄Method (Gorman, Cin DNA Cloning:A NewApproach 2:143-190 (1985)) separately with the pRK5 carrier, with pRK5-hML or with pRK5-ML ₁₅₃(pRK5-ML ₁₅₃Being to utilize PCR to introduce terminator codons and produce in residue 153 back of hML) transfection spends the night.In conditioned medium, cultivated 36 hours as described in example 1 above then, analyze the hormesis that Ba/F3-mpl cell proliferation (A) or vitro human megalokaryocyte generate (B).Utilize ¹²⁵I is radiolabeled at the special glycoprotein GPII of megalokaryocyte _bThe mouse IgG monoclonal antibody quantitative analysis megalokaryocyte of IIIa generates (as Grant etc., Blood 69:1334-1339 (1987) is described).Utilize McDon-ald, T.P. the rebound thrombocyte described in the Proc.Soc.Exp.Biol.Med.144:1006-10012 (1973) generate determination and analysis partially purified reorganization ML (rML) to body in thrombopoietic effect (C).Partially purified rML makes from the conditioned medium that 200ml contains the ML that recombinates.Substratum by equilibrated 2mlBlue-Separose post in PBS, is washed post with PBS, with the PBS wash-out that contains 2M urea and 2M NaCl.Active elution fraction is dialysed into PBS, make 1mg/ml concentration with no endotoxic BSA.Every ml sample contains the intracellular toxin that is less than 1 unit.Give injected in mice 64,000,32,000 or the rML of 16,000 units or independent vehicle.Every group of 6 mouse.Provide the mean value and the standard deviation of each group.Utilize the relatively 2 tool tail T-test of intermediate value to measure the p value.

Figure 13 has compared people mpl ligand isoforms and the effect of varient in the Ba/F3-mpl cell proliferating determining.As described in the embodiment 1 when the various thinning ratio to hML, simulation test, hML2, hML3, hML (R153A, R154A) and hML ₁₅₃Measure.

Figure 14 A, 14B and 14C have shown derivation aminoacid sequence (SEQ ID NO:1) and the human gene group DNA's encoding sequence (SEQ ID NO:11) of people mpl part (hML) or people TPO (hT-PO).Beginning at every row is Nucleotide and amino acid numbering.

Figure 15 has shown the 293-rhML of purifying ₃₃₂293-rhML with purifying ₁₅₃SDS-PAGE.

Figure 16 has shown the derivation aminoacid sequence (SEQ ID NO:13) of the open reading frame of nucleotide sequence: cDNA coding (SEQ ID NO:12) and certain mouse ML isotype.This maturation mouse mpl ligand isoforms contains 331 amino-acid residues, lacks 4 than the total length mML that supposes, therefore is expressed as mML2.The Nucleotide numbering is in the beginning of every row.Begin above sequence, to be the amino-acid residue numbering from Ser1.Underscore marks potential N-glycosylation site.Stain with the sequence top marks cysteine residues.

Figure 17 shows the cDNA sequence (SEQ IDNO:14) of this mouse ML isotype (mML) and the protein sequence (SEQ ID NO:15) of supposition.The Nucleotide numbering is at every line start.The amino acid numbering is above sequence, from Ser1.This maturation mouse mpl ligand isoforms contains 335 amino-acid residues, and it is believed that it is total length mpl part, is designated as mML.With dashed lines marks signal peptide sequence, marks possible cleavage site with arrow.5 ' and 3 ' non-translational region represent with lowercase.The two places disappearances (mML2 and mML3) that variable splicing produces mark with underscore.Mark 4 cysteine residues with a stain.7 potential N-glycosylation sites add collimation mark and go out.

Figure 18 has compared people ML isotype hML3 (SEQ ID NO:9) and mouse ML isotype mML3 (SEQ ID NO:16) deduced amino acid.The supposition aminoacid sequence and the mouse mpl ligand sequence of people mpl part are come into line.Identical amino acid is added frame, and the neutral gear with dashed lines of introducing for alignment marks.At every line start amino acid is numbered.

Figure 19 has compared mouse ML (SEQ ID NO:17), the supposition aminoacid sequence of the ripe ML isotype of pig ML (SEQ ID NO:18) and people ML (SEQ ID NO:6).Aminoacid sequence is come into line, wherein have with dashed lines to mark the neutral gear of introducing for alignment.Number to amino acid at every line start, add collimation mark and go out identical residue.Mark potential N-glycosylation position with the square frame that adds shade, mark the cysteine residue with point.The two basic aminoacids motifs of conservative property in expression potential proteolytic enzyme cutting site are carried out underscore.4 aminoacid deletion (ML2) that all occur in three kinds mark with the runic square frame.

Figure 20 has shown the cDNA sequence (SEQ IDNO:19) and the supposition ripe protein sequence (SEQ ID NO:18) of certain boar ML isotype (pML).This pig mpl ligand isoforms contains 332 amino-acid residues, and it is believed that it is the pig mpl part of total length, called after pML.At every line start is the Nucleotide numbering.Above sequence, number, from Ser1 to amino-acid residue.

Figure 21 has shown the cDNA sequence (SEQID NO:20) of certain boar ML isotype (pML2) and the maturation protein sequence (SEQ ID NO:21) of supposition.This pig mpl ligand isoforms contains 328 amino-acid residues, lacks 4, called after pML2 than total length pig mpl part.Number to Nucleotide at every line start.Above sequence, number, from Ser1 to amino-acid residue.

Figure 22 has compared the derivation aminoacid sequence (SEQ ID NO:21) of the pig ML isotype of the derivation aminoacid sequence (SEQID NO:18) of total length pig ML isotype pML and called after pML2.The supposition aminoacid sequence of pML and the sequence of pML2 are come into line.Add frame for identical amino-acid residue, dotted line is expressed as alignment and the neutral gear of introducing.Number to amino acid at every line start.

Figure 23 has shown that being used for transfecting host CHO-DP12 cell produces CHO-rhTPO ₃₃₂Plasmid pSV15.ID.LL.MLORF (" total length " or TPO ₃₃₂) related characteristics.

Figure 24 has shown that being used for transfecting host CHO-DP12 cell produces CHO-rhTPO ₁₅₃Plasmid pSV15.ID.LL.MLEPO-D (" brachymemma " or TPO ₁₅₃) related characteristics.

Figure 25 A, 25B and 25C have shown E.coli-rhTPO (Met ^-1, 153) in the normal mouse body to the effect of thrombocyte (A), red corpuscle (B) and white corpuscle (C).Inject PBS damping fluid or 0.3 μ g E.coli-rhTPO (Met every day for two groups 6 one group female C57 B6 mouse ^-1, 153) and (100 μ l sc.).At the 0th day and 3-7 days, extract 40 μ l blood by the socket of the eye hole.This blood sample is diluted in 10ml immediately and is purchased in the thinner and on SerronoBaker Hematology Analyzer 9018 and obtains full blood count.Data are expressed as the means standard deviation of mean value.

Figure 26 A, 26B and 26C have shown E.coli-rhTPO (Met ^-1, 153) in the mouse body of sublethal exposure to the effect of thrombocyte (A), red corpuscle (B) and white corpuscle (C).Use from ¹³⁷Two groups 10 female C57 B6 mouse of the 750cGy gamma-rays sublethal exposure of Cs, and inject PBS damping fluid or 0.3 μ g E.coli-rhTPO (Met every day ^-1, 153) and (100 μ l sc.).At the 0th day and later some pitch time, extract 40 μ l blood by the socket of the eye hole.This blood sample is diluted in 10ml immediately and is purchased in the thinner and on Serrono BakerHematology Analyzer 9018 and obtains full blood count.Data are expressed as the means standard deviation of mean value.

Figure 27 A, 27B and 27C have shown CHO-rhTPO ₃₃₂In the normal mouse body to (A) thrombocyte, (B) red corpuscle and (C) leukocytic effect.Inject PBS damping fluid or 0.3 μ g CHO-rhTPO every day for two groups 6 one group female C57 B6 mouse ₃₃₂(100 μ l sc.).At the 0th day and 3-7 days, extract 40 μ l blood by the socket of the eye hole.This blood sample is diluted in 10ml immediately and is purchased in the thinner and on Serrono Baker HematologyAnalyzer 9018 and obtains full blood count.Data are expressed as the means standard deviation of mean value.

Figure 28 has shown the various forms of rhTPO dose response curves that obtain from various clones.The rhTPO that takes from following clone is drawn dose response curve: from the hTPO of CHO (from the total length form of Chinese hamster ovary cell) ₃₃₂HTPO _Met ^-1 ₁₅₃(E-coli deutero-clipped form has a N-end methionine(Met)); HTPO ₃₃₂(from the total length TPO of people's 293 cells); Met-less 155 E.coli are (from the clipped form (rhTPO of E.coli ₁₅₅), do not have terminal methionine(Met)).With various rhTPO respectively to the C57B6 injected in mice 7 days of 6 one group of each group.Extract 40 μ l blood from the socket of the eye hole every day and carry out full blood count.The data of expression are the usefulness of pretending most that finding appeared at the 7th day on the figure, have only an exception (Met153E-coli).And in aforesaid " Met 153E-coli " group, pretend most with appearing at the 5th day.Data are expressed as the means standard deviation of mean value.

Figure 29 has shown rhTPO of the total length that relatively produces and " brachymemma " form and active dose response curve from the E.coli clipped form in Chinese hamster ovary celI.Inject all kinds rhTPO of 0.3 μ g every day to 6 one group C57B6 female mice.At 2-7 days, extract 40 μ l blood by the socket of the eye hole and carry out full blood count.Treatment group is the clipped form TPO rhTPO from E.coli ₁₅₃Total length TPO rhTPO ₃₃₂(mixed composition) contains about 80-90% total length and 10-20% clipped form; TPO ₃₃₂(30K component)=from the brachymemma component of former mixed preparation purifying; TPO ₃₃₂(70K component)=from the total length TPO component of former mixed preparation purifying.Data are expressed as the means standard deviation of mean value.

Figure 30 is the synoptic diagram that the KIRA ELISA experiment of TPO is measured in expression.The figure illustrates the relevant portion of MPL/Rse.gD mosaic and parental generation acceptor and the relevant flow chart of steps (left side of figure) of final structure (right side of figure) and expression mensuration.

Figure 31 is that the KIRA ELISA in each step in the representation program measures schema.

Figure 32 A-32L provides the nucleotide sequence (SEQ ID NO:22) of the pSVI17.ID.LL expression vector that is used to express Res.gD in embodiment 17.

Figure 33 is the diagram of preparation plasmid pMP1.

Figure 34 is the diagram of preparation plasmid pMP21.

Figure 35 is the diagram of preparation plasmid pMP151.

Figure 36 is the diagram of preparation plasmid pMP202.

Figure 37 is the diagram of preparation plasmid pMP172.

Figure 38 is the diagram of preparation plasmid pMP210.

Figure 39 is the tabulation from 5 best TPO cloning by expressions (SEQ ID NO:23,24,25,26,27 and 28) in the pMP210 plasmid library.

Figure 40 is the diagram of preparation plasmid pMP41.

Figure 41 is the diagram of preparation plasmid pMP57.

Figure 42 is the diagram of preparation plasmid pMP251.

Detailed description 1. definition of the present invention

As Yi in situation, when following word Yong Yu specification, embodiment and claims, has described definition under Yi.

" chaotropic agent " Zhi Yi Zhong is in aqueous solution Zhong and reaches while being fit to concentration, and energy Yin plays the compound of protein steric configuration and conformation change, and this variation is to realize by Zhi small part ground, destroying the power of maintaining the normal secondary of protein Zheng and tertiary structure. The a little compounds of Zhe comprise: urea, guanidine hydrochloride and sodium sulfocyanate etc. Usually could have an effect to the conformation of protein in the time of Yao asking a little compounds of Zhe to reach the high concentration of 4-9M.

property term as the Yi of the protein of Yi cell mass secretion of " cell factor " expression You, this proteinoid Zuo is other cells of intercellular amboceptor Zuo Yong Yu. Zhu Ru lymphokine, monokine and traditional polypeptide hormone etc. comprising: the growth hormone IGF, human growth hormone (HGH), N-methionyl human growth hormone (HGH), BGH, parathormone, thyroxine, Yi island element, Yi island element Yuan, relaxins, relaxation precipitinogen, glycoprotein hormones is (as follicle-stimulating hormone [FSH], thyroid-stimulating hormone [TSH], luteotropin [LH]), hemopoieticgrowth factor, LGF, fibroblast growth factor, prolactin, choriomammotropin, cachectin (TNF-α and TNF-β), mullerian inhibitory substance, the related Tai of mouse gonadotropic hormone, Yi Zhi element, activin, VEGF, integrin, nerve growth factor (as NGF-β), PDGF, TGF TGFs (as TGF-α and TGF-β), IGF-I and-II, hematopoietin (EPO), bone-inducing factor, interferon is (as α, β, IFN-γ), colony stimulating factor (CSFs) is (as macrophage colony stimulatory factor [M-CSF], granulocyte-macrophage colony stimutaing factor [GM-CSF], granulocyte colony stimulating factor [G-CSF]), interleukins (IL ' s) (as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12) and other polypeptide factors (as leukocyte inhibitory factor [LIF], stem cell factor [SCF] and complete part). " cell factor " term herein represents the various protein that obtain from the right source of Zi or recombinant cell culture thing. similarly, Zhe term also comprises having bioactive equivalent, for example its amino acid sequence Zhong Yi or a plurality of amino acid differences or type of glycosylation and degree difference.

" mpl part ", " mpl ligand polypeptide ", " ML ", " TPO " or " TPO " be Alternate in this article, but and comprises any polypeptide with the characteristic of Yu mpl, being combined. Mpl is Yi the member of cytokine receptor superfamily Zhong, and possesses the biological characteristics of ML as described below. Wherein the typical biological characteristics of Yi Zhong be the nucleotides that can promote mark (as³The H-thymidine) mix Ba/F3 cell DNA Zhong that Yong people mpl P Zhuan dyes, that Yi relies IL-3. Another kind of typical biological characteristics is that experiment Zhong is analyzed in the rebound of Zai mouse Xue platelet, can promote³⁵S mixes the Xue platelet of Xun ring. Zhe definition Zhu Yaoed certain polypeptide of Zhi, this polypeptide is separated to from mpl part source (as undergrown Zhu Xue slurry) or other sources (as comprise other animal species of the people), perhaps standby by Chong Zu or synthetic method Zhi, this definition also has comprised the various ways that comprises functional derivatives, fragment, allele, isotype and analog thereof simultaneously.

" mpl part fragment " or " TPO fragment " Zhi have removed Yi Yi section individual or a plurality of amino acid residues or sugar unit, that Zi so produces ripe total length mpl part or TPO sequence Zhong. Wherein, the removal of amino acid residue can occur in any position on the Tai chain, comprises that N end, C end or Tai are inner. This fragment Zhi possesses the Yi Zhong biological characteristics identical Yu the mpl part less. The continuous sequence of few 10,15,20,25,30 or 40 amino acid residues of a You Yi Zhi as this mpl part fragment Yi, Yu the mpl part that separates from mammal, comprise the part of separation from undergrown Zhu Xue slurry or the mpl part of people or mouse, it is its hematopoietin (EPO) domain for You, and sequence is identical. The representative example You hML of N terminal fragment₁₅₃Or TPO (Met^-11-153)。

" mpl ligand variant body " or " mpl ligand sequence variant " represents the following bioactive mpl part that has in this article, and it infers mpl part or people's part sequence that 100% ground You is not identical sequence, that be separated to from recombinant cell culture thing or undergrown Zhu Xue slurry Yu having as shown in Fig. 1 (SEQ ID NO:1). Usually, Yi the amino acid sequence Yu the mpl part that is separated to from undergrown Zhu Xue slurry or ripe mouse or people's part and fragment identical (seeing Fig. 1 [SEQ ID NO:1]) thereof with bioactive mpl ligand variant body Ying Zhi rare 70%, reach better at least about 75% Ze, better at least about 80% Ze, also Yao is good at least about 85%, even also Yao is good, good the most identical at least about 95% at least about 90%.

" chimeric mpl part " expression You total length mpl part or one or a plurality of segment compositions or be incorporated into the less important heterologous polypeptide of Yi bar or one or a plurality of fragment Zhong and Yi Zhong polypeptide that Zu becomes. The rare Yi Zhong of this chimera Zhi biological characteristics is identical Yu the mpl part. Be cell factor, immunoglobulin (Ig) or its fragment as that less important polypeptide Yi.

" the mpl part of separation ", " high-purity mpl part " and the mpl part of homogeneous " basic " are used interchangeably, and expression, from the mpl part that certain mpl ligand sources purifying or You Chong Zu or synthetic method obtain, wherein is substantially free of other Tai or protein. (1) the cup type sequenator of Yong Zhuan or very business-like commercially available Protein Sequencer or file according to the application that improving one's methods of rear announcement obtained Zhi and be less 15 from N end or certain intersegmental part amino acid sequence, the amino acid residue of 20 preferably, perhaps (2) Yong Coomassie brilliant blue or the argentation of preferably Yonging, under the non-reduced or reducing condition of Zai, the Yi sds polyacrylamide gel electrophoresis reaches homogeneity. Here, homogeneity represents that the pollution of foreign protein is lower than 5%.

" biological characteristics " Yi word Zai Yu " mpl part " or " the mpl part that separates " are when using, and expression has You mpl part (no matter being Zi the conformation after right or sex change) or its fragment Zhi connects or indirectly Yin or TPO activity or vivo effect function or antigenicity function or the activity exercised. Effector function comprises that it is that the Zhuan of Zeng Zhi signal leads to mpl in conjunction with the excitation of activity, mpl and the anti-Zuo Yong of Jie You in conjunction with active and any Zai body, comprise copy, the adjusting of DNA regulatory function, other cell factor BAs, acceptor (it is cell factor for You) activation, forward or negative regulation, Growth of Cells or differentiation etc. The antibody that Yi natural mpl part of energy Yu You of antigenicity functional representation Yong You obtains plays epi-position or the antigen site of cross reaction. To be it separate Yu You the antibody that the undergrown Zhu Xue slurry of Zi mpl part obtains to the major antigen sexual function of mpl ligand polypeptide is combined, and its compatibility Zhi rare 10⁶L/ mole. Usually, this compatibility Zhi is 10 less⁷L/mole. Under optimal cases, to be Yi Zhong possess Yu You the polypeptide that antibody that the mpl part of above-mentioned certain effector function obtains combines to the mpl ligand polypeptide with antigen active. Yong Yu determines that the antibody of " biologically active " is the rabbit polyclonal antibody that obtains by the following method: Zai Freund ' s Freund's complete adjuvant Zhong at first, the standby mpl part that separates from recombinant cell culture thing or undergrown Zhu Xue slurry of Zhi, then hypodermic injection Zhi is for thing, Zai is to the standby thing Yi Zeng strong immune response of intraperitoneal injection Zhi, and tiring of Zhi Zhi mpl ligand antibody reaches platform area.

" biologically active " Yi word Zai Yu " mpl part " or " the mpl part that separates " are when using, represent Yi Zhong mpl part or polypeptide described herein, it shows TPO activity or possesses from certain effector function of the Zhu plasma separation or the mpl part expression of You recombinant cell culture thing of ateliosis. Yi basic function of the mpl part herein or polypeptide be combined Yu mpl and can promote mark nucleotides (³The H-thymidine) mix Ba/F3 cell DNA Zhong that Yong people mpl P Zhuan dyes, that Yi relies IL-3. Mpl part herein or another effector function of polypeptide are that Zai mouse Xue platelet rebound mensuration Zhong can promote³⁵S mixes the Xue platelet of Xun ring. Also having the effector function of the mpl part of Yi Yi Zhi is to promote the vitro human megakaryocytopoiesis, and available radiolabeled Zhen carries out quantitatively the monoclonal antibody of megacaryocyte glycoprotein GPIIbIIIa.

About " the amino acid sequence identity percentage " of mpl ligand sequence, the amino acid residue that represents in this article candidate sequence Zhong Yu possess as shown in Fig. 1 (SEQ ID NO:1) amino acid infer sequence, from undergrown Zhu Xue slurry the identical percentage of residue of that separate or mpl ligand sequence Zhong mouse or people's part. In order to reach the largest percentage of sequence homogeneity, in case of necessity, but Zai relatively before makes sequence linearisation and Yin enter breach. In the time of relatively, any preservative replacement not Zuo be that the Yi of sequence homogeneity partly analyzes. The N end of mpl ligand sequence, C end or inner Yan stretch, lack or insert and be considered to not Ying sound sequence homogeneity or homology. Yin this, Yi typically has a bit bioactive mpl ligand polypeptide and is considered to have identical sequence, comprises front mpl part Yuan, mpl part Yuan and ripe mpl part.

" microsequencing of mpl part " but the enough sensitive suitable standardization programs of the arbitrary Zu of You complete. The method Zhong of Zai Yi Zhe Yang, the You sds gel or Zui after the high-purity polypeptide Yong that obtains of high performance liquid chromatography (HPLC) the step 470A model Applied Biosystems gas phase sequenator that has been equipped with Yi platform 120A phenylthiohydantoin (PTH) amino-acid analyzer move Edman (phenyl isothiocyanate) direct Sequencing of degrading by Zi. In addition, after You chemicals (as Xiuization cyanogen, Qiang amine, 2-nitro-5-thiocyanic acid phenyl ester) or enzyme (as tryptose enzyme, Collagenase, SP) digestion and purifying (as HPLC), resulting mpl part fragment also can similarly check order. ChromPerfect data system (Justice Innovations, Palo Alto, CA) is used in the amino acid whose analysis of PTH. The people Zai J.Chromatography such as sequence decipher You Henzel, 404:41-52[1987] computer of the VAX 11/ 785 Digital Equipment Co. that mention of Zhong completes. Perhaps, a HPLC Zu can be divided on Zai 5-20%SDS polyacrylamide gel after electrophoresis, electrotransfer is (ProBlott Zhi pvdf membrane, AIB, Foster City, CA), then Yong coomassie brilliant blue staining (Mat-surdiara, J.Biol.Chem., 262:10035-10038[1987]). The specific proteins of differentiating by dyeing cuts out from stain, and Yong gas phase sequenator as above carries out the N end sequencing. Protein sequence for inside, first HPLC Zu is divided empty (SpeedVac) Zhong of Zai Zhen dry, then the buffer solution Zhong Chong that Zai is suitable is outstanding, Zai Yong Xiuization cyanogen, special enzyme Lys-C (the Wako Chemicals of lysine, Richmond, VA) or the special enzyme Asp-N (Boehringer Mannheim, Indianapolis, IN) of aspartic acid digest. After digestion, before the order-checking of the Tai chain Yi form of mixtures that obtains or Zai gas phase order-checking Yi, carry out the HPLC separation Zhong the C4 Zhu of a Zai Yi 0.1%TFA (trifluoracetic acid) propyl alcohol gradient.

The Xue platelet number of " decrease of platelet " Zhi Xue Ye Zhong is less than 150 * 10⁹/ every liter.

" thrombopoietic activity " refers to that Yi Zhong can accelerate megacaryocyte or megacaryocyte precursor Zeng Zhi, differentiation and/or the ripe biologically active that becomes the thrombocytopoiesis form. But this biologically active Yong several different methods is measured, measure as in body, the rebound of mouse Xue platelet is synthetic, Yong Zhen carries out Xue platelet cell surface antigen You to the antiplatelet immunoassays [anti-GPIIbIIIa] of human leukemia megakaryoblast system [CMK] and leads analysis, and the polyploidy You of Yi and Yi megakaryoblast system (DAMI) leads.

" TPO " is (TPO) that Yi Zhong has thrombopoietic activity or can Zeng adds the compound of mammalian blood serum Zhong Xue platelet number. Can add to few 10% endogenous Xue platelet number by Zeng in the better situation of TPO Zai, 50% Ze is better, and under the Zai optimal cases, can make the Xue platelet number of human blood Zhong bring up to 150 * 10⁹On/every liter of Yi.

" the mpl ligand nucleic acid of separation " is Yi section RNA or DNA, wherein contain and be no less than 16, preferably 20 or how sequential nucleotide base, this sequence possesses following features: can encode and have bioactive mpl part and segment thereof, complementary Yu RNA or DNA, or can keep stable bond under Yu RNA or DNA Za friendship and Zai gentleness and the tight condition of Yan. This RNA or this Zhi of DNA Ying are not polluted by the pollution sources nucleic acid of common its combination of Yu of Yi the right Yuan Zhong of Zai Zi less, and preferably are substantially free of other mammalian rnas or DNA. Here, " Zhi is not polluted from the pollution sources nucleic acid of its combination by Yi less usually " comprises following situation: nucleic acid is present in Yuan Zhong or the right cell Zhong of Zi but chromosome position of living in is different, or be arranged in flank, usually Zai source cell Zhao less than nucleotide sequence. Yi example of the mpl ligand nucleic acid that separates is Yi section RNA or DNA, and coding has bioactive mpl part, and is identical Yu the sequence of the mpl of people, mouse or Zhu part Zhi rare 75%, preferably Zhi few 80%, more preferably Zhi lacks 85%, and also Yao is that 90%, Zui is 95% goodly goodly.

" control sequence " Yong Yu relates to while expressing, and is illustrated in Yi specific host You body Zhong, is that the Yi section operationally is connected in coded sequence and this coded sequence is expressed necessary DNA sequence. The control sequence that is suitable for the Yuan nucleus comprises promoter, optional certain operon sequence, ribosome bind site, may also have the modern unknown sequences of a little Zhi of Yi. Control sequence You promoter, polyadenylic acid signal and the enhancer of Zhen nucleus Yi Zhi.

" be operably connected " while relating to nucleic acid, Yi this nucleic acid of of distinguishing the flavor of is in position relevant on function to another nucleotide sequence. For example, if the precursor protein that certain DNA expresses has participated in the secretion of polypeptide, Zhe DNA that expresses presequence or secretion leader is operably connected with regard to the DNA Yu expressing this polypeptide so; If Yi promoter or enhancer ring the Zhuan record You Ying of coded sequence, it is to be operably connected Yu this coded sequence; If perhaps Yi ribosome bind site is placed in Yi promotion translation on Yi coded sequence, between Zhi them, also is operably connected so. Usually, " be operably connected " and Yao ask adjoining between Zhi the DNA sequence dna that is connected. , for the secretion leader, ask adjoining and be in readable state. But it is contiguous that enhancer might not be asked. Connection is to have linked by suitable restriction site. If the site of Zhe Yang does not exist, Ze completes according to routine techniques Yong synthetic oligonucleotides adapter or joint.

During certain Yuan spare of " external source " Zhi, expression comes from extracellular nucleotide sequence, perhaps Yu cell homology, but a certain position Zhong of Zai host cell nucleotides can not find this Yuan spare usually.

" cell ", " clone " and " cell culture " be used interchangeably in this article, and this definition has comprised all sub-generation of Yi cell or clone. Yin this, the term that is similar to " Zhuanization body " and " transformant " has comprised that Yuan godmother cell and Yan thereof give birth to culture, and does not consider the number of times that goes down to posterity. Ying be somebody's turn to do understanding, You Yu You leads or spontaneous mutation, not all identical DNA sequence dnas of Zi generation You. Those have Yuan beginning transformant identical function and the same also of bioactive sudden change offspring that Yu filters out and are included. When attempting certain special representation of Zhi, this word based on context content is understood.

" Zhi grain " is the ring-shaped DNA molecule of Yi Zhong energy self-replacation, and it has independently replication origin. Herein, Yong " p " heel capitalization and/or numeral represent. Zui archiplasm grain source herein is to obtain or program disclosed according to Yi builds as basis take above-mentioned two Zhong Zhi grains from public approach in commercially available, the hard-core situation of Zai. In addition, other suitable Zhi grains are as known in the art, and for the technical staff as Yi, are apparent.

" restriction enzyme digestion ", for DNA, is illustrated under the catalytic action of Yi Zhong enzyme, the di-phosphate ester bond fission of DNA inside, and the Zuo Yong Zhi of enzyme is limited to the specific site on DNA sequence dna. This enzyme is called " restriction endonuclease ". The special DNA sequence dna of each restriction endonuclease identification Yi section, i.e. " restriction site " of dual symmetry. Various restriction enzyme used herein can be purchased, and can use according to reaction condition, confactor and other conditions that supplier provides. Usually, the following abbreviated form of restriction enzyme Yong represents: other Zi matrixs of first capitalization heel show microorganism, therefrom obtain restriction enzyme, are then Yi the specific enzymes of numeral. The condition of restriction enzyme digestion is generally, and Zai is 20 microlitre cushioning liquid Zhong Yue, and 1 microgram Zhi grain or DNA fragmentation add Yue 1-2 unit's enzyme. It is fixed that the suitable buffer that specific restriction enzyme is used as and the amount You Zao business processed of substrate are Zhied. Conventional step is 37 ℃ of incubations Yue 1 hour, but Ying is changed according to supplier's suggestion. After incubation, proteins and peptides Yong phenol and chloroform extracting are removed, and the method for digested nucleic acid Ze Yong Yi alcohol precipitation reclaims from buffer solution. After restriction enzyme digestion, can 5 ' terminal phosphate be hydrolyzed and be used as the Yong bacterial alkaline phosphatase, Yi prevents two restriction enzyme simple stage property ends " cyclisation " of Yi DNA fragmentation or forms Yi closed hoop and the insertion of other DNA fragmentations of Zu Zhi Zai restriction site. Unless what You was other Yao ask, otherwise do not need to carry out the dephosphorylation of 5 ' end after Zhi grain digestion. Dephosphorylized conventional steps and reagent can be referring to the Zhu of the people such as Sambrook institute " molecular cloning: laboratory manual " Zhong 1.56-1.61 parts [New York:Cold Spring Har-bor Laboratory Press, 1989].

After restriction enzyme digestion, " recovery " of DNA fragmentation or " separation " refer to by polyacrylamide or agarose electrophoresis, digestion product be separated, differentiate the purpose fragment according to the marker DNA fragment comparative electrophoresis mobility Yu Yi Zhi molecular weight, cut out the gel piece that contains the purpose fragment, and the DNA of this gel piece Zhong is separated. Zhe program Yi is through being held by Zhang widely. For example, can be referring to people such as Lawn, Nucleic Acids Res., 9:6103-6114[1981] people such as Yi and Goeddel, Nucleic Acids Res., 8:4057[1980].

" Southern analysis " or " Southern blotting " be Zhe Yang Yi Zhong method: DNA or the Zu compound that contains DNA after digestion with restriction enzyme, the existence of handing over the real DNA sequence dna of Zheng by the oligonucleotides Yu known mark or DNA fragmentation Za. The step that Southern analyzes is for common Yi time: agarose gel electrophoresis DNA isolation digestion product, make the DNA sex change after electrophoretic separation, and the DNA Zhuan that will obtain moves on on nitrocellulose filter, nylon membrane or other suitable films, analyzes the hybridisation events of penetrating property of Yu mark, biotin labeling or enzyme labelled probe. Referring to people Zhu Zuo (the same) Zhong 9.37-9.52 parts such as Sambrook.

" Northern analysis " or " Northern blotting " Ze are that Yi Zhong is by the method Yu Yi known probe such as oligonucleotides, DNA fragmentation, cDNA and fragment thereof or RNA fragment Za friendship evaluation RNA sequence. But the mark Yong radio isotope of probe (as³²P), biotin or enzyme. Usually, after agarose or polyacrylamide gel electrophoresis separation, RNA to be analyzed is transferred to Yu Probe Hybridization on nitrocellulose filter, nylon membrane or other suitable films. The standard technique of using is widely Zhi road of this area Zhong. 7.39-7.52 part referring to people Zhu Zuo (the same) Zhong such as Sambrook.

" connection " is to form the process of phosphodiester bond between two nucleic acid fragments. The connection request of two fragments end each other mates mutually. In some situation of Zai, the coupling between end can connect realization by the digestion Zhi of restriction endonuclease. But after Zai restriction endonuclease digestion Yi, the staggered end Zhuan that must will usually produce becomes blunt end and make it more appropriate to connect. For producing blunt end, must be with 15 ℃ of Yu suitable buffer Zhong of DNA Zai, Yong 10 unit DNA polymerase i Klenow fragments or T4 archaeal dna polymerase and Zai 4 Zhong deoxyribonucleoside triphosphates exist down to few Zuo Yong 15 minutes. Then, the extracting of Yong phenol-chloroform and Yi alcohol precipitation are carried out purify DNA. The molal quantity ground Zhi Yu solution Zhong such as DNA fragmentation with Yong Yu connection. Solution Zhong also Ying contains atriphos (ATP), ligase buffer solution and Yi Zhong ligase such as T4 DNA ligase, and its content Yue every 0.5 micrograms of DNA adds Yue 10 units. When certain Zai body was connected, first Yao made the linearisation of Zai body by the digestion of suitable restriction enzyme as DNA. Then, Yong bacterium phosphatase or calf small intestine phosphatase are processed the linearisation fragment, and Yi prevents that Zai connection procedure Zhong from the Zi body occurring connect.

" Zhi is standby " DNA represents isolated plasmid dna from the host cell culture from cell. Extensive and small-scale Zhi grain Zhi is standby is the DNA Preparation Method processed of commonly using, referring to the 1.25-1.33 part of people Zhu Zuo (the same) Zhong such as Sambrook. But the DNA Yong this area Zhong after Zhi the is standby widely method in Zhi road carrys out purifying, referring to 1.40 parts of people Zhu Zuo (the same) Zhong such as Sambrook.

" oligonucleotides " is synthetic strand or double-stranded polydeoxyribonucleotide short chain of Yong known chemistry. The chemical synthesis process You of Yi Zhi: use solid phase technique (referring to the EP 266 that delivered on May 4th, 1988,032) or by thing between dezyribonucleoside H-Lin acid esters Zhong (referring to people such as Froehler, Nucl.Acids Res., 14:5399-5407[1986]) phosphotriester, phosphite or phosphoramidite chemical method. Other method You: the oligonucleotides on the moving primer method Yi of polymerase chain reaction as described below and other Zi and solid support is synthetic. The narration of all these methods sees the people such as Engel, Agnew.Chem.Int.Ed. Engl., 28:716-734 (1989). If the Zheng of known nucleotide sequence or obtained the complementary nucleic acid sequence of coding strand, just can use said method. Another kind selects Ze to be, if Yi Zhi target amino acid sequence, Ze can infer potential nucleotide sequence by the best codon corresponding according to each amino acid residue. Oligonucleotides after synthetic carries out purifying by polyacrylamide gel.

" polymerase chain reaction " or abbreviation " PCR " are that Yi Zhong is with process or the technology of the specific nucleic acid of trace (RNA and/or DNA) amplification. Referring to the U.S. Patent number 4,683,195 of announcing on July 28th, 1987. Usually, need to obtain the sequence situation from end or the end outside of section interested, Yi designs Oligonucleolide primers. The Yin thing is identical or similar to the complementary strand sequence of template to be amplified, makes 5 ' terminal nucleotide of two Yin things to combine Yu amplification material end. PCR can enough specific RNA sequences that increases, a Zheng specific DNA sequence dna of genomic DNA Zhong, record cDNA, bacteriophage or the plasmid sequence etc. that obtain from Zong cell RNA Zhuan. Referring to people such as Mullis, Cold Spring Harbor Symp.Quant. Biol., 51:263 (1987); The people such as Erlich edit, PCR Technology, (Stock-ton Press, NY, 1989). In this article, PCR is considered to the nucleic acid polymerase reaction method of Yi Zhong (but not being unique Yi Zhong) amplification of nucleic acid specimen, and the nucleic acid Zuo that comprises Yong Yi Zhi is that Yin thing and Yong Yi Zhong nucleic acid polymerase increase or produce specific nucleic acid fragment.

Wash under " the tight condition of Yan " Zhi (1) Zai LIS and high temperature, 0.015M NaCl/0.001SM natrium citricum/0.1% dodecyl sodium sulfate (SDS) during as 50 ℃, perhaps (2) Zai crossover process Zhong uses the denaturants such as formamide, as the formamide Zhong of 50% (v/v) under 42 ℃, contains the sodium phosphate buffer of 0.1% bovine serum albumin(BSA)/0.1%Ficoll/0.1% polyvinylpyrrolidone/50mM pH6.5 and 750mM NaCl, 75mM natrium citricum; You is as 50% formamide, 5 * SSC (0.75M NaCl under 42 ℃, 0.075M natrium citricum), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5 * Denhardt ' s solution, the Gui of ultrasonic processing essence DNA (50 ug/ml), 0.1%SD and 10% dextran sulfate, and use 0.2 * SSC and 0.1%SDS washing under 42 ℃ of Zai.

Wash solution and hybridization conditions that the Zhi such as " Zhong the tight condition of Yan " uses are lower than the tight condition of above-mentioned Yan, and (as temperature, ionic strength and SDS percent concentration), referring to people Zhu Zuo (the same) such as Sambrook. Yi example of the tight conditions of Yan such as Zhong is that 37 ℃ of the solution Zhong that Zai contains composition under Yi are incubated overnight, the shearing Gui of 20% formamide, 5 * SSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 * Denhardt ' s solution, 10% dextran sulfate and 20 ug/ml sex change essence DNA, wash filter membrane under 37-50 ℃ of Yong 1 * SSC Zai subsequently. Those skilled in the art understand how according to similar conditions such as temperature, ionic strength of adjusting Yin element such as probe length.

" antibody " is (Abs) glycoprotein of You same structure feature with " immunoglobulin (Ig) (Igs) ". Antibody is used as to specific antigen You specific binding, and immunoglobulin (Ig) comprises antibody and other Yu antibody class like but there is no the molecule of antigentic specificity. For example, the latter's the low-level generation of polypeptide chain You lymphatic system Yi and You myeloma Yi enhanced level produce.

" natural antibody and immunoglobulin (Ig) " be 150,000 daltonian Yi Yuan tetramer glycoprotein Yue normally. Its two identical light chain of You (L) and two identical Chong chains (H) form. Every the L chain all is combined by covalent disulfide bonds Yu Yi bar H chain, and the disulfide bond number between Zhi two Chong chains of different Immunoglobulin Isotypes is different. Every light chain and Chong chain be the regularly spaced intrachain disulfide bond bridge of You separately. Yi variable region (V of Yi end You of every Chong chain_H), the some constant regions of heel. Yi variable region (V of Yi end You of every light chain_L), Yi constant region of other end You. The constant region of light chain Yu first constant region of Chong chain side by side, and the variable region of light chain Yu the variable region of Chong chain side by side. Believe You Yi interface that the You particular amino acid residue forms between Zhi the variable region of Zai light chain and Chong chain. (referring to people such as Clothia, J.Mol.Bi-ol., 186:651-663[1985]; Novotny and Haber, Proc.Natl.Acad. Sci.USA, 82:4592-4596[1985]).

The specific part sequence of the various antibody variable regions of term " variable " Zhi is generally different, thereby be used as making between every specific antibodies and its specific antigen, produces specific binding. Yet, the variable region Zhong of Zai antibody, the changeability of sequence is not what be evenly distributed. Changeability concentrates on three sections that are called complementary determining region (CDRs) or hypervariable region of every light chain and variable region of heavy chain. Variable region higher conservative part Ze is called framework (FR). Every natural light chain and four Zhu of Chong chain You Yao be the FR district of beta sheet configuration, three of You circlewise or some situation of Zai under the CDRs district of forming section beta sheet structure connect. The CDRs district of every chain closely flocks together by the FR district, and with the CDRs of other chains, form the antigen binding site of antibody (referring to the Sequneces of Proteins of Immuno-logical Interest of the Zhu of the people such as Kabat institute, National Institute of Health, Bethesda, MD [1987]). Constant region not Zhi connects the combination that participates in antigen and antibody, but can show multiple effector function, as the participation of the bad cell toxicant Zhong antibody of Zai antibody Yi.

Behind papain digestion antibody, produce two identical Fabs " Fab " and remnants " Fc " fragment.Each Fab fragment has an antigen binding site, and the Fc fragment is easy to crystallization.Pepsin antibody then obtains " a F (ab ') ₂" fragment, have two antigen binding sites, and still can crosslinked antigen.

" Fv " is the minimum antibody fragment that has complete antigen recognition and binding site.This is a kind of variable region and dimer of forming by non-covalent combination closely of the variable region of a heavy chain by a light chain.In its configuration, at V _H-V _LDimeric surface, three CDRs of each variable region interact and form an antigen binding site.These six CDRs have determined the antigen-binding specificity of antibody jointly.But, even single variable region (or be specific to half Fv that certain antigenic CDRs form by three) also have the ability of identification and conjugated antigen, though its affinity is lower than whole binding site.

The Fab fragment has also comprised the constant region of light chain and first constant region (CH1) of heavy chain.Fab ' fragment and segmental different being of Fab, the former the C-terminal in heavy chain CH1 district many several residues, comprise one or more halfcystines from antibody hinge region.Fab '-SH represents to carry a free sulfydryl on the halfcystine of Fab ' constant region in this article.F (ab ') ₂Antibody fragment has the Fab ' form of halfcystine hinge to produce with a pair of centre at first.In addition, also have some chemical couplings between antibody fragment.

According to the aminoacid sequence of constant region, " light chain " from vertebrates antibody (immunoglobulin (Ig)) can be divided into two classes clearly: κ type and λ type.

According to the aminoacid sequence of CH, immunoglobulin (Ig) can be divided into different types.Mainly contain five kinds of immunoglobulin class: IgA, IgD, IgE, IgG and IgM, what wherein have can also be further divided into subclass (isotype), as IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2.The constant region of dissimilar heavy chain immunoglobulins then is called respectively accordingly: α, δ, ε, γ and μ.The subunit structure and the 3-d modelling of different immunoglobulin classes have been known quite clearly.

Term " antibody " is during from broadest use, wherein comprises single-minded monoclonal antibody (comprising stimulant and antagonist antibodies) specially, has the specific antibody compositions of multi-epitope and antibody fragment (as Fab, F (ab ') ₂And Fv), as long as they possess desired biological activity.

Term " monoclonal antibody " refers to certain antibody of obtaining in this article from a basic homologous antibody population.Promptly except the spontaneous mutation of minute quantity, each antibody that constitutes colony is all identical.Monoclonal antibody high special ground is directly corresponding to single-minded antigen site.And, being different from conventional (polyclone) antibody preparations, these prepared products generally comprise directly at a plurality of antibody of a plurality of antigenic determinants (epi-position), and every kind of monoclonal antibody is directly at the single-minded determinant on the antigen.Except their specificity, the superiority of monoclonal antibody also is embodied in and can cultivates to synthesize by enough hybridomas, and can not polluted by other immunoglobulin (Ig)s.Modifier " monoclonal " has illustrated that the characteristic of this antibody is the antibody population that comes from basic homogeneous, and does not represent any special method of acquisition needs of antibody.For example, the monoclonal antibody of using among the present invention can use hybridoma technology (at first by Kohler ﹠amp; Milstein is at Nature, 256:495[1975] in describe) or recombinant DNA technology be prepared (referring to U.S. Patent number 4,816, people such as 567[Cabilly]).

Monoclonal antibody herein also comprises " chimeric " antibody (immunoglobulin (Ig)) especially.The chimeric antibody heavy chain is or/and the part on the light chain and the identical or homology of corresponding sequence from antibody a certain specific species or that belong to a certain specific antibodies class or subclass; And remaining another part with from another specific species or belong to the identical or homology of corresponding sequence of the antibody of another specific antibodies class or subclass.The fragment of chimeric antibody also has this situation, as long as they can show required biological activity.(referring to people such as people's such as Cabilly U.S. Patent number 4,816,567 and Morrison, Proc.Natl.Acad.Sci.USA, 81:6851-6855[1984]).

" humanization " form of non-human (as mouse) antibody is that a kind of gomphosis immunoglobulin, immunoglobulin chain or its fragment are (as Fv, Fab, Fab ', F (ab ') ₂Or other antigens of antibody are in conjunction with subsequence), wherein have few non-human immunoglobulin sequences.Usually, humanized antibody is human normal immunoglobulin (receptor antibody), wherein the residue of the complementary determining region of acceptor (CDR) is replaced by the residue of the CDR of other species (donor antibody), and as the residue of the CDR of mouse, rat or rabbit, they have required specificity, affinity and capacity.Sometimes, the Fv framework residue of human normal immunoglobulin is also replaced by corresponding non-human residue.Even humanized antibody also can contain neither from the also non-residue from donor CDR or frame sequence of receptor antibody.This class is modified the function that can further improve and optimize antibody.Usually, humanized antibody has comprised all variable region (at least one, general two), wherein whole or most CDR district is corresponding to the CDR district of non-human immunoglobulin (Ig), and whole or most FR district is the FR district of human normal immunoglobulin consensus sequence.Humanized immunoglobulin preferably also has at least a portion constant region for immunoglobulin (Fc), generally is the human normal immunoglobulin constant region.Further details can be referring to people such as Jones, Nature, 321:522-525[1986]; People such as Reichman, Nature, 332:323-329[1988] and Presta, Curr.Op.Struct.Biol., 2:593-596[1992].

" non-immunogenic in the human body " refers to that the polypeptide that will be present in the pharmaceutically acceptable carrier maybe contacts with the dosage that the treatment effect is arranged this polypeptide with suitable tissue, behind one section suitable hide these (as 8 to 14 day), use this polypeptide once more, do not have supersensitivity and the resistance state of appearance at this polypeptide.II. preferred example of the present invention

The present invention preferentially selects for use some to be called as the basic homologous polypeptide of mpl part or thrombopoietin (TPO).A member of these polypeptide and recipient cell factor superfamily, mpl has binding characteristic, and have the Nucleotide that promotes mark ( ³The H-thymidine) mixes biological nature in Ba/F3 cell DNA personnel selection mpl P transfection, that IL-3 relies on.The mpl part of more preferably selecting for use is separated mammalian proteins matter, and it has hematopoiesis, and especially megalokaryocyte generates or thrombopoietic activity; That is to say, can promote immature megalokaryocyte or megalokaryocyte precursor propagation, maturation and/or be divided into sophisticated thrombocyte generation form.The polypeptide that override of the present invention is selected for use is people mpl part and the fragment thereof that hematopoiesis, megalokaryocyte generation or thrombopoietic activity are arranged.Selectively make these people mpl parts non-glycosylated.Other the people mpl parts that can preferentially select for use have: be called as hML ₁₅₃Or hTPO ₁₅₃HML " EPO " structural domain; Be called as hML ₂₄₅Or hTPO ₂₄₅The hML clipped form; Have shown in Fig. 1 (SEQ ID NO:1) aminoacid sequence, be called as hML, hML ₃₃₂Or hTPO ₃₃₂Ripe full-length polypeptide; And the displacement varient hML (R153A, R154A) of biologically active.

The polypeptide that the present invention can preferentially select has biological or immunocompetent mpl ligand variant body in addition, and they are selected from hML2, hML3, hML4, mML, mML2, mML3, pML and pML2.

The polypeptide that the present invention can preferentially select also has the mpl ligand variant body of biologically active, itself and following mpl part relatively have at least 70% aminoacid sequence identical: people mpl part (seeing Fig. 1 [SEQ ID NO:1]), mouse mpl part (seeing Figure 16 [SEQID NOS:12 and 13]), reorganization pig mpl part (seeing Figure 19 [SEQ ID NO:18]) or from the isolating mpl part of undergrown porcine blood plasma.Homology reach at least 75% better, reach at least 80% and also will get well, reach at least 85% better again, reach at least 90% even also will get well, reach at least 95% the best.

From the isolating mpl part of undergrown porcine blood plasma following characteristics are arranged:

(1) partially purified this part is with phosphate buffered saline buffer (PBS) wash-out from gel filter post, and its molecular weight is 60,000-70, and 000, PBS or contain 0.1%SDS, or contain 4MMgCl ₂

(2) activity of this part can be destroyed by PRONASE A;

(3) this part keeps stable when low pH value (2.5), 0.1%SDS and 2M urea;

(4) based on combining with multiple lectin post, this part is a kind of glycoprotein;

(5) molecular weight 25,000-35, this part of 000 high purity can be from irreducibility SDS-polyacrylamide gel wash-out.During a small amount of actives, the molecular weight of wash-out is about 18,000-22,000 and 60,000;

(6) molecular weight 28,000-31,000 high purity part in reductibility SDS-polyacrylamide gel with the doublet isolated in form;

(7) 18,000-22, the N-terminal sequence of 000,28,000 and 31,000 bands is the same: SPAPPACDPRLLNKLLRDDHVLHGR (SEQ ID NO:29);

(8) following affinity column can supply part combination and wash-out:

Blue-Sepharose，

CM Blue-Sepharose，

MONO-Q，

MONO-S，

LcA-Sepharose,

Wheat germ agglutinin (WGA)-Sepharose,

Concanavalin A-Sepharose,

Ether 650m Toyopearl,

Butyl 650m Toyopearl,

Phenyl 650m Toyopearl and

Phenyl-Sepharose.

The mpl ligand polypeptide that more can preferentially select for use is by the people's gene group or have the cDNA coding of aminoacid sequence shown in Fig. 1 (SEQ IDNO:1).

Other mpl ligand polypeptides with natural bioactive that the present invention preferentially selects for use comprise: preceding mpl part is former, the mpl part is former, sophisticated mpl part, mpl part fragment and glycosylation varient thereof.

Also have the preferred polypeptide of some the present invention, comprise mpl ligand sequence varient and mosaic.Usually, the two all is the mpl ligand variant body of biologically active, its aminoacid sequence have at least 70% with people mpl part or identical from the isolating mpl part of undergrown porcine blood plasma.Homologous sequence reaches at least 75%, 80%, 85%, 90%, 95% respectively, and then preferred rank improves successively.An example of preferred mpl ligand variant body is hML N-terminal structural domain varient (because of its sequence and erythropoietin homology are called " EPO structural domain ").Preferred hML EPO structural domain comprises preceding 153 amino-acid residues of ripe hML, also is known as hML ₁₅₃A kind of alternative preferred hML sequence variant has one or more alkalescence or two alkaline amino acid residue to be substituted by non-alkaline amino acid residue (as: hydrophobic, neutral, tart, aromatic, glycine, proline(Pro) etc.) at the C-terminal structural domain.In a kind of preferred hML C-terminal structural domain sequence variant, the arginine of 153 and 154 positions is substituted by L-Ala, and this varient is called hML ₃₃₂(R153A, R154A).Another preferred hML varient hML ₃₃₂Or hML ₁₅₃, the residue of its 111-114 position (QLPP or LPPQ) lacks or is substituted by other tetrad peptides (as AGAG or similar sequence).Above-mentioned deletion mutantion is known as Δ 4hML ₃₃₂Or Δ 4hML ₁₅₃

The mosaic of preferentially selecting for use is that mpl part and fragment (being defined as follows) thereof merge with heterologous polypeptide and fragment thereof.For example, hML ₁₅₃Can merge to improve serum half-life with the IgG fragment, also can obtain an enhanced thrombopoietic activity or chimeric hematopoletic bioactive molecule with IL-3, G-CSF or EPO fusion.

Another alternative preferred people mpl part mosaic is " a ML-EPO structural domain mosaic ", its constructional feature is that the hML residue of one or more (but not being whole) N-terminal 153-157 position is substituted by people EPO residue (possible arrangement is [SEQ ID NO:7] as shown in figure 10).In the hML mosaic of about 153-166 the residue length of this example, single or combination residue is added into or replaces the hML sequence in the people EPO sequence, and the position of replacement is corresponding to the arrangement shown in Figure 10 [SEQ ID NO:7].The typical composite sequence that inserts hML N end parts is included on 24-27, the 38-40 of EPO and the 83-85 position one or more N-glycosylation sites; Parents matchmaker's property α spiral tube one or more that also comprise the prediction of four 9-22,59-76,90-107 and 132-152 positions that are arranged in EPO; And other comprise the zone of high conservative, as N-terminal district, C-terminal district and 44-52 residue position (EPO) (with reference to seeing people such as Wen, Blood, 82:1507-1516[1993] and people such as Boissel, J.Biol.Chem., 268 (21): 15983-15993[1993]).Expect that this " ML-EPO structural domain mosaic " unite and have thrombocyte to generate the biological activity of an erythropoiesis (TEPO).

Other preferred polypeptides of the present invention comprise mpl part fragment, the continuous sequence that contains at least 10,15,20,25,30 or 40 amino-acid residue length, this sequence and described separation in this article from the sequence of undergrown porcine blood plasma mpl part or people mpl part identical (see Table 14, embodiment 24).A kind of preferred mpl part fragment is people ML[1-X], wherein X represents 153,164,205,207,217,229 or 245 (seeing 1-X residue sequence part among Fig. 1 [SEQ ID NO:1]).Other preferred mpl part fragments comprise chemistry or the enzymolysis or the postdigestive product of the mpl part of those purifying.

The method that another preferred aspect of the present invention is a purifying mpl ligand molecular.The principle of this method is: according to mpl ligand molecular to be purified optionally absorption make immobilized receptor polypeptides such as the mpl ligand sources contact mpl that contains the mpl ligand molecular or mpl fusion polypeptide in immobilized receptor polypeptides; The washing immobilization support is to remove non-absorption material; With elution buffer the molecule on the immobilized receptor polypeptides is eluted then and reach purifying.Perhaps, the mpl ligand sources is a blood plasma, and preferred immobilization acceptor is the mpl-IgG syzygy.

The reconstitution cell culture is as the mpl ligand sources, and the mpl part content in its substratum or the cell lysate will be higher than blood plasma or other natural origins usually.In this case, use the above-mentioned affine method of mpl-IgG immunity still feasible, but also nonessential, available conventional method of purifying protein as known in the art.In brief, the preferred purification process that produces basic homology mpl part comprises: remove small fragments such as host cell or crack fragment with centrifugal or hyperfiltration process; Selectively use the protein that is purchased to concentrate filter membrane and come proteins concentrate; Then, by following one or more steps the mpl part is separated from other impurity: immunity is affine, ion-exchange (as DEAE or contain carboxymethyl or the matrix of sulfonic acid n-propyl group), Blue-Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S; LcA-Sepharose; wheat germ agglutinin-Sepharose; concanavalin A-Sepharose; ether Toypearl; butyl Toypearl; phenyl Toypearl; albumin A-Sepharose; SDS-polyacrylamide gel electrophoresis; reversed-phase HPLC (as the silica gel of band aliphatic group) or dextrane gel molecular sieve size exclusion chromatogram, and ethanol or ammonium sulphate precipitation.All can add proteinase inhibitor such as fluoridizing methylsulfonic acid in any one of above-mentioned steps, with the arrestin hydrolytic action.

In another preferred example, provide a kind of can with mpl part bonded separation antibody.The mpl part separation antibody of preferentially selecting for use be monoclonal (Kohler and Milstein, Nature, 256:495-497[1975]; Campbell, Laboratory Techniquesin Biochemistry and Molecular Biology, people such as Burdon edit, 13 volumes, Elisevier Science Publisrers, Amsterdam[1985]; With people such as Huse, Science, 246:1275-1281[1989]).The affinity of preferred mpl part separation antibody and mpl part should be 10 at least ⁶Liter/mole.The affinity of antibodies reaches 10 ⁷During liter/mole, more should preferentially select for use.The most preferential situation is, antibody is at the mpl part of the above-mentioned effector function of tool and produce.Can be selectively merge with another polypeptide with mpl part bonded separation antibody, this antibody or its syzygy can be used as immobilized mpl polypeptide, with the mpl part from above-mentioned source separation and purification to.In this example was further paid the utmost attention to, the invention provides the method for mpl part in the outer or body of a kind of detection bodies: contact antibody with suspicious sample such as the serum sample that contains part, whether detection combination took place.

In further preferred example, the invention provides a kind of coding mpl part and segmental isolated nucleic acid molecule thereof, this nucleic acid molecule can detect composition with one and mark or remove mark.The present invention also provides a kind of nucleic acid molecule, and its sequence is complementary or can hybridize with it under tight or moderate stringent condition with the sequence of nucleic acid molecules that contains coding mpl part.Preferred mpl ligand nucleic acid is the RNA or the DNA of the mpl part of coding biologically active, its coded product and people mpl part have at least 75% sequence identical, identical sequence reaches at least 80%, 85%, 90%, 95% respectively, and the degree of priority of then selecting for use improves successively.Preferred isolated nucleic acid molecule is the dna sequence dna of the mpl part of coding biologically active, and its source has: (a) the coding region DNA of Mammals mpl ligand gene (as containing the dna molecular and the fragment thereof of nucleotide sequence shown in Fig. 1 [SEQ ID NO:2]); (b) at least can be under the moderate stringent condition and (a) DNA of described DNA hybridization; And (c) because of degeneracy of genetic code resulting (a) or (b) the degeneracy DNA of described DNA.The described herein new mpl part of our expectations is a member of ligand family or cytokine family, has suitable sequence identity, so that be lower than under the moderate stringent condition, their DNA (or its complementary strand or fragment) can be hybridized with the DNA shown in Fig. 1 (SEQ ID NO:2).Therefore, the present invention further aspect comprised and being lower than under the moderate stringent condition, with the DNA of the DNA hybridization of coding mpl ligand polypeptide.

In further preferred example of the present invention, nucleic acid molecule is the cDNA of coding mpl part, is included on the reproducible carrier, and wherein cDNA operationally is incorporated into the control sequence that can discern with this carrier host transformed.This respect has comprised further again with this carrier transformed host cells with cDNA and has produced a kind of method of the mpl part that effect is arranged that this method comprises: the expression of cDNA in the transformed host cell culture of coding mpl part and therefrom reclaiming.The mpl part that obtains with this method is homologous people mpl part substantially preferably.The preferred host cell that produces the mpl part is Chinese hamster ovary (CHO) cell.

The present invention has also further comprised the especially mammiferous method of thrombocyte generation minimizing of a treatment immunological disease or hematopoiesis disease, comprises the mpl part that this Mammals is imposed effective therapeutic dose.Selectively, the mpl part can with cytokine, especially G CFS or interleukin-bonded form administration.Preferred G CFS or interleukin-comprise: complete part, LIF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9 or IL-11.III. preparation method

Some authors think that for a long time hematoblastic generation is the specific humoral factor control by multispectral system.Suppose to think, two kinds of different active cells factors that are called as megakaryocyte colony stimulating factor (meg-CSF) and thrombopoietin regulated that megalokaryocyte generates and the thrombocyte nucleus formation (referring to people such as Williams, J.Cell Physiol., 110:101-104[1982]; People such as Williams, Blood Cell, 15:123-133[1989]; With people such as Gor-den, Blood, 80:302-307[1992]).According to this hypothesis, meg-CSF promotes the propagation of precursor megakaryocytes, and thrombopoietin mainly influences the maturation of more noble cellss and final thrombocyte discharges.Since the sixties, after lot of documents has been put down in writing and the thrombopenia phenomenon occurred, can bring out and form in the blood plasma of animal and human's class, serum and the urine meg-CSF and TPO activity thing (as people such as Odell, Proc.Soc.Exp.Biol.Med., 108:428-431[1961]; People such as Nakeff, Acta Haematol., 54:340-344[1975]; Specter, Proc.Soc.Exp.Biol., 108:146-149[1961]; People such as Schreiner, J.Clin.Invest., 49:1709-1713[1970]; Ebbe, Blood 44:605-608[1974]; People such as Hoffman, N.En-gl.J.Med., 305:533[1981]; People such as Straneva, Exp.Hematol., 17:1122-1127[1988]; People such as Mazur, Exp.Hematol., 13:1164[1985]; People such as Mazur, J.Clin.Invest., 68:733-741[1981]; People such as Sheiner, Blood 56:183-188[1980]; People such as Hill, Exp.Hema-tol., 20:354-360[1992]; And people such as Hegyi, Int.J.Cell Cloning, 8:236-244[1990]).According to report, the pedigree under these two kinds of active factores be different from known cytokine (referring to people such as Hil R.J, Blood 80:346[1992]; People such as Er-ickson-Miller C.L., Brit.J.Haematol., 84:197-203[1993]; People such as Straneva J.E., Exp.Hematol., 20:4750[1992]; And people such as Tsukada J., Blood 81:866-867[1993]).So far, the work of purification meg-CSF and thrombopoietin is not also succeeded from thrombocytopenic blood plasma or urine.

With above-mentioned from thrombopenia blood plasma observed phenomenon consistent, we find that the underdevelopment porcine blood plasma (APP) that the pig that crosses from radiation treatment obtains can generate at stimulated in vitro people megalokaryocyte.We find that this stimulating activity can be stopped by the extracellular soluble outskirt of c-mpl, thereby confirm that APP is a potential source of the general mpl part of being thought (ML).We successfully have been purified into the mpl part from APP, and amino acid sequence information has been used to separate mouse, pig and people MLcDNA.These ML have with the sequence that comes from erythropoietin, and have meg-CSF and thrombopoietin sample activity simultaneously.

1. from blood plasma, identify and purifying mpl part

As above-mentioned, there is report to show that the underdevelopment blood plasma from different plant species has the activity that promotes that in-vitro blood cells generates; But, also do not have to find from blood plasma, to separate the example of the hematopoietic stimulation factor in the former report.A source of underdevelopment blood plasma is the pig through the processing of irradiation property.This undergrown porcine blood plasma (APP) can the stimulated in vitro human blood cell generate.Judge whether contain the mpl part among the APP, can be by measuring ³The H-thymidine mixes the Ba/F3 cell of personnel selection mpl P transfection (step is seen Fig. 2) and analyzes its effect.APP can promote ³The H-thymidine mixes the Ba/F3-mpl cell, rather than Ba/F3 control cells (the mpl P transfection of promptly not choosing).In addition, in normal porcine blood plasma, do not find similar activity.The The above results explanation, APP contains one or more factors, can perhaps be exactly the native ligand of this receptor by proliferation signal of mpl acceptor transduction.Handle APP capable of blocking with solubility mpl-IgG this viewpoint has been supported in the discovery of Ba/F3-mpl cell accelerating effect further.

Actives is a kind of protein among the APP, because PRONASE A, DTT or heating can destroy APP activity (Fig. 3).This actives also can not be dialysed.But it is stable that this actives keeps when low pH value (pH2.5,2 hours), and adsorb and wash-out on some lectin affinity columns, and demonstrating is a kind of glycoprotein.For further clear and definite this actives structure and characteristic thereof, can utilize the mpl-IgG mosaic from APP, to carry out affinity purification.

The treatment process of APP can be according to the scheme among embodiment 1 and the embodiment 2.In brief, the mpl part can use hydrophobic interaction chromatogram (HIC), fixed dye chromatogram and mpl-affinity chromatography to come purifying.Reclaim each step of this actives and see Fig. 4, the purifying multiple in each step sees Table 1.By the mpl-affinity column, the rate of recovery about 10% that this actives is total.The actives peak value component (F6) of mpl-affinity column wash-out has an appointment 9.8 * 10 ⁶The activity of unit/milligram.5 liters of APP purifying about 4 * 10 ⁶(from 0.8 unit/milligram to 3.3 * 10 ⁶Unit/milligram), protein content has then diluted 83 * 10 ⁶Doubly (restraining 3 micrograms) from 250.Estimation ligand specificity's activity of wash-out from the mpl-affinity column is about 3 * 10 ⁶Unit/milligram.

Table 1

The purifying of mpl part

Sample	Volume mls	The protein mg/ml	Units per ml	Unit	Specific activity unit/milligram	Output %	The purifying multiple
								APP	5,000	50	40	200,000	0.8	-	1
Phenyl	4,700	0.8	40	200,000	50	94	62
								Biue-Sep. mpl (liter)	640	0.93	400	256,000	430	128	538
(Fxns)5-7)	12	5×10 -4	1666	20,000	3,300,000	10	4,100,000

Measure definite protein by Bradford.The estimation of the protein concn of mpl eluate component 5-7 is to dye painted intensity in the sds gel according to silver.A unit definition is to cause maximum promoted 50% of Ba/F3-mpl cell proliferation.

Under reductive condition, analyze the component of wash-out in the mpl-affinity column, can find to exist the some protein (see figure 5) by SDS-polyacrylamide gel electrophoresis (4-20%, Novex gel).It is about 66,000,55,000,30,000,28,000 and 18 to dye the protein molecular weight of demonstration with high strength silver, 000-22,000.For proving which protein has promoted the propagation of Ba/F3-mpl cell culture, can as described in the embodiment 2 with they wash-outs from gel.

This experimental result shows that most actives molecular weight that elute are 28 from proteinaceous gel thin slice, 000-32, and 000, and molecular weight 18,000-22, the actives of 000 (Fig. 6) is less.A visible protein molecular weight is 30,000,28,000 and 18 in these zones, 000-22,000.In order to identify and obtain the sequence of isolating protein in this gel area (promptly 30,28 and the band of 18-22 kilodalton), these protein, and are checked order with method as described in embodiment 3 on PVDF by electroblotting.The N-terminal sequence that obtains is as shown in table 2.

Table 2

Mpl part N-terminal sequence

30 kDa 1 5 10 15 20 25 (S)P A P P A(C)D P R L L N K L L R D D (H/S)V L H (G)R L	(SEQ ID NO：30)
		28 kDa 1 5 10 15 20 25 (S)P A P P A X D P R L L N K L L R D D (H)V L(H)G R	(SEQ ID NO：31)
18-22 kDa 1 5 10 X P A P P A X D P R L X (N) (K)	(SEQ ID NO：32)

Computer-aided analysis shows that these aminoacid sequences are newfound.Because all these three kinds of sequences all are identical, can think this 30 kilodalton, the protein of 28 kilodaltons and 18 kilodaltons is relevant, perhaps is exactly multi-form with a kind of new protein.And this protein may be exactly a kind of mpl part that exists naturally.Because this actives can (28,000-32,000) separate in the same zone of the SDS-of 4-20% polyacrylamide gel electrophoresis.In addition, when with Superose 12 (Pharmacia company) when post is made gel filtration chromatography, partially purified part and 17,000-30, the material of 000 molecular weight moves together.Believe that the different molecular weight form of part is the result of proteolysisization or glycosylation difference, or the result that modification caused before and after other translations.

As previously mentioned, antisense people mplRNA can stop enrichment CD34 ⁺Megalokaryocyte in people's marrow culture of parent cell generates, but does not influence differentiation people's (the same) such as () Methia of other hematopoietic cell lineages.This result represents that perhaps the mpl acceptor plays a role in Megakaryocytic in-vitro multiplication and atomization.In order further to illustrate the effect of mpl part in megalokaryocyte generates, the effect of APP in vitro human megalokaryocyte generative process that can compare APP and remove the mpl part.The effect that APP generates for people's megalokaryocyte generates of analyzing by the liquid suspension megalokaryocyte and improves one's methods and measure, and embodiment 4 is seen in the description of method.During this is analyzed, at the front and back of mpl-IgG affinity chromatography APP handler's peripheral stem cell (PSC).GPII _bIII _aThe promoter action that megalokaryocyte is generated is by antibody ¹²⁵I-anti-II _bIII _aCome quantitative (see figure 7).As shown in Figure 7,10% APP can cause about 3 times promoter action, but the APP that removes the mpl part does not have effect.Obviously, the APP that removes the mpl part can not induce the propagation of Ba/F3-mpl cell.

In another experiment, respectively the 0th, 2, soluble human mpl-IgG added in 4 days and to contain in the culture of 10%APP, can in and APP promoter action (see figure 8) that people's megalokaryocyte is generated.These results show that the mpl part plays a role in the adjusting that people's megalokaryocyte generates, therefore also may be useful to the treatment of thrombocytopenia.

2.mpl the molecular cloning of part

According to from 30 kilodaltons, the amino terminal amino acid sequence (seeing above-mentioned table 2) that 28 kilodaltons and 18-22 kilodalton protein obtain has designed and has used two degeneracy oligonucleotide primer assembly-uses in by pcr amplification pig genomic dna.Can think quite reasonablely that if the amino terminal amino acid sequence is encoded by single exon, the length of so correct PCR product should be 69bp.Found that the dna fragmentation of this length and subclone are to pGEMT.PCR Oligonucleolide primers and three cloned sequences that obtain see embodiment 5.Be equal to sequence (the 9-17 residue of 28 and 30 kilodalton pig protein sequences shown in seeing above) fully by the aminoacid sequence of the peptide of part coding between the PCR primer (PRLLNKLLR[SEQ ID NO:33]) to obtaining behind the pig part N-terminal protein sequencing.

A kind ofly be used to screen the human gene group DNA library according to PCR fragment sequence synthetic oligonucleotide.An oligonucleotide that is called 45 aggressiveness of pR45 designs and synthesizes out according to the PCR fragments sequence.This oligonucleotide has following sequence: 5 ' GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-TTT-ATT-TAG-GAG-TCG 3 ' (SEQ ID NO:34)

With reference to embodiment 6, this deoxy-oligonucleotide is used to screen the human gene group DNA library that is implemented in the λ gem12 phage under low tight hybridization and wash conditions.Choose positive colony, the purifying plaque is analyzed with restriction map and southern blotting.390bp EcoRl-XBal fragment with the hybridization of 45 aggressiveness is subcloned on pBluescriptSK-.This clone's dna sequencing shows that the DNA of people's homologue of coding pig mpl part is separated.Human DNA sequence and aminoacid sequence such as Fig. 9 (SEQ ID NO:3 and 4) of inferring according to this.The predicted position of intron is represented by arrow in the genome sequence, and marks the exon (" exon 3 ") of a deduction.

Synthesized oligonucleotide according to people's " exon 3 " sequence corresponding to exon 3 ' end and 5 ' terminal sequence.From the different people tissue, prepare template DNA, carry out the PCR reaction with these two primers.The size of desired correct PCR product is 140bp.After in 12% polyacrylamide gel electrophoresis, analyzing the PCR product, can detect the big or small dna fragmentation of expectation in the cDNA library of adult's kidney, 293 fetal kidney cell constructions and among the cDNA of human fetal liver preparation.

Then, screen the fetal livers cDNA library (7 * 10 that is implemented in the λ DR2 phage with 45 same aggressiveness oligonucleotide ⁶Individual clone), this 45 aggressiveness oligonucleotide is used for screening people's gene group library and fetal livers cDNA library under low tight assorted also condition.Select positive colony, the purifying plaque, and measure to insert segmental size with PCR.Selection contains 1.8kb and inserts segmental clone with for further analysis.Utilization embodiment 7 described programs, the aminoacid sequence that obtains the nucleotide sequence of people mpl part (hML) and infer according to this.These sequences see Fig. 1 (SEQ ID NOS:1 and 2).

3. the structure of people mpl part (hML)

People mpl part (hML) cDNA sequence (Fig. 1 [SEQ ID NO:2]) is made of 1774 Nucleotide, and a poly A tail is arranged.It comprises 3 of 5 of 215 Nucleotide ' end non-translated sequence and 498 Nucleotide ' end non-translational region.Supposition initiator codon at nucleotide position 216-218 place is positioned at the consensus sequence of the initial requirement of eukaryotic translation.Open reading frame originates in nucleotide position 220, has 1059 Nucleotide, and code length is the polypeptide chain of 353 amino-acid residues.The N-terminal height of this predicted amino acid sequence is hydrophobic, may be corresponding to certain signal peptide.The aminoacid sequence of prediction is carried out Computer Analysis (people such as vonHeijne, Eur.J.Biochem .133:17-21[1983]) be presented between residue 21 and 22, the potential cleavage site of a signal peptidase is arranged.Can produce the mature polypeptide of 332 amino-acid residue length in this locational cutting, its starting point is the N-terminal sequence of purifying from the mpl of porcine blood plasma part.Predict these 332 amino-acid residue length parts without about 38 kilodaltons of glycosylation molecular weight.Have 6 potential glycosylation sites and 4 cysteine residues.

Mpl ligand sequence and Genbank sequence library are relatively found: 23% identical (Figure 10 [SEQ ID NOS:6 and 7]) arranged between aminoterminal 153 residues of sophisticated people mpl part and erythropoietin (hEPO).If the consideration preservative replacement, hML and hEPO reach 50% in this regional similarity.HML and hEPO have 4 halfcystines.Wherein, comprise that 3 in 4 halfcystines of first and last are guarded in hML.The site-directed mutagenesis experimental result shows, first of erythropoietin and the needs formation disulfide linkage (Wang, people such as F.F., Endocrinology 116:2286-2292) of last halfcystine according to its function.Similarly, first of hML may also form an important disulfide linkage with last halfcystine.In hML, there is not conservative glycosylation site.All potential N-glycosylation sites of hML polypeptide all are positioned at half place of nearly C-terminal.

Similar with hEPO, the both not total polyadenylic acid sequence A AUAAA of hML mRNA does not have regulatory element AUUUA yet, AUUUA appears at 3 of many cytokines ' end non-translational region, and be considered to influence the stability (people such as Shawet, Cell, 46:659-667[1986]) of mRNA.The analysis of Northern blotting has shown that single 1.8kb hML rna transcription thing level is low in fetus and adult liver.After the long period exposure, can in adult's kidney, detect the more weak band of identical size.Through comparing, people's erythropoietin is expressed in fetal livers, and when hypoxemia occurs, in adult's kidney and liver, express (people such as Jacobs, Nature, 313:804-809[1985] and people such as Bondurant, Molec.Cell.Biol., 6:2731-2733[1986]).

The importance in hML C-terminal district is still waiting further elaboration.According to the existence of 6 potential N-glycosylation sites and this part can with lectin affinity column bonded characteristics, this zone of hML may be by glycosylation.In the experiment of some gel wash-outs, it is about 60,000 that we observe molecular weight that actives differentiates, perhaps represented the glycosylation molecule of total length.Therefore, perhaps the C-terminal district has play a part to stabilize and increase the round-robin hML transformation period.With regard to erythropoietin, it has complete biological activity without glycosylated form external, but compares with the glycosylation erythropoietin, and the transformation period of blood plasma obviously shortens (people such as Takeuchi, J.Biol.Chem., 265:12127-12130[1990]; People such as Narhi, J.Biol.Chem., 266:23022-23026[1991] and people such as Spivack, Blood, 7:90-99[1989]).The C-terminal structural domain of hML has the aminoacid sequence [the Arg-Arg motif in 153-154 and 245-246 position] of two two alkalescence, can be used as potential processing site.Cutting in these sites may separate exactly from the ML of APP 30,28, the source of 18-22 kilodalton form.Very importantly, the appearance of Arg153-Arg154 sequence is the erythropoietin spline structure territory of and then ML.These phenomenon explanations, perhaps the ML of total length is a kind of precursor protein, produces sophisticated part through limited proteolysis.

4. the isotype of people mpl part and varient

The isotype of people mpl part or splicing form can detect by the PCR of adult liver.In brief, synthetic corresponding to the hML encoding sequence two ends and the primer of the interior region of selection.As described in embodiment 10, these primers are used to by RT-PCR amplification adult liver RNA.Except the total length form that is called hML, also observe or infer other three kinds of forms, be called hML 2, hML3 and hML4.The mature amino acid sequence of these four kinds of isotypes of all that infer is shown in Figure 11 (SEQ ID NOS:6,8,9 and 10).There are 116 nucleotide deletions at 700 places to hML3 in the position, have caused amino acid whose disappearance and frameshit.The mature polypeptide of 265 amino acid lengths of cDNA coding is from the 139th amino-acid residue divergence of hML sequence.At last, hML4 existing in the site 618 back 12 nucleotide deletions (also in mouse and pig sequence find [seeing below]), the disappearance of the 116bp that sees hML3 is also arranged.Though in the people, also be not separated to the clone who has only 12bp (being right after nucleotide site 619) disappearance, but this hML2 of being called as isotype may exist, because in mouse and pig, all identified such isotype (seeing below), also because have been found that its with the hML4 that lacks 116 Nucleotide together with.

Whether the Arg153-Arg154 of two alkalescence is fabricated with the ML that judges total length necessary by biologically active by " EPO structural domain " clipped form of two alanine residue alternate hML replacement mutation bodies and hML.The two alkaline sequence replacement mutation body and function embodiment 10 described PCR method of Arg153-Arg154 that are called hML (R153A, R154A) make up." EPO structural domain " clipped form hML153 by introduce a terminator codon behind Arg153, also is to make up with PCR.

5. the expression of recombinant human mpl part (rhML) in human embryo kidney (293) cell of transient transfection

People cDNA coding mpl part in order to confirm to clone under the control of cytomegalovirus immediate early promoter, uses expression vector pRK5-hML or pRK5-hML153, expresses part in Mammals 293 cells.Have been found that the supernatant liquor that obtains from the human embryonic kidney 293 cell of transient transfection can promote ³The H-thymidine mixes the Ba/F3-mpl cell, but is not parental generation Ba/F3 cell (Figure 12 A).Only the substratum with 293 cells of pRK carrier transfection does not then have this activity.In substratum, add mpl-IgG and can eliminate this promoter action (data do not show).These results show, clone's the cDNA a kind of functional people ML (hML) that encodes.

The clipped form hML153 of hML is expressed in 293 cells, can judge separately " EPO structural domain " whether can in conjunction with and activate mpl.The supernatant liquor of transfectional cell has similar activity to the supernatant liquor of the cell of expressing total length hML (Figure 12 A), and the C-terminal structural domain of expression ML is not necessary for combination and the activation of c-mpl.

6.mpl part promotes megalokaryocyte to generate and thrombocyte generates

Two kinds of forms of reorganization hML, the rhML of total length and the rhML153 of brachymemma can both go into megalokaryocyte in external promotion and generate (Figure 12 B).When not adding other external source hemopoieticgrowth factors, can observe this effect.Except IL-3, ML is unique this active hemopoieticgrowth factor that shows in detection.In our analysis, generation does not all have effect (data do not show) to megalokaryocyte when detecting separately for IL-11, IL-6, IL-1, erythropoietin, G-CSF, IL-9, LIF, complete part (KL), M-CSF, OSM and GM-CSF.This result has shown that ML has megalokaryocyte and promotes activity, has also pointed out the effect of ML in the megalokaryocyte generation is regulated.

In mouse thrombocythemia rebound is analyzed, thrombopoietic activity thing in the blood plasma of trouble thrombocytopenia animal demonstrates and promotes thrombocyte to generate (McDonald, Proc.Soc.Exp.Biol.Med., 14:1006-1001[1973] and people such as Mcdonald, Scand.J.Haematol., 16:326-334[1976]).In this model, use special antiplatelet sera to cause the acute thrombocyte of mouse and lack, the thrombocythemia rebound in causing predicting.This immune thrombocythemia type mouse for the thrombopoietin sample actives of external source than the more responsive (McDonald of common mouse, Proc.Soc.Exp.Biol.Med., 14:1006-1001[1973]), just as the mouse of removing hypoxemia to the susceptibility of erythropoietin than the strong (people such as McDonald of common mouse, J.Lab.Clin.Med., 77:134-143[1971]).In order to judge whether in vivo stimulating platelet generation of rML, partially purified rhML is injected in the mouse of thrombocythemia rebound.Then, metering platelet count and being incorporated in the thrombocyte ³⁵S.The rML of 64,000 or 32,000 units is expelled to significantly platelet increasing generation in the mouse body, compares with only being injected into the contrast mouse of composing the row agent, platelet count increases about 20% (corresponding p=0.0005 and 0.0001), mixes hematoblastic ³⁵S increases about 40% (p=0.003) (Figure 12 C).This irritation level uses the observed situation of IL-6 mutually than (data do not show) in this model with us.Only the rML with 16,000 units handles the generation of stimulating platelet significantly.These results show that therefore ML has possessed thrombopoietin sample activity with the generation of dose-dependent mode stimulating platelet.

Breed with above-mentioned hML isotype construction rotaring redyeing 293 cell and with Ba/F3-mpl and to take determination and analysis supernatant liquor (seeing Figure 13).In analysis, hML2, hML3 do not have to show the activity that can measure, but hML (R153A, activity R154A) is similar to hML and hML153, and this result shows, active neither necessary in the processing of the two basic sites of Arg153-Arg154 for it, neither be disadvantageous.

7. megalokaryocyte generates and the mpl part

Had hypothesis to think, megalokaryocyte generate the adjusting be subjected to the cell multiplex level (people such as Williams, J.Cell Physiol., 110:101-104[1982] and people such as William, Blood Cell, 15:123-133[1989]).The main foundation of this hypothesis is, the propagation of some hemopoieticgrowth factor stimulating megakaryocyte precursor, and other mainly influence its maturation.Results suggest ML herein plays a role with propagation and maturation factor simultaneously.There are a series of evidences to support the hormesis of ML to megalokaryocyte precursor propagation.At first, APP can be in the propagation and the maturation of stimulated in vitro people megalokaryocyte precursor, and its hormesis can be suppressed (Fig. 7 and Fig. 8) fully by mpl-IgG.In addition, the inhibition that the c-mpl antisense oligonucleotide forms megakaryocyte colony (people such as Methia, Blood, 82:1395-1401[1993]) and intracellular proliferation signal can be transduceed discovery (people such as Skoda in its transfected object of c-mpl, EMBO, 12:2645-2653[1993] and people such as Vigon, Oncogene, 8:2607-2615[1993]) shown all that also ML stimulates proliferation.Interim when all of megalokaryocyte differentiation, c-mpl express significantly (people such as Methia, Blood, 82:1395-1401[1993]) and reorganization ML in vivo rapidly the ability that generates of stimulating platelet shown that all ML also influences maturation.Owing to can obtain the ML that recombinates, just can carry out careful assessment in the effect that megalokaryocyte generates and thrombocyte generates in regulating, and it be to the potential impact of other hematopoietic cell lineages to it.

8. the separation of people mpl part (TPO) gene

Under low tight or high stringent condition, use a people cD-NA 3 ' to hold the corresponding fragment of half sequence with coding mpl part, screening contains the people's gene group library in λ-Gem12 phage of being implemented in of pR45, and is separated to the human gene group DNA clone who contains the TPO gene.Two overlapping λ clones that cross over 35kb are separated.Two overlapping fragmentses (BamH1 and EcoRI) that contain complete TPO gene are carried out subclone and order-checking.

The structure of people's gene is to contain 6 exons in the 7kb genomic dna.All exons all consistent with the intron fillet (Shapiro, people such as M.B., Nucl.Acids Res.15:7155[1987]) with the consensus motif of mammalian genes.Exons 1 and exon 2 contain 4 initial amino acids of 5 ' end non-translated sequence and signal peptide.Preceding 26 amino acid of the residue of secretion signal and mature protein are encoded by exon 3.About 50 amino acid in whole carboxyl structure territory and 3 ' non-translational region and erythropoietin spline structure territory are by exon 6 codings.4 amino acid that relate to observed disappearance in hML-2 (hTPO-2) are by 5 of exon 6 ' end coding.

Southern blotting analyst genomic dna shows that the TPO gene exists with single copy form.The position of this gene on karyomit(e) is positioned karyomit(e) 3q27-28 by fluorescence in situ hybridization (FISH).

9.293 the expression and purification of cell TPO

Be described in detail among the embodiment 19 how from 293 cell preparation and purifying ML or TPO.In simple terms, obtain cDNA with pRK5-hmpl I by PCR exactly corresponding to the whole open reading frame of TPO.PCR product purification rear clone is gone into plasmid pRK5tkneo (a kind of carrier derived from pRK5, through transforming to express neomycin resistance gene under the thymidine kinase promoter control) restriction site Clal and Xbal between, to obtain carrier pRK5tkneo.ORF (a kind of carrier of the whole open reading frame of encoding).

The carrier of second kind of coding EPO homeodomain can be made with identical method, but uses different PCR primers, and the construction that obtains at last is pRK5-tkneoEPO-D.

These two kinds of constructions are transfected into the human embryonic kidney cell by the CaPO4 method, pick out the clone of neomycin resistance and make it to grow into and converge.In conditioned medium, assess ML among these clones by the Ba/F3-mpl proliferation assay ₁₅₃Or ML ₃₃₂Expression.

RhML ₃₃₂Purifying as described in the embodiment 19.In simple terms, with 293-rhML ₃₃₂Conditioned medium be splined on Blue-Sepharose (Pharmacia company) post, then with the damping fluid washing that contains 2M urea.The wash-out of the post damping fluid that contains 2M urea and 1MNaCl.Aggregate behind the Blue-Sepharose post wash-out directly is splined on the WGA-Sepharose post again, and with the 10 times of column volume damping fluids washing that contains 2M urea and 1MNaCl, wash-out is with same damping fluid, but contains N-acetyl-D-glycosamine of 0.5M.The eluate of WGA-Sepharose is splined on the C4-HPLC post again, and (Synchrom Inc.), and comes wash-out with discontinuous propyl alcohol gradient.By SDS-PAGE, the 293-rhML of purifying ₃₃₂The district moves with a broadband at the 68-80 of gel kilodalton.

RhML ₁₅₃Purifying also as described in the embodiment 19.In simple terms, the conditioned medium of rhML153 such as rhML ₃₃₂The same ground separates on the Blue-Sepharose post.The eluate of Blue-Sepharose post directly is splined on mpl affinity column as previously described.The rhML that obtains from the mpl affinity column ₁₅₃Eluate, with rhML ₃₃₂Under the identical condition, reach homogeneity with the C4-HPLC column purification.By SDS-PAGE, the rhML of purifying ₁₅₃Be separated into 2 master tapes and 2 time band, molecular weight is about 18,000-22,000 (seeing Figure 15).

10. mouse mpl part

One obtains, uses after the gel-purified by PCR corresponding to the dna fragmentation of people mpl part coding region ³²P-dATP and ³²The P-dCTP mark.This probe is used to screen 10 of the mouse liver cDNA library that is implemented in the λ GT10 phage ⁶Individual clone.One contains 1443 base pairs and inserts the separated and order-checking of segmental mouse clone (Figure 16 [SEQ ID NOS:12 and 13]).Supposition initiator codon at nucleotide position 138-141 place is positioned at the consensus sequence (Kozak, M.J.Cell Biol., 108:229-241[1989]) of the initial requirement of eukaryotic translation.The open reading frame of 1056 Nucleotide is understood in this sequence table, indicates that it can produce main length is 352 amino acid translation products.At the two ends of this open reading frame, have 5 ' non-translational region of 137 Nucleotide of end and 247 Nucleotide of 3 ' end.Behind 3 ' end non-translational region, do not have poly A tail, represent that this clone may be imperfect.The aminoacid sequence N-terminal height of prediction is hydrophobic may represent a signal peptide.Computer Analysis (von Heijne, G.Eur.J.Biochem.133:17-21[1983]) show that the potential cutting broken site of a signal peptidase is arranged between residue 21-22.The cutting that takes place in this position produces the polypeptide of sophisticated 331 amino acid (35 kilodalton), is called mML ₃₃₁(owing to following reason is also referred to as mML2).This sequence has 4 halfcystines and 7 potential N-glycosylation sites, and the former guards in people's sequence, has 5 to guard in people's sequence among the latter.And the same with hML, all 7 potential N-glycosylation sites all are positioned at half place of c-terminal of protein.

When comparing with people ML, can observe in " the EPO structural domain " of these ML, the aminoacid sequence of Nucleotide and deduction thereof all has very big homogeny.Yet, if the aminoacid sequence arrangement of people and mouse ML deduction is made comparisons, can between the 111-114 of mouse sequence residue, find the disappearance of a tetrapeptide, corresponding to 12 nucleotide deletions behind the nucleotide position 618 in people's (seeing above) and pig (seeing below) cDNA.Correspondingly, check that other clones are to detect possible mouse ML isotype.The polypeptide that 335 amino acid whose deductions of a clones coding are arranged wherein contains the tetrapeptide LPLQ of disappearance.This form is believed to be the mouse ML of total length, is called mML or mML ₃₃₅The nucleotide sequence of mML and the aminoacid sequence of deduction thereof are seen Figure 17 (SEQ ID NOS:14 and 15).This cDNA clone is made of 1443 base pairs, after connect a poly A tail.It has the open reading frame of a 1068bp, and respectively there is the non-translational region sequence of 134 bases and 241 bases its both sides at 5 ' end and 3 ' end.The initiator codon of supposing is positioned at nucleotide position 138-140 place.356 amino acid whose protein of open reading frame coding, wherein begin 21 highly hydrophobic and the function of similar secretion signal arranged.

At last, the third clone that separates again, checked order contains 116 nucleotide deletions corresponding to bML3.This mouse isotype is named is mML3.The aminoacid sequence that these two kinds of isotypes are inferred is relatively seen Figure 18 (SEQ ID NOS:9 and 16).

The homogeny of the whole aminoacid sequence of people and mouse ML is 72% (Figure 19 [SEQID NOS:6 and 17]), but this homology is not to distribute equably.The conservative property (homology 86%) that is called " EPO structural domain " (human amino acid sequence 1-153, mouse aminoacid sequence 1-149) zone is better than proteinic C-terminal zone (homology 62%).Perhaps, this shows further that having only " EPO structural domain " is important to proteinic biological activity.Enjoyably, in two two basic aminoacids motifs that find in hML, two alkaline motifs (residue position 153-154) that only are right after " EPO structural domain " are found in the mouse sequence.This phenomenon has been supported a kind of like this possibility: perhaps the ML of total length has represented a precursor protein matter, produces sophisticated part through limited proteolysis.Also possible, the proteolysis between Arg153-Arg154 helps to decompose hML.

As described in embodiment 1, expression vector that contains the mML complete encoding sequence by transient transfection in 293 cells.The conditioned medium of these cells can stimulate ³The H-thymidine mixes the Ba/F3 cell of expressing mouse or people mpl.But parental generation (few mpl) clone there is not effect.This shows, functional part (mpl) that can activate mouse and people ML acceptor of mouse ML cDNA coding of clone.

11. pig mpl part

As described in embodiment 13, pig ML (pML) cDNA separates by RACE PCR.In kidney, found and subclone the PCR cDNA product of a 1342bp.Through order-checking, find to have several clones pig mpl part of 332 amino-acid residues of encoding, be called pML or pML ₃₃₂These clones' nucleotide sequence and deduced amino acid thereof are shown in Figure 20 (SEQ ID NOS:18 and 19).

In addition, identified second kind of form (seeing Figure 21 [SEQ ID NO:21]), be called pML2, its encoded protein matter has 4 amino-acid residue disappearances (228 amino-acid residues).Compare pML and pML2 as can be known, except tetrapeptide QLPP disappearance corresponding to residue 111-114, two kinds of forms just the same (seeing Figure 22 [SEQ ID NOS:18 and 21]).Observed 4 aminoacid deletion all are the same positions that accurately occurs in putative protein matter in mouse and pig ML cDNA.

Compare the aminoacid sequence (Figure 19 [SEQ IDNOS:6,17 and 18]) of the ripe ML supposition of people, mouse and pig, the result shows, on the whole sequence homogeny, is 72% between mouse and the people, is 68% between mouse and the pig, and is 73% between pig and the people.At N-terminal half place of ML (EPO homeodomain), homology increases significantly.Between any two kinds of above species, this regional homology is 80% to 84%, and at half place (carbohydrate structure territory) of carboxyl terminal, homogeny only has 57% to 67%.At the C-terminal of erythropoietin homeodomain, two basic aminoacids motifs of representing proteolytic enzyme cutting position are arranged.This locational this motif is (Figure 19 [the SEQ ID NOS:6,17 and 18]) that guards between three species.Second the two basic site that occurs at people's sequence location 245-246 place do not occur in the sequence of pig or mouse.Mouse and pig ML sequence have 4 halfcystines, all remain in people's the sequence.In mouse part and pig ML, potential N-glycosylation site has 7 and 6 respectively, wherein in the sequence that 5 remain in the people.In addition, all potential N-glycosylation sites all are positioned at half place of protein carboxyl terminal.

12. expression and the purifying of Chinese hamster ovary (CHO) cell TPO

The expression vector that is used for transfection CHO cell is called: pSV15.ID.LL.MLORF (total length or TPO332) and pSV15.ID.LL.MLORF-D (brachymemma or TPO153).The correlation properties of these plasmids are seen Figure 23 and Figure 24.

The process of transfection is as described in the embodiment 20.In simple terms, obtain cDNA by PCR corresponding to the whole open reading frame of TPO.PCR product purification rear clone is gone between two restriction sites (Clal and Sall) of pSV15.ID.LL plasmid, thereby obtains carrier pSV15.ID.LL.MLORF.Second kind of construction corresponding to the EPO homeodomain produces with the same manner, but uses different antisense primer (EPOD.Sal).The final construction of the carrier of the EPO homeodomain of coding TPO is called pSV15.ID.LL.MLEPO-D.

These two kinds of constructions make it linearizing with Notl digestion, and are transfected into Chinese hamster ovary cell (CHO-DP12 cell, EP delivered on March 15th, 307,247 1989) by electroporation.10 ⁷Individual cell is made electroporation having 10,25 or during 50mg DNA with BRL electroporation apparatus (350 volts, 330 millifarads are hanged down electric capacity), (Andreason, G.L.J.Tissue Cult.Meth.15,56[1993]).After the transfection second day, cell selected to beat in the substratum (not containing high concentration glucose DMEM-F12 50:50, the 2mM glutamine of glycine, the calf embryo serum of 2-5% dialysis) at DHFR spare.After 10 to 15 days, single colony is transferred to 96 flat boards, make it to grow to and converge.Assess ML153 or the expression (as embodiment 1 as described in) of ML332 in conditioned medium with the Ba/F3-mpl proliferation assay from these clones.

The step of separation and purifying TPO is seen embodiment 20 from the Chinese hamster ovary celI nutrient solution of results.In simple terms, the Chinese hamster ovary celI nutrient solution of gathering in the crops (HCCF) is splined on Blue-Sepharose post (Pharmcia company), the ratio of last sample is every liter of about 100 liters of HCCF of resin.Then, earlier with the damping fluid washing column of 3 to 5 times of column volumes, what continue contains the damping fluid washing column of 2.0M urea with 3 to 5 times of column volumes.Then, the damping fluid that contains 2.0M urea and 1.0MNaCl with 3 to 5 times of column volumes again comes wash-out TPO.

The Blue-Sepharose wash-out aggregation that will contain TPO is splined on Wheat Germ (wheat germ) lectin-Sepharose post (Pharmacia company), comes balance columns in the ratio of 8 to 16 milliliters of Blue-Sepharose elutriants of every milliliter of resin with the Blue-Sepharose elution buffer.The washing of post is with the level pad of 2 to 3 times of column volumes.The wash-out of TPO adopts 2 to 5 times of column volumes to contain the damping fluid of 2.0M urea and 0.5M N-acetyl-D-glycosamine.

Subsequently, will contain the wheat germ agglutinin eluate acidifying of TPO, and add C ₁₂E ₈Making final concentration is 0.04%.Again the solution that obtains is splined on the C4 reversed-phase column, with 0.1%TFA, 0.04%C ₁₂E ₈Balance, loading capacity are every milliliter of about 0.2 to 0.5mg protein of resin.

Containing 0.1%TFA and 0.04%C ₁₂E ₈Acetonitrile two-phase linear gradient in elute protein, and behind SDS-PAGE, obtain a kind of aggregation.

Then, with the aggregation among C4 dilution, and be to filter thoroughly with the damping fluid of 6 times of volumes on the ultra-filtration membrane of Amicon YM and so on of 10,000 to 30,000 Dalton molecular weights making it to block.The saturating filtrate that obtains can be directly used in later operation or concentrate further through ultrafiltration.Usually, saturating filter/concentrated solution final concentration is that 0.01% tween-80 is regulated.

Then, all or part of filter/concentrated solution that will be equivalent to column volume 2% to 5% is splined on Sephacryl S-300 HR post (Pharmacia company), and balance is carried out chromatogram then with the damping fluid that contains 0.01% tween-80.The TPO component of degraded product that does not contain polymkeric substance and proteolysis is with after obtain aggregation behind the SDS-PAGE.The aggregation that obtains is stored in 2-8 ℃ after filtering.

13. in microorganism, transform with induce TPO synthetic method and therefrom separate, purifying and refolding synthetic TPO

Being structured in of intestinal bacteria TPO expression vector has detailed description among the embodiment 21.In simple terms, design plasmid pMP21, pMP151, pMP41, pMP57 and pMP202 express preceding 155 amino acid of TPO in a short homing sequence downstream, and this homing sequence is variant because of different constructions.This homing sequence mainly provides high-caliber translation initiation and fast purifying.Designed again plasmid pMP210-1 ,-T8 ,-21 ,-22 ,-24 ,-25 expresses preceding 153 amino acid in TPO initial methionine downstream, the difference of these plasmids only is the use at preceding 6 the amino acid whose codons of TPO.After the C-terminal of TPO extends two amino acid among the pMP210-1, obtain pMP251.Under the inducing of trp promoter, all above-mentioned plasmids will produce TPO expression (Yansura, D.G. etc. in the high-caliber cell in intestinal bacteria, Methods in Enzymology[Goedded, D.V., editor] 185:54-60, Academic Press, SanDiego[1990]).Plasmid pMP 1 and pMP172 are the transition things in the above-mentioned TPO cell inner expression plasmid construction.

Pass through CaCl ₂The heat-shocked method, with above-mentioned TPO expression plasmid transformed into escherichia coli (Mandel, M. etc., J.Mol.Biol., 53:159-162, [1970]), embodiment 21 is seen in the description of other conversion processes.In simple terms, at first transformant is cultivated in 37 ℃ and reached about 2-3 until the culture optical density(OD).The dilution culture, and under aeration condition, after the growth, add acid.Then, allow culture continue under aeration condition growth after 15 hours, the centrifuging collecting cell.

Embodiment 22 and embodiment 23 the people TPO that produces bioactive, refolding has been described and segmentally separate, purifying and refolding step; These methods can be applicable to comprise the recovery that N-terminal and C-terminal extend any TPO varient of form.Other steps that are suitable for refolding recombinant chou or synthetic TPO can be referring to following patent: Builder etc., the U.S. patent No. 4,511,502; Jones etc., the U.S. patent No. 4,512,922; Olson, the U.S. patent No. 4,518,526 and Builder etc., the U.S. patent No. 4,620,948; Recovery and refolding method have wherein roughly been narrated with a large amount of recombinant proteins of insoluble formal representation in intestinal bacteria.

A. the recovery of insoluble TPO

To express microorganism such as intestinal bacteria by any suitable plasmid-encoded TPO, fermentation under certain condition requires the TPO can be with insoluble " refractile body " heavy mud.Selectively at first use the cell lysis buffer solution washed cell.Usually, the cells of about 100 grams are resuspended in the cell lysis buffer solution of 10 times of volumes (as 10mMTris, 5mM EDTA, pH8), and with the Polytron homogenizer with 5000 * g, 30 minutes condition eccentric cell.Then, use such as routine techniques lysing cell such as tension force shock, supersound process, pressure cycling, chemistry or enzyme process.For example, above-mentioned cell precipitation thing through washing can be resuspended in by homogenizer in the another kind of cell lysis buffer solution of 10 times of volumes, and according to the explanation of manufacturers with cell suspending liquid flow through LH Cell Disrupter (LH Inceltech company) or Microflu-idizer (Microfluidics International company).The particulate matter that contains TPO is separated from liquid phase, and selectively with any suitable liquid scrubbing.For example, a kind of suspension of cell parallel off thing can 5, centrifugal 30 minutes of 000g, and it is centrifugal selectively to carry out the second time after resuspended, obtains the refractile body precipitation through washing at last.These precipitations through washing can be used or stored frozen (as-70 ℃) immediately.

B. the solubilising of monomer TPO and purifying

Insoluble TPO comes solubilising with the solubilising damping fluid in the refractile body precipitation.The solubilising damping fluid contains chaotropic agent, plays shock absorption usually under alkaline pH, and contains the reductive agent that can improve monomer TPO output.Representational chaotropic agent has: urea, Guanidinium hydrochloride and Sodium Thiocyanate 99.And preferred chaotropic agent is a Guanidinium hydrochloride.Concentration from agent is generally 4-9M, is preferably 6-8M.The pH value of solubilising damping fluid is maintained in the 7.5-9.5 scope by suitable damping fluid, is preferably 8.0-9.0, is preferably 8.0 the most.Also contain reductive agent in the preferred solubilising damping fluid to help to form the monomeric form of TPO.Appropriate reductant comprises free sulfydryl organic compound such as (RSH).Representational reductive agent comprises dithiothreitol (DTT) (DTT), dithioerythritol (DTE), mercaptoethanol, gsh (GSH), cysteamine and halfcystine.Preferred reductive agent is dithiothreitol (DTT) (DTT).Selectively, the solubilising damping fluid can comprise a kind of oxygenant (as oxygen molecule) of gentleness and a kind of sulphite to form monomer TPO by sulphiting.In this example, the TPO-S-sulfonate that obtains later on again in potential buffer solution (as GSH/GSSG) refolding to form suitable folding TPO.

TPO protein need be further purified, usual method such as centrifugal, gel filtration chromatography and reversed-phase column chromatography.

With the method for giving an example, can obtain the monomer TPO of suitable output through the following steps.Refractile body precipitation is resuspended in the solubilising damping fluid (20mM Tris, pH8,6-8M guanidine and 25mM DTT) of about 5 times of volumes, stirs and spent the night in 1-3 hour or 4 ℃, make TPO protein solubilising.Also can use the urea (6-8M) of high density, but compare with guanidine, output will be hanged down.Behind the solubilising, solution centrifugal 30 minutes at 30,000 * g obtains containing the transparent supernatant liquor of sex change monomer TPO.Then, (setting flow velocity is 2 ml/min for Phar-macia company, 2.6 * 60cm) enterprising circumstances in which people get things ready for a trip spectrums at Superdex 200 gel columns with supernatant liquor.Proteinic elution requirement is 20mM sodium phosphate pH6.0,10mMDTT.Merge to collect the component that contains between 160 to 200 milliliters of the sex change monomer TPO protein elutriants.TPO protein is further purified on semipreparative reversed-phase column (2 * 20cm VYDAC).Sample is with sample on 5 ml/min, and the balance of post usefulness contains the 0.1%TFA (trifluoroacetic acid) of 30% acetonitrile.Proteinic wash-out adopts the linear gradient (in 60 minutes, from 30% to 60%) of acetonitrile.The reductibility protein of purifying is eluted when concentration is about 50% acetonitrile.Eluate is used for refolding to obtain the TPO varient of biologically active.

C. refolding TPO is to produce biologically active form

The TPO solubilising and be further purified after, will obtain biologically active form by the refolding of sex change monomer TPO in potential buffer solution.Because the height of TPO is tired (during about 3pg/ml, can reach half of maximal stimulation effect in Ba/F3 mensuration), it is possible obtaining biologically active substance by multiple different damping fluid, washing agent and redox condition.But, in most of the cases, can only obtain a small amount of suitable folding material (＜10%).With the program of manufacturer recommendation, wish that the refolding product that obtains is at least 10%, it is better to reach 30%-50%, reaches then best more than 50%.The washing agent that can produce a certain amount of suitable folding product at least that has been found that has a lot: Triton X-100, dodecyl-beta-maltose, CHAPS, CHAPSO, SDS, sarkosyl, polysorbas20, tween 80, Zwittergent and other.But wherein highly preferred washing agent all belongs to CHAPS family (CHAPS and CHAPSO), and best results and the energy limit protein matter of CHAOS family washing agent in the refolding reaction is assembled and the formation of unsuitable disulfide linkage.The highly preferred concentration of CHAPS is greater than 1%.For obtaining production peak, need the sodium-chlor of optimal concentration 0.1 to 0.5M.In order to be limited in the oxygenizement of finding in some prepared products by metal catalytic (and congregation), can in potential buffer solution, add EDTA (1-5mM).The suitableeest refolding condition needs concentration greater than 15% glycerine.In order to obtain production peak, also must in potential buffer solution, contain a redox couple that constitutes by oxidation and the organic sulfydryl of reduction (RSH).Suitable redox couple has: mercaptoethanol, gsh (GSH), cysteamine, halfcystine and their corresponding oxidised forms.Preferred redox couple is gsh (GSH): oxidized glutathione (GSSH) or cysteamine: halfcystine.And highly preferred redox couple is gsh (GSH): oxidized glutathione (GSSH).Usually, when the mol ratio of redox centering oxide compound is equal to or greater than reduzate, can obtain higher output.The optimum pH of these TPO varient refoldings is between 7.5 and about 9.The tolerance concentration of organic solvent (as ethanol, acetonitrile, methyl alcohol) is 10-15% or lower.The organic solvent of greater concn has increased the amount of improper folded form.Tris and mercapto phthalate buffer can work usually.4 ℃ of incubations also can increase the suitable folding amount of TPO.

Through behind first C4 step purifying, the refolding output of TPO is generally 40-60% (according to the reduction that is used for the refolding reaction and the amount of sex change TPO).Though because a large amount of precipitation and in TPO refolding process the proteinic interference output of non-TPO can reduce, when prepared product purification degrees lower (for example: direct after Superdex 200 posts or initial refractile body extracting), can access active substance.

Because TPO contains 4 cysteine residues, this protein may form 3 kinds of different disulfide linkage forms:

Form 1: disulfide linkage is between cysteine residues 1-4 and 2-3

Form 2: disulfide linkage is between cysteine residues 1-2 and 3-4

Form 3: disulfide linkage is between cysteine residues 1-3 and 2-4.

In the initial experiment of determining the refolding condition, several different proteinic peaks of TPO that contain have been obtained by the C4 reversed-phase column chromatography.In these peaks, have only one in Ba/F3 measures, to show tangible biological activity.Subsequently, optimize the refolding condition with this form of preferential generation.Under these conditions, the malfolding form of all monomer TPO that obtain from solubilization step is lower than 10-20%.

Use mass spectroscopy and protein sequencing (from N-terminal, according to this to the halfcystine numbering), the disulfide linkage form of having determined biologically active TPO is 1-4 and 2-3.The cross-linked form of this halfcystine is with the disulfide linkage form unanimity of known relevant erythropoietin molecule.

D. the biological activity of recombinant chou, refolding TPO

In body and isolated measuring, the TPO of refolding and purifying has activity.For example in Ba/F3 measures, the TPO (Met of 3.3pg/ml (0.3pm) ^-11-153) hormesis that thymidine is mixed the Ba/F3 cell reach peaked half.In enzyme-linked immunosorbent assay (ELISA) based on the mpl acceptor, when concentration is 1.9ng/ml (120pM), activity reach peaked half.In intact animal and bone marrow depression animal through the nearly X ray processing that causes death, refolding TPO (Met ^-11-153) stimulate new thrombocyte to generate (, can be observed activity) efficiently in that dosage is low when reaching the 30ng/ mouse.In other forms of TPO after the above-mentioned steps refolding, also can observe similar biological activity.

14. measure the method for TPO activity

The measurement of biologically active pdgf can be passed through several different methods, for example embodiment 1 described Ba/F3 mpl part is measured, the thrombocyte rebound is synthetic in the body measures, induce mensuration (referring to Sato etc. with the platelet cell surface antigen measured at the antiplatelet immunity (anti-GPIIbIIIa) of human leukemia megakaryoblast system (CMK), Brit.J.Heamatol., 72:184-190[1989]) the inducing of polyploidization in (also generate referring to the described liquid suspension scavenger cells of embodiment 4 and measure) and the megakaryoblast system (DAMI) (referring to Ogura etc., Blood (1), 72:49-60[1988]).From immature, do not have that DNA synthetic scavenger cell is ripe to be comprised for the process of the identifiable scavenger cell of form mostly: occur organoid in the kytoplasm, obtain membrane antigen (GPIIbIIIa) and the endoreduplication described in background of invention and release thrombocyte.Need the special ripe Megakaryocytic promotor (as the mpl part) of pedigree to induce the variation of prematurity scavenger cell at least a portion in the ripening process, thereby cause discharging thrombocyte and relax thrombopenia.Therefore, having designed some tests and detects the appearance that immature macrophage system is these parameters in CMK and the DAMI cell.CMK measures (embodiment 4) and detects special thrombocyte mark GPII _bIII _aAnd the appearance of thrombocyte release.DAMI measures (embodiment 15) and then detects endoreduplication, because the increase of the ploidy sophisticated sign that is scavenger cell.Discernible scavenger cell has 2N, 4N, 8N, 16N, 32N equimultiple value.At last, the rebound of mouse thrombocyte is measured (embodiment 16) and be can be used to proof in the body, uses the increase that test compounds (being the mpl part) will cause platelet count herein.

Develop two kinds of isolated measuring methods in addition again and detected the TPO activity.First kind is that kinases receptors activates (KIRA) enzyme-linked immunosorbent assay, wherein, detect the tyrosine phosphorylation (referring to embodiment 17) of Rse with enzyme-linked immunosorbent assay with mpl-Rse mosaic transfection CHO cell and after chimeric mpl partly is exposed to the mpl part.Second kind of enzyme-linked immunosorbent assay that is based on acceptor wherein caught and mpl part bonded people Chimerical receptor mpl-IgG to be measured by rabbit anti-human igg's enzyme-linked immunosorbent assay plate with bag.A biotin labeled rabbit polyclonal antibody of mpl part is used to detect bonded mpl part, and measures with embodiment 18 described streptavidin-peroxidase.

15. normal mice of handling with TPO and biological respinse in the Asia causes death the body of radiating mouse

The normal mice and the radiating mouse that causes death through the Asia are all used and brachymemma handle with TPO total length, and wherein TPO separates from Chinese hamster ovary (CHO) cell, intestinal bacteria and HEKC (293).The two kinds of forms of TPO that obtain from these three kinds of hosts can both stimulate the thrombocyte of mouse to generate, and still, separate having shown the strongest body internal reaction from the total length TPO of CHO.These results show that the suitableeest activity in vivo may carry out suitable glycosylation at the C-terminal structural domain.

(a) intestinal bacteria-rhTPO (Met ^-1, 153)

Every day, " Met " form (methionine(Met) of 1 position adds preceding 153 residues of people TPO) of the EPO structural domain that intestinal bacteria (referring to embodiment 23) are produced is injected into normal female C57 B6 mouse, the explanation of method such as Figure 25 A, 25B and 25C.These figure show, by intestinal bacteria produce and with the TPO of aforesaid method refolding not the glycosylation clipped form can stimulate that platelet counts increases by 2 times in the normal mice, and do not influence red corpuscle and leukocytic quantity.

As the explanation of Figure 26 A, 26B and 26C, every day with same molecule be injected into the inferior radiation that causes death ( ¹³⁷Cs) female C57 B6 mouse, but the answer of stimulating platelet and improving the standard, but red corpuscle or white corpuscle are not had effect.

(b)CHO-rhTPO ₃₃₂

As the explanation of 27A, 27B and 27C, will be injected into female C57 B6 mouse every day by the TPO total length form that CHO produces, and the platelet counts of normal mice be increased by 500, but the quantity of red corpuscle and white corpuscle ball is not had effect.

(c) CHO-rhTPO ₃₃₂Intestinal bacteria-rhTPO (Met ^-1, 153); 293-rhTPO ₃₃₂And intestinal bacteria-rhTPO ₁₅₅

As the explanation of Figure 28, handle normal mice with rhTPO and make up dose response curve, wherein rhTPO is from different clone (CHO-rhTPO ₃₃₂Intestinal bacteria-rhTPO (Met ^-1, 153); 293-rhTPO ₃₃₂And intestinal bacteria-rhTPO ₁₅₅).This figure shows, the generation that the tested form of all of this molecule can both stimulating platelet, but the total length form that CHO produces has the strongest activity in vivo.

(d) CHO-rhTPO ₁₅₃CHO-rhTPO " brachymemma " and CHO-rhT-PO ₃₃₂

Equally, as the explanation of Figure 29, with CHO (CHO-rhTPO ₁₅₃CHO-rhT-PO " brachymemma " and CHO-rhTPO ₃₃₂) rhTPO of the various ways that produces handles normal mice and make up dose response curve.This figure shows, all generations of stimulating platelet of the tested CHO form of all of this molecule, but 70 kilodalton forms of total length have the strongest activity in vivo.

16.mpl the general reorganization preparation of part and varient

Preferential employing standard reorganization program prepares the mpl part: cultivate the cell with the nucleic acid transfection (generally using a kind of expression vector transformant) of expressing the mpl part, produce the mpl ligand polypeptide, reclaim polypeptide from cell.But, can think that also the generation of mpl part is by homologous recombination or reorganization production method, wherein the latter is a cell of controlling elements being introduced the DNA that has contained coding mpl part.For example, a strong promoter/enhancer element, suppressor gene or external source transcription regulatory element can be inserted into the genome of specifying host cell, the vicinity of on position and direction are enough to influence the transcribing of DNA of the required mpl ligand polypeptide of coding.The controlling elements mpl part of not encoding, and that this DNA is a host cell is original.Then, can screen as required, perhaps obtain the cell that expression level strengthens or lowers to obtain producing the cell of receptor polypeptides of the present invention.

Therefore, the present invention expects a kind of method of the mpl of generation part, requires this method a transcription regulatory element can be inserted the cellular genome that contains the mpl ligand nucleic acid molecule, and the enough vicinities and the direction of on position are influential to transcribing of this nucleic acid molecule.Perhaps further step should comprise that also cultivation contains the cell of transcription regulatory element and nucleic acid molecule.The present invention also expects a kind of host cell, and the endogenous mpl ligand nucleic acid molecule that wherein contains operationally combines with the external source control sequence of being discerned by host cell.

A. the encode separation of DNA of mpl ligand polypeptide

The DNA of coding mpl ligand polypeptide can obtain from any cDNA library, but the preparation in this cDNA library is also can be with the tissue of detection level expression from be sure oing to contain the mpl ligand mRNA.The mpl ligand gene also can obtain from certain genome dna library or by the external oligonucleotide of complete Nucleotide or aminoacid sequence is synthetic.

With the probe screening library that designs, to obtain target gene and encoded protein matter thereof.For the cDNA expression library, suitable probe comprise can discern and specificity in conjunction with the mono-clonal or the polyclonal antibody of mpl part.For the cDNA library, suitable probe comprises the oligonucleotide of the about 20-80 of a length base, can encode from the known or unknown portions of the mpl Ligand cDNA of identical or different species; The complementation or homology cDNA or its fragment that also comprise the same or similar gene of encoding.The proper probes in screening-gene group DNA library include, but is not limited to encode oligonucleotide, cDNA or their fragment and/or homologous gene group DNA or its fragment of same or similar gene.Standard program described in the 10-12 chapter of Sambrook etc. (the same) can be used as with the selected probe screening cDNA or the guidance of genomic library.

Another method of separating coding mpl ligand gene is PCR, as described in (the same) the 14th parts such as Sambrook.This method need use can with the oligonucleotide probe of the DNA hybridization of coding mpl part.Select the tactful as described below of oligonucleotide.

Putting into practice a preferred method of the present invention is, with the cDNA library of the oligonucleotide sequence screening of selecting meticulously from multiple tissue, preferably those tissues are people or pig kidney (adult or embryo) or hepatic cell line.For example, people's embryo hepatic cell line cDNA screens with oligonucleotide probe in the library.Perhaps, screen with oligonucleotide probe in people's gene group library.

The oligonucleotide sequence that is selected as probe should have enough length, and will have enough accuracies to make false positive reach minimum.Usually, the actual nucleotide sequence that uses is the zone design according to mpl part codon Feng Yu minimum.This oligonucleotide may be degeneracy in one or several position.For screening library the species that are the unknowns from a preferred codon use, the use of degeneracy oligonucleotide is extremely important.

This oligonucleotide must be labeled, thereby it can be detected when hybridizing with the DNA in screened library.The preferred method of mark is (as γ with ATP ³²P) and 5 ' end of polynueleotide kinase radio-labeling oligonucleotide.But additive method also can be used for labeled oligonucleotide, includes, but is not limited to vitamin H or enzyme labelling.

The most interesting is the to encode mpl ligand nucleic acid of total length mpl ligand polypeptide.In some preferred example, its nucleotide sequence has comprised natural mpl part signal sequence.Use the cDNA or the genomic library of deduced amino acid screening selection, can obtain having the nucleic acid of all proteins encoding sequence.

The variant amino acid sequence body of B. natural mpl part

The preparation method of the variant amino acid sequence body of mpl part is: suitable Nucleotide is changed to introduce among the mpl part DNA or by the external of required mpl ligand polypeptide synthesize.For example this class varient comprises disappearance, insertion or the displacement of residue in the pig mpl part aminoacid sequence.For example can be by in body or stripped proteolysis cutting action or by the clone and express a fragment or the DNA of coding total length mpl part removes the C-terminal part of ripe total length mpl part, thus the varient of a biologically active obtained.As long as final construction has required biological activity, therefore disappearance, insertion and displacement arbitrary combination can be formed final construction.Perhaps, amino acid whose variation can also change the post-treatment process of translating of mpl part, for example changes the quantity and the position of glycosylation site.For the design of mpl part variant amino acid sequence body, mutational site and emergent properties are by will reformed mpl part characteristic determining.The change in mutational site can be whenever next or a series of at every turn, for example (1) effect of reaching as required at first conservatively, then more radically makes one's options and replaces; (2) remove the target residue; (3) near specified site, insert similar or inhomogeneous residue, perhaps will select the combination of 1-3.

As Cunningham and Wells, Science is at 244:1081-1085[1989] described in, " alanine scanning mutagenesis " is to determine that some residue of mpl ligand polypeptide or zone are a process useful of the optimum position of mutagenesis.Here, can determine a residue or one group of target residue (as charged residues such as arginine, aspartic acid, Histidine, Methionin and L-glutamic acid) and can be replaced (preferably L-Ala or poly-L-Ala), but as replace also better with neutral or electronegative amino acid by any amino acid.The result who replaces has influenced in amino acid and the cell or the interaction of extracellular ambient water solution environmental.Displacement is produced these structural domains of functional sensitivity, can make a variation it is refining by introduce further other at replacement site.Therefore, when being predetermined the site of introducing a variant amino acid sequence, then the character of sudden change itself does not need scheduled.For example, optimize the sudden change behavior in order to specify site at certain, the mpl ligand variant body of implementing L-Ala scanning or random mutagenesis at target codon or target region place and filtering out expression is to obtain required active best of breed.

In making up the variant amino acid sequence body, two main variables are arranged: the location in mutational site and the character of sudden change.For example, the varient of mpl ligand polypeptide comprises the varient of mpl ligand sequence, and may represent the allelotrope (not needing the operation of mpl part DNA) or the predetermined mutant forms (dna mutation is become undiscovered allelotrope of occurring in nature or varient) of natural formation.Usually, the position of sudden change and character are selected according to mpl part characteristic to be finished.

Sequential amino acid deletion 1-30 the residue of having an appointment usually, about 1-10 residue is better, and the disappearance of residue generally is a successive.Perhaps, the sequential amino acid deletion of mpl part may comprise part or all of C-terminal glycosylation structural domain.The disappearance of aminoacid sequence also may comprise one or several in initial 6 n terminal residues of mature protein.Selectable aminoacid deletion comprises the one or more residues in the one or more rings zone that is present between " helical bundle ".The successive disappearance is generally the even number residue, but the disappearance of single or odd number residue also belongs to this category.Can introduce disappearance to modify the activity of mpl part at the low homologous region that has between the mpl part of most sequence identities.Perhaps, people mpl part and have and other Mammals mpl ligand polypeptides of people mpl part overwhelming majority sequence identities between low homologous region, also can introduce disappearance.Introducing disappearance, more may make the biological activity of this mpl part that more significant variation takes place with certain Mammals mpl ligand polypeptide zone of the basic homologous of another Mammals mpl part.Choose the residue number of consecutive miss, to keep the tertiary structure in the mpl ligand effect structural domain, as βZhe Die or α spiral.

The insertion of aminoacid sequence comprises the fusion of amino and/or C-terminal, and its length can be from the polypeptide of hundred of residues to even more residues; The interior insertion of sequence that has also comprised simultaneously single or multiple amino-acid residues.Insert (being the insertion in the ripe mpl ligand sequence) 1 to 10 residue of generally having an appointment in the sequence, 1 to 5 residue is better, and 1 to 3 residue is preferably arranged.A typical fusion occurs between mpl part or its fragment and another cytokine or its fragment.The terminal example that inserts comprises the ripe mpl part with the terminal methionyl of a N-, the artificial constructed thing of the direct expression of ripe mpl part in the reconstitution cell culture and the fusion of an allos N-terminus signal sequence and ripe mpl ligand molecular N-end, and the latter can promote recombinant host to secrete sophisticated mpl part.The sort signal sequence is usually from specified host cell kind, and homology with it.Suitable sequence comprises the bleb gD of colibacillary STII or Ipp, zymic alpha factor and viral signal such as mammalian cell.

The insertion varient of other mpl ligand moleculars comprises mpl part N-end or C-end and has the syzygy (syzygy of promptly using is an external source for the host) of immunogenic polypeptide that immunogenic polypeptide has the enzyme or the Yeast protein of bacterial peptide such as β Sumylact L or intestinal bacteria trp locus coding; The syzygy that also comprises the protein and the C-terminal of long half time, the former is as constant region for immunoglobulin (or other immunoglobulin domains), albumin or ferritin, referring to the WO 89/02922 that delivered on April 6th, 1989.

The 3rd class varient is the amino-acid substitution varient.In these varients, have at least the amino-acid residue of a mpl ligand molecular to be removed and insert a different residue in the original place.The most interested site of displacement mutagenesis comprises position and the following position that is confirmed to be mpl ligand activity site.In such position, because volume, the charged or hydrophobicity of side chain are different substantially with the amino acid in other analogues; But for selected site, also there are the sequence identity of height in multiple mpl ligand species and/or certain mpl part member between the analogue of multiple animal.

In other interested sites, identical from the specific residue of the mpl part of multiple family member or animal species.Replace in conservative relatively mode in these sites, and especially those are in the sequence position of containing 3 other identical conservative sites at least.Such conservative substitution is entitled as preferred metathetical part referring to table 3 acceptance of the bid.If such displacement causes bioactive variation, will introduce how substantial variation so and filter out product, these substantial variation are referring to typical replacing section in the table 3 or with reference to following further describing the amino acid type.

The preferred displacement of the typical displacement of residue Ala (A) Val that table 3 is original; Leu; Ile ValArg (R) Lys; Gln; Asn LysAsn (N) Gln; His; Lys; Arg GlnAsp, (D) Glu GluCys, (C) Ser SerGln, (Q) Asn AsnGlu, (E) Asp AspGly, (G) Pro ProHis, (H) Asn; Gln; Lys; Arg ArgIle (I) Leu; Val; Met; Ala; Phe;

Nor-leucine LeuLeu (L) nor-leucine; Ile; Val;

Met；Ala；Phe IleLys(K) Arg；Gln；Asn ArgMet(M) Leu；Phe；Ile LeuPhe(F) Leu；Val；Ile；Ala LeuPro(P) Gly GlySer(S) Thr ThrThr(T) Ser SerTrp(W) Tyr TyrTyr(Y) Trp；Phe；Thr；Ser PheVal(V) Ile；Leu；Met；Phe；

Ala nor-leucine Leu

Mpl ligand function or the conforming basic modification of immunity are finished by selecting displacement, these displacements have significant difference according to its effect of keeping following situation: (a) structure of polypeptide main chain in the replacement areas, for example one folds or helical conformation, (b) charging property or the hydrophobicity of target site punishment, or (c) volume of side chain.According to general side chain characteristic, the residue of natural formation is divided into following type:

(1) hydrophobicity: nor-leucine, methionine(Met), L-Ala, Xie Ansuan, leucine, Isoleucine

(2) neutral hydrophilic: halfcystine, Serine, Threonine

(3) acidity: aspartic acid, L-glutamic acid

(4) alkalescence: asparagine, glutamine, Histidine, Methionin, arginine

(5) influence the residue of chain direction: glycine, proline(Pro)

(6) aromatic series: tryptophane, tyrosine, phenylalanine

Nonconservative displacement must be to replace residue in another type by a residue in the above-mentioned type.Such displacement residue also can be introduced into the preservative replacement site or preferably introduce (non-conservation) site that keeps.

In an example of the present invention, hope can make the one or more proteolytic enzyme cuttings site inactivation that exists in the molecule.These sites are determined by checking amino acid sequence coded, for example for trypsinase, will be sought Methionin or arginine residues.After having determined proteolytic enzyme cutting site, cutting does not have effect to proteolytic enzyme just to make them by the following method: the displacement of target residue is used for metathetical residue preferably alkaline residue such as glutamine or hydrophobic residue such as Serine; Remove this residue; Insert a proline(Pro) after perhaps being right after this residue.

In another example in, any methionine residues except the initial methionine residues of signal sequence and be positioned at each this methionine residues N-end or terminal about 3 residues of C-with any residue all by another residue displacement (preferably according to table 3) or be removed.Perhaps, near this site, insert about 1 to 3 residue.

Any cysteine residues of keeping the suitable conformation of mpl part that relates to also can be replaced, and with the oxidative stability that improves molecule with prevent unusual crosslinkedly, is used for the metathetical residue and is generally Serine.Have been found that from the epo structural domain of N-terminal first and the 4th halfcystine are essential for keeping suitable conformation, but second and the 3rd are nonessential.Correspondingly, second and the 3rd halfcystine can be replaced in the epo structural domain.

The nucleic acid molecule of coding mpl ligand variant body can prepare with several different methods known in the art.These methods include, but is not limited to separate (for the variant amino acid sequence body that forms naturally) or mutagenesis prepares, a PCR mutagenesis and varient that has prepared or the unmanifest bodily form formula of mpl ligand polypeptide carried out cassette mutagenesis with oligonucleotide mediated (or location) from natural source.

Oligonucleotide mediated mutagenesis is a preferred method of displacement, disappearance and the insertion varient of preparation mpl part DNA.Referring to Adelman etc., DNA, 2:183[1983], this technology is widely known by the people.In simple terms, the oligonucleotide by the required sudden change of will encoding and a dna profiling hybridization change mpl part DNA, and template is to contain not becoming or the plasmid of natural DNA sequence or the single stranded form of phage of mpl part.After the hybridization, use the synthetic complete second chain that is complementary to template of archaeal dna polymerase, thus, this chain can mix Oligonucleolide primers, and encodes for variation selected among the mpl part DNA.

Usually, use length to have the oligonucleotide of 25 Nucleotide at least.The suitableeest oligonucleotide has 12 to 15 Nucleotide, and on the Nucleotide both sides of encoding mutant, all complementary fully with template.This has just guaranteed that this oligonucleotide can correctly hybridize with the single stranded DNA template molecule.This oligonucleotide can use on this area known technology easily synthetic, referring to Crea etc., and Proc.Natl.Acad.Sci.USA, 75:5765[1978].

Dna profiling is produced by carrier, these carriers can be the derivatives (M13mp18 that is purchased and M13mp19 are suitable carriers) of phage M13 carrier, also can be through duplicating the single stranded phage that obtains, referring to Viera etc., Meth.Enzymol., 153:3[1987].Therefore, the DNA of needs sudden change can be inserted in above-mentioned a kind of carrier, thereby produce single-stranded template.The generation of single-stranded template is referring to Sambrook etc., 4.21-4.41 part among the MolecularCloning:A Laboratory Manual (Cold Spring Harbor LaboratoryPress, NY 1989).

Perhaps, the single stranded DNA template can obtain by the double-stranded plasmid of sex change or other DNA with standard technique.

Under suitable hybridization conditions, in single-stranded template natural dna sequence dna is changed (as producing the variant amino acid sequence body) oligonucleotide hybridization.Then, add certain archaeal dna polymerase, normally the Klenow fragment of dna polymerase i is synthesized the complementary strand of template, and this oligonucleotide is then as the synthetic primer.So, just formed a heteroduplex molecule, the mutant form of a DNA chain encoding mpl part wherein, another chain (primary template) then encode the mpl part natural, do not become sequence.Again this heteroduplex molecule is transformed into proper host cell, normally prokaryotic cell prokaryocyte such as intestinal bacteria JM101.Cell is coated agarose plate with it after outgrowth, and uses ³²The radiolabeled Oligonucleolide primers of P screens, and contains the bacterium colony of mutant DNA with evaluation.The proteinic carrier of suitable generation is removed and inserted to saltation zone, and this class expression vector is as transforming appropriate host usually.

Aforesaid method can be modified, be that two chains of plasmid all contain sudden change to produce the homoduplex molecule.The mode of modifying is as described below.Single stranded oligonucleotide is annealed with above-mentioned single-stranded template.The mixture of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), these three kinds of deoxynucleotides of deoxyguanosine triphosphate (dGTP) is combined with the sulfo-deoxycytidine triphosphate that is called as dCTP-(aS) (can obtain from Amersham company).Mixture is added template-oligonucleotide complex.In the mixture that obtains, add after the archaeal dna polymerase, just obtained a DNA chain identical, wherein except the mutating alkali yl with template.In addition, in new synthetic DNA chain, dCTP-(aS) has replaced dCTP, protects the digestion of its unrestricted restriction endonuclease.

Use suitable restriction enzyme on the template strand of this heteroduplex, to produce a breach, then, template strand is digested with ExoIII nuclease or other suitable nucleases that does not act on the zone, mutational site.Behind the reaction terminating, obtain the molecule of a part strand.After adding all 4 kinds of deoxynucleoside triphosphates, ATP and dna ligases, use archaeal dna polymerase with synthetic complete dna homology duplex molecule.As mentioned above, this homoduplex molecule can be transformed into proper host cell such as intestinal bacteria JM101.

The DNA of the mpl part mutant of an above amino-acid substitution of coding can be by some generation the in the several method.If the position of metathetical amino acid on polypeptide chain is nearer each other, can be with an oligonucleotide with they simultaneous mutations, all anticipations are by metathetical amino acid as long as this oligonucleotide can be encoded.But if these amino acid whose positions have certain distance (by more than 10 amino acid separately) each other, the oligonucleotide that produces single all anticipation variations of encoding is just more difficult.Can use one in following two alternatives to solve.

First method, produce an isolating oligonucleotide come corresponding each by metathetical amino acid.This oligonucleotide is annealed with single-stranded template DNA, from the amino-acid substitution of all anticipations of template synthetic second DNA codified.

Second method needs twice or the mutant of more mutagenesis circulation to obtain envisioning.Working cycle for the first time is identical with above-mentioned single mutation type: wild-type DNA is as template, and the oligonucleotide of first anticipation amino-acid substitution of encoding is annealed with template, produces the heteroduplex DNA molecule then.The mutant DNA of the circulation second time mutagenesis for the first time circulation generation of mutagenesis is as template.So this template has contained one or more sudden changes.Then, oligonucleotide of other anticipation amino-acid substitutions of coding are annealed with this template, then just obtained that a DNA chain can be encoded for the first time and the circulation of mutagenesis for the second time in sudden change.The DNA that obtains so just can be as template, and so in circulation for the third time.

PCR mutagenesis also is applicable to the amino acid variation body that produces the mpl ligand polypeptide.Though following discussion only refers to DNA, should understand this technology and be equally applicable to RNA.Round pcr generally includes following program (referring to Erlich, 61 to 70 pages of chapters and sections that the same R.Higuchi shows): among the PCR with a small amount of template DNA as initial substance, use the primer that Light Difference is arranged with the sequence of template DNA respective regions, can produce a large amount of relatively specific DNA fragments, its sequence is only variant in primer position and the template different with template.To introduce a plasmid DNA in order suddenling change, to need primer of design and sudden change location overlap and contain this sudden change; The sequence of another primer must be identical with a part of sequence of plasmid complementary strand, but this sequence can be positioned any position on the plasmid DNA.But the sequence of second primer preferably is positioned apart from 200 Nucleotide of first primer sequence the whole DNA amplification region that is connected with primer that obtains at last easily to be checked order.As mentioned above, pcr amplification uses a pair of primer to produce certain a large amount of dna fragmentations, and the sudden change position that the difference of this dna fragmentation is may be in primer special also might be in other positions, because duplicating of template produces fallibility sometimes.

If template and product ratio are very low, then the dna fragmentation of overwhelming majority generation has mixed the sudden change of anticipation.Use the standard DNA technology, this product can be substituted as the respective regions on the plasmid of pcr template.Sudden change on disconnected position can be introduced simultaneously, introducing method can be to use second kind of primer of a mutant, also can be to be connected in carrier segments simultaneously with the different mutant primer work PCR second time and two PCR fragments that will obtain by three step (or more) ligations.

In a specific example of PCR mutagenesis, template plasmid DNA (1 microgram) is through the digestion of restriction enzyme and linearizing, and the part of this restriction enzyme beyond plasmid DNA is amplified the zone has only single recognition site.This product adding of 100 nanograms is contained in the PCR mixture of PCR damping fluid, is in the PCR mixture of 50 microlitres in final volume, and GeneAmp is arranged ^In the matched reagent (from Perkin-Elmer Cetus company, Norwalk, CT and Emeryville, CA) four kinds of deoxynucleoside triphosphates and two kinds of each 25 picomole of Oligonucleolide primers.Cover reaction mixture with 35 microlitre mineral oil.Reaction mixture is 100 ℃ of sex change after 5 minutes, places on ice and add 1 microlitre thermus aquaticus (Taq) archaeal dna polymerase (every microlitre 5 units, available from Perkin-Elmer Cetus company) under the mineral oil reservoir.Then, reaction mixture is placed DNA Thermal Cycler instrument (available from Perkin-Elmer Cetus company), following setting program:

2 minutes, 55 ℃

30 seconds, 72 ℃ was 19 circulations of following steps then:

30 seconds, 94 ℃

30 seconds, 55 ℃ and

30 seconds, 72 ℃.

After above EP (end of program), the reaction bottle takes out thermal cycler instrument, water is transferred in the new bottle, and phenol/chloroform extracting (volume 50: 50), ethanol sedimentation, and by standard program recovery DNA.Then, this product is done suitable processing and make it to insert carrier.

Cassette mutagenesis is the another kind of method for preparing varient, and its technical basis is referring to Wells etc., Gene, 34:315[1985].Initial substance is the plasmid (or other carriers) that contains the mpl part DNA that will be suddenlyd change.In mpl part DNA, the codon that will be suddenlyd change is determined.Each side in the mutational site of determining all must have single restriction endonuclease sites.If such restriction site does not exist, can use above-mentioned oligonucleotide mediated mutafacient system to be introduced into the correct position of mpl part DNA.After restriction site is incorporated into plasmid, make it linearizing at these sites cutting plasmids.With the synthetic dna sequence dna between the restriction site but contain the double chain oligonucleotide of predetermined sudden change of encoding of standard program.Article two, chain is synthetic respectively, and makes it hybridization with standard technique.This double chain oligonucleotide is called as box.This box is designed to contain and the terminal compatible 3 ' end and 5 of linearization plasmid ' end, makes it directly to be connected with plasmid.At this moment, this plasmid has contained mpl part dna sequence dna.

C. the insertion of replicable vector amplifying nucleic acid

Nucleic acid (as cDNA or genomic dna) coding is natural or variation mpl ligand polypeptide inserts reproducible carrier and is used for further clone (amplification of DNA) or expression.Many available support are arranged, and the selection of suitable carrier is a basis: (1) whether this carrier will be used for DNA cloning or expression, and (2) are inserted into nucleic acid size and (3) of carrier will be by this carrier transformed host cells.Each carrier can contain multiple components according to its function (amplification of DNA or expression) with host cell that it adapts.The composition of carrier generally includes (but being not limited to) following one or more: signal sequence, replication origin, one or more marker gene, enhancer element, promotor and transcription termination sequence.

(i) signal sequence composition

Mpl part of the present invention can not only directly be expressed, and can with the fusion formal representation of heterologous polypeptide, this heterologous polypeptide is signal sequence or other polypeptide that a specificity cleavage site is arranged at the N-of mature protein or polypeptide end preferably.Usually, this signal sequence can be a composition of carrier, or inserts the part of the mpl part DNA of carrier.Host cell (can be ruptured by a signal peptidase) must be discerned or belong to selecteed allos signal sequence by host cell.For the prokaryotic host cell that can not discern or not have natural mpl part signal sequence, the chosen prokaryotic signal sequence of this signal sequence substitutes, for example selected signal sequence from one group of alkaline phosphatase, penicillinase, Ipp or thermally-stabilised enterotoxin 1 I leader.Secrete for zymic, the example that can substitute the natural signals sequence has: the signal described in the WO90/13646 of yeast invertase, alpha factor, acid phosphatase leader, Candida albicans glucoamylase (EP 362,179 that publish April 4 nineteen ninety) or publication on November 15 nineteen ninety.For mammiferous expression, natural signals sequence (promptly instructing the mpl part leader sequence that natural mammalian cell secretion mpl part is done in the body) is gratifying, though other mammiferous signal sequences also may be suitable, for example: from other mpl ligand polypeptides or from the signal sequence of the identical mpl part of a different animals kind, from the signal sequence of a mpl part, from signal sequence and the viral secretory leader such as the single dominance combination of the bleb gD signal of the secrete polypeptide of identical or relevant species.

(ii) replication origin composition

Expression and cloning vector all contain a nucleotide sequence, and this sequence can be duplicated carrier in the host cell of one or several selection.Usually, in cloning vector, this sequence makes carrier be independent of the host chromosome cell DNA and duplicates, and contains replication orgin or autonomy replication sequence.This sequence is distributed widely among bacterium, yeast and the virus.Replication origin from the pBR322 plasmid is suitable for most of gram negative bacteriums; 2 μ plasmid starting points are suitable for yeast; Multiple viral starting point (SV40, polyomavirus, adenovirus, VSV or BPV) is then useful to the cloning vector in the mammalian cell.Usually, mammalian expression vector does not need replication origin composition (will use the SV40 starting point usually, for no other reason than that it contains early promoter).

Most of expression vectors are " shuttling back and forth " carriers, and promptly they can duplicate at least one class organism but can transfectedly express in another kind of organism.For example, a carrier is cloned in intestinal bacteria, then same carrier transfection is expressed in yeast or mammalian cell, even it can not be independent of host cell chromosome and duplicate.

Equally can be by inserting the host genome DNA amplification.This process can be by easily finishing as the host with bacillus, for example: add a dna sequence dna that is complementary to certain sequence on the vaccae genomic dna in carrier.With this carrier transfection bacillus, homologous recombination between the mpl part DNA of generation bacillus gene group and insertion.Yet the recovery of the genomic dna of coding mpl part is more complicated than the recovery of external source replicating vector, because need the digestion of restriction enzyme to cut mpl part DNA.

(iii) select the gene composition

Expression and cloning vector should contain one selects gene, also claims selective marker.This genes encoding transformed host cell is being selected the survival and the necessary protein of growing on the substratum.Can not on substratum, survive without containing the carrier transformed host cells of selecting gene.The typical protein of genes encoding of selecting has: (a) provide microbiotic or other toxin resistances, as penbritin, Xin Meisu, methotrexate or tsiklomitsin, (b) complementary auxotrophic defective or (c) provide the important nutrition that does not have in the complex medium is as the gene of coding bacillus D alanine racemase.

An example of select planning is to use medicine to stop the growth of host cell.Those are expressed the protein of a drug resistance with heterologous gene success cell transformed and can survive under selection condition.The medicine that several examples that this advantage is selected use is Xin Meisu (South-ern etc., J.Molec.Appl.Genet.1:327[1982]), mycophenolic acid (Mullgan etc., Science, 209:1422[1980]) or Totomycin (Sugden etc., Mol.Biol., 5:410-413[1985]).Above-mentioned three examples use bacterial gene to express resistance to suitable medicine under eucaryon control, and corresponding medicine is G418 or Xin Meisu (Geneticin), xgpt (mycophenolic acid), Totomycin.

The example of the selected marker of other suitable mammalian cells is marks that those energy identification of cell compositions contain the mpl ligand nucleic acid, as dihydrofolic acid (DHFR) or thymidine kinase.The mammalian cell transformant is under the selection pressure, has only transformant underlinedly to adapt to and survive owing to containing.In substratum, change continuously under the condition of concentration of selective reagents, the transformant of cultivating is imposed selective pressure, consequently cause selecting the DNA of gene and coding mpl ligand polypeptide all to be amplified.In such amplification procedure, in the reconstitution cell karyomit(e) of continuous passage, the gene that increases because of the necessary a kind of protein requirement amount of growing repeats with the series connection form.The DNA of amplification increases mpl part synthetic amount.

For example, the evaluation of cell of selecting gene transformation with DHFR is cultivated all transformant at first by in the culture that contains a kind of DHFR competitive antagonist methotrexate (Mtx).When utilization wild-type DHFR, lacking the active Chinese hamster ovary of DHFR (CHO) clone is a kind of proper host cell, about its preparation and propagation, referring to Urlaub and Chasin, Proc.Natl.Acad.Sci.USA, 77:4216[1980].Then, transformant is exposed to the Mtx that concentration increases.This has caused other DNA that constitute expression vectors of the synthetic and consequent of multiple copied DHFR gene to increase as the copy number of the DNA of coding mpl part.If used the mutant DHFR gene (EP 117,060) that Mtx is had the height resistance, endogenous DHFR is arranged, this amplification technique is also applicable to any other appropriate host, as ATCC No.CCL61 CHO-K1.Perhaps, with coding mpl part, wild-type dhfr protein matter and another kind of selected marker.As the dna sequence dna conversion of aminoglycoside 3 ' transphosphorylase or the host cell [the wild-type host of especially containing endogenous DHFR] of cotransformation, can be selected by the cell growth in containing the culture of selective agent, wherein, the selective agent that is used for selected marker is an aminoglycoside antibiotics etc., as kantlex, Xin Meisu or G418.Referring to the U.S. patent No. 4,965,199.

Be suitable for using in the yeast one select gene be the trp1 gene that comes across yeast plasmid YRp7 (Stinchcomb etc., Nature, 282:39[1979]; Kingsman etc., Gene, 7:141[1979] or Tschemper etc., Gene, 10:157[1980]).The trp1 gene offers the yeast mutant strain of in tryptophane energy for growth disappearance with a selective marker, for example ATCC No.44076 or PEP4-1 (Jones, Genetics, 85:12[1977]).So the trpl disappearance in the yeast host cell gene group provides a kind of effect environment, is used for detecting conversion by the growth under no tryptophane situation.Similarly, yeast strain of Leu2 defective (ATCC No.20,622 or 38,626) and known plasmid complementation of carrying the Leu2 gene.

(iv) promotor composition

Expression and cloning vector contain a promotor, the mpl ligand nucleic acid of can being discerned by host organisms and be operably connected usually.Promotor is a non-translated sequence, operationally connects with a structure gene (usually about 100 to 1, between the 000bp), and is positioned at the upstream (5 ') of the structure gene initiator codon that control specific nucleic acid sequence such as mpl ligand nucleic acid sequence transcribe and translate.This promotor is divided into induction type and composing type two classes usually.Inducible promoter can be controlled DNA and transcribe, and makes transcriptional level begin to increase, this effect be with in the culture condition some change as a kind of nutraceutical existence whether or variation of temperature occur.Now, having known can be by a large amount of promotor of many potential host cell identifications.Digestion through Restriction Enzyme is excised promotor from source DNA, isolating promoter sequence is inserted carrier, thereby makes the be operably connected DNA of coding mpl part of these promotors.Natural mpl part promotor and many allogeneic promoters can both be used for instructing amplification and/or the expression of mpl part DNA.But allogenic promotor is more better, because compare with natural mpl part promotor, they can allow stronger transcribing and higher mpl ligand expression output usually.

The promotor that is applicable to prokaryotic hosts comprises beta lactamase and lactose promoter systems (Chang etc., Nature, 275:615[1978]), alkaline phosphatase, tryptophane (trp) promoter systems (Goeddel, Nucleic Acid Res., 8:4057[1980] and EP 36,776) and hybrid promoters such as tac promotor (deBoer etc., Proc.Natl.Acad.Sci.USA, 80:21-25[1983]).Yet other known bacterium promotors are suitable.Their nucleotide sequence is delivered, and therefore, uses joint or adapter that required restriction site is provided, and skilled staff operationally connects their the DNA (siebenlist etc., Cell, 20:269[1980]) of coding mpl part.The promotor that is used for bacterial system also contains a Shine-Dalgarno (S.D.) sequence, the DNA of the coding mpl ligand polypeptide that is operably connected.

Eukaryotic cell also has promoter sequence.In fact, all there is an AT enrichment region at about 25 to 30 base places, all eukaryotic gene transcription initiation site upstreams.At 70 to 80 base places, many genetic transcription starting points upstream, found that also another section sequence is called the CXCAAT district, wherein X can be any Nucleotide.Perhaps, at 3 ' end of most of eukaryotic genes the sequence of AATAAA being arranged, is the signal that adds poly A tract at encoding sequence 3 ' end.All these sequences can be inserted carrier for expression of eukaryon suitably.

The example of the suitable initiating sequence that the yeast host uses has: glycerol 3-phosphate acid kinase (Hitzeman etc., J.Biol.Chem., 255:2073[1980]) or other glycolytic ferments (Hess etc., J.Adv.Enzyme Reg., 7:149[1968] and Holland, Biochemistry, 17:4900[1978]) promotor, as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvic carboxylase, phosphofructokinase, glucose-6-phosphate isomerase, the 3-phoshoglyceric acid mutase, pyruvate kinase, triosephosphate isomerase, glucose phosphate isomerase and glucokinase.

The inducible promoter of the advantage of additionally transcribing is promptly controlled and had to other yeast promotors by growth conditions, be the promoter region of following material: alcohol dehydrogenase 2, isomery cytochrome C, acid phosphatase, the degrading enzyme of being correlated with nitrogen metabolism, metallothionein(MT), glyceraldehyde-3-phosphate dehydrogenase and the enzyme relevant with the semi-lactosi use with maltose.About the suitable carrier that uses in yeast expressed and promotor further describe referring to Hitzeman etc., EP 73,657A.It also is favourable that the yeast enhanser uses with the yeast promotor.

For instance, the mpl part of carrier is transcribed by promotor and is controlled in the mammalian host cell, the promotor here is from viral genome, (on July 5th, 1989 published as polyomavirus, fowlpox virus, UK 2,211,504), adenovirus (as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a kind of retrovirus, hepatitis B virus, simian virus 40 (V40) preferably; From allogenic mammalian promoter, as actin promoter and immunoglobulin promoter; From the heat-shocked promotor; Also can be from the common mpl ligand sequence relevant, as long as these promotors are compatible with host cell systems with the mpl part.

Early stage and the late promoter of SV40 virus can be used as a SV40 restriction fragment and obtains easily, wherein also contain SV40 virus replication initiation (Fiers etc., Na-ture, 273:113[1978]; Mulligan and Berg, Science, 209:1422-1427[1980]; Pavlakis etc., Proc.Natl.Acad.Sci.USA, 78:7398-7402[1981]).The cytomegalovirus immediate early promoter can be used as HindIII E restriction fragment and obtains (Greenway etc., Gene, 18:355-360[1982]) easily.The description that with the bovine papilloma virus is carrier system of expressible dna in mammalian hosts is referring to the U.S. patent No. 4,419,446.U.S. the patent No. 4,601, and 978 is an improvement of this system.Other also can be with reference to Gray etc., Nature, among the 295:503-508 about the expression of coding type II interferon cDNA in simian cells; Reyes etc., Na-ture, 297:598-601[1982] in about under the control of the single dominance combination of bleb viral thymidine kinase promoter, the expression of human cDNA in the mouse cell; Canaani and Berg, Proc.Natl.Acad.Sci.USA, 79:5166-5170[1982] in about people β 1 interferon gene in mouse of cultivating and the expression in the rabbit cell; And Gorman etc., Proc.Natl.Sci.USA, 79:6777-6781[1982] in about being promotor with Rous sarcoma virus long terminal repeat, the expression of bacterium CAT sequence in CV-1 ape and monkey nephrocyte, chick embryo fibroblast, Chinese hamster ovary cell, HeLa cell and mouse NIH-3T3 cell.

(v) enhanser composition

In the present invention, often increase the transcribing of DNA of coding mpl part in the more high eukaryotic cell by in carrier, inserting an enhancer sequence.Enhanser is the cis-acting elements of DNA, and about 100 to 300bp usually, and acting on one increases its promotor of transcribing.The position and the direction of enhanser are relatively independent, at 5 of transcription unit ' end (Laimis etc., Proc.Natl.Acad.Sci.USA, 78:993[1981]) and 3 ' end (Lusky etc., Mol.Cell Bio., 3:1108[1983]) discovery all arranged, encoding sequence (people such as Osborne at an intron (Banerji etc., Cell, 33:729[1983]) and itself, Mol.Cell Bio., 4:1293[1984]) among discovery is also arranged.The enhancer sequence of known many mammalian genes (globin, elastoser, albumin, α-fetoprotein and Regular Insulin).But normally used enhanser is from eukaryotic cell virus.For example, at the SV40 enhanser (100-270bp) of replication orgin late gene one side, the sub-enhanser of cytomegalovirus early promoter is at the polyoma enhanser and the adenovirus enhanser of replication orgin late gene one side.Referring to Yaniv, Nature strengthens element about the eukaryotic promoter activatory among the 297:17-18.Enhanser can be spliced to the carrier that is arranged in mpl part encoding sequence 5 ' end or 3 ' end, but preferred site is at 5 of promotor ' end.

(vi) Transcription Termination composition

The expression vector that uses in the eukaryotic host cell (yeast, fungi, insect, plant, animal, people or other multicellular organisms contain karyocyte) also contains and stops transcribing the sequence required with stable mRNA.Usually, this sequence is from 5 ' end, once in a while from 3 of eukaryotic cell or viral DNA or cDNA ' end non-translational region.The nucleotide fragments that partly is transcribed into polyadenylic acid at the untranslated of coding mpl ligand mRNA is contained in these zones.

(the vii) structure of carrier and analysis

By the standard interconnection technique, make up and contain one or more suitable carrier in the above listed composition.Isolating plasmid or dna fragmentation are cut, adapt and be connected, with the required plasmid of form generation of anticipation.

Connect mixture transformed into escherichia coli K12 bacterial strain 294 (ATCC No.31,446) for analyzing the correct sequence of determining to make up plasmid, using, according to circumstances select successful transformant with penbritin or tetracyclin resistance.With digestion with restriction enzyme preparation, analyze in the transformant plasmid and/or with Messing etc., Nucleic Acids Res., 9:309[1981] or Maxam etc., Methods in Enzymology, 65:499[1980] in the method order-checking.

(viii) transient expression carrier

Useful especially expression vector can provide the transient expression of the DNA of coding mpl ligand polypeptide in the mammalian cell in enforcement of the present invention.Usually, transient expression requires to use the expression vector that energy effectively duplicates in host cell, and it effectively duplicates the many copies that make accumulation expression vector in the host cell, synthesizes the required polypeptide by expression vector codes thus high-levelly.Referring to Sambrook etc., the same, pp.16.17-16.22.Transient expression system is made up of suitable expression vector and host cell, can identify the cloned DNA encoded polypeptides easily and promptly screen this polypeptide to obtain required biology and physiological property.Therefore, to have the analogue and a varient of the bioactive mpl ligand polypeptide of mpl ligand polypeptide for evaluation in the present invention particularly useful for transient expression system.

(ix) example of suitable vertebrate cells carrier

In the recombinant vertebrate cell culture, other suitable methods, carrier and the host cell of synthetic mpl part be referring to Gething etc., Nature, 293:620-625[1981]; Mantei etc., Nature, 281:40-46[1979]; Levinson etc., EP 117,060 and EP 117,058.PRK5 (EP 307, the 247 U.S. patent No.s 5,258,287) or pSV16B (PCT publication number WO 91/08291) are the useful especially plasmids that mammalian cell cultures is expressed the mpl part.D. the selection of host cell and conversion

Prokaryotic cell prokaryocyte mentioned above, yeast or higher eucaryotic cells all are the clones or express the suitable host cell of carrier herein.Suitable prokaryotic cell prokaryocyte comprises eubacterium such as Gram-positive or negative bacterium, for example pseudomonas, salmonella typhimurium or emplastic serratias such as bacillus such as intestinal bacteria, Bacillus subtilus, Pseudomonas aeruginosa.Though other bacterial strain such as intestinal bacteria B, intestinal bacteria X1776 (ATCC No.31,537) and intestinal bacteria W3110 (ATCC No.27,325) also are suitable, intestinal bacteria 294 (ATCC No.31,446) are preferred escherichia coli cloning hosts.These examples only are to be used for explanation, are not that the expression proper host cell is only limited to these.Preferably, the amount of secretory host cell proteolytic ferment is minimum.In addition, the body outer clone method also is suitable for as PCR or other nucleic acid polymerase reactions.

Except prokaryotic cell prokaryocyte, eukaryotic microorganisms such as filamentous fungus or yeast are mpl part code carrier appropriate host.Yeast saccharomyces cerevisiae or common bread yeast are the most frequently used in eucaryon host microorganism such as low.But usually available a series of other genus, kind and bacterial strain can be used for herein, as pombe fission yeast bacterium (Beach and Nurse, Na-ture, 290:140[1981]); The EP 139 that on May 2nd, 1985 delivered, 383), kluyveromyces spp host (the U.S. patent No. 4,943,529) as K.lactis (Louven-court etc., J.Bacteriol., 737[1983]), K.fragilis, K.bulgaricus, K. thermoduric bacteria and K.marxianus, yarrowia (EP 402,226), (EP 183,070 for Pichia pastoris; Sreekrishna etc., J.Basic Microbiol., 28:265-278[1988]), white silk yeast, (EP 244 for Trichoderma reesia (trichoderma), 234), Neurospora crassa (Case etc., Proc.Natl.Acad.Sci.USA, 76:5259-5263[1979]) and filamentous fungus such as chain spore enzyme genus, Penicillium, Tolypocladi-um (WO 91/00357 that on January 10th, 1991 published) and Aspergillus host such as Aspergillus nidulans (Ballance etc., Biochem.Biophys.Res.Commun., 112:284-289[1983]; Tilburn etc., Gene, 26:205-221[1983]; Yelton etc., Proc.Sci.USA, 81:1470-1474[1 984]) and aspergillus niger (Kelly and Hynes, EMBO J., 4:475-479[1985]).The suitable host cell of expressing glycosylation mpl part is from multicellular organism.This class host cell can be finished complicated processing and tool glycosylation activity.In principle, no matter be from vertebrates or invertebrates culture, any higher eucaryotic cells culture is an available.The example of invertebral zooblast has plant and insect cell.Identified a large amount of baculovirus strains and varient and corresponding insect and allowed host cell, as fall army worm (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), drosophila melanogaster (fruit bat) and silkworm.Referring to Luckow etc., Bio/Technology, 6:47-55[1988]; Miller etc., Genetic Engineering, editors such as Setlow, Vol.8 (Plenum publishes, 1986), pp277-279 and Maeda etc., Na-ture, 315:592-594[1985].Can obtain many virus strain that are used for transfection from public approach, as the Bm-5 strain system of L-1 varient and the silkworm NPV of autographa california NPV, these viruses can be by used herein, especially for the transfection of fall army worm cell.

The plant cell cultures of cotton, corn, potato, soybean, morning glory, tomato and tobacco can be used as the host.Usually, the transfection of vegetable cell is by with the Agrobacterium tumefaciems incubation, and wherein, the latter has been contained mpl part DNA through operation.By the incubation of vegetable cell with Agrobacterium tumefaciems, the DNA of coding mpl part transfers to and makes it to be subjected to transfection among the vegetable cell host, and expresses mpl part DNA under appropriate condition.In addition, can obtain adjusting and the signal sequence compatible, as rouge alkali synthetase promoter and polyadenylic acid signal sequence with vegetable cell.Referring to Depicker etc., J.Mol.Appl.Gen., 1:561[1982].In addition, can activate or increase the transcriptional level of expressive gene of plant the plant tissue that contains recombinant DNA from T-DNA780 upstream area of gene separated DNA fragment.Referring to the EP 321,196 that published on June 21st, 1989.

But the most interesting is vertebrate cells.In recent years, the propagation of vertebrate cells in substratum (tissue culture) has become a kind of routine operation (TissueCulture, Academic Press, Kruse and Patterson, editors[1973]).The monkey kidney CV1 clone (COS-7, ATCC CRL 1651) that useful mammalian host cell line has SV40 to transform; The human embryonic kidney cell line (grow in 293 or 293 subclone cells of suspension culture, referring to Graham etc., J.Gen virol., 36:59[1977]); Baby hamster kidney cell (BHK, ATCC CCL 10); Chinese hamster ovary cell/-DHFR (CHO, Urlaub and Chasin, Proc.Natl.Acad.Sci.USA, 77:4261[1980]); The mouse podocyte (TM4, Mather, Biol.Reprod., 23:243-251[1980]); Monkey-kidney cells (CV1 ATCC CCL 70); African green monkey kidney cell (VERO-76, ATCC CRL-1587); People's laryngeal cancer cell (HELA, ATCCCCL 2); Madin-Darby canine kidney(cell line) (MDCK, ATCC CCL 34); Buffalo mouse liver cell (BRL3A, ATCC CRL 1442); Human pneumonocyte (W138, ATCC CCL 75); Human liver cell (Hep G2, HB 8065); (MMT 060562, ATCCCCL51) for mouse milk tumour; The TRI cell (Mather etc., Annals N.Y.Acad.Sci.383:44-68[1982]); The MRC5 cell; FS4 cell and Bel7402 (Hep G2).

With above-mentioned expression vector of the present invention or cloning vector transfection, transformed host cell preferably, in the nutritional medium of routine, cultivate and cooperate inducible promoter, select the gene of transformant or the required sequence of amplification coding.Whether no matter in fact to have encoding sequence to express, transfection refers to that host cell obtains an expression vector.For masterful technique person, the method that a large amount of transfections are arranged is known, as calcium phosphate and electroporation.Usually, when in host cell, finding any sign of this carrier function, can confirm the transfection success.

Transform expression DNA is introduced a kind of biology, this DNA can be duplicated with outer composition of karyomit(e) or chromosomal integration form.According to the host cell that is used, utilization is fit to the standard technique of this cell and finishes conversion.As Sambrook etc., the same 1.82 parts are described, have the cell of cell walls to carry out calcium with calcium chloride to prokaryotic cell prokaryocyte and other usually and handle.The certain plants transformation infects by Agrobacterium tumefaciems, referring to shaw etc., Gene, 23:315[1983] and the WO 89/05859 that published on June 29th, 1989.In addition, also available supersound process transfection plant.Referring to the WO 91/00358 that published on January 10th, 1991.For the mammalian cell that does not have cell walls, use Graham and van derEb at Virology, 52:456-457[1978] in the calcium phosphate precipitation method better.Axel has narrated the generalized case that the mammalian cell host system transforms in the U.S. patent No. 4,399,216 of promulgation on August 16 nineteen eighty-three.Carry out usual method that yeast transforms referring to Van Solingen etc., J.Bact., 130:946[1977] and Hsiao etc., Proc.Natl.Acad.Sci. (USA), 76:3829[1979].But also available additive method is introduced cell with DNA, merges as nucleic acid injection, electroporation or protoplasma.E. the cultivation of host cell

As Sambrook etc., the general introduction of (the same), the prokaryotic cell prokaryocyte that is used for producing the mpl ligand polypeptide among the present invention is cultivated in suitable medium.

The mammalian host cell that is used for producing the mpl ligand polypeptide among the present invention can be cultivated in multiple substratum.Substratum that is purchased such as Ham ' s F10 (sigma company), MEM ([MEM] sigma company), RPMI-1640 (sigma company) and the improved Eagle ' of Dul-beccos s substratum ([MEM] sigma company) all are fit to the cultivation of host cell.In addition, any substratum described in the following document can be used as the substratum of host cell: Ham and Wallace, Meth.Enz., 58:44[1979], Barnes and Sato.Anal.Biochem., 102:255[1980], U.S. Patent number 4,767,704; 4,657,866; 4,927,762 or 4,560,655; WO 90/03430; WO 87/00195; United States Patent (USP) Re.30,985 or the copending U.S.S.N.07/592 that files simultaneously October 3 nineteen ninety, 107 or 07/592,141, all files are incorporated herein by reference.As required can be at hormone supplemented in any one of these substratum and/or other somatomedins (as Regular Insulin, Transferrins,iron complexes or Urogastron), salt (as sodium-chlor, calcium, magnesium and phosphoric acid salt), damping fluid (as HEPES), nucleosides (as adenosine and thymidine), microbiotic (as the medicine gentamicin), trace element (final concentration is at the inorganics of micromole's level usually) and the glucose or the suitable energy.Those skilled in the art know, also can add other any essential fill-ins of proper concn.The culture condition of temperature, pH value and so on before be used to select those conditions of expression host cell identical, this also is clearly to general those of skill in the art.

Host cell comprises that the cell of vitro culture and the intravital cell F. of host animal detect gene amplification/expression

In a sample, the measurement of gene amplification and/or expression can be directly by following method: conventional Southern trace that quantification of mrna is transcribed and Northern blotting (Thomas, Proc.Natl.Acad.Sci.USA, 77:5201-5205[1980]), Dot blot (DNA analysis) or in situ hybridization, and according to the suitable label probe of sequence use that provides.Have many marks to be used, great majority are radio isotope, especially ³²P.But, also can use other technologies, as the Nucleotide of biotin modification is introduced polynucleotide.Then, as the site in conjunction with avidin or antibody, the latter can be by many kinds of mark institute marks, as radionuclide, fluorescent agent, enzyme or other analogues with vitamin H.Perhaps, the antibody that to discern specific duplex be can use, DNA duplex, RNA duplex, DNA-RNA heteroduplex body or DNA-protein duplex comprised.Conversely, but antagonist carries out mark, and analyzes at duplex and surface bonding place, when forming duplex from the teeth outwards, can detect and duplex bonded antibody.

The also available immunization method of expression of gene is measured, and comes the expression of direct quantitative gene product as the analysis with the immunohistochemical staining of tissue slice and cell culture or body fluid.In the immunohistochemical staining technology, generally prepare cell sample with dehydration and fixing means, then react the mark of use such as enzyme labelling, fluorescent mark, luminescent marking and analogue in the position that can detect mark usually with the traget antibody that is specific to the coupling gene product.Be applicable to that a responsive especially staining technique of the present invention sees Hsu etc., Am.J.Clin.Path., 75:734-738[1980].

The antibody that can be used for immunohistochemical staining and/or sample liquids analysis can be mono-clonal or polyclone, can prepare in any Mammals.Usually, the preparation of antibody is passed through at natural mpl ligand polypeptide or synthetic peptide, and wherein, the latter synthesizes according to the dna sequence dna that provides herein, and further narration sees below.The purifying of G.mpl ligand polypeptide

Though when directly expressing, also can from the host cell lysate, reclaim the mpl part, had better from substratum, reclaim with the form of secrete polypeptide there not being secretion signal.

When being expressed in the reconstitution cell of mpl part in inhuman source, do not contain people's source protein matter or polypeptide in this mpl part fully.But, still be necessary purifying mpl part from other recombinant cell protein matter or polypeptide, to obtain and the basic homologous prepared product of mpl part itself.The first step, centrifugal culture or lysate are to remove small cell debris.Then, separatory membrane and soluble protein part.Perhaps, can use commercially available protein to concentrate filter membrane (as Amicon or Millipore Pellicon ultrafiltration unit).So, whether combine according to the mpl part with film, can partly and the membrane portions be purified to the mpl part from the soluble protein of cultivating lysate.Subsequently, by saltouing and using the exchange or the chromatography program of multiple gel matrix, the mpl part is purified from impurity soluble protein and polypeptide.These matrix comprise: acrylamide, agarose, dextran, Mierocrystalline cellulose and other materials commonly used in protein purification.The typical color spectrometry program that is applicable to protein purification comprises: immunity affine (as anti-hmpl part Mab), acceptor is affine (as mpl-IgG or albumin A Sepharose), hydrophobic interaction chromatogram (HIC) is (as ether, butyl or phenyl Toyopearl), phytohemagglutinin chromatography (as concanavalin A Sepharose and root of Szemao crotalaria phytohemagglutinin Sepharose), size exclusion (as Sephadex G-75), positively charged ion and anion-exchange column (as DEAE or carboxymethyl and sulfopropyl Mierocrystalline cellulose) and RPLC (RP-HPLC) are (as referring to Urdal etc., J.Chromatog., 296:171[1984] described in, with the human IL-2 of two successive RP-HPLC step purification of Recombinant).Other available purification steps comprise: ethanol sedimentation, ammonium sulfate precipitation, chromatofocusing method, preparation sds polyacrylamide gel electrophoresis and other similar approach.

Consider any qualitative basic variation of generation, have residue disappearance, insertion and metathetical mpl ligand variant body can use the method recovery same with natural mpl part.For example, the preparation of the syzygy of a mpl part and another protein or polypeptide such as bacterium or virus antigen can promote purifying; Available immune affinity column absorption fusion polypeptide contains in the post should antigenic antibody.In the combination of a remaining immune epitope, immune affinity columns such as the anti-mpl part of rabbit polyclonal post can be in order to absorption mpl ligand variant body by at least.Perhaps, available affinity chromatography purifying mpl part uses wherein that (CA) or the mpl-IgG of similar resin link coupled purifying, this method is widely used for Bio-Rad, Richmond with the Affi-Gel 10 of immobilization (preferably).Phenmethyl sulfuric acid fluorine proteinase inhibitor such as (PMSF) can be used for the Degradation of arrestin enzymolysis in purge process, and can add the growth that antibiosis usually prevents the accidental pollution thing.One skilled in the art will recognize that be fit to natural mpl part purification process perhaps needs revise to adapt to and express the variation of mpl part and varient character thereof in the reconstitution cell culture.The covalent modification of H.mpl ligand polypeptide

The covalent modification that has comprised the mpl ligand polypeptide in the category of the present invention.The natural mpl part and the variant amino acid sequence body of mpl part can both be by covalent modifications.Mpl part fragment is a type of covalent modification in the category of the present invention.Amino-acid residue can prepare easily up to about 40 variation mpl ligand polypeptide fragment, and its method has chemosynthesis or enzyme process or chemical chop total length or variation mpl ligand polypeptide.By the reaction of mpl part and segmental target amino acid residue and a kind of organic derivating agent, the mpl part and the segmental covalent modification thereof of other types are introduced molecule, this reagent can react with the residue of the side chain of selecting or N-or C-end.

Cysteinyl residue usually with α halogenated acetic acids (with corresponding amine) as chloracetic acid or chloro-acetamide reaction, with acquisition carboxymethyl or carboxylic acid amides methyl-derivatives.Cysteinyl residue derive from the reaction of following material, the propionic acid of bromine trifluoroacetone, α-bromo-β-(5-imidozoyl-), p chloromethylbenzoic acid acetyl, N-alkyl maleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, right-chloromercuri-benzoate, 2-chlorine mercury-4-nitrophenol or chloro-7-oil of mirbane-2-oxa-1, the 3-diazole.

Deriving of histidyl-residue by when the pH5.5-7.0 and the reaction of tetra-sodium diethyl ester, because this reagent has relative specificity to the histidyl-side chain.Also available para-bromop-henacyl bromide thing; The Kraft base acid sodium of 0.1M when preferred reaction conditions is pH6.0.

The anhydride reaction that relies amine acyl and n terminal residue and succsinic acid or other carboxylic acids.The derivatization of using these reagent to carry out can play the effect that counter-rotating relies amine acyl residue electric charge.Other are applicable to that the derivative reagent that contains amino residue has imido-ester such as subunit picolinimidate, pyridoxal phosphate, pyridoxal, chlorine hydroborate, trinitro-benzene-sulfonic acid, O-methyl-isourea, 2,4-diacetylmethane and contain the transaminase catalyzed reaction of glyoxylate.

The modification of spermine acyl residue by with the reaction of one or several conventional reagent, as phenyl glyoxylyl, 2,3-dimethyl diketone, 1,2-cyclohexanedione and triketohydrindene hydrate.Because the high pKa value of guanidine functional group, deriving of arginine residues need be carried out being reflected under the alkaline condition.In addition, these reagent can also react with the group and the arginic ε amino of Methionin.

The specificity of tyrosyl residue modify by with the reaction of aromatic diazonium compound or tetranitromethane, its main purpose is special mark is incorporated into network aminoacyl residue.In most of the cases, use N-acetyl imidazole and tetranitromethane correspondingly to obtain O-acetyl tyrosyl kind and 3-nitro-derivative.With ¹²⁵I or ¹³¹I iodate tyrosyl residue is used for the labelled protein of radioimmunoassay with preparation, and aforesaid chloramine-T method is applicable to this operation.

The carboxyl side group of aspartoyl and glutamy is by modifying selectively with the reaction of carbodiimide (R-N=C=N-R '), wherein, R is different alkyl with R ', as 1-ring ethyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethyl amyl group) carbodiimide.In addition, by with the reaction of ammonium ion, aspartoyl and glutamy residue change into asparaginyl and glutaminyl residue.

Can be used for the mpl part is linked on the water-insoluble supporting dielectric or surface that uses in the anti-mpl ligand antibody purification process with deriving of carrying out of bifunctional reagent.Vice versaad.Linking agent commonly used comprises 1,1-two (diazonium ethyl)-2-phenylethane, glutaraldehyde, N-hydroxy-succinamide ester such as 4-azidosalicylic acid ester, the difunctional imines ester of homology comprise that the double amber imide ester is as 3,3 '-dithio bis (succinimide propionic acid) and bifunctional maleimide be as two-N-dimaleoyl imino-1, the 8-octane.Derivative reagent such as methyl-3-[(p-azidophenyl) dithio] propioimidate produces and can form crosslinked photosensitive intermediate under illumination.Perhaps, the substrate of the water-fast medium of reaction such as cyanogen bromide activated carbohydrate and reaction such as U.S. Patent number 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537 and 4,330, described in 440, can be used for proteinic fixing.

Asparaginyl and the frequent deamination of glutaminyl residue and become corresponding aspartoyl and glutamy residue.The deamination of these residues is to carry out under neutrality or alkaline condition.The deamination form of these residues is within category of the present invention.

Other modifications comprise: the hydroxylation of proline(Pro) and Methionin, the phosphorylation of seryl or threonyl residue hydroxyl, (the T.E.Creighton that methylates of Methionin, arginine and Histidine side chain α amino, Proteins:Structure and Molecular Properties, W.H.Freeman ﹠amp; Co., San Francisco, pp.79-86[1983]), the amidation of the acetylize of N-terminal amino group and any C-terminal carboxyl(group).

The Natively glycosylated form that changes polypeptide is the mpl ligand polypeptide covalent modification of another type in the category of the present invention.The variation here refers to remove the carbohydrate part of one or more natural mpl parts and/or add the glycosylation site that does not have in one or more natural mpl parts.

The glycosylation of polypeptide is N-combination or O-combination normally.N-is in conjunction with referring to being connected of carbohydrate part and an asparagine residue side chain.The carbohydrate part is a tripeptide sequence with the enzymatic recognition sequence that combines of l-asparagine side chain: l-asparagine-X-Serine and l-asparagine-X-Threonine, wherein X represents any amino acid except that proline(Pro).Therefore, during in occurring these tripeptide sequences in the polypeptide one, also just formed a potential glycosylation site simultaneously.The glycosylation of O bonded refers to sugar and the combining of hydroxyamino acid, and wherein, the former is as N-acetylgalactosamine, semi-lactosi or wood sugar, and the latter is generally Serine or Threonine, and 5-oxyproline or 5-hydroxylysine are also available.

In the mpl ligand polypeptide, add glycosylation site and generally be by changing aminoacid sequence, make it to contain one or more above-mentioned tripeptide sequences (for N-bonded glycosylation site).This change also can be gone into one or more Serines or threonine residues (for O-bonded glycosylation site) by adding in natural mpl ligand sequence or displacement.For convenience of some, preferably the variation by dna level changes mpl part aminoacid sequence, and especially undergoing mutation in the base of preliminary election by the DNA that makes coding mpl ligand polypeptide obtains translating amino acid needed codon.The method of dna mutation can be carried out for the content of " the variant amino acid sequence body of mpl part " according to above-mentioned title.

Another method that increases the carbohydrate partial amt in the mpl part is to make glycosyl and polypeptide coupling by chemistry or enzyme process.The advantage of these programs is: they do not require at one N-or O-are produced polypeptide in conjunction with glycosylation in the competent host cell.According to the link coupled form, carbohydrate can be connected to (a) arginine and Histidine, (b) free carboxyl, (c) sulfydryl of free sulfydryl such as halfcystine, (d) hydroxyl of free hydroxyl such as Serine, Threonine or oxyproline, (e) aromatic residue of aromatic residue such as phenylalanine, tyrosine or tryptophane, the perhaps amide group of (f) glutamine.These methods can be referring to the WO 87/05330 and the in Aplin and Wriston that published on September 11st, 1987, CRC Crit.Rev.Biochem., pp.259-306[1981].

Can remove carbohydrate part in the mpl ligand polypeptide with chemistry or enzyme process.The de-glycosylation of chemistry need place polypeptide trifluoromethayl sulfonic acid or similar compounds.This processing will cause except that in conjunction with the great majority the carbohydrate (N-acetyl-glucosamine or N-acetylgalactosamine) or all carbohydrates fractures, but to not influence of polypeptide chain.The deglycosylated method of chemistry can be referring to Hakimuddin etc., Arch.Biochem.Biophy., 259:52[1987] and Edge etc., Anal.Biochem., 118:131[1981].The fracture of the enzyme process of carbohydrate part can be used multiple endogenous or external source Glycosylase in the polypeptide, referring to Thotakura etc., and Meth.enzy-mol., 138:350[1987].

Can prevent the glycosylation of potential glycosylation site with the compound tunicamycin, referring to Buskin etc., J.Biol.Chem., 257:3105[1982].Tunicamycin can stop the formation of protein-N-glycosidic link.

The another kind of type of mpl part covalent modification is by one in mpl ligand polypeptide and the multiple nonprotein polymer is linked to each other, as polyoxyethylene glycol, polypropylene glycol or polyoxy alkane, referring to U.S. Patent number 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.Be known as the mpl ligand polypeptide of polymerization with the covalently bound mpl ligand polypeptide of above-mentioned polymer.

In the preferred case, need carry out some to the mpl part that reclaims and screen and select best varient, in order to combine with mpl and to have above-mentioned immunity and/or a biological activity.Can screen following properties: the stability in reconstitution cell culture or blood plasma (as rifting), for a mpl member's high-affinity to proteolysis resistant, oxidative stability, with the output that improves by excretory ability and other similar characteristics.For example, certain variation of immune property as to specifying the affinity of antibody, is measured by the competitive immunization analysis in the mpl ligand polypeptide.Potential variation of other of protein or polypeptide character such as redox or thermostability, hydrophobicity or to the susceptibility of proteolysis Degradation can be analyzed with methods known in the art.17. general method Antibody Preparation (i) polyclonal antibody of the antibody of preparation mpl part

Mpl ligand polypeptide or segmental Polyclonal Antibody Preparation normally are expelled to mpl part and an adjuvant repeatedly subcutaneous (sc) or intraperitoneal (ip) in certain animal body.Be to conjugate in and have immunogenic protein of great use, as keyhole  hemocyanin, serum albumin, thyroglobulin or Trypsin inhibitor SBTI by the species of immunity with containing the mpl part of target amino acid sequence or fragment.Connecting needs to use a kind of difunctional or derivative reagent, as maleimide benzoyl sulfosuccinimide ester (by the cysteine residues conjugated), N-hydroxy-succinamide (passing through lysine residue), glytaralde-hyde, succinyl oxide, SOCl ₂Or R ¹N=C=NR, wherein R and R ¹It is different alkyl.

By mpl ligand polypeptide or fragment, have immunogenic conjugate or derivative comes immune animal, be by with the peptide of 1 milligram or 1 microgram or conjugate (corresponding to rabbit or mouse) mixes with the Freund ' s Freund's complete adjuvant of 3 times of volumes and with the solution intradermal injection in a plurality of positions.After one month, with 1/5 to 1/10 peptide or conjugate and the Freund ' s Freund's complete adjuvant of amount were blended in the subcutaneous injection of a plurality of positions with booster immunization originally.After 7 to 14 days, with tiring of mpl ligand antibody in animal bloodletting and the serum analysis.Continue booster immunization and arrive platform area until the curve of tiring.Preferably, use the conjugate booster immunization of identical mpl part, but can conjugate to a kind of different protein also/or by a kind with cross-linking reagent.Conjugate also can be used as the protein blend zoarium and makes in the reconstitution cell culture.In addition, aggregating agent prepared therefrom such as alum can be in order to improve immunne response.(ii) monoclonal antibody

Mono-clonal can be from the group of a basic homologous antibody, and promptly external except the minimum spontaneous mutation of the probability of occurrence that may exist, each antibody that constitutes group all is identical.Therefore, qualifier " mono-clonal " is meant the characteristic of antibody, is not the mixture that refers to independent antibody.

For example, mpl part MONOCLONAL ANTIBODIES SPECIFIC FOR can be with hybridoma method or recombinant DNA method among the present invention, and the former is by Kohler and Milstein, Nature, 256:495[1975] at first describe, the latter is referring to U.S. Patent number 4,816,567 (Cabilly etc.).

In hybridoma method, with an aforesaid method mouse of immunity or other appropriate host animal such as hamster, to draw lymphocyte, these lymphocytes produce the antibody that maybe can produce and can combine with the protein specific as immunity.Perhaps, can be at external immune lymphocyte.Then, use a kind of suitable fusogen such as polyoxyethylene glycol, lymphocyte is fused to forms hybridoma (Goding, Monoclonal Antibodies:Princi-ples and Practice among the myeloma cell, pp.59-103[Academic Press, 1986]).

The hybridoma for preparing is like this inoculated in suitable medium and grown, preferably contain the growth that can suppress the parental generation myeloma cell of not merging or one or more materials of survival in the substratum.For example, if parental generation myeloma cell lacks hypoxanthine phosphoribosyltransferase (HGPRT or HPRT), usually just can make and contain xanthoglobulin, aminopterinum and Thymine deoxyriboside (HAT substratum) in the hybridoma substratum, thereby suppress the growth of HGPRT deficient cells substantially.

Preferred myeloma cell's characteristics are: the antibody-producting cell that can merge effectively, support to select is with stable high level expression antibody with to substratum sensitivities such as HAT.Wherein, preferred myeloma cell line is a rat bone marrow tumour system, as from Salk Institute Cell Distri-bution Center, San Diego, the MOPC-21 of California USA and MPC-11 mouse tumour cell and from American Type Culture Collection, Rockville, the SP-2 cell of Maryland USA.Also on the books with human myeloma and mouse-people's allos myeloma cell line be used to produce human monoclonal antibodies (Kozbor, J.Im-munol., 133:3001[1984]; Brodeur etc., Monoclonal AntibodyProduction Techniques and Application, pp.51-63, MarcelDekker, Inc., New York, 1987).

Substratum to hybridoma growth is analyzed, with the output of the monoclonal antibody of determining direct corresponding mpl part.In the preferred case, the binding specificity of the monoclonal antibody that hybridoma produces is decided by immuno-precipitation or stripped binding analysis such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of monoclonal antibody can be used for example Scatchard analysis ofMunson; Pollard, Anal.biochem., 107:220[1980] described in method decide.

Determined hybridoma can produce have required specificity, affinity and/or active antibody after, can by the Method of Limited Dilution program will clone subcloning and with standard method make it the growth (Goding, the same).This purpose suitable medium be can reach and Dulbec-co ' s Modified Eagle ' s substratum or RPMI-1640 substratum comprised.In addition, hybridoma can be grown with the form of ascites tumour in animal body.

Subclone excretory monoclonal antibody is suitably separated with substratum, ascites or serum, separate and adopt conventional immunoglobulin purification procedure such as albumin A Sepharose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography.

Use conventional procedure can separate easily and check order coding monoclonal antibody of the present invention DNA (as by use can with the gene specific bonded oligonucleotide probe of encode murine antibody heavy chain and light chain).Hybridoma of the present invention is a preferred source as this DNA.In case it is separated, DNA can be presented in the expression vector, then with carrier transfection host cell such as monkey kidney COS cell, Chinese hamster ovary (CHO) cell or no longer produce the myeloma cell of immunoglobulin (Ig) is to obtain the synthetic of monoclonal antibody in the recombinant host cell.Also can modify DNA, as with the sequence of homologous sequence permutation encoding people's heavy chain and the constant region of light chain of mouse (Cabilly etc., the same; Proc.Nat.Acad.Sci. such as Morrison, 81:6851[1984]) or the encoding sequence of all or part NIg polypeptide chain is covalently bonded in the encoding sequence of immunoglobulin (Ig).

Usually, such NIg polypeptide chain is used to replace the constant region of a kind of antibody of the present invention, perhaps, be used to replace the variable region of an antigen binding site of a kind of antibody of the present invention, to form a chimeric bivalent antibody, one of them antigen binding site is specific to the mpl part, and another then is specific to different antigen.

Chimeric or hybrid antibody also can use known protein matter synthetic chemistry method in external preparation, comprises that those relate to the method for linking agent.For example, can use disulfide exchange reaction or make up an immunotoxin by forming a thioester bond.Comprise imino-thiolate and methyl-4-mercaptobutyrimidate for reaching the required suitable reagent of this purpose.

Because the needs of diagnosis, antibody of the present invention can be surveyed part with one usually and mark.To this unique requirement that can survey part is can directly or indirectly generate one can survey signal.For example, this can survey part can be a radio isotope, as ³H, ¹⁴C, ³²P, ³⁵S or ¹²⁵I, fluorescence or chemiluminescence compound, as fluorescein isothiocyanate, rhodamine or luciferin, labelled with radioisotope, as ¹²⁵I, ³²P, ¹⁴C or ³H, perhaps certain enzyme is as alkaline phosphatase, beta galactosidase enzyme or horseradish peroxidase.

Any known method that can survey part that antibody individually can be conjugated in this area all can be used, and comprises the method described in the following document: Hunter etc., Nature, 144:945[1962]; David etc., Biochemistry, 13:1014[1974]; Pain etc., J.Immunol.Meth., 40:219[1981] and Nygren, J.Hisrochem.andCytochem., 30:407[1982].

Antibody of the present invention can be used for any known analytical procedure, as competitive binding analysis, direct or indirect sandwich assay and immunoprecipitation analysis.Referring to Zola, Monoclonal Anti-bodies:A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).

Competitive binding analysis is according to a standard substance that is labeled (can be mpl part or its immune response part) and the ability of laboratory sample assay (mpl part) competition with the antibodies of limited quantity.The amount of the standard substance of the amount of mpl part and binding antibody is inversely proportional in the laboratory sample.For the amount of faster decision bonded standard substance, usually will the competition before or after curing antibody, thereby can easily the standard substance and the assay of binding antibody be separated from free standard substance and assay.

Sandwich assay relates to the use of two antibody, and the difference that each antibody can both be bonded to testing protein (mpl part) causes immune position, i.e. epitope.In sandwich assay, the laboratory sample assay is attached to first antibody that is fixed in a solid support, and subsequently, second antibodies is in assay, so just formed an insoluble ternary complex.Referring to David and Greene, U.S. Patent number 4,376,110.Second antibody can itself can be surveyed part mark (directly sandwich assay) or measure (sandwich assay indirectly) with the AIA that can be surveyed the part mark.For example, elisa assay is a type of sandwich assay, and the part surveyed wherein is a kind of enzyme (as a horseradish peroxidase).(iii) humanized antibody and people's antibody

The method of the non-human body antibody of humanization is to be widely known by the people in this area.The amino-acid residue that the source, one or more inhuman source of introducing is arranged in formerization of people antibody usually.The amino-acid residue in these inhuman sources often is known as " input " residue, generally can distinguish from one " input ".The elementary operation of humanization process according to Winter and co-worker's thereof method (Jones etc., Nature, 321:522-525[1986]; Riechmann etc., Nature, 332:323-327[1988]; Verhoeyen etc., Science, 239:1534-1536[1988]), replace corresponding human antibody sequence with mouse CDRs or CDR sequence.So this " humanization " antibody belongs to chimeric antibody (Cabilly etc., the same), wherein, the sequence that is less than a whole person variable region is replaced by the corresponding sequence from inhuman species.In fact, humanized antibody generally is that the number of C DR residue of people's antibody and some possible FR residues are replaced by the residue from similar site in the murine antibody.

Be reducing antigenicity, is very important to the selection of the people variable region that is used to prepare humanized antibody heavy chain and light chain.According to so-called " the suitableeest " method, to the sequence of the complete library screening murine antibody variable region of known people's variable region sequences.So, with the immediate human sequence of mouse sequence be taken as humanized antibody skeleton (FR) (Sims etc., J.Immunol., 151:2296[1993]; Chothia and Lesk, J.Mol.Biol., 196:901[1987]).Another method is used a special skeleton, and this skeleton is a special hypotype of heavy chain or light chain from the consensus sequence of everyone antibody.Identical skeleton can be used for several different humanized antibodies (Carter, Proc.Natl.Acad.Sci.USA, 89:4285[1992]; Presta etc., J.Immnol., 151:2623[1993]).

The more important thing is that humanized antibody should keep the biological nature to antigenic high-affinity and other optimizations.In order to reach this target, according to a preferred method, the preparation of humanized antibody is passed through: the three-dimensional model with parental generation and humanization sequence is analyzed parental generation sequence and multiple conceptual humanization product.Three-dimensional immunoglobulin (Ig) model is very common, and is very familiar for those skilled in the art.Can obtain the possible three-dimensional conformation structure that computer program shows candidate's immunoglobulin sequences of selecting.By checking that these displaying contents just can analyze residue may act in candidate's immunoglobulin sequences function, promptly residue is analyzed in conjunction with the influence of its antigenic capacity candidate's immunoglobulin (Ig).By this method, can from total and list entries, select the FR residue, to obtain required antibody characteristic, as increasing affinity to target antigen.Usually, the CDR residue directly and influence the antigen combination basically most of the time.Further details is referring to the U. S. application series number 07/934,373 of filing on August 21st, 1992, and this document has partly continued the application serial no 07/715,272 of filing on July 14th, 1991.

Perhaps, can produce transgenic animal (as mouse) now, can be after immunization, when lacking endogenous immunoglobulin (Ig) product, generate the complete storehouse of people's antibody.For example, such description has been arranged, the inhibition fully that the homozygous deletion of heavy chain of antibody joining region (JH) gene in chimeric and germ line mutation mouse can cause endogenous antibody to generate.In this germ line mutation type mouse, ethnic group is changing over to and will causing producing people's antibody according to antigenic attack of immunoglobulin gene arrangement.Referring to Jakobovits etc., Proc.Natl.Acad.Sci.USA, 90:2551-255[1993]; Jakobovits etc., Natjuer, 362:255-258[1993]; Bruggermann etc., Year in Immune., 7:33[1993].People's antibody also can show in the library and generate in phage (Hoogenboom and Winter, J.Mol.biol.227,381[1991]; Marks etc., J.Mol.Biol.222,581[1991]).(iv) bispecific antibody

Bispecific antibody is monoclonal, and preferably people or humanized antibody have binding specificity at least two different antigens.The method for preparing bispecific antibody known in the art.

Traditionally, the recombinant products of bispecific antibody is based on two coexpressions that immunoglobulin (Ig) weight-light chain is right, and wherein, two heavy chains have different specificity (Millstein and Cuello, Nature, 305:537-539[1983]).Because the combination at random of heavy chain immunoglobulin and light chain, these hybridomas (tetrad knurl) produce the potential mixture that 10 different antibodies molecules are arranged, but wherein have only one correct dual specificity structure is arranged.Generally come this correct molecule of purifying, but purge process is quite heavy, and yield is low by the affinity chromatography step.Similarly program is referring to PCT publication number WO 93/08829 (publication on May 13rd, 1993) and Traunecker etc., EMBO, 10:3655-3659[1991].

According to a different but better method, the antibody variable region and the constant region for immunoglobulin sequence that will have required binding specificity merge.The position of merging is the constant region of heavy chain immunoglobulin preferably, wherein contains part hinge area, CH2 and CH3 district at least.Preferably make and contain light chain and at least one syzygy, occur in conjunction with first CH in required site.The DNA of coding heavy chain immunoglobulin syzygy and (if needed) light chain immunoglobulin be inserted into independently expression vector and by cotransfection in the appropriate host biology.Like this, just provide in the adjusting of three mutual ratios of polypeptide chain in to each example to have very big elasticity, and the unequal ratio of these three polypeptide chains can obtain best output in the structure.But, when the expression of the polypeptide chain of at least two equal proportions causes high yield or when ratio is not particularly important, just the encoding sequence of two or all three polypeptide chains might be inserted in the expression vector.In a preferred example of present method, constituting of bispecific antibody having the hybrid immunoglobulins heavy chain that possesses first binding specificity and a hybrid immunoglobulins heavy chain-light chain is being arranged to (second binding specificity is provided) on other one arm on the one arm.Have been found that this unsymmetrical structure has promoted required dual specificity composition to separate from the compound chain of unwanted immunoglobulin (Ig), as having only when on half dual specificity molecule a light chain immunoglobulin being arranged, it is easier to separate.Present method is referring to the copending patent application serial numbers 07/931,811 of filing on August 17th, 1992.

The further details that generates bispecific antibody is referring to Suresh etc., Methodsin Enzymology, 121:210[1986] etc.(v) allos conjugated antibodies

The allos conjugated antibodies also belongs within the category of the present invention.The allos conjugated antibodies is made of two covalently bound antibody.For example the effect of this antibody makes immune system cell infect (PCT publication number WO 91/00360 and WO 92/00373 at unwanted cells (U.S. Patent number 4,676,980) and treatment HIV; EP 03089).The allos conjugated antibodies can use traditional cross-linking method to make.Suitable cross-linking reagent is widely known by the people in this area, can wherein also include a series of crosslinking technologicals referring to U.S. Patent number 4,676,980.IV. megalokaryocyte generates the application of albumen mpl part in treatment

The hematopoietic effect subfunction is arranged and be called as here that megalokaryocyte generates or biological activity mpl part that thrombocyte generates albumen (TPO) can be used for aseptic medication preparation prescription, generate and thrombopoietic activity with the megalokaryocyte that promotes patient, this kind patient is impaired for being generated by thrombocyte, isolate or destroy and increase the thrombopenia that causes.Synthetics of the present invention can be treated thrombopenia relevant hypoplastic bone marrow (as the underdevelopment anaemia after chemotherapy or the bone marrow transplantation) and various illness such as disseminated intravascular coagulation (DIC), immunity thrombopenia (comprising HIV inductive or non-HIV inductive ITP), chronic idiopathic thrombopenia, congenital thrombopenia, osteomyelodysplasia and thrombus thrombopenia effectively.In addition, these megalokaryocytes generation albumen also can be used for treating under myeloproliferative blood platelet disorder and inflammation condition and the iron deficiency thrombocyte situation.

Megalokaryocyte of the present invention generates and the preferable use of thrombocyte generation albumen (TPO) is: the marrow that is used for leukemia or solid tumor poisons the chemotherapy, hypoplastic bone marrow, the spy that learn treatment, be used for from body or allogeneic bone marrow transplantation sends out sexual dysgenesis anaemia, congenital thrombopenia and immunity thrombopenia.

Megalokaryocyte of the present invention generates the treatment that albumen can also be used for some other diseases, activates platelet defect or the destruction that causes as medicine, poisoning or artificial surface.In these cases, press for a kind of compound and can promote new " not destroying " hematoblastic " outflow ".A more complete table of effectively using is referring to " background " (the same), especially the reference mentioned of (a) to (f) part and this paper.

When megalokaryocyte generation albumen of the present invention is used for the treatment of above-mentioned disease or situation, can use separately, also can use with other cytokine, erythropoietin, interleukin-, somatomedin or antibodies.Therefore, this compound that presses for can have the protein of thrombopoietic activity or peptide to be used in combination with other, as G-CSF, GM-CSF, LIF, M-CSF, IL-1, IL-3, erythropoietin (EPO), complete part, IL-6 and IL-11.

Megalokaryocyte of the present invention generates proteic preparation to carry out in a mixture that contains pharmaceutically acceptable carrier.The administration of therapeutic composition is by intravenous injection or through nose or lung.As required, but these compositions also parenteral or subcutaneous administration.When administration systematically, therapeutic composition should not contain pyrogen, and is in pH, isotonicity and all suitable parenteral of stability and can accepts in the solution.These conditions are known in the art.In brief, prepare the dosage formulation of compound of the present invention after, the compound that required purity will be arranged and physiologically acceptable carrier, compose the row agent or stablizer stores or administration with mixing.Aspect dosage that uses and concentration, these materials are nontoxic for acceptor, also comprise damping fluid such as phosphoric acid salt, Citrate trianion, acetate and other organic acid salts in these materials; Antioxidant such as xitix; Lower molecular weight (being less than 10 residues approximately) peptide such as poly arginine, protein such as serum albumin, gelatin or immunoglobulin (Ig); Hydrophobicity polymer such as polyvinylpyrrolidone; Amino acid such as glycine, L-glutamic acid, aspartic acid or arginine; Monose, disaccharides and other carbohydrates such as Mierocrystalline cellulose and derivative, glucose, seminose or dextrin; Sequestrant such as EDTA; Sugar alcohol such as N.F,USP MANNITOL or sorbose; Counterion such as sodium and/or nonionogenic tenside such as tween, Propiram Buddhist nun gram or polyoxyethylene glycol.

Put into practice needs according to feasible pharmacy, it is compound that about 0.5 to 500 milligram megalokaryocyte is generated protein (existing with free acid or alkali form or pharmacy acceptable salt form) compound or mixture and physiologically acceptable media, carrier, the agent of tax row, tamanori, sanitas, stablizer, seasonings or the like.In these combination groups, the amount of active ingredient should be able to can obtain proper dosage in specified scope.

Adding the required aseptic composition of injection is according to traditional pharmacy practice.For example, perhaps need active ingredient to be dissolved in media or suspend, media such as water, crude vegetal such as sesame, peanut or Oleum Gossypii semen or synthetic fat media such as ethyl oleic acid or analogue.Damping fluid, sanitas, antioxidant and analogue can be attached to wherein according to feasible pharmacy practice.

The suitable example of sustained release preparation comprises the semipermeability matrix of the solid hydrophobic polymer that contains polypeptide, and this matrix exists with tangible form, as film or minigel.The example of sustained-release matrix comprises polyester, hydrogel is [as Langer etc., J.biomed.Mater.Res., 15:167-277[1981] and Langer, Chem.Tech., 12:98-105[1982] described in poly-(2-hydroxyethyl-methylacrylic acid) or polyvinyl alcohol], polylactide (U.S. Patent number 3,773,919, EP 58,481), multipolymer (the Sid-man etc. of L-L-glutamic acid and γ ethyl-L-L-glutamic acid, Biopolymers, 22:547-556[1983]), nondegradable ethylene-vinyl acetate (Langer etc., the same), degradable lactic acid-oxalic acid multipolymer such as Lupron De-pot ^TM(Injectable microspheres of forming by lactic acid-oxalic acid multipolymer and leuprolide acetate) and poly D-(-)-3-hydroxybutyric acid (EP 133,988).

Polymer as ethylene-vinyl acetate and lactic acid-oxalic acid can make the release of molecule surpass 100 days, and some hydrogel discharges protein in the short period of time.If the protein that is installed in the capsule keeps segment length's time in vivo, these protein are exposed to ground sex change or gathering under the wet condition in the time of perhaps can be as 37 ℃, and cause bioactive lose and possible immunogenicity changes.According to related mechanism, can design rational strategy and keep proteinic stable.For example, if finding accumulative mechanism is because the S-S key that the exchange of intermolecular disulfide bond forms, stable realization just can be passed through: additives that modification hydroxyl residue, freeze-drying from acid solution, controlling moisture, use are suitable and develop special polymer matrix composition.

The slowly-releasing megalokaryocyte generates protein composition and has comprised that also the megalokaryocyte in the liposome generates albumen.Contain preparation method that megalokaryocyte generates proteic liposome according to: DE 3,218,121; Epstein etc., Proc.Natl.Acad.Sci.USA, 82:3688-3692[1985]; Hwang etc., Proc.Natl.Acad.Sci.USA, 77:4030-4034[1980]; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent number 4,485,045 and 4,544,545 and EP 102,324.Usually, liposome is little (about 200-800 dust) single layer type, and content of cholesterol is greater than about 30 moles of % in its liquid contents, and this is one and is adjusted to the ratio that best megalokaryocyte generates protein for treatment.

How much being determined according to the consideration of multiple factor by the doctor of dosage, known can the pharmaceutically-active factor of change have: the mode of the type of disease and severity, body weight, sex, diet, administration and time, the other drug of using and other relevant clinical factors.Usually, the consumption of every day is between per kilogram of body weight 0.1 to 100 microgram.More suitably, consumption is between per kilogram of body weight 0.1 to 50 microgram.More suitably, the consumption at initial stage 1 between 5 micrograms/kg/day.Perhaps, the scope of dosage is identical with other cytokines, especially G-CSF, GM-CSF and EPO.The effective dose of treatment can be by exsomatizing or determining at body method.

Embodiment

Under situation about not further specifying,, can believe that persons skilled in the art can farthest utilize and realize the present invention by the embodiment that uses following institute to describe and illustrate.Following embodiment is the fabulous concrete manifestation of the present invention, but scope of the present invention is not limited to these.

Embodiment 1

The partial purification of pig mpl part

Pig collection by normal or aplastic anemia lacks hematoblastic blood plasma.By using the 4mEV linear accelerator that the irradiation that pig carries out 900cGy is made pig underdevelopment.Postradiation pig continued to keep 6-8 days by intramuscular injection cefazolin.Under general anesthetization, take out their whole blood then, anticoagulant heparin, and under 1800 * g, produced in centrifugal 30 minutes and lack hematoblastic blood plasma.The megalokaryocyte stimulating activity peaks after 6 days in irradiation.

Take from the aplastic porcine blood plasma of irradiation back pig and handle, and under room temperature, stirred 30 minutes with 4M NaCl.By on Sorvall RC3B with 3800rpm (rev/min) centrifugal removing the precipitation of gained, and supernatant liquor is moved to the 10mMNaPO that a usefulness contains 4M NaCl ₄In the equilibrated Phenyl-Toyoperal post (220ml).This post washs to A with this damping fluid ₂₈₀＜0.05 also with redistilled water (dH ₂O) wash-out.The protein peak of wash-out is dissolved to conductivity 15mS with redistilled water and goes up sample to in PBS equilibrated (Blue-Sepharose) post (240ml).Then, post is with 5 times of volume PBS that contain 2M urea respectively and 10mM NAPO ₄(pH7.4) washing.With the 10mMNaPO that contains 2M urea and 1M NaCl ₄(pH7.4) with protein wash-out from post.The protein peak of wash-out is handled with EDTA and the Pefa-bloc (Boehinges Mannheim company) of 0.01% n-octyl glucoside (n-octyl β-D-glucose pyranoside) and 1mM, directly be splined on placed in-line CD4-IgG (Capon then, D, Nature 337:525-531[1989 such as .J.]) and mpl-IgG Ultralink (Pierce) post (stating as follows).When sample be loaded into and mpl-IgG (4ml) post by with the PBS of 10 times of volumes with after containing the PBS washing and glycine-HCl wash-out of 2M NaCl with 0.1M pH2.25, removal CD4-IgG post (2ml).Elution fraction is feared in the 1M of 1/10 volume Tris-HCl (pH8.0).

Ice the protein ingredient of analyzing by mpl-affinity column wash-out by under reductive condition, carrying out SDS-PAGE (4-10%, Novex glue) electricity.There is some protein in result's demonstration.Protein has proved that by argentation the molecular weight of some protein is 66,000,55,000,30,000,28,000 and 14,000.In order to determine which kind of can promote the propagation of Ba/F3-mpl cell culture in these protein, these albumen are with method wash-out from glue of description among the following embodiment 2.

The Ultralink affinity column

10-20mg is dissolved in Ul-tralink resin (Pierce) coupling (according to the explanation of manufacturers) of mpl-IgG or CD4-IgG and 0.5 gram of PBS.

The structure of mpl-IgG and expression

The chimeric molecule in the human IgG1's of whole ectodomains of a kind of people of comprising mpl (amino acid/11-491) Fc zone is expressed in 293 cells.The cDNA fragment of the amino acid/11-491 of a coding people mpl is by carrying out PCR and obtain and checking order from people's megalokaryocyte CMK cell cDNA storehouse.Insert a Cla1 site at 5 ' end, 3 ' end inserts a BstE II site.Because 2 BstE II sites are arranged in the mpl of dna encoding ectodomain, this PCR product is partly digested the upstream of rear clone in the IgG1Fc coding region between Cla1 and the BstEII on the Bluescropt carrier.The BstE11 site that mpl PCR product 3 ' end is introduced is the framework that is used for being positioned at the Fc zone mpl ectodomain.The construction of gained is gone between the ClaI of pRK5-tkneo carrier and the XbaI site and by calcium phosphate method to be transfected into 293 HEKC by subclone.Screen transformant and separate single clone by G418 with 0.4mg/ml.The mpl-IgG that determines separating clone by the special ELISA of end user Fc expresses.Best cloning by expression has the mpl-IgG expression level of 1-2mg/ml.

Ba/F3 IgG P express cell

CDNA corresponding to people mpl P complete coding region territory is cloned into pRK5-tkneo, makes it linearizing and passes through electroporation (1 * 10 with NotI then ⁷Cell, 9605F, 250 volts) it is transfected among the clone Ba/F3 of IL-3 dependence, after three days, begin screening with 2mg/ml G418.Screen clone set or one by the limiting dilution in 96 orifice plates.The cell of screening remains among the RPMI that contains 15%FBS, 1mg/ml G418,20mM glutamine, 10mM HEPES and 100 μ g/ml Pen-Strep.By using anti-mpl P rabbit polyclonal antibody to carry out the expression that facs analysis is determined the mpl P in the screening and cloning.

Ba/F3 mpl part detects

The detection of mpl part as shown in Figure 2.For the existence of the mpl part of determining various sources, mpl P Ba/F3 cell when lacking IL-3 with 5 * 10 ⁵The density of cell/ml in the humidity incubator (37 ℃, 5% CO ₂And air) cultivated 24 hours.The hungry processing of IL-3 back places cell on 96 well culture plates with the density of per 200 μ l substratum, 50,000 cells, wherein contains or do not contain the sample of wash-out, cultivates 24 hours in cell culture incubator.At last 6-8 hour, 20 μ l did not have the RPMI substratum but contain 1 μ Ci ³The serum of H-thymidine is added in each hole.Use 96 hole GF/C screen plate collecting cells then, and wash with water 5 times.Filtrate is counted with PackardTop Count calculating instrument in the presence of 40 μ l scintillating liquids (Microsciut 20).

Embodiment 2

Highly purified pig mpl part

The glue elution step

The mpl part of equal-volume affinity purification (wash-out is from the component 6 of mpl-IgG post) and 2 times of Laemmli sample buffers mixed at room temperature under the situation of not going back the original reagent existence also are splined in the Novex 4-2% polyacrylamide gel as quickly as possible.Sample need not heat.In contrast, there is not the sample buffer of part at contiguous swimming lane electrophoresis.Gel at 4-6 ℃ in 135 volts of electrophoresis 2 hours 15 minutes.It when electrophoretic buffer begins room temperature.Gel shifts out from gel box and gel slab places the one side that shifts out gel.

The following glue of will founding is replicated on the nitrocellulose membrane: a nitrocellulose filter is carefully placed on the gel face of exposure by after moistening with sterilized water, drives bubble away.Fiducial mark is placed on nitrocellulose membrane and the gel slab, so that carry out the reorientation of replica exactly after dyeing.After about 2 minutes, carefully remove nitrocellulose filter, seal gel and place refrigerator with plastic film.Nitrocellulose filter dyes with Biorad ' s fold tolal prorcin stain, at first with film at 3 * 10ml 0.1%Tween 20+0.5M NaCl, stir among+0.1M Tris-HCl the pH7.5 above 45 minutes, follow in 3 * 10ml pure water above 5 minutes.Add gold stain then and band occurs until standard substance.Use the replica on the water rinse film again, be placed on the plastic film of gel, carefully align with fiducial mark, the position of Novex standard model and line indication need the part of cutting on the mark gel.Discard nitrocellulose membrane and plastic film, cut gel along indicatrix with a sharp razor.The part of being cut should surpass can be used for determining the cutting part position when sample lane is colored with convenient glue.After removing cutting part, remaining glue carries out that silver dyes and the position of measurement standard sample and cutting part.Determine and the corresponding molecular weight of cutting part by the Novex standard model.

12 gel section are placed in the groove of electroelution instrument of Biorad model 422.The 12-14K molecular weight blocks membrane cover and is used in the groove.50mM bicarbonate of ammonia and 0.05%SDS (approximately pH7.8) are elution buffers.About 1 hour of (4-6 ℃) precooling in the cold house before use of one liter of damping fluid.The gel cutting part carries out Xian in 10ma/ groove (initial 40 volts) and takes off in 4-6 ℃ of cold house.Wash-out approximately needs 4 hours.Carefully remove groove and remove liquid on the hole with pipettor.Remove the liquid that blocks on the membrane cover and preserve with pipettor, the pure water of 50 mul aliquots is placed on the membrane cover, remove after stirring the SDS dissolution of crystals.These liquid are with the above liquid of preserving.Whole elution samples volumes of each gel section are about 300-500 μ l.Sample is placed in the 10mm Spec-trapor 412-14K that soaks a few hours in advance in pure water and blocks in the dialysis tubing.They descend per six samples to 600ml phosphate buffered saline buffer (PBS approximately is the 4mM potassium ion) dialysed overnight at 4-6 ℃.Replaced buffering dialysis 2.5 hours in second day, from dialysis tubing, remove sample, and place miniature centrifuge tube, centrifuge tube is put 1 hour on ice after, in 14,000 2pm centrifugal 3 minutes, carefully on the SDS precipitation, shift out supernatant, supernatant is placed on ice about 1 hour then or longer and recentrifuge 4 minutes, supernatant is diluted in the phosphate buffered saline buffer to carry out vitality test, and remaining sample is frozen in-70 ℃.

Embodiment 3

Pig part micrometering preface

The component 6 (2.6ml) that is obtained by the mpl-IgG affinity column goes up concentrated at Microcon-10 (Amicou).In order to prevent that the mpl part is adsorbed in Microcon, add in the component 6 with 1%SDS rinsing film and with 5 μ l 10%SDS.Concentrate back (2 μ l) on Microcon, (20 μ l) adds in the component 6 with 2 times of sample buffers, and all volumes (40 μ l) are added in the single swimming lane of 4-20% acrylamide gradient gel (Novex).Gel is pressed Novex degree electrophoresis.Then with gel balance 5 minutes, electroblotting in 10mM 3-cyclohexyl amino-1-propanesulfonic acid (CAPS) damping fluid (pH11.0) that comprises 10% methyl alcohol.Electroblotting to Immobilon-PSQ film (Millipore) carried out 45 minutes, in carrying out in Biohad Trans-Blot transfer call under the 250mA constant current.Pvdf membrane dyeed 1 minute in being dissolved in 0.1% Xylene Brilliant Cyanine G R-250 solution of 40% methyl alcohol, 0.1% acetate.Decoloured 2-3 minute with 10% acetate that is dissolved in 50% methyl alcohol then.Unique visible is at the molecular weight 18 of trace, 000-35, and the protein molecular weight in 000 scope is 30,000,28,000 and 22,000.

30,28 and the protein band of 22KDa be carried out order-checking, automatic protein checks order on the 470 class apploed Biosystem sequenator that is equipped with a PTH analyser.Sequenator is improved to the sample (rodrignez, J.Chromatogr., 350:217-225 (1985)) that injects 80-90%.Acetone (about 12 μ l/l) is added into solvent orange 2 A and absorbs with balance ultraviolet (UV).The protein of electroblotting checks order in the Blott film.Protein peak enters Justice lnnovation software by NelsonAnalytical 970 interfaces.Sequence deciphers and analyzes on VAX5900 the quantity (square brackets are interior) of carrying out (Henzel et al., J.Chromatogr., 404:41-52 (1987)) N-end sequence (with uncertain residue in the letter representation bracket) and gained material and the results are shown in Table 2 '.

Table 2 '

Mpl part amino terminal sequence

30 kDa [1.8pmol] 1 5 10 15 20 25 (S)P A P P A(C)D P R L L N K L L R D D(H/S)V L H(G)R L	(SEQ ID NO：30)
		28 kDa [0.5pmol] 1 5 10 15 20 25 (S)P A P P A X D P R L L N K L L R D D(H)V L(H)G R	(SEQ ID NO：31)
18-22 kDa [0.5pmol] 1 5 10 X P A P P A X D P R L X(N)(K)	(SEQ ID NO：32)

Embodiment 4

The liquid suspension megalokaryocyte generates and detects

People's peripheral stem cell (PSC) (taking from the patient through agreeing) dilutes 5 times with IMDM nutrient solution (Gibco company), under room temperature with 800 * g centrifugal 15 minutes then, cell precipitation was suspended in the IMDM substratum again and is plated on that 60% Percoll goes up (density is 1.077mg/ml) (Pharmacia company) and in 800 * g centrifugal 30 minutes.The low density monocyte that is positioned at the interface is drawn out of and washes secondary with IMDM, is laid on then that (density is 1-2 * 10 among the IMDM that contains 30%FBS (final volume is 1ml) ⁶Cell/ml), place 24 hole tissue culturing plates (Costar company).App or the mpl part that lacks APP are added to 10%, and culture (37 ℃ contain 5%CO in the humidity incubator ₂And air) cultivated 12-14 days.Continue cultivation cultivating in the presence of the 10%APP that fate containing 0.5 μ g mpl-IgG at 0,2,4 o'clock.Remove APP in the mpl part by the mpl-IgG affinity column.

For the megalokaryocyte in these fluid suspension culture things of quantitative analysis generates, adopt improved method such as Solberg and utilize radiolabeled at GPII _bIII _aThe HP1-1D (referring to Grant, B etc., Blood 69:1334-1339 (1987)) of mouse IgG monoclonal antibody (HP1-1D) (Mayo Cilinic is provided by doctor Nichols) 100 μ g use 1mCi Na ¹²⁵(Biorad, Richmond CA) carry out radio-labeling to I according to the enzyme pearl method (Enzymobeads) that manufacturers illustrates.Radiolabeled HP1-1D is stored among the PBS that contains 0.01% n-octyl-glucoside, deposits in-70 ℃.Typical specificity vigor be 1-2 * ⁶Cpm/ μ g (by 12.5% trichloroacetic acid precipitation time surpass 95%).

The fluid suspension culture thing carries out three times in each experimental procedure.After cultivating in 12-14 days, the 1ml culture is transferred in the Eppendorf pipe of 1.5ml and under room temperature with 800 * g centrifugal 10 minutes.The cell precipitation of gained is resuspended among the 100 μ l PBS that contain 0.02%EDTA and 20% calf serum.The detection damping fluid of 50 μ l (contains 10ng ¹²⁵1-HP1-1D) be added in the culture of resuspension and under room temperature, hatched 60 minutes, shake occasionally.Then, by coming collecting cell at room temperature centrifugal 10 minutes with 800 * g.And wash 2 times with the detection damping fluid.Precipitation was counted 1 minute with gamma counter (Packazd company).Non-specific binding is determined by add the cold HP1-1D of 1 μ g (60 minutes) before the HP1-1D that adds mark.Specificity is in conjunction with passing through with total ¹²⁵The combination that the combination of 1-HD1-1D deducts in the presence of excessive non-absolute altitude HP1-1D is determined.

Embodiment 5

Oligonucleotide PCR primer

Based on the amino-acid sequence by the amino-end of 30kDa, 28kDa and 20kDa gained, the degeneracy oligonucleotide is designed to the primer (seeing Table 4) of polymerase chain reaction (PCR).Two primer collection compounds are synthesized, just 10 aggressiveness aggregation coded amino acid residue 2-8 (mpl 1), antisense 21 aggressiveness aggregation coded amino acid 18-24 (mpl 2).

Table 4

The degeneracy oligonucleotide primer aggregation

Mpl 1:5 ' CCN GCN CCN CCN GCN TGY GA 3 ' (2.048-times of degeneracy)	(SEQ ID NO：35)
		Mpl 2:5 ' NCC RTG NAR NAC RTG RTC RTC 3 ' (2.048-times of degeneracy)	(SEQ ID NO：36)

Be used as the template of PCR by the isolating pig genomic dna of pig peripheral blood lymphocyte.The reaction system of 50 μ l comprises: 0.8 μ g is dissolved in pig genomic dna, 50mM KCl, the 3mM MgCl among the 10mM Tris-HCl (pH8.3) ₂, 100 μ g/ml BSA, 400 μ M dNTPs, each primer collection compound of 1 μ M and 2.5 Taq of unit polysaccharases.The original template sex change be 94 ℃ 8 minutes, then 35 circulations be 94 ℃ 45 seconds, 55 ℃ 1 minute, 72 ℃ 1 minute.Connecing 72 ℃ after last extended 10 minutes.PCR product electrophoretic separation on 12% polyacrylamide gel is also differentiated with the dyeing of bromination second silver.If aminoterminal amino acid is encoded by single password, correct PCR product should be 69 bases.A dna fragmentation of this size is gone into pGEMT (Promega company) by wash-out from glue and subclone.Three clones' sequence sees the following form 5.

Table 5

69bp pig group group dna fragmentation

gemT3

5′CCAGCGCCGC CAGCCTGTGA CCCCCGACTC CTAAATAAAC TGCCTCGTGA 3′GGTCGCGGCG GTCGGACACT GGGGGCTGAG GATTTATTTG ACGGAGCA CTTGACCACGTT CAGCACGGC[69 bp](SEQ ID NO：37) ACTGGTGCAA GTCGTGCCG (SEQ ID NO：38)
	gemT7 5′CCAGCACCTC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA 3′GGTCGTGGAG GCCGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCA CTCGACCACGTC CATCACGGC[69 bp](SEQ ID NO：39) GCTGGTGCAG GTAGTGCCG (SEQ ID NO：40)
gemT9 P R L L N K L L R(SEQ ID NO：32) 5′CCAGCACCGCCGGCATGTGA CCCCCGACTCCTAAATAAACTGCTTCGTGACG 3′GGTCGTGGCGGCCGTACACTGGGGGCTGAGGATTTATTTGACGAAGCA CTGCATCATGTCTATCACGGT 3′(SEQ ID NO：41) TAGTACAGATAGTGCCA 5′(SEQ ID NO：42)

The base that underscore is arranged in the sequence is the position of PCR primer.These results have confirmed 30kDa, 29kDa and the resulting N-end sequence of the proteinic amino acid 9-17 of 20kDa and have pointed out the single password coding of this sequence by pig DNA.

Embodiment 6

People mpl ligand gene

Based on the result of embodiment 5, a deoxy-oligonucleotide that is called as 45 aggressiveness of pR45 is designed and synthesizes to be used for screening group group library.45 aggressiveness have following sequence:

5′GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-TTT-ATT-TAG-GAG-TCG 3′

(SEQ ID No：28)

This oligonucleotide (γ ³²P)-ATP and T4 kinases carry out ³²P-mark and three things screen people's group group library under low tight hybridization and wash conditions (embodiment 7).Positive colony is selected and the purifying plaque, analyzes by restriction endonuclease map and Southern trace.The clone ^#4 selectedly go out further to analyze.

A 2.8kb can be gone into pBlupscript sk-by subclone with the assorted Bam HI-XbaI fragment also of 45 aggressiveness.By using the special Oligonucleolide primers of pig mpl part dna sequence dna that this clone's dna sequence dna is carried out the part order-checking.The sequence of gained proves, and is separated with the human DNA sequence of pig mpl ligand homologous.An EcoRI restriction site in the sequence is determined, thereby can separate the EcoRI-XbaI fragment of a 390bp from the Bam HI-XbaI fragment of 2.8kb, and subclone is gone into pBluescript sk-.

This segmental two strands is all checked order, human DNA sequence and deduced amino acid such as Fig. 9 ( SEQ ID NOS 3 and 4).Indicate the introne position of predicting in the group group sequence with haircut, and determined the exon (" exon 3 ") of a supposition.

Detection to the predicted amino acid sequence has confirmed first amino acid that serine residue is ripe mpl part, this with come to the same thing by amino acid direct analysis gained.The predicted amino acid that is right after this codon upstream sequence is likely and relates to ripe mpl part excretory signal sequence.The coding region of this signal sequence may be interrupted by an intron at nucleotide position 68 places.

On 3 ' direction, exon terminates in Nucleotide 196.One section 42 amino acid whose sequence of this exons coding, wherein 16 may be the part of signal sequence, 26 is the part of the ripe mpl part of people.

Embodiment 7

Total length people mpl Ligand cDNA

Based on the sequence (embodiment 6) of people " exon 3 ", Synthetic 2 and " exon 3 " sequence 3 ' and the corresponding nondegenerate oligonucleotide of 5 ' end.

Table 6

People cDNA nondegenerate PCR Oligonucleolide primers

Forward primer: 5 ' GCT AGC TCT AGA AAT TGC TCC TCG TGG TCA TGC TTC T3 '	(SEQ ID NO： 43)
		Reverse primer: 5 ' CAG TCT GCC GTG AAG GAC ATG G 3 '	(SEQ ID NO： 44)

These two primers are used to PCR reaction, and template DNA wherein is to come from different cDNA libraries or the 1ngQuick Clone cDNA (Clonefeoh company) that derives from different tissues by as described in Example 5.The expection size of correct PCR product is 140bp.After on 12% polyacrylamide gel the PCR product being analyzed, a dna fragmentation that derives from the expection size in adult's kidney cDNA library is determined, and this cDNA library is respectively from adult kidney, 293 tire nephrocytes and people's tire liver (ClonerdchCat. ^#7171-1).

The 45 aggressiveness oligonucleotide in screening people's gene group library are used to screen a λ DR ₂In tire liver cDNA library.Use the T4 polynueleotide kinase with (γ ³²-P)-the ATP labeled oligonucleotide.Under low tight hybridization conditions, the library is screened.Film is prehybridization 2 hours at first, spends the night with probe hybridization in 42 ℃ in 20% methane amide, 5 * SSC, 10 * Denhardt ' s, 0.05M sodium phosphate (pH6.5), 0.1% trisodium phosphate, 50 μ g/ml salmon sperm dnas then.Use 2 * SSC rinsing film then, in 0.5 * SSC, 0.1%SDS, washing film once under 42 ℃.The exposure of film and kodak X-Ray sheet is spent the night then.Select positive colony purifying plaque, with λ DR2 (clonetech cat. ^#6475-1) oligonucleotide of BamHI-XbaI both sides carries out PCR and determines the segmental size of insertion among the clone.The phage liquid storage of 5 μ l is used as template.Initial sex change be 94 ℃ 7 minutes.Carried out the amplification of 30 round-robin then (94 ℃ 1 minute; 52 ℃ of 1 minute and 72 ℃ 1.5 minutes), extend at last 72 ℃ 15 minutes, clone ^#Fl2b has the insertion fragment of a 1.8kb and selectedly goes out further to analyze.

Obtain to be contained in plasmid pDR2 (clonetech, λ DR2 and pDR2 clone and expression system, laboratory manual, p42) (clonerech, λ DR2 and pDR2 clone and expression system in the λ DR2 bacteriophage arm according to the explanation of manufacturers.Laboratory manual p29-30).With BamHI and XbaI plasmid pDR2-FL2b is carried out restriction enzyme analysis and show that a BamHI restriction site is arranged in the insertion fragment of about 650 positions.With BanHI-XbaI plasmid is digested, will insert fragment and cut out 2 fragments, one is 0.65kb, and another is 1.15kb.Three kinds of different templates that obtained by plasmid pDR2-FL2b are carried out sequencing, with AB1371 (applied Biosystems, Foster City, California) automatic fluorescent DNA sequenator, the standard method of the dideoxyribonucleoside triphosphate terminator (dyestuff-terminator) of use dye marker and traditional synthetic step are moved primer (Sanger etc., Proc.Natl.Acad, Sci.USA, 74:5463-5467 (1977)); Smith etc., Natwre, 321:674-679 (1986)) carry out double-stranded plasmid DNA order-checking.The direct survey of the pcr amplification product of plasmid is filled by using traditional primer and dyestuff-terminator to be reflected on the AB1373 sequenator and is undertaken.Single-stranded template produces (DNASTAR company, Madison, Wisconsin) (Burland etc., Nucl, AcidsRes., 21 with M13 Janus carrier; 3385-3390 (1993)).BamHI-XbaI (1.15kb) and BamHI (0.65kb) separate from plasmid pDR2-FL2b from section and obtain, and in the presence of deoxynucleotide, its fragment end is filled and led up with the T4 archaeal dna polymerase.Subclone is gone into the SmaI site of M13 Janus then.With the general and reverse primer of the M13 of dye marker or the step is moved primer and the dyestuff terminator checks order by standard program.Use the step move primer and standard dideoxy terminator chemistry (Sanger etc., Proc.Natl.Acad, Sci, USA, 74:5463-5467 (1977)), ³³(United States Biochen-ital Corp., Clevcland Ohio), carry out artificial sequencing reaction by strand M13 DNA for the α-dATP of P mark and Sequenase.(Gene Codes Corporation, Ann Ar-bor Michigem) carries out the dna sequence dna distributional analysis with Sequencher V2.1b12.The hML sequence of Nucleotide and derivation is seen Fig. 1 (SEQ ID NO:1).

Embodiment 8

The separation of people mpl part (TPO) gene

The people's gene group library among λ-Gem12 is screened the genomic dna cloning of separation of human TPO gene with the pR45 plasmid, pR45 is an oligonucleotide probe that preamble had been described, it under (seeing embodiment 7) under the low stringent condition or height stringent condition all can with one corresponding to people mpl Ligand cDNA 3 ' half fragment hybridization (from BamHI site to 3 ' end).The overlapping clone that to isolate two spans be 35kb.Subclone contains two overlapping fragmentses (BamHI and EcoRI) and the order-checking of whole TPO gene.The structure of this people's gene constitutes (Figure 14 A, B and C) by 6 exons in the 7kb genomic dna.The fillet of all exon all consistent (Shapiro, M.B. etc., Nucl.Acids Res.15:7155 (1987)) with mammiferous consensus sequence.Exons 1 and exon 2 contain initial 4 amino acid of 5 ' non-translated sequence and signal peptide.Preceding 26 amino acid of the rest part of secretion signal and maturation protein are encoded by exon 3.Complete carboxyl structure territory and 3 ' untranslated structural domain and erythropoietin spline structure territory～50 amino acid are by exon 6 codings.4 amino acid relevant with observed disappearance in ML-2 (hTPO-2) are by 5 of exon 6 ' end coding.

Embodiment 9

The transient expression of people mpl part (hML)

Insert fragment for the total length that subclone is contained in the pDR2-FL2b, with XbaI fully, partly digest with BamHI then plasmid digestion.The gel electrophoresis purifying inserts segmental dna fragmentation corresponding to 1.8kb and its subclone gone into pRK5 (pRK5-hmpl 1) (relevant pRK5 structure referring to U.S. Patent No. 5,258,287) and makes under its regulation and control that place the cytomegalovirus immediate early promoter.DNA from construction pRK5-hmpl 1 prepares by the PEG method, and being transfected into human embryo kidney (HEK) 293 cells, this cell is incubated in the improved Eagle substratum of the Dulbecco that is supplemented with F-12 nutritional blend, 20mM Hepes (pH7.4) and 10% foetal calf serum (DMEM).(Gorman, C. (1985) is in DNA Cloning: A Practical Approach (Glover, D.M. compile) Vol.II, pp.143-190, IRL Press, Woshing-ton, D.C.) transfectional cell with calcium phosphate method.After the transfection 36 hours, in proliferation assay, analyze the activity (embodiment 1) in the transfectional cell supernatant liquor.Only with the supernatant liquor of 293 cells of pRK carrier transfection to Ba/F3 or Ba/F3-mpl cell have no stimulation (Figure 12 A).With the supernatant liquor of pRK5-hmpl 1 cells transfected the Ba/F3 cell is not had effect but greatly stimulated the propagation (as 12A) of Ba/F3-mpl cell, show this cDNA a kind of active people mpl of function part that has of having encoded thus.

Embodiment 10

People mpl ligand isoforms hML2, hML3 and hML4

In order to identify the alternative splicing form of hML, synthesized corresponding to each terminal primer of hML encoding sequence.These primers are used to RT-PCR with amplification adult liver RNA.In addition, made up the inner primer (as follows) of selected target area flank and carry out same use.The direct order-checking of PCR product end has disclosed accurately (sees Fig. 1 (SEQ ID NO:1) corresponding to separating from one section unique sequence of people's embryo liver gene library cDNA sequence.But, show a kind of multiplexed sequence form near one section zone of EPO domain C end (at the middle part of PCR product), show to exist possible splicing variants in this zone.In order to separate these splicing variants, the primer of the target area flank that provides in the table 7 is used as the template of adult liver cDNA in PCR.

Table 7

People ML isotype PCR primer

phmpllcdna.3el： 5′TGTGGACTTT AGCTTGGGAG AATG 3′	(SEQ ID NO.45)
		Pbx4.f2： 5′GGTCCAGGGA CCTGGAGGTT TG 3′	(SEQ ID NO.46)

The PCR product is gone into M13 with flat terminal subclone.There are 3 kinds of ML isotypes at least in order-checking announcement to each subclone.One of them is that hML (is also referred to as hML ₃₃₂), it is the longest form, and is accurately corresponding with the sequence of separating from embryo liver library.(Figure 11 (SEQ IDNO:6,8,9,10) provided listed from the longest hML to the shortest hML-4 the sequence of these 4 kinds of people mpl ligand isoforms.

Embodiment 11

The structure of people Mpl ligand isoforms and substituted varient and transient expression

HML2, hML3 and hML (R153A, R154)

Utilize recombinant PCR technology (Russell Higuchi, PCR Protocols, Aguide to Methods and Applications, Acad.Press, M.A.Innis, D.H.Gelfand, J.J.Sninsky ﹠amp; T.J.White compiles), by hML rebuild isotype hML2 and hML3 and substituted variant hML (R153A, R154).

In all structures, used " outward " primer is listed in table 8, and " overlapping " primer is listed in table 9.

Table 8

Outer primer

Cla.FL.F2： 5′ATCGATATCG ATAGCCAGAC ACCCCGGCCA G 3′	(SEQ ID NO：47)
		HMPLL-R： 5′GCTAGCTCTA GACAGGGAAG GGAGCTGTAC ATGAGA 3′	(SEQ ID NO：48)

Table 9

Overlapping primer

hML-2：MLΔ4.F：5′CTCCTTGGAA CCCAGGGCAG GACC3′ MLΔ4.R：5′GGTCCTGCCC TGGGTTCCAA GGAG3′	(SEQ ID NO：49) (SEQ ID NO：50)
		hML-3：MLΔ116+：5′CTGCTCCGAG GAAAGGACTT CTGGATT 3′ MLΔ116-：5′AATCCAGAAG TCCTTTCCTC GGAGCAG 3′	(SEQ ID NO：51) (SEQ ID NO：52)
hML(R153A、R154A)：RR-KO-F：5′CCCTCTGCGT CGCGGCGGCC CCACCCAC 3′ RR-KO-R：5′GTGGGTGGGG CCGCCGCGAC GCAGAGGG 3′	(SEQ ID NO：53) (SEQ ID NO：54)

All pcr amplifications all use the Pfu archaeal dna polymerase (Stratagene company) of cloning, and carry out with following condition: initial template sex change is 94 ℃, 7 minutes, circulate then 30 times (94 ℃, 1 minute; 55 ℃, 1 minute; 72 ℃, 1.5 minutes).Last is taken turns at 72 ℃ and extended 10 minutes.With ClaI-XbaI digestion PCR end product, the gel electrophoresis purifying also is cloned into pRK5tkneo.With various constructions rotaring redyeing 293 cell as previously mentioned, with Ba/F3-mpl proliferation assay clear liquid analytically.In this was measured, hML-2 and hML-3 did not show detectable activity, but (R153A, activity R154A) is similar to hML to show that the processing in the alkaline position of this pair is not active necessary (seeing Figure 13) to hML.

Embodiment 12

Mouse mpl Ligand cDNA

MML, mML-2 and mML-3

The separation of mML cDNA

Utilize PCR to obtain one section dna fragmentation corresponding to the full coding region of people mpl part, gel electrophoresis purifying and utilization have ³²P-dATP and ³²The random priming that P-dCTP exists carries out mark.This probe is used for screening λ GT10 mouse liver cDNA library 10 ⁶Clone (Clontech cat#ML3001a).

Get the filter membrane that duplicates at 35% methane amide, 5 * SSC, 10 * Denhardt ' s, 0.1%SDS, spend the night with probe hybridization in the salmon sperm DNA of 0.05M sodium phosphate (pH6.5), 0.1% trisodium phosphate, 100 μ g/ml ultrasonic grinding.In 2 * SSC the rinsing filter membrane, 42 ℃ of washings are once in 0.5 * SSC, 0.1%SDS then.The purifying plaque inserts the Eco RI position that the fragment subclone is gone into Bluestript SK-plasmid to obtain hybridizing phage with cDNA.Insertion segmental " LD " clone who picks out band 1.5kb is used for further analysis, as described above people ML cDNA is carried out the two strands order-checking.Figure 14 (SEQ ID NO:1 and 11) provides LD clone's nucleotide sequence and deduced amino acid.Long 331 amino-acid residues of ripe ML sequence from this clone of deriving are defined as mML ₃₃₁(or be called mML-2 for some reason hereinafter).In the EPO of these ML spline structure territory, observed the height consistence of nucleotide sequence and deduced amino acid.But, during with the derivation aminoacid sequence alignment of the ML of people and mouse, the sequence table of mouse is revealed a tetrapeptide disappearance between people's residue 111-114, one section 12 nucleotide deletion behind 618 Nucleotide all seeing corresponding to the cDNA people's (seeing above) and pig (seeing below).So, checked that other clone is to search possible mouse ML isotype.Clone " L7 " has the insertion fragment of a 1.4kb of one section 335 deduced amino acid sequence that comprises " losing " tetrapeptide LPLQ.This form it is believed that it is total length mouse ML, is called as mML or mML ₃₃₅Figure 16 (SEQ ID NO:12 and 13) has provided Nucleotide and the deduced amino acid of mML.At last, isolate clone " L2 " and order-checking.This clone has the disappearance of 116 Nucleotide corresponding with hML3, so called after mML3.Figure 16 has shown the comparison of the derivation aminoacid sequence of the two isotype.The expression of reorganization mML

Basic identical described in the preparation of expression vectors of mouse ML and the embodiment 8.The clone of coding mML and mML-2 is gone into pRK5tkneo by subclone, and this is the expression vector that can express under CMV promotor and the regulation and control of SV40 polyadenylation signal.Utilize calcium phosphate method that the expression vector mML pRK5tkneo and the mML2pRK5tkneo transient transfection of gained are gone into 293 cells.Behind the transient transfection, in conditioned medium, cultivated 5 days.Cell is maintained in the high glucose DMEM substratum that is supplemented with 10% foetal calf serum.The expression of mouse mpl (mmpl) in the Ba/F3 cell

Obtain the stable cell lines of expressing c-mpl with mmpl pRK5tkneo transfection, basic identical with the method that people mpl is carried out described in the embodiment 1.In brief, utilize electroporation (5 * 10 ⁶Individual cell, 250V, 960 μ F) will contain expression vector (the 20 μ g of the whole coding sequence (Skoda, R.C. etc., EMBO are (1993) J.12:2645-2653) of mouse mpl; Linearizing) be transfected into the Ba/F3 cell, carry out neomycin resistance with 2mg/ml G418 then and select.With the anti-mouse mpl-IgG of rabbit antiserum(antisera), estimate the expression of mpl by flow cytometry.The Ba/F3 cell is maintained at from RPMI 1640 substratum as the WEHI-3B cell in IL-3 source.As described in embodiment 1, in transfection analyze the supernatant liquor of 293 cells of use by oneself mML and mML-2 transient transfection in the Ba/F3 cell of mmpl and hmpl.

Embodiment 13

Pig mpl Ligand cDNA

PML and pML-2

Utilize RACE PCR to separate pig ML (pML) cDNA.In brief, according to the exon sequence of coding purifying, design one section oligomerization dT primer and 2 sections Auele Specific Primers from the N-terminal pig ML of aplastic anemia porcine blood serum ML gene.Obtain the cDNA for preparing by in the various aplastic porcine tissues, and amplification.In kidney, found the PCR cDNA product of a kind of 1342bp and with its subclone.Several clones are checked order, find the pig mpl part (not comprising complete secreting signal peptide) of their encoding matures.This cDNA is found one section 332 amino acid whose maturation protein pML with sequence shown in Figure 18 (SEQ ID NO:9 and 16) of coding ₃₃₂

Method: pML gene and cDNA separate

Screen the genomic clone that pig genomic library among the EMBL3 (Clontech Inc) separates pig ML gene with pR45.The screening of gene library is described with embodiment 7 substantially.Isolate several clones and the exon that coding is same as the ML aminoacid sequence that derives from purifying is checked order.Utilize RACE PCR improvement program to obtain pig ML cDNA.According to two sections Auele Specific Primers of pig ML gene order design.Substantially as previously mentioned, the mRNA that separates polyadenylation by the kidney of aplastic anemia pig.Prepare cDNA with the BamdT primer reverse transcription directly relative with the poly A tail of mRNA.

(BamdT：5′GACTCGAGGA TCCATCGATT TTTTTTTTTT TTTTT 3′)

(SEQ ID NO：55)

With the specific h-forward-1 of ML primer

(h-forward-1:5 ' GCTAGCTCTA GAAATTGCTC CTCGTGGTCA TGCTTCT 3 ')

(SEQ ID NO：43)

With the BAMAD primer

(BAMAD：5′GACTCGAGGA TCCATCG 3′)

(SEQ ID NO:56) be (50mM KCl, 1.5mM MgCl, 10mM Tris pH8.0,0.2mM dNTP in the 100ml reaction volume, 0.05U/ml Amplitaq polysaccharase [Perkin ElmerInc.]) carrying out PCR just increases and (circulates 28 times: 95 ℃ 60 seconds, 58 ℃ 60 seconds, 72 ℃ 90 seconds).With ClaI digestion PCR product,, be connected on the Bluestript SK-carrier (StratageneInc.) of using ClaI and KpnI cutting then with phenol-chloroform (1: 1) extracting, ethanol sedimentation.Cultivate under the room temperature after 2 hours, 1/4 connection mixture is introduced directly into and uses second species specific preceding-1 primer:

(forward-1:5 ' GCTAGCTCTA GAAGCCCGGC TCCTCCTGCC TG 3 ')

(SEQ ID NO:57) and T3-21 (with near the one section oligonucleotide of one section sequence bonded the polyclone zone in the Bluestript SK-carrier):

5′CGAAATTAAC CCTCACTAAA G 3′

(SEQ ID NO:58) second takes turns PCR (circulating as previously mentioned 22 times).The PCR product that obtains digests with XbaI and ClaI, and subclone is gone into Bluestript SK-.The several clones that derive from independent PCR reaction are checked order.

Repeated again once, identified second kind of form pML-2, its coding is with the protein (seeing Figure 21 [SEQ ID NO:21]) of 4 amino-acid residues disappearances (328 amino-acid residues).The relatively demonstration of pML and pML-2 aminoacid sequence, a kind of form in back is in full accord except the QLPP tetrapeptide of disappearance corresponding to residue 111-114 (comprising end value) (sees Figure 22 [SEQ ID NO:18 and 21].Observed 4 aminoacid deletion appear at identical part in the predicted protein in mouse, people and pig.

Embodiment 14

Thrombopoietin (TPO) is to platelet antigen

GPII _bIII _aThe CMK test of the inducing action of expressing

The CMK cell is maintained in RMPI 1640 substratum (Sigma company) that are supplemented with 10% foetal calf serum and 10mM glutamine.In the preparatory stage of test, harvested cell washs, and it is being mended with the 5mg/l Sigma I8405 resuspended one-tenth 5 * 10 in the serum-free GIF substratum of 10mg/l apotransferrin, 1 * trace element ⁵Cell/ml.In one 96 hole flat undersides, in each hole, add 100 μ l TPO standard or laboratory samples by the suitable proportion dilution.100 μ l CMK cell suspending liquids are added each hole, with plate at 37 ℃, 5%CO ₂Incubator in cultivated 48 hours.After the cultivation, plate is centrifugal 5 minutes at 4 ℃ with 100rpm.Abandoning supernatant adds FITC conjugated GPII in each hole _bIII _aMono-clonal 2D2 antibody.4 ℃ cultivate 1 hour after, test panel centrifugal again 5 minutes with 1000rpm.Discard and contain the not supernatant liquor of binding antibody, adding 200 μ l 0.1%BSA-PBS washing lotions in each hole.0.1%BSA-PBS washing step triplicate.On FASCAN, utilize the single-parameter analysis method analysis of cells of measuring relative intensity of fluorescence.

Embodiment 15

The DAMI that carries out thrombopoietin (TPO) by mitogen activation in the mensuration DAMI nucleus on 96 hole microtiter plates measures

The DAMI cell is maintained in the IMDM+10% horse serum (Gibco company) that is supplemented with 10mM glutamine, 100ng/ml penicillin G and 50 μ g/ml Streptomycin sulphates.The test preparatory stage, harvested cell, the washing, and in the IMDM+1% horse serum resuspended one-tenth 1 * 10 ⁶Cell/ml.In test panel at the bottom of one 96 hole circles, 100 μ l TPO standards or specimen are added the DAMI cell suspending liquid.Then at 37 ℃, 5%CO ₂Cultivated 48 hours in the incubator.After the cultivation, test panel is centrifugal 5 minutes with 1000rpm, 4 ℃ on Sorvall 6000B whizzer.Abandoning supernatant repeats the washing step of 200 μ l PBS-0.1%BSA.Add the ice-cold 70% ethanol-PBS of 200 μ l and fix cell, and utilize ventilation resuspended.After 15 minutes, test panel centrifugal 5 minutes with 2000rpm adds 1mg/mlRNAse (RNA enzyme) and the 0.05%Tween-20 that 150 μ l contain 0.1mg/ml iodine third ingot (propidim iodide) in each hole 4 ℃ of cultivations., measure dna content by flow cytometry and change after 1 hour 37 ℃ of cultivations.Following mensuration and quantitative polyploidy:

The polyploid of normalizing is than (the cell % of＞G2+M/＜the cell % of G2+M) of (the cell % of＞G2+M/＜the cell % of G2+M)/contrast of (NPR)=add TPO

Embodiment 16

The body build-in test of thrombopoietin

(mouse thrombocyte rebound test)

Be used in the thrombopoietic body ³⁵The mensuration that S determines

Gave C57BL6 mouse (deriving from Charles River) intraperitoneal (IP) injection 1ml sheep anti mouse thrombocyte serum (6amps) to produce thrombopenia at first day.At the 5th day and the 6th day, give the mouse IP double injection factor or PBS in contrast.At the 7th day, intravenous injection was dissolved in 30 μ Ci Na of 0.1ml physiological saline ₂ ³⁵SO ₄, measure derive from receive treatment and mouse blood sample in contrast in the injected dose ³⁵S mixes per-cent in the circulation thrombocyte.Simultaneously thrombocyte and the white corpuscle taken from the socket of the eye hole blood sample of back are counted.

Embodiment 17

Carry out the KIRA ELISA of thrombopoietin (TPO) by measuring mpl-Rse.gD Chimerical receptor phosphorylation

Vigon etc. (PNAS, USA 89:5640-5644 (1992)) have disclosed people mpl acceptor.Contain the membrane spaning domain (TM) of mpl acceptor ectodomain (ECD) and Rse and the Chimerical receptor of born of the same parents' intracellular domain (ICD) (Mark etc., J.of Biol.Chem.269 (14): 10720-10728[1994]) and be used to KIRA ELISA described herein with a kind of carboxyl terminal tail (flag) polypeptide.About illustrating of this test can be referring to Figure 30 and 31.

(a) capture the preparation of agent

Produce monoclonal anti gD (clone 5B6) (Paborsky etc., Protein Engineering 3 (6): 547-553[1990]) with a kind of peptide of taking from herpes simplex virus glycoprotein D.The storing solution of purifying is transferred to 3.0mg/ml, pH7.4 in phosphate buffered saline buffer (PBS), be divided into the equal portions of 1.0ml, be stored in-20 ℃.

(b) preparation of anti-phosphotyrosine antibody

(Lake Placid NY) buys the monoclonal anti phosphotyrosine antibody and promptly clones 4G10, and (Cleveland OH) carries out biotin labeling for Biotin-X-NHS, Research Organics to utilize long-armed vitamin H-N-hydroxyl succinic diamide from UBI.

(c) part

Utilize recombinant technology as herein described to prepare the mpl part.The mpl part of purifying is made storing solution be stored in 4 ℃.

(d) preparation of Rse.gD nucleic acid

The synthetic double chain oligonucleotide is used to rebuild the encoding sequence of people Rse C 10 amino acid of end (880-890) and add 21 amino acid in addition, wherein contains epitope and terminator codon of antibody 5B6.Table 10 has been represented the ultimate sequence of this fusion gene composite part.

Table 10

The synthetic double-stranded part of people Rse fusion gene

Coding strand 5 '-TGCAGCAAGGGCTACTGCCACACTCGAGCTGCGCAGATGCTAGCCTCAAG ATGGCTGATCCAAATCGATTCCGCGGCAAAGATCTTCCGGTCCTGTAGAAGCT-3 '	(SEQ ID NO：59)
		Non-coding (antisense) chain 5 '-AGCTTCTACAGGACCGGAAGATCTTTGCCGCGGAATCGATTTGGATCAGC CATCTTGAGGCTAGCATCTGCGCAGCTCGAGTGTGGCAGTAGCCCTTGCTGCA-3 '	(SEQ ID NO：60)

This synthetic DNA is connected with the 1-880 amino acid whose cDNA of HindIII position with coding people Rse at PstI, the PstI site originates in the 2644th Nucleotide of disclosed people Rse cDNA sequence, the HindIII site then at expression vector pSV17.ID.LL (referring to Figure 32, SEQ ID NO:22) in the polylinker, is built into expression plasmid pSV.ID.Rse.gD after the connection.In brief, this expression plasmid contains one two cistron transcription product originally, wherein contain by 5 ' shearing DHFR encoding sequence that donor and 3 ' shearing acceptor intron montage position is defined, and be thereafter the sequence of coding Rse.gD.Total length (non-montage) messenger RNA(mRNA) contains the DHFR as initial open reading frame, so, can produce the dhfr protein that is used to select the stable conversion body.

(e) preparation of mpl-Rse.gD nucleic acid

The expression plasmid pSV.ID.Rse.gD that modifies as above generation is to generate plasmid pSV.ID.M.tmRd6, and this plasmid contains the encoding sequence of the people mplECD (amino acid/11-491) that merges with Rse.gD (amino acid 429-911) membrane spaning domain and born of the same parents' intracellular domain.At Mark etc., in the two-step pcr cloning reaction described in the J.Biol.Chem.267:26166-26171 (1992), with synthetic a part of encoding sequence and the Rse.gD encoding sequence that has oligonucleotide to connect people mpl ectodomain.The used primer of the first step PCR reaction pair mplcDNA template is M1:

(5′-TCTCGCTACC GTTTACAG-3′)

(SEQ ID NO：61)

And M2:

(5′-CAGGTACCCA CCAGGCGGTC TCGGT-3′)

(SEQ ID NO：62)

To the used primer of Rse template is R1:

(5′-GGGCCATGAC ACTGTCAA-3′)

(SEQ ID NO：63)

And R2:

(5′-GACCGCCACC GAGACCGCCT GGTGGGTACC TGTGGTCCTT-3′)

(SEQ ID NO：64)。

The PvuII-SmaI of this fusion linker partly is used to make up the total length Chimerical receptor.

(f) transformation

Be opened into linear pSV.ID.tm-Rd6 electroporation DP12.CHO cell (EP307 that on March 15th, 1989 was announced, 247) in the single NotI position of plasmid framework.After phenol/chloroform extracting, ethanol sedimentation DNA also is resuspended among 20 μ l, 1/10 TrisEDTA.Then, before with 400V, 330 μ f electroporations with 10 μ g DNA and 10 ⁷Individual CHO DP12 cell was hatched on ice 10 minutes.Before the flat board of non-selective substratum upper berth, cell was put in ice 10 minutes again.After 24 hours, clone for the cell feed supplement to select stable DHFR+ with the substratum of free nucleic acid.

(g) be used for the selecting of transformant of KIRA ELISA

By SDS-PAGE the separated portion of whole cell pyrolysis liquid is carried out the western trace with the antibody 5B6 that detects gD decision base and identify the clone who expresses mpl/Rse.gD.

(h) substratum

Cell is in F12/DMEM (Gibco/BRL, Life Technologies, Grand Island, NY) middle growth in 50: 50.Substratum mended with 10% saturating filterable FBS (Hy-Clone, Logan, Utah), 25mM HEPES and 2mM L-glutamine.

(i)KIRA ELISA

The DP12.CHO cell that mpl-Rse.gD is transformed adds flat 96 well culture plates, and each hole is 100 μ l substratum (3 * 10 ⁴Individual/lattice), at 37 ℃, 5%CO ₂Middle overnight incubation.In morning next day, decantation is removed supernatant liquor, and culture plate is placed on the paper handkerchief.Every hole adds and contains laboratory sample or 200,50,12.5,3.12,0.78,0.19,0.048 or the 50 μ l substratum of 0ng/ml mpl.37 ℃ of irritation cells 30 minutes, the decantation abandoning supernatant pressed dry culture plate on paper handkerchief more gently.For lysing cell and dissolve Chimerical receptor, every hole adds 100 μ l lysis buffers.Lysis buffer is by the 150mM NaCl solution that contains 50mM HEPES (Gibco), 0.5%Triton-X 100 (Gibco), 0.01% Thiomersalate, 30KIU/ML Trypsin inhibitor,Trasylol (ICN Biochemicals, Aurora, OH), 1mM 4-(2-amino-ethyl)-benzenesulfonyl hydrogen fluoride (AEBSF; ICN Biochemicals), 50 μ M leupeptin (ICN Biochemicals) and 2mM sodium vanadate (Na ₃VO ₄Sigma Chemi-cal Co.St.Louis MO) forms pH7.5.(Bell-co Istruments, Vineland NJ) went up under the room temperature mild stirring 60 minutes to culture plate at the culture plate vibrator.

In the time of dissolved cell, be coated with 4 ℃ of 5B6 monoclonal antibodies of spending the night and (in the 50mM carbonate buffer solution, be made into 5.0 μ g/ml, pH9.6,100 μ l/ lattice) ELISA microtiter plate (Nunc Maxiscorp, Inter Med, Denmark) supernatant liquor that inclines, on paper handkerchief, press dry, (contain 0.5%BSA (Intergen Compa-ny, Purchansa with 150 μ l/ holes sealing damping fluid, NY) and the PBS of 0.01% Thiomersalate) sealing 60 minutes under the room temperature, mild stirring simultaneously.After 60 minutes, (Inc.Sterling is VA) with lavation buffer solution (PBS that contains 0.05%Tween-20 and 0.01% Thiomersalate) washing 6 times for ScanWasher300, Skatron Instruments with automatic plate washing device for the test board that is coated with anti-gD56.

The lysate (85 μ l/ lattice) that will contain dissolved MPL/Rse.gD from the cell culture microtiter well is transferred in the ELISA hole that is coated with anti-gD56 and is closed, and hatches under the room temperature 2 hours, simultaneously mild stirring.With the unconjugated mpl-Rse.gD of lavation buffer solution flush away, and in every hole, add 100 μ l and be diluted in dilution buffer liquid by 1: 18000 and (contain 0.5%BSA, 0.05%Tween-20, the PBS of 5mMEDTA and 0.01% Thiomersalate) the biotin labeled 4G10 in (anti-Tyrosine O-phosphate), promptly every hole adds 56ng/ml.After hatching 2 hours under the room temperature, wash plate and every hole add by be diluted at 1: 60000 horseradish peroxidase (HRPO) conjugated Streptavidin in the dilution buffer liquid (the Zymed laboratory, S.San Francisco, CA).Under the room temperature, mild stirring was hatched 30 minutes.Free avidin conjugate is by flush away, and every hole adds the freshly prepd substrate solution of 100 μ l (tetramethyl-benzidine (TMB); 2 component substrate covers are joined reagent; Kirkegaard and Perry, Gaighersburg, MD).Reacted 10 minutes, and added 100 μ l/ hole 1.0mMH then ₃PO ₄The color development stopping reaction.Read the value (ABS at 450nm place with reference to the absorbancy at 650nm place _450/650), use be with Macintosh Centris650 (apple computer, Cupertino, CA) and DeltaSoft software (BioMetallics, Inc, Princeton, NJ) Kong Zhi vmax plate reader (Molecular Divices, Plao Alto CA).

With 200,50,12.5,3.12,0.78,0.19,0.048 or 0ng/ml mpl ligand stimulation dp12.trkA, B or C.gD cell, utilize the DeltaSoft program to be depicted as ng/mlTPO to average A BS _450/650The typical curve of ± standard deviation.Utilize interpolation technique on typical curve, to read sample concentration, be expressed as the ng/mlTPO activity by absorbancy.

The mpl-part is found and can activates the mpl-Rse.gD Chimerical receptor with concentration dependent or ligand specificity's mode.In addition, mpl-Rse.gD KIRA-ELISA is found the human serum (showing) that can tolerate up to 100% or 100% blood plasma (not shown), thereby makes this test can be used for screening easily patient and pK sample.

The TPO that 293-produces ₃₃₂Typical curve

The summary of TPO EC50 ' s

TPO type (cell)	EC50(wt/v)	EC50 (mole)
			People TPO 322 (293)	2.56ng/ml	67.4pM
Mouse TPO 322 (293)	3.69ng/ml	97.1pM
			People TPO 153 (293)	～41ng/ml	～1.08nM
People TPO 155 (E.coli)	0.44ng/ml	11.6pM
			People TPO 153met (E.coli)	0.829ng/ml	21.8pM

Embodiment 18

The ELISA that is used for thrombopoietin based on acceptor

The elisa plate bag is by with the rabbit F (ab ') in the pH9.6 carbonate buffer solution ₂Anti-human IgG (Fc) spends the night in 4 ℃ of placements.With sealing under the 0.5% bovine serum albumin room temperature that is dissolved among the PBS 1 hour.On test board, add and contain Chimerical receptor, mpl-IgG, the fermentation container cutting and hatched 2 hours.In test board, add standard substance (by the TPO of 293 cells generation ₃₃₂, its concentration is determined by quantitative amino acid analysis) the twice serial dilution and the sample of serial dilution in 0.5% bovine serum albumin, 0.05%Tween20, hatched 2 hours.With the albumin A of purifying, the TPO that in E.coli, produces ₁₅₅Biotin labeled rabbit antibody test bonded TPO (hatching 1 hour), add Streptavidin-peroxidase (hatching 30 minutes) then and with 3,3 ', 5,5 '-tetramethyl benzidine is a substrate.Read the absorbancy at 450nm place.Between per step, all want the washing test plate.In order to carry out data analysis, utilize typical curve of 4 parametric line fit procedure matches of Kaleidagraph.Concentration by the typical curve calculation sample.

Embodiment 19

The preparation of expression and purification 1.293 fibrocyte expression vectors of 293 cell TPO

By being the PCR of primer, can obtain a cDNA corresponding to the whole open reading frame of TPO with following oligonucleotide.

Table 11

293 PCR primers

Cla.FL.F：5′ATC GAT ATC GAT CAG CCA GAC ACC CCG GCC AG 3′	(SEQ ID NO：65)
		hmpII-R：5′GCT AGC TCT AGA CAG GGA AGG GAG CTG TAC ATG AGA 3′	(SEQ ID NO：48)

In the reaction that adds pfuDNA polysaccharase (Stratagene company), PRK5-hmplI (referring to embodiment 9) is used as template.After 94 ℃ of initial sex change in 7 minutes, continuous with 25 amplification cycles (94 ℃, 1 minute; 55 ℃, 1 minute and 72 ℃, 1 minute).Last extends to 72 ℃, 15 minutes.Purified pcr product and between the restriction site Clal of plasmid pRK5tkneo and Xbal clone's product to obtain carrier pRK5tkneo.ORF, pPK5tkneo modifies the carrier that obtains behind the pRK5, can express neomycin resistance under the control of thymidine kinase promoter.Second construction corresponding to the epo homeodomain generated by same method, but used primer forward is Cla.FL.F, is reversed:

Arg.STOP.Xba：5′TCT AGA TCT AGA TCA CCT GAC GCA GAG GGT GGA CC 3′

(SEQ ID NO：66)

Last construction is called pRK5-tkneoEPO-D.The sequence of these two constructions is referring to embodiment 7.2. the transfection of HEKC

Use CaPO ₄Method with these two construction transfections in HEKC, referring to embodiment 9.After the transfection 24 hours, in the G418 of 0.4mg/ml, begin to select clone to neomycin resistance.After 10 to 15 days, one colony transferred in 96 orifice plates and make it to grow to converge.ML from these clones ₁₅₃And ML ₃₃₂Expression in conditioned medium can be assessed (referring to embodiment 1) by the Ba/F3-mpl proliferation assay.3.rhML ₃₃₂Purifying

With 293-rhML ₃₃₂Substratum is splined on equilibrated Blue-Sepharose in 10mM sodium phosphate pH7.4 (buffer A) (pharmacia company) post.Subsequently, each is with the buffer A and the buffer A washing column that contains the 2M urea of 10 times of column volumes.Then, with the buffer A wash-out post that contains 2M urea and 1MNaCl.Then, the wash-out aggregation with the Blue-Sepharose post directly is splined on the post with buffer A equilibrated wheat germ agglutinin Sepharose.The washing of wheat germ agglutinin Sepharose post contains the buffer A of 2M urea and 1MNaCl with 10 times of column volumes, and wash-out is with same damping fluid but also contain 0.5M N-acetyl-D-glycosamine.The eluate of wheat germ agglutinin Sepharose post is splined on equilibrated C4 performance liquid chromatographic column (synchrom company) in 0.1%TFA.The C4 performance liquid chromatographic column is to come wash-out (0-25%, 25-35%, 35-70%) with discontinuous propyl alcohol gradient.Find rhML ₃₃₂At 28-30% propyl alcohol gradient region by wash-out.By sds polyacrylamide gel electrophoresis, the rhML that is purified ₃₃₂Move (referring to Figure 15) in the 68-80 of gel kilodalton zone with a broadband.4.rhML ₁₅₃Purifying

293-rhML ₁₅₃Conditioned medium with rhML ₃₃₂Identical mode is separated on Blue-Sepharose.Identical with aforesaid method, the Blue-Sepharose eluate directly is splined on a mpl affinity column.With above-mentioned rhML ₃₃₂Under the identical condition, use a rhML that C4 performance liquid chromatographic column purifying elutes from the mpl affinity column ₁₅₃, make it to reach homogeneity.By sds polyacrylamide gel electrophoresis, again with the rhML that is purified ₁₅₃Be separated into two master tapes and twice band, molecular weight is about 18,000-21,000 (referring to Figure 15).

Embodiment 20

The expression vector that uses in the following electroporation scheme that is described in of the TPO expression and purification 1.CHO expression vector of CHO is specified:

PSV15.ID.LL.MLORF (total length or hTPO332) and

PSV15.ID.LL.MLEPO-D (brachymemma or hTPO153).About the character of these plasmids is seen Figure 23 and Figure 24.2.CHO preparation of expression vectors

By being the PCR of primer, can obtain a cDNA corresponding to the whole open reading frame of TPO with the oligonucleotide shown in the table 12.

Table 12

CHO expression vector PCR primer

Cla.FL.F2 5′ATC GAT ATC GAT AGC CAG ACA CCC CGG CCA G 3′	(SEQ ID NO：47)
		ORF.Sal 5′AGT CGA CGT CGA CGT CGG CAG TGT CTG AGA ACC 3′	(SEQ ID NO：67)

In the reaction that adds pfuDNA polysaccharase (Stratagene company), PRK5-hmplI (referring to embodiment 7 and 9) is used as template.After initial sex change in 94 ℃, 7 minutes, continuous with 25 amplification cycles (94 ℃, 1 minute; 55 ℃, 1 minute and 72 ℃, 1 minute).Last extends to 72 ℃, 15 minutes.Purified pcr product and between the restriction site Clal of plasmid pSV15.ID.LL and Sall clone's product to obtain carrier pSV15.ID.LL.MLORF.Second construction corresponding to the epo homeodomain generated by same method, but used primer forward is Cla.FL.F2, is reversed:

EPOD.Sal 5′AGT CGA CGT CGA CTC ACC TGA CGC AGA GGG TGG ACC 3′

(SEQ ID NO:68) last construction is called as pSV15.ID.LL.MLEPO-D.The sequence of these two constructions is all referring to embodiment 7 and 9.

In essence, the encoding sequence of total length and brachymemma part is introduced into the multiple clone site of CHO expression vector pSV15.ID.LL.This plasmid contain early promoter/enhanser zone of SV40, modification montage unit of containing mouse DHFR cDNA, one introduce required gene multiple clone site (here, the sequence of TPO is described), polyadenylation signal and the replication orgin of a SV40, and be used in the bacterium that plasmid is selected and the beta lactamase gene of amplification.3. set up stable express recombinant people TPO ₃₃₂And TPO ₁₅₃The method of Chinese hamster ovary celI system

The description of a.CHO parental cell system

Host CHO (Chinese hamster ovary) clone of the TPO of being used for developed by molecule described herein is known as CHO-DP12 (referring to the EP 307,247 that published on March 15th, 1989).For the clone who obtains insulin requirements is reduced, with a carrier transfection parent line of expressing preproinsulin (CHO-K1 DUX-B11 (DHFR-)-, agree through Dr.L.Chasin, obtain from the Dr.Frank Lee of Stanford University), from clone, select this mammal cell line.These cells also are the DHFR defectives, and by in the growth that lacks on the substratum that nucleosides replenishes (glycine, xanthoglobulin and thymidine), the selection that DHFR cDNA carrier sequence is existed and obtain the clone.This selective system as stably express Chinese hamster ovary celI system is usually employed.

B. transfection method (electroporation)

TPO ₃₃₂And TPO ₁₅₃Express cell system produces (referring to as Andreason, G.L.J.Tiss.Cult.Meth., 15,56[1993]) through electroporation transfection DP12 cell, and correspondingly uses linearization plasmid pSV15.ID.LL.MLORF or pSV.ID.LL.MLEPO-D.The reaction of three Restriction Enzymes has been set up in the cutting of each plasmid: 10 micrograms, 25 micrograms and 50 microgram carriers and enzyme NOTI, and with the molecular biology method of standard.This restriction site only finds once in carrier, be positioned at linearizing zone 3 ' and TPO part transcription unit beyond (referring to Figure 23).Setting up 100 microlitre reaction systems and 37 ℃ is incubated overnight.Second day, with phenol-chloroform-primary isoamyl alcohol (50: 49: 1) extracting once and on dry ice about 1 hour with ethanol sedimentation.Then, separate and dry collecting precipitation thing through 15 minutes microcentrifugations.Linearizing DNA is resuspended in 1: 1 substratum of Ham ' s DMEM-F12 that 50 microlitres have replenished standard antibiotic and 2mM glutamine.

Collect the DP12 cell of suspension growth, in the substratum washing that is used for resuspended DNA once and finally with per 750 microlitres 10 ⁷The concentration of cell is resuspended in the same substratum.The aliquots containig of cell (750 microlitre) and each linearizing DNA mixture be incubation 1 hour at room temperature together, transfers to then in the BRL electroporation groove.Then, each reaction mixture of electroporation in the standard BRL electroporation apparatus of 350 volts, 330 microfarads and low electric capacity.After electroporation, cell can be placed in instrument 5 minutes again, cultivated again on ice 10 minutes then.In cell transfer to the 60 millimeter Tissue Culture Dish with electroporation, wherein contain 5 milliliters of standard perfect medium (High glucose DMEM-F12 50: 50 that are used to cultivate Chinese hamster ovary celI, do not contain glycine, replenished 1X GHT, 2mM glutamine and 5% foetal calf serum), and at 5%CO ₂Grow overnight in the cell cultures thermostat container.

C. select and screening method

Second day, cell with standard method from flat board by trysinization and be transferred to and contain (Ham ' s DMEM-F12 150 millimeters tissue culture wares that DHFR selects substratum, 1: 1 substratum, as mentioned above, replenished 2% or 5% dialysis foetal calf serum, but lack glycine, xanthoglobulin and thymidine, this is that we employed standard DHFR selects substratum).Cell from each 60 millimeters flat board is applied to 5/150 flat board subsequently again.Then, cell is at 37 ℃/5%CO ₂Middle incubation 10 to 15 days (subculture is changed in the centre) is up to cloning appearance and reaching the size that is fit to transfer to 96 orifice plates.After having spent 4 to 5 days, clone is transferred on 96 orifice plates with the aseptic transfer pipet that is set to 50 milliliters.Cell is grown to converge back (common 3 to 5 days), the trysinization culture dish also copies two copies of original culture dish.In refrigerator, and the cell in each hole all is diluted among the DMSO that 50 microlitres contain 10%FCS these two copies by short term stored.By measuring based on the Ba/F3 cell activity, the TPO that sample in the conditioned medium that did not contain serum in 5 days is measured in the confluent growth hole from the 3rd culture dish expresses.Based on high-expression clone recovery from store of this mensuration, and extended in 150 millimeters T-flasks of two confluent growths, be used for transfer for the adaptability that suspends, the cell culture group measuring and store and carry out again.

D. scheme increases

The several the highest clone of tiring that obtains by above-mentioned selection is with after standard methotrexate amplification method generates the higher clone who tires.The Chinese hamster ovary celI clone is expanded and coats 10 centimetres of culture dish, 4 methotrexate concentration (as 50nM, 100nM, 200nM and 400nM) is wherein arranged, 2 to 3 cell quantity (every wares 10 ⁵, 5 * 10 ⁵With 10 ⁶Individual cell).Then, with 37 ℃/5%CO of these cultures ₂Incubation is further analyzed until cloning to form and be suitable for transferring to 96 orifice plates.The several high clone that tires who obtains from this selection is added in the methotrexate of greater concn (as 600nM, 800nM, 1000nM and 1200nM) again and as previously mentioned, resistance clone can form and be transferred to and be used in 96 orifice plates measuring.4. culture expression recombinant human TPO ₃₃₂And TPO ₁₅₃Stable Chinese hamster ovary celI system

The cell that stores is thawed, and not containing serum or containing in the substratum of serum, with the cell growth method of standard cell mass is expanded.After expanding to enough cell densities, washed cell is to remove the cell culture medium that does not re-use.Then, with any standard method culturing cell, comprise batch culture, fed batch cultivation or be not less than under 5% the condition cultured continuously at 25-40 ℃, pH neutral, dissolved oxygen content and assemble until composing type excretory TPO.Subsequently, with the centrifugal mechanical means that waits with cell culture fluid and cellular segregation.5. from the purifying of the recombinant human TPO of CHO nutrient solution

The cell culture fluid of collecting (HCCL) directly is splined on Blue Sepharose 6Fast Flow post (Pharmacia company), the balance of post sodium phosphate and the 0.15M NaCl of 0.01M pH7.4, when about 50 milliliters/hour/square centimeters of linear rate of flow, go up sample by the ratio of every liter of about 100 liters of HCCL of resin.Then, come washing column with 3 to 5 times of column volume balance liquids, continuous 0.01M sodium phosphate pH7.4,2.0M urea and 1.0MNaCl with 3 to 5 times of column volumes.

Then, the Blue Sepharose aggregation that will contain TPO is splined on wheat germ agglutinin Sepharose 6MB post (Pharmacia company), the balance of post 0.01M sodium phosphate pH7.4,2.0M urea and 1.0MNaCl, when about 50 milliliters/hour/square centimeters of linear rate of flow, go up sample by the ratio of every milliliter of about 8 to 16 milliliters of Blue Sepharose aggregations of resin.Subsequently, with the level pad washing column of 2 to 3 times of column volumes.Then, the wash-out of TPO is with 0.01 sodium phosphate pH7.4,2.0M urea and the 0.5MN-acetyl-D-glycosamine of 2 to 5 times of column volumes.

Then, the final concentration of wheat germ agglutinin aggregation is adjusted to 0.04%C ₁₂E ₈With 0.1% trifluoracetic acid (TFA).The aggregation that obtains is splined on C4 reversed-phase column (Vydac214TP1022), the balance of post 0.1%TFA and 0.04%C ₁₂E ₈, when flow velocity is 157 milliliters/hour/square centimeters, by sample on every milliliter of about 0.2 to 0.5 milligram of proteinic ratio of resin.

Protein is containing 0.1%TFA and 0.04%C ₁₂E ₈Acetonitrile two-phase linear gradient in wash-out.The first acetonitrile linear gradient by 0-30% in 15 minutes constitutes.The second acetonitrile linear gradient by 30-60% in 60 minutes constitutes.At acetonitrile concentration about 50% o'clock, TPO was by wash-out.On the basis of sds polyacrylamide gel electrophoresis, obtain an aggregation.

Then, 0.01M sodium phosphate pH7.4 and 0.15M NaCl dilution C4 aggregation with 2 times of volumes, and with the 0.01M sodium phosphate pH7.4 of about 6 times of volumes and 0.15M Na-Cl at an Amicon YM or filter thoroughly on the saturating filter membrane of class that one 10,000 to 30,000 Dalton molecular weight blocks arranged.The saturating filtrate that obtains can with after ultrafiltration further concentrate.Saturating filtrate/concentrated solution is adjusted to the final concentration of one 0.01% tween 80.

Subsequently, all or part of filtrate/the concentrated solution that equals summation column volume 2-5% is splined on Sephacryl S-300 HR post (Phamacia company), the balance of post is with 0.01M sodium phosphate, pH7.4,0.15M NaCl and 0.01% tween 80, and carries out stratographic analysis with 17 milliliters/hour/square centimeters speed.The TPO that contains aggregate-free or proteolytic degradation product component is gathered by sds polyacrylamide gel electrophoresis.The aggregation that obtains filters with 0.22 μ filter paper, Millex-GV or analogue, and is stored in 2-8 ℃.

Embodiment 21

TPO albumen synthetic transforms and the structure of inducing 1. intestinal bacteria TPO expression vectors in the intestinal bacteria

Plasmid pMP21, pMP151, pMP41, pMP57 and pMP202 are designed to be expressed in a short leader downstream, preceding 155 amino acid of TPO, and this weak point leader changes because of different constructions.This leader mainly provides high-caliber translation initiation and fast purifying.Plasmid pMP210-1 ,-T8 ,-21 ,-22 ,-24 and-25 is designed to be expressed in preceding 153 amino acid of TPO in an initial methionine downstream, their difference only is the usage to preceding 6 the amino acid whose codons of TPO, and wherein plasmid pMP251 is the derivative that obtains at TPO C-terminal two amino acid of extension of pMP210-1.Under the inducing of trp promoter, all above-mentioned plasmids can produce high-caliber TPO cell inner expression (Yansura, D.G. etc. in intestinal bacteria, Method in Enzymology (Goeddel, D.V., editor) 185:54-60, A-cademic Press, San Diego[1990]).Plasmid pMP1 and pMP172 are the intermediates in the above-mentioned TPO cell inner expression plasmid construction.

(a) plasmid pMP1

Plasmid pMP1 is preceding 155 amino acid whose one excretion vectors of TPO, and its structure is by 5 dna fragmentations are linked together as shown in figure 33.First fragment wherein is to have removed the segmental pPho21 carrier of short MluI-BamHI.PPho21 is phGH1 (Chang, C.N. etc., Gene 55:189-196[1987]) a derivative, wherein human growth hormone gene is by intestinal bacteria phoA gene substitution, and in the encoding sequence of the STII signal sequence at amino acid 20-21 place, inserted a Mlul restriction site.

In ensuing two fragments, one is the DNA HinfI-PstI fragment from 258 base pairs of pRK5-hmpl I (embodiment 9), coding TPO amino acid/11 9-103, another is the synthetic DNA of following coded amino acid 1-18: 5 '-CGCGTATGCCAGCCCGGCTCCTCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGC TTCGTG

ATACGGTCGGGCCGAGGAGGACGAACACTGGAGGCTCAGGAGTCATTTGACGAAGCACTGA-5′

(SEQ ID NO：69)

(SEQ ID NO:70) these two fragments connect in advance with the T4 dna ligase, then cut with PstI.The 4th fragment is a PstI-HaeIII fragment from 152 base pairs of pRK5hmpl I, the amino acid of the 104-155 position of coding TPO.Last fragment is a StuI-BamHI fragment from 412 base pairs of pdh108, contains as above-mentioned transcription terminator λ (Scholtissek, S. etc., NAR 15:3185[1987]).

(b) plasmid pMP21

Plasmid pMP21 designs for preceding 155 amino acid of expressing TPO, and one of this expression process need comprises the help of 13 amino acid whose leaders of part STII signal sequence.As shown in figure 34, the structure of this plasmid is by 3 dna fragmentations are linked together.Wherein first is to have removed the segmental carrier pVEG31 of short XbaI-SphI.Carrier pVEG is pHGH207-1 (de Boer; H.A. etc.; in Pro-moter Structure and Function (Rodriguez; R.L.and Chamber-lain, M.J., editor); 462; Praeger, New York[1982]) a derivative, human growth hormone gene is wherein substituted (same carrier segments can obtain from plasmid after a while) by vascellum esoderma growth factor gene.

The second section that connects is the synthetic DNA duplex that following sequence is arranged:

5′-CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAA

TTAATACTTTTTCTTATAGCGTAAAGAAGAATTGCGC-5′

(SEQ ID NO：71)

(SEQ ID NO:72) last fragment is the MluI-SphI fragment from 1072 base pairs of pMP1,155 amino acid of coding TPO.

(c) plasmid pMP151

Plasmid pMP151 designs for preceding 155 amino acid of the TPO that expresses a leader downstream, and this leader is made up of 7 amino acid of STII signal sequence, 8 Histidines and a factor Xa broken site.As shown in figure 35, pMP151 makes up by the connection of three dna fragmentations.Wherein first is exactly the above-mentioned segmental carrier pVEG31 of XbaI-SphI that removes.Second is the synthetic DNA duplex that following sequence is arranged: 5 '-CTAGAATTATGAAAAAGAATATCGCATTTCATCACCATCACCATCACCATCACATC GAAGGTCGTAGCC

TTAATACTTTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTTCCAGCAT-5′

(SEQ ID NO：73)

Last is the BglI-SphI fragment from 1064 base pairs of pMP11 for (SEQ ID NO:74), 154 amino acid of coding TPO.Except the variation of some codons in STII signal sequence (this fragment can obtain from pMP1), plasmid pMP11 and pMP1 are just the same.

(d) plasmid pMP202

Factor Xa broken site in leader was substituted by a zymoplasm broken site, plasmid pMP202 and expression vector pMP151 were closely similar.As shown in figure 36, pMP202 makes up by connecting 3 dna fragmentations.Wherein first is exactly the above-mentioned segmental pVEG31 of short XbaI-SphI that removes.Second is the dna double spiral that following sequence is arranged:

5′-CTAGAATTATGAAAAAGAATATCGCATTTCATCACCATCACCATCACCATCACATCGAA

CCACGTAGCC

TTAATACTTTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTT

GGTGCAT-5′ (SEQ ID NO：75)

(SEQ ID NO:76) last fragment is the BglI-SphI fragment from 1064 base pairs of above-mentioned plasmid pMP11.

(e) plasmid pMP172

Plasmid pMP172 is preceding 153 amino acid whose one excretion vectors of TPO, also is the intermediate that makes up pMP210.As shown in figure 37, the preparation of pMP172 is by linking together 3 dna fragmentations.Wherein first is the carrier pLS32lamB that has removed short EcoRI-HindIII part.Second is the EcoRI-HgaI fragment from 946 base pairs of above-mentioned plasmid pMP11.Last fragment is following synthetic DNA duplex.5′-TCCACCCTCTGCGTCAGGT (SEQ ID NO：77)

GGAGACGCAGTCCATCGA-5′(SEQ ID NO：78)

(f) plasmid pMP210

Plasmid pMP210 designs in order to be expressed in preceding 153 amino acid of the TPO behind the translation initiation methionine(Met).In fact this plasmid is made into a plasmid storehouse, and wherein each in preceding 6 codons of TPO is all the 3rd position random variation.As shown in figure 38, this plasmid is made up by connection by three dna fragmentations.Wherein first is as the above-mentioned segmental carrier pVEG31 of short XbaI-SphI that removes.Second is the synthetic DNA duplex, at first use archaeal dna polymerase (Klenow) to handle, digest with XbaI and HinfI then, coding initial methionine and preceding 6 random cipher of TPO, its sequence is as follows: 5 '-GCAGCAGTTCTAGAATTATGTCNCCNGCNCCNCCNGCNTGTGACCTCCGA

ACACTGGAGGCT

GTTCTCAGTAAA (SEQ ID NO：79)

The 3rd fragment of CAAGAGTCATTTGACGAAGCACTGAGGGTACAGGAAG-5 ' (SEQ ID NO:80) is the HinfI-SphI fragment from 890 base pairs of pMP172, the 19-153 amino acid of coding TPO.

About 3700 clones' plasmid pMP210 storehouse is transformed on high tsiklomitsin (50 mcg/ml) the LB flat board again to select high translation initiation clone (Yansura, D.G. etc., Methods:A Companion to Methods in Enzymology 4:151-158[1992]).In 8 colonies that are incubated at high tsiklomitsin flat board, select 5 TPO and express the best dna sequencing that carries out, its result is (SEQ ID NOS:23,24,25,26,27 and 28) as shown in figure 39.

(g) plasmid pMP41

Plasmid pMP41 designs in order to express preceding 155 amino acid of TPO, and wherein TPO has incorporated the leader of forming by connecing a factor Xa broken site behind 7 amino acid whose STII signal sequences.As shown in figure 40, this plasmid makes up by 3 dna fragmentations are linked together.Wherein first is as the segmental carrier pVEG31 of the short XbaI-SphI of above-mentioned excision.Second is synthetic DNA duplex described as follows: 5 '-CTAGAATTATGAAAAAGAATATCGCATTTATCGAAGGTCGTAGCC (SEQ ID NO:81)

A last fragment of TTAATACTTTTTCTTATAGCGTAAATAGCTTCCAGCAT (SEQ ID NO:82) is the BglI-SphI fragment from 1064 base pairs of above-mentioned plasmid pMP11.

(h) plasmid pMP57

Plasmid pMP57 expresses preceding 155 amino acid of TPO in the leader downstream that is made of 9 amino acid whose STII signal sequences and two basic site Lys-Arg.This pair basic site provides a method of removing leader with protease A rgC.As shown in figure 41, this plasmid makes up by the connection of three dna fragmentations.Wherein first is the above-mentioned segmental carrier pVEG31 of short XbaI-SphI that removes.Second is synthetic DNA duplex as follows:

5′-CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAAACGTAGCC (SEQ ID NO：83)

The 3rd fragment of TTAATACTTTTTCTTATAGCGTAAAGAAGAATTTGCAT-5 ' (SEQ ID NO:84) is the BglI-SphI fragment from 1064 base pairs of above-mentioned plasmid pMP11.

(i) plasmid pMP251

Plasmid pMP251 is the derivative of pMP210-1, wherein, and at two amino acid of C-terminal adding of TPO.As shown in figure 42, this plasmid is connected by two dna fragmentations and makes up.Wherein first is the above-mentioned segmental pMP21 of short XbaI-SphI that removes.The second section that connects is the XbaI-ApaI fragment from 316 base pairs of pMP210-1.2. have the colibacillary conversion of TPO expression vector and induce

Use calcium chloride heat-shocked method (Mandel, M. etc., J.Mol.Biol., 53:159-162, [1970]), above-mentioned TPO expression plasmid can be used for transformed into escherichia coli bacterial strain 44C6 (w3110 tonA _ΔRpoH _TNLon _ΔClpP _ΔGalE).At first, make transformant at 37 ℃ with contain in the LB substratum of 50 mcg/ml Pyocianils and grow, reach about 2 to 3 up to the optical density(OD) (600 nanometer) of culture.Then, in the M9 substratum that contains 0.49% casamino acids (w/v) and 50 mcg/ml Pyocianils, the LB substratum is diluted 20 times.After 1 hour, it is 50 mcg/ml that the adding indole-3-acetic acid makes final concentration in growth under 30 ℃ of ventilation conditions.Then, make culture continued growth 15 hours under 30 ℃ and ventilation condition, use the centrifuging collecting cell at last.

Embodiment 22

The generation of biological activity TPO (Met-11-153) in the intestinal bacteria

Refolding TPO (the Met of following generation biologically active ^-1Program 1-153) can be applied to comprise the recovery (referring to embodiment 23) that N and C-terminal extend other TPO varients of form similarly.A. insoluble TPO (Met ^-1Recovery 1-153)

With the TPO (Met of aforesaid method fermentation expression by plasmid pMP210-1 coding ^-1Intestinal bacteria 1-153).Usually, use the Polytron homogenizer, make about 100 gram cells be resuspended in 1 liter of (10 times of volumes) cell lysis buffer solution (10mM Tris, 5mM EDTA, pH6), and with 5,000 * g eccentric cell 30 minutes.To be resuspended in 1 liter of cell lysis buffer solution through the cell precipitation of washing with the Polytron homogenizer once more, and, make cell suspending liquid pass through a LH Cell Disrupter (LH Inceltech company) or a Microfluidizer (Microgluidics International company) according to service manual.With suspension 5,000g is after centrifugal 30 minutes, and is resuspended once more and centrifugal to obtain a refractile body precipitation through washing.This precipitation through washing needs use immediately or refrigerated storage in-70 ℃.B. monomer TPO (Met ^-1Solubilising 1-153) and purifying

The precipitation that is obtained by above-mentioned steps is resuspended in the 20mMTris pH8 of 5 times of volumes (calculating by weight), adds 6-8M guanidine and 25mM DTT (dithiothreitol (DTT)), and under 4 ℃, stirs 1 to 3 hour or spent the night to make TPO protein solubilising.Also can use the urea (6-8M) of high density, but compare with guanidine, the output that obtains usually is lower.After the solubilising, 30,000xg centrifugal solution 30 minutes is to obtain containing the proteinic supernatant liquor of sex change monomer TPO.Then, supernatant liquor is gone up chromatogram with the flow velocity of 2 ml/min at Superdex * 200 gel-filtration columns (Pharmacia company, 2.6 * 60 centimetres), and with the 200mM sodium phosphate pH6.0 wash-out that contains 10mM DTT.The component that contains monomer sex change TPO of wash-out is collected between 160 and 200 milliliters.TPO protein is gone up at semipreparative C4 reversed-phase column (2 * 20 centimetres of VYDAC) and is further purified.Sample is splined on 0.1%TFA (trifluoracetic acid) the equilibrated post that contains 30% acetonitrile with the speed of 5 ml/min.Linear gradient (30-60% in 60 minutes) elute protein with acetonitrile.When about 50% acetonitrile concentration, purifying go back crude protein by wash-out.This product obtains bioactive TPO varient after refolding.C. biological activity TPO (Met ^-1Generation 1-153)

About 20 milligrams of monomers, reductibility and sex change TPO protein in 40 milliliters of 0.1%TFA/50% acetonitriles are diluted in 360 milliliters of refolding damping fluids, wherein preferably contain following reagent: 50mM Tris0.3M NaCl5mM EDTA2%CHAPS washing agent 25% glycerine 5mM oxidized glutathione 1mM reduced glutathione pH regulator to 8.3

After mixing, 4 ℃ of following mild stirring refolding damping fluids 12 to 48 hours, thus obtain TPO production peak with correct disulfide linkage form (vide infra) refolding.Then, 0.2%TFA makes solution acidicization with final concentration, through 0.45 or 0.22 micron filter paper filtering solution, and adds the acetonitrile of 1/10 volume.This solution directly pumped into immediately the C4 reversed-phase column and with the refolding TPO (Met of identical gradient elution purifying mentioned above ^-11-153).Under these conditions, have bioactive refolding TPO about 45% o'clock of acetonitrile concentration by wash-out.The TPO form of unsuitable disulfide linkage is eluted earlier.Through the assessment of sds gel and the analysis of C4 reverse-phase chromatography, final TPO (Met ^-11-153) purity is greater than 95%.For zooscopy, the material of C4 purifying is dialysed can hold damping fluid into physiological.Use contains the isotonic buffer solution (10mM sodium acetate pH5.5,10mM sodium succinate pH5.5 or 10mM sodium phosphate pH7.4) of 150mM NaCl and 0.01% tween 80.

Because the height of TPO in Ba/F3 measures tired (reach during about 3 pg/ml maximal stimulation half), might use many different damping fluids, washing agent and redox condition to obtain biologically active substance.But, in most of the cases, can only obtain a small amount of (＜10%) suitably folding material.For commercial manufacturing processed, wish that the output at least 10%, 30 to 50% of refolding is better, then best greater than 50%.Many different washing agents (Triton X-100, dodecyl-β-maltoside, CHAPS, CHAPSO, SDS, sarcosyl, polysorbas20 and tween 80, Zwittergent3-14 and other washing agents) have been assessed to supporting the usefulness of high refolding output.In these washing agents, only find that CHAPS family (CHAPS and CHAPSO) can be used for assembling and the formation of unsuitable disulfide linkage in refolding reaction limit protein matter usually.The level that is higher than 1%CHAPS works most.Best output needs sodium-chlor, and its optimum level is between 0.1M and 0.5M.Adding EDTA (1-5mM) can limit some the metal catalytic oxidation effects (and congregation) in some prepared product.Glycerol concentration greater than 5% provides the suitableeest refolding condition.In order to obtain the highest output, must add oxidation and reduced glutathione or oxidation and reduction halfcystine simultaneously as redox couple.Usually, equal or during more than reductive agent, can be observed higher output at the oxygenant of redox centering.The optimum pH of these TPO varient refoldings is between 7.5 and about 9.Concentration be 10 to 15% or lower organic solvent (as ethanol, acetonitrile and methyl alcohol) can be tolerated.Higher levels of organic solvent increases the amount of inappropriate folded form.Tris and phosphate buffered saline buffer are normally useful.4 ℃ of incubations also can obtain the higher level of suitably folding TPO.

40 to 60% refolding output (according to reduction that is used for the refolding reaction and sex change TPO amount) is arranged in the TPO prepared product by first C4 step purifying usually.In the prepared product of low-purity (as directly after Superdex200 post or initial refractile body extracting) can obtain active substance, though can be because too much precipitating action and the proteinic interference of non-TPO and output is reduced in the TPO refolding process.

Because TPO (Met ^-11-153) contain 4 cysteine residues, it may generate this proteinic three different disulfide linkage forms:

Form 1: disulfide linkage is between cysteine residues 1-4 and 2-3

Form 2: disulfide linkage is between cysteine residues 1-2 and 3-4

Form 3: disulfide linkage is between cysteine residues 1-3 and 2-4.

In the initial trial of determining the refolding condition, the proteinic different peaks of several TPO of containing have been gone out by the C4 reversed phase chromatography separation.Determined wherein to have only a peak that significant biological activity is arranged with the Ba/F3 assay method.Subsequently, adjust the refolding condition to obtain the production peak of this form.Under these conditions, among the whole monomer TPO that obtain, the false folding form is lower than 10-20%.

By mass spectroscopy and protein sequencing, determined that the disulfide linkage form of biological activity TPO is 1-4 and 2-3 (being form 1).The aliquots containig of multiple C4 detached peaks (5-10 nmole) is with tryptic digestion (trypsinase and proteinic mol ratio are 1: 25).Before or after the DTT reductive action,, analyze digestion mixture by the auxiliary laser desorption mass spectrometry of matrix.After reductive action, detect the agglomerate of separating peptide corresponding to the big pancreatin of great majority of TPO.In non-reduced sample, some in these agglomerates have disappeared and new agglomerate can be observed.The agglomerate at new peak corresponds essentially to the number that the right single pancreatin that relates to disulfide linkage is separated peptide.Therefore, the disulfide linkage form that might confirm refolding, reorganization, bioactive TPO clearly is 1-4 and 2-3.This is consistent with the known disulfide linkage form of relevant erythropoietin molecule.D. the reorganization, refolding TPO (Met ^-1Biological activity 1-153)

TPO (the Met of refolding and purifying ^-11-153) in vivo with in the external mensuration activity is arranged all.In Ba/F3 measured, when 3.3 pg/ml (0.3pM), the stimulation of mixing the thymidine of Ba/F3 cell reached half of maximum.In ELISA, during 1.9 nanograms/milliliter, reach half of maximum activity based on the mpl acceptor.In bone marrow depression animal normal and that handle with the low X ray that causes death, TPO (Met ^-11-153) have and stimulate new thrombopoietic high potentiality (can see activity when reaching 30 nanograms/mouse in that dosage is low).

Embodiment 23

The generation of the active TPO varient of other biological in the intestinal bacteria

The three kinds of different TPO varients that become biologically active form with refolding that are purified that produce in intestinal bacteria hereinafter are provided.

(1) incorporated the N-end structure territory (residue 1-155) of TPO from the MLF-13 residue of the signal sequence STII of bacterium.The sequence that obtains is:

MKKNIAFLLNAYASPAPPAC……CVRRA

(SEQ ID NO:85) leader sequence wherein is the underscore part, and C ... C representative is from halfcystine 7 to halfcystine 151.The TPO radioiodination effect that the structure of this varient provides a tyrosine to be used for acceptor and biological study.

(2) the H8MLF-7 residue of being made up of STII sequence, 8 histidine residues and factor Xa enzymatic breaking sequence IEGR is incorporated the N-end structure territory (residue 1-155) of TPO.Its sequence is:

MKKNIAFHHHHHHHHIEGRSPAPPAC……CVRRA

(SEQ ID NO:86) leader sequence wherein is the underscore part, and C ... C representative is from halfcystine 7 to halfcystine 151.When being purified with refolding, this varient available enzyme factor Xa handles, and factor Xa ruptures after the arginine residues of sequence IEGR, is 155 residues to produce a length, TPO varient that natural silk propylhomoserin-terminal amino acid is arranged.

(3) incorporated the N-terminal structural domain of TPO except the responsive sequence IEPR of a zymoplasm, T-H8MLF-is with the method preparation identical with above-mentioned varient (2).The sequence that obtains is:

MKKNIAFHHHHHHHHIEPRSPAPPAC……CVRRA

(SEQ ID NO：87)

Leader sequence wherein is the underscore part, and C ... C representative is from halfcystine 7 to halfcystine 151.After purifying and refolding, this varient of available Thrombin treatment is to obtain the TPO natural N-terminal varient of 155 residue length.Recovery, solubilising and the purifying of A. monomeric biological activity TPO varient (1), (2) and (3)

In intestinal bacteria, can express all varients.As about TPO (Met ^-1Described in the embodiment 22 1-153), most of varients in refractile body, have been found.Can use and embodiment 22 described identical program recovery, solubilising and purifying monomer TPO varient.Use and TPO (Met ^-11-153) used identical refolding condition can obtain 30 to 50% ultimate production.After refolding, with acetonitrile gradient as indicated above, by the C4 reverse-phase chromatography among the 0.1%TFA, purification TPO varient.According to the assessment that Ba/F3 measures, all TPO varients (in their non-proteolytic form) have biological activity, reach half of maximum activity during 2-5pM.B. produce the varient (2) of the terminal TPO of true N-(1-155) and the proteolysis process of (3)

Above-mentioned varient (2) and (3) but be designed to have the leading peptide of an enzymic fracture before the normal-terminal amino acid residue of TPO.After the refolding and purifying of above-mentioned varient (2) and (3), each is all by suitable enzymic digestion.For each varient, remove acetonitrile in the anti-phase step of C4 by in solution, being blown into the stream nitrogen of getting a breathing space.Subsequently, as mentioned below, with factor Xa or two kinds of varients of Thrombin treatment.

For TPO varient (2), the 1M Tris damping fluid of pH8 is added the solution that does not contain acetonitrile, making final concentration is 50mM, and if necessary, with pH regulator to 8.Add NaCl and CaCl ₂, make concentration should be 0.1M and 2mM mutually.It is 1: 25 to 1: 100 that adding factor Xa (New EnglandBiolabs company) makes the mol ratio of enzyme and varient.Room temperature incubation sample made fracture reach maximum value in 1 to 2 hour, can assess out this maximum value by showing the migration and variation on the sds gel that leader sequence loses.Subsequently, use and above-mentioned correct identical gradient and the condition of purifying that folds varient, by C4 reverse-phase chromatography purification reaction mixture.By these conditions, Duan Lie varient B does not separate with the varient (2) of fracture.-terminal amino acid is SPAPP, and the terminal leader sequence of expression N-is successfully removed.Factor Xa also produces the internal break of different quantities in the TPO structural domain; After the arginine residues of position 118 generates an extra N-end sequence TTAHKDP (SEQ ID NO:88), can observe fracture.On non-reduced sds gel,, can observe about 17,000 daltonian single bands corresponding to the varient of factor Xa fracture; On the reduction sds gel,, can observe molecular weight about 12,000 and 5,000 daltonian two bands corresponding to fracture at arginine 118.This observation has confirmed that also two parts of this molecule connect together by the disulfide linkage between first and the 4th halfcystine, and this deduction with trysinization experimental result mentioned above is consistent.In the Ba/F3 biological assay, purifying TPO (1-155) the variation physical efficiency of removing the terminal leader sequence of N-and having an internal break reaches half of maximum activity 0.2 to 0.3pM the time.

Concerning varient (3), the digestion damping fluid contains 50mM Tris, 2%CHAPS, 0.3M NaCl, 5mM EDTA and people or the thrombin of beef (Calbiochem company) of pH8, and wherein latter's (enzyme) and the proteinic weight ratio of TPO varient are 1: 25 to 1: 50.Digestion is under the room temperature 2-6 hour.Sds gel by above-mentioned factor Xa cleavage reaction is assessed the digestion process.Usually, occur in this time greater than the fracture of 90% leader.The TPO that obtains purifies with aforesaid C4 reversed-phase column and demonstrates required N-end by amino acid sequencing.Have to seldom amount (＜5%) as the above-mentioned internal break of factor Xa between arginine-Threonine key that pass through.The TPO protein that obtains has high biological activity, and in Ba/F3 measured, 0.2-0.4pM albumen mass-energy reached half of the highest response value.In the enzyme-linked immunosorbent assay based on the mpl acceptor, the protein purification of 2-4 nanograms/milliliter (120-240pM) can reach half of the highest response value, and the usefulness of complete varient in two mensuration that contains leader sequence all will reduce by 5 to 10 times.For zooscopy, the damping fluid that the fracture protein of HPLC purifying is dialysed and can be held into physiological wherein contains 10mM sodium succinate or the 10mM sodium acetate of pH5.5 or the sodium phosphate of pH7.4 of 150mM NaCl, 0.01% tween 80 and pH5.5.By HPLC and sds gel, the protein of purifying can be stablized several weeks in 4 ℃ of preservations.In normal and myelosuppressive mouse, this has the purifying TPO of true N-end sequence to have high activity, reaches under the low dosage of 30 nanograms/mouse low, can promote hematoblastic generation.

Embodiment 24

Synthetic mpl part

Though use recombinant methods people mpl part (hML) usually, also can connect to synthesize by the enzymatic of synthetic peptide fragment, its method is as mentioned below.The synthetic production of hML allows mixing of alpha-non-natural amino acid or complex functionality thing such as polyoxyethylene glycol.In the past, designed the mutant of a serine protease subtilisin DPN.Make up withered grass ligase enzyme (subtiligase) (S221C/P225A), can be in order to connection peptides ester (Abrahmsen etc., Biochem., 30:4151-4159[1991]) in the aqueous solution effectively.Now, show that synthetic peptide can be connected and produce long peptide and protein such as the ribonuclease A (Jackson etc., Science, [1994]) with enzymatic activity according to priority by enzymatic.This technology makes us be able to the long protein of chemosynthesis, and former, long protein can only make with recombinant DNA technology.More detailed details is as mentioned below.

Use the synthetic hML of withered grass ligase enzyme ₁₅₃General strategy shown in scheme 1.With the complete de-protected peptide corresponding to protein C-terminal fragment is starting point, and N-end has been protected, and the terminal activated ester of C-peptide adds with the withered grass ligase enzyme.After reaction finishes, isolate product by RPLC, and blocking group is removed from the N-end.Connect next peptide fragment and go and protect and use a series of peptides to come this process of repetition until obtaining whole length protein.This process is similar to such solid phase method: N-end is protected, the terminal peptide that is activated of C-is connected to the N-end of previous peptide, and makes protein synthetic according to the direction of C → N.Though because each pairing will cause the adding of 50 residues nearly and product in that to connect the back each time separated, the high-purity protein of length can be synthesized with a rational output.

The strategy of the synthetic hML of scheme 1. usefulness withered grass ligase enzymes

The R-NH-peptide ₂-CO-R '+H ₂The N-peptide ₁-CO ₂

↓ 1) withered grass ligase enzyme

The R-NH-peptide ₂-CO-NH-peptide ₁-CO ₂

↓2)Zn/CH ₃CO ₂H

H ₂The N-peptide ₂-CO-NH-peptide ₁-CO ₂

↓ 3) repeat 1+2

H ₂The N-peptide ₃-CO-NH-peptide ₂-CO-NH-peptide ₁-CO ₂

According to withered grass ligase enzyme sequence-specific and to the understanding of hML biological activity " EPO structural domain " aminoacid sequence, we are with hML ₁₅₃Be divided into 7 fragments of 18 to 25 residues of length.The connection tetrapeptide of synthetic test is in order to determine the suitable connection juncture of 18 to 25 aggressiveness.Table 13 has shown the result that these tests connect.

Table 13

HML tests connection.In 22 ℃, 100mM Wheat flavone, dissolving donor and nucleophile peptide, making concentration is 10mM.From the storing solution of 1.6mg/ml (about 70 μ M), take out ligase enzyme and add solution, making final concentration is 10 μ M.Connection procedure can spend the night.Output is calculated according to the connection percentage with respect to the donor hydrolase polypeptide.

The site	Donor (glc-K-NH2)	Nucleophilic WSG-NH2	Hydrolysis %	Connect %
					1(23/24)	HVLH (SEQ ID NO：89)	SRLS (SEQ ID NO：90)	92	08
(22/23)	SHVL (SEQ ID NO：91)	HSRL (SEQ ID NO：92)	48	52
					2(46/47)	AVDF (SEQ ID NO：93)	SLGE (SEQ ID NO：94)	22	78
3(69/70)	AVTL (SEQ ID NO：95)	LLEG (SEQ ID NO：96)	53	47
					4(89/90)	LSSL (SEQ ID NO：97)	LGQL (SEQ ID NO：98)	95	05
(88/89)	C(acm)LSS (SEQ ID NO：99)	LLGQ (SEQ ID NO：100)	00	00
					(90/91)	SSLL (SEQ ID NO：101)	GQLS (SEQ ID NO：102)	45	55
(88/89)	CLSS (SEQ ID NO：103)	LLGQ (SEQ ID NO：100)	90	10
					5(107/108)	LQSL (SEQ ID NO：104)	LGTQ (SEQ ID NO：105)	99	01
(106/107)	ALQS (SEQ ID NO：106)	LLGT (SEQ ID NO：107)	70	30
					6(128/129)	NAIF (SEQ ID NO：108)	LSFQ (SEQ ID NO：109)	60	40

According to these experiments, the connection peptides shown in the table 14 should be connected effectively by the withered grass ligase enzyme.All need a suitable N-terminal blocking group to prevent that self from connecting for each donor ester peptide.We select different nicotinoyl (iNOC) blocking group (Veber etc.; J.Org.Chem.; 42:3286-3289[1977]); because it is water miscible; can be impregnated in the final step of solid-phase peptide synthetic, and it is being used for protecting and the anhydrous HF that peptide separates from solid-phase resin can kept stable.In addition, under weak reductive condition (zinc/acetate), it can be removed from peptide after connecting each time, thereby generates a free N-terminal that is used for ligation subsequently.Oxyacetate-lysyl-acid amides (glc-K-NH ₂) ester is used as and activates the C-end, it is according to being that former experiment shows that it can be by withered grass ligase enzyme acidylate (Abrahmsen etc., Biochem., 30:4151-4159[1991]) effectively.Protected by iNOC and used the solid phase method of standard to synthesize by glc-K-amide activated Toplink, its main points see scheme 2.Then, peptide is linked in sequence until generating complete protein, and end product external by refolding.According to the homology of EPO, believe that can to form disulphide between cysteine residues 7 and 151 and 28 and 85 right.Only need in well-oxygenated environment, to stir this reduzate several hrs, just can make the disulphide oxidation.The refolding product also will contain the component merging and the freeze-drying preservation of active protein subsequently with the high performance liquid chromatography purifying.In addition, available diverse ways protect disulphide be controlled at specific disulphide between a series of oxygenizements.With ethanamide methyl (acm) group the protection of halfcystine 7 and 151 has been guaranteed 28 and 85 oxygenizement.Subsequently, the acm group can be removed and with residue 7 and 151 oxidations.Otherwise, make in a series of oxygenizements of needs folding when correct, can be with residue 28 and 85 usefulness acm protection and oxidation.Perhaps, halfcystine 28 and 85 can be by another natural or alpha-non-natural amino acid displacement except that halfcystine, to guarantee the correct oxidation of halfcystine 7 and 151.

Table 14. synthesizes the employed peptide fragment fragment of complete hML with the withered grass ligase enzyme

By the formula (I) include compounds having the illustrative embodiments included below, Example bag Including (E) - isomer and (Z) - isomer compounds and mixtures thereof are self-evident, In only its representatives listed here, does not in any way limit the invention: 2 - (3 - acetylamino-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indol-3 - Yl) acrylate; 2 - (3 - phenyl-propionylamino) -3 - (2 - ethyl-4, 6 - dichloro-indol-3 - Yl) acrylate; 2 - (3 - butyrylamino) -3 - (2 - ethyl-4, 6 - dichloro-indol-3 - Yl) acrylate; 2 - (3 - benzamido-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (3 - (N-methoxycarbonylamino)-phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (3 - (N-ethoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (3 - (N-propoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (3 - (N-isopropoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4 ,6 - Dichloro-indol-3 - yl) acrylate; 2 - (3 - (N-butoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (3 - methyl sulfonyl aminophenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (3 - amino-ethanesulfonyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (3 - C sulfonylamino) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (3 - butanesulfonylamino) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (3 - benzenesulfonylamino-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (4 - acetylamino-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indol-3 - Yl) acrylate; 2 - (4 - benzamido-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (4 - (N-methoxycarbonylamino)-phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (4 - (N-ethoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (4 - (N-isopropoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4 ,6 - Dichloro-indol-3 - yl) acrylate; 2 - (4 - methylsulfonyl-aminophenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (4 - aminophenyl benzenesulfonyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (2 - acetylamino-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indol-3 - Yl) acrylate; 2 - (2 - benzamido-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (2 - (N-methoxycarbonylamino)-phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (2 - (N-ethoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (2 - (N-isopropoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4 ,6 - Dichloro-indol-3 - yl) acrylate; 2 - (2 - methyl sulfonyl aminophenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (2 - amino-phenyl-sulfonyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (3 - acetylamino-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indol-3 - Yl) acrylate; 2 - (3 - phenyl-propionylamino) -3 - (2 - ethyl-4, 6 - dichloro-indol-3 - Yl) acrylate; 2 - (3 - butyrylamino) -3 - (2 - ethyl-4, 6 - dichloro-indol-3 - Yl) acrylate; 2 - (3 - benzamido-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (3 - (N-methoxycarbonylamino)-phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (3 - (N-ethoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (3 - (N-propoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (3 - (N-isopropoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4 ,6 - Dichloro-indol-3 - yl) acrylate; 2 - (3 - (N-butoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (3 - methyl sulfonyl aminophenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (3 - amino-ethanesulfonyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (3 - C sulfonylamino) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (3 - butanesulfonylamino) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (3 - benzenesulfonylamino-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (4 - acetylamino-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indol-3 - Yl) acrylate; 2 - (4 - benzamido-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (4 - (N-methoxycarbonylamino)-phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (4 - (N-ethoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (4 - (N-isopropoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4 ,6 - Dichloro-indol-3 - yl) acrylate; 2 - (4 - methylsulfonyl-aminophenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (4 - aminophenyl benzenesulfonyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (2 - acetylamino-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indol-3 - Yl) acrylate; 2 - (2 - benzamido-phenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (2 - (N-methoxycarbonylamino)-phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (2 - (N-ethoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4, 6 - Dichloro-indol-3 - yl) acrylate; 2 - (2 - (N-isopropoxycarbonyl-amino) phenyl) -3 - (2 - ethyl-4 ,6 - Dichloro-indol-3 - yl) acrylate; 2 - (2 - methyl sulfonyl aminophenyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (2 - amino-phenyl-sulfonyl) -3 - (2 - ethyl-4, 6 - dichloro-indole - ³ -) acrylate; 2 - (3 - acetylamino-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic Acid) acrylic acid; 2 - (3 - phenyl-propionylamino) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic Acid) acrylic acid; 2 - (3 - butyrylamino) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic Acid) acrylic acid; 2 - (3 - benzamido-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic) acid; 2 - (3 - (4 - methoxy-benzoyl amino) phenyl) -3 - (4,6 - dichloro-indole -3 - Yl 2 - carboxylic acid) acrylic acid; 2 - (3 - (4 - chloro-benzoyl) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (3 - (4 - methyl-benzoyl amino) phenyl) -3 - (4,6 - dichloro-indole - ³ - 2 - carboxylic acid) acrylic acid; 2 - (3 - (4 - fluoro-benzoyl) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (3 - (4 - trifluoromethyl) phenyl) -3 - (4,6 - dichloro-indole - ³ - 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-methoxycarbonylamino)-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (3 - (N-ethoxycarbonyl-amino) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (3 - (N-propoxycarbonyl-amino) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (3 - (N-isopropoxycarbonyl-amino) phenyl) -3 - (4,6 - dichloro-indole - ³ - 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-butoxycarbonyl-amino) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (3 - methyl sulfonyl aminophenyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic) acid; 2 - (3 - amino-ethanesulfonyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic) acid; 2 - (3 - C sulfonylamino) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic) acid; 2 - (3 - butanesulfonylamino) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic) acid; 2 - (3 - benzenesulfonylamino-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic) acid; 2 - (3 - (4 - methoxy) phenyl) -3 - (4,6 - dichloro-indole -3 - Yl 2 - carboxylic acid) acrylic acid; 2 - (3 - (4 - chlorophenyl) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (3 - acetylamino-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic Acid) acrylamide; 2 - (3 - phenyl-propionylamino) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic Acid) acrylamide; 2 - (3 - butyrylamino) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic Acid) acrylamide; 2 - (3 - benzamido-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic acid) acrylamide; 2 - (3 - (4 - methoxy-benzoyl amino) phenyl) -3 - (4,6 - dichloro-indole -3 - Yl 2 - carboxylic acid) acrylamide; 2 - (3 - (4 - chloro-benzoyl) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylamide; 2 - (3 - (4 - methyl-benzoyl amino) phenyl) -3 - (4,6 - dichloro-indole - ³ - 2 - carboxylic acid) acrylamide; 2 - (3 - (4 - fluoro-benzoyl) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylamide; 2 - (3 - (4 - trifluoromethyl) phenyl) -3 - (4,6 - dichloro-indole - ³ - 2 - carboxylic acid) acrylamide; 2 - (3 - (N-methoxycarbonylamino)-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylamide; 2 - (3 - (N-ethoxycarbonyl-amino) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylamide; 2 - (3 - (N-propoxycarbonyl-amino) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylamide; 2 - (3 - (N-isopropoxycarbonyl-amino) phenyl) -3 - (4,6 - dichloro-indole - ³ - 2 - carboxylic acid) acrylamide; 2 - (3 - (N-butyl-carbonyloxy) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylamide; 2 - (3 - methyl sulfonyl aminophenyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic acid) acrylamide; 2 - (3 - amino-ethanesulfonyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic acid) acrylamide; 2 - (3 - C sulfonylamino) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic acid) acrylamide; 2 - (3 - butanesulfonylamino) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic acid) acrylamide; 2 - (3 - benzenesulfonylamino-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic acid) acrylamide; 2 - (3 - (4 - methoxy) phenyl) -3 - (4,6 - dichloro-indole -3 - Yl 2 - carboxylic acid) acrylamide; 2 - (3 - (4 - chlorophenyl) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylamide; 2 - (4 - acetylamino-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic Acid) acrylic acid; 2 - (4 - phenyl-propionylamino) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic Acid) acrylic acid; 2 - (4 - butyrylamino) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic Acid) acrylic acid; 2 - (4 - benzamido-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic) acid; 2 - (4 - (N-methoxycarbonylamino)-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (4 - (N-ethoxycarbonyl-amino) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (4 - (N-propoxycarbonyl-amino) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (4 - (N-isopropoxycarbonyl-amino) phenyl) -3 - (4,6 - dichloro-indole - ³ - 2 - carboxylic acid) acrylic acid; 2 - (4 - methylsulfonyl-aminophenyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic) acid; 2 - (4 - aminophenyl benzenesulfonyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic) acid; 2 - (2 - acetylamino-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic Acid) acrylic acid; 2 - (2 - benzamido-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic) acid; 2 - (2 - (N-methoxycarbonylamino)-phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (2 - (N-ethoxycarbonyl-amino) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (2 - (N-propoxycarbonyl-amino) phenyl) -3 - (4,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (2 - (N-isopropoxycarbonyl-amino) phenyl) -3 - (4,6 - dichloro-indole - ³ - 2 - carboxylic acid) acrylic acid; 2 - (2 - methyl sulfonyl aminophenyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic) acid; 2 - (2 - amino-phenyl-sulfonyl) -3 - (4,6 - dichloro-indol-3 --2 - Carboxylic) acid; 2 - (3 - acetylamino-phenyl) -3 - (5,6 - dichloro-indol-3 --2 - carboxylic Acid) acrylic acid; 2 - (3 - benzamido-phenyl) -3 - (5,6 - dichloro-indol-3 --2 - Carboxylic) acid; 2 - (3 - (N-methoxycarbonylamino)-phenyl) -3 - (5,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (3 - (N-ethoxycarbonyl-amino) phenyl) -3 - (5,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (3 - (N-propoxycarbonyl-amino) phenyl) -3 - (5,6 - dichloro-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (3 - (N-isopropoxycarbonyl-amino) phenyl) -3 - (5,6 - dichloro-indole - ³ - 2 - carboxylic acid) acrylic acid; 2 - (3 - methyl sulfonyl aminophenyl) -3 - (6 - chloro-indol-3 - yl 2 - carboxylic acid) acrylic acid; 2 - (3 - benzenesulfonylamino-phenyl) -3 - (6 - chloro-indol-3 - yl 2 - carboxylic acid) Acid; 2 - (3 - acetylamino-phenyl) -3 - (6 - chloro-indol-3 - yl 2 - carboxylic acid) C Acid; 2 - (3 - benzamido-phenyl) -3 - (6 - chloro-indol-3 - yl 2 - carboxylic acid) Acid; 2 - (3 - (N-methoxycarbonylamino)-phenyl) -3 - (6 - chloro-indol-3 - yl - 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-ethoxycarbonyl-amino) phenyl) -3 - (6 - chloro-indol-3 - yl - 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-propoxycarbonyl-amino) phenyl) -3 - (6 - chloro-indol-3 - yl - 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-isopropoxycarbonyl-amino) phenyl) -3 - (6 - chloro-indol-3 - yl 2 - carboxylic acid) acrylic acid; 2 - (3 - methyl sulfonyl aminophenyl) -3 - (6 - chloro-indol-3 - yl 2 - carboxylic acid) Acid; 2 - (3 - benzenesulfonylamino-phenyl) -3 - (6 - chloro-indol-3 - yl 2 - carboxylic acid) Acid; 2 - (3 - acetylamino-phenyl) -3 - (indol-3 - yl 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-methoxycarbonylamino)-phenyl) -3 - (indol-3 --2 - carboxylic Acid) acrylic acid; 2 - (3 - methyl sulfonyl aminophenyl) -3 - (indol-3 - yl 2 - carboxylic acid) acrylic acid; 2 - (3 - benzenesulfonylamino-phenyl) -3 - (indol-3 - yl 2 - carboxylic acid) Acid; 2 - (3 - acetylamino-phenyl) -3 - (7 - ethyl-5 - bromo-indol-3 --2 - Carboxylic acid) acrylic acid; 2 - (3 - benzamido-phenyl) -3 - (7 - ethyl-5 - bromo-indol-3 - yl - 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-methoxycarbonylamino)-phenyl) -3 - (7 - ethyl-5 - bromo-indole -3 - Yl 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-ethoxycarbonyl) phenyl) -3 - (7 - ethyl-5 - bromo-indole -3 - Yl 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-propoxycarbonyl-amino)-phenyl) -3 - (7 - ethyl-5 - bromo-indole -3 - Yl 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-isopropoxycarbonyl-amino)-phenyl) -3 - (7 - ethyl-5 - bromo-indole Indole -3 --2 - carboxylic acid) acrylic acid; 2 - (3 - methyl sulfonyl aminophenyl) -3 - (7 - ethyl-5 - bromo-indol-3 - yl - 2 - carboxylic acid) acrylic acid; 2 - (3 - benzenesulfonylamino-phenyl) -3 - (7 - ethyl-5 - bromo-indol-3 - yl - 2 - carboxylic acid) acrylic acid; 2 - (3 - acetylamino-phenyl) -3 - (5 - ethyl-7 - bromo-indol-3 --2 - Carboxylic acid) acrylic acid; 2 - (3 - benzamido-phenyl) -3 - (5 - ethyl-7 - bromo-indol-3 - yl - 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-methoxycarbonylamino)-phenyl) -3 - (5 - ethyl-7 - bromo-indole -3 - Yl 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-ethoxycarbonyl-amino) phenyl) -3 - (5 - ethyl-7 - bromo-indole -3 - Yl 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-propoxycarbonyl-amino)-phenyl) -3 - (5 - ethyl-7 - bromo-indole -3 - Yl 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-isopropoxycarbonyl-amino)-phenyl) -3 - (5 - ethyl-7 - bromo-indole Indole -3 --2 - carboxylic acid) acrylic acid; 2 - (3 - methyl sulfonyl aminophenyl) -3 - (5 - ethyl-7 - bromo-indol-3 - yl - 2 - carboxylic acid) acrylic acid; 2 - (3 - benzenesulfonylamino-phenyl) -3 - (5 - ethyl-7 - bromo-indol-3 - yl - 2 - carboxylic acid) acrylic acid; 2 - (3 - acetylamino-phenyl) -3 - (5 - Fluoro-7 - chloro-indol-3 --2 - Carboxylic) acid; 2 - (3 - benzamido-phenyl) -3 - (5 - Fluoro-7 - chloro-indol-3 --2 - Carboxylic acid) acrylic acid; 2 - (3 - (N-methoxycarbonylamino)-phenyl) -3 - (5 - Fluoro-7 - chloro-indole - ³ - 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-ethoxycarbonyl-amino) phenyl) -3 - (5 - Fluoro-7 - chloro-indole - ³ - 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-propoxycarbonyl-amino)-phenyl) -3 - (5 - Fluoro-7 - chloro-indole - ³ - 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-isopropoxycarbonyl-amino)-phenyl) -3 - (5 - Fluoro-7 - chloro-indole -3 - Yl 2 - carboxylic acid) acrylic acid; 2 - (3 - methyl sulfonyl aminophenyl) -3 - (5 - Fluoro-7 - chloro-indol-3 --2 - Carboxylic acid) acrylic acid; 2 - (3 - benzenesulfonylamino-phenyl) -3 - (5 - Fluoro-7 - chloro-indol-3 --2 - Carboxylic acid) acrylic acid; 2 - (3 - acetylamino-phenyl) -3 - (7 - fluoro-5 - chloro-indol-3 --2 - Carboxylic) acid; 2 - (3 - benzamido-phenyl) -3 - (7 - fluoro-5 - chloro-indol-3 --2 - Carboxylic acid) acrylic acid; 2 - (3 - (N-methoxycarbonylamino)-phenyl) -3 - (7 - fluoro-5 - chloro-indole - ³ - 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-ethoxycarbonyl) phenyl) -3 - (7 - fluoro-5 - chloro-indole - ³ - 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-propoxycarbonyl-amino)-phenyl) -3 - (7 - fluoro-5 - chloro-indole - ³ - 2 - carboxylic acid) acrylic acid; 2 - (3 - (N-isopropoxycarbonyl-amino)-phenyl) -3 - (7 - fluoro-5 - chloro-indol- -3 - Yl 2 - carboxylic acid) acrylic acid; 2 - (3 - methyl sulfonyl aminophenyl) -3 - (7 - fluoro-5 - chloro-indol-3 --2 - Carboxylic acid) acrylic acid; 2 - (3 - benzenesulfonylamino-phenyl) -3 - (7 - fluoro-5 - chloro-indol-3 --2 - Carboxylic acid) acrylic acid; 2 - (3 - acetylamino-phenyl) -3 - (5,7 - dibromo-indol-3 --2 - carboxylic Acid) acrylic acid; 2 - (3 - benzamido-phenyl) -3 - (5,7 - dibromo-indol-3 --2 - Carboxylic) acid; 2 - (3 - (N-methoxycarbonylamino)-phenyl) -3 - (5,7 - dibromo-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (3 - (N-ethoxycarbonyl-amino) phenyl) -3 - (5,7 - dibromo-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (3 - (N-propoxycarbonyl-amino) phenyl) -3 - (5,7 - dibromo-indol-3 --2 - carboxylic acid) acrylic acid; 2 - (3 - (N-isopropoxycarbonyl-amino) phenyl) -3 - (5,7 - dibromo-indol - ³ - 2 - carboxylic acid) acrylic acid; 2 - (3 - methyl sulfonyl aminophenyl) -3 - (5,7 - dibromo-indol-3 --2 - Carboxylic) acid; 2 - (3 - benzenesulfonylamino-phenyl) -3 - (5,7 - dibromo-indol-3 --2 - Carboxylic acid) acrylic acid. ...

Finishing the condition that peptide connects is 25 ℃, 100mM Wheat flavone pH8 (prepared fresh and through the vacuum filtration exhaust of 5 μ M filter paper).Usually, the C-terminal fragment is dissolved in the damping fluid (2-5mM peptide), and adding withered grass ligase enzyme 10 * storing solution (in the 100mM Wheat flavone of pH8, concentration 1mg/ml) makes final enzyme concn reach about 5 μ M.3 to 5 moles excessive glc-K-NH ₂Activated donor peptide adds with insoluble solid form subsequently and makes 25 ℃ of mixture static.The monitoring that connects is by the anti-phase C18 efficient liquid phase chromatographic analysis (CH in 0.1% trifluoracetic acid ₃CN/H ₂The O gradient).Connect high-efficient liquid phase chromatogram purification and the freeze-drying of product by preparation.The going of different nicotinoyl (iNOC) protected by will being stirred with protected peptide by the zinc bits of hydrochloric acid activation in acetate.The zinc bits are by removing by filter, and acetate is then by vacuum-evaporation.The Toplink that obtains is directly used in next step connection and repeats said process.Synthetic hML ₁₅₃Can be connected in synthetic or reorganization hML with the described step of similar above _154-332Thereby, produce synthetic or semisynthetic total length hML.

Synthetic hML has a lot of advantages with respect to recombinant chou.Can introduce non-natural side chain to improve usefulness or specificity.Can mix polymer function thing such as polyoxyethylene glycol, with the perdurability of increase effect.For example, a step or multistep connect finish before or after, polyoxyethylene glycol can be connected to the lysine residue (table 14) of individual sections.The responsive peptide bond of proteolytic enzyme can be removed or change to improve stability in vivo.In addition, can synthesize heavy atom derivative to help to determine structure.

Scheme 2. is used for the solid phase synthesis of the peptide fragment of fragment connection

A) lysyl-to methylbenzene hydrylamine (MBHA) resin 1 (0.63meq./gm., Advanced ChemTech) in methylacetamide (DMA) solution and 25 ℃ the time, stir 1 hour with bromoacetic acid (5eq.) and di-isopropyl carbodiimide (5eq.), thereby obtain bromoacetic acid derivative 2.B), stir 24 hours amino acid with two yellow soda ash, thereby obtain corresponding oxyacetate-phenylalanyl-aminoacyl-resin 3 with the one Boc protection of esterification with a large amount of washing resins of DMA and in dimethyl formamide (DMF) and 50 ℃ the time.Glycyl resin DMF (3 *) and methylene dichloride (CH ₂Cl ₂) (3 *) washing also can be in the room temperature preservation several months.Subsequently, resin 3 can be splined on an automatic peptide synthesizer (Ap-plied Biosystems 430A) and use standard solid-phase program (5) that peptide is extended.C) with the CH of 45% trifluoroacetic acid ₂Cl ₂Solution is removed N-α-Boc group.D) Boc protection amino acid (5eq.) subsequently use in advance benzotriazole-1-yl-oxy-tris-(dimethylamino) phosphorus hexafluorophosphate among the DMA (BOP, 4eq.) and N-methylmorpholine (NMM 10eq.) activates also coupling 1 to 2 hour.E) remove last N-α-Boc group (TFA/CH ₂Cl ₂) obtaining 4, and as mentioned before,, introduce different nicotinoyl (iNOC) blocking group by stirring 24 hours with the different nicotinoyl of 4--2-4-dinitrobenzene carbonate (3eq.) and NMM (6eq.) in DMA and 25 ℃ the time.F) handled 1 hour by 0 ℃ of anhydrous HF (5% methyl-phenoxide/5% ethyl-methyl sufide), make the peptide fracture and go protection, thereby obtain the iNOC protection, oxyacetate activated peptide 5, and with anti-phase C18 high performance liquid chromatography (CH ₃CN/ water gradient, 0.1%TFA) purifying.The evaluation of all substrates is finished by mass spectroscopy.

* * * * * * * * * * * * * * * * * * * * * *

The realizability of replenishing

According to the reference and the parent material of above-mentioned explanation and easily acquisition, can realize the content of the present invention that claims propose.But, the applicant is at AmericanType Culture Collection, Rockville, Md., preserved following clone among the USA (ATCC):

Intestinal bacteria, DH10B-pBSK-hmpl I 1.8, ATCC registration number CRL69575, on February 24th, 1994 preserved;

Plasmid, pSV15.ID.L L.MLORF, the ATCC registration number was preserved on December 2nd, 75958,1994; With

CHO DP-12 cell, ML 1/50 MCB (mark #1594), ATCC registration number CRL11770, on December 6th, 1994 preserved.

This preservation thing is under the regulation of Budapest Treaty, is subjected to the international approval that microorganism preserves, and is that purpose is made with patented procedure and rule (Budapest Treaty) thereof.This can keep great-hearted culture 30 years from having guaranteed to preserve certainly.In Bu-dapest Treaty specified period, according to the agreement between applicant and the ATCC, can obtain required biology from ATCC, wherein ATCC should guarantee unrestricted availability under the assurance of relevant United States Patent (USP).The availability that is saved bacterial strain can not be interpreted as: violate by any government according to its patent law during granted entitlements to application of the present invention.

* * * * * * * * * * * * * * * * * * *

Though description of the invention must be in conjunction with preferred example and specific operability embodiment, but after having read aforesaid explanation, only otherwise depart from spirit of the present invention and category, those skilled in the art can realize the displacement of described multiple variation, Equivalent and the change of subject matter herein.Therefore, the present invention's method that can realize be not only herein special those that describe.Thereby, wish that the protection of being given by written patent only is subjected to the restriction of this paper appended claims and Equivalent thereof herein.

All reference contents that are cited are in this article all taken this in the lump as a reference.

Sequence table (1) general information:

(i) applicant: Genetech, Inc.

Eaton，Dan L.

de Sauvage，Frederic J.

(ii) denomination of invention: thrombopoietin

(iii) sequence number: 144

(iv) mailing address:

(A) address: Genentech, Inc.

(B) street: 460 Point San Bruno Blvd

(C) city: South San Francisco

(D) state: California

(E) country: the U.S.

(F) postcode: 94080

(v) computer-reader form:

(A) recording medium type: 3.5 inches, the 1.44Mb floppy disk

(B) computer: IBM PC compatible type

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

(D) software: WinPatin (Genentech)

(vi) the application's data:

(A) application number: 08/374540

(B) applying date: the 03-5 month-1995

(C) classification:

(vii) request for data formerly:

(A) application number: PCT/US94/14553

(B) applying date: the 28-12 month-1994

(vii) request for data formerly:

(A) application number: 08/249376

(B) applying date: the 25-5 month-1994 (vii) request for data formerly:

(A) application number: 08/223263

(B) applying date: the 04-4 month-1994 (vii) request for data formerly:

(A) application number: 08/196689

(B) applying date: the 15-2 month-1994 (vii) request for data formerly:

(A) application number: 08/348658

(B) applying date: the 02-12 month-1994 (vii) request for data formerly:

(A) application number: 08/185607

(B) applying date: the 21-1 month-1994 (vii) request for data formerly:

(A) application number: 08/348657

(B) applying date: the 02-12 month-1994 (vii) request for data formerly:

(A) application number: 08/176553

(B) applying date: the 03-1 month-1994 (viii) lawyer/proxy's information:

(A) name: Winter, Daryl B.

(B) registration number: 32,637

(C) folder/file number: P0871P5 (ix) communication information:

(A) phone: 415/225-1249

(B) fax: 415/952-9881

(C) information of fax: 910/371-7168 (2) SEQ ID NO:1:

(i) sequence signature:

(A) length: 353 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:1:Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr1 5 10 15Ala Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu

20 25 30Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser

35 40 45Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val

50 55 60Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln

65 70 75Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu

80 85 90Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr

95 100 105Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu

110 115 120Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro

125 130 135Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu

140 145 150Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu

155 160 165Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr

170 175 180Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu

185 190 195Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser

200 205 210Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly Phe

215 220 225Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu

230 235 240Asp Gln Ile Pro Gly Tyr Leu Asn Arg Ile His Giu Leu Leu Asn

245 250 255Gly Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly

260 265 270Ala Pro Asp Ile Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro

275 280 285Pro Asn Leu Gln Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro

290 295 300Thr Gly Gln Tyr Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr

305 310 315Pro Val Val Gln Leu His Pro Leu Leu Pro Asp Pro Ser Ala Pro

320 325 330Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn Thr Ser Tyr Thr His

335 340 345Ser Gln Asn Leu Ser Gln Glu Gly

The information of 350 353 (2) SEQ ID NO:2:

(i) sequence signature:

(A) length: 1795 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ, ID, NO:2:TCTTCCTACC, CATCTGCTCC, CCAGAGGGCT, GCCTGCTGTG, CACTTGGGTC, 50CTGGAGCCCT, TCTCCACCCG, GATAGATTCC, TCACCCTTGG, CCCGCCTTTG, 100CCCCACCCTA, CTCTGCCCAG, AAGTGCAAGA, GCCTAAGCCG, CCTCCATGGC, 150CCCAGGAAGG, ATTCAGGGGA, GAGGCCCCAA, ACAGGGAGCC, ACGCCAGCCA, 200GACACCCCGG, CCAGAATGGA, GCTGACTGAA, TTGCTCCTCG, TGGTCATGCT, 250TCTCCTAACT, GCAAGGCTAA, CGCTGTCCAG, CCCGGCTCCT, CCTGCTTGTG, 300ACCTCCGAGT, CCTCAGTAAA, CTGCTTCGTG, ACTCCCATGT, CCTTCACAGC, 350AGACTGAGCC, AGTGCCCAGA, GGTTCACCCT, TTGCCTACAC, CTGTCCTGCT, 400GCCTGCTGTG, GACTTTAGCT, TGGGAGAATG, GAAAACCCAG, ATGGAGGAGA, 450CCAAGGCACA, GGACATTCTG, GGAGCAGTGA, CCCTTCTGCT, GGAGGGAGTG, 500ATGGCAGCAC, GGGGACAACT, GGGACCCACT, TGCCTCTCAT, CCCTCCTGGG, 550GCAGCTTTCT, GGACAGGTCC, GTCTCCTCCT, TGGGGCCCTG, CAGAGCCTCC, 600TTGGAACCCA, GCTTCCTCCA, CAGGGCAGGA, CCACAGCTCA, CAAGGATCCC, 650AATGCCATCT, TCCTGAGCTT, CCAACACCTG, CTCCGAGGAA, AGGTGCGTTT, 700CCTGATGCTT, GTAGGAGGGT, CCACCCTCTG, CGTCAGGCGG, GCCCCACCCA, 750CCACAGCTGT, CCCCAGCAGA, ACCTCTCTAG, TCCTCACACT, GAACGAGCTC, 800CCAAACAGGA, CTTCTGGATT, GTTGGAGACA, AACTTCACTG, CCTCAGCCAG, 850AACTACTGGC, TCTGGGCTTC, TGAAGTGGCA, GCAGGGATTC, AGAGCCAAGA, 900TTCCTGGTCT, GCTGAACCAA, ACCTCCAGGT, CCCTGGACCA, AATCCCCGGA, 950TACCTGAACA, GGATACACGA, ACTCTTGAAT, GGAACTCGTG, GACTCTTTCC, 1000TGGACCCTCA, CGCAGGACCC, TAGGAGCCCC, GGACATTTCC, TCAGGAACAT, 1050CAGACACAGG, CTCCCTGCCA, CCCAACCTCC, AGCCTGGATA, TTCTCCTTCC, 1100CCAACCCATC, CTCCTACTGG, ACAGTATACG, CTCTTCCCTC, TTCCACCCAC, 1150CTTGCCCACC, CCTGTGGTCC, AGCTCCACCC, CCTGCTTCCT, GACCCTTCTG, 1200CTCCAACGCC, CACCCCTACC, AGCCCTCTTC, TAAACACATC, CTACACCCAC, 1250TCCCAGAATC, TGTCTCAGGA, AGGGTAAGGT, TCTCAGACAC, TGCCGACATC, 1300AGCATTGTCT, CATGTACAGC, TCCCTTCCCT, GCAGGGCGCC, CCTGGGAGAC, 1350AACTGGACAA, GATTTCCTAC, TTTCTCCTGA, AACCCAAAGC, CCTGGTAAAA, 1400GGGATACACA, GGACTGAAAA, GGGAATCATT, TTTCACTGTA, CATTATAAAC, 1450CTTCAGAAGC, TATTTTTTTA, AGCTATCAGC, AATACTCATC, AGAGCAGCTA, 1500GCTCTTTGGT, CTATTTTCTG, CAGAAATTTG, CAACTCACTG, ATTCTCTACA, 1550TGCTCTTTTT, CTGTGATAAC, TCTGCAAAGG, CCTGGGCTGG, CCTGGCAGTT, 1600GAACAGAGGG, AGAGACTAAC, CTTGAGTCAG, AAAACAGAGA, AAGGGTAATT, 1650TCCTTTGCTT, CAAATTCAAG, GCCTTCCAAC, GCCCCCATCC, CCTTTACTAT, 1700CATTCTCAGT, GGGACTCTGA, TCCCATATTC, TTAACAGATC, TTTACTCTTG, 1750AGAAATGAAT, AAGCTTTCTC, TCAGAAAAAA, AAAAAAAAAA, AAAAA, 1795, (2) SEQ, ID, the information of NO:3:

(i) sequence signature:

(A) length: 42 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:3:Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala Arg Leu Thr Leu1 5 10 15Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys

20 25 30Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu

The information of 35 40 42 (2) SEQ ID NO:4:

(i) sequence signature:

(A) length: 390 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GAATTCCTGG AATACCAGCT GACAATGATT TCCTCCTCAT CTTTCAACCT 50CACCTCTCCT CATCTAAGAA TTGCTCCTCG TGGTCATGCT TCTCCTAACT 100GCAAGGCTAA CGCTGTCCAG CCCGGCTCCT CCTGCTTGTG ACCTCCGAGT 150CCTCAGTAAA CTGCTTCGTG ACTCCCATGT CCTTCACAGC AGACTGGTGA 200GAACTCCCAA CATTATCCCC TTTATCCGCG TAACTGGTAA GACACCCATA 250CTCCCAGGAA GACACCATCA CTTCCTCTAA CTCCTTGACC CAATGACTAT 300TCTTCCCATA TTGTCCCCAC CTACTGATCA CACTCTCTGA CAAGAATTAT 350TCTTCACAAT ACAGCCCGCA TTTAAAAGCT CTCGTCTAGA 390 (2) SEQ ID NO: 5 information about:

(i) sequence signature:

(A) length: 390 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TCTAGACGAG AGCTTTTAAA TGCGGGCTGT ATTGTGAAGA ATAATTCTTG 50TCAGAGAGTG TGATCAGTAG GTGGGGACAA TATGGGAAGA ATAGTCATTG 100GGTCAAGGAG TTAGAGGAAG TGATGGTGTC TTCCTGGGAG TATGGGTGTC 150TTACCAGTTA CGCGGATAAA GGGGATAATG TTGGGAGTTC TCACCAGTCT 200GCTGTGAAGG ACATGGGAGT CACGAAGCAG TTTACTGAGG ACTCGGAGGT 250CACAAGCAGG AGGAGCCGGG CTGGACAGCG TTAGCCTTGC AGTTAGGAGA 300AGCATGACCA CGAGGAGCAA TTCTTAGATG AGGAGAGGTG AGGTTGAAAG 350ATGAGGAGGA AATCATTGTC AGCTGGTATT CCAGGAATTC 390 (2) SEQ ID NO: 6 of the message:

(i) sequence signature:

(A) length: 332 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:6:Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu1 5 10 15Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro

20 25 30Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp

35 40 45Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala

50 55 60Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met

65 70 75Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu

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

95 100 105Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala

110 115 120His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu

125 130 135Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu

140 145 150Cys Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr

155 160 165Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly

170 175 180Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser

185 190 195Gly Leu Leu Lys Trp Gln Gln Gly Phe Arg Ala Lys Ile Pro Gly

200 205 210Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly Tyr

215 220 225Leu Asn Arg Ile His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe

230 235 240Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser

245 250 255Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly

260 265 270Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu

275 280 285Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu His

290 295 300Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser

305 310 315Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gln Asn Leu Ser Gln

320 325 330Glu Gly

The information of 332 (2) SEQ ID NO:7:

(i) sequence signature:

(A) length: 166 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:7:Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr1 5 10 15Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala

20 25 30Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys

35 40 45Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala

50 55 60Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu

65 70 75Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro

80 85 90Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu

95 100 105Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser

110 115 120Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala

125 130 135Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg

140 145 150Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp

The information of 155 160 165Arg166 (2) SEQ ID NO:8:

(i) sequence signature:

(A) length: 328 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:8:Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu1 5 10 15Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro

20 25 30Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp

35 40 45Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala

50 55 60Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met

65 70 75Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu

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

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

110 115 120Asn 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

140 145 150Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu

155 160 165Thr Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr

170 175 180Asn Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys

185 190 195Trp Gln Gln Gly Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln

200 205 210Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly Tyr Leu Asn Arg Ile

215 220 225His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe Pro Gly Pro Ser

230 235 240Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser Gly Thr Ser Asp

245 250 255Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr Ser Pro Ser

260 265 270Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu Phe Pro Leu Pro

275 280 285Pro Thr Leu Pro Thr Pro Val Val Gln Leu His Pro Leu Leu Pro

290 295 300Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn

305 310 315Thr Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu Gly

The information of 320 325 328 (2) SEQ ID NO:9:

(i) sequence signature:

(A) length: 265 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:9:Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu1 5 10 15Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro

20 25 30Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp

35 40 45Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala

50 55 60Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met

65 70 75Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu

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

95 100 105Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala

110 115 120His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu

125 130 135Arg Gly Lys Asp Phe Trp Ile Val Gly Asp Lys Leu His Cys Leu

140 145 150Ser Gln Asn Tyr Trp Leu Trp Ala Ser Glu Val Ala Ala Gly Ile

155 160 165Gln Ser Gln Asp Ser Trp Ser Ala Glu Pro Asn Leu Gln Val Pro

170 175 180Gly Pro Asn Pro Arg Ile Pro Glu Gln Asp Thr Arg Thr Leu Glu

185 190 195Trp Asn Ser Trp Thr Leu Ser Trp Thr Leu Thr Gln Asp Pro Arg

200 205 210Ser Pro Gly His Phe Leu Arg Asn Ile Arg His Arg Leu Pro Ala

215 220 225Thr Gln Pro Pro Ala Trp Ile Phe Ser Phe Pro Asn Pro Ser Ser

230 235 240Tyr Trp Thr Val Tyr Ala Leu Pro Ser Ser Thr His Leu Ala His

245 250 255Pro Cys Gly Pro Ala Pro Pro Pro Ala Ser

The information of 260 265 (2) SEQ ID NO:10:

(i) sequence signature:

(A) length: 261 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:10:Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu1 5 10 15Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro

20 25 30Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp

35 40 45Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala

50 55 60Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met

65 70 75Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu

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

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

110 115 120Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Asp

125 130 135Phe Trp Ile Val Gly Asp Lys Leu His Cys Leu Ser Gln Asn Tyr

140 145 150Trp Leu Trp Ala Ser Glu Val Ala Ala Gly Ile Gln Ser Gln Asp

155 160 165Ser Trp Ser Ala Glu Pro Asn Leu Gln Val Pro Gly Pro Asn Pro

170 175 180Arg Ile Pro Glu Gln Asp Thr Arg Thr Leu Glu Trp Asn Ser Trp

185 190 195Thr Leu Ser Trp Thr Leu Thr Gln Asp Pro Arg Ser Pro Gly His

200 205 210Phe Leu Arg Asn Ile Arg His Arg Leu Pro Ala Thr Gln Pro Pro

215 220 225Ala Trp Ile Phe Ser Phe Pro Asn Pro Ser Ser Tyr Trp Thr Val

230 235 240Tyr Ala Leu Pro Ser Ser Thr His Leu Ala His Pro Cys Gly Pro

245 250 255Ala Pro Pro Pro Ala Ser

The information of 260 261 (2) SEQ ID NO:11:

(i) sequence signature:

(A) length: 7849 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CCCAGCCTCC TTTCTCTTGT TCCCTGGTCA TGCCTGCCTC CCTGTCTCCT 50 GTCTCTCCCT CCCACACACA CCCACTATCC TCCCAGCTAT CCCTACACCC 100 TCCTTCCTAA TCTTGGGAGA CATCTCGTCT GGCTGGACGG GAAAATTCCA 150 GGATCTAGGC CACACTTCTC AGCAGACATG CCCATCCTTG GGGAGGAGGA 200 ACAGGAGAGA GCCTGAGGAA GTTCTGGGGG ACAGGGGGAT GATGGGATCA 250 AGGTCAGGCC AGGAAGCCCC TGAGGACAGA GACTGTGGGG AGACTGGGAC 300 TGGGAAGAAA GCAAAGGAGC TAGAGCCAGG GCCAAAGGAA AAGGGGGGCC 350 AGCAGGGAGG TATTTGCGGG GGAGGTCCAG CAGCTGTCTT TCCTAAGACA 400 GGGACACATG GGCCTGGTTA TTCCTCTTGT CACATGTGGA ACGGTAGGAG 450 ATGGAAGACG GAGACAGAAC AAGCAAAGGA GGGCCCTGGG CACAGAGGTC 500 TGTGTGTGTA GCCATCCAAG CCACTGGACC CCAGCAGACG AGCACCTAAG 550 CTCAGGCTTA ACCCAGTGCA CGTGTGCGCA CATACATGTG CCCCGCACCT 600 GACAGTCCAC TCAACCCGTC CAAACCCTTT CCCCATAACA CCAACCCATA 650 ACAGGAGATT TCTCTCATGT GGGCAATATC CGTGTTCCCA CTTCGAAAGG 700 GGGAATGACA AGATAGGACT CCCTAGGGGA TTACAGAAAG AAAAGCAGGA 750 AAGCAAGCAT CCTGTTGGAT TTCAGCAGCA GGTATGATGT CCAGGGAAAA 800 GAAATTTGGA TAGCCAGGGA GTGAAAACCC CACCAATCTT AAACAAGACC 850 TCTGTGCTTC TTCCCCAGCA ACACAAATGT CCTGCCAGAT TCCTCCTGGA 900 AAAAAACTTC TGCTCCTGTC CCCCTCCAGG TCCCAGGTTG CCCATGTCCA 950 GGAAAAGATG GATCCCCCTA TCCAAATCTT CTCCGTGGTG TGTGTGGGTG 1000 GAGGAGTGGA CCCTGGTCCA GGCAGGGGCT CCAGGGAAGA GAAGGCGTCA 1050 CTTCCGGGGG CCTTCACCAG TGTCTGGTGG CTCCCTTCTC TGATTGGGCA 1100 GAAGTGGCCC AGGCAGGCGT ATGACCTGCT GCTGTGGAGG GGCTGTGCCC 1150 CACCGCCACA TGTCTTCCTA CCCATCTGCT CCCCAGAGGG CTGCCTGCTG 1200 TGCACTTGGG TCCTGGAGCC CTTCTCCACC CGGTGAGTGG CCAGCAGGGT 1250 GTGGGGTTAT GTGAGGGTAG AAAGGACAGC AAAGAGAAAT GGGCTCCCAG 1300 CTGGGGGAGG GGCAGGCAAA CTGGAACCTA CAGGCACTGA CCTTTGTCGA 1350 GAAGAGTGTA GCCTTCCCAG AATGGGAGGA GCAGGGCAGA GCAGGGGTAG 1400 GGGGTGGGGT GCTGGTTTCT GAGGGACTGA TCACTTACTT GGTGGAATAC 1450 AGCACAGCCC TGGCTGGCCC TAAGGAAAGG GGACATGAGC CCAGGGAGAA 1500 AATAAGAGAG GGAGCTGCAC TTAGGGCTTA GCAAACACAG TAGTAAGATG 1550 GACACAGCCC CAATCCCCAT TCTTAGCTGG TCATTCCTCG TTAGCTTAAG 1600 GTTCTGAATC TGGTGCTGGG GAAGCTGGGC CAGGCAAGCC AGGGCGCAAG 1650 GAGAGGGTAA TGGGAGGAGG GCCCACTCAT GTTGACAGAC CTACAGGAAA 1700 TCCCAATATT GAATCAGGTG CAAGCCTCTT TGCACAACTT GTGAAAGGAG 1750 GAGGAAGCCA TGTGGGGGGT CCTGTGAAGG AACCGGAAGG GGTTCTGCCA 1800 AGGGGGCAGG GAGGCAGGTG TGAGCTATGA GACAGATATG TTAGTGGGCG 1850 CCTAAGACAA GGTAAGCCCC TAAGGTGGGC ATCACCCAGC AGGTGCCCGT 1900 TCCTGGGCAG CTGGTCTCAG GAAGGAAGTC CCAGAACTGT TAGCCCATCT 1950 CTTGGCCTCA GATAATGGAG TATTTCAGGA CTTGGAGTCC AAAGAAAAGC 2000 TCCAGTGGCT TTATGTGTGG GGGTAGATAG GGAAAGAATA GAGGTTAATT 2050 TCTCCCATAC CGCCTTTTAA TCCTGACCTC TAGTGGTCCC AGTTACAGCT 2100 TTGTGCAGTT CCCCTCCCCA GCCCCACTCC CCACCGCAGA AGTTACCCCT 2150 CAACATATTG CGCCCGTTTG CCAGTTCCTC ACCCAGGCCC TGCATCCCAT 2200 TTTCCACTCT CTTCTCCAGG CTGAAGCCAC AATACTTTCC TTCTCTATCC 2250 CCATCCCAGA TTTTCTCTGA CCTAACAACC AAGGTTGCTC AGAATTTAAG 2300 GCTAATTAAG ATATGTGTGT ATACATATCA TGTCCTGCTG CTCTCAGCAG 2350 GGGTAGGTGG CACCAAATCC GTGTCCGATT CACTGAGGAG TCCTGACAAA 2400 AAGGAGACAC CATATGCTTT CTTGCTTTCT TTCTTTCTTT CTTTCTTTTT 2450 TTTTTTTTGA GACGGAGTTT CACTCTTATT GCCCAGGCTG GAGTGCAATG 2500 GTGCGATCTC GGCTCACCAC AAACCTCCGC CTCCCAGGTA CAAGCGATTC 2550 TCCTGTCTCA GCCTCCCAAG TAGCTTGGAT TACAGGCATG AGCCACCACA 2600 CCCTGCTAGT TTTTTTGTAT TTCGTAGAGC CGGGGTTTCA CCATGTTAGT 2650 GAGGCTGGTG GCGAACTCCT GACCTCAGGT GATCCACCCG CCTTGGACTC 2700 CCAAAGTGCT GGGATTACAG GCATGAGCCA CTGCACCCGG CACACCATAT 2750 GCTTTCATCA CAAGAAAATG TGAGAGAATT CAGGGCTTTG GCAGTTCCAG 2800 GCTGGTCAGC ATCTCAAGCC CTCCCCAGCA TCTGTTCACC CTGCCAGGCA 2850 GTCTCTTCCT AGAAACTTGG TTAAATGTTC ACTCTTCTTG CTACTTTCAG 2900 GATAGATTCC TCACCCTTGG CCCGCCTTTG CCCCACCCTA CTCTGCCCAG 2950 AAGTGCAAGA GCCTAAGCCG CCTCCATGGC CCCAGGAAGG ATTCAGGGGA 3000 GAGGCCCCAA ACAGGGAGCC ACGCCAGCCA GACACCCCGG CCAGAATGGA 3050 GCTGACTGGT GAGAACACAC CTGAGGGGCT AGGGCCATAT GGAAACATGA 3100 CAGAAGGGGA GAGAGAAAGG AGACACGCTG CAGGGGGCAG GAAGCTGGGG 3150 GAACCCATTC TCCCAAAAAT AAGGGGTCTG AGGGGTGGAT TCCCTGGGTT 3200 TCAGGTCTGG GTCCTGAATG GGAATTCCTG GAATACCAGC TGACAATGAT 3250 TTCCTCCTCA TCTTTCAACC TCACCTCTCC TCATCTAAGA ATTGCTCCTC 3300 GTGGTCATGC TTCTCCTAAC TGCAAGGCTA ACGCTGTCCA GCCCGGCTCC 3350 TCCTGCTTGT GACCTCCGAG TCCTCAGTAA ACTGCTTCGT GACTCCCATG 3400 TCCTTCACAG CAGACTGGTG AGAACTCCCA ACATTATCCC CTTTATCCGC 3450 GTAACTGGTA AGACACCCAT ACTCCCAGGA AGACACCATC ACTTCCTCTA 3500 ACTCCTTGAC CCAATGACTA TTCTTCCCAT ATTGTCCCCA CCTACTGATC 3550 ACACTCTCTG ACAAGAATTA TTCTTCACAA TACAGCCCGC ATTTAAAAGC 3600 TCTCGTCTAG AGATAGTACT CATGGAGGAC TAGCCTGCTT ATTAGGCTAC 3650 CATAGCTCTC TCTATTTCAG CTCCCTTCTC CCCCCACCAA TCTTTTTCAA 3700 CAGAGCCAGT GCCCAGAGGT TCACCCTTTG CCTACACCTG TCCTCCTGCC 3750 TGCTGTGGAC TTTAGCTTGG GAGAATGGAA AACCCAGATG GTAAGAAAGC 3800 CATCCCTAAC CTTGGCTTCC CTAAGTCCTG TCTTCAGTTT CCCACTGCTT 3850 CCCATGGATT CTCCAACATT CTTGAGCTTT TTAAAAATAT CTCACCTTCA 3900 GCTTGGCCAC CCTAACCCAA TCTACATTCA CCTATGATGA TAGCCTGTGG 3950 ATAAGATGAT GGCTTGCAGG TCCAATATGT GAATAGATTT GAAGCTGAAC 4000 ACCATGAAAA GCTGGAGAGA AATCGCTCAT GGCCATGCCT TTGACCTATT 4050 CCYGTTCAGT CTTCTTAAAT TGGCATGAAG AAGCAAGACT CATATGTCAT 4100 CCACAGATGA CACAAAGCTG GGAAGTACCA CTAAAATAAC AAAAGACTGA 4150 ATCAAGATTC AAATCACTGA AAGACTAGGT CAAAAACAAG GTGAAACAAC 4200 AGAGATATAA ACTTCTACAT GTGGGCCGGG GGCTCACGCC TGTAATCCCA 4250 GCACTTTGGG AGGCCGAGGC AGGCAGATCA CCTGAGGGCA GGAGTTTGAG 4300 AGCAGCCTGG CCAACATGGC GAAACCCCGT CTCTACTAAG AATACAAAAT 4350 TAGCCGGGCA TGGTAGTGCA TGCCTGTAAT CCCAGCTACT TGGAAGGCTG 4400 AAGCAGGAGA ATCCCTTGAA CCCAGGAGGT GGAGGTTGTA GTGAGCTGAG 4450 ATCATGCCAA TGCACTCCAG CCTGGGTGAC AAGAGCAAAA CTCCGTCTCA 4500 AAAAGAAAAA AAAATTCTAC ATGTGTAAAT TAATGAGTAA AGTCCTATTC 4550 CAGCTTTCAG GCCACAATGC CCTGCTTCCA TCATTTAAGC CTCTGGCCCT 4600 AGCACTTCCT ACGAAAAGGA TCTGAGAGAA TTAAATTGCC CCCAAACTTA 4650 CCATGTAACA TTACTGAAGC TGCTATTCTT AAAGCTAGTA ATTCTTGTCT 4700 GTTTGATGTT TAGCATCCCC ATTGTGGAAA TGCTCGTACA GAACTCTATT 4750 CCGAGTGGAC TACACTTAAA TATACTGGCC TGAACACCGG ACATCCCCCT 4800 GAAGACATAT GCTAATTTAT TAAGAGGGAC CATATTAAAC TAACATGTGT 4850 CTAGAAAGCA GCAGCCTGAA CAGAAAGAGA CTAGAAGCAT GTTTTATGGG 4900 CAATAGTTTA AAAAACTAAA ATCTATCCTC AAGAACCCTA GCGTCCCTTC 4950 TTCCTTCAGG ACTGAGTCAG GGAAGAAGGG CAGTTCCTAT GGGTCCCTTC 5000 TAGTCCTTTC TTTTCATCCT TATGATCATT ATGGTAGAGT CTCATACCTA 5050 CATTTAGTTT ATTTATTATT ATTATTTGAG ACGGAGTCTC ACTCTATCCC 5100 CCAGGCTGGA GTGCAGTGGC ATGATCTCAA CTCACTGCAA CCTCAGCCTC 5150 CCGGATTCAA GCGATTCTCC TGCCTCAGTC TCCCAAGTAG CTGGGATTAC 5200 AGGTGCCCAC CACCATGCCC AGCTAATTTT TGTATTTTTG GTAGAGATGG 5250 GGTTTCACCA TGTTGGCCAG GCTGATCTTG AACTCCTGAC CTCAGGTGAT 5300 CCACCTGCCT CAGCCTCCCA AAGTGCTGGG ATTACAGGCG TGAGCCACTG 5350 CACCCAGCCT TCATTCAGTT TAAAAATCAA ATGATCCTAA GGTTTTGCAG 5400 CAGAAAGAGT AAATTTGCAG CACTAGAACC AAGAGGTAAA AGCTGTAACA 5450 GGGCAGATTT CAGCAACGTA AGAAAAAAGG AGCTCTTCTC ACTGAAACCA 5500 AGTGTAAGAC CAGGCTGGAC TAGAGGACAC GGGAGTTTTT GAAGCAGAGG 5550 CTGATGACCA GCTGTCGGGA GACTGTGAAG GAATTCCTGC CCTGGGTGGG 5600 ACCTTGGTCC TGTCCAGTTC TCAGCCTGTA TGATTCACTC TGCTGGCTAC 5650 TCCTAAGGCT CCCCACCCGC TTTTAGTGTG CCCTTTGAGG CAGTGCGCTT 5700 CTCTCTTCCA TCTCTTTCTC AGGAGGAGAC CAAGGCACAG GACATTCTGG 5750 GAGCAGTGAC CCTTCTGCTG GAGGGAGTGA TGGCAGCACG GGGACAACTG 5800 GGACCCACTT GCCTCTCATC CCTCCTGGGG CAGCTTTCTG GACAGGTCCG 5850 TCTCCTCCTT GGGGCCCTGC AGAGCCTCCT TGGAACCCAG GTAAGTCCCC 5900 AGTCAAGGGA TCTGTAGAAA CTGTTCTTTT CTGACTCAGT CCCACTAGAA 5950 GACCTGAGGG AAGAAGGGCT CTTCCAGGGA GCTCAAGGGC AGAAGAGCTG 6000 ATCTACTAAG AGTGCTCCCT GCCAGCCACA ATGCCTGGGT ACTGGCATCC 6050 TGTCTTTCCT ACTTAGACAA GGGAGGCCTG AGATCTGGCC CTGGTGTTTG 6100 GCCTCAGGAC CATCCTCTGC CCTCAGCTTC CTCCACAGGG CAGGACCACA 6150 GCTCACAAGG ATCCCAATGC CATCTTCCTG AGTTTCCAAC ACCTGCTCCG 6200 AGGAAAGGTG CGTTTCCTGA TGCTTGTAGG AGGGTCCACC CTCTGCGTCA 6250 GGCGGGCCCC ACCCACCACA GCTGTCCCCA GCAGAACCTC TCTAGTCCTC 6300 ACACTGAACG AGCTCCCAAA CAGGACTTCT GGATTGTTGG AGACAAACTT 6350 CACTGCCTCA GCCAGAACTA CTGGCTCTGG GCTTCTGAAG TGGCAGCAGG 6400 GATTCAGAGC CAAGATTCCT GGTCTGCTGA ACCAAACCTC CAGGTCCCTG 6450 GACCAAATCC CCGGATACCT GAACAGGATA CACGAACTCT TGAATGGAAC 6500 TCGTGGACTC TTTCCTGGAC CCTCACGCAG GACCCTAGGA GCCCCGGACA 6550 TTTCCTCAGG AACATCAGAC ACAGGCTCCC TGCCACCCAA CCTCCAGCCT 6600 GGATATTCTC CTTCCCCAAC CCATCCTCCT ACTGGACAGT ATACGCTCTT 6650 CCCTCTTCCA CCCACCTTGC CCACCCCTGT GGTCCAGCTC CACCCCCTGC 6700 TTCCTGACCC TTCTGCTCCA ACGCCCACCC CTACCAGCCC TCTTCTAAAC 6750 ACATCCTACA CCCACTCCCA GAATCTGTCT CAGGAAGGGT AAGGTTCTCA 6800 GACACTGCCG ACATCAGCAT TGTCTCATGT ACAGCTCCCT TCCCTGCAGG 6850 GCGCCCCTGG GAGACAACTG GACAAGATTT CCTACTTTCT CCTGAAACCC 6900 AAAGCCCTGG TAAAAGGGAT ACACAGGACT GAAAAGGGAA TCATTTTTCA 6950 CTGTACATTA TAAACCTTCA GAAGCTATTT TTTTAAGCTA TCAGCAATAC 7000 TCATCAGAGC AGCTAGCTCT TTGGTCTATT TTCTGCAGAA ATTTGCAACT 7050 CACTGATTCT CTACATGCTC TTTTTCTGTG ATAACTCTGC AAAGGCCTGG 7100 GCTGGCCTGG CAGTTGAACA GAGGGAGAGA CTAACCTTGA GTCAGAAAAC 7150 AGAGAAAGGG TAATTTCCTT TGCTTCAAAT TCAAGGCCTT CCAACGCCCC 7200 CATCCCCTTT ACTATCATTC TCAGTGGGAC TCTGATCCCA TATTCTTAAC 7250 AGATCTTTAC TCTTGAGAAA TGAATAAGCT TTCTCTCAGA AATGCTGTCC 7300 CTATACACTA GACAAAACTG AGCCTGTATA AGGAATAAAT GGGAGCGCCG 7350 AAAAGCTCCC TAAAAAGCAA GGGAAAGATG TTCTTCGAGG GTGGCAATAG 7400 ATCCCCCTCA CCCTGCCACC CCAAACAAAA AAGCTAACAG GAAGCCTTGG 7450 AGAGCCTCAC ACCCCAGGTA AGGCTGTGTA GACAGTTCAG TAAAGACAGG 7500 ACCTGGATGT GACAGCTGAG CAAACAGCTA GAGCTTTGGC AGCTCAGCAG 7550 GAGGCTTTGC CAGGCATGGA CGCCTGCCTC CCTCCTGTGG AGGTCAGGAG 7600 GAAGTGCAGG AAGTGGCATG AGTCAGGCTC CTTGAGCTCA CACAGCAGGA 7650 GAACAAGTAC AAGTCAAGTA CAAGTTGAAG GCTCATTTCC CAGTTCCCGC 7700 AAATGCATCT AAAAAGCAGC TCTGTGTGAC CACCATAAAC TCTGCTAGGG 7750 GATCTCTAAA AAGGAGTCAG GCTTATGGGG CTTTGCAAAT AAGTGCTGCC 7800 TTGGTGCTCA GGAAAAGGTT TGTGTTGCAC AAAACACAAA TTCCACTGC 7849 (2) SEQ ID NO: 12 information about: ...

(i) sequence signature:

(A) length: 1443 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ, ID, NO:12:GAGTCCTTGG, CCCACCTCTC, TCCCACCCGA, CTCTGCCGAA, AGAAGCACAG, 50AAGCTCAAGC, CGCCTCCATG, GCCCCAGGAA, AGATTCAGGG, GAGAGGCCCC, 100ATACAGGGAG, CCACTTCAGT, TAGACACCCT, GGCCAGAATG, GAGCTGACTG, 150ATTTGCTCCT, GGCGGCCATG, CTTCTTGCAG, TGGCAAGACT, AACTCTGTCC, 200AGCCCCGTAG, CTCCTGCCTG, TGACCCCAGA, CTCCTAAATA, AACTGCTGCG, 250TGACTCCCAC, CTCCTTCACA, GCCGACTGAG, TCAGTGTCCC, GACGTCGACC, 300CTTTGTCTAT, CCCTGTTCTG, CTGCCTGCTG, TGGACTTTAG, CCTGGGAGAA, 350TGGAAAACCC, AGACGGAACA, GAGCAAGGCA, CAGGACATTC, TAGGGGCAGT, 400GTCCCTTCTA, CTGGAGGGAG, TGATGGCAGC, ACGAGGACAG, TTGGAACCCT, 450CCTGCCTCTC, ATCCCTCCTG, GGACAGCTTT, CTGGGCAGGT, TCGCCTCCTC, 500TTGGGGGCCC, TGCAGGGCCT, CCTAGGAACC, CAGGGCAGGA, CCACAGCTCA, 550CAAGGACCCC, AATGCCCTCT, TCTTGAGCTT, GCAACAACTG, CTTCGGGGAA, 600AGGTGCGCTT, CCTGCTTCTG, GTAGAAGGTC, CCACCCTCTG, TGTCAGACGG, 650ACCCTGCCAA, CCACAGCTGT, CCCAAGCAGT, ACTTCTCAAC, TCCTCACACT, 700AAACAAGTTC, CCAAACAGGA, CTTCTGGATT, GTTGGAGACG, AACTTCAGTG, 750TCACAGCCAG, AACTGCTGGC, CCTGGACTTC, TGAGCAGGCT, TCAGGGATTC, 800AGAGTCAAGA, TTACTCCTGG, TCAGCTAAAT, CAAACCTCCA, GGTCCCCAGT, 850CCAAATCTCT, GGATACCTGA, ACAGGACACA, CGGACCTGTG, AATGGAACTC, 900ATGGGCTCTT, TGCTGGAACC, TCACTTCAGA, CCCTGGAAGC, CTCAGACATC, 950TCGCCCCGAG, CTTTCAACAA, AGGCTCCCTG, GCATTCAACC, TCCAGGGTGG, 1000ACTTCCTCCT, TCTCCAAGCC, TTGCTCCTGA, TGGACACACA, CCCTTCCCTC, 1050CTTCACCTGC, CTTGCCCACC, ACCCATGGAT, CTCCACCCCA, GCTCCACCCC, 1100CTGTTTCCTG, ACCCTTCCAC, CACCATGCCT, AACTCTACCG, CCCCTCATCC, 1150AGTCACAATG, TACCCTCATC, CCAGGAATTT, GTCTCAGGAA, ACATAGCGCG, 1200GGCACTGGCC, CAGTGAGCGT, CTGCAGCTTC, TCTCGGGGAC, AAGCTTCCCC, 1250AGGAAGGCTG, AGAGGCAGCT, GCATCTGCTC, CAGATGTTCT, GCTTTCACCT, 1300AAAAGGCCCT, GGGGAAGGGA, TACACAGCAC, TGGAGATTGT, AAAATTTTAG, 1350GAGCTATTTT, TTTTTAACCT, ATCAGCAATA, TTCATCAGAG, CAGCTAGCGA, 1400TCTTTGGTCT, ATTTTCGGTA, TAAATTTGAA, AATCACTAAT, TCT, 1443, (2) SEQ, ID, the information of NO:13:

(i) sequence signature:

(A) length: 352 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:13:Met Glu Leu Thr Asp Leu Leu Leu Ala Ala Met Leu Leu Ala Val1 5 10 15Ala Arg Leu Thr Leu Ser Ser Pro Val Ala Pro Ala Cys Asp Pro

20 25 30Arg Leu Leu Asn Lys Leu Leu Arg Asp Ser His Leu Leu His Ser

35 40 45Arg Leu Ser Gln Cys Pro Asp Val Asp Pro Leu Ser Ile Pro Val

50 55 60Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln

65 70 75Thr Glu Gln Ser Lys Ala Gln Asp Ile Leu Gly Ala Val Ser Leu

80 85 90Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Glu Pro Ser

95 100 105Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu

110 115 120Leu Leu Gly Ala Leu Gln Gly Leu Leu Gly Thr Gln Gly Arg Thr

125 130 135Thr Ala His Lys Asp Pro Asn Ala Leu Phe Leu Ser Leu Gln Gln

140 145 150Leu Leu Arg Gly Lys Val Arg Phe Leu Leu Leu Val Glu Gly Pro

155 160 165Thr Leu Cys Val Arg Arg Thr Leu Pro Thr Thr Ala Val Pro Ser

170 175 180Ser Thr Ser Gln Leu Leu Thr Leu Asn Lys Phe Pro Asn Arg Thr

185 190 195Ser Gly Leu Leu Glu Thr Asn Phe Ser Val Thr Ala Arg Thr Ala

200 205 210Gly Pro Gly Leu Leu Ser Arg Leu Gln Gly Phe Arg Val Lys Ile

215 220 225Thr Pro Gly Gln Leu Asn Gln Thr Ser Arg Ser Pro Val Gln Ile

230 235 240Ser Gly Tyr Leu Asn Arg Thr His Gly Pro Val Asn Gly Thr His

245 250 255Gly Leu Phe Ala Gly Thr Ser Leu Gln Thr Leu Glu Ala Ser Asp

260 265 270Ile Ser Pro Gly Ala Phe Asn Lys Gly Ser Leu Ala Phe Asn Leu

275 280 285Gln Gly Gly Leu Pro Pro Ser Pro Ser Leu Ala Pro Asp Gly His

290 295 300Thr Pro Phe Pro Pro Ser Pro Ala Leu Pro Thr Thr His Gly Ser

305 310 315Pro Pro Gln Leu His Pro Leu Phe Pro Asp Pro Ser Thr Thr Met

320 325 330Pro Asn Ser Thr Ala Pro His Pro Val Thr Met Tyr Pro His Pro

335 340 345Arg Asn Leu Ser Gln Glu Thr

The information of 350 352 (2) SEQ ID NO:14:

(i) sequence signature:

(A) length: 1536 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ, ID, NO:14:GAGTCCTTGG, CCCACCTCTC, TCCCACCCGA, CTCTGCCGAA, AGAAGCACAG, 50AAGCTCAAGC, CGCCTCCATG, GCCCCAGGAA, AGATTCAGGG, GAGAGGCCCC, 100ATACAGGGAG, CCACTTCAGT, TAGACACCCT, GGCCAGAATG, GAGCTGACTG, 150ATTTGCTCCT, GGCGGCCATG, CTTCTTGCAG, TGGCAAGACT, AACTCTGTCC, 200AGCCCCGTAG, CTCCTGCCTG, TGACCCCAGA, CTCCTAAATA, AACTGCTGCG, 250TGACTCCCAC, CTCCTTCACA, GCCGACTGAG, TCAGTGTCCC, GACGTCGACC, 300CTTTGTCTAT, CCCTGTTCTG, CTGCCTGCTG, TGGACTTTAG, CCTGGGAGAA, 350TGGAAAACCC, AGACGGAACA, GAGCAAGGCA, CAGGACATTC, TAGGGGCAGT, 400GTCCCTTCTA, CTGGAGGGAG, TGATGGCAGC, ACGAGGACAG, TTGGAACCCT, 450CCTGCCTCTC, ATCCCTCCTG, GGACAGCTTT, CTGGGCAGGT, TCGCCTCCTC, 500TTGGGGGCCC, TGCAGGGCCT, CCTAGGAACC, CAGCTTCCTC, TACAGGGCAG, 550GACCACAGCT, CACAAGGACC, CCAATGCCCT, CTTCTTGAGC, TTGCAACAAC, 600TGCTTCGGGG, AAAGGTGCGC, TTCCTGCTTC, TGGTAGAAGG, TCCCACCCTC, 650TGTGTCAGAC, GGACCCTGCC, AACCACAGCT, GTCCCAAGCA, GTACTTCTCA, 700ACTCCTCACA, CTAAACAAGT, TCCCAAACAG, GACTTCTGGA, TTGTTGGAGA, 750CGAACTTCAG, TGTCACAGCC, AGAACTGCTG, GCCCTGGACT, TCTGAGCAGG, 800CTTCAGGGAT, TCAGAGTCAA, GATTACTCCT, GGTCAGCTAA, ATCAAACCTC, 850CAGGTCCCCA, GTCCAAATCT, CTGGATACCT, GAACAGGACA, CACGGACCTG, 900TGAATGGAAC, TCATGGGCTC, TTTGCTGGAA, CCTCACTTCA, GACCCTGGAA, 950GCCTCAGACA, TCTCGCCCGG, AGCTTTCAAC, AAAGGCTCCC, TGGCATTCAA, 1000CCTCCAGGGT, GGACTTCCTC, CTTCTCCAAG, CCTTGCTCCT, GATGGACACA, 1050CACCCTTCCC, TCCTTCACCT, GCCTTGCCCA, CCACCCATGG, ATCTCCACCC, 1100CAGCTCCACC, CCCTGTTTCC, TGACCCTTCC, ACCACCATGC, CTAACTCTAC, 1150CGCCCCTCAT, CCAGTCACAA, TGTACCCTCA, TCCCAGGAAT, TTGTCTCAGG, 1200AAACATAGCG, CGGGCACTGG, CCCAGTGAGC, GTCTGCAGCT, TCTCTCGGGG, 1250ACAAGCTTCC, CCAGGAAGGC, TGAGAGGCAG, CTGCATCTGC, TCCAGATGTT, 1300CTGCTTTCAC, CTAAAAGGCC, CTGGGGAAGG, GATACACAGC, ACTGGAGATT, 1350GTAAAATTTT, AGGAGCTATT, TTTTTTTAAC, CTATCAGCAA, TATTCATCAG, 1400AGCAGCTAGC, GATCTTTGGT, CTATTTTCGG, TATAAATTTG, AAAATCACTA, 1450AAAAAAAAAA, AAAAAAAAAA, AAAAAAAAAA, AAAAAAAAAA, AAAAAAAAAA, 1500AAAAAAAAAA, AAAAAAAAAA, AAAAAAAAAA, AAAAAA, 1536, (2) SEQ, ID, the information of NO:15:

(i) sequence signature:

(A) length: 356 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:15:Met Glu Leu Thr Asp Leu Leu Leu Ala Ala Met Leu Leu Ala Val1 5 10 15Ala Arg Leu Thr Leu Ser Ser Pro Val Ala Pro Ala Cys Asp Pro

20 25 30Arg Leu Leu Asn Lys Leu Leu Arg Asp Ser His Leu Leu His Ser

35 40 45Arg Leu Ser Gln Cys Pro Asp Val Asp Pro Leu Ser Ile Pro Val

50 55 60Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln

65 70 75Thr Glu Gln Ser Lys Ala Gln Asp Ile Leu Gly Ala Val Ser Leu

80 85 90Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Glu Pro Ser

95 100 105Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu

110 115 120Leu Leu Gly Ala Leu Gln Gly Leu Leu Gly Thr Gln Leu Pro Leu

125 130 135Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Leu Phe Leu

140 145 150Ser Leu Gln Gln Leu Leu Arg Gly Lys Val Arg Phe Leu Leu Leu

155 160 165Val Glu Gly Pro Thr Leu Cys Val Arg Arg Thr Leu Pro Thr Thr

170 175 180Ala Val Pro Ser Ser Thr Ser Gln Leu Leu Thr Leu Asn Lys Phe

185 190 195Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Ser Val Thr

200 205 210Ala Arg Thr Ala Gly Pro Gly Leu Leu Ser Arg Leu Gln Gly Phe

215 220 225Arg Val Lys Ile Thr Pro Gly Gln Leu Asn Gln Thr Ser Arg Ser

230 235 240 Pro Val Gln Ile Ser Gly Tyr Leu Asn Arg Thr His Gly Pro Val

245 250 255Asn Gly Thr His Gly Leu Phe Ala Gly Thr Ser Leu Gln Thr Leu

260 265 270Glu Ala Ser Asp Ile Ser Pro Gly Ala Phe Asn Lys Gly Ser Leu

275 280 285Ala Phe Asn Leu Gln Gly Gly Leu Pro Pro Ser Pro Ser Leu Ala

290 295 300Pro Asp Gly His Thr Pro Phe Pro Pro Ser Pro Ala Leu Pro Thr

305 310 315Thr His Gly Ser Pro Pro Gln Leu His Pro Leu Phe Pro Asp Pro

320 325 330Ser Thr Thr Met Pro Asn Ser Thr Ala Pro His Pro Val Thr Met

335 340 345Tyr Pro His Pro Arg Asn Leu Ser Gln Glu Thr

The information of 350 355 356 (2) SEQ ID NO:16:

(i) sequence signature:

(A) length: 241 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:16:Ser Pro Val Ala Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu1 5 10 15Leu Arg Asp Ser His Leu Leu His Ser Arg Leu Ser Gln Cys Pro

20 25 30Asp Val Asp Pro Leu Ser Ile Pro Val Leu Leu Pro Ala Val Asp

35 40 45Phe Ser Leu Gly Glu Trp Lys Thr Gln Thr Glu Gln Ser Lys Ala

50 55 60Gln Asp Ile Leu Gly Ala Val Ser Leu Leu Leu Glu Gly Val Met

65 70 75Ala Ala Arg Gly Gln Leu Glu Pro Ser Cys Leu Ser Ser Leu Leu

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

95 100 105Gly Leu Leu Gly Thr Gln Leu Pro Leu Gln Gly Arg Thr Thr Ala

110 115 120His Lys Asp Pro Asn Ala Leu Phe Leu Ser Leu Gln Gln Leu Leu

125 130 135Arg Gly Lys Asp Phe Trp Ile Val Gly Asp Glu Leu Gln Cys His

140 145 150Ser Gln Asn Cys Trp Pro Trp Thr Ser Glu Gln Ala Ser Gly Ile

155 160 165Gln Ser Gln Asp Tyr Ser Trp Ser Ala Lys Ser Asn Leu Gln Val

170 175 180Pro Ser Pro Asn Leu Trp Ile Pro Glu Gln Asp Thr Arg Thr Cys

185 190 195Glu Trp Asn Ser Trp Ala Leu Cys Trp Asn Leu Thr Ser Asp Pro

200 205 210Gly Ser Leu Arg His Leu Ala Arg Ser Phe Gln Gln Arg Leu Pro

215 220 225Gly Ile Gln Pro Pro Gly Trp Thr Ser Ser Phe Ser Lys Pro Cys

The information of 230 235 240Ser241 (2) SEQ ID NO:17:

(i) sequence signature:

(A) length: 335 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:17:Ser Pro Val Ala Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu1 5 10 15Leu Arg Asp Ser His Leu Leu His Ser Arg Leu Ser Gln Cys Pro

20 25 30Asp Val Asp Pro Leu Ser Ile Pro Val Leu Leu Pro Ala Val Asp

35 40 45Phe Ser Leu Gly Glu Trp Lys Thr Gln Thr Glu Gln Ser Lys Ala

50 55 60Gln Asp Ile Leu Gly Ala Val Ser Leu Leu Leu Glu Gly Val Met

65 70 75Ala Ala Arg Gly Gln Leu Glu Pro Ser Cys Leu Ser Ser Leu Leu

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

95 100 105Gly Leu Leu Gly Thr Gln Leu Pro Leu Gln Gly Arg Thr Thr Ala

110 115 120His Lys Asp Pro Asn Ala Leu Phe Leu Ser Leu Gln Gln Leu Leu

125 130 135Arg Gly Lys Val Arg Phe Leu Leu Leu Val Glu Gly Pro Thr Leu

140 145 150Cys Val Arg Arg Thr Leu Pro Thr Thr Ala Val Pro Ser Ser Thr

155 160 165Ser Gln Leu Leu Thr Leu Asn Lys Phe Pro Asn Arg Thr Ser Gly

170 175 180Leu Leu Glu Thr Asn Phe Ser Val Thr Ala Arg Thr Ala Gly Pro

185 190 195Gly Leu Leu Ser Arg Leu Gln Gly Phe Arg Val Lys Ile Thr Pro

200 205 210Gly Gln Leu Asn Gln Thr Ser Arg Ser Pro Val Gln Ile Ser Gly

215 220 225Tyr Leu Asn Arg Thr His Gly Pro Val Asn Gly Thr His Gly Leu

230 235 240Phe Ala Gly Thr Ser Leu Gln Thr Leu Glu Ala Ser Asp Ile Ser

245 250 255Pro Gly Ala Phe Asn Lys Gly Ser Leu Ala Phe Asn Leu Gln Gly

260 265 270Gly Leu Pro Pro Ser Pro Ser Leu Ala Pro Asp Gly His Thr Pro

275 280 285Phe Pro Pro Ser Pro Ala Leu Pro Thr Thr His Gly Ser Pro Pro

290 295 300Gln Leu His Pro Leu Phe Pro Asp Pro Ser Thr Thr Met Pro Asn

305 310 315Ser Thr Ala Pro His Pro Val Thr Met Tyr Pro His Pro Arg Asn

320 325 330Leu Ser Gln Glu Thr

The information of 335 (2) SEQ ID NO:18:

(i) sequence signature:

(A) length: 332 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:18:Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu1 5 10 15Leu Arg Asp Ser His Val Leu His Gly Arg Leu Ser Gln Cys Pro

20 25 30Asp Ile Asn Pro Leu Ser Thr Pro Val Leu Leu Pro Ala Val Asp

35 40 45Phe Thr Leu Gly Glu Trp Lys Thr Gln Thr Glu Gln Thr Lys Ala

50 55 60Gln Asp Val Leu Gly Ala Thr Thr Leu Leu Leu Glu Ala Val Met

65 70 75Thr Ala Arg Gly Gln Val Gly Pro Pro Cys Leu Ser Ser Leu Leu

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

95 100 105Asp Leu Leu Gly Met Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala

110 115 120His Lys Asp Pro Ser Ala Ile Phe Leu Asn Phe Gln Gln Leu Leu

125 130 135Arg Gly Lys Val Arg Phe Leu Leu Leu Val Val Gly Pro Ser Leu

140 145 150Cys Ala Lys Arg Ala Pro Pro Ala Ile Ala Val Pro Ser Ser Thr

155 160 165Ser Pro Phe His Thr Leu Asn Lys Leu Pro Asn Arg Thr Ser Gly

170 175 180Leu Leu Glu Thr Asn Ser Ser Ile Ser Ala Arg Thr Thr Gly Ser

185 190 195Gly Phe Leu Lys Arg Leu Gln Ala Phe Arg Ala Lys Ile Pro Gly

200 205 210Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly His

215 220 225Gln Asn Gly Thr His Gly Pro Leu Ser Gly Ile His Gly Leu Phe

230 235 240Pro Gly Pro Gln Pro Gly Ala Leu Gly Ala Pro Asp Ile Pro Pro

245 250 255Ala Thr Ser Gly Met Gly Ser Arg Pro Thr Tyr Leu Gln Pro Gly

260 265 270Glu Ser Pro Ser Pro Ala His Pro Ser Pro Gly Arg Tyr Thr Leu

275 280 285Phe Ser Pro Ser Pro Thr Ser Pro Ser Pro Thr Val Gln Leu Gln

290 295 300Pro Leu Leu Pro Asp Pro Ser Ala Ile Thr Pro Asn Ser Thr Ser

305 310 315Pro Leu Leu Phe Ala Ala His Pro His Phe Gln Asn Leu Ser Gln

320 325 330Glu Glu

The information of 332 (2) SEQ ID NO:19:

(i) sequence signature:

(A) length: 1026 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ, ID, NO:19:AGCCCGGCTC, CTCCTGCCTG, TGACCCCCGA, CTCCTAAATA, AACTGCTTCG, 50TGACTCCCAT, GTCCTTCACG, GCAGACTGAG, CCAGTGCCCA, GACATTAACC, 100CTTTGTCCAC, ACCTGTCCTG, CTGCCTGCTG, TGGACTTCAC, CTTGGGAGAA, 150TGGAAAACCC, AGACGGAGCA, GACAAAGGCA, CAGGATGTCC, TGGGAGCCAC, 200AACCCTTCTG, CTGGAGGCAG, TGATGACAGC, ACGGGGACAA, GTGGGACCCC, 250CTTGCCTCTC, ATCCCTGCTG, GTGCAGCTTT, CTGGACAGGT, TCGCCTCCTC, 300CTCGGGGCCC, TGCAGGACCT, CCTTGGAATG, CAGCTTCCTC, CACAGGGAAG, 350GACCACAGCT, CACAAGGATC, CCAGTGCCAT, CTTCCTGAAC, TTCCAACAAC, 400TGCTCCGAGG, AAAGGTGCGT, TTCCTGCTCC, TTGTAGTGGG, GCCCTCCCTC, 450TGTGGCAAGA, GGGCCCCACC, CGCCATAGCT, GTCCCGAGCA, GCACCTCTCC, 500ATTCCACACA, CTGAACAAGC, TCCCAAACAG, GACCTCTGGA, TTGTTGGAGA, 550CAAACTCCAG, TATCTCAGCC, AGAACTACTG, GCTCTGGATT, TCTCAAGAGG, 600CTGCAGGCAT, TCAGAGCCAA, GATTCCTGGT, CTGCTGAACC, AAACCTCCAG, 650GTCCCTAGAC, CAAATCCCTG, GACACCAGAA, TGGGACACAC, GGACCCTTGA, 700GTGGAATTCA, TGGACTCTTT, CCTGGACCCC, AACCCGGGGC, CCTCGGAGCT, 750CCAGACATTC, CTCCAGCAAC, TTCAGGCATG, GGCTCCCGGC, CAACCTACCT, 800CCAGCCTGGA, GAGTCTCCTT, CCCCAGCTCA, CCCTTCTCCT, GGACGATACA, 850CTCTCTTCTC, TCCTTCACCC, ACCTCGCCCT, CCCCCACAGT, CCAGCTCCAG, 900CCTCTGCTTC, CTGACCCCTC, TGCGATCACA, CCCAACTCTA, CCAGTCCTCT, 950TCTATTTGCA, GCTCACCCTC, ATTTCCAGAA, CCTGTCTCAG, GAAGAGTAAG, 1000GTGCTCAGAC, CCTGCCAACT, TCAGCA, 1026, (2) SEQ, ID, the information of NO:20:

(i) sequence signature:

(A) length: 1014 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ, ID, NO:20:AGCCCGGCTC, CTCCTGCCTG, TGACCCCCGA, CTCCTAAATA, AACTGCTTCG, 50TGACTCCCAT, GTCCTTCACG, GCAGACTGAG, CCAGTGCCCA, GACATTAACC, 100CTTTGTCCAC, ACCTGTCCTG, CTGCCTGCTG, TGGACTTCAC, CTTGGGAGAA, 150TGGAAAACCC, AGACGGAGCA, GACAAAGGCA, CAGGATGTCC, TGGGAGCCAC, 200AACCCTTCTG, CTGGAGGCAG, TGATGACAGC, ACGGGGACAA, GTGGGACCCC, 250CTTGCCTCTC, ATCCCTGCTG, GTGCAGCTTT, CTGGACAGGT, TCGCCTCCTC, 300CTCGGGGCCC, TGCAGGACCT, CCTTGGAATG, CAGGGAAGGA, CCACAGCTCA, 350CAAGGATCCC, AGTGCCATCT, TCCTGAACTT, CCAACAACTG, CTCCGAGGAA, 400AGGTGCGTTT, CCTGCTCCTT, GTAGTGGGGC, CCTCCCTCTG, TGCCAAGAGG, 450GCCCCACCCG, CCATAGCTGT, CCCGAGCAGC, ACCTCTCCAT, TCCACACACT, 500GAACAAGCTC, CCAAACAGGA, CCTCTGGATT, GTTGGAGACA, AACTCCAGTA, 550TCTCAGCCAG, AACTACTGGC, TCTGGATTTC, TCAAGAGGCT, GCAGGCATTC, 600AGAGCCAAGA, TTCCTGGTCT, GCTGAACCAA, ACCTCCAGGT, CCCTAGACCA, 650AATCCCTGGA, CACCAGAATG, GGACACACGG, ACCCTTGAGT, GGAATTCATG, 700GACTCTTTCC, TGGACCCCAA, CCCGGGGCCC, TCGGAGCTCC, AGACATTCCT, 750CCAGCAACTT, CAGGCATGGG, CTCCCGGCCA, ACCTACCTCC, AGCCTGGAGA, 800GTCTCCTTCC, CCAGCTCACC, CTTCTCCTGG, ACGATACACT, CTCTTCTCTC, 850CTTCACCCAC, CTCGCCCTCC, CCCACAGTCC, AGCTCCAGCC, TCTGCTTCCT, 900GACCCCTCTG, CGATCACACC, CAACTCTACC, AGTCCTCTTC, TATTTGCAGC, 950TCACCCTCAT, TTCCAGAACC, TGTCTCAGGA, AGAGTAAGGT, GCTCAGACCC, 1000TGCCAACTTC, AGCA, 1014

(2) information of SEQ ID NO:21:

(i) sequence signature:

(A) length: 328 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:21:Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu1 5 10 15Leu Arg Asp Ser His Val Leu His Gly Arg Leu Ser Gln Cys Pro

20 25 30Asp Ile Asn Pro Leu Ser Thr Pro Val Leu Leu Pro Ala Val Asp

35 40 45Phe Thr Leu Gly Glu Trp Lys Thr Gln Thr Glu Gln Thr Lys Ala

50 55 60Gln Asp Val Leu Gly Ala Thr Thr Leu Leu Leu Glu Ala Val Met

65 70 75Thr Ala Arg Gly Gln Val Gly Pro Pro Cys Leu Ser Ser Leu Leu

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

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

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

125 130 135Arg Phe Leu Leu Leu Val Val Gly Pro Ser Leu Cys Ala Lys Arg

140 145 150Ala Pro Pro Ala Ile Ala Val Pro Ser Ser Thr Ser Pro Phe His

155 160 165Thr Leu Asn Lys Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr

170 175 180Asn Ser Ser Ile Ser Ala Arg Thr Thr Gly Ser Gly Phe Leu Lys

185 190 195Arg Leu Gln Ala Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln

200 205 210Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly His Gln Asn Gly Thr

215 220 225His Gly Pro Leu Ser Gly Ile His Gly Leu Phe Pro Gly Pro Gln

230 235 240Pro Gly Ala Leu Gly Ala Pro Asp Ile Pro Pro Ala Thr Ser Gly

245 250 255Met Gly Ser Arg Pro Thr Tyr Leu Gln Pro Gly Glu Ser Pro Ser

260 265 270Pro Ala His Pro Ser Pro Gly Arg Tyr Thr Leu Phe Ser Pro Ser

275 280 285Pro Thr Ser Pro Ser Pro Thr Val Gln Leu Gln Pro Leu Leu Pro

290 295 300Asp Pro Ser Ala Ile Thr Pro Asn Ser Thr Ser Pro Leu Leu Phe

305 310 315Ala Ala His Pro His Phe Gln Asn Leu Ser Gln Glu Glu

The information of 320 325 328 (2) SEQ ID NO:22:

(i) sequence signature:

(A) length: 5141 base pairs

(B) type: nucleic acid

(C) characteristic of stock: two strands

(D) topological framework: linearity

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: TTCGAGCTCG CCCGACATTG ATTATTGACT AGAGTCGATC GACAGCTGTG 50 GAATGTGTGT CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA 100 GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAG GTGTGGAAAG 150 TCCCCAGGCT CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA 200 GTCAGCAACC ATAGTCCCGC CCCTAACTCC GCCCATCCCG CCCCTAACTC 250 CGCCCAGTTC CGCCCATTCT CCGCCCCATG GCTGACTAAT TTTTTTTATT 300 TATGCAGAGG CCGAGGCCGC CTCGGCCTCT GAGCTATTCC AGAAGTAGTG 350 AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG CAAAAAGCTA GCTTATCCGG 400 CCGGGAACGG TGCATTGGAA CGCGGATTCC CCGTGCCAAG AGTGACGTAA 450 GTACCGCCTA TAGAGCGATA AGAGGATTTT ATCCCCGCTG CCATCATGGT 500 TCGACCATTG AACTGCATCG TCGCCGTGTC CCAAAATATG GGGATTGGCA 550 AGAACGGAGA CCTACCCTGG CCTCCGCTCA GGAACGAGTT CAAGTACTTC 600 CAAAGAATGA CCACAACCTC TTCAGTGGAA GGTAAACAGA ATCTGGTGAT 650 TATGGGTAGG AAAACCTGGT TCTCCATTCC TGAGAAGAAT CGACCTTTAA 700 AGGACAGAAT TAATATAGTT CTCAGTAGAG AACTCAAAGA ACCACCACGA 750 GGAGCTCATT TTCTTGCCAA AAGTTTGGAT GATGCCTTAA GACTTATTGA 800 ACAACCGGAA TTGGCAAGTA AAGTAGACAT GGTTTGGATA GTCGGAGGCA 850 GTTCTGTTTA CCAGGAAGCC ATGAATCAAC CAGGCCACCT TAGACTCTTT 900 GTGACAAGGA TCATGCAGGA ATTTGAAAGT GACACGTTTT TCCCAGAAAT 950 TGATTTGGGG AAATATAAAC CTCTCCCAGA ATACCCAGGC GTCCTCTCTG 1000 AGGTCCAGGA GGAAAAAGGC ATCAAGTATA AGTTTGAAGT CTACGAGAAG 1050 AAAGACTAAC AGGAAGATGC TTTCAAGTTC TCTGCTCCCC TCCTAAAGCT 1100 ATGCATTTTT ATAAGACCAT GGGACTTTTG CTGGCTTTAG ATCCCCTTGG 1150 CTTCGTTAGA ACGCGGCTAC AATTAATACA TAACCTTATG TATCATACAC 1200 ATACGATTTA GGTGACACTA TAGATAACAT CCACTTTGCC TTTCTCTCCA 1250 CAGGTGTCCA CTCCCAGGTC CAACTGCACC TCGGTTCTAA GCTTCTGCAG 1300 GTCGACTCTA GAGGATCCCC GGGGAATTCA ATCGATGGCC GCCATGGCCC 1350 AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC 1400 AAATTTCACA AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT 1450 CCAAACTCAT CAATGTATCT TATCATGTCT GGATCGATCG GGAATTAATT 1500 CGGCGCAGCA CCATGGCCTG AAATAACCTC TGAAAGAGGA ACTTGGTTAG 1550 GTACCTTCTG AGGCGGAAAG AACCAGCTGT GGAATGTGTG TCAGTTAGGG 1600 TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA 1650 TCTCAATTAG TCAGCAACCA GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG 1700 GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CATAGTCCCG 1750 CCCCTAACTC CGCCCATCCC GCCCCTAACT CCGCCCAGTT CCGCCCATTC 1800 TCCGCCCCAT GGCTGACTAA TTTTTTTTAT TTATGCAGAG GCCGAGGCCG 1850 CCTCGGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC 1900 CTAGGCTTTT GCAAAAAGCT GTTACCTCGA GCGGCCGCTT AATTAAGGCG 1950 CGCCATTTAA ATCCTGCAGG TAACAGCTTG GCACTGGCCG TCGTTTTACA 2000 ACGTCGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG 2050 CACATCCCCC CTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT 2100 CGCCCTTCCC AACAGTTGCG TAGCCTGAAT GGCGAATGGC GCCTGATGCG 2150 GTATTTTCTC CTTACGCATC TGTGCGGTAT TTCACACCGC ATACGTCAAA 2200 GCAACCATAG TACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG 2250 TGGTTACGCG CAGCGTGACC GCTACACTTG CCAGCGCCCT AGCGCCCGCT 2300 CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG 2350 TCAAGCTCTA AATCGGGGGC TCCCTTTAGG GTTCCGATTT AGTGCTTTAC 2400 GGCACCTCGA CCCCAAAAAA CTTGATTTGG GTGATGGTTC ACGTAGTGGG 2450 CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG AGTCCACGTT 2500 CTTTAATAGT GGACTCTTGT TCCAAACTGG AACAACACTC AACCCTATCT 2550 CGGGCTATTC TTTTGATTTA TAAGGGATTT TGCCGATTTC GGCCTATTGG 2600 TTAAAAAATG AGCTGATTTA ACAAAAATTT AACGCGAATT TTAACAAAAT 2650 ATTAACGTTT ACAATTTTAT GGTGCACTCT CAGTACAATC TGCTCTGATG 2700 CCGCATAGTT AAGCCAACTC CGCTATCGCT ACGTGACTGG GTCATGGCTG 2750 CGCCCCGACA CCCGCCAACA CCCGCTGACG CGCCCTGACG GGCTTGTCTG 2800 CTCCCGGCAT CCGCTTACAG ACAAGCTGTG ACCGTCTCCG GGAGCTGCAT 2850 GTGTCAGAGG TTTTCACCGT CATCACCGAA ACGCGCGAGG CAGTATTCTT 2900 GAAGACGAAA GGGCCTCGTG ATACGCCTAT TTTTATAGGT TAATGTCATG 2950 ATAATAATGG TTTCTTAGAC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG 3000 CGGAACCCCT ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC 3050 TCATGAGACA ATAACCCTGA TAAATGCTTC AATAATATTG AAAAAGGAAG 3100 AGTATGAGTA TTCAACATTT CCGTGTCGCC CTTATTCCCT TTTTTGCGGC 3150 ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG AAAGTAAAAG 3200 ATGCTGAAGA TCAGTTGGGT GCACGAGTGG GTTACATCGA ACTGGATCTC 3250 AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT 3300 GATGAGCACT TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTGATG 3350 ACGCCGGGCA AGAGCAACTC GGTCGCCGCA TACACTATTC TCAGAATGAC 3400 TTGGTTGAGT ACTCACCAGT CACAGAAAAG CATCTTACGG ATGGCATGAC 3450 AGTAAGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT AACACTGCGG 3500 CCAACTTACT TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT 3550 TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT GGGAACCGGA 3600 GCTGAATGAA GCCATACCAA ACGACGAGCG TGACACCACG ATGCCAGCAG 3650 CAATGGCAAC AACGTTGCGC AAACTATTAA CTGGCGAACT ACTTACTCTA 3700 GCTTCCCGGC AACAATTAAT AGACTGGATG GAGGCGGATA AAGTTGCAGG 3750 ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT GCTGATAAAT 3800 CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA 3850 GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC 3900 AACTATGGAT GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA 3950 TTAAGCATTG GTAACTGTCA GACCAAGTTT ACTCATATAT ACTTTAGATT 4000 GATTTAAAAC TTCATTTTTA ATTTAAAAGG ATCTAGGTGA AGATCCTTTT 4050 TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG TTCCACTGAG 4100 CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT 4150 CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT 4200 GGTTTGTTTG CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG 4250 GCTTCAGCAG AGCGCAGATA CCAAATACTG TCCTTCTAGT GTAGCCGTAG 4300 TTAGGCCACC ACTTCAAGAA CTCTGTAGCA CCGCCTACAT ACCTCGCTCT 4350 GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG TCGTGTCTTA 4400 CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC 4450 TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC 4500 CGAACTGAGA TACCTACAGC GTGAGCATTG AGAAAGCGCC ACGCTTCCCG 4550 AAGGGAGAAA GGCGGACAGG TATCCGGTAA GCGGCAGGGT CGGAACAGGA 4600 GAGCGCACGA GGGAGCTTCC AGGGGGAAAC GCCTGGTATC TTTATAGTCC 4650 TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG TGATGCTCGT 4700 CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG 4750 TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC CTGCGTTATC 4800 CCCTGATTCT GTGGATAACC GTATTACCGC CTTTGAGTGA GCTGATACCG 4850 CTCGCCGCAG CCGAACGACC GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG 4900 GAAGAGCGCC CAATACGCAA ACCGCCTCTC CCCGCGCGTT GGCCGATTCA 4950 TTAATCCAGC TGGCACGACA GGTTTCCCGA CTGGAAAGCG GGCAGTGAGC 5000 GCAACGCAAT TAATGTGAGT TACCTCACTC ATTAGGCACC CCAGGCTTTA 5050 CACTTTATGC TTCCGGCTCG TATGTTGTGT GGAATTGTGA GCGGATAACA 5100 ATTTCACACA GGAAACAGCT ATGACCATGA TTACGAATTA A 5141 (2) SEQ ID NO: 23 information about: ...

(i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:23:ATGTCNCCNG CNCCNCCNGC N 21 (2) SEQ ID NO:24:

(i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:24:ATGTCTCCAG CGCCGCCAGC G 21 (2) SEQ ID NO:25:

(i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:25:ATGTCGCCTG CTCCACCTGC T 21 (2) SEQ ID NO:26:

(i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:26:ATGTCGCCAG CGCCACCAGC C 21 (2) SEQ ID NO:27:

(i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:27:ATGTCCCCAG CCCCACCCGC A 21 (2) SEQ ID NO:28:

(i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:28:ATGTCGCCAG CGCCGCCAGC G 21 (2) SEQ ID NO:29:

(i) sequence signature:

(A) length: 25 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:29:Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu1 5 10 15Leu Arg Asp Asp His Val Leu His Gly Arg

The information of 20 25 (2) SEQ ID NO:30:

(i) sequence signature:

(A) length: 26 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:30:Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu1 5 10 15Leu Arg Asp Asp Xaa Val Leu His Gly Arg Leu

The information of 20 25 26 (2) SEQ ID NO:31:

(i) sequence signature:

(A) length: 25 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:31:Ser Pro Ala Pro Pro Ala Xaa Asp Pro Arg Leu Leu Asn Lys Leu1 5 10 15Leu Arg Asp Asp His Val Leu His Gly Arg

The information of 20 25 (2) SEQ ID NO:32:

(i) sequence signature:

(A) length: 14 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:32:Xaa Pro Ala Pro Pro Ala Xaa Asp Pro Arg Leu Xaa Asn Lys1 5 10 14 (2) SEQ ID NO:33:

(i) sequence signature:

(A) length: 9 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:33:Pro Arg Leu Leu Asn Lys Leu Leu Arg1 59 (2) SEQ ID NO:34:

(i) sequence signature:

(A) length: 45 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:34:GCCGTGAAGG ACGTGGTCGT CACGAAGCAG TTTATTTAGG AGTCG 45 (2) SEQ ID NO:35:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:35:CCNGCNCCNC CNGCNTGYGA 20 (2) SEQ ID NO:36:

(i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:36:NCCRTGNARN ACRTGRTCRT C 21 (2) SEQ ID NO:37:

(i) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:37:CCAGCGCCGC CAGCCTGTGA CCCCCGACTC CTAAATAAAC TGCCTCGTGA 50TGACCACGTT CAGCACGGC 69 (2) SEQ ID NO:38:

(i) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:38:GGTCGCGGCG GTCGGACACT GGGGGCTGAG GATTTATTTG ACGGAGCACT 50ACTGGTGCAA GTCGTGCCG 69 (2) SEQ ID NO:39:

(i) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:39:CCAGCACCTC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA 50CGACCACGTC CATCACGGC 69 (2) SEQ ID NO:40:

(i) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:40:GGTCGTGGAG GCCGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCACT 50GCTGGTGCAG GTAGTGCCG 69 (2) SEQ ID NO:41:

(i) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:41:CCAGCACCGC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA 50CGATCATGTC TATCACGGT 69 (2) SEQ ID NO:42:

(i) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:42:GGTCGTGGCG GCCGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCACT 50GCTAGTACAG ATAGTGCCA 69 (2) SEQ ID NO:43:

(i) sequence signature:

(A) length: 37 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:43:GCTAGCTCTA GAAATTGCTC CTCGTGGTCA TGCTTCT 37 (2) SEQ ID NO:44:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:44:CAGTCTGCCG TGAAGGACAT GG 22 (2) SEQ ID NO:45:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:45:TGTGGACTTT AGCTTGGGAG AATG 24 (2) SEQ ID NO:46:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:46:GGTCCAGGGA CCTGGAGGTT TG 22 (2) SEQ ID NO:47:

(i) sequence signature:

(A) length: 31 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:47:ATCGATATCG ATAGCCAGAC ACCCCGGCCA G 31 (2) SEQ ID NO:48:

(i) sequence signature:

(A) length: 36 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:48:GCTAGCTCTA GACAGGGAAG GGAGCTGTAC ATGAGA 36 (2) SEQ ID NO:49:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: the information of SEQ ID NO:49:CTCCTTGGAA CCCAGGGCAG GACC 24 (2) SEQ ID NO:50:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:50:GGTCCTGCCC TGGGTTCCAA GGAG 24 (2) SEQ ID NO:51:

(i) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:51:CTGCTCCGAG GAAAGGACTT CTGGATT 27 (2) SEQ ID NO:52:

(i) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:52:AATCCAGAAG TCCTTTCCTC GGAGCAG 27 (2) SEQ ID NO:53:

(i) sequence signature:

(A) length: 28 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:53:CCCTCTGCGT CGCGGCGGCC CCACCCAC 28 (2) SEQ ID NO:54:

(i) sequence signature:

(A) length: 28 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:54:GTGGGTGGGG CCGCCGCGAC GCAGAGGG 28 (2) SEQ ID NO:55:

(i) sequence signature:

(A) length: 35 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:55:GACTCGAGGA TCCATCGATT TTTTTTTTTT TTTTT 35 (2) SEQ ID NO:56:

(i) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:56:GACTCGAGGA TCCATCG 17 (2) SEQ ID NO:57:

(i) sequence signature:

(A) length: 32 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:57:GCTAGCTCTA GAAGCCCGGC TCCTCCTGCC TG 32 (2) SEQ ID NO:58:

(i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:58:CGAAATTAAC CCTCACTAAA G 21 (2) SEQ ID NO:59:

(i) sequence signature:

(A) length: 103 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:59:TGCAGCAAGG GCTACTGCCA CACTCGAGCT GCGCAGATGC TAGCCTCAAG 50ATGGCTGATC CAAATCGATT CCGCGGCAAA GATCTTCCGG TCCTGTAGAA 100GCT 103 (2) SEQ ID NO:60:

(i) sequence signature:

(A) length: 103 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:60:AGCTTCTACA GGACCGGAAG ATCTTTGCCG CGGAATCGAT TTGGATCAGC 50CATCTTGAGG CTAGCATCTG CGCAGCTCGA GTGTGGCAGT AGCCCTTGCT 100GCA103 (2) SEQ ID NO:61:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:61:TCTCGCTACC GTTTACAG 18 (2) SEQ ID NO:62:

(i) sequence signature:

(A) length: 25 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:62:CAGGTACCCA CCAGGCGGTC TCGGT 25 (2) SEQ ID NO:63:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:63:GGGCCATGAC ACTGTCAA 18 (2) SEQ ID NO:64:

(i) sequence signature:

(A) length: 40 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:64:GACCGCCACC GAGACCGCCT GGTGGGTACC TGTGGTCCTT 40 (2) SEQ ID NO:65:

(i) sequence signature:

(A) length: 32 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:65:ATCGATATCG ATCAGCCAGA CACCCCGGCC AG 32 (2) SEQ ID NO:66:

(i) sequence signature:

(A) length: 35 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:66:TCTAGATCTA GATCACCTGA CGCAGAGGGT GGACC 35 (2) SEQ ID NO:67:

(i) sequence signature:

(A) length: 33 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:67:AGTCGACGTC GACGTCGGCA GTGTCTGAGA ACC 33 (2) SEQ ID NO:68:

(i) sequence signature:

(A) length: 36 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:68:AGTCGACGTC GACTCACCTG ACGCAGAGGG TGGACC 36 (2) SEQ ID NO:69:

(i) sequence signature:

(A) length: 62 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:69:CGCGTATGCC AGCCCGGCTC CTCCTGCTTG TGACCTCCGA GTCCTCAGTA 50AACTGCTTCG TG 62 (2) SEQ ID NO:70:

(i) sequence signature:

(A) length: 61 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:70:ATACGGTCGG GCCGAGGAGG ACGAACACTG GAGGCTCAGG AGTCATTTGA 50CGAAGCACTG A 61 (2) SEQ ID NO:71:

(i) sequence signature:

(A) length: 37 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:71:CTAGAATTAT GAAAAAGAAT ATCGCATTTC TTCTTAA 37 (2) SEQ ID NO:72:

(i) sequence signature:

(A) length: 37 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:72:TTAATACTTT TTCTTATAGC GTAAAGAAGA ATTGCGC 37 (2) SEQ ID NO:73:

(i) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:73:GTAGAATTAT GAAAAAGAAT ATCGCATTTC ATCACCATCA CCATCACCAT 50CACATCGAAG GTCGTAGCC 69 (2) SEQ ID NO:74:

(i) sequence signature:

(A) length: 62 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:74:TTAATACTTT TTCTTATAGC GTAAAGTAGT GGTAGTGGTA GTGGTAGTGT 50AGCTTCCAGC AT 62 (2) SEQ ID NO:75:

(i) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:75:CTAGAATTAT GAAAAAGAAT ATCGCATTTC ATCACCATCA CCATCACCAT 50CACATCGAAC CACGTAGCC 69 (2) SEQ ID NO:76:

(i) sequence signature:

(A) length: 62 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:76:TTAATACTTT TTCTTATAGC GTAAAGTAGT GGTAGTGGTA GTGGTAGTGT 50AGCTTGGTGC AT 62 (2) SEQ ID NO:77:

(i) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:77:TCCACCCTCT GCGTCAGGT 19 (2) SEQ ID NO:78:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:78:GGAGACGCAG TCCATCGA 18 (2) SEQ ID NO:79:

(i) sequence signature:

(A) length: 62 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:79:GCAGCAGTTC TAGAATTATG TCNCCNGCNC CNCCNGCNTG TGACCTCCGA 50GTTCTCAGTA AA 62 (2) SEQ ID NO:80:

(i) sequence signature:

(A) length: 49 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:80:ACACTGGAGG CTCAAGAGTC ATTTGACGAA GCACTGAGGG TACAGGAAG 49 (2) SEQ ID NO:81:

(i) sequence signature:

(A) length: 45 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:81:CTAGAATTAT GAAAAAGAAT ATCGCATTTA TCGAAGGTCG TAGCC 45 (2) SEQ ID NO:82:

(i) sequence signature:

(A) length: 38 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:82:TTAATACTTT TTCTTATAGC GTAAATAGCT TCCAGCAT 38 (2) SEQ ID NO:83:

(i) sequence signature:

(A) length: 45 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:83:CTAGAATTAT GAAAAAGAAT ATCGCATTTC TTCTTAAACG TAGCC 45 (2) SEQ ID NO:84:

(i) sequence signature:

(A) length: 38 base pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:84:TTAATACTTT TTCTTATAGC GTAAAGAAGA ATTTGCAT 38 (2) SEQ ID NO:85:

(i) sequence signature:

(A) length: 25 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:85:Met Lys Lys Asn Ile Ala Phe Leu Leu Asn Ala Tyr Ala Ser Pro1 5 10 15Ala Pro Pro Ala Cys Cys Val Arg Arg Ala

20 25(2)SEQ ID NO：86：

(i) sequence signature:

(A) length: 31 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:86:Met Lys Lys Asn Ile Ala Phe His His His His His Hi3 His His1 5 10 15Ile Glu Gly Arg Ser Pro Ala Pro Pro Ala Cys Cys Val Arg Arg

20 25 30Ala31(2)SEQ ID NO：87：

(i) sequence signature:

(A) length: 31 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:87:Met Lys Lys Asn Ile Ala Phe His His His His His His His His1 5 10 15Ile Glu Pro Arg Ser Pro Ala Pro Pro Ala Cys Cys Val Arg Arg

20 25 30Ala31(2)SEQ ID NO：88：

(i) sequence signature:

(A) length: 7 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:88:Thr Thr Ala His Lys Asp Pro1 57 (2) SEQ ID NO:89:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:89:His Val Leu His1 4 (2) SEQ ID NO:90:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:90:Ser Arg Leu Ser1 4 (2) SEQ ID NO:91:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:91:Ser His Val Leu1 4 (2) SEQ ID NO:92:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:92:His Ser Arg Leu1 4 (2) SEQ ID NO:93:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:93:Ala Val Asp Phe1 4 (2) SEQ ID NO:94:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:94:Ser Leu Gly Glu1 4 (2) SEQ ID NO:95:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:95:Ala Val Thr Leu1 4 (2) SEQ ID NO:96:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:96:Leu Leu Glu Gly1 4 (2) SEQ ID NO:97:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:97:Leu Ser Ser Leu1 4 (2) SEQ ID NO:98:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:98:Leu Gly Gln Leu1 4 (2) SEQ ID NO:99:

(i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:99:Cys Xaa Leu Ser Ser1 5 (2) SEQ ID NO:100:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:100:Leu Leu Gly Gln1 4 (2) SEQ ID NO:101:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:101:Ser Ser Leu Leu1 4 (2) SEQ ID NO:102:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:102:Gly Gln Leu Ser1 4 (2) SEQ ID NO:103:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:103:Cys Leu Ser Ser1 4 (2) SEQ ID NO:104:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:104:Leu Gln Ser Leu1 4 (2) SEQ ID NO:105:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:105:Leu Gly Thr Gln1 4 (2) SEQ ID NO:106:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:106:Ala Leu Gln Ser1 4 (2) SEQ ID NO:107:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:107:Leu Leu Gly Thr1 4 (2) SEQ ID NO:108:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:108:Asn Ala Ile Phe1 4 (2) SEQ ID NO:109:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:109:Leu Ser Phe Gln1 4 (2) SEQ ID NO:110:

(i) sequence signature:

(A) length: 22 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:110:Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu1 5 10 15Leu Arg Asp Ser His Val Leu

20 22(2)SEQ ID NO：111：

(i) sequence signature:

(A) length: 24 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:111:His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr1 5 10 15Pro Val Leu Leu Pro Ala Val Asp Phe

20 24(2)SEQ ID NO：112：

(i) sequence signature:

(A) length: 23 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:112:Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln1 5 10 15Asp Ile Leu Gly Ala Val Thr Leu

20 23(2)SEQ ID NO：113：

(i) sequence signature:

(A) length: 21 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:113:Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr1 5 10 15Cys Leu Ser Ser Leu Leu

20 21(2)SEQ ID NO：114：

(i) sequence signature:

(A) length: 16 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:114:Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln1 5 10 15Ser16 (2) SEQ ID NO:115:

(i) sequence signature:

(A) length: 22 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:115:Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His1 5 10 15Lys Asp Pro Asn Ala Ile Phe

20 22(2)SEQ ID NO：116：

(i) sequence signature:

(A) length: 25 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:116:Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met1 5 10 15Leu Val Gly Gly Ser Thr Leu Cys Val Arg

20 25(2)SEQ ID NO：117：

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:117:Met Pro Pro Ala1 4 (2) SEQ ID NO:118:

(i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:118:Met Ala Pro Pro Ala1 5 (2) SEQ ID NO:119:

(i) sequence signature:

(A) length: 6 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:119:Met Pro Ala Pro Pro Ala1 56 (2) SEQ ID NO:120:

(i) sequence signature:

(A) length: 7 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:120:Met Ser Pro Ala Pro Pro Ala1 57 (2) SEQ ID NO:121:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:121:Ala Pro Pro Ala1 4 (2) SEQ ID NO:122:

(i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:122:Pro Ala Pro Pro Ala1 5 (2) SEQ ID NO:123:

(i) sequence signature:

(A) length: 6 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:123:Ser Pro Ala Pro Pro Ala1 56 (2) SEQ ID NO:124:

(i) sequence signature:

(A) length: 4 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:124:Val Arg Arg Ala1 4 (2) SEQ ID NO:125:

(i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:125:Val Arg Arg Ala Pro1 5 (2) SEQ ID NO:126:

(i) sequence signature:

(A) length: 6 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:126:Val Arg Arg Ala Pro Pro1 56 (2) SEQ ID NO:127:

(i) sequence signature:

(A) length: 7 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:127:Val Arg Arg Ala Pro Pro Thr1 57 (2) SEQ ID NO:128:

(i) sequence signature:

(A) length: 8 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:128:Val Arg Arg Ala Pro Pro Thr Thr1 58 (2) SEQ ID NO:129:

(i) sequence signature:

(A) length: 9 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:129:Val Arg Arg Ala Pro Pro Thr Thr Ala1 59 (2) SEQ ID NO:130:

(i) sequence signature:

(A) length: 10 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:130:Val Arg Arg Ala Pro Pro Thr Thr Ala Val1 5 10 (2) SEQ ID NO:131:

(i) sequence signature:

(A) length: 11 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:131:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro1 5 10 11 (2) SEQ ID NO:132:

(i) sequence signature:

(A) length: 12 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:132:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser1 5 10 12 (2) SEQ ID NO:133:

(i) sequence signature:

(A) length: 13 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:133:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg1 5 10 13 (2) SEQ ID NO:134:

(i) sequence signature:

(A) length: 14 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:134:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr1 5 10 14 (2) SEQ ID NO:135:

(i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:135:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser1 5 10 15 (2) SEQ ID NO:136:

(i) sequence signature:

(A) length: 16 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:136:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser1 5 10 15Leu16 (2) SEQ ID NO:137:

(i) sequence signature:

(A) length: 17 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:137:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser1 5 10 15Leu Val

17(2)SEQ ID NO：138：

(i) sequence signature:

(A) length: 18 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:138:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser1 5 10 15Leu Val Leu

18(2)SEQ ID NO：139：

(i) sequence signature:

(A) length: 19 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:139:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser1 5 10 15Leu Val Leu Thr

19(2)SEQ ID NO：140：

(i) sequence signature:

(A) length: 20 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:140:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser1 5 10 15Leu Val Leu Thr Leu

20(2)SEQ ID NO：141：

(i) sequence signature:

(A) length: 21 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:141:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser1 5 10 15Leu Val Leu Thr Leu Asn

20 21(2)SEQ ID NO：142：

(i) sequence signature:

(A) length: 22 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:142:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser1 5 10 15Leu Val Leu Thr Leu Asn Glu

20 22(2)SEQ ID NO：143：

(i) sequence signature:

(A) length: 23 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:143:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser1 5 10 15Leu Val Leu Thr Leu Asn Glu Leu

20 23(2)SEQ ID NO：144：

(i) sequence signature:

(A) length: 24 amino acid

(B) type: amino acid

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO:144:Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser1 5 10 15Leu Val Leu Thr Leu Asn Glu Leu Pro

20 24

Claims (23)

1. a TPO (1-332) or TPO (1-153) mpl ligand polypeptide is characterized in that, it can obtain by the method that comprises the following step:

(i) use based on the oligonucleotide of genome sequence shown in Figure 9 screening people's gene group library, isolating the genomic dna that contains mpl part exons coding sequence shown in Figure 9 and these all the other exons of gene,

(ii) this DNA is inserted expression vector,

(iii) use this carrier transfection mammalian cell, and expressing gene and

(iv) from cell culture medium, reclaim TPO (1-332) or TPO (1-153) mpl ligand polypeptide.

2. a TPO (1-332) or TPO (1-153) mpl ligand polypeptide is characterized in that, it can obtain by the method that comprises the following step:

(i) from genomic library, isolate and dna sequence dna

5’GCCCTGAAGGACGTGGTCGTCACGAAGCAGTTTATTTAGGAGTCG3’

The group group DNA of hybridization, it contains the exon of coding mpl ligand polypeptide,

(ii) this DNA is inserted expression vector,

(iii) use this carrier transfection mammalian cell, and expressing gene and

(iv) from cell culture medium, reclaim TPO (1-332) or TPO (1-153) mpl ligand polypeptide.

3. a TPO (1-332) or TPO (1-153) mpl ligand polypeptide is characterized in that, it can obtain by the method that comprises the following step:

(i) identify the suitable R NA cell source of people mpl part, and prepare one or more cDNA library with this RNA,

(iii) use labeled oligonucleotide, differentiate and isolate the clone who contains mpl part encoding sequence by the screening by hybridization library based on encoding sequence shown in Figure 9,

(iv) this coding DNA is inserted the expression vector that is adapted at expressing in the mammalian cell,

(v) use this carrier transfection mammalian cell, and express cDNA and

(vi) from cell culture medium, reclaim TPO (1-332) or TPO (1-153) mpl ligand polypeptide.

4. a TPO (1-332) or TPO (1-153) mpl ligand polypeptide is characterized in that, it can obtain by the method that comprises the following step:

(i) use the mRNA that extracting goes out from human kidney cells to prepare one or more cDNA library,

(ii) use labeled oligonucleotide, differentiate and isolate the clone who contains mpl part encoding sequence by the screening by hybridization library based on encoding sequence shown in Figure 9,

(iii) this coding DNA is inserted the expression vector that is adapted at expressing in the mammalian cell,

(iv) use this carrier transfection mammalian cell, and express cDNA and

(v) from cell culture medium, reclaim TPO (1-332) or TPO (1-153) mpl ligand polypeptide.

5. the TPO (1-332) or TPO (1-153) the mpl part of isolating, a basic homogeneous is characterized in that,

(a) nucleosides of this ligand stimulation mark ( ³The H-thymidine) mixes among the DNA of IL-3 dependent form Ba/F3 cell of personnel selection mpl P transfection;

(b) this part is measured moderate stimulation in the thrombocyte rebound ³⁵S mixes the round-robin thrombocyte;

(c) this part is stable in pH2.5,0.1%SDS and 2M urea;

(d) this part is a glycoprotein;

(e) the N-terminal sequence of this polypeptide is

SPAPPACDPRLLNKLLRDDHVLHGR or

SPAPPACDLRVLSKLLRDSHVLHSRL。

6. part as claimed in claim 5, it is characterized in that, it can obtain by hydrophobic interaction chromatogram, curing dyestuff chromatogram and mpl affinity chromatograph from undergrown protoplasma, and the SDS-PAGE under reductive condition discloses, its apparent molecular weight is 18-22kD, 28kD or 30kD.

7. an isolating TPO (1-332) or TPO (1-153) mpl ligand polypeptide, it is characterized in that, it is by such nucleic acid encoding, this nucleic acid has under the moderate stringent condition and the sequence that has before Fig. 9 Nucleotide 119, be SEQ ID NO:4 or SEQ ID NO:5, the sequence of nucleic acid hybridization.

8. as described isolating TPO of arbitrary claim among the claim 1-7 (1-332) or TPO (1-153) mpl part, it is characterized in that its biologically active.

9. as described TPO of arbitrary claim (1-332) among the claim 1-7 or TPO (1-153) mpl part, it is characterized in that it is derived from the mankind.

10. as described TPO of arbitrary claim (1-332) among the claim 1-7 or TPO (1-153) mpl part, it is characterized in that it is derived from inhuman Mammals.

11., it is characterized in that it is nonglycosylated as described TPO of arbitrary claim (1-332) among the claim 1-7 or TPO (1-153) mpl part.

12. the nucleic acid molecule of isolating, coding above-mentioned arbitrary claim described polypeptide.

13. the nucleic acid described in claim 12 is characterized in that, it operationally is connected in promotor.

14. an expression vector is characterized in that, it contains the described nucleic acid of claim 12, and this nucleic acid operationally is connected in the promotor of host cell identification.

15. a host cell is characterized in that, it transforms with the described nucleic acid of claim 12, thereby can express this nucleic acid, produces TPO (1-332) or TPO (1-153) mpl ligand polypeptide.

16. a method is characterized in that, it is included in expresses the described recombinant nucleic acid of claim 12 in the proper host cell, produce the mpl ligand polypeptide.

17. method as claimed in claim 16 is characterized in that, reclaims TPO (1-332) or TPO (1-153) mpl ligand polypeptide from host cell or host cell substratum.

18. method, it is characterized in that, under the regulation and control of promotor/enhancer element, inhibition or external source transcriptional regulatory element, in cell, express original mpl ligand gene, wherein these elements are inserted into cellular genome, and the vicinity of its position and direction are enough to the transcribing of DNA of influence coding TPO (1-332) or TPO (1-153) mpl ligand polypeptide.

19. a composition is characterized in that, it contains the described mpl part of arbitrary claim among the claim 1-11, TPO (1-332) or TPO (1-153) mpl part that the method for perhaps available claim 16 obtains, and pharmaceutically acceptable carrier.

20. method, it is characterized in that, comprise that with described TPO of arbitrary claim (1-332) or TPO (1-153) mpl part among the claim 1-11 perhaps TPO (1-332) or TPO (1-153) the mpl part with the method acquisition of claim 16 is used to prepare medicament.

21. the composition that composition as claimed in claim 19 or available claim 20 method obtain is characterized in that, also contains the reagent of organizing under being selected from of effective therapeutic dose: cytokine G CFS and interleukin-.

22. composition as claimed in claim 21 is characterized in that, this reagent is selected from: LIF, G-CSF, GM-CSF, M-CSF, Epo, IL-1, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9 and IL-11.

23. the purposes of described TPO of arbitrary claim (1-332) or TPO (1-153) mpl part is characterized in that among the claim 1-11, is used to prepare mpl ligand specificity antibody.

Images