RU2194074C2 - Polypeptide thrombopoietin (tpo), dna encoding polypeptide tpo (versions), method of preparing polypeptide (versions), pharmaceutical composition, method of treatment (versions), antibody against polypeptide tpo - Google Patents

Abstract

FIELD: genetic engineering, molecular biology, medicine, pharmacy. SUBSTANCE: polypeptide thrombopoietin (TPO) shows biological activity of specific stimulation or enhancement of platelets formation. Amino acid sequence of TPO is given in description as well as DNA encoding polypeptide TPO with amino acid sequence given in description. Polypeptide TPO is obtained by its expression in suitable host followed by its isolation. Pharmaceutical composition for treatment of platelet-associated disorders comprises effective amount of polypeptide TPO and acceptable carrier. Pharmaceutical composition is administrated to subject in effective amount for treatment of platelet-associated disorders, for example, thrombocytopenia. Immunization of animal with polypeptide TPO is carried out and antibodies raised against this polypeptide are isolated. Invention ensures to perform effective treatment of disorders associated with platelets. EFFECT: improved method of preparing and treatment. 34 cl, 34 dwg, 9 tbl

Description

 This application is a partial continuation of U.S. Patent Application Serial (unknown), filed January 31, 1995, pending resolution, which is a partial continuation of U.S. Patent Application Serial 08/361811, filed December 22, 1994, pending a decision, which is a partial continuation of U. S. Patent Application Serial N 08/320300, filed October 11, 1994, pending a decision, which is a partial continuation of U.S. Patent Application Serial 08/278083, filed July 20, 1994, pending, which is a partial continuation of US Patent Application Serial 08/221020, filed April 1, 1994, revoked, which is a partial continuation of US Patent Application Serial 08/212164, filed March 14, 1994 being recalled.

Field of Invention
This invention relates to a new protein having activity of specific stimulation or increase in platelet formation in vivo or enhance the proliferation and differentiation of megakaryocyte progenitor cells, the DNA sequence encoding this protein, and a method for producing it.

BACKGROUND OF THE INVENTION
Megakaryocytes are large cytoplasm-rich multinucleated cells that produce platelets and can be found mainly in the bone marrow. Megakaryocytes come from pluripotent hematopoietic (hematopoietic) stem cells in the bone marrow. The primitive pluripotent stem cell differentiates to some extent into megakaryocyte progenitor cells, which give rise to several generations of megakaryocytes. Precursor cells of megakaryocytes multiply and differentiate into megakaryocytes. Then megakaryocytes undergo polyploidization and cytoplasmic maturation and ultimately secrete their nuclear-free cytoplasmic fragments, namely, platelets, into the blood circulation. On average, 2-4 thousand platelets are formed from one mature megakaryocyte. Although the mechanism of platelet formation is still unclear in many respects, it is believed that megakaryocytes are usually localized on the protein surface of the sinus endothelium in the bone marrow and produce cytoplasmic processes that propagate into the sinusoid, where platelet fragmentation occurs.

 There are many unclear points regarding the mechanism of platelet formation, although it is possible that megakaryocytes are present locally in the dermal layer of the membrane of the venous sinus of the bone marrow and that the cytoplasm passes through the dermal membrane, forming a string-like protrusion on the inner wall of the venous sinus, resulting in platelet ejection.

 It has been suggested that there is a specific function for the formation and regulation of platelet formation in the hematopoiesis of megakaryocytes. In healthy people and animals, the number of effective platelets remains, although it is known that with the introduction of antibodies to platelets to healthy animals, for example, the number of platelets decreases sharply, then their number temporarily increases to a level higher than the usual level, and finally they return to normal level. It is also known in the clinic area that a decrease in the number of platelets (thrombocytopenia) or an increase in the number of platelets (thrombocytosis) is observed even with normal amounts of red blood cells and white blood cells.

 Sometimes the most important function of a platelet is the formation of a blood clot in a hemostatic (hemostatic) mechanism. If the hemostatic mechanism does not function properly due to a decrease in the number of platelets, a tendency is formed in the direction of hemostasis.

 It has been suggested that there is a specific regulatory mechanism for megakaryocytopoiesis and thrombopoiesis. Platelets are maintained in effective amounts in healthy people and normal animals. However, it is known that with the introduction of platelet antibodies to a normal animal, the platelet count decreases sharply for a short period of time, after which it begins to increase and temporarily exceeds the normal level, but eventually returns to the normal level. It is known that in the clinical field, a decrease in the number of platelets (thrombocytopenia) or an increase in their number (thrombocytosis) occurs even with normal amounts of red blood cells and white blood cells. However, to date, there have been no reports of success in the isolation and identification of a specific regulatory factor involved in the formation of platelets (for example, like erythropoietin in the formation of red blood cells).

 The most important function of platelets is the formation of a blood clot in the hemostatic (hemostatic) mechanism. A tendency to bleeding occurs when the normal function of the hemostatic mechanism is impaired by thrombocytopenia. In the field of radiation therapy and chemotherapy for cancerous tumors, thrombocytopenia caused by bone marrow suppression is a lethal complication, and platelet transfusion is performed in such patients to prevent bleeding. Platelet transfusion is also used after bone marrow transplantation or hypoplastic anemia.

 Platelets for use in such transfusion are prepared by thrombocytophoresis from the blood of healthy blood donors, however, such platelets for use in transfusion have a short shelf life and may be contaminated with a bacterial infection. Platelet transfusion represents a possible risk of dangerous viruses entering the patient, such as human immunodeficiency virus (H IV) or various hepatitis viruses, the risk of producing antibodies specific for the histocompatibility complex antigen (H L A) of transfused platelets or the transplant versus host reaction "(GVHD) due to an admixture of lymphocytes in the platelets used for transfusion.

 As a result, it will be very useful if in patients with thrombocytopenia it will be possible to stimulate the internal formation of platelets and at the same time their dependence on platelet transfusion can be reduced. In addition, if thrombocytopenia in cancer patients can be corrected or prevented, such treatments can be carried out more safely, the intensity of treatment can be increased, and further improvement in the anticancer effect can be expected.

 For these reasons, a number of intensive studies have been carried out to isolate and identify specific regulatory factors involved in the regulation of the formation of megakaryocytes and platelets. According to in vitro studies, the regulatory factors that control megakaryocytopoiesis are roughly divided into the following two factors (see Williams et al., J, Cell Physiol., Vol. 110, pp. 101-104, 1982). Colony-stimulating factor megakaryocytes (Meg-CSF) is a regulatory factor that stimulates the proliferation and differentiation of CFU-MK with the formation of colonies of megakaryocytes in a semi-solid culture medium. Another regulatory factor, called the megakaryocyte potentiation factor (Meg-Pot), megakaryocyte stimulation factor, thrombocytopoiesis stimulation factor or the like, acts mainly on immature or mature megakaryocytes, enhancing their differentiation and maturation. Meg-Pot is found in combination with Meg-CSF activity in some cases. In addition, since platelet counts increased with the administration of serum or plasma taken from experimentally induced animals with thrombocytopenia to other normal animals, it was suggested that there was a humoral factor called thromboetin (TPO) that could enhance platelet formation in vivo.

 In recent years, some cytokines whose genes have been cloned have been tested for their ability to stimulate megakaryocytopoiesis and thrombocytopoiesis. Human IL-3 stimulated colony formation of human megakaryocytes (Bruno et al., Exp. Hematol., Vol. 16, pp. 371-377, 1988) and, at least in monkeys, increasing platelet counts (Donahue et al., Science Vol. 241, p. 1820, 1988). However, since IL-3 is a factor that acts on the proliferation and differentiation of all hematopoietic cells, it should be distinguished from specific regulatory factors that regulate megakaryocytopoiesis and platelet formation. Human IL-6 did not detect Meg-CSF activity, but acted on immature megakaryocytes and then enhanced their differentiation into mature megakaryocytes (Williams et al., Exp. Hematol., Vol 18, p.69, 1990). In vivo administration of IL-6 induced platelet formation and increased maturation and caused a shift towards higher ploidy of bone marrow megakaryocytes in primates, but also caused side effects such as weight loss, acute phase protein induction (Asano et al., Blood, vol. 78, pp. 1467-1475, 1991). Human IL-11 did not detect Meg-CSF activity, but showed Meg-Pot activity and enhanced platelet formation in mice (Neben et al., Blood, vol. 81, pp. 901-908, 1993). In addition, human LIF (an inhibitor of lymphocyte migration) significantly increased platelet counts in primates (Mauer et al., Blood, vol. 81, pp. 326-3233, 1993), but its effect in vitro on megakaryocytes was weak (Burstein et al ., J, Cell. Physiol., Vol. 153, pp. 305-312, 1992).

 Although the clinical use of these cytokines as platelet enlargement factors can be expected, their functions are not specific to a number of megakaryocyte generations and they cause side effects. Therefore, it was required to find a platelet-increasing factor, which is specific for the megakaryocyte-platelet system and causes less side effects, for clinical use.

 Meg-CSF, Meg-Pot, or TPO activity was detected in the serum, plasma, or urine of patients with thrombocytopenia or animals or in the culture supernatant of some cultured human cell lines. However, it is currently unknown whether these activities are due to the presence of a single factor or a combination of several factors and whether they differ from known cytokines.

 Hoffman et al. found that the sera of patients with hypoplastic anemia and amegakaryocytic thrombocytopenic purpura contained Meg-CSF activity, which significantly increased the formation of colonies of human megakaryocytes (Hoffman et al., N. Eng. J. Med., vol. 305, pp. 533-538, 1981). After that, Mazur et al. reported that Meg-CSF activity is present in the serum of patients with aplastic anemia and it differs from both IL-3 and GM-CSF (Mazur et al., Blood, vol. 76, pp. 290-297, 1990). Similar Meg-CSF activity was found in the sera of cancer patients who received intensive cytotoxic chemotherapy and bone marrow transplant patients (Mazur et al., Exp. Hematol., Vol. 12, pp.624-628, 1984; de Alarcon and Schmieder , Prog. Clin. Bio. Res., Vol. 215, pp. 335-340, 1986).

Hoffman et al. reported that Meg-CSF with an average mol. weighing 46,000 was purified from the plasma of patients with hypomegakaryocytic thrombocytopenia (Hoffman et al. J. Clin. Invest., vol 75, pp. 1174-1182, 1985), but further studies showed that this material was not of such a purity level that allowed accurate amino acid sequencing (Hoffman, Blood, vol. 74, pp. 1196-1212, 1989). A substance having TPO-like activity that enhanced the incorporation of ⁷⁵ Se-selenomethionine into newly formed platelets in mice was partially purified from the plasma of thrombocytopenic patients or the urine of patients with idiopathic thrombocytopenic purpura (of unclear origin) (1TP). The average mol. the mass of plasma derived factor was 40,000 (Grossi et al., Hematologica, vol. 72, pp. 291-295, 1987; Vannucchi et al., Leukemia, vol. 2, pp. 236-240, 1988).

 Meg-CSF and TPO-like activity were also found in urine samples from patients with aplastic anemia and severe ITP (Kawakita et al., Br. J. Haematol., Vol. 48, pp. 609-615, 1981; Kawakita et al. , Blood, vol. 556-560, 1983). Kawakita et al. also reported that Meg-CSF activity detected in the urine extract of patients with aplastic anemia, found an average mol. a mass of 45,000, determined by gel filtration under dissociating conditions (Kawakita et al., Br. J. Haematol., vol. 62, pp. 715-722, 1986). Erikson-Miller et al. they also reported the purification of Meg-CSF from similar urine samples, but did not provide information on its structure (Erikson-Miller et al., "Blood Cell Growth Factors: their present and future use in hematology and oncology" ed. by Murphy, AlphaMed Press , Dayton, Ohio, pp. 204-220, 1992). Turner et al. purified megakaryocyte stimulation factor (MSF) having Meg-CSF activity from the urine of patients with transplanted bone marrow and cloned its gene (Turner et al., Blood, vol. 78, p. 1106, 279a, 1991, (abstr., Suppl .1)). This MSF has a pier. weight 28000-35000. The identity of this factor with Meg-CSF, found so far in serum and plasma samples of thrombocytopenic patients, and its activity in increasing platelet counts have yet to be elucidated.

 A substance having TPO-like activity with mol. weighing 32,000, was purified from the culture supernatant of the line of human embryonic kidney cells (HEK cells) and its biological and biochemical properties were extensively studied, but its structure is still unknown (McDonald et al., J. Lab. Clin. Hed., vol. 106, pp. 162-174, 1985; McDonald, Int. J. Cell Cloning, vol. 7, pp. 139-155, 1989). Other researchers, in contrast, reported that the main activity in HEK-conditioned air, which enhances the maturation of megakaryocytes in vitro, is due to known cytokines, namely IL-6 and EPO (withy et al., J., Cell. Physiol., Vol 15, pp. 362-372, 1992).

Regarding animal derived factors, Evatt et al. reported that TPO-like activity, enhancing the incorporation of ⁷⁵ Se-selenomethionine into newly formed platelets in rabbits and mice, was found in plasma of thromocytopenic rabbits induced by injection of serum against platelets (Evatt et al., J. Lab. Clin. Med.,. vol. 83, pp. 364-371, 1974). In addition, a number of similar studies have been published from the 60s to the 70s (e.g., Odell et al., Proc. Soc. Biol. Med., Vol. 108, pp. 428-431, 1961; Evatt and Levin, J. Clin. Invest., Vol. 48, pp. 1615-1626; Harker, Am. J. Physiol., Vol. 218, pp. 1376-1380, 1970; Shreiner and Levin. J. Clin, Invest, vol. 49, pp. 1709-1713, 1970; and Penington, Br. Med. J. vol. 1, pp. 606-608, 1970). Evatt et al., And Hill and Levin partially purified TPO-like activity from plasma of thrombocytopenic rabbits (Evatt et al., Blood, vol. 54, pp. 377-388, 1979; Hill and Levin, Exp. Hematol., Vol .14, pp.752-759, 1986).

 After this, the purification of this factor was continued, activity in enhancing the differentiation and maturation of megakaryocytes in vitro was checked, namely, Meg-Pot activity, it was found that the average mol. mass was 40000-46000, determined by gel filtration (Keller et al., Exp. Hematol., vol. 16, pp. 262-267, 1988; Hill et al., Exp. Hematol., vol. 20, pp. 354 -360, 1992). Since IL-6 activity was not detected in the plasma of rabbits with severe acute thrombocytopenia induced by the administration of anti-platelet antiserum, it was suggested that this TPO-like activity is due to a factor other than IL-6 (Hill et al., Blod, vol. 80, pp. 346-351, 1992).

 Tayrien Rosenberg also refined the factor having an average mol. mass 15,000, from plasma of thrombocytopenic rabbits and culture supernatant of HEK cells, which stimulates the formation of platelet factor 4 in the rat megakaryocyte cell line, but no information was provided on its structure (Tayrien and Rosenberg, J. Biol., vol. 262, pp. 3262- 3268, 1987).

 In addition, Nakeff detected Meg-CSF activity in the serum of thrombocytopenic mice induced by the administration of anti-platelet antiserum (Nakeff, Experimental Nematology Today ed. By Baum and Ledney, Springer-Verlag, NY, pp. 111-123, 1977). On the other hand, serum from thrombocytopenic rabbits enhanced the maturation of megakaryocytes (Keller et al., Exp. Hematol., Vol. 16, pp. 262-267, 1988; Hill et al., Exp. Hematol., Vol. 17, pp. 903-907, 1989) and stimulated the morphological change of megakaryocytes into platelets (Leven and Yee, Blood, vol. 69, pp. 1046-1052, 1989), but did not have detectable Meg-CSF activity.

 Miura et al. found Meg-CSF activity in plasma of rats that became thrombocytopenic under the influence of sublethal irradiation of the whole body (Miura et al., Blood, vol. 63, pp. 1060-1066, 1984), and suggested that the in vivo induction of Meg-CSF activity is associated with a decrease in megakaryocytes, but not with a decrease in platelets, since this activity did not change during platelet transfusion (Miura et al., Exp. Hematol vol. 16, pp. 139-144, 1988), Mazur and South found Mef-CSF activity in serum dogs sublethally irradiated and reported that this factor has an average mol. a mass of 175,000 when measured by gel filtration (Mazur and South, Exp. Hematol., vol. 13, pp. 1164-1172, 1985). In addition to the above, factors derived from serum, plasma, and urine have been reported by other researchers, for example, Straneva et al. , (Straneva et al., Exp. Hematol., Vol. 15, pp. 657-663, 1987).

 Thus, as described above, various activities that stimulate megakaryocytopoiesis and thrombocytopoiesis were found in biological samples obtained from thrombocytopenic patients and animals, but the isolation of these factors, their biochemical and biological identification and determination of their properties were not achieved due to their extremely small quantities in natural sources such as blood and urine.

Summary of the invention
The objectives of this invention are the isolation of TPO protein from natural sources and its identification, and the TPO protein has the activity of stimulating or increasing platelet formation in vivo and / or enhancing the proliferation and differentiation of megakaryocyte progenitor cells (hereinafter referred to as "TPO activity"), and the isolation of the gene, encoding TPO protein, and providing a method for producing this protein in a homogeneous form and in large quantities by recombinant DNA technology. Success in achieving these goals will replace or reduce the frequency of platelet transfusion currently in use, and such a new protein will also be used to treat and diagnose platelet-related disorders.

Thus, the present invention relates to (i) a purified and isolated DNA sequence encoding a protein with TPO activity selected from the group consisting of:
(a) the DNA sequences shown in SEQ ID 194, 195 and 196, or their complementary chains, and
(b) DNA sequences hybridizing under stringent conditions with the DNA sequences of (a), or fragments thereof, and
(c) DNA sequences that would hybridize with the DNA sequences of points (a) and (c), if not for the degeneracy of the genetic code,
(ii) a method for producing a protein with TPO activity, comprising the steps of:
growing under suitable nutritional conditions prokaryotic or eukaryotic host cells transformed for transfection with the indicated DNA sequence so that expression of the protein is possible, and
the selection of the target protein product obtained by expression of the specified DNA sequence, and
(iii) a protein product obtained by expression in a prokaryotic or eukaryotic host cell of the indicated DNA sequence.

 In addition, the present invention relates to a pharmaceutical composition containing an effective amount of a protein with TPO activity, and a method for treating platelet-related disorders, in particular thrombocytopenia, comprising administering said protein to patients with such disorders.

Brief Description of the Drawings
FIG. 1 shows Sephacryl S-200HR gel filtration chromatography on Phenyl Sepharose 6 FF / LS F2 derived from XRP.

 Figure 2 shows Capcell Pak C1 reverse phase chromatography of UMS-pack CN-AP TPO-active fraction obtained from a low molecular weight TPO (Sephacryl S-200HR F3) XRP sample.

 FIG. 3 shows an analysis in SDS-PAGE of Capcell Pak C1 TPO-active fraction (PA) from a low molecular weight TPO XRP sample.

 Figure 4 shows the peptide maps on C18 HPLC with reverse phase rat TPO isolated by SDS-PAGE (SDS-PAGE). Peptide fragments were obtained by systematic hydrolysis using three proteases.

 FIG. 5 shows TPO activity obtained from XRP in the rat CFU-MK test system.

 6 shows the construction of an expression vector pEF18S.

 FIG. 7 shows TPO activity in the culture supernatant of COSI cells into which pEF18S-A2α was introduced in the rat CFU-MK test system.

 FIG. Figure 8 shows TPO activity in the culture supernatant of COS1 cells into which pEF18S-HL34 was introduced in the rat CFU-MK test system.

 FIG. 9 shows TPO activity in the culture supernatant of COS1 cells into which pHT1-231 was introduced in the rat CFU-MK test system.

 Fig. 10a shows TPO activity in the culture supernatant of COS1 cells into which pHTF1 was introduced in the rat CFU-MK test system.

 Fig. 10b shows TPO activity in the culture supernatant of COS1 cells into which pHTF1 was introduced in the M-07e test system.

 FIG. 11 shows a restriction map of the λHGT1 clone and the pHGT1 pEFHGTE construct.

(E: EcoR1, H: Hind 111, S; Sal1)
12a shows TPO activity in the culture supernatant of COS1 cells into which pEFHGTE was introduced in the rat CFU-MK test system.

 12b shows TPO activity in the culture supernatant of COS1 cells into which pEFHFTE was introduced in the M-07e test system.

 Fig. 13a shows TPO activity in the culture supernatant of COS1 cells into which pHT1-211 1, pHT1-191 1 or pHT1-171 2 was introduced, in the rat CFU-MK test system.

 13 b shows TPO activity in the culture supernatant of COS1 cells into which pHT1-163 2 was introduced in a rat CFU-MK test system.

 FIG. 14 shows TPO activity in the culture supernatant of COS1 cells into which pHT1-211 1, pHT1-191 1, pHT1-171 2, or pHT1-163 2 was introduced, in test system M-07e.

 FIG. 15 is a chromatogram obtained by reverse phase chromatography (Vydac column) for purification of human TPO from the culture supernatant of CHO cells into which pDEF202-hTPO-P1 vector expressing TPO was introduced.

 FIG. 16 is a photograph showing the separation in SDS-PAGE (SDS page-SDS) of human TPO purified from the culture supernatant of CHO cells into which pDEF2020 hTPO-P1 was introduced and TPO was expressed.

 FIG. 17 is a chromatogram obtained by reverse phase chromatography for purification of human TPO from E. coli, into which pCFM536 / h 6T (1-163) was introduced to express TPO.

 FIG. 18 is a photograph showing the separation in SDS-PAGE (SDS page-SDS) of a variant of human TPO (1-163) isolated purified from E. coli into which pCFM536 / h6T (1-163) was introduced and TPO was allowed to be expressed.

 FIG. 19 shows an elution pattern of hTPO163 on a Superdex 75 pg column in connection with the purification of hTPO 163 from the culture supernatant as starting material obtained by transfection of the human TPO expressing plasmid pDEF202 hTPO163 into CHO cells. The amount of protein was determined at 220 nm.

 FIG. 20 shows an SDS-PAGE analysis of standard pTPO163 eluted from a 75 pg Superdex column while purifying hTPO163 from the culture supernatant as starting material obtained by transfection of human TPO expressing plasmid pDEF202-hTPO163 into CHO cells. The hTPO163 protein was stained with silver on a gel.

 Fig.21 shows the structure of the expression vector pSMT201.

 FIG. 22 shows a graph depicting the TPO activity determined by the M-07e test in the culture supernatant of COS7 cells into which βGL-TPO, 3 / TPO or 09 / TPO were injected and then expressed.

 FIG. 23 is a graph depicting TPO activity determined by the M-07e test in the supernatant of COS7 cell cultures into which a derivative with an insertion or deletion of human TPO was introduced, followed by expression.

 24 shows an increased platelet count in mice that were administered with TPO via intravenous and subcutaneous injections.

 25 shows a dose-dependent increase in platelet count in mice after subcutaneous injection of TPO.

 FIG. 26 shows the TPO-induced increase in platelet count after treatment with 5-FU mice to induce thrombocytopenia.

 FIG. 27 shows the TPO-induced increase in platelet count after treatment of mice with nimustine hydrochloride to induce thrombocytopenia.

 FIG. Figure 28 shows TPO-induced platelet count increase after bone marrow transplantation.

 FIG. 29 shows the TPO-induced increase in platelet count after x-ray irradiation of mice to induce thrombocytopenia.

 FIG. 30 shows a dose-dependent increase in platelet count after administration of truncated TPO (amino acids 1-163 in SEQ 1D 6).

 FIG. 31 shows an increase in platelet count after administration of truncated TPO (amino acids 1-63 in SEQ 1D 6) after administration of nimustine hydrochloride to induce thrombocytopenia.

 Fig. 32 shows that an increase in the concentration of Mp1-X added to the culture system of human megakaryocytes leads to increasing blocking of megakaryocyte development.

FIG. 33 represents TPO activity determined in Test M-07e, TPO derivatives [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹³³ ] - TPO (1-163), [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Pro ¹⁴⁸ ] TPO (1-163) [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹¹⁵ ] TPO (1-163) expressed in E. coli.

Fig. 34 describes TPO activity determined in the M-07e test, TPO derivatives [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹²⁹ ] - TPO (1-163), [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹⁴³ ] TPO (1-163), [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Leu ⁸² ] TPO (1-163), [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Leu ¹⁴⁶ ] TPO (1-163) [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ⁵⁹ ] TPO (1-163) expressed in E. coli.

DETAILED DESCRIPTION OF THE INVENTION
This invention specifically provided thrombopoietin polypeptides (TPO) having biological activity of specific stimulation or increased platelet formation and containing the amino acid sequence 1-332 of SEQ 1D 6, or derivatives thereof. Illustrative polypeptides include polypeptides that consist of amino acid sequences 1-163 of SEQ 1D 6, 1-232 SEQ 1D 6, 1-151 of SEQ 1D 6, a mature amino acid sequence of SEQ 1D 2, 4 and 6, including polypeptides, having deleted 1-6 amino acids of the NH ₂ -terminal. Additional illustrative polypeptides of this invention include [Thr ³³ , Thr ³³³ , Ser ³³⁴ , Ile ³³⁵ , Gly ³³⁶ , Tyr ³³⁷ , Pro ³³⁸ , Tyr ³³⁹ , Asp ³⁴⁰ , Val ³⁴¹ , Pro ³⁴² , Asp ³⁴³ , Tyr ²⁴⁴ , Ala ³⁴⁵ , Gly ³⁴⁶ , Val ³⁴⁷ , His ³⁴⁸ , His ³⁴⁹ , His ³⁵⁰ , His ³⁵¹ , His ³⁵² , His ³⁵³ ,] TPO, [Asn ²⁵ , Lys ²³¹ , Thr ³³³ , Ser ³³⁴ , Ile ³³⁵ , Gly ³³⁶ , Tyr ³³⁷ , Pro ³³⁸ , Tyr ³³⁹ , Asp ³⁴⁰ , Val ³⁴¹ , Pro ³⁴² , Asp ³⁴³ , Tyr ³⁴⁴ , Ala ³⁴⁵ , Gly ³⁴⁶ , Val ³⁴⁷ , His ³⁴⁸ , His ³⁴⁹ , His ³⁵⁰ , His ³⁵¹ , His ³⁵² , His ³⁵³ ] TPO, [Asn ²⁵ ] TPO [Thr ³³ ] TPO. Other polypeptides of this invention include TPO derivatives polypeptides [ΔHis ³³ ] TPO (1-163), [ΔArg ¹¹⁷ ] TPO (1-163), [ΔGly ^116] TPO (1-163), [His ³³ , Thr ³³ ', Pro ³⁴ ] TPO (1-163), [His ³³ , Ala ³³ ', Pro ³⁴ ] TPO (1-163), His ³³ , Gly ³³ ', Pro ³⁴ , Ser ³⁸ ] TPO (1-163), [Gly ¹¹⁶ , Asn ¹¹⁶ ', Arg ¹⁷⁷ ] TPO (1-163), [Gly ¹¹⁶ , Ala ¹¹⁶ ', Arg ¹⁷⁷ ] TPO (1-163), [Gly ¹¹⁶ , Gly ¹¹⁶ ', Arg ¹¹⁷ ] -TPO (1-163 ), [Ala ¹ , Val ³ , Arg ¹²⁹ ] TPO (1-163), [Ala ¹ , Val ³ , Arg ¹³³ ] -TPO (1-163), [Ala ¹ , Val ³ , Arg ¹⁴³ ] TPO (1 -163), [Ala ¹ , Val ³ , Leu ⁸² ] -TPO (1-163), [Ala ¹ , Val ³ , Leu ¹⁴⁶ ] TPO (1-163), [Ala ¹ , Val ³ , Pro ¹⁴⁸ ] - TPO (1-163), [Ala ¹ , Val ³ , Arg ⁵⁹ ] TPO (1-163) and [Ala ¹ , Val ³ , Arg ¹¹⁵ ] -TPO (1-163).

The TPO polypeptides of this invention also include those polypeptides that are covalently linked to a polymer, preferably polyethylene glycol. In addition, the TPO polypeptides of the invention may include the amino acids [Met ^-2 -Lys ^-1 ], [Met ^-1 ] or [Gly ^-1 ]. The invention provides DNAs that encode a TPO polypeptide and the derivatives described above, which DNAs are provided in the form of cDNA, genomic DNA, and synthetic DNAs.

The invention also provides methods for producing the TPO polypeptide described above, comprising the steps of expressing a polypeptide encoded by the DNA of the present invention in a suitable host, and isolating the TPO polypeptide. If the expressed TPO polypeptide is a Met- ^2- Lys ^-1- polypeptide, then these methods include the step of cleaving the Met- ^1- Lys ^-1 from the isolated TPO polypeptide.

The invention also provides methods for producing TPO polypeptides, such as polypeptides fused to glutathione S-transferase (GST). The DNA encoding the amino terminal GST polypeptide, the thrombin recognizing peptide, and the TPO polypeptide are introduced into a suitable host, the fusion polypeptide is isolated and the GST portion is removed by treatment with thrombin. The resulting TPO polypeptides have a [Gly ^-1 ] structure.

 Additionally provided are prokaryotic and eukaryotic host cells transformed or transfected with the DNA sequence according to the invention in such a way that the host cells are able to express a polypeptide having biological activity to stimulate or increase platelet formation.

 The pharmaceutical compositions of this invention contain an effective amount of TPO polypeptide or its derivative in combination with a pharmaceutically acceptable carrier and are suitable for use in the treatment of platelet-related disorders, in particular for the treatment of thrombocytopenia, for example, due to chemotherapy, radiation therapy or bone marrow transplantation. The invention provides appropriate methods of treatment.

 Finally, this invention provides antibodies specifically immunoreacting with TPO polypeptides and their derivatives described above. Such antibodies are useful in methods for isolating and quantifying TPO of the polypeptides of the invention.

 According to this invention, a new DNA sequence is provided that encodes a protein having TPO activity (hereinafter referred to as the DNA sequence of the present invention). The DNA sequence of the present invention contains a DNA sequence that encodes an amino acid sequence of DNA that encodes the amino acid sequence shown in SEQ 1D 2, 4 or 6 of the attached list of sequences.

 Also, the DNA sequence of the present invention is called the DNA sequence encoding a partially modified (substitution, deletion, insertion or addition) version of the above amino acid sequence shown in SEQ 1D 2, 4 or 6, provided that such modifications do not violate TPO activity. Those. DNA sequences encoding TPO derivatives are also included in this invention.

 In other words, the DNA sequence of the present invention includes DNA sequences encoding protein molecules whose amino acid sequences are essentially the amino acid sequences shown in SEQ 1D 2, 4 or 6. The phrase "amino acid sequences are essentially the amino acid sequences shown in SEQ 1D 2, 4 or 6 "as used herein means that these amino acid sequences include sequences represented by SEQ 1D 2, 4 or 6, as well as sequences represented by SEQ 1D 2, 4 or 6, which have a partial modification, such as substitution, deletion, insertion, addition, etc., provided that such modifications do not violate the TPO activity.

 Further, the DNA sequence of the present invention consists essentially of a DNA sequence encoding a protein with TPO activity.

 The expression "DNA sequence encoding an amino acid sequence" includes all DNA sequences that may have degeneracy in the nucleotide sequences.

The DNA sequence of the present invention also includes the following sequences:
(a) DNA sequences represented by SEQ 1D 7, 194, 195 and 196, or chains complementary to them,
(c) DNA sequences that hybridize under stringent conditions to the DNA sequences defined in (a), or fragments thereof, or
(c) DNA sequences that would hybridize to DNA sequences (a) and (c), if not for the degeneracy of the genetic code.

In other words, the DNA sequence of the present invention also includes the following sequences:
(a) a DNA sequence that is integrated into the vector pEF18S-A2α (deposit FERM BP-4565) included in the E. coli strain DH5. vector pHT1-231 (deposit FERM BP-4564) included in the E. coli strain DH5. vector pHTGI (deposit FERM BP-4617) included in E. coli strain DH5. vector pHGT1 (deposit FERM BP-4616) included in E. coli strain DH5 and encodes the amino acid sequence of a protein having TPO activity, or
(c) DNA sequences that hybridize (under stringent conditions) with the DNA sequences described in (a), or fragments thereof, and encode the amino acid sequence of a protein having TPO activity.

 As used herein, representative "stringent" hybridization conditions are those that are used in the PCR examples of DNA amplification of this invention using degenerate and / or unique oligonucleotide primers (probes). See also, for example, Chapters 11 and 14 of Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) Unit 2.10 Current Protocols in Molecular Biology, Ausubel et al., Eds. Current Protocols, USA, 1993).

 Also the sequence of the present invention is a DNA sequence that encodes a protein with TPO activity and contains a nucleotide sequence encoding the positions 1-163 of the amino acid sequence represented by SEQ 1D N 6.

 Such DNA sequences may also contain a restriction enzyme cleavage site and / or an additional DNA sequence at the initiation, termination, or intermediate site, which facilitate the construction of easily expressed vectors. When using a non-mammalian host, a preferred codon for gene expression in that host may be included.

 An example of the DNA sequence of this invention is a cDNA molecule obtained by isolation of mRNA from mammalian cells, including humans, followed by screening of this cDNA in the usual way from a cDNA library obtained in a known manner. In this case, mRNA sources can be McA-R H8994 cell line derived from rat hepatocytes, NTS cells, H411-E cells, rat liver, kidneys, rat brain and small intestine, human liver, etc.

 Another example of the DNA sequence of the present invention is a genomic DNA molecule, which is obtained by screening it in the usual way from a genomic library obtained in a known manner from mammalian cells, including humans. In this case, the sources of genomic DNA may be chromosomal DNA preparations obtained from humans, rats, mice, etc.

 A DNA sequence encoding a TPO derivative can be obtained from a cDNA sequence encoding a protein with TPO activity as described above by modifying this cDNA using site-directed mutagenesis, thereby partially modifying the corresponding amino acid sequence.

 After determining the amino acid sequence or DNA sequence of a protein having TPO activity, in the present invention, the DNA sequence encoding a partially modified amino acid sequence can be easily obtained by chemical synthesis.

 The DNA sequence of the present invention is valuable material for the large-scale production of a protein having TPO activity using various recombinant DNA methods.

 The DNA sequence of the present invention is also applicable as a labeled probe for isolation of a gene encoding a related TPO protein, as well as cDNA and genomic DNA encoding TPO of other mammalian species. It is also applicable in the gene therapy of humans and other mammalian species. In addition, the DNA sequence of the present invention is applicable for the development of transgenic species of small nutrients that can be used as a eukaryotic host for large-scale production of TPO (Palmiter et al., Science, vol. 222, pp. 809-814, 1983).

 The invention also provides a vector that integrates the above DNA sequence encoding a protein having TPO activity, host cells transformed with this vector, and a method for producing a protein with TPO activity, comprising culturing host cells and separating and purifying an expressed protein having TPO activity .

 Examples of host cells useful in this case are prokaryotic cells such as E. coli cells and the like, and eukaryotic cells such as yeast, insects, mammals and the like. Illustrative examples of mammalian cells are COS cells, Chinese hamster ovary (CEO) cells, C-127 cells, hamster baby kidney (BWK) cells, and the like. Illustrative examples of yeast are baker's yeast (Saccharomyces cerevisiae), methanol-assimilating yeast (Pichia pastoris), and the like. Illustrative examples of insect cells are cultured silkworm cells, etc.

 As for the vectors for use in the transformation of these host cells, pKS30 (Shimatake H. and M. Rosenberg, Nature, 292, 128-132, 1981), pTrC99A (Amann E. et al., Gene, 69, 301-315, 1988) and the like. For transformation of mammalian cells, pSV2-neo (Southern and Berg. J. Mol. Appl. Genet., 1, 327-341, 1982), pCAGGS (Niwa et al., Gene, 108, 193-200, 1991) can be used, pcDL-SRα296 (Takebe et al., Mol. Cell. Biol. 8, 466-472, 1988) or the like. For yeast cells, pG-1 (Schena M. and Yamamoto K.R., Science, 241, 965-967, 1988) or the like can be used. To transform the silkworm, a transducing vector for a recombinant viral construct, for example, pAC373, can be used (Luckow et al., Bio / Technology, 6, 47-55, 1988).

 If required, each of these vectors may contain the beginning of replication, a marker (s) of selection, a promoter, etc., as well as an RNA splicing site, a polyadenylation signal, etc. in the case of vectors for use in eukaryotic cells.

 As for the start of replication, a sequence obtained, for example, from SV40, adenovirus, bovine papilloma virus, or the like, can be used in mammalian cell vectors. In vectors for E. coli cells, a sequence derived from ColE1, R factor, F factor or the like can be used. In vectors for yeast cells, a sequence derived from 2 μm DNA, ARS1 or the like can be used.

 As for promoters for gene expression, in mammalian cell vectors, promoters derived, for example, from retrovirus, polyoma virus, adenovirus, SV40, and the like, can be used. A promoter derived from the bacteriophage λ, for example, the trp, lpp, lac or tac promoter, can be used in vectors for E. coli cells. The ADH, PHO5, GPD, PGK, or MAFα promoter can be used in baker's yeast cell vectors, and the AOX1 promoter or the like. in vectors for methanol-assimilating yeast. A promoter derived from nuclear polyhedrosis virus can be used in silkworm vectors.

 Typical examples of selectable markers useful in mammalian cell vectors are the neomycin resistance gene (neo), the thymidine kinase (TK) gene, the dihydrofolate reductase gene (DHFR), the E. coli xanthine-guanine phosphoribosyltransferase gene (ECOgpt), and the like. Illustrative examples of selectable markers useful in vectors for E. coli cells are the kanamycin resistance gene, ampicillin resistance gene, tetracycline resistance gene, etc., and the leu2, Trp1, Ura3 genes, etc. are markers for yeast cells. P.

 Obtaining a protein with TPO activity using appropriate combinations of host vector systems can be accomplished by transforming the appropriate host cells of the recombinant DNA obtained by inserting a gene of the invention into a suitable site of the specified vector, culturing the resulting transformant, followed by separation and purification of the target polypeptide from the obtained cells or culture medium or filtrate. Commonly used tools and procedures can be used for this method in combination.

 When expressing the target gene, the original signal sequence can be modified or replaced by a signal sequence derived from another protein in order to obtain a homogeneous N-terminus of the expressed product. Homogenization of the N-terminus can be performed by modifying (replacing or adding) amino acid residues at or near the N-terminus. In the case of expression using E. coli as a host cell, for example, a lysine residue may be added in addition to the methionine residue.

 The new protein of the present invention having TPO activity (hereinafter referred to as “the protein of the present invention”) includes proteins, each of which contains the amino acid sequence shown in SEQ 1D 2, 4 or 6. Derivatives of TPO, the amino acid sequences of which are partially modified (by substitution, deletion , insertion or addition) are also included in this invention, provided that the TPO activity is not disturbed by such a modification.

 In other words, the protein of the present invention includes protein molecules, the amino acid sequences of which are essentially the amino acid sequences shown in SEQ 1D 2, 4 or 6.

 The expression "amino acid sequences are essentially the amino acid sequences shown in (or represented by) SEQ 1D 2, 4 or 6," as used herein, means that said amino acid sequences include the sequences shown in SEQ 1D 2, 4 or 6, as well as the sequences shown in SEQ 2, 4 or 6, which have a partial modification, such as substitution, deletion, insertion, addition, etc., provided that such modifications do not violate the TPO activity.

 The protein of the present invention includes a protein containing positions 7-151 of the amino acid sequence shown in SEQ 1D 6 and having TPO activity. Also, the protein of the present invention is a protein that has TPO activity and contains positions 1-163 of the amino acid sequence represented by SEQ 1D 6.

 Examples of other TPO derivatives of the invention include a derivative whose in vivo stability and longevity have been improved by amino acid modification (substitution, deletion, insertion or addition), a derivative in which at least one potential glycosylation has been altered by deletion or addition, a derivative in which at least one cysteine residue has been deleted or replaced with another amino acid residue (for example, an alanine or serine residue).

 Preferably, the protein of the present invention is characterized in that it is separated and purified from host cells transformed with a recombinant vector containing a cDNA molecule, a genomic DNA molecule, or a DNA fragment obtained by chemical synthesis.

 When intracellular expression is performed using a bacterium, such as E. coli as a host, a protein is obtained in which the initiating methionine residue is added to the N-terminal side of the protein molecule having TPO activity, which is also included in this invention. Depending on the host used, the resulting protein with TPO activity may be glycosylated or non-glycosylated, and each of these cases is included in this invention.

 The protein of the present invention also includes naturally occurring TPO-active proteins purified and isolated from natural sources, such as cell culture medium having TPO activity, urine, serum or human plasma.

 A method for purifying SRW from such natural sources is also included in this invention. This purification method can be performed by applying one or a combination of commonly used protein purification steps, such as ion exchange chromatography, lectin affinity chromatography, triazine dye affinity chromatography, hydrophobic chromatography, gel chromatography, reverse phase chromatography, heparin affinity chromatography , sulfated gel chromatography, hydroxyapatite chromatography, isoelectric focusing, metal chelate chromatography, preparative electrophoresis, gel electrophoresis with isoelectric focusing, etc. A cleaning process in which certain methods are used in combination using the physicochemical properties of the SRW, which can be derived from examples of this application, is also included in this invention. In addition, affinity chromatography using antibodies capable of recognizing TPO can also be used. Further, it was found that TPO is a ligand for MPl (de Sauvage et al., Nature, 369; 533-538 (1984); Bartley et al., Cell, 77: 1117-1124 (1994); Kaushansky et al., Nature, 369: 565-568 (1984), by which TPO can be purified using an affinity gel column to which MPl is linked. More specifically, an example of such a column is a MPl-X column prepared by linking a resin to the extracellular region of Mpl (MPl-X) obtained by recombinant DNA methods using CHO cells as a host (Bartley et al., Supra).

 As described herein, the TPO polypeptides of this invention are further distinguished by their ability to bind to the MPl receptor and specifically to its extracellular (soluble) domain.

Also included in the invention is a protein encoded by a portion of DNA that is complementary to the protein coding strand of the human cDNA or the TPO gene genomic DNA sequence, namely, the “complementary inverted protein” described
Tramontano et al. (Nucl. Acids Res., Vol. 12, pp. 5049-5059, 1984).

Also included in the invention is a protein of the invention that is labeled with a detectable marker, for example 125 ₁ , or biotinylated, providing a reagent useful for detecting and quantifying TPO or TPO receptor expressing cells in solid samples, such as tissues, and liquid samples, such as blood, urine, etc.

 The biotinylated protein of the present invention is applicable if it is bound to immobilized streptavidin to remove megakaryocytes from the bone marrow during autologous bone marrow transplantation. It is also applicable if it is bound to immobilized streptavidin to concentrate autologous or allogeneic megakaryocyte cells during autologous or allogeneic bone marrow transplantation. A conjugate of TPO with a toxin, such as ricin, diphtheria toxin or the like, with a radioactive isotope is applicable in antitumor therapy and in the conditioning of bone marrow transplantation.

 The invention also provides a nucleic acid material that is useful for tagging with a detectable marker, including a radioactive marker or non-radioactive marker, such as biotin, or when used in a hybridization procedure to detect the position of the human TPO gene and / or related gene family on a chromosome map. Such material is also applicable for proving abnormalities in the human TPO gene at the DNA level, and it can be used as a genetic marker to confirm abnormalities in adjacent genes.

 The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a protein of the invention together with a useful and effective diluent, antiseptic agent, solubilizing agent, emulsifier, adjuvant and / or carrier. The term "therapeutically effective amount" as used herein means an amount that provides a therapeutic effect for the claimed conditions and routes of administration and conditioning. Such a composition is used in the form of a liquid, lyophilized or dried preparation. It contains a diluent selected from various buffers (Tris-HCl, acetate buffer and phosphate buffer, for example) having different pH values and ionic strength, anti-surface absorption additive, such as albumin or gelatin, a surfactant such as Tween 20 , Tween 80, Pluronic F68 or a bile salt, a solubilizing agent such as glycerin or polyethylene glycol, an antioxidant such as ascorbic acid or sodium metabisulfite, an antiseptic agent such as thimerosal, benzyl alcohol or paraben, and wear spruce or tonicity agent, such as lactose or mannitol. A composition comprising covalently linking a protein of the present invention to a polymer such as polyethylene glycol, chelating the protein with metal ions, incorporating the protein into a granular preparation or on a surface consisting of a polymer compound such as polylactic acid, polyglycolic acid or hydrogel, or including beam in liposomes, microemulsions, micelles, single-layer or multi-layer vesicles, "shadows" of red blood cells or spheroblasts. Such a composition will affect physical conditions, solubility, stability, in vivo release rate and in vivo TPO clearance.

 The choice of composition is carried out depending on the physical and chemical properties of the used TPO-active protein. The invention also provides a granular composition in which the granules are coated with a polymer, such as poloxamer or poloxamine, and TPO, which binds to antibodies for tissue-specific receptors, ligands or antigens, or to tissue-specific receptor ligands. Other examples of the compositions of this invention are granular compositions that have a protective coating and contain a protease inhibitor or penetration enhancer for use in various modes of administration, such as parenteral, pulmonary, transnasal and oral administration.

 A pharmaceutical composition containing a protein of the present invention can be administered several times a day, usually in an amount of 0.05 μg - 1 mg / kg (based on TPO protein) of body weight depending on the condition and gender of the patient, route of administration, etc. .

 The pharmaceutical composition containing the protein of the present invention can be administered at a dose of 25,000-500,000 active ingredient (in units of relative activity obtained using test M-07e, which will be described later) per kg of body weight once or several times a day for 1 -7 days a week, depending on the symptoms, gender of the patient and route of administration.

 The authors of this application have confirmed that with respect to the C-terminal side of human TPO, it can be noted that activity is maintained when amino acid residues up to the 152nd amino acid in the amino acid sequence shown in SEQ 1D 6 are deleted, and as for the N-terminal side, activity preserved even if amino acid residues up to the 6th residue of the amino acid sequence are deleted.

 Thus, a protein having TPO activity, containing the amino acid sequence from the 7th to the 151st amino acid residue of SEQ 1D 6, and modified (by substitution, deletion, insertion or addition) in other parts, can preferably be used as an effective ingredient in this study as well as unmodified protein. A more preferred TPO derivative is a derivative having the amino acid sequence 1-163 of the sequence SEQ 1D 6.

 A composition comprising at least one of additional hematopoietic factors such as EPO, G-CSF, GM-CSF, M-CSL, IL-1, IL-2, IL-3, IL-4, IL-5, IL- 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, LIF and SCF, in addition to the protein of the present invention, is also included in this invention.

 The protein of the present invention is applicable to the treatment of various thrombocytopenic diseases, either individually or in combination with other additional hematopoietic factors. Examples of other additional hematopoietic factors are EPO, G-CSF, GM-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 8, IL-9, IL-10, IL-11, IL-12, IL-13, LIF, and SCF.

 There are many diseases characterized by platelet-related disorders, such as thrombocytopenia, caused by impaired platelet formation or shortened platelet life (caused by the destruction or consumption of platelets) that can be treated with the protein of the present invention. For example, it can be used to enhance platelet recovery in a thrombocytopenic patient whose disease is caused by congenital hypoplastic anemia with pancytopenia and other abnormalities (Fanconi syndrome) or hypoplastic anemia caused by chemotherapy, radiation, myelodysplastic syndrome, acute myelocytosis, and cystic myelocytoma brain. Thrombocytopenia caused by a shortened platelet or megakaryocyte life span includes idiopathic thrombocytopenic purpura, hypoplastic anemia, acquired immunodeficiency syndrome (AIDS), multiple intravascular coagulation syndrome, and thrombotic thrombocytopenia. In addition, the protein can also be used in the case of platelet autotransfusion, in which TPO is administered to the patient before surgery to increase the number of the patient’s own platelets and, therefore, an increased platelet count is used as transfused platelets at the time of surgery.

 Another use of the protein of the present invention is the treatment of diseases associated with a temporary decrease or destruction of platelets caused by other chemical or pharmaceutical drugs or therapeutic treatments. SRW can be used to enhance the release of new "intact" platelets in such patients.

 Further, the present invention relates to antibodies specific for TPO. The protein of the present invention can be used as antigens, and the corresponding antibodies include both monoclonal and polyclonal antibodies and chimeric antibodies, namely, "recombinant" antibodies obtained by conventional methods. To obtain such anti-TPO antibodies, human TPO itself can be used as an antigen or, alternatively, a partial peptide of human TPO can be used as an antigen. When using antibodies to the antigen of such a peptide, the epitope can be determined by specification of the antigenic region. On the other hand, when using antibodies to the antigenic protein itself (TPO), the antigenic region can be determined by analysis of the epitope of the antibody. In such cases, these antibodies, each of which has a zitope so determined, can be used for fractionation, detection, quantification and purification of various SRW, which differ in their properties, for example, the type of added sugar, the length of the peptide chains, etc.

 The TPO peptide containing the amino acid sequence selected in this way is synthesized and bound to a suitable carrier protein, for example, albumin, KLH (fissurelli hemocyanin) or the like, by covalent binding to prepare an immunoreactive antigen. Alternatively, a peptide of the type of a peptide with multiple antigens (MAP) is obtained for use as an antigen according to the method of Tam (Proc.Natl. Acad. Sci. (USA) 85: 5409-5423, 1983). Then, the antigen thus prepared, together with an adjuvant or the like. used to immunize mammals, birds, etc., which are commonly used to produce antibodies such as rabbits, mice, rats, goats, sheep, chickens, hamsters, horses, guinea pigs, etc. From the animal thus immunized, its antiserum and cells from which the polyclonal antibodies were obtained were obtained. When using a peptide as an antigen, the epitope can be determined by the specification of the antigenic region. In this case, the regions of various TPO molecules having different mol. masses are screened and identified by immunological engineering. For example, a peptide deletion region can be screened and identified. In addition, an antibody gene or part of this gene is cloned from cells expressing the target antibody to produce an antibody molecule that has been expressed using genetic engineering.

 Compared to a polyclonal antibody, which contains various antibodies specific for different antigenic determinants (epitopes), a monoclonal antibody is specific for only one antigenic determinant on an antigen. A specific TPO antibody is applicable to improve the selectivity and specificity of diagnostics based on the antigen-antibody reaction and the analytical test method, as well as in the separation and purification of SRW. In addition, such antibodies can be used to neutralize or remove TPO from serum. Monoclonal antibodies are also useful for detecting and quantifying TPO, for example, in whole blood serum.

 The anti-human TPO antibody of the present invention can be used as a ligand in affinity chromatography for purification and isolation of human TPO. To fix the antibody so that it can be used in affinity chromatography, any conventional methods for fixing various enzymes can be used. For example, a method using a CNBr carrier is activated — activated Sepharose 4B (manufactured by Pharmacia Fine Chemicals Co.) or the like. To purify human TPO using a fixed anti-human TPO antibody, the fixed antibody is loaded into a column and the liquid containing human TPO is passed through this column. As a result of this operation, a large amount of human SRW is adsorbed onto the carrier in a column. Suitable elution solvents are, for example, glycine-HC1 buffer (pH 2.5), sodium chloride solution, propionic acid, dioxane, ethylene glycol, chaotropic salt, guanidine hydrochloride, urea, etc. By elution with a similar solvent, highly purified human TPO is eluted.

 The antibody of the present invention can be used to determine human TPO by immunochemical quantitative analysis, in particular, enzyme-linked immunosorbent assay carried out according to the method of solid-phase "sandwich".

 The advantage of monoclonal antibodies is that they can be produced by hybridoma cells in a medium that does not contain any other immunoglobulin molecules. Monoclonal antibodies can be obtained from culture supernatants of hybridoma cells or from murine ascites induced by intraperitoneal injection of hybridoma cells. The hybridoma method, originally disclosed by Kohler Milstein (Eur.J. Immunol. 6: 511-519, 1976), can be widely used to produce hybrid cell lines that have high levels of monoclonal antibodies for a number of specific antigens.

 One or more steps that are commonly used for protein purification (affinity chromatography, for example Protein A affinity chromatography, Protein G affinity, can be combined to isolate the desired antibody from the material thus obtained containing the antibody, such as antiserum, etc. chromatography, Avid gel chromatography, fixed anti-immunoglobulin gel chromatography, etc. as well as cation exchange chromatography, anion exchange chromatography, affinity chromatography using lectin, adsorption chromatography dye chromatography, hydrophobic chromatography, gel permeation chromatography, reverse phase chromatography, hydroxyl-apatite chromatography, fluorine-apatite chromatography, metal chelate chromatography, isoelectric focusing chromatography, distribution chromatography, isoelectric focusing electrophoresis, etc.). In addition to these methods, an affinity purification method using an antigen can also be applied in which a carrier gel or membrane is prepared to which the human TPO protein itself or a peptide containing an antigen region or part of this region, i.e. a molecule capable of recognizing the target antibody is added to the carrier or membrane thus obtained, containing the antibody material, so that the target antibody is adsorbed on the carrier or membrane. Thereafter, the antibody adsorbed in this manner is eluted and recovered under suitable conditions.

 The present invention also allows the use of endogenous DNA sequences encoding a TPO polypeptide to produce large quantities of polypeptide products. For example, in vitro and ex vivo homologous recombination methods can be used to transform host cells to provide expression or enhanced expression of polypeptides. Preferred host cells are human cells (e.g., liver, bone marrow, etc.) into which a promoter or enhancer sequence is introduced using flanking sequences that are homologous to the target region in the cell genome, resulting in or enhanced TPO expression of the polypeptide. See, for example, U.S. Letters Patent 5,272,071, published by PCT WO 90/14092, WO 91/06666, WO 91/09955.

The invention also provides methods for producing the TPO polypeptide described above, comprising the steps of expressing a polypeptide encoded by the DNA of the invention in a suitable host and isolating the TPO polypeptide. If the expressed TPO polypeptide is a Met ^-2 - Lys ^-1 polypeptide, such methods may further include the step of cleaving the Met ^-2 - Lys ^-1 from the isolated TPO polypeptide.

Also provided are methods for producing TPO polypeptides having a [Gly ^-1 ] structure, comprising the steps of introducing into a suitable host cell a DNA encoding a glutathione S-transferase polypeptide, 5 ′ with respect to sequences encoding a TPO polypeptide, wherein GST and TPO polypeptide encoding the sequences are separated by DNA encoding a thrombin recognizing polypeptide, isolating the GST-TPO expression product and treating the expressed polypeptide with thrombin to remove the GST amino acids. The resulting TPO polypeptides have a [Gly ^-1 ] structure.

 The invention is described below in detail.

 (A) Purification of rat TPO, analysis of partial amino acid sequences of purified rat TPO and analysis of the biological characteristics of purified rat TPO.

 The authors of this invention first tried to purify a protein (rat TPO), which has the activity of enhancing the proliferation and differentiation of rat CFU-MK. In this purification study, many errors were made in the purification procedures, such as the selection of various natural food sources, the selection of chromatographic gels, and separation methods. As a result, the authors were able to purify a protein having TPO activity from the blood plasma of thrombocytopenic rats induced by X-ray or γ-ray irradiation using TPO activity as a marker in the rat CFU-MK test, which will be described later in the "Standard Example", and determine the partial amino acid sequences of the purified protein ("Examples 1 and 2").

 The biological characteristics of the plasma derived TPO from plasma were also determined in Example 3.

 The basics of the procedures from purification of rat TPO to determination of partial amino acid sequences of the purified protein are described below.

 (i) A blood plasma sample was obtained from approximately 1100 thrombocytopenic rats induced by X-ray or γ-ray and subjected to chromatography on Sephadex G-25, anion exchange chromatography (Q-Sepharose FF) and lectin chromatography (WGA-Agarose) in this order, obtaining TPO-active fraction adsorbed on a WGA-Agarose.

 (ii) The resulting adsorbed TPO-active fraction was then subjected to triazine dye affinity chromatography (TSK AF-BLUE 650 MN), hydrophobic chromatography (Phenyl Sepharose 6 FF / LS), and gel chromatography (Sephacryl S-200 HR) in this sequence. Since TPO activity was divided into 4 peaks (F1 (fraction 1) as the highest molecular weight fraction, then F2, F3 and F4) by gel filtration on Sephacryl S-200 HR, each of the TPO-active fractions F2 and F3 was concentrated to obtain a sample F2 SRW with high mol. weight and samples F3 with low molecular weight SRW, which were used separately in the subsequent stages of purification.

 (iii) Sample F3 of low molecular weight TPO was subjected to preparative reverse phase chromatography (YMC-Pack PRO-TEIN-RP), reverse phase chromatography (YMC-Pack CN-AP), reverse phase chromatography (Capcell Pack CI) in the indicated sequence. When the TPO-active fraction thus obtained was deposited on SDS-PAGE for electrophoresis and the TPO-active substance was extracted from the gel, the presence of TPO activity was detected in the band corresponding to the average mol. masses approximately 17000-19000 under non-reducing conditions.

 (iv) Subsequently, all TPO-active fractions were applied to SDS-PAGe gel under non-reducing conditions and transferred onto a PVDF membrane. By carrying out systematic limited enzymatic hydrolysis of the protein on the PVDF membrane to the peptide fragments, the partial amino acid sequences of the rat TPO protein were determined. Based on information about the amino acid sequences of two peptide fragments, the rat TPO gene was cloned.

 (v) Separately, a high molecular weight TPO sample F2 obtained with Sephacryl S-200 HR was purified in the same manner as a low molecular weight TPO sample F3. When the TPO-active fraction obtained by reverse phase chromatography (Capcell Pack C1) was applied to the SDS-PAGE gel and the TPO-active substance was extracted from the gel, the presence of TPO activity was detected in the band corresponding to the average mol. masses approximately 17000-22000 under non-reducing conditions.

(B) Specialization of rat TPO producing cells, production of mRNA, and construction of rat cDNA library
Since rat TPO purified in this way was obtained from blood plasma, it was necessary to screen organs or cells as sources of mRNA for use in cDNA cloning. As a result, TPO activities in various organs and supernatants of cell cultures were screened based on the biochemical and biological properties of the rat plasma-derived TPO. As a result of TPO activity, almost equal to the activity of rat plasma-derived TPO were found in the cultural supernatant of McA-RH8994, HTC and H4-11-E rat hepatocyte cell lines and in the supernatant of rat primary hepatocyte culture (Example 4).

 On the other hand, the expression vector pEF18S was constructed from 2 expressing vectors pME189 and pEFBOS (Example 5). The construction of this vector made it possible to easily clone cDNA using a highly efficient expression vector having multiple cloning sites that can be used to integrate insertions.

In addition to the expression vector indicated above, plasmid vectors pUC and pBR and vectors based on phage λ were mainly used to construct cDNA libraries.
McA-RH8994 cells were cultured, homogenized with the addition of a guanidine thiocyanate solution, and then subjected to CSC1 density gradient centrifugation to obtain total RNA (Example 6).

 RNA production can also be carried out using the phenolic method (with hot phenol), a method using acidic guanidinium-phenol-chloroform, and the like.

After poly (A) ⁺ RNA was separated from total RNA using oligo-dT-immobilized latex particles, the first cDNA strand was synthesized using reverse transcriptase using oligo-dT as a primer to which Notl restriction enzyme recognition sequence was added, followed by synthesis second cDNA strand using RNase H and E. coli DNA polymerase 1. EcoRl was added to the thus obtained double-stranded cDNA, the obtained cDNA was ligated with the mammalian cell expression vector pEF18S constructed in Example 5, which was digested with Notl and EcoRl, and then the ligated cDNA was transformed into competent cells of E. coli strain DH5 to construct a cDNA library (example 7).

(C) Obtaining (cloning) of a rat TPO cDNA fragment using PCR
The DNA sequence was derived from the partial amino acid sequence of rat TPO purified from rat blood plasma for the synthesis of degenerate primers used in the polymerase chain reaction (PCR). Primers for use in PCR can also be obtained based on a different amino acid sequence than the position of the primers used here. High degeneracy primers can also be used without inosine. In addition, primers with reduced degeneracy can be designed by using a codon highly applicable in rats (Wada et al., Nucleic Acids Res., Vol. 18, p. 2367-2411, 1990).

 When plasmid DNA was extracted from the entire portion of the cDNA library obtained as described above and PCR was performed using the extracted DNA as a template, a band of approximately 330 bp was found, which was then determined by nucleotide sequencing as a DNA fragment (A1 fragment), encoding part of rat TPO (example 8).

(E) Screening of rat TPO cDNA using PCR, rat TPO cDNA sequence and confirmation of TPO activity
The cDNA library obtained as described above was divided into pools, each of which contained approximately 10,000 clones, and plasmid DNA was extracted from each of the 100 pools. When conducting PCR using each pool of plasmid DNA as a template and primers newly synthesized based on the nucleotide sequence of the A1 fragment, bands were found in 3 pools that were considered specific. One of these 3 pools was divided into sub-pools, each of which contained approximately 900 clones. Plasmid DNA was purified from 100 subpools for PCR as described. As a result, a specific band was found in 3 sub pools. One of these pools was divided into sub-pools, each of which contained 40 clones, and finally each clone was screened using PCR in the manner described. As a result, clone pEF18S-A2α was isolated, which apparently encodes rat TPO cDNA (examples 9 and 10).

 When analyzing the nucleotide sequence of this clone, it was found that it encodes a partial amino acid sequence of a protein purified from rat blood plasma, confirming, therefore, the strong likelihood that this clone contains rat TPO cDNA (Example 10).

 Upon purification of plasmid DNA from clone pEF18S-A2α, obtained as described above, and transfection into COS 1 cells, TPO activity was detected in the supernatant of the transfected cell culture. This confirmed that clone pEF18S-A2α contains cDNA encoding rat TPO (Example 11).

(E) Detection of TPO mRNA in various rat tissues
The expression of TPO mRNA in rat tissues was analyzed using PCR. Specific expression was found in the brain, liver, small intestines, and kidneys (Example 12).

(F) Construction of a human cDNA library
Based on the results of Examples 4 and 12, the liver was selected as the starting tissue for cloning human TPO cDNA. Subsequently, a cDNA library was constructed using commercial mRNA obtained from a healthy human liver. Using pEF-18S as a vector, as in the case of the rat cDNA library, cDNAs were synthesized by similar methods, obtaining a library of directionally cloned cDNAs using Not1 and EcoR1 restriction enzymes. The library obtained by introducing vector ligated cDNA into E. coli DH5 contained approximately 1,200,000 clones (Example 13).

(G) Obtaining (cloning) a cDNA fragment of human TPO using PCR
Several primers for PCR were synthesized based on the nucleotide sequence of clone pEF18S-A2α encoding rat TPO cDNA. In the synthesis of cDNA using commercial mRNA obtained from normal human liver and PCR using these primers and cDNA as a template, a band of approximately 620 bp was observed. When analyzing the nucleotide sequence, it was found that this clone contains a DNA fragment that has a homology of approximately 86% with rat TPO cDNA, thus confirming the strong likelihood that it is part of the gene encoding human TPO (Example 14).

(H) Screening of human TPO cDNA using PCR, human TPO cDNA sequence and confirmation of TPO activity
The human cDNA library obtained as described above was amplified, divided into pools, each of which contained approximately 10,000 clones, and plasmid DNA was extracted from each of 90 pools. When conducting PCR using each pool of plasmid DNA molecules as a template and primers newly synthesized based on the nucleotide sequence of the human TPO fragment obtained in Example 14, possible bands were found in 3 pools. One of these pools was divided into subpools, each of which contained 5000 clones, and plasmid DNA was purified from each of the 90 subpools. When PCR was performed using each pool of plasmid DNA as a template in the same way, possible bands were detected in 5 sub pools. When dividing one of these pools into sub-pools, each of which contained 250 clones, purifying plasmid DNA from each of the 90 sub-clones and performing PCR, possible bands were found in 3 sub-clones. When dividing one of these pools into subpools, each of which contained 30 clones, purifying plasmid DNA from each of 90 subpools and performing PCR, possible bands were found in 3 subpools. After that, 90 colonies were isolated from one of these subpools, and plasmid DNA purified from each of these colonies was subjected to PCR, finally obtaining a clone called HL34 (Example 15).

 When analyzing the nucleotide sequence of plasmid DNA in this clone, it was found that this clone contains cDNA having approximately 84% homology with rat TPO cDNA (Example 16).

 When purifying cloned plasmid DNA and transfecting it into COS1 TPO cells, activity was detected in the culture supernatant of transfected cells. Thus, it was confirmed that this plasmid clone contains cDNA encoding human TPO (Example 17).

 However, this cDNA is apparently an artificial cloning product, because no termination codon was found in this clone and a sequence similar to a poly (A) tail was found at its 3'-end. Subsequently, an expression vector was constructed that encoded the amino acid sequence, with the exception of the portion corresponding to a sequence similar to a poly (A) tail. When expressing the vector so constructed in COS 1 TPO cells, activity was detected in the obtained culture supernatant (Example 18).

 Using PCR, a DNA fragment was obtained in the 3'-terminal region of human TPO to analyze the structure of full-sized cDNA.

 The determination of the nucleotide sequence of this fragment revealed that it partially overlaps with the cDNA carried by the HL34 clone obtained in Example 15. It was also expected that a full-sized human TPO cDNA could contain an open reading frame and encode a protein consisting of 353 amino acids, including signal a sequence of 21 amino acids (example 19).

 In addition to the cloning method described above, cloning of human TPO cDNA can be achieved by colony hybridization or plaque hybridization using a rat TPO cDNA fragment as a probe, using a library constructed using pUC or pBR vectors based on phage λ or the like . When constructing a degenerate probe, inosine can be used to reduce the degree of degeneracy. If there is a test method capable of specifically detecting TPO activity or having high sensitivity, expression cloning can be carried out using a library, as was done in this invention.

 However, since the content of human TPO RNA encoding is apparently extremely small in the normal human liver, as will be described later in Example 15 (the content calculated from the results of this example was in the ratio of 1: 3000000), hybridization screening, in which the synthesized oligonucleotide or rat or human TPO cDNA fragment as a probe will be very difficult to perform since the number of clones or plaques to be processed becomes very large and the sensitivity and specificity of the method hybridizations are lower than the sensitivity and specificity of the PCR method. In fact, the inventors of this invention hybridized colonies of two million clones in a normal human liver cDNA library prepared as described in Example 13 using a rat TPO cDNA fragment as a probe, but failed to obtain a human TPO cDNA clone.

(I) Reconstruction of cDNA derived from normal human liver
Since the HL34 clone obtained in Example 15 apparently contains an incomplete cDNA, the cDNA library was reconstructed using a commercial preparation of poly (A) ⁺ RNA obtained from normal human liver to obtain complete human TPO cDNA. This library (hTPO-F1), obtained by introducing a vector ligated cDNA into E. coli DH5, contained 1.0 x 10 ⁶ transformants (Example 20).

(J) Screening of a TPO cDNA Clone, Sequencing and Expression of Human TPO CDNA and Confirmation of TPO Activity
PCR primers were synthesized based on the nucleotide sequence (SEQ 1D 3) of the partial cDNA obtained in Example 14 and the nucleotide sequence (SEQ 1D 196) of the full human TPO cDNA predicted in Example 19.

 The human liver cDNA library (hTPO-F1) constructed in Example 20 was divided into 3 pools (pools 1-3). PCR was performed using plasmid DNA obtained from each pool as a template and synthesized primers. As a result, using a plasmid DNA obtained from pool 3, a DNA fragment having the expected size was amplified. Then pool 3 was divided into subpools, each of which contained 15,000 transformants, and screened using PCR, as described above. As a result, amplification of DNA having the expected size was detected in 6 out of 90 pools. When dividing one of these positive pools into sub-pools, each of which contained 1000 clones, plasmid DNA extraction and PCR using the same method did not observe DNA amplification. This was thought to be caused by low plasmid DNA extraction due to weaker growth of the target clone compared to the growth of other clones. Therefore, the initial pool 3 was propagated on 100L B plates with an inoculum size such that 4,100 colonies grew on each L B plate, and a replicate cup was obtained from each of the plates so inoculated. When conducting PCR of these DNAs extracted from colonies growing on this plate, amplification of the expected band was observed in one of 100 sub-pools.

 Two filter-replicas were prepared from each cup of this subpool, and colony hybridization was performed using a labeled probe (fragment EcoR1 / BamH1 of plasmid pEF18S-HL34). As a result, only one signal was positive. Colonies were collected from the original plate and again inoculated onto L B plate. DNA samples were obtained from 50 colonies growing on this plate and subjected to PCR, finally obtaining a clone called pHTF1 (Example 21). Methods such as hybridization, PCR, and expression cloning were mainly used in cDNA clone screening, and combinations of these methods increased screening efficiency and sensitivity or reduced screening labor. When determining the nucleotide sequence of the pHTP1 clone thus prepared, it was found that the pHTF1 clone had an open reading frame and the amino acid sequence of the protein encoded (potentially) by this open reading frame completely coincided with the deduced human TPO amino acid sequence (SEQ 1D 6). The nucleotide sequence differed from the deduced sequence (SEQ 1D 196) at 3 positions, but these differences did not cause changes in amino acids. It has been confirmed that the human TPO protein contains 353 amino acid residues, including a signal sequence of 21 amino acids. Upon receipt of plasmid DNA from the thus obtained pHTF1 clone and its transfection into COS 1 TPO cells, activity was detected in the culture supernatant (Example 23).

(K) Screening of human TPO chromosomal DNA using spot hybridization, sequencing and expression of human TPO chromosomal DNA and confirmation of TPO activity
Genomic library obtained from prof. T. Yamamoto (Gene Research Center, Tohoku University) was plated on 18 NZYM plates in such an inoculum size that one cup contained 30,000 phage particles, and from each of the 18 dishes thus prepared two filter replicas were made. Human TPO cDNA fragment (base position numbers 178-1025 in SEQ 1D 7) were amplified by PCR and purified. Spot hybridization was performed as a probe with this purified ³² P labeled fragment. 13 positive spots were obtained. The spots were collected from the original plates and inoculated again on NZYM cups in that size re inoculum, that 1000 spots were formed on each dish, two replica filters were prepared from each dish for hybridization of the spots under the conditions described above. As a result, positive signals were detected on all filters of 13 groups. One spot (plaque) was removed from each of the obtained phage DNA plates: Phase DNA samples thus obtained from 13 clones were checked for the presence of a cDNA coding region using PCR. Five of the 13 clones appear to contain the complete amino acid coding region predicted from this cDNA. Subsequently, one of these clones (λ HGT1 clone) was selected and analyzed by Southern blotting (using the probe indicated above). Since, in the case of Hind 111 cleavage, a single band of approximately 10 kb was observed, the DNA of this clone was cleaved and Hind 111 was subjected to agarose gel electrophoresis. Strip 10 kb excised from the gel, purified and subcloned into the pUC13 cloning fluctor, finally obtaining a clone called pHGT1 (Example 24).

 When determining the nucleotide sequence of the pHGT1 clone thus obtained, it was found that the chromosomal DNA carried by this clone contains the entire coding region for the protein predicted in Example 19, and the nucleotide sequence of this coding region completely coincides with the sequence of the predicted nucleotide sequence (SEQ 1D 196) .

 In addition, the region corresponding to the amino acid coding exon contained 4 introns and the nucleotide sequence was different from the sequence of the complete cDNA of the pHTF1 clone obtained in Example 21 (SEQ 1D 7).

 When determining the nucleotide sequences of the 4 remaining clones among the 5 chromosomal DNA clones independently selected by screening, screening revealed that 2 of the 4 clones were identical to the pHGT1 clone and the other 2 clones were similar to the pHGT1 clone, except that they had a different a nucleotide within a 3'-non-coding region as observed with the pHTF1 clone (Example 25).

 The EcoRI fragment in the thus obtained plasmid clone pHGT1 was ligated with the expression vector PEF18S to obtain the expression plasmid pEFHGTE of human TPO. After receiving this plasmid DNA (pEFHGTE) and transfecting it into COS 1 cells, TPO activity was detected in the culture supernatant (Example 26).

(L) Obtaining deletion derivatives of human TPO, their expression in COS1 cells and confirmation of TPO activity
The results of examples 18 and 22 revealed that the human TPO protein could exhibit its biological activity even after removal of its carboxy-terminal third. Thus, for further analysis of biologically active parts, experiments were carried out with deletion derivatives. A number of expression plasmids were prepared using PCR using the DNA of the plasmid clone pHT1-231 obtained in Example 18 as a template and the synthesized oligonucleotides as primers.

 Expression plasmids were obtained that contained DNA encoding the deletion derivatives of human TPO without the carboxyl terminal region of the TPO protein, i.e. deletion derivatives encoding the positions 1-211, 1-191, 1-171 and 1-163 of the amino acid sequence. When preparing plasmid DNA from each of these clones and transfecting it into COS 1 TPO cells, activity was detected in each culture supernatant (Example 27).

 When derivatives having a number of deletions of C-terminal amino acid residues to position 151 and other derivatives having a corresponding deletion of N-terminal amino acid residues to the 6th, 7th and 12th positions and activity after their expression in COS were constructed 1 cell was measured to analyze the TPO-bearing region in great detail; it turned out that the activity became undetectable by deletion of amino acid residues of the N-terminal side to position 7 or by deletion of amino acid residues of the C-terminal side to Provisions 151 (Examples 28 and 29).

 Derivatives of a protein having TPO activity can be obtained by modifying (deletion, substitution, insertion, or addition) of a cDNA encoding this protein. For such a modification, methods such as PCR, site-directed mutagenesis and chemical synthesis can be used.

(M) Expression of human TPO cDNA in CHO cell and TPO purification
An expression vector, pHTP1, was constructed that can express cDNA encoding the deduced human TPO amino acid sequence shown in SEQ 1D 6 in animal cells (Example 30).

 To construct the vector pDEF202-hTPO-P1, the cDNA region in pHTP1 was used for its expression in the CHO cell (Example 31).

 As a result of transfection of this vector into CHO cells and subsequent selection of transfected cells, transfected cells were obtained in which an expression vector carrying human TPO cDNA was integrated into the chromosomes of CHO cells (Example 32).

 In Example 32, large-scale cultivation of the CHO cell line (CHO 28-30 cells resistant to 25 nM MTX) producing human TPO was performed. Cells were obtained by transfection of human TPO expressing plasmid pDEF202-hTPO-P1 in CHO cells (Example 55).

 From the culture supernatant, 100 L was purified by human TPO (Example 56).

 Alternatively, human TPO was purified from the culture supernatant in Example 55 by another method (Example 57).

(N) Expression of human TPO cDNA in X63.6.5.3. cells and confirmation of its activity
Using the TPO cDNA region in the pBLTEN plasmid obtained in Example 30, an expression vector BMСGSneo-hTPO-P1 was constructed for use in X63.6.5.3. cells (example 33).

 After transfection X63.6.5.3. cells with this vector transformants were obtained in which the human expression cDNA encoding expression vector was integrated into their chromosomes. When culturing these TPO cells, activity was detected in the culture supernatant (Example 34).

(O) Expression of large quantities of human TPO in COS 1 cells and purification, measurement of mol. mass and biological characteristics of human SRW
The pHTP1 expression vector prepared in Example 30 was transfected into COS 1 cells to obtain a large amount (approximately 40 L total) of the culture supernatant containing the expressed product (Example 35).

 TPO purification was performed from approximately 7 L of cell-free culture supernatant of COS 1 cells obtained in Example 35 containing TPO derived from the pHTP1 expression vector. High TPO activity could be obtained using cation exchange chromatography steps, WGA column chromatography and reverse phase column chromatography (Example 36).

 Partially purified in this way TPO from the culture supernatant of COS 1 cells was used to measure mol. mass and analysis of biological activity (examples 37 and 38).

(P) Expression of human TPO in E. coli
The pGEX-2T / h vector (1-174) was constructed for use in expression of a fusion protein (called "GST-TPO- (1-174)") of glutathione-S-transferase and human TPO (amino acid residues 1-174) in E .coli. In this case, part of the nucleotide sequence of human TPO cDNA (approximately half of the 5'-side zone) was replaced by the preferred E. coli codons (Example 39).

 GST-TPO (1-174) was expressed in E. coli, the resulting cells were destroyed and then solubilized GST-TPO (1-174) contained in the precipitated fraction. Then, by combining various stages, including testing the conditions for the placement of TPO, testing the cleaning conditions (affinity column with glutathione, cation exchange column, etc.) and cleaving the GST region with thrombin, it was possible to partially purify a protein containing the amino acid sequence of TPO. This protein was confirmed to detect TPO activity in rat CFU-MK test system (Examples 40 and 41).

 The pCFM536 / h6T (1-163) vector was also designed for use in expression of a mutated human type TPO protein in E. coli (called "h6T (1-163")), in which the Ser residue is at position 1 and the Ala residue is at position 3 Human TPO (amino acid residues 1-163) were respectively replaced by Ala and Val, and both Lys and Met were respectively added at positions -1 and -2. The entire portion of the nucleotide sequence of human TPO cDNA (corresponding to amino acid residues 1-163) carried by this vector was replaced by codons preferred for E. coli (Example 42).

 h6T (1-163) was expressed in E. coli, the resulting cells were lysed, and then h6T (1-163) contained in the precipitated fraction was solubilized. Protein folding conditions were tested and the protein containing the TPO amino acid sequence was partially purified. It was confirmed that this protein exhibits TPO activity in rat CFU-MK test system (Examples 43 and 44).

 In addition, the pCPM536 / hMK vector (1-163) was constructed for use in expression of a mutated human TPO protein (called "hMKT (1-163)" in E. coli), in which the Lys residue and Met residue were respectively added to the positions -1 and -2. This hMKT (1-163) was expressed in E. coli as described in Example 43, and the expressed protein was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane and then subjected to analysis of the N-terminal amino acid sequence, confirming that this protein contains the described amino acid sequence (example 52).

 In addition, the pCFM536 / hMKT vector (1-332) was constructed for expression in E. coli of a mutated human TPO protein in which the Lys residue was added at position -1 and the Met residue at position -2 of human TPO (amino acid positions 1-332) (called "hMKT (1-322)"). This hMKT (1-332) was expressed inside E. coli in the same manner as in Example 42, and its expression was confirmed by Western blotting using antibodies against the human TPO peptide obtained in Example 45 below (Example 66).

(Q) Preparation of Anti-TPO Peptide Antibodies and Preparation of Anti-TPO Peptide Antibody Column
Rabbit polyclonal anti-TPO peptide antibodies were prepared using synthesized peptides corresponding to the three partial regions of the rat TPO amino acid sequence defined in Example 10. It was confirmed that these antibodies can recognize rat and human TPO molecules. In addition, peptides corresponding to 6 partial regions of the human TPO amino acid sequence shown in SEQ 1D 6 (or SEQ 1D 7) were synthesized and then used to produce rabbit polyclonal antibodies against TPO peptides. It was shown that the obtained antibodies can recognize human TPO (example 45).

 TPO can be purified by affinity column chromatography using a column packed with certain molecules having binding affinity for TPO, such as anti-TPO antibodies, TPO receptors, and the like. An anti-TPO antibody column was first prepared by linking the anti-TPO antibody peptide obtained in Example 45 (Example 46).

(R) Purification of human TPO expressed in COS 1 cells using a column of antibodies against TPO peptide and mol. mass and biological characteristics of SRW
Using the culture supernatant from transfected COS 1 cells (pHTP1 expression vector), partially purified TPO was obtained as a starting material, which was applied to a column with anti-TPO antibodies. Since TPO activity was detected in the adsorbed fraction, this fraction was then subjected to reverse phase column chromatography to purify the protein and determine its mol. mass and biological characteristics (example 47).

(S) Confirmation of the activity of a partially purified human TPO sample expressed in COS 1 cells
The TPO active fraction purified in Example 36, namely, the TPO sample purified to the Capcell Pak Cl 300A column stage from the culture supernatant obtained by transfection of COS 1 cells with the pHTP1 expression vector, was tested for its biological activity to determine if it could increase the number platelets in a living body (example 48).

Also, the crude TPO fraction obtained on a cation exchange column from 33 L of culture supernatant obtained by transfection of COS 1 cells with pHTP1 expression vector was checked for its biological activity,
to determine if it increases the number of platelets in the body (example 49).

(T) Expression of human TPO chromosomal DNA in CHO cells and confirmation of TPO activity
The pDEF202-ghTPO vector was designed for use in human TPO chromosome expression in CHO cells (Example 50).

 When this vector was introduced into CHO cells, a transformant cell was obtained in which the vector expressing the chromosomal DNA was integrated into its chromosome. When culturing this cell clone, TPO activity was detected in the culture supernatant (Example 51).

(U) Partial purification and confirmation of the activity of the human TPO mutated type, expressed in E. coli
The mutated human TPO obtained from the human TPO nucleotide sequence encoding the human clone pCFM536 / h6T (1-163) and expressed in E. coli was re-laid using guanidine hydrochloride and glutathione to determine if the thus obtained h6T (1- 163) platelet-increasing action in a living body (Example 53).

 The mutated type human TPO obtained from the nucleotide sequence encoding the human TPO clone, pCFM536 / h6T (1-163), and expressed in E. coli, was re-assembled using N-lauroyl sarcosinate sodium and copper sulfate and then cation exchange chromatography to determine increases whether the thus purified h6T (1-163) platelet count in a living body (Example 54).

 In addition, the laying and cleaning of the human TPO mutated type, h6T (1-163) was carried out using other procedures (examples 60 and 61).

(V) Expression of human TPO cDNA in insect cells and identification of TPO activity
A recombinant virus for the expression of human TPO in insect cells was obtained as described in Example 58 and expressed in Sf 21 insect cells, after which TPO activity was identified in the culture supernatant (Example 59).

(W) Expression of human TPO (amino acid positions 1-163) in CHO cells and its purification
The pDEF202-hTPO163 vector was designed to express human TPO protein (called "hTPO163") having an amino acid sequence of 1-163 in the human TPO amino acid sequence shown in SEQ 1D 7. The expression vector pDEF202-hTPO163 was transfected into CHO cells and thus obtained hTPO 163 producing CHO cell line was cultivated in large-scale production. HTPO163 was purified from the supernatant of this culture (Examples 62-65).

(X) Obtaining substituted derivatives of human TPO
A derivative (called "N3 / TPO") was obtained in which Arg-25 and Glu-231 human TPO were replaced by Asn and Lys, respectively, as well as a derivative (called "09 / TPO") in which His-33 was replaced on Thr.

 The plasmid encoding each derivative was transfected into COS 7 cells, which were then cultured. TPO activity was detected in the supernatant of this culture.

 Derivatives were obtained in which one amino acid h6T (1-163) was replaced by another amino acid, using the E. coli TPO expression system, activity was detected in each derivative (Example 94).

(Y1) Preparation of Insertion or Deletion Derivatives of Human TPO
The amino acid was introduced into or deleted from hTPO163 to prepare insertion or deletion derivatives, after which COS7 cells were transfected with plasmids encoding the resulting derivatives. TPO activity was detected in the culture supernatant of these COS 7 cells (Example 68).

 A method for measuring TPO activity (in vitro test system) used in the present invention is described below as a “Standard Example”.

(Z) Obtaining and purification of antibodies against human TPO
Polyclonal antibodies are prepared using peptide fragments of human TPO (Example 69) and then used in Western blotting (Example 70) and in the construction of affinity columns with antibodies against TPO (Example 71).

(AA) In vivo Human TPO Activity
Purified human TPO is administered to mice with induced thrombocytopenia and changes in platelet count are observed compared to the control group (Example 72) - (Example 79). Pharmaceutical compositions are described that are potentially useful in the treatment of thrombocytopenia (Example 80) - (Example 89).

(BB) TPO activity as a function of receptor binding
A test is provided in which TPO activity is determined in units of specific binding to the MP1 receptor (Example 90) - (Example 93).

Standard example
A. Test for determination of rat megakaryocyte progenitor cells (rat CFU-MK test system) (liquid culture system)
Megakaryocytes include and store serotonin in cytoplasmic dense granules using an active energy-dependent process (Fedorko, Lab, Invest., Vol. 36, pp. 310-320, 1977). This phenomenon can already be observed in small mononuclear acetylcholinesterase-positive cells, which are believed to be located between CFU-MK and distinguishable megakaryocytes (Bricker and Zuckerman, Exp. Hematol., Vol. 12, p. 672, 1984). The amount of serotonin incorporated increases in response to an increase in megakaryocyte size (Schick and Weinstein, J. Lab. Clin. Med. Vol. 98, pp. 607-615, 1981).

In addition, it is known that such a phenomenon is specific only for megakaryocytes among bone marrow cells (Schick and Weinstein, J. Lab. Clin. Med., Vol. 98, pp. 607-615, 1981). In this test system, highly enriched rat CFU-MK cells (Gp11b / 111a ⁺ CFU-MK fraction, which will be described below) are cultured in the presence of test samples and the incorporation of ¹⁴ C-serotonin ( ¹⁴ C-hydroxytryptamine-creatine sulfate, ¹⁴ C-5HT ) to megakaryocytes growing from CFU-MK.

The advantages of this test system are that indirect effects of impurity cells (for example, the formation of Meg-CSF activity by impurity cells stimulated by a substance other than the target factor, or the formation of a factor by impurity cells that undergo a combined effect of interest factor) can be reduced, since the percentage of CFU-MK in the cells of the Gp11b / 111a ⁺ CFU-MK fraction is very high (compare the following [test method]), while the number of contaminating (impurity) cells is small. In addition, suitable culture conditions can be maintained for a relatively long period, since the total number of cells seeded in one well is very small. Another advantage is that the number of mature large megakaryocytes grown from CFU-MK in the presence of an active sample during the cultivation period can be visually detected under a phase contrast microscope, which allows a qualitative assessment of the presence and degree of activity. The results of the qualitative assessment are in good agreement with the results of a quantitative determination based on the incorporation of ¹⁴ C-serotonin. Thus, the reliability of quantification can be further improved by the simultaneous application of a qualitative assessment.

Test method:
Highly enriched rat CFU-MK cells (C-p11b / 111a ⁺ CFU-MK fraction) used in this assay were prepared using a slightly modified method (Miyazaki et al. (Exp. Hematol., Vol. 20, pp. 855-861, 1992).

Briefly, the femur and tibia were removed from Wist rats (males, 8-12 weeks old) to prepare a suspension of bone marrow cells, as described. A suspension medium was used to prepare a suspension of bone marrow cells (a solution consisting of 13.6 mm trisodium salt of citric acid, 11.1 mm glucose, 1 mm adenosine, 1 mm theophylline, 10 mm HEPES (pH 7.25), 0.5 % bovine serum albumin (hereinafter referred to as BSA) (Hanks balanced solution: hereinafter referred to as "HATCH solution"), which is a slightly modified version of the megakaryocyte separation medium reported by Levine Fedoroko (Levine and Fedoroko, Blood, vol. 50, pp. 713- 725, 1977). Thus obtained suspension of bone marrow cells is layered to interrupt grained density gradient of Percoll (density: 1.050 g / ml / 1.063 g / ml / 1.082 g / ml), which was prepared by diluting the Percoll solution (Pharmacia) NATSN solution, and centrifuged at 20 ^o C at 400 xg for 20 minutes after centrifugation. cells are removed between layers with a density of 1.063 and 1.082 g / ml After washing, the extracted cells are suspended in Dulbecco's culture medium modified by Iskov (hereinafter referred to as 1MDM culture medium) containing 10% fetal calf serum (hereinafter referred to as FCS), placed in plastic cups tissue culture (100 mm) and cultured at 37 ^o C for 1 hour in an incubator with 5% CO ₂ . After incubation, non-adherent cells are isolated and cultured again in plastic dishes at 37 ^{° C.} for 1 hour. Non-adherent cells are suspended in HATCH solution and incubated for 1 hour on bacteriological Petri dishes (100 mm) on which mouse monoclonal antibodies against rat platelets Gp11b / 111a ⁺ , P55 antibodies were previously adsorbed (Miyazaki et al., Thromb. Res.,. vol. 59, pp. 941-953, 1990).

After incubation, non-adherent cells are removed by thorough washing with HATCH solution and the remaining cells adsorbed by immobilized P55 antibodies are pipetted and harvested. Usually, 3-4 • 10 ⁵ cells from one rat are obtained. The cell fraction thus obtained contains highly enriched rat CFU-MK (hereinafter referred to as the Gr11b / 111a ⁺ CFU-MK fraction). This cell fraction is known to contain approximately 5-10% CFU-MK based on measurement using a colony analysis system in the presence of a saturating concentration of rat IL-3. A hybridoma capable of producing the aforementioned P55 antibodies was deposited under Deposit FERM BP-4563 National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology. Ministry of International Trade and Industry, February 14, 1994.

Then, the cells of the thus obtained Gp11b / 111a ⁺ CFU-MK fraction were suspended in 1MDM culture medium containing 10% FCS and distributed in 10 ⁴ cells per well of a 96-well tissue culture plate. A standard sample (this will be discussed in more detail below) or a test sample was then added to each well, bringing the final volume of the medium to 200 μl per well. The thus prepared plate was placed in a CO ₂ incubator and incubated for 4 days at 37 ^° C. On the 4th day of incubation, 0.1 μCi (3.7 KBq) of ¹⁴ C-serotonin was added to each well 3 hours before completion cultivation and final incubation is continued at 37 ^o C. After 3 hours of incubation, the plate is centrifuged at 1000 rpm for 3 minutes and the resulting supernatant is removed by suction. 200 μl of PBS containing 0.05% EDTA was added to each well, and the plate was centrifuged to rinse by removing the resulting supernatant. This washing step is repeated one more time. 200 μl of 2% Triton X-100 is added to the pellets thus obtained in each well and the plate is pumped on a tablet mixer for approximately 5-10 minutes to completely lyse the cells. Parts of 150 μl of the thus obtained cell lysates were transferred to a commercial solid scintillator in the form of a cap (Ready Cap; Beckman), which was then left overnight at 50 ^{° C.} in an oven to dry the lysate. The next day the Ready Cap placed in a glass vial for radioactivity measurement ¹⁴ C using a liquid scintillation counter.

In this case, almost the same results can be obtained in the test of incorporation of ¹⁴ C-serotonin and in the measurement of acetylcholinesterase activity in the mentioned cell lysate according to the method of Ishibashi and Burstein (Blood, vol. 67, pp. 1512-1514, 1986).

Standard Samples:
First, the blood plasma of thrombocytopenic rats was prepared for use in the preparation of standard samples as follows.

 Normal male Wistar rats (7-8 week old) were converted to thrombocytopenic by intravenous administration of P55 antibodies at a dose of 0.5 mg per animal, twice with an interval of approximately 24 hours. About 24 hours after the second injection, blood was collected from the abdominal aorta under ether anesthesia using a 10 ml syringe into which 1 ml of 3.8% (v / v) trisodium citric acid was injected as an anticoagulant. The blood sample thus collected was poured into plastic centrifuge tubes and centrifuged at 1200 g for 10 minutes to obtain a blood plasma fraction. The thus obtained blood plasma fraction was again centrifuged at 1200 g for 10 minutes and the obtained blood plasma fraction was collected, avoiding the collection of cells and platelets, etc., and combined (the thus obtained blood plasma is hereinafter referred to as TRP). Calcium-treated TRP (standard sample c), the active fraction of the WGA agarose column (standard sample W) or the active fraction of Phenyl Sepharose 6 FF / LS (standard sample P) obtained from TRP in accordance with the procedure of Example 1 were dialyzed extensively against a sufficient volume 1MDM culture medium and used as test standards.

 In the rat TPO purification process described in Example 1, standard sample C was used at an early stage, but then it was replaced with standard sample W halfway thereafter and then standard sample P. The specific activity of standard sample C was defined as 1 and the relative activities of standard samples W and P was calculated based on this setting. The relative activity of each test sample was determined by comparing the dose-response curve of the standard sample with the curves of the test samples. The relative activity of the test sample was determined as n if its activity is n times higher than the activity of a standard sample C.

B. Test system with colony counting
In this test system, bone marrow cells were cultured in a semi-solid culture medium in the presence of the test sample, and Meg-CSF activity was measured by counting the number of megakaryocyte colonies resulting from the proliferation and differentiation of CFU-MK.

Test method
(a) In the case of using undivided rat bone marrow cells, etc.

1 ml of the final volume of 1MDM culture medium containing undivided rat bone marrow cells, cells obtained at each stage of separation of the Gp11b / 111a ⁺ CFU-MK test system A or Gp11b / 111a ⁺ CFU-MK fractions, and 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol and 0.3% agar (AGAR NOBLE manufactured by D1FCO) were placed in a plastic tissue culture dish (35 mm) and cultured at room temperature at 37 ^{° C.} incubator with CO ₂ . Typically, the number of cells per cup was adjusted to 2-4 • 10 ⁵ in the case of undivided bone marrow cells, 2-5 • 10 ⁴ in the case of cells at the stage of Percoll density gradient or depletion by attachment, or 0.5-2 • 10 ³ in the case of cells fractions Gp11b / 111a ⁺ CFU-MK. After 6-7 days of cultivation, agar discs were separated from the plates and placed on glass slides (76 mm • 52 mm). To dry each agar disc, a piece of a 50 m nylon mesh and filter paper were placed on the disc in this sequence. Agar glasses thus dried were fixed by heating for 5 minutes at 50 ^° C on a hot plate and soaked for 2-4 hours in a staining solution of acetylcholinesterase according to the Jackson method (Blood, vol. 42, pp. 413-421, 1973). If sufficient staining of megakaryocytes was observed, the agar glass was washed with water, dried, subjected to post-staining with Harris hematoxylin solution for 30 seconds, washed with water and then dried in air. Colonies of megakaryocytes were considered 3 or more closely grouped acetylcholinesterase-positive cells.

(c) In the case of the use of undivided mouse bone marrow cells
The colony count test was performed as described in (a) using 2-4 x 10 ⁵ cells per dish.

(c) In the case of the use of human bone marrow cells or blood cells of the human spinal cord
You can use human bone marrow cells or blood cells of the human spinal cord as such or in the form of CFU-MK fractions enriched as follows.

First, bone marrow fluid or spinal cord blood is infused with Lymphoprep (manufactured by Daiichi Kagaku Co., Ltd.) and centrifuged, after which the leukocyte fraction obtained in the interphase is recovered. From the cell fraction thus extracted, the cells to which the biotinylated monoclonal antibodies specific for human surface antigens (CD2, CD11, CD19) bind are removed using beads connected to avidin. The cells removed by magnetic beads are mainly B cells, T cells, macrophages and part of granulocytes. The remaining cells are stained with FITC-labeled anti-CD34 antibodies and PE-labeled anti-HLA-DR antibodies. The CD34 and HLA-DR positive fractions are then recovered using a cell sorter (e.g. ELITE manufactured by COULTER). CFU-MK cells are concentrated in this fraction (hereinafter referred to as the XD34 ⁺ DR ⁺ CFU-MK fraction). The test with the calculation of human cell colonies is carried out in the same way as in the case of rat bone marrow cells, except that 3-5 • 10 ³ cells of CD34 ⁺ DR ⁺ CFU-MK fraction are used per cup and a mixture of 12.5% plasma is used Human AB and 12.5% FCS instead of 10% FCS. The cultivation period for the formation of colonies of megakaryocytes is 12-14 days. To detect megakaryocytes, immunological staining of megakaryocytes is carried out according to the method using alkaline phosphatase antibodies against alkaline phosphatase with murine monoclonal antibodies specific for the megakaryocyte surface antigen Gp11b / 111a (e.g., Teramura et al., Exp. Hematol., Vol. 16, pp. 843-848, 1988) and count colonies consisting of 3 or more megakaryocytes.

(C) Test system with megakaryoblast cell lines (M-07e test)
M-07e cells, the cell line of human megakaryoblasts, proliferate in response to GM-CSF, IL-3, SCF, IL-2, and the like. (Avanzi et al., J. Cell. Physiol., Vol. 145, pp. 458-464, 1990). Since these cells also respond to TPO, they are applicable in a replacement test system for a rat CFU-MK test system.

Test method:
M-07e cells retained in the presence of GM-CSF are harvested, washed thoroughly, and then suspended in 1MDM culture medium containing 10% FCS. The resulting suspension of M-07e cells was poured in portions of 10 ⁴ cells into the wells of a 96-well tissue culture plate, and then a standard sample or test sample was added to each well, bringing the final volume to 200 μl per well. The thus obtained plate was placed in an incubator with 5% CO ₂ and incubated for 3 days at 37 ^° C. After 3 days of cultivation, 1 μCi (37 KBq) of ³ H-thymidine was added to each well 4 hours before completion of the culture. After cultivation, cells are collected on glass fiber filters using a cell harvester to measure ³ H radioactivity using a liquid scintillation counter (e.g., Beta Plate manufactured by Pharmacia).

D. Test system (Ba / F3 test) using a mouse pro-B cell line
The mouse Ba / F3 pro-B cell line is known as a cell line that proliferates in response to IL-3, IL-4, and the like. (Palacios et. Al., Cell, vol. 41, pp. 727-734). Since its sub-strain BF-TE22 is capable of cell proliferation in response not only to IL-3 and IL-4, but also to TPO, it can be used in a test system that replaces the rat CFU-MK test or the human megakaryoblast cell line test (M-07e test) as follows. Briefly, BF-TE22 cells growing in 1MDM medium (1MDM: G1BCO) in the presence of 1 ng / ml mouse IL-3 are recovered, washed three times with 1MDM and suspended in 1MDM medium containing 10% FCS. The cell suspension is then poured into the wells of a 96-well tissue culture plate at a density of 1 × 10 ⁴ cells per well, then a standard TPO solution or test sample is added to each well and the final volume is adjusted to 200 μl per well. The resulting microplate is incubated for 2-3 days in an incubator with 5% CO ₂ . On day 2 and day 3, 1 μCi (37 KBq) of ³ H-thymidine was added to each well and cultivation continued for another 4 hours. Finally, cells are collected on a glass fiber filter using a cell harvester to measure ³ H radioactivity using a liquid counter. In this test system, almost all cells cultured in media without TPO activity die and therefore do not include ³ H-thymidine. However, cells cultured in media containing TPO activity show active proliferation depending on the concentration of TPO and include ³ H-thymidine. In addition, the results of this test are parallel to the results of the M-07e and CFU-MK tests.

 The following examples further illustrate the invention.

Example 1-1 Purification of rat TPO from blood plasma of thrombocytopenic rats induced by the administration of anti-platelet antibodies. Obtaining blood plasma of thrombocytopenic rats induced by the introduction of antibodies against platelets
TPP was obtained as a purification source from approximately 1000 rats according to the procedure described in "Standard Example" A above, which describes the rat megakaryocyte progenitor cell test system (CFU-MK).

Purification of TPO rats from TRP
Initially, purification was performed using TRP from approximately 1000 rats, assuming that the TPO content in TRP should be a maximum of 1 in 3 million, and a little less than 1 pmol of partially purified rat TPO was obtained. Although this is difficult, an attempt was made to analyze its partial amino acid sequences and three partial amino acid sequences were obtained, but with low accuracy. These sequences coincided or were close to the amino acid sequence of a serine protease inhibitor (SP1) produced in the liver. It is not clear from these data whether TPO has a structure similar to that of a serine protease inhibitor, or whether these sequences are derived from sequences of impurity proteins other than TPO. Using these undefined sequences, the TPO gene was cloned from the rat cDNA library, but could not obtain potential TPO coding genes. After that, intensive studies were conducted on the basis of the assumption that this failure was due to a low degree of purity and an insufficient amount of the analyzed sample. To obtain a purified final sample required for amino acid analysis, the purification was carried out in accordance with the process described in the following example 1-2. Unexpectedly, the results of Example 1-2 showed that the TPO content in TPP or XRP (this will be described below) was as extremely low as 1: 100000000 - 1 bln. total plasma proteins, so complete purification of the SRW was extremely difficult.

Example 1-2. Purification of rat SRW from blood plasma of thrombocytopenic rats induced by irradiation of the whole body with X-rays or γ-rays [Production of blood plasma of thrombocytopenic rats induced by irradiation of the whole body with X-rays or γ rays]
Normal male Wister rats (7-8 week old) were converted to thrombocytopenic by whole-body irradiation with X-ray or γ-rays at a sublethal dose (6-7 Gy). Blood was collected from rats on the 14th day after irradiation and a blood plasma fraction (hereinafter referred to as XRP) was prepared as described above for the preparation of TRP.

 In this case, XRP (approximately 8 L) was obtained from 1100 rats and used as starting material (source) for purification.

Purification of SRW rats from HRP
A blood plasma sample of 1,100 rats was too large for a single treatment, as its total protein content reached 493,000 mg. Therefore, the blood plasma sample was divided into 11 series, each corresponding to approximately 100 rats, in the following stages of purification (1) - (4). In further stages of purification (5) - (7), the sample was divided into 6 groups. In and after the purification step (8), a sample, roughly purified, from a total blood plasma of approximately 1100 rats was used. A typical example of each purification step is described below for the case of series (XW9) and group (XB6).

At all stages of TPO purification, activity was measured using the rat CFU-MK test system described in the Standard Example. Unless otherwise indicated, all purification steps were carried out at 4 ^° C, with the exception of reverse phase chromatography and gel filtration on Superdex 75 pg in the presence of a surfactant, which was carried out at room temperature. Protein determinations were performed in the Coomassie dye binding test (reagent system manufactured by PIERCE, cat. 23236X) or by the method using bicinchoninic acid (reagent system manufactured by PIERCE, cat. 23225).

 The cleaning scheme is shown in table 1.

(1) Treatment with calcium chloride, centrifugation and treatment with rat plasma protease inhibitor
In the case of the XW9 series
XRP (742 ml, protein concentration 54.8 mg / ml, total protein 40 686 mg) corresponding to about 100 rats, stored at -80 ^° C, was thawed and poured into polypropylene centrifuge tubes (manufactured by Nalgene). Calcium chloride powder was added in each tube to a final concentration of 100 mM. After incubation overnight at 4 ^{° C., the} resulting mixture was centrifuged at 8,000 rpm for 60 minutes. To the obtained TPO-active supernatant (742 ml, protein concentration 54.9 mg / ml, total protein 40 740 mg), the p-APMSF protease inhibitor ((p-aminodiphenyl) methanesulfonyl fluoride, hydrochloride manufactured by WAko Pure Chemical Industries, Ltd., was added. Cat. 010-10393) to a final concentration of 1 mM. The sample thus obtained was used in a further buffer exchange step on a Sephadex G-25 column.

 In the described manner, calcium chloride / p-APMSF was treated with all XP obtained from 1100 rats (total volume 8184 ml, total protein 493000 mg) by dividing into 11 series, each of which corresponded to approximately 100 rats, and separately processing these series. The resulting 11 samples were processed separately in a subsequent purification step on a Sephadex G-25 column.

(2) Sephadex G-25 (buffer change)
In the case of the XW9 series
The supernatant obtained after the calcium treatment of step (1) (742 ml, protein concentration 54.9 mg / ml, total protein 40 740 mg) was applied at a flow rate of 40-70 ml / min to a Sephadex G-25 column (manufactured by Pharmacia Biotech , cat. 17-0033-03, diameter 11.3 cm, packed column height 47 cm), balanced in advance with 20 mM Tris-HC1, pH 8. Elution was carried out with the same buffer. A portion of 1300 ml of the eluate was discarded before elution of the protein. When UV absorption was detected in the eluates, the fractions were collected until the conductivity reached 500 μS / cm, thereby extracting the TPO-active protein fraction, now containing 20 mM Tris-HC1, pH 8 (1377 ml, protein concentration 27.56 mg / ml total protein 37882 mg, protein yield 93%). The relative activity of SRW in this fraction was 2.3.

 As a result of processing on a Sephadex G-25 column of all series of samples, a total of 21117 ml of TPO-active fraction was obtained (total protein 480300 mg, average relative activity 1, total activity 864600).

(3) Q-Sepharose FF (strong anion exchange chromatography)
In the case of the XW9 series
The TPO-active fraction obtained in step (2) by treatment with Sephadex G-25 (1377 ml, protein concentration 27.5 mg / ml, total protein 37841 mg, relative activity 2.3) was applied to Q-Sepharose FF (Pharmacia Biotech , Cat. 17-0510-01, diameter 5 cm, column height 27 cm) at a flow rate of 40 ml / min, 20 mM Tris-HC1 (pH 8) was eluted and the F1 fraction passing through the column was collected (3949 ml, concentration protein 0.98 mg / ml, total protein 3870 ml, relative activity 0).

 Then, the buffer was replaced with 20 mM Tris-HCl (pH 8) containing 175 mM NaCl to elute the TPO-active fraction F2 (4375 ml, protein concentration 5.36 mg / ml).

 Finally, the F3 fraction (1221 ml, protein concentration 3.9 mg / ml, total protein 4783 mg, relative activity 3.8) was eluted with 20 mM Tris-HCl (pH 8) containing 1000 mM NaCl. The total protein in the TPO-active fraction of F2 was 23440 mg and the yield of F2 protein at this stage was 61.9%. In addition, the relative activity of SRW increased to 6.8.

 As a result of applying all series of TPO-active fractions after Sephadex G-25 Q-Sepharose FF, a total of 35842 ml of TPO-active fraction F2 was obtained (total protein 314384 mg, average relative activity 9, total activity 2704000).

(4) Wheat germ agglutinin (WGA) -Agarose (pectin affinity chromatography)
In the case of the XW9 series
The TRO-active fraction F2 obtained in step (3) using Q-Sepharose FF was divided into 3 parts and applied to WGA-Agarose (manufactured by Honen Corp. Cat. 800273, diameter 5 cm, height 22.5 cm) at a speed flow 5 ml / min and was suirable Dulbecco isotonic phosphate buffer (DPBS). the F1 fraction passing through the column was collected (9336 ml, protein concentration 2.30 mg / ml, total protein 21407 mg, relative activity 6.9).

 The buffer was then replaced with a 20 mM buffer containing sodium phosphate (pH 7.2) containing 0.2 M N-acetyl-D-glucosamine (GlcNAc manufactured by Nacalai tesque Cat. 005-20), 150 mM NaCl and 0.02 % sodium azide, and the resulting eluates were combined and concentrated using an ultrafiltration element (Omega Ultrasette, nominal molecular weight cut-off of 8000, manufactured by Filtron), resulting in a TPO-active fraction F2 (2993 ml, protein concentration 0.376 mg / ml).

The total protein in the TPO-active fraction was 1125 mg and the yield of F2 protein of this stage was 4.8%. In addition, the relative activity of TPO increased to 101. The thus obtained fraction F2 was stored at -80 ^o C.

 As a result of applying all series of TPO-active Q-Sepharose FF fractions to WGA-Agarose, a total of 33094 ml of WGA-Agarose TPO-active fraction F2 was obtained (total protein 15030 mg, average relative activity 132, total activity 1987000).

(5) TSK - gel AF-BLUE 650 MN (affinity chromatography with triazine dye)
In the case of group XB6
WGA-Agarose, absorbing TPO-active fraction of the XW9 series, and WGA-Agarose, absorbing TPO-active fraction of the F2 XW9 series, obtained in stage (4), corresponding to the initial 215 rats, were combined as the XB6 group (5947 ml, protein concentration 0.388 mg / ml, total protein 2319 mg, relative activity 150).

 5974 ml from the pooled sample was mixed with 0.85 mol of NaCl per 1000 ml (296.76 g in total) to obtain 6132 ml of a solution having a final NaCl concentration of 0.822 M and the resulting solution was applied at a flow rate of 7 ml / min on TSK - AF-BLUE gel 650 MH column (TOSOH CORP. Cat. 08705, diameter 5 cm, column height 23 cm), which was pre-equilibrated with 20 mM sodium phosphate buffer (pH 7.2) containing 1 M NaCl.

 After completion of the application, the protein was eluted with 20 mM sodium phosphate buffer (pH 7.2) containing 1 M NaCl at a flow rate of 10 ml / min. The resulting eluates were combined and concentrated using an ultrafiltration element (Omega Ultrasette, nominal molecular weight cut-off of 8000) to obtain the passing fraction F1 (543 ml, protein concentration 2.05 mg / ml), total protein 1112 mg, relative activity 31).

 Then, the elution buffer was replaced with 2 M NaSCN to obtain the eluted TSK-gel AF-BLUE 650 MN absorbed TPO-active fraction F2 (1427 ml, protein concentration 0.447 mg / ml).

 The total protein in the TPO-active fraction of F2 was 638 mg and the yield of F2 protein in this stage was 27.5%. In addition, the relative activity of SRW increased to 1500.

 As a result of applying all WGA-Agarose groups of absorbed TPO-active F2 fractions to TSK-gel AF-BLUE 650 MN, a total of 10655 ml of TPO-active F2 fraction was obtained (total protein 4236 mg, average relative activity 905, total activity 3834000).

(6) Phenyl Sepharose 6 FF / LS (hydrophobic chromatography)
In the case of group XB6
1424 ml from the TPO-active fraction F2 from TSK-gel AF-BLUE 650 MN (1424 ml, protein concentration 0.447 mg / ml, total protein 638 mg, relative activity 1500) were mixed with 1.5 mol of ammonium sulfate powder per 1000 ml ( 282.2 g in total), obtaining a solution with a final concentration of ammonium sulfate 1.35 M.

 The resulting solution was applied at a flow rate of 7 ml / min to a Phenyl Sepharose 6 FF (Low Sub) column (Pharmacia Biotech, Cat. 17-0965-05, diameter 5 cm, column height 10 cm), which was balanced in advance with 50 mM sodium -phosphate buffer (pH 7.2) containing 1.5 M ammonium sulfate. After application was complete, elution was performed using 36 mM sodium phosphate buffer containing 0.8 M ammonium sulfate at a flow rate of 10 ml / min. The resulting eluates (approximately 3160 ml) were combined and concentrated using an ultrafiltration element (Omega Ultrasette, nominal molecular weight cut-off 8000) to obtain the F1 fraction (485 ml, protein concentration 0.194 mg / ml, total protein 94.2 mg, relative activity 0 )

 Then, the elution buffer was replaced with 20 mM sodium phosphate buffer (pH 7.2) to obtain the eluted TPO-active fraction F2 (approximately 2500 ml). The eluted fraction was concentrated using an ultrafiltration element (Omega Ultrasette) and a sample was taken for analysis. At this stage, the protein concentration and the total protein of the TPO-active fraction F2 (220 ml) were 1.45 mg / ml and 319 mg, respectively, and the yield of F2 protein at this stage was 50.0%. The relative activity of SRW was 1230.

 As a result of the application of all groups of TPO-active F2 fractions from TSK-gel AF-BLUE 650 MN on Phenul Sepharose 6 FF / LS, a total of 1966 ml of F2 fractions were obtained (total protein 2762 mg, average relative activity 847, total activity 2339000).

(7) Sephacryl S-200 HR (gel chromatography)
In the case of group XB6 (see figure 1)
The TPO active fraction F2 from Phenul Sepharose 6 FF / LS (217 ml, protein concentration 1.45 mg / ml, total protein 315 mg, relative activity 1230) was mixed with 144.8 ml of a 5 M NaCl solution, obtaining 362 ml of solution with the final concentration of NaCl 2 M, and the resulting solution was concentrated to approximately 50 ml using an ultrafiltration device with a UM3 membrane (76 mm in diameter, Amicon Corp.).

 An equal volume (50 ml) of 8 M urea was added to this solution, yielding approximately 100 ml of a solution containing 1 M NaCl and 4 M urea as final concentrations. It was concentrated to about 80 ml, a sample of 88.78 ml was taken and applied to a Sephacryl S-200 HR column (Pharmacia Biotech, Cat. 17-0584-01, 7.5 cm in diameter and 100 cm in height).

After that, PBS was eluted at a flow rate of 3 ml / min, and the eluates after an empty volume of 1200 ml were collected in 45 ml portions in 60 polypropylene tubes. The test was carried out every two tubes and the remaining tubes were stored at -85 ^° C until the processing of all samples obtained from 1100 rats was completed. Based on the obtained analysis results, fractions of the ХВ6 group were grouped as follows.

(F1) tube numbers 1-15 (fractions near empty volume, mol. Mass 94000 or more)
(F2) tube numbers 16-26 (mol. Masses 94000-33000)
(F3) tube numbers 27-44 (mol. Mass 33000-3000)
(F4) tube numbers 45-55 (mol. Mass 3000 or less)
Thus, all groups of F2 fractions (TPO-active) obtained using Phenyl Sepharose 6 FF / LS were separately applied to Sephacryl S-200 HR, analyzed and stored at -85 ^o C. After completion of this treatment of all groups and immediately before subsequent by reverse phase chromatography (YMC-Pack PROTEIN-RP), these stored samples were thawed and concentrated using an ultrafiltration device with an UM 3 membrane (76 mm in diameter, Amicon Corp.) to obtain the following two samples. This concentrated sample of the TPO-active fraction F2 obtained after Sephacryl S-200 HR is hereinafter referred to as “sample F2 of high molecular weight TPO”, and F3 is called “sample F3 of low molecular weight TPO”.

For convenience, the F2 sample of high molecular weight TPO and the F3 sample of low molecular weight TPO are combined fractions of different elution zones in gel chromatography, and the terms "high molecular weight" and "low molecular weight", therefore, cannot denote their true mol. mass (see table I)
A low molecular weight TPO sample F3 and a high molecular weight TPO sample F3 were separately subjected to the following purification steps.

 Purification of the F3 low molecular weight TPO sample is described in the following steps (8) to (11).

(8) YMC-Pack PROTEIN-RP (reverse phase chromatography)
A low molecular weight TPO sample F3 (total protein 50.3 mg, protein concentration 0.184 mg / ml, relative activity 20,000, total activity 1,007,000, total volume 274 ml) obtained in step (7) was mixed with solvent A (0.025% trifluoroacetic acid ( TFA)) and solvent B (1-propanol containing 0.025% TRA) to obtain a solution having a total volume of 508.63 ml and final concentrations of propanol, TFA and protein of approximately 20%, 0.012% and 0.0989 mg / ml, respectively . Insoluble material formed during the preparation of this solution was separated by centrifugation and the resulting supernatant was divided into two parts of 254.3 ml (25.2 mg of protein) and applied at a flow rate of 2 ml / min to a column packed with YMC-Pack PROTEIN-RP (UMS, Cat. A-PPR-33-03-15, 3 cm in diameter, 7.5 cm column height), which was previously equilibrated with 30% B. The precipitate formed by centrifugation was dissolved in 20 ml of 20 mM sodium acetate (pH 5.5) containing 5 mM CHAPS (3- [3-cholamidopropyl sulfonate) dimethylammonio] -1-propane manufactured by Dojindo Laboratorie s, cat. 75612-03-3) and were also applied to this column.

 After applying the sample, approximately 50 ml of solvent (solvents A: B = 3: 1) was passed through the column to obtain a fraction passing through the column. Then, the developing program was started (120 minutes of a linear gradient from 30% B to 45% B) and the eluates were collected in 36 polypropylene tubes of 10 ml in each tube. This process was repeated again for the remaining sample using the same fraction collection tubes, thereby obtaining a total of 36 tubes, each containing 20 ml of eluate. The fraction passing through the column was immediately concentrated to 20 ml using an ultrafiltration device with an UM 3 membrane (76 ml in diameter, manufactured by Amicon Corp.).

0.1 ml of the fraction passing through the column and of each of 20 ml fractions of tubes 1-36 were mixed with 20 μl of 5% VA, dried by evaporation, dissolved in 0.25 ml of 1MDM test medium and then analyzed to identify TPO-active fractions. As a result, TPO activity was detected in the fractions of tubes numbered 17-27 (range of propanol concentrations 36.0-43.0%), which were combined and used as YMC-Pack PROTEIN-RP TPO-active fraction F2 obtained from a low molecular weight sample F3 SRW. This fraction was stored at -85 ^° C until it was used in a subsequent step using YMC-Pack CN-AP.

YMC-Pack PROTEIN-RP TPO-active fraction F2 obtained from sample F3 low molecular weight TPO
Total volume - 220 ml
Protein concentration - 0.0130 mg / ml
Total protein - 2.85 mg
Relative activity - 130,000
Total activity - 371,000
(9) YMC-Pack CN-AP (reverse phase chromatography)
214.9 ml of YMC-Pack PROTEIN-RP TPO-active fraction F2 obtained from low molecular weight TPO sample F3 (total protein 2.79 mg, protein concentration 0.0130 mg / ml, relative activity 130,000, total activity 36300) obtained from stage (8), mixed with 0.6 ml of 50% glycerol and concentrated to 1.8 ml. This concentrate was then made up to a volume of 5 ml containing 20% or less propanol and approximately 6% glycerol.

 The concentrate was divided into 5 parts for use in the next column operation (0.555 mg of protein and 1 ml of volume for each operation). Each of the samples thus separated was applied at a flow rate of 0.6 ml / min onto a column packed with YMC-Pack CN-AP (UMC, Cat. AP-513, 6 mm in diameter and 250 mm in height), which was previously balanced 15 % B, using 0.1% TFA as solvent A and 0.05% TFA containing 1-propanol as solvent B. After application, the concentration of propanol was increased from 15% B to 25% B and a linear gradient of 65 minutes was performed from 25 % B to 50% B. After completion of the final (5th) operation, 1 ml of a solution having the same composition, with the exception of protein, was applied to Lonkila and treated in the same manner to retrieve TPO activity retained by the column. Since the same polypropylene tubes were used to collect the eluates in 6 operations, a total of 44 tubes were obtained, each containing 7.2 ml of eluate.

30 μl of each of the fractions thus obtained (1/240 part of each fraction) was mixed with 20 μl of 5% BSA, dried by evaporation, dissolved in 0.24 ml of 1MDM test medium and then analyzed to identify TPO-active fractions. As a result, high TPO activity was detected in fractions
tubes with numbers 28-33 (37.0-42.0% - range of propanol concentrations), which were combined and used as the YMC-Pack CN-AP main TPO-active fraction FA obtained from sample F3 of low molecular weight TPO.

YMC-Pack CN-AP TPO-active fraction of FA obtained from sample F3 of low molecular weight TPO
Total volume - 43.20 ml
Protein concentration - 0.00863 mg / ml
Total protein - 0.373 mg
Relative activity - 800,000
Total activity - 298,400
(10) Capcell Pak C1 300 (reverse phase chromatography)
43.12 ml from 43.20 ml of the TPO-active fraction of FA obtained from the F3 sample of low molecular weight TPO (total protein 0.372 mg, protein concentration 0.00863 mg / ml, relative activity 800000, total activity 297500) obtained in stage (9 ), mixed with 0.2 ml of 50% glycerol and concentrated to obtain 0.1 ml of glycerol solution.

 This solution was mixed with 2 ml of a solution of solvent A (0.1% TFA): solvent B (1-propanol containing 0.05% TFA) = 85:15 (15% B) to obtain 2.1 ml of a sample containing approximately 14% propanol, approximately 4.8% glycerol, and 0.177 mg / ml protein. The sample thus obtained was applied to a column packed with Capcell Pak C1 300A (manufactured by Shiseido Co., Ltd., Cat. C1 TYPE: SG300A; 4.6 mm in diameter and 250 mm in height), which was previously balanced with 15% B, and showed 65 minutes with a linear gradient from 27% V to 38% V at a flow rate of 0.4 ml / min.

 The eluates were collected in 72 polypropylene tubes of 0.6 ml in each tube.

 3 μl of each of the fractions thus obtained (1/200 part of each fraction) was mixed with 20 μl of 5% BSA, the medium was replaced with 225 μl of 1MDM test medium and the fraction diluted 75 times was analyzed.

1 μl of each fraction (1/600 fraction) was taken for use in electrophoresis, dried by evaporation and treated at 95 ^{° C. for} 5 minutes with 10 μl of non-reducing sample buffer for SDS-PAGE. The sample thus treated was subjected to SDS-PAGE electrophoresis using 15-25% SDS-polyacrylamide preformed gel (manufactured by Daiichi Pure Chemicals Co., Ltd) and then silver stained from the 2D-Silver Stain-11 "DAICI" kit (manufactured by Daiichi Pure Chemicals Co., Ltd., Cat. 167997, hereinafter referred to as Silver Staining Kit). As markers pier. the masses used / DAIICHI / -111 low molecular weight markers (manufactured by Daiichi Pure Chemicals Co., Ltd., cat. 181061, hereinafter referred to as "DPC111").

 As a result of this analysis, significant TPO activity was detected in the fractions of tubes with numbers 35-43 (the range of propanol concentrations was 30.0-32.5%). Of these, fractions with tube numbers 36-42 (range of propanol concentrations 30.5-32.0%) were combined and used as the main TPO-active fraction FA. These results are shown in FIG.

 When the protein content determined from the chromatogram was compared with these analysis results, it was found that the fraction thus obtained had a total protein of 39.6 μg, a protein concentration of 9.4 μg / ml, a relative activity of 4890000 and a total activity of 193600. Study of SDS distributions -PAGE TPO-active fractions with numbers 36-42 detected the presence of a band whose staining density correlates with the strength of activity. In addition, the apparent pier. the mass of this unrestored strip was 17000-19000 when compared with mol. masses of standard proteins with known mol. masses on the same gel that were recovered, which shows that this band is a strong candidate for SRW.

(11) Extraction of TPO-active protein from electrophoresis gel (15% SDS-PAGE)
An example of the analysis of TPO-active fraction FA
From 4200 μl of the TPO-active fraction of FA obtained from the F3 sample of low molecular weight TPO (total protein 39.6 μg, protein concentration 9.4 μg / ml, relative activity 4890000, total activity 193600) obtained in stage (10), 5, 5 μl (1/764 fractions) for use in the extraction of the active protein and 2.5 μl (1/1680 fractions) for use in silver staining were transferred to the appropriate sample tubes, dried by evaporation, treated with 10 μl of non-reducing sample buffer for SDS- PAGE at 37 ^o C for 1 hour and then left to stand up to 18 hours at room temperature ur, which contributed to the SDS reaction.

A pre-stained low-range marker (Bio-Rad Laboratories, Inc., 161-0305) and the aforementioned DPC111 were used as molar markers. masses. Using gel microplates, electrophoresis on 15% SDS-PAG of these samples was carried out at 4 ^{° C.} according to the conventional method (Laemmli, Nature, vol. 227, pp. 680-685, 1970). After electrophoresis, the parts of the gel intended for silver staining were immediately excised with a scalpel from the gel, placed in a fixative solution and then stained using the silver staining kit mentioned above.

Apart from this, the entire range of pier. the gel mass for detecting activity was cut with a scalpel into 34 sections and each section having a width of 1.5-2.5 mm was broken using the slightly modified Kobayashi method (Kobayashi, M., Seikagaku (Biochemistry), vol. 59, N9 , 1987, published by the Japanese Biochemical Society). Each gel section, thus broken into small fragments, was mixed with 0.3 ml of extraction buffer (20 mM Tris-Hcl, pH 8, 500 mM NaCl, 0.05% BSA) and pumped for 6 hours at 4 ^° C to enhance extraction.

To this suspension was added 500 mM potassium phosphate (pH 6.8) to a final concentration of 20 mM. After 1 hour of shaking at 4 ^{° C., the} resulting mixture was transferred to an Ultra Free C3GV 0.22 μm filtration device (Millipore Corp., UFC3 OGVOS) and centrifuged at 1000 g (4000 rpm) for 15 minutes to remove precipitated SDS and recover the resulting supernatant . The filtrate was transferred to an Ultra Free C3-LGC ultrafiltration device (nominal cut-off molar mass 10000, Millipore Corp., UFC3 LGC 00) and centrifuged at 3000 g (7000 rpm). When the volume of the concentrated solution reached approximately 50 μl, 300 μl of 20 mM sodium phosphate buffer (pH 7.2) was added and ultrafiltered again.

 Ultrafiltration using 300 μl of 20 mM sodium phosphate buffer was repeated twice to remove the remaining SDS. After this, a similar step was repeated for exchange onto the culture medium for sample preparation (300 μl), which was then sterilized and subjected to measurement of TPO activity.

 Three protein bands were clearly detected when stained with silver, which showed average mol. masses of approximately 17000-19000, 14000 and 11000, according to DPC111 markers mol. mass

 In the preceding stage (10), a strip having a correlation between the strength of activity and the staining density and having an average mol. a mass of approximately 17000-19000 was observed in electrophoresis of the TPO-active fraction of the Capcell Pak C1 column. In the experiment of this stage (11), TPO activity was detected only in the band with an average mol. mass of approximately 17000-19000 (see figure 3).

 Based on the above results, it was confirmed that the TPO-active protein was purified in the active fraction of the Capcell Pak C1 300A column to a detectable level on an electrophoresis gel. Based on the silver staining density of this band obtained with 15% SDS-PAGE, it was determined that the amount of TPO protein candidate (average molar mass of approximately 17000-19000) in the total TPO-active fraction is approximately 1.7 μg.

 Purification of the F2 sample of high molecular weight SRW is described in the following steps (12) to (15).

(12) YMC-Pack PROTEIN-RP (reverse phase chromatography)
A high molecular weight TPO sample F2 (total protein 257 mg, protein concentration 0.894 mg / ml, relative activity 7840, total activity 2015000, total volume 287 ml) obtained in stage (7) was mixed with 95.8 ml (1/3 of the sample volume ) solvent B (1-propanol containing 0.025% TFA) to obtain a solution having a total volume of 383 ml and final concentrations of propanol, TFA and protein of approximately 25%, 0.006% and 0.671 mg / ml, respectively. A solution of 0.025% TFA was used for solvent A. The insoluble material formed during the preparation of the applied sample was separated by centrifugation and the resulting supernatant was divided into six parts of 62.3 ml (42.8 ml of protein) and applied at a flow rate of 2 ml / min per column packed with YMC-Pack PROTEIN-RP (UMS, Cat. A-PPR-33-03-15, 3 cm in diameter and 7.5 cm in height), which was previously balanced 30% B. The precipitate obtained by centrifugation , dissolved in 10 ml of 20 mm sodium acetate (pH 5.5) containing 5 mm CHAPS, and also applied to this column.

 After applying the sample, approximately 50 ml of solvent (solvents A: B = 3: 1) passed through the column to obtain a fraction passing through the column. Then the developing program was started (120 min linear gradient from 30% B to 45% B), and the eluates were collected in 24 polypropylene tubes of 15 ml in each tube. This process was repeated for 6 separated samples using the same fraction collection tubes, thereby obtaining a total of 24 tubes, each of which contained 90 ml of eluate. The fraction passing through the column and the fraction of tube 1 were combined and immediately concentrated to 90 ml using an ultrafiltration device with a UM3 membrane (76 mm in diameter, Amicon Corp.).

0.3 ml of the fraction passing through the column plus fractions of tube 1 and each of the fractions of tubes 2-24 were mixed with 10 μl of 5% BSA, dried by evaporation, dissolved in 0.30 ml of IMDM test culture medium and then analyzed to identify TPO-active fractions. As a result, TPO activity was detected in the fractions of tubes with numbers 10-15 (range of propanol concentrations 34.0-39.5%), which were combined and used as YMC-Pack PROTEIN-RP TPO-active fraction F2 obtained from a high molecular weight sample F2 SRW. This fraction was stored at -85 ^° C until use in the subsequent step of the YMC-Pack CN-AP.

YMC-Pack PROTEIN-RP TPO-active fraction F2 obtained from high molecular weight TPO sample F2
Total volume - 540 ml
Protein Concentration - 0.021 mg / ml
Total protein - 11.4 mg
Relative activity - 227000
Total activity - 2588000
(13) Superdex 75 pg (gel chromatography in the presence of CHAPS)
538.2 ml of the high molecular weight TPO YMC-Pack PROTEIN-RP TPO active fraction F2 originating from the F2 sample (total protein 11.3 mg; protein concentration 0.021 mg / ml; relative activity 227000, total activity 2565000) obtained in stage (12 ), mixed with 0.6 ml of 50% glycerol and concentrated by evaporation. Then, 18 ml of 20 mMCHAPS was added, followed by stirring and incubation for 41 hours at 4 ^° C. After this, the first sample was applied to a column packed with Hiload 26/60 Superdex 75 pg (manufactured by Pharmacia Biotech, Cat. 17-1070-01, 2.6 cm in diameter and 60 cm in height) and eluted with DPBS containing 5 mM CHAPS at a flow rate of 1 ml / min. Parts of 4 ml of each sample (protein concentration 0.466 mg / ml, protein 1.86 mg) were applied to this column.

 In this case, the Superdex 75 column operation was repeated 6 times by dividing the entire YMS-Pack PROTEIN-RP TPO-active fraction into 6 parts. The eluates of each operation were collected in 45 tubes, receiving at the end of 45 fractions, each of which contained 30 ml of eluate after completion of 6 column operations.

 0.1 ml of each of the fractions thus obtained was mixed with 10 μl of 5% BSA, dried by evaporation, dissolved in 0.25 ml of IMDM and then analyzed to identify TPO-active fractions. As a result, TPO activity was detected in the fractions of tubes with numbers 13-31 (molar mass range from 78000 to 3000), which were combined and used as a high molecular weight TPO Superdex 74 pg TPO active fraction F2 from the F2 sample.

Obtained from the F2 sample of high molecular weight TPO Superdex 75 pg TPO-active fraction F2
Total volume - 540 ml
Protein concentration - 0.00216 mg / ml
Total protein 1.17 mg
Relative activity - 1750000
Total activity - 2041000
(14) YMC-Pack CN-AP (reverse phase chromatography)
513.2 ml of 540 ml obtained from the F2 sample of high molecular weight TPO Superdex 75 pg TPO-active fraction F2 (molar mass 78000-3000) (total protein 1.11 mg, protein concentration 0.00216 mg / ml, relative activity 1750000, total activity 1,943,000) obtained in step (13) was mixed with 1/10 volume of solvent B (1-propanol containing 0.05% TFA) and applied at a flow rate of 0.6 ml / min to a column packed with a YMC-Pack CN-AP (manufactured by UMC, cat. AR-513, 6 mm in diameter and 250 mm high), which was previously balanced 15% B, using solvent A (0.1% TFA) and solvent B. After on Eseniya propanol concentration was increased from 15% B to 25% B and 65 carried minute linear gradient from 25% B to 50% B.

 Before chromatography on the described column, 1/20 of the total sample applied was tested to confirm the correct extraction of activity. After that, the remaining 19/20 samples were divided into 2 samples for the column operation twice. In other words, the column operation was repeated three times. Since the same tubes were used to collect eluates in 3 operations, a total of 44 tubes were obtained, each containing 3.6 ml of eluate.

 5 ml of each of the fractions thus obtained (1/720 part of each fraction) was mixed with 0.25 ml of IMDM and then analyzed to identify TPO-active fractions. As a result, TPO activity (quite strong) was detected in the fractions of tubes with numbers 24-30 (range of propanol concentrations 36.0-42.0), which were combined and used as the main TPO from the F2 sample of high molecular weight TPO YMC-Pack CN-AP -active fraction of FA.

Obtained from F2 high molecular weight TPO sample YMC-Pack CN-AP TPO active fraction FA
Total volume - 25.20 ml
Protein Concentration - 0.0246 mg / ml
Total protein - 0.620 mg
Relative activity - 700,000
Total activity - 434,000
(15) Capcell rak C1 300A (reverse phase chromatography)
24.66 ml of 25.20 ml of high molecular weight TPO YMC-Pack CN-AP TPO-active fraction FA obtained from sample F2 (total protein 0.606 mg, protein concentration 0.0246 mg / ml, relative activity 700,000, total activity 424000), obtained in stage (14), was mixed with 0.4 ml of 50% glycerol and concentrated by evaporation.

 In this way, 2 ml of a concentrated sample were obtained with a propanol concentration of several%, a glycerol concentration of 10% and a protein concentration of 0.303 mg / ml. It was applied to a column packed with Capcell Pak C1 300A (manufactured by Shiseido Co., Ltd., Cat. C1 TYPE: SG300A, 4.6 mm in diameter and 250 mm in height), which was previously equilibrated with 15% B using solvent A (0.1% TFA) and solvent B (1-propanol containing 0.05% TFA) and showed 65 minutes with a linear gradient from 27% B to 38% B at a flow rate of 0.4 ml / min. Eluates were collected in 72 polypropylene tubes of 0.6 μl in each tube.

 0.75 μl from each of the fractions thus obtained (1/800 of each fraction) was mixed with 20 μl of 5% BSA, the medium was replaced with 225 μl of IMDM and the fractions thus diluted 300 times were analyzed.

Samples for electrophoresis were prepared from 2 μl of each fraction (1/300 fraction), dried by evaporation and treated at 95 ^{° C.} for 5 minutes with 10 μl of non-reducing sample buffer for SDS-PAGE. The sample thus treated was subjected to SDS-PAGE using a 15-25% SDS-polyacrylamide preformed gel (manufactured by Daiichi Pure Chemicals Co., Ltd.) and then stained using a silver staining kit. The DPCIII described above was used as a mole marker. masses.

 As a result of the described analysis, significant TPO activity was detected in the fractions of tubes with numbers 33-39 (range of propanol concentrations 29.5-31.5%). Fractions of 34-39 tubes (range of propanol concentrations 30.0-31.5%) were pooled and used as the main TPO-active fraction A. Using the SDS-PAGE distribution of the main TPO-active fraction, a band was found whose staining density correlates with the strength activity, within the average mol. masses 17000-19000 under non-reducing conditions, which was similar to the band in the case of the low molecular weight TPO TPO-active fraction obtained from sample F3 described in stage (10).

Example 2. Analysis of partial amino acid sequences of purified TPO rats
The amino acid sequence analysis of rat TPO candidate protein in Capcell Pak C1 300A of the TPO active fraction of FA obtained in the purification step (10) of Example 1 was carried out according to the Iwamatsu procedure (Iwamatsu et al., "Shin Kiso Seikagaku Jikken-ho (New Basic Biochemical Experiments) ", vol. 4, pp. 33-84, pub. Maruzen; Iwamatsu, A, Seikagaku (Biochemistry): vol. 63, No. 2, pp. 139-143, 1991; Iwamatsu, A., Electrophoresis, vol 13, pp. 142-147, 1992): T. E. the sample was subjected to SDS-PAGE and electrically transferred to a polyvinylidene difluoride (PVDF) membrane. After recovery and S-alkylation of the protein on the PVDF membrane, the protein thus treated was digested into peptide fragments by systematic and stepwise hydrolysis using restriction enzymes in situ with three proteases, and the obtained peptide fragments were separated and purified by reverse phase chromatography at each cleavage stage and their amino acid sequences analyzed using a highly sensitive method for determining amino acids. Further this process is described in detail.

An example of analysis of the candidate TPO protein in the low molecular weight TPO Capcell Pak C1 300A obtained from the F3 sample of the TPO FA fraction
(1) Concentration of Capcell Pak C1 300A fraction TPO FA
From 4200 μl of the low molecular weight TPO TPO active fraction FA obtained from the F3 sample from the Capcell Pak C1 300A column (tube numbers 36-42) obtained in stage (10) (total protein 39.6 μg, protein concentration 9.4 μg / ml, relative activity 4890000, total activity 193600), 4151 μl (98.8% of the whole fraction) were used in the analysis of amino acid sequences. The total protein calculated from the chromatogram was 39.1 μg, of which 1.6 μg was the estimated TPO protein stained with silver after SDS-PAGE and having an average mol. a mass of approximately 17000-19000.

 This sample was mixed with glycerol and concentrated by evaporation to obtain 5 μl of glycerol solution. A non-reducing buffer for SDS-PAGE and 1 M Tris-HCl (pH 8) was added to adjust its pH to obtain approximately 25 μl of a sample containing 200 mM Tris-HCl (pH 8), 50 mM Tris-HCl (pH 6, 8), 1.1% SDS, 2 mM EDTA, 0.02% bromophenol blue, and 30 g glycerol.

The sample thus obtained was kept at room temperature for 14 hours and then treated at 60 ^{° C.} for 5 minutes to carry out the reaction with SDS without excessive heating.

(2) Electrophoresis
Gel microplates were prepared (4.0% acrylamide concentrating gel and 15% acrylamide separating gel) and SDS-PAGE was performed at room temperature for 2 hours at a constant current of 12.5 mA and then at 17.5 mA. Pre-stained Low Range Marker (Bio-Rad, 161-0305) and DOCIII were used as markers mol. mass Immediately after electrophoresis, the resulting protein molecules were transferred onto a PVDF membrane (see next step).

 Part of this sample was also used in another electrophoresis using 15-25% polyacrylamide preformed gel (Multi Gel 15/25 manufactured by Daiichi Pure Chemicals Co., Ltd., 211072) under non-reducing conditions or after restoration of the sample with dithiothreitol (DTT). When the obtained gel was stained using the silver staining kit described above, it was confirmed that mol. the weight of the estimated TPO protein band was approximately 19,000 under reducing conditions, and the purity of the estimated TPO protein from the Capcell Pak C1 300A column was only a few percent. In addition, since the mobility of this band varied under non-reducing and reducing conditions, it was suggested that the candidate protein contains at least one disulfide bond.

(3) Transfer of protein to a PVDF membrane by electro-blotting and band detection
Protein Transfer to the PVDF ProBlott Membrane, Applied Biosystems; cat. 400994) was carried out for 1 hour at a direct current of 160 mA (11-17 V) using a semi-dry transfer device (Model KS-8460 manufactured by Marysol). A solution consisting of 0.3 M Tris and 20% methanol (pH 10) was used as an anolyte, a solution consisting of 25 mM Tris and 20% methanol (pH 10.4) was used as a solution for the membrane and the solution consisting of 25 mM Tris, 40 mM aminocaproic acid and 20% methanol (pH 10.4) as catholyte.

 When staining the membrane transferred in this way with a staining solution of Ponceau S (0.1) g Ponceau S and 1 ml of acetic acid in 100 ml of water), many bands were found and it was confirmed that the putative TPO protein had a mol. weight 19000. This strip was excised for use in subsequent steps of peptide fragment formation.

(4) Formation of peptide fragments, peptide mapping and analysis of amino acid sequences
To perform systematic (directed) fragmentation of the putative TPO protein transferred onto the membrane and S-alkylated after reduction on the PVDF membrane, stepwise limited enzymatic hydrolysis was carried out using the following three proteases.

 First cleavage: lysyl endopeptidase (Achromobacter lyticus m497-1 manufactured by Wako Pure Chemical Industries, Ltd., cat. 129-02541).

Second Cleavage: ASp-N Endoproteinase (manufactured by Boeringer-Mannheim Corp., Cat. 1054 589)
Third Cleavage: Trypsin-TRSC (manufactured by Worthington Biochemical Cat. 3740)
Peptide fragments obtained by each enzymatic digestion were loaded onto a reverse phase Wakosil-11 5C18 C18 column (Wako Pure Chemical Industries, Ltd., 2.0 mm in diameter and 150 mm in length) and eluted with solvent A (0 , 05% TFA) and solvent B (isopropanol: acetonitrile = 7: 3, 0.02% TFA) for 30 min with a linear gradient from 1% B to 50% V at a flow rate of 0.25 ml / min and at a column temperature of 30 ^o C. In this way, peptide mapping was done (see FIG. 4). The resulting peptide fragments were recovered and subjected to Erdman cleavage using a gas phase amino acid sequencer (PPSQ-2 manufactured by Shimadzu Corp.). Thereafter, each amino acid obtained in turn from a sequencer whose N-terminus was connected to PTH was identified using C18 reverse phase column chromatography using isocratic elution. The results are presented in table II.

 In the sequences shown in Table II, the sequences shown in parentheses at the N-ends are amino acid residues that can be deduced from targeted enzymatic cleavage.

(5) Analysis of the homology of the obtained amino acid sequences (search for homology)
In order to find out if the thus obtained amino acid sequences are contained in a known reported protein or whether a protein having similar sequences exists, they were analyzed using the analysis sequence of the Mac Vector program (Kodak International Biotechologies, Inc.). For information on known proteins or known genes, the Entrez Release 6 database (National Center of Biotechnology Information, National Library of Medicine, National Inctitute of Health, USA, published on August 15, 1993) was used.

It includes the following databases:
.Entrez Release 6 database
.NCB1-GenBank, August 15, 1993 (Release 78.0)
.EMBL, July 15, 1993 (Release 35.0 plus updates)
.DDBL, July 15, 1993
.SWISS-PROT, April 1993 (Release 25.0)
.PIR, June 30, 1993 (Release 37.0)
.PDB, April 1993
.PRF, May, 1993
.dbEST, July 15, 1993 (Release 1.10)
.US and European Patents
As a result, the (K) DSFLADVK AP12 sequence completely coincided with the internal KDSFLADVK sequence of the rat corticosteroid binding globulin precursor (CBG) [PIR data base accession No. A40066; Smith and Hammond; "Rat Corticosteroid Binding Globulin: The Primary Structure and Levels of Liver mRNA under Various Physiological Conditions," Mol. Endocrinol., (1989), 3, 420-426].

 Further detailed research revealed that the KQYYESE sequence (SEQ 1D No. 193), which is highly similar to the (K) XYYESZ sequence (X = A, S, G, M or Q and Z = E or K) AP3, is contained in rat CBG- amino acid sequence. In rat CBG, sequences corresponding to the AP12 and AP3 sequences are linked together to form the internal amino acid sequence KDSFLADVKQYYESE (SEQ 1D # 190).

 As for the amino acid sequences of fragments other than AP12 and AP3, no protein or gene was found whose similarities could be discussed.

Example 3. Analysis of the biological characteristics of SRW obtained from blood plasma of thrombocytopenic rats
(1) In the rat CFU-MK test system (liquid culture system)
As a typical example, FIG. 5 shows a dose response curve for a TPO sample partially purified from plasma of thrombocytopenic rats (TPO active fraction F2 from a YMC PackProtein-RP column described in the purification step (8) of Example 1-2) . Periodic observation of cultured cells under a microscope revealed the generation and maturation of megakaryocytes, namely an increase in cell size, possibly together with an increase in the number of megakaryocytes. As a particularly significant change, the process of formation of megakaryocytes was detected on the 4th day, which was the last day of cultivation (they were difficult to recognize on the 3rd day). This formation process is called the cytoplasmic formation process (Leven and Yee, Blood, vol. 69, pp. 1046-1052, 1987) or the formation of prothrombocytes (Torr et al., Blood, vol. 76, pp. 912-924, 1990) , which are considered the structure of platelet precursors differentiated further from mature megakaryocytes, and these prothrombocytes are considered the final stage of megakaryocyte differentiation observed so far in vitro. Since such a morphological change was observed with high frequency when using only a TOP sample, it is possible that this factor alone can stimulate the proliferation and differentiation of CFU-MK, it can produce mature megakaryocytes and can ultimately release platelets.

(2) In the colony count test system
When testing a TPO sample, partially purified from blood plasma of thrombocytopenic rats, using a colony colony test system using undivided rat bone marrow cells, cells of each separation / concentration stage, or Gp11b / 111a ⁺ CFU-MK TPO fraction obtained from rat plasma, stimulated the formation of colonies of megakaryocytes. Compared to megakaryocyte colonies induced by other cytokines, such as IL-3, mouse GM-CSF or human EPO, TPO-induced megakaryocyte colonies were different in that each colony consisted of a small number of megakaryocytes, but each megakaryocyte was larger, which meant great maturity. In addition, since TPO did not cause colony formation or caused a small colony formation of other cell lines, Meg-CSF activity of TPO can be considered as megakaryocyte-specific. Based on these facts, it is obvious that TPO has different biological properties compared with the properties of other cytokines, such as rat IL-3, mouse GM-CSF and human EPO, and exhibits unique Meg-CSF activity.

A sample of TPO, partially purified from blood plasma of thrombocytopenic rats, also showed Meg-CSF activity on CD34 ⁺ , DR ⁺ cell fractions obtained from human bone marrow cells or human spinal cord blood cells, and induced significant numbers of human megakaryocyte colonies, indicating the fact that this factor has no species specificity.

Example 4. Specialization of rat TPO producing cells
(1) Screening of TPO-producing rat organs
Rat TPO-producing organs were screened and specialized in order to provide a source of mRNA for use in cloning the rat TPO gene. First, bone marrow, lungs, liver and spleen were periodically taken from rats made by thrombocytopenic administration of P55 antibodies, their cells (organ sections in the case of lungs and liver) were cultured, and the activities in the obtained culture supernatant were tested using a test system with rat CFU-MK. However, this initial attempt did not give clear results. Then, a further attempt was made to cultivate the liver cells (hepatocytes) obtained by collagenase perfusion from the liver of thrombocytopenic rats induced by the administration of P55 antibodies, taking into account the message indicating the relationship between the liver and the formation of TPO in rats (Siemensma et al., J. Lab. Clin Med., Vol. 86, pp. 817-833, 1975).

 When the obtained culture supernatant was applied to a WGA-Agarose column, followed by fractionation of the adsorbed fraction on a reverse phase Vydac phenyl column, a very similar activity was found at the same position of the plasma-derived TPO in the rat CFU-MK test system. Weak but the same activity was found in the culture supernatant of normal rat hepatocytes. These results suggest that the liver is probably one of the TPO producing organs.

(2) Screening of Production of Rat TPO Cell Lines
Based on the above results, rat TPO producing cell lines were screened. First, each of the 20 rat liver-derived cell lines was cultured in an appropriate subcultural liquid medium until the cells almost reached the fusion stage. Then, the culture medium was replaced with the same appropriate medium supplemented with 5% FCS and cultivation was continued for 3 days. When each of the obtained culture supernatants was partially purified in accordance with the procedure described in stage (1), to investigate the presence of TPO formation, TPO activity was distinctly detected in three cell line derived from parenchymal liver cells of rats, namely McA-RH8994 cells ( ATCC CRL 1602, Becker et al., "0ncodevelopmental Gene Expression", ed. By Fishman and Sell, Academic Press, NY, pp. 259-270, 1976; purchased from Dainippon Pharmaceutical Co., Ltd.), H4-11- E cells (ATCC CRL 1548, Pitot et al., Nat. Cancer Int. Monogr., Vol. 13, pp. 229-245, 1964; purchased from Dainippon Pharmaceutical Co., Ltd.) and HTS cells (Thompson et al. , Pr oc. Natl. Acad. Sci, USA, vol. 56, pp. 296-303: 1966 purchased from Dainippon Pharmaceutical Co., Ltd.).

(3) Detailed analysis of TPO activities in McA-RH8994 cells, H4-11-E cells and NTS cells
The biochemical and biological properties of TPO-active proteins secreted from these three rat cell lines were studied in detail and compared with the properties of plasma-derived TPO.

McA-RH8994 cells were suspended in alpha-MEM (-) liquid culture medium containing 10% FCS and transferred to a plastic microculture flask (175 cm ² ) at 1 x 10 ⁶ cells per flask. After 3 days of culturing at 37 ^{° C.} in a CO ₂ incubator (5%), this medium was replaced with IMDM culture medium containing 5% FCS, and cultivation was continued for another 3 days to obtain the resulting culture supernatant. H4-11-E cells were suspended in Eagle's modified Dulbecco method (liquid medium) (glucose 4.5 g / l, hereinafter referred to as DMEM) containing 10% FCS and transferred to plastic flasks (175 cm ² ) at 5 • 10 ⁵ cells per flask. After 3 days of culturing at 37 ^{° C.} in an incubator with 5% CO _{2, the} medium was replaced with IMDM containing 5% FCS, and cultivation was continued for another 3 days to obtain the resulting culture supernatant. NTS cells were also suspended in DMEM liquid culture medium containing 5% FCS and transferred to plastic flasks (175 cm ² ) at 2.5 x 10 ⁵ cells per flask. After 3 days of culturing at 37 ^{° C.} in an incubator with 5% CO _{2, the} medium was replaced with IMDM culture medium containing 5% FCS, and cultivation was continued for another 3 days to recover the obtained culture supernatant.

 Using 2 L of the culture supernatant thus obtained from each of these three cell lines, a partial purification of the TPO obtained from the cell lines was carried out in accordance with the procedure for the purification of TPO from XRP described in Example 1-2. The results obtained are described below.

 First, each culture supernatant was concentrated approximately 6-fold using an ultrafiltration device and the medium was replaced with 20 mM Tris-HCl buffer (pH 8.0) using a Sephadex G-25 column. The resulting eluate was applied to a Q-Sepharose FF column, the column was washed with 20 mM Tris-HCl (pH 8.0) and then the adsorbed fraction was eluted with 20 mM Tris-HCl (pH 8.0) containing 175 mM NaCl. The fraction thus eluted was loaded onto a WGA-Agarose column, the column was washed with PBS, and the adsorbed fraction was eluted with 20 mM sodium phosphate (pH 7.2) containing 0.2 M GlcNAc and 0.15 M NaCl. The resulting eluate was applied to a TSK-gel AF-BLUE 650MH column, the column was washed with 20 mM sodium phosphate (pH 7.2) containing 1 M NaCl, and then the adsorbed fraction was eluted with 2 M NaSCN. The resulting eluate was applied to a Phenyl-Sepharose 6 FF / LS column, the column was washed with 50 mM sodium phosphate (pH 7.2) containing 1.5 M ammonium sulfate, followed by washing with 0.8 M ammonium sulfate containing 36 mm sodium phosphate, and then the adsorbed fraction was eluted with 20 mM sodium phosphate (pH 7.2). The fraction thus obtained was concentrated and then fractionated using a reverse phase Vydac Protein C4 column (The Separation Group, Cat. 214TP51015, diameter 1 cm, height 15 cm). Those. this sample was applied to a C4 column, previously equilibrated with 20% B, and then eluted for 90 minutes with a linear gradient from 20% B to 40% B at a flow rate of 1 ml / min, using 0.1% TFA as developing solvent A and 1-propanol containing 0.05% TFA as the developing solvent B. As a result, each of these TPO-active proteins obtained from these cell lines was eluted at a concentration of 1-propanol of 30-43%.

When measuring TPO activities in samples of each purification step using a rat CFU-MK test system, the results showed that the TPO activities of these 3 cell lines gave similar distributions to those obtained from XRP TPO (compare Example 1-2). The relative activity, activity yield, etc., at each purification step, measured in a rat CFU-MK test system, are shown in Table 2. In addition, when measuring TPO activities in eluates from a reverse phase end column in a test system with rat CFU-MK TPO activities of 3 cell lines and activity of XRP derived TPO were detected at the same peaks. When conducting a test with the counting of colonies of TPO-active fractions from a reverse phase column using non-attached cells obtained in the depletion stage by attachment to separate and concentrate rat CP11b / 111a ⁺ CFU-MK, the distribution of elution, MEG-CSF activity were very similar to the distributions TPO activity measured in a rat CFU-MK test system (see table 3).

 In addition, similar to the TPO obtained from XRP, each of the TPO activities of these 3 cell lines did not form (or formed very few) colonies of other cell lines.

 Each of the combined active fractions from the reverse phase column was subjected to SDS-PAGE according to the procedure described in example 1-2. When extracting protein from the obtained gel and measuring TPO activity in a test system with rat CFU-MK, it was found that the average mol. the masses of XRP-derived TPO, obtained from McA-RH8994 TPO cells and obtained from H4-11-E TPO cells were 17000-22000, 33000-39000 and 31000-38000, respectively, and obtained from the STS of TPO cells detected average mol. masses 17000-22000 and 28000-35000.

 Thus, these results showed that the TPO proteins produced by McA-RH8994 cells, H4-11-E cells and NTS cells are equivalent to those obtained from XRP TPO in their biological properties, although their biochemical properties are slightly different, in particular, in relation to mol .mass. A noteworthy discovery in this invention is the possibility that the TPO-active protein molecule contained in the blood may be a partial cleavage product at its specific or disordered site in its producing cells or after its secretion from producing cells. This also suggests the possible existence of TPO genes (mRNA and cDNA) having different lengths.

Example 5. Construction of an expression vector (pEF18S) for a cDNA library
A vector into which a DNA fragment can be easily integrated and which can affect the high expression efficiency of cloned cDNA has been designed for use in cloning and expression of TPO cDNA. Those. The pEF18S expression vector was constructed from the pME18S expression vector by substituting the SRα promoter with the promoter of the elongation factor Iα (EFIα) promoter, which is known as the promoter of high expression (see FIG. 6). The promoter of the elongation factor Iα was obtained by partial cleavage of 1 μg of the pEF-BOS expression vector (Mizushima et al., Nucleic Acids Res., Vol. 18, p. 5322, 1990) with the restriction enzymes Hind111 and EcoRI, after which the cleavage product was subjected to electrophoresis in 2 % agarose gel (FMC BioProducts) to isolate a DNA fragment of approximately 1200 bp and purification of this DNA fragment using Prep-A-Gene DNA Purification Kit (Bio-Rad Laboratories, Inc .; kit for use in selective DNA purification, which is performed by adsorption of porous silicon dioxide matrix based on the procedure reported by Willis et al., In Bio Tehniques, vol. 9, pp. 92-99, 1990), 100 ng of the thus obtained DNA fragment was ligated with 50 ng of the pME18S expression vector (Liu et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 8957-8961, 1993), which was previously cleaved by HindIII and EcoRI. Cells of a commercial host strain, a highly competent strain of DH 5 E. coli (Toyobo Co., Ltd .; competent strain of E. coli obtained by a modified method of Hanahan et al., J. Mol. Biol. Vol. 166, pp. 557-580, 1983), transformed so constructed vector. After culturing the transformed cells, 12 colonies were randomly selected and plasmid DNA was purified from each colony. Altogether, 10 clones containing the target plasmid (pEF18S) were obtained by analysis of the restriction enzyme cleavage distributions of these DNA molecules. After that, a clone selected from them was cultured to obtain a large amount of this plasmid DNA.

Purification of plasmid DNA was carried out mainly in accordance with the procedure described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Those. clone. pEF18S obtained as described above was cultured overnight at 37 ^{° C.} in 50 ml of LV medium (1% Bacto-tryptone, 0.5% Basto-yeast extract, 0.5% NaCl) containing 50 μg / ml ampicillin , and the obtained cells were collected by centrifugation and suspended in 4 ml of a TEG-lysozyme solution (25 mM Tris-HCl, pH 8, 10 mM EDTA, 50 mM glucose, 0.5 lysozyme). To it was added 8 ml of 0.2N NaOH / 1% SDS (solution) and then 6 ml of a 3 M potassium solution / 5 M acetate to thoroughly suspend the cells. After centrifugation of this suspension, the resulting supernatant liquid was treated with a phenol / chloroform mixture (1: 1) mixed with the same volume of isopropanol, and then centrifuged. The resulting precipitate was dissolved in a TE solution (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) and treated with RNase and then with a phenol / chloroform mixture (1: 1), followed by precipitation with ethanol. The resulting precipitate was again dissolved in a TE solution, to which NaCI and polyethylene glycol 3000 were successively added to their final concentration of 0.63 M and 7.5%, respectively. After centrifugation, the precipitate was dissolved in a TE solution and precipitated with ethanol. Approximately 300 μg of plasmid DNA was obtained in this way. 100 μg of this DNA was completely digested with restriction enzymes EcoRl and Notl and electrophoresed on 0.8% agarose gel (manufactured by FMS BioProducts) and the fragments of the vector thus extracted were purified using Prep-AGene DNA purification kit (Bio-Rad Laboratories, Inc.) , obtaining approximately 55 μg of plasmid DNA, which was used in the subsequent construction of the cDNA library.

Example 6. Purification of mRNA from McA-RH8994 cells
McA-RH8994 cells that showed relatively high activity in the colony count test of Example 4 were selected as material for use in rat TPO cDNA cloning and were subjected to the following experiments.

Isolation of total RNA was carried out mainly in accordance with the procedure described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Those. McA-RH8994 was grown to fusion in 15 culture dishes 90 mm in diameter. After removing the liquid medium, the cells in each dish were carefully suspended in 0.8 ml of a 5 M guanidine solution (5 M guanidine thiocyanate, 5 mM sodium citrate (pH 7.0), 0.1 M β-mercaptoethanol, 0.5% sodium sarcosyl sulfate) and the resulting suspensions in all plates were collected in one tube and the total volume was adjusted to 29 ml with guanidine solution. The resulting mixture, which became viscous due to cell disruption, was subjected to approximately 20 repetitions of suction / discharge with a 20 ml syringe equipped with a 21G needle until the mixture ceased to be viscous. 18 ml of 5.7 M CSC1-0.1 M EDTA (pH 7.5) was used as a pad in a polyallomeric centrifuge tube for use in a Beckman SW28 rotor, and the above mixture in an amount of approximately 20 ml was carefully layered onto this pad in such a way so that the layers are not disturbed and the tube is almost full. The tube thus obtained was centrifuged for 20 hours at 25,000 rpm at 20 ^° C. The resulting precipitate was washed twice with a small amount of 80% ethanol, dissolved in TE solution, extracted with phenol / chloroform (1: 1), and then precipitated with ethanol with 1 / 10 volumes of 3 M sodium acetate and 25 volumes of ethanol. Approximately 2.5 mg of total RNA from approximately 10 ⁸ cells was obtained in this way.

Purification of poly (A) ⁺ RNA from total RNA was performed using Oligotex ^™ -DT30 (Super) (manufactured by Japan Synthetic Rubber / Nippon Roche; oligodd molecules are immobilized on the latex particles by covalent binding, and purification of poly (A) ⁺ PHK can be achieved similarly with how this is achieved using an oligo dT column, which is usually used for this purpose). The result was 20 μg of poly (A) ⁺ RNA from approximately 500 μg of total RNA.

Example 7. Construction of a rat cDNA library
A double-stranded cDNA having an EcoRl recognition site at its 5'-end and a Notl recognition site at its 3'-end was synthesized from 5 μg of poly (A) ⁺ RNA obtained as described in Example 6 using the Time cDNA synthesis kit Saver ^™ (manufactured by Pharmacia; cDNA synthesis kit based on the modified method of Okayama and Berg, Mol. Cell. Biol., Vol. 2, pp. 161-170, 1982) and DIRECTIONAL CLONING TOOLBOX (manufactured by Pharmacia; kit from
primer 5'-AACTGGAAGAATTCGCGGCCGCAGGAA (T) ₁₈ -3 '(SEQ 1D No. 15) containing the Notl recognition sequence for use in cDNA synthesis, and 5'-AATTCGGCACGAG-3' adapters (SEQ 1D no 16) 5'-CTCGTGTGCCG- 3 '(SEQ 1N No. 17) for use in adding an EcoRl recognition sequence). Thus synthesized cDNA was ligated with 1.2 μg of pEF18S expression vector (see Example 5), which was previously digested with EcoRl and Notl, and transformed into 8.4 ml of the aforementioned highly competent E. coli DH5 (manufactured by Toyobo Co., Ltd.) . As a result, 5.3 • 10 ⁵ transformants were obtained.

Example 8. Obtaining (cloning) cDNA of a rat TPO fragment using PCR
530000 clones of the cDNA of the Mca-RH8994 library constructed in Example 7 were cultured overnight in 50 ml of LB medium containing 50 μg / ml ampicillin, and the plasmid McA-RH8994 of the cDNA library was purified from the obtained cells by centrifugation using QIAGEN-tip 100 (manufactured by DIAGEN; anion exchange column with silica for DNA purification). Received approximately 200 μg of plasmid DNA.

Synthesized two antisense nucleotide primers AP18-1R and AP8-2R, which correspond to the amino acid sequence of the peptide fragment AP8 described in example 2, and two sense nucleotide primers EFIα-1 and EFIα-2, which correspond to the first intron of human elongation factor Iα of the plasmid vector pEF -18S (see Fig.6) used to obtain a cDNA library. These primers were synthesized using a 394DNA / RNA synthesizer (manufactured by Applied Biosystems, a synthesizer based on the β-cyanoethyl amidite method) and purified using an OPC column for purifying synthetic DNA (manufactured by Applied Biosystems; a reverse phase silica gel column for use in purification of synthetic DNA having trityl groups). Each of the thus synthesized and purified DNA molecules was dissolved in a TE solution to a final concentration of 50 μM and stored at -20 ^° C until their use. The synthetic oligonucleotides used in the following procedure were synthesized and purified in the same way.

 Primers AP8-1R and AP8-2R are mixed primers consisting of 17 continuous nucleotides that include deoxyinosine, as reported by Takahashi et al. (Proc. Natl. Acad. Sci. USA, vol. 82, pp. 1231-1935, 1985).

Primers EFIα-1 and EFIα-2, consisting of 21 and 20 nucleotides, respectively, were synthesized based on the nucleotide sequences of positions 1491-1512 and 1513-1532 of the genomic sequence reported by Uetsuki et al. (J. Biol. Chem., Vol. 264, pp. 5791-5798, 1989). (see table III)
With 3 μg McA-RH8994 plasmid cDNA library as template, AP8-1R (500 pmol) and EFIα-1 (100 pmol) as primers and GeneAmp ^TM PCR reagent kit with AmpliTaq ^TM DNA polymerase (manufactured by Takara Shuzo Co., Ltd., a set of thermostable Taql polymers, reaction buffer and dNTP for use in PCR) was performed by PCR using the GeneAmp ^TM PCR system (Perkin-Elmer; thermoblock for PCR) (100 μl volume, heating at 95 ^o C 2 min, total 35 cycles, each cycle consists of denaturation at 95 ^o C for 1 minute, hybridization (annealing) at 40 ^o C for 1 minute and synthesis at 72 ^o C for 1 minute, and final incubation at 72 ^{° C.} for 7 minutes). To improve the specificity of the DNA fragments thus amplified, further PCR was performed (100 μl volume, heating at 95 ^° C for 2 minutes, 35 cycles in total, each cycle consisting of denaturation at 95 ^° C for 1 minute, hybridization at 45 ^° C in for 1 minute and synthesis at 72 ^° C for 1 minute, and final incubation at 72 ^° C for 7 minutes) using 1 μl of the resulting PCR reaction solution as a matrix and EFlα-2 (100 pmoles) and AP8-IR ( 500 pmoles) as primers.

The reaction solution thus obtained was subjected to electrophoresis on a 2% agarose gel (manufactured by FMC BioProducts) to isolate a DNA fragment of approximately 330 bp as the main PCR product, which was then purified using the aforementioned Prep-A-Gene DNA purification kit (manufactured by Bio-Rad Laboratories, Inc.) using T4 DNA ligase (manufactured by Life Technologies), the DNA fragment thus purified was subcloned into the pCR vector ^TM 11 (a vector for use in TA cloning of PCR products manufactured by Invitrogen). Since the thermostable polymerase used in PCR has terminal transferase activity, the amplified DNA fragment was directly subcloned into the pCR ^TM 11 vector, which has a 5'-dT sticky end, using the property of this enzyme to add one deoxyadenylic acid to the 3'-end of PCR amplified DNA Plasmid DNA molecules were purified from 28 clones randomly selected from the obtained clones using QIAGEN-tip100 (DIAGEN), and their nucleotide sequences were determined using a 373A DNA sequencer (Fluorescent sequencer manufactured by Applied Biosystems) using the Taq Dye Deoxy ^TM Terminater Cycle Seguening kit Kit (Applied Biosystems; kit for use in determining the nucleotide sequence using fluorescent dyes based on the dideoxy method of Senger et al. (Proc. Natl. Acad. Sci. USA, vol. 74, pp. 5463-5467, 1977). When a DNA fragment encoding three amino acids The residues (Ile / Thr / Ser) -Val-Pro of the AP8 amino acid sequence shown above adjacent to the AP8-2R primer were selected from the DNA fragments obtained in this way and its full length was sequenced; it contained 261 kb cDNA Positions 173-175 of this cDNA fragment encoded methionine and the coding frame coincided with the amino acid frame of AP8. Since the sequence wedged in position 173-175 of the DNA fragment coincided with the Kozak sequence (Kozak, M., Cell, vol. 44, pp. 238-292, 1986), one might think that this DNA fragment consists of a translation initiation region encoding the N-terminus of rat TPO protein. This DNA fragment was named A1. The nucleotide sequence of the A1 fragment, including the vector sequence, and the amino acid sequence deduced from it are shown in the list of sequences (SEQ 1D No. 1), attached here.

Example 9. Screening of a rat TPO cDNA clone using PCR
The cDNA library was divided into pools of approximately 10,000 clones, cultured overnight in 1 ml of the aforementioned LB medium containing 50 μg / ml ampicillin, and then the plasmid DNA was extracted using an automated P1-100 plasmid DNA extraction device (VER-3.0 , Kurabo Industries, Ltd., an automated plasmid DNA extraction device based on a modified alkaline SDS method described in Molecular Cloning, Sambrook et. Al., Cold Spring Harbor Laboratory Press, 1989). The following two oligonucleotides based on the A1 fragment of the cDNA were synthesized and purified separately.

 5'-CGAGGGTGTACCTGGGTCCTG-3 '(SEQ 1D No. 31) (sense sequence of positions 1-17 of the sequence SEQ 1D No. 1; CGAG denotes the adapter sequence 5'-CAGAGTTAGTCTTGCGGTGAG-3' (SEQ 1D No. 32) (antisense sequence of positions 212-232 SEQ 1D sequence No. 1).

Using 1/30 of the volume of each plasmid DNA sample obtained as described above as a template, the oligonucleotides thus synthesized as primers and the GeneAmp ^TM PCR reagent kit with AmpliTaq ^TM DNA polymerase (manufactured by Takara Shuzo Co., Ltd.) PCR was performed using GeneAmp ^™ PCR System 9600 (PERKIN-ELMER) (30 cycles in total, each cycle consisting of denaturation at 94 ^° C for 30 seconds, hybridization at 66 ^° C for 30 seconds and synthesis at 72 ^° C for 1 minutes). As a result, a specific band of 236 kb was detected. in 3 out of 100 pools. When dividing one of these pools into subpools of approximately 900 clones for extraction of plasmid DNA and PCR as described, a specific band was found in 3 out of 100 subpools. Upon further separation of these subpools into pools of 40 clones and the same screening process, a specific band was found in 3 out of 100 tested pools. One of the candidate pools was cultured on an LV plate (LB medium containing 15% agar) with the addition of 50 μg / ml ampicillin and each of the formed colonies was subjected to extraction of plasmid DNA and PCR in the described manner. As a result, a positive band was detected in 2 out of 100 tested clones.

Example 10. Sequencing of rat TPO cDNA
Purification of plasmid DNA was carried out mainly in accordance with the procedure described in Molecular Cloning (Sambrook et. Al., Cold Spring Harbor Laboratory Press, 1989). Each of the two obtained clones of Example 9 was cultured overnight in 50 ml of LB medium containing 50 μg / ml ampicillin, and approximately 300 μg of plasmid DNA was obtained after purification as described in Example 6.

The thus obtained plasmid DNA was sequenced by the method described in Example 8 using the set of ^{Taq Dye Deoxy TM Terminater Cycle Sequening Kit} (Applied Biosystems) to determine the complete nucleotide sequence comprising a fragment A1. As a result, the nucleotide sequences of these two clones completely coincided with each other, indicating that they are the same clone. One of these clones was selected and the plasmid of this clone was named pEF18S-A2α. Its nucleotide sequence and the deduced amino acid sequence are shown in the sequence list (SEQ 1D No. 2).

 The sequence presented in the sequence list (SEQ 1D No. 2) has clear characteristics. The sequence in the direction of 5'-3 'from 172 bp The 5'-non-translating region encodes an amino acid sequence rich in hydrophobic amino acids, which is thought to be a signal sequence for protein secretion consisting of 21 amino acids starting with methionine. This protein contains 126 amino acid residues. The 3'-untranslated sequence contains 1025 nucleotides and a poly A tail after the termination codon (TAA). This protein contains a sequence that corresponds to the amino acid sequence AP8 analyzed in Example 2 (amino acid residues 1-12 in SEQ 1D No. 2), but does not contain a consensus sequence for N-glycosylation. The nucleotide sequence 1624-1629, located near the end of the 3'-untranslated sequence, differs from the consensus sequence and is, apparently, a potential polyadenylation sequence.

 The vector pEF18S-A2α carried by E. coli strain DH5 was deposited by the inventors on February 14 under deposit FERM BP-4565 at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Techology, Ministry of International Trade and Industry, Japan.

Example 11. Expression of rat TPO cDNA in COS 1 cells and confirmation of TPO activity
Transfection of the plasmid pEF18S-A2α into COS 1 cells was carried out as follows by a slight modification of the DEAE-dextran method involving chloroquine treatment (Sompayrac et al., Proc. Natl. Acad. Sci. USA, vol. 78, pp. 7575-7578, 1981 ; Luthman et al., Nucl. Acids Res., Vol. 11, pp. 1295-1308, 1983). COS 1 cells (ATCC CRL 1650) were suspended in the above DMEM containing 10% FCS, placed in a plastic tissue culture dish (100 mm) and cultured at 37 ^{° C.} in an incubator with 5% CO ₂ until the cells did not reach the stage of approximately 40% merger. Separately, plasmid pEF18S-A2α (10 μg) dissolved in 30 μl of HBS (21 mm HEPES, 145 mm NaCl, pH 7.1) was added to 4 ml of DMEM culture medium, 500 μg / ml DEAE-dextran (Pharmacia) was added, 80 μM chloroquine (Sigma Chemical Co.), 8% (v / v) HBS and 9% (v / v) Nu serum (Collaborative Research, Inc.). The mixture thus prepared was added to cultured COS 1 cells, which were washed twice with DMEM culture medium, and cultivation was continued for another 5 hours in an incubator with 5% CO ₂ . After that, the culture supernatant in this dish was removed by suction and the remaining cells in the dish were washed twice with DMEM culture medium, 15 ml of DMEM medium containing 10% FCS were added and cultured at 37 ^° C for 3-5 days in an incubator with 5% CO ₂ to extract the obtained culture supernatant.

 The culture supernatant thus obtained was extensively dialyzed against IMDM culture medium and evaluated in a rat CFU-MK test system. TPO activity was detected in a dose-dependent manner in the culture supernatant of COS 1 cells in which the plasmid pEF18S-A2α was expressed (Fig. 7). Similar to the case of XRP-derived TPO, many megakaryocytes formed elongated cytoplasmic formations on the 4th day of cultivation. In contrast, TPO activity was not detected in the culture supernatate of COS 1 cells into which only plasmid pEF18S was introduced (Fig. 7). In the M-07e test system, the proliferation activity of M-07e cells was also found in a dose-dependent manner in the culture supernatant of COS 1 cells in which plasmid pEF18S-A2α was expressed, but not in the culture supernatant of COS 1 cells in which plasmid pEF18S was expressed . These results indicate that A2α contains cDNA encoding a protein having TPO activity.

 Then, a sample of partially purified TPO was prepared to test the ability of this TPO activity to enhance platelet formation in vivo. First, COS 1 cells transfected with plasmid pEF18S-A2α were cultured for 3 days in a serum-free culture medium in which 0.2 mg of BSA was added, giving approximately 5.8 L of serum-free culture supernatant. To it was added a pAPMSS protease inhibitor to a final concentration of 1 mM and the mixture was filtered using a 0.22 μm filter. To 5793 ml of the obtained filtrate (protein concentration 0.229 mg / ml, total protein 1326 mg, relative activity 1000, total activity 1326000) 0.85 moles of NaCl per 1000 ml (288 g total) were added, getting 5849 ml of a solution containing 0.822 M NaCl . The solution thus obtained was applied at a flow rate of 7 ml / min to a TSK-gel AF-BLUE 650 MN column (Tosoh Corp., Cat. 08705, diameter 5 cm and height 6 cm), which was previously equilibrated with 20 mM sodium phosphate (pH 7.2) containing 1 M NaCl. After application was complete, approximately 7900 ml of eluate passed through the column, eluting with 20 mM sodium phosphate (pH 7.2) containing 1 M NaCl. This eluate was collected together and concentrated using an ultrafiltration device (Omega Ultrasette, nominal molecular weight cut-off of 8000) manufactured by Filtron) to obtain an F1 fraction passing through the column (460 ml, protein concentration 1.11 mg / ml, total protein 973 mg, relative activity 16.3). Then, the eluting solution was replaced with 2 M NaSCN and the TSK-gel AF-BLUE 650 MN TPO fraction eluted in such a way that was absorbed in such a way, F2 (2840 ml) was concentrated to 6.81 ml using an ultrafiltration device with an UM 3 membrane (76 mm in diameter, Amicon Corp.). The total protein in this TPO-active fraction of F2 was 12.5 mg and the yield of F2 protein at this stage was 0.62%. Then F2 was applied to a HiLoad 26/60 Superdex 200 pg column (Pharmacia Biotech, Cat. 17-1071-01, diameter 2.6 cm and height 60 cm) and developed with 20 mM sodium acetate (pH 5.5) containing 50 mM NaCl, at a flow rate of 1 ml / min. After the onset of TPO manifestation, activity was detected in the eluates in the range from 194 to 260 ml after application. These eluates were combined to give the Superdex 200 pg TPO active fraction F2 (66 ml, protein concentration 0.112 mg / ml, total protein 7.41 mg, relative activity 142860, total activity 1058600). Then, buffer A (20 mM sodium acetate, pH 5.5) and buffer B (20 mM sodium phosphate, pH 7.2, containing 500 mM NaCl) and the TPO-active fraction were prepared at a flow rate of 1 ml / min per strong a RESOURCE S cation exchange column (Pharmacia Biotech, Cat. 17-1178-01, 0.64 cm in diameter and 3 cm high), which was previously balanced with 100% A. After that, elution was performed at a flow rate of 0.3 ml / min, with a 40 min linear gradient from 100% A to 100% B. When analyzing the activity of TPO, activity was found in a wide range of eluates (from 5% B to 32% B). These TPO-active eluates were combined and concentrated using an ultrafiltration device with an UM 3 membrane (diameter 25 mm, Amicon Corp.), obtaining RESOURCE S TPO-active fraction F2 (1.65 ml, protein concentration 4.74 mg / ml, total protein 7.82, relative activity 71400, total activity 558600).

 A partially purified TPO sample was injected subcutaneously for 5 consecutive days (474 μg (100 μl) / mouse / day) ICR in male mice (9 weeks old), platelet counts of which were measured on the day before administration. As a control, BSA (200 μg (100 μl) - / mouse / day) or a buffer used as a solvent for partially purified TPO (100 μl / mouse / day) was administered in the same manner. The day after the last injection, blood was collected from the heart of each mouse to measure platelet counts using a hemocytometer (F800, Toa Iyo Denshi). In mice injected with partially purified TPO, platelet counts were observed, approximately 2.14 times greater than platelet counts prior to administration. When compared with control groups, platelet numbers in mice that were administered partially purified TPO were approximately 1.74 times higher than platelet numbers in BSA-treated mice and approximately 1.90 times higher than in buffer-treated mice. These results showed that TPO enhances platelet formation in vivo. Furthermore, since the amount of immunosuppressive acidic protein (1AP), known as the acute phase protein of mice, did not increase in mice injected with partially purified TPO, this strongly suggests that TPO is different from IL-6 and IL-11 in terms of induction acute phase protein.

Example 12. Detection of TPO mRNA in rat tissues
To localize the expression of TPO mRNA, tissues in the rat body were extracted from various rat tissues. A total of 6 rats were irradiated with X-rays, as described in Example 1, and various tissues (brain, thymus, lungs, heart, spleen, small intestine, kidneys, testes, and bone marrow cells) were excised from these rats on 11th to 14th day after irradiation and immediately frozen in liquid nitrogen. Extraction of total RNA was performed using an ISOGEN RNA isolation reagent (manufactured by Wako Pure Chemical Industries, Ltd.). The amount of ISOGEN was determined according to the weight of the frozen tissue sample and the sample with the added reagent was treated with a homogenizer (Physcotron ^R NS-60, N1T1-ON Medical & Physical Instruments MFG.Co., Ltd) until the tissue was completely destroyed (approximately 45-60 seconds at 10,000 rpm) min). The tissue homogenate was then extracted with total RNA using a procedure based on a method with a mixture of acidic guanidinium phenol-chloroform (Chomczynski et al. (Anal. Biochem., Vol. 162, pp. 156-159, 1987). As a result, 1, 1-5.6 mg of total RNA from the corresponding tissues.

Using Oligotex ^™ -dT30 (Super) (manufactured by Japan Synthetic Rubber / Nippon Roche) 20 μg poly (A) ⁺ RNA was purified from approximately 500 μg total RNA.

Using an arbitrary primer, the first cDNA strand was synthesized per 1 μg of poly (A) ⁺ RNA obtained from each tissue. Those. 1 μg of poly (A) ⁺ RNA was dissolved in 10 μl of sterile water, incubated at 70 ^o C for 15 minutes and then quickly cooled. To this solution was added 75 pmoles of arbitrary primer (Takara Shuzo Co., Ltd.), 10 U of RNase inhibitor (Boehringer-Mannheim Corp.), 50 mM Tris-Hcl (pH 8.3), 75 mM KCl, 3 mM MgCl ₂ and 200 E Super Script ^TM 11 (reverse transcriptase manufactured by Life Technologies). The resulting solution (total volume 20 μl) was incubated at 37 ^° C for 1 hour and the resulting reaction solution was incubated at 70 ^° C for 10 minutes to inactivate the enzyme and stored at -20 ^° C until use.

 New PCR primers were synthesized based on the rat TPO cDNA sequence obtained in Example 10. The synthesized primer sequences were as follows.

 rТРО-1: 5'-ССТ GTC CTG CTG CCT GCT GTG-3 '(SEQ 1D No. 33) (positions 347-367 in SEQ 1D No. 2).

 rTPO-N: 5'-TGA AGT TCG TCT CCA ACA ATC-3 '(SEQ 1D No. 34) (antisense primer corresponding to the provisions of 1005-1025 in SEQ 1D No. 2).

Using 1/10 volume of each of the synthesized cDNA solution as a template, 1 μM of each of the synthesized rTPO-1 and rTPO-N as primers and the GeneAmp ^™ PCR Reagent Kit with AmpliTaq ^™ DNA polymerase (manufactured by Takara Shuzo Co., Ltd .) PCR was performed in a volume of 100 μl using a GeneAmp ^™ PCR System 9600 (PERKIN-ELMER) and heating at 95 ^° C for 2 minutes, repeating a total of 30 cycles, each cycle consisting of denaturation at 95 ^° C for 1 minute hybridization at 57 ^o C for 1 minute and synthesis at 72 ^o C for 1 minute, followed by final incubation for 7 minutes ut. By electrophoresis of the resulting reaction solutions on a 2% agarose gel (FMC BioProducts) and testing of amplified bands, bands that seemed specific for TPO mRNA were found on gels originating from the brain, liver, small intestine, and kidneys. These results show that the expression of TPO mRNA in rats occurs in the tissues of these organs, although its quantitative expression cannot be estimated. While discussing the possibility of the existence of such a method of expression in the human body and the results of example 4, we can assume that the liver is a suitable starting material for obtaining human TPO cDNA.

Example 13. Construction of a cDNA library obtained from normal human liver
Based on the results of Example 12, the liver was selected as the starting material for use in cloning human TPO cDNA. In the same manner as described in Example 7, a double-stranded cDNA having an EcoRl recognition site at its 5'-end and a Notl recognition site at its 3'-end was synthesized from 5 μg of commercial poly (A) ⁺ RNA obtained from normal human liver (manufactured by Clontech; the product was extracted based on the method with Chomczynski et al. acid guanidinium-phenol-chloroform mixture) using Time Saver ^™ cDNA synthesis Kit (kit) (manufactured by Pharmacia) and DIRECTIONAL CLONING TOOLBOX (manufactured by Pharmacia). The cDNA thus obtained was ligated with 1.2 μg of pEF18S expression vector, which had previously been digested with EcoRl and Notl, and transformed into 8.4 ml of Competent E. coli strain DH5 (Toyobo Co., Ltd.). As a result, 1.2 • 10 ⁶ transformants were obtained.

Example 14. Obtaining (cloning) a fragment of human TPO cDNA using PCR
Using arbitrary primers, the first cDNA strand was synthesized from 1 μg of commercial poly (A) ⁺ RNA obtained from normal human liver (manufactured by Clontech). Those. 1 μg of poly (A) ⁺ RNA was dissolved in 10 μl of sterile water, incubated at 70 ^o C for 15 minutes and then quickly cooled. To this solution were added 75 pmoles of arbitrary primers (Takara Shuzo Co., Ltd.), 10 U RNase inhibitor (Boehringer-Mannheim Corp.), 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl ₂ and 200E reverse transcriptase Super Script ^™ (manufactured by Life Technologies). The solution thus obtained (total volume 20 μl) was incubated at 37 ^° C for 1 hour and the resulting reaction solution was incubated at 70 ^° C for 10 minutes to inactivate the enzyme and stored at -20 ^° C until use.

 PCR primers were synthesized based on the cDNA of rat TPO cDNA sequence (SEQ 1D 2). The sequences of the synthesized primers were as follows.

 rTPO-AIN: 5'-ATG GAG CTG ACT GAT TTG CTC -3'- (SEQ 1D 35) (positions 173-193 in SEQ 1D 2).

 rTPO-N: 5'-TGA AGT TCG TCT CCA ACA ATC-3 '(SEQ 1D 36) antisense primer corresponding to the provisions of 1005-1025 in SEQ 1D 2).

Using 1/10 volume of the synthesized cDNA solution as a template, 1 μM of each of the rTPO-A1N and rTPO-N synthesized in this way as primers and a GeneAmp ^TM PCR reagent kit with AmpliTaq ^TM DNA polymerase (manufactured by Takara Shuzo Co., Ltd .) PCR was performed with a volume of 100 μl, using GeneAmp ^™ PCR System 9600 (PERKIN-ELMER) and heating at 95 ^° C for 2 minutes, repeating a total of 35 cycles consisting of denaturation at 95 ^° C for 1 minute, hybridization at 40 ^o C for 1 minute and synthesis at 72 ^o C for 1 minute, followed by a final incubation at 72 ^o C. echenie 7 minutes.

The reaction solution thus obtained was subjected to electrophoresis on a 2% agarose gel (manufactured by FMC BioProducts) to isolate a DNA fragment of approximately 620 bp as the main PCR product, which was then purified using the above Prep-A-Gene DNA Purification Kit (Bio-Rad Laboratories, Inc.). The nucleotide sequence of the thus purified DNA fragment was immediately determined by the DNA sequencer 373A (Applied Biosystems) using the aforementioned Taq Dye Deoxy ^TM Terminater Cycle Sequening Kit (manufactured by Applied Biosystems). The nucleotide sequence so determined, except for the primer part, and the amino acid sequence deduced from it are shown in the sequence list (SEQ 1D 3).

 This DNA fragment, with the exception of the primer sequences, has a length of 580 kb. When comparing this human cDNA, 86% homology was found with the nucleotide sequence of rat cDNA, which indicates that this DNA fragment encodes part of human TPO.

Example 16. Screening of a clone of human TPO cDNA using PCR
The corresponding human TPO primers for PCR were synthesized based on SEQ 1D 3. The sequences of the primers thus synthesized were as follows.

 hTPO-I: 5'-TTG TGA CCT CCG AGT CCT CAG-3 '(SEQ 1D 37) (positions 60-80 in SEQ 1D 3).

 hTPO-j: 5'-TGA CGC AGA GGG TGG ACC STC-3 '(SEQ 1D 38) (antisense primer corresponding to the provisions of 479-499 in SEQ 1D 3).

 The human cDNA library constructed in Example 13 was amplified and divided into pools (each pool contained approximately 100,000 clones), cultured overnight in 1 ml LB medium containing 50 μg / ml ampicillin, and then plasmid DNA was extracted from them using automated P1-100 plasmid isolation device (VER-3.0 manufactured by Kurabo Industries, Ltd.). The extracted DNA was dissolved in TE solution.

Using a 5% extracted DNA sample as template, 1 μM of each of the synthesized oligonucleotides (hTPO-I and hTPO-j) as primers and a set of reagents for PCR GeneAmp ^™ with AmpliTaq ^™ DNA polymerase (Tukara Shuzo Co., Ltd) PCR was performed in 20 μl volume using a PCR 9600 GeneAmp ^{™ system} (manufactured by PERKIN-ELMER) (35 cycles total, each cycle consisting of denaturation at 95 ^° C for 1 minute, hybridization at 59 ^° C for 1 minute, and synthesis at 72 ^o C for 1 minute, followed by final incubation at 72 ^o C for 7 minutes). As a result, a specific band was found in 3 of the 90 pools used. One of these 3 pools was divided into subpools, each of which contained approximately 5,000 clones, and plasmid DNA was purified from 90 subclones to perform PCR as described. As a result, a specific band was found in 5 sub pools. One of these 5 pools was divided into sub-pools, each of which contained 250 clones, and plasmid DNA was extracted from 90 sub-pools, with a specific band being found in 3 sub-pools. One of these 3 pools was divided into sub-pools, each of which contained 30 clones, and plasmid DNA was purified from 90 sub-pools to perform PCR as described. As a result, a specific band was found in 3 sub-pools. One of the candidate pools was cultured on the described LB plate containing 50 μg / ml ampicillin, and each of the 90 colonies thus obtained was extracted with plasmid DNA and PCR as described. As a result, at the end, clone HL34 was obtained.

Example 16. Sequencing of human TPO cDNA
Purification of plasmid DNA was carried out mainly in accordance with the procedure described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Clone HL 34 was cultured overnight in 50 ml of LV medium containing 50 μg / ml ampicillin, and the resulting cells were collected by centrifugation and suspended in 4 ml of the above TEG-lysozyme solution. To them was added 8 ml of 0.2 N NaOH / 1% SDS and then 6 ml of a solution of 3 M potassium / 5 M acetate to thoroughly suspend the cells. After centrifugation of this suspension, the resulting supernatant was treated with a phenol / chloroform mixture (1: 1), mixed with the same volume of isopropanol, and then centrifuged. The resulting precipitate was dissolved in a TE solution and treated with RNase and then with a phenol / chloroform mixture (1: 1), followed by ethanol precipitation. The resulting precipitate was again dissolved in a TE solution, to which NaCl and polyethylene glycol 3000 were added to final concentrations of 0.63 M and 7.5%, respectively. After centrifugation, the precipitate was dissolved in a TE solution and precipitated with ethanol. Approximately 300 μg of pEF18S-HL34 plasmid DNA was obtained in this way.

The plasmid DNA thus purified was used in a 373A DNA sequencer (Applied Biosystems) to determine its complete nucleotide sequence using the Taq Dye Deoxy ^TM Terminater Cycle Sequening Kit (Applied Biosystems). The nucleotide sequence thus determined and the amino acid sequence derived from it are shown in the list of sequences (SEQ 1D 4). In this case, oligonucleotides synthesized based on the nucleotide sequence of SEQ 1D 3 and synthetic oligonucleotides constructed on the basis of the internal sequence obtained by sequencing analysis were used as primers for determining the nucleotide sequence.

 As a result, it was proved that the plasmid clone pEF18-S-HL-34 contains a cDNA fragment of 861 bp and contains highly homologous sequences with the amino acid sequences AP8 (amino acid numbers 1-12 in SEQ 1D 4) and TP2 / TP3 (amino acids 157-162 in SEQ 1D 4) analyzed in Example 2. Apparently, this DNA fragment encodes an open reading frame starting at position 25, but does not have a termination codon and contains a poly A-tail of a similar sequence of 76 bases at its 3'-end. Its amino acid sequence, consisting of 253 amino acid residues, found 84% homology with the corresponding part of rat TPO cDNA (part of the amino acid sequence consisting of 147 residues shown in SEQ 1D 2). Subsequently, it was found that this clone is a DNA fragment encoding a portion of human cDNA that corresponds to rat TPO cDNA. Due to the lack of a termination codon and the presence of a poly A-tail of a similar sequence at its 3'-end, this clone, apparently, is not a complete cDNA, but is an artificial product obtained during the procedure for constructing a cDNA library.

Example 17. Expression of human TPO cDNA in COS 1 cells and confirmation of TPO activity
Transfection of the plasmid clone pEF18S-HL-34 obtained in the described manner into COS 1 cells was carried out according to the method of Example 11. That is, transfection was performed with 10 μg of plasmid DNA according to the DEAE-dextran method, which involves treatment with chloroquine. These COS 1 cells were cultured for 3-5 days and then received a supernatant.

 The resulting culture supernatant was extensively dialyzed against IMDM culture medium and its activity was evaluated in a rat CFU-MK test system. TPO activity was detected in a dose-dependent manner in the culture supernatant of COS 1 cells in which plasmid pEF18S-HL-34 was expressed (Fig. 8). After 4 days of cultivation, many megakaryocytes formed elongated cytoplasmic protrusions. In contrast, TPO activity was not detected in the culture supernatant of COS 1 cells in which only the plasmid pEF18S was expressed (Fig. 8). In the test system with M-07e cells, the activity of enhancing the proliferation of M-07e cells was found only in the supernatant of COS 1 cells in which the plasmid pEF18S-HL-34 was expressed as a result of its transfection. These results showed that pEF18S-HL34 contains a gene that encodes a protein having TPO activity. In addition, it was shown that human TPO acts on progenitor cells of rat megakaryocytes (i.e. there is no species specificity).

Example 18. Expression of cDNA deletion type human TPO and confirmation of TPO activity
Clone HL34 obtained in Example 15 contained a cDNA having a poly A-tail similar to a continuous sequence at its 3'-end, which is not present in rat TPO cDNA, and therefore is apparently an artificial product of this experiment. As a result, a cDNA molecule was obtained from which a poly A-tail-like sequence was deleted to check whether the protein expressed by this deletion cDNA has TPO activity. Deletion cDNA was obtained using PCR. The primer sequences used for PCR were those in which the restriction enzyme recognition site was added to the 5'-end (EcoRI k hTPO5 and Notl and two stop codons TAA and TGA k hTPO3).

 hTPO5: 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3 '(SEQ 1D 170) (positions 1-21 in SEQ 1D 4).

 hTRO3: 5'-TTT GCG GCC GCT CAT TAT TCG TGT ATC CTG TTC AGG TAT CC-3 '(SEQ 1D 171) (antisense primer corresponding to the provisions of 757-780 in SEQ 1D 4).

Using 1 μg of plasmid DNA of the plasmid clone pEF18S-HL34 obtained in Example 16 as a template, 10 μM of each of the synthesized hTPO5 and hTPO3 as primers and a set of reagents for PCR GeneAmp ^TM with AmpliTaq ^TM DNA polymerase (Takara Shuzo Co. , Ltd.,) PCR was performed with a volume of 100 μl using a 9600 GeneAmp ^™ PCR system (PERKIN-ELMER) and a total of 15 cycles were repeated, each of which consisted of denaturation at 95 ^° C for 1 minute, hybridization at 65 ^° C in for 1 minute and synthesis at 72 ^o C for 1 minute, followed by final incubation for 7 minutes. The approximately 800 bp band thus obtained was digested with restriction enzymes EcoRl and Notl, purified and then subcloned into the expression vector pEF18S, which was pretreated with the same restriction enzymes. Of the obtained transformants, 5 clones containing a DNA fragment of approximately 800 bp were selected to obtain a large amount of plasmid DNA in accordance with the procedure described in example 5. The full length of the amplified region of approximately 800 BP each plasmid was subjected to nucleotide sequence analysis, obtaining its complete identity with the nucleotide sequence analyzed in example 16 (positions 1-780 in SEQ 1D 4).

The clone thus obtained was named pHT1-231. Vector pHT1-231 carried by E. coli strain DH5 was deposited by the inventors on February 14, 1994 under deposit FERM BP-4564 at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan . Transfection of the plasmid thus obtained into COS1 cells was carried out according to the method of Example 11. That is, transfection was performed with 10 μg of plasmid DNA according to the DEAE-dextran method, which involves treatment with chloroquine. COS 1 cells were cultured for 3-5 days at 37 ^{° C.} and then the supernatant was collected.

 The culture supernatant thus obtained was extensively dialyzed against IMDM culture medium and evaluated in a rat CFU-MK test system. TPO activity was detected in the culture supernatant of COS 1 cells in which the plasmid pHT1-231 was expressed (Fig. 9). On the 4th day of cultivation, many megakaryocytes formed elongated cytoplasmic protrusions. In contrast, TPO activity was not detected in the culture supernatant of COS 1 cells in which only pEF18S was expressed (FIG. 9). In the M-07e test system, the activity of enhancing the proliferation of M-07e cells was found only in the culture supernatant of COS 1 cells in which the plasmid pHT1-231 was expressed. These results showed that pHT1-231 contains cDNA that encodes a protein having TPO activity.

Example 19. Obtaining 3'-terminal region of human TPO cDNA using PCR
Clone HL34 obtained in Example 15 contained a cDNA having a poly-A-tail-like sequence, based on which it was suggested that its 3'-terminal region was incomplete. Therefore, an attempt was made to obtain a full-sized 3'-terminal region using PCR. The following 4 types of 5-side primers for PCR were synthesized based on the sequences defined in Example 16:
hTPO-H: 5'-AGC AGA ACC TST STA GTC STS-3 '(SEQ 1D 39) (positions 574-594 in SEQ 1D 4).

 hTPO-K: 5'-ACA CTG AAC GAG CTC CCA AAC-3 '(SEQ 1D No. 4) (positions 595-615 in SEQ 1D 4).

 hTPO-N: 5'-AAC TAC TGG CTC TGG GCT TCT-3 '(SEQ 1D No. 41) (positions 660-680 in SEQ 1D 4).

 hTPO-O: 5'-AGG GAT TCA GAG CCA AGA TTC-3 '(SEQ 1D No. 42) (positions 692-712 in SEQ 1D 4).

 The following 3 ′ side primers containing mixed nucleotides at 4 bases of the 3 ′ end were synthesized to amplify cDNA from the start of the poly A portion. An anchor primer was also synthesized without a mixed nucleotide.

hTPO3mix: 5'-TAG CGG CCG C (T) ₁₇ G GGG-3 '(SEQ 1D No. 43).

 A AAA-3 '(SEQ 1D No. 44).

 With TTT-3 '(SEQ 1D No 45).

 CCC-3 '(SEQ 1D No. 46).

hTPO3anchor: 5'-TAG CGG CCG C (T) ₁₁ -3 '(SEQ 1D No 47).

The first cDNA strand was synthesized with 1 μg of commercial poly (A) ⁺ RNA (Clontech) obtained from normal human liver, as in Example 14, but using 0.5 μg of oligo dT primer (it is included in the Time Saver ^TM cDNA synthesis kit, manufactured by Pharmacia). Using 1/10 volume of the synthesized cDNA solution as a template, 20 μM hTRO-H primer and 10 μM hTPO3mix primer and CeneAmp ^™ PCR reagent kit with AmpliTaq ^™ DNA polymerase (manufactured by Takara Shuzo Co., Ltd.), PCR was performed with 50 μl using PCR system 9600 GeneAmp ^TM (PERKIN-ELMER) with heating at 96 ^o C for 2 minutes, repeating a total of 10 cycles, each of which consists of thermal denaturation at 96 ^o C for 1 minute, hybridization at 48 ^o C at 72 ^o for 1 minute and synthesis at 72 ^o C for 1 minute, followed by a final incubation with for 7 minutes.

The second PCR was carried out using 1/10 of the solution volume obtained in the first PCR as a matrix, 20 μM hTPO-K primer and 10 μM hTPO3mix primer with a volume of 50 μl, heated at 96 ^° C for 2 minutes and repeated in general 10 cycles, each of which consisted of thermal denaturation at 96 ^° C for 1 minute, hybridization at 63 ^° C for 1 minute and synthesis at 72 ^° C for 1 minute, followed by final incubation at 72 ^° C for 7 minutes.

The third PCR was performed using 1/10 of the volume of solution obtained with the second PCR as a matrix, 20 μM hTPO-N primer and 10 μM hTPO3mix primer with a volume of 50 μl, heated at 96 ^° C for 2 minutes and repeated in general 10 cycles, each of which consisted of thermal denaturation at 96 ^° C for 1 minute, hybridization at 63 ^° C for 1 minute and synthesis at 72 ^° C for 1 minute, followed by final incubation at 72 ^° C for 7 minutes.

The fourth PCR was performed using 1/10 of the volume of the solution obtained in the third PCR as a matrix, 20 μM hTPO-O primer and 10 μM hTPO3mix primer with a volume of 50 μl and heating at 96 ^° C for 2 minutes, repeating in general 10 cycles, each of which consisted of thermal denaturation at 96 ^° C for 1 minute, hybridization at 63 ^° C for 1 minute and synthesis at 72 ^° C for 1 minute, followed by final incubation at 72 ^° C for 7 minutes.

The fifth PCR was performed using 1/10 of the volume of the solution obtained in the fourth PCR as a matrix, 20 μM hTPO-O primer and 20 μM hTRO3anchor primer with a volume of 50 μl, heated at 96 ^° C for 2 minutes, repeating in total 10 cycles, each of which consisted of thermal denaturation at 96 ^° C for 1 minute, hybridization at 58 ^° C for 1 minute and synthesis at 72 ^° C for 1 minute, followed by final incubation at 72 ^° C for 7 minutes .

The reaction solution thus obtained was subjected to electrophoresis on a 2% agarose gel (FMC BioProducts) to isolate a DNA fragment of approximately 600 bp. as the main PCR product, which was then purified using the aforementioned Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad Laboratories, Inc.). The nucleotide sequence of the thus purified DNA fragment was immediately determined using a 373 A DNA sequencer (Applied Biosystems) using the Taq Dye Deoxy ^TM Terminator Cycle Sequening Kit (Applied Biosystems). The specific nucleotide sequence and the derived amino acid sequence are shown in the sequence list (SEQ 1D 5).

 This DNA fragment has a nucleotide sequence encoding 130 amino acids, starting with the hTPO-O primer, followed by a sequence of more than 180 nucleotides at the 3'-end (nucleotides after position 577 could not be determined). The amino acid sequence starting with glycine matches the amino acid sequence of positions 203-232 in SEQ 1D 4. The nucleotide sequence of positions 1-94 also matches the nucleotide sequence of positions 692-785 in SEQ 1D 4.

Example 20. Reconstruction of a cDNA library obtained from normal human liver
Since the HL34 clone obtained in Example 15 contained a poly-A-tail-like sequence directly at its 3'-end of its open reading frame without termination codons and therefore was apparently an unnatural cDNA synthesis product, the cDNA library was reconstructed using 5 μg commercially obtained from normal human liver poly (A) ⁺ RNA (a drug manufactured by Clontech). The cDNA synthesis was performed using SuperScript ^TM Lambda System for the synthesis of cDNA and λ Cloning Kit SuperScript ^TM 11 RNase N ^- (both manufactured by LIFE TECHNOLOGIES). Poly (A) ⁺ RNA was heat denatured and then added to 20 μl of a reaction solution containing the Notl oligo dT recognition sequence as a primer included in the kit (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl ₂ , 1 mM DTT, 1 mM dNTP mix, 200 U SuperScript ^TM 11 RNase H ^- ), followed by incubation at 37 ^° C for 60 minutes. After synthesis of the second cDNA chain (2 hours of incubation at 16 ^° C in 150 μl of a reaction solution containing 25 mM Tris-HCl, pH 8.3, 100 mM KCl, 5 mM MgCl ₂ , 250 μM dNTP mix, 5 mM DTT, 40 U DNA polymerase 1E.coli, 2 U E. coli H RNase H and 10 U E. coli DNA ligase) were added 10 U T4 DNA polymerase and the resulting mixture was incubated at 16 ^{° C.} for 5 minutes. The reaction solution was heated at 65 ^{° C.} for 10 minutes and extracted with the same volume of a phenol / chloroform mixture. CDNA molecules having a length of less than 400 bp were removed using a SizeSep ^™ 400 rotating column (low molecular weight DNA removal column attached to a TimeSaver ^™ cDNA synthesis kit, Pharmacia). After addition of the EcoRl Adapter (attached to the Directional Cloning Toolbox, Pharmacia), the thus-treated sample was digested with Notl and again applied to the same column to remove low molecular weight DNA. The double-stranded cDNA (1.3 μg) thus synthesized, having the EcoRl recognition sequence at its 5'-end and Notl recognition sequence at its 3'-end, was ligated with the expression vector pEF18S, previously digested with EcoRl and Notl, and then transformed into 9 , 2 ml of Competent E. coli strain DH5 (Toyobo Co., Ltd.).

Thus obtained library of human liver cDNA (hTPO-F1) contained 1.0 • 10 ⁶ transformants.

Example 21. Screening of a clone of TPO cDNA from the library of human liver ctNA hTPO-F1
Primers corresponding to human TPO cDNA for PCR were synthesized based on SEQ 1D 3 and 6, presented in the list of sequences. The sequences of the synthesized primers were as follows.

 hTPO-1: 5'-TTG TGA CCT CCG AGT CCT CAG-3 '(SEQ 1D No. 48) (positions 60-80 in SEQ 1D 3).

 hTPO-KU: 5'-AGG ATG GGT TGG GGA AGG AGA-3 '(SEQ 1D No. 49) (antisense primer corresponding to the sequence of positions 901-921 in SEQ 1D 6).

The human liver hTPO-F1 cDNA library (1.0 x 10 ⁶ transformants) constructed in Example 20 was divided into 3 pools (pools 1-3) and these pools were frozen. PCR was performed using 2 μg of plasmid DNA obtained from each pool as a template and 1 μM of each of the synthesized oligonucleotides (hTPO-1 and hTPO-KU) as primers. Using a PCR reagent kit GeneAmp ^™ with AmpliTaq ^™ DNA polymerase (Takara Shuzo Co. Ltd.), PCR was performed in 100 μl volume using a PCR system 9600 GeneAmp ^™ (PERKIN-ELMER) (35 cycles in total, each of which consisted of denaturation at 95 ^° C for 1 minute, hybridization at 59 ^° C for 1 minute and synthesis at 72 ^° C for 1 minute, followed by final incubation at 72 ^° C for 7 minutes). As a result, the DNA fragment having the expected size was amplified in the current case when the plasmid DNA obtained from pool 3 was used. Pool 3 was then divided into subpools, each of which contained 15,000 transformants, and these subpools were cultured overnight in 1 ml of LV medium containing 50 μg / ml ampicillin, followed by extraction of plasmid DNA using an automated device for the isolation of plasmids P1-100. The DNA thus extracted was dissolved in a TE solution and 5% of the resulting solutions were used as matrices for PCR using the same primers and under the conditions described above. As a result of amplification, DNA with the expected size was detected in 6 out of 90 pools. One of these positive pools was divided into sub-pools, each of which contained 1000 clones, plasmid DNA was extracted and PCR was performed as described above. At the same time, DNA amplifications were observed. The density of the bands of DNA fragments on the electrophoresis gels after a series of PCRs became smaller and smaller as the pools were further divided, which was apparently due to the low plasmid DNA extraction due to the weak growth of the target clone. Therefore, another screening was performed by colony hybridization using an initial pool of 3.

Pool 3 was distributed on 100 LB agar plates 15 cm in diameter with an inoculum size such that 4,100 colonies grew on each plate. After receiving a replica dish from each of the dishes thus inoculated, one of the duplicate dishes was cultured at 37 ^{° C. for} 6 hours to extract the colonies grown on the dish and to extract plasmid DNA samples. When conducting PCR of these DNA samples, as described above, amplification of a band equivalent to the expected size was observed in one of 100 sub-pools. Two filter replicas were prepared from the cup of this subpool using BIODYNE ^TM TRANSFER MEMBRANE (PALL). These filters were denatured by soaking them in 10% SDS for 10 minutes, in 0.5N NaOH / 1.5 M NaCl for 10 minutes, and in 0.5 M Tris-HCl (pH 8.0) / 1.5 M NaCl for 10 minutes in the indicated sequence, followed by 30 minutes of drying in air and subsequent heating for 1 hour at 80 ^o C in a vacuum oven. The resulting filters were washed with 6 x SSC (obtained by diluting a 20 x SSC stock solution consisting of 175.3 g of NaCl and 88.2 g of Na citrate dissolved in 1 L of water, pH 7.0) with the addition of 1% SDS. Prehybridization of the filters washed in this way was carried out by incubating them at 42 ^{° C.} for 30 minutes with pumping in 30 ml of a reaction solution consisting of 50% formamide, 5 x SSC, 5 x Denhardt solution (obtained from 50 x Denhardt solution containing 5 g Ficoll 5 g of polyvinylpyrrolidone and 5 g of bovine serum albumin fraction V in 500 ml of water), 1% SDS and 20 μg / ml salmon sperm DNA.

After prehybridization, the reaction solution was exchanged with 30 ml of a hybridization solution of the same composition, mixed with a probe labeled with [α- ³² P] - CTP (Amersham) and then incubated with shaking at 42 ^° C for 20 hours. The labeled probe used in this experiment was an EcoRl / BamH1 fragment of plasmid pEF18S-HL34, which was obtained by purification of a part of SEQ 1D 4 from its 5'-end to position 458 and labeling of this purified part using random primers with the Megaprime DNA Labeling System ( Amersham kit based on the method described in Anal. Biochem. 132, 6-13, 1983). The filters thus treated were washed in a 2 x SSC / 0.1% SDS solution at 42 ^° C for 30 minutes and then in a 0.2 x SSC / 0.1% SDS solution at 42 ^° C for 30 minutes. Then these filters were subjected to 16 hours of autoradiography at -70 ^° C using an intensifying screen X-OMAT ^TM AR5 film (Eastman Kodak). As a result, a single signal was observed, considered as positive. Colonies approximately corresponding to this signal were collected from the original plate and plated again on a 10 cm agar plate. 50 colonies grown on this plate were cultured separately and their DNA samples were subjected to PCR using hTRO-1 and hTRO-KU primers as described above conditions. As a result, amplification of a band equivalent to the expected size was detected in only one clone, which was named pHTF1.

Example 22. The determination of the nucleotide sequence of the clone pHTF1 cDNA of human TPO
Purification of plasmid DNA was carried out mainly according to the procedure described in Molecular Cloning (Sambrook et al. Cold Spring Harbor Laboratory Press, 1989). The pHTF1 clone was cultured overnight in 50 ml LB medium containing 50 μg / ml ampicillin. The resulting cells were collected by centrifugation and suspended in 4 ml of a solution of TEG-lysozyme. To them was added 8 ml of a solution of 0.2 N NaOH / 1% SDS and then 6 ml of a solution of 3 M potassium / 5 M acetate to thoroughly suspend the cells. After centrifugation of this suspension, the resulting supernatant was treated with a phenol / chloroform mixture (1: 1) mixed with the same volume of isopropanol, and then centrifuged. The resulting precipitate was dissolved in a TE solution and treated with RNase and then with a phenol / chloroform mixture (1: 1), followed by ethanol precipitation. The resulting precipitate was again dissolved in a TE solution, to which NaCl and polyethylene glycol 3000 were then added to their final concentrations of 0.63 M and 7.5%, respectively. After centrifugation, the precipitate was dissolved in a TE solution and precipitated with ethanol. Approximately 300 μg of plasmid DNA pHTF1 was obtained in this way.

The plasmid DNA thus purified was used in the aforementioned 373A DNA sequencer (Applied Biosystems) to determine its complete nucleotide sequence using the Taq Dye Deoxy ^TM Terminator Cycle Sequening Kit (Applied Biosystems). The nucleotide sequence thus determined and the amino acid sequence derived from it are shown in the sequence list (SEQ 1D 7). Oligonucleotides synthesized based on the nucleotide sequence of SEQ 1D 6 and on the internal sequence obtained by analysis of their sequencing were used as primers in the determination of the nucleotide sequence.

 As a result, it was confirmed that the pHTP1 clone contains a cDNA fragment of 1721 bp. and has high homology with the amino acid sequences AP8 (amino acid numbers 1-12 in SEQ 1D 7) and TP2 / TP3 (amino acid numbers 157-162 in SEQ 1D 7) analyzed in Example 2. This DNA fragment appears to have 5 'is a non-coding region of 101 bases, an open reading frame, consisting of 353 amino acid residues, starting with a methionine residue encoded at nucleotide positions 102-104, and ending with a glycine residue encoded at nucleotide positions 1158-1160, the following termination codon ( TAA) and 3'- non-coding region ty of 531 bases and a poly A tail sequence of 30 bases. The amino acid sequence of the protein that can be encoded by this open reading frame completely coincided with the predicted human TPO amino acid sequence shown in SEQ 1D 6. The pHTF1 cDNA sequence was larger than the probable cDNA sequence shown in SEQ 1D 6 and contains 77 additional bases at the 5'-end and 347 additional bases at the 3'-end before the poly A-tail sequence. Also, this nucleotide sequence differed from SEQ 1D 6 at three positions. T.E. A (position 84), A (position 740) and G (position 1198) in SEQ 1D 7 were C, T and A in SEQ 1D 6, respectively. Only the mutation at position 740 entered the protein coding region, but this mutation did not cause changes in the amino acid, since bases A and T were the third base of the threonine codons. Although the reason for these changes in the bases is currently unclear, analysis of the plasmid clone pHTF1 confirmed that the human TPO protein contained 353 amino acid residues, including a signal sequence consisting of 21 amino acids. Like the mass of a mature protein without a signal sequence was determined to be 35466.

 The pHTF1 vector in E. coli strain DH5 was deposited by the inventors on March 24, 1994 under FERM BP-4617 at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Techology, Ministry of International Trade and Industry, Japan.

Example 23. The expression of the clone pHTF1 cDNA of human TPO in COS 1 cells and confirmation of TPO activity
Transfection of COS1 cells with the plasmid clone pHTF1 thus obtained was carried out according to the method of Example 11. That is, transfection was carried out with 10 μg of plasmid DNA by DEAE-dextran method, involving treatment with chloroquine. Transfected COS 1 cells were cultured for 3 days at 37 ^{° C.} and the culture supernatant was collected.

 The culture supernatant was extensively dialyzed against IMDM culture medium and evaluated in rat CFU-MK assay system. TPO activity was detected in a dose-dependent manner in the supernatant of COS 1 cells in which the plasmid pHTFl was expressed (Fig. 10a). In contrast, TPO activity was not detected in the culture supernatant of COS 1 cells in which only the plasmid pEF18S was expressed (Fig. 10a). Similar results were obtained in the M-07e test system. The supernatant of COS 1 cells in which the plasmid pHTF1 was expressed significantly increased the proliferation of M-07e cells in a dose-dependent manner (Fig. 10b). These results showed that pHTF1 contains a gene encoding a protein having TPO activity.

Example 24. Cloning of human TPO chromosomal DNA
Cloning of human TPO chromosomal DNA was performed using human TPO cDNA as a probe. The genomic library used in cloning was a gift from Professor T. Yamamoto of Gene Research Center, Tohoku University (library reported by Sakal et al. In J. Biol. Chem, 269, 2173-2182, 1994, constructed by partial cleavage of human chromosomal DNA restriction enzyme Sau3A1 and ligation of the partial cleavage product with the BamH1 site of the phage vector Lambda EMBL3 manufactured by Stratagene). Screening from this library using human TPO cDNA as a probe was performed mainly according to the method described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Using E. coli LE392 as a host, this library was inoculated onto a plate (15 cm) containing NZYM (10 g NZ amine, 5 g NaCl, 5 g Bacto yeast extract, 2 g MgSO ₄ • 7H ₂ O and 15 g agar in 1 liter of water (pH 7.0) in such an inoculum size that one cup contained 30,000 phage particles.Two filter replicas were obtained from each of the 18 plates obtained using BIODYME ^™ A TRANSFER MEMBRANE (manufactured by PALL.). The filters were denatured by impregnation. them in 0.5 N NaOH / 1.5 M NaCl for 10 minutes and then in 0.5 M Tris-HCl (pH 8.0) / 1.5 M NaCl for 10 minutes, followed by drying in air in current 3 0 minutes and hot drying at 80 ^{° C.} for 1.5 hours in a vacuum brake The prehybridization was carried out by incubating the filters thus treated at 42 ^{° C.} for 1 hour in 500 ml of a reaction solution consisting of 50% formamide, 5x SSC, 5 x Denhardt's solution, 1% SDS and 20 μg / ml salmon sperm DNA For use as a probe, the human TPO cDNA fragment (base position numbers 178-1025 in SEQ 1D 7) was PCR amplified and purified and the purified fragment was labeled ³² P using the kit for labeling arbitrary DNA primers (kit for DNA labeling, Takara Shuzo based on the random primer method described in Anal.Biochem. 132, 6-13, 1983). The primer sequences used in this PCR were as follows.

 hTPO-1: 5'-TTG TGA CCT CCG AGT CCT CAG-3 '(SEQ 1D 50) (positions 178-198 in SEQ 1D 7).

 hTPO-N: 5'-AGG GAA GAG CGT ATA CTG TCC-3 '(SEQ 1D 51) (antisense primer corresponding to the sequence of positions 1005-1025 in SEQ 1D 7).

Using an isotope-labeled probe, hybridization was carried out at 42 ^{° C.} for 20 hours in 500 ml of a reaction solution having the same composition as the composition of the prehybridization solution. The resulting filters were washed 3 times in a solution of 2 x SSC / 0.1% SDS at room temperature for 5 minutes and then once in a solution of 0.1 x SSC / 0.1% SDS at 68 ^° C for 1 hour. Then the filters were subjected to radio-autography at -79 ^° C using an intensifying screen and X-OMAT ^TM AR5 film (Eastman Kodak). As a result, 13 positive signals were received. Plaques approximately corresponding to each of the positive signals were collected from the original plates and again inoculated onto NZYM plates (15 cm) in such an inoculum size that 1000 plaques formed on each plate. Two filter replicas were prepared from each of the obtained plates for hybridization under the conditions described above. As a result, positive signals were detected on all filters from 13 groups. One plaque was removed from each of the obtained plates to obtain phage DNA using the plate lysate method described in Molecular Cloning. Phage DNA samples thus obtained from 13 clones were checked for the presence of cDNA of the coding region using PCR using the following primers.

 hTPO-L: 5'-GGC CAG CCA GAC ACC CCG GCC-3 '(SEQ 1D 52) (positions 1-21 in SEQ 1D 6).

 hTPO-F: 5'-ATG GGA GTC ACG AAG CAG TTT-3 '(SEQ 1D 53) (antisense primer corresponding to the sequence of positions 137-147 in SEQ 1D 6).

 hTPO-V: 5'-AAC CTT ACC CTT CCT GAG ACA-3 '(SEQ 1D 55) (antisense primer corresponding to the sequence of positions 1070-1090 in SEQ 1D 6).

When conducting PCR using these primer combinations, 5 of the 13 clones appeared to contain the complete amino acid coding region prompted from this cDNA. Chromosomal DNA molecules containing these 5 clones had a similar length of approximately 20 bp and found almost the same distribution of fragments in a preliminary restriction analysis using restriction enzymes. Therefore, one of these clones (λ HGTl clone) was selected and analyzed by Southern blotting. Those. 1 μg of clone λ HGTl DNA was digested completely with restriction enzyme EcoRl or Hind111 and applied to a 0.8% agarose electrophoresis gel and the gel strips were transferred to BIODYNE ^TM A TRANSFER MEMBRANE (PALL). The resulting filter was dried in air for 30 minutes and annealed for 2 hours at 80 ^o C in vacuum lay. Prehybridization was carried out by incubating the filter thus treated at 42 ^{° C.} for 1 hour in 50 ml of a reaction solution consisting of 50% formamide, 5 x SSC, 5 x Denhardt solution, 1% SDS and 20 μg / ml salmon sperm DNA. For use as a probe, the human TPO cDNA fragment (base position numbers 178-1025 in SEQ 1D 7) was amplified by PCR and the purified fragment was labeled with ³² P using the Random Prime DNA Labeling Kit (kit manufactured by Takara Shuzo). Using an isotope-labeled probe, hybridization was carried out at 42 ^{° C.} for 20 hours in 50 ml of a reaction solution having the same composition as the prehybridization solution. The resulting filter was washed 3 times in 2 x SSC / 0.1% SDS - solution at room temperature for 5 minutes and then 1 time in a solution of 0.1 x SSC / 0.1% SDS at 68 ^o C for 1 hour. The filter was then subjected to 16 hours autoradiography at -70 ^° C using an intensifying screen and X-OMAT ^™ AR5 film (Eastman Kodak). as a result, one lane of approximately 10 kb was observed. in the case of Hindlll cleavage. Therefore, 10 μg of clone λ HGTl DNA was digested with Hindlll and electrophoresed on a 0.8% agarose gel and a 10 kbp band. excised from the gel, purified using Prep-A-Gene DNA Purification Kit (Bio-Rad), and subcloned into the pUC13 cloning vector (Pharmacia), which had previously been digested with Hind111. In this case, the highly competent E. coli strain DH5 (TOUOVO) was used as the host strain.

 Of the clones thus obtained, a clone was selected containing a Hind111 fragment of 10 kb and was designated pHGT1.

 A restriction map of the phage clone λ HGT1 is shown in FIG. 11.

Example 25. Determination of the nucleotide sequence of the chromosomal clone pHGT1 TPO person
The cultivation of the pHGTl clone and the purification of plasmid DNA was carried out mainly according to the method described in Molecular Cloning (Sambrook et al., Cold Spring Narbo Laboratory Press, 1989). The pHGT1 clone was cultured overnight in 50 ml LB medium containing 50 μg / ml ampicillin. The resulting cells were collected by centrifugation and suspended in 4 ml of the TEG-lysozyme solution described above. To them was added 8 ml of a solution of 0.2 N NaOH / 0.1% SDS and then 6 ml of a solution of 3 M potassium / 5 M acetate to thoroughly suspend the cells. After centrifugation of this suspension, the resulting supernatant was treated with a phenol / chloroform mixture (1: 1) mixed with an equal volume of isopropanol, and then centrifuged. The resulting precipitate was dissolved in a TE solution and treated with RNase and then with a phenol / chloroform mixture (1: 1), followed by ethanol precipitation. The resulting precipitate was again dissolved in a TE solution, to which NaCl and polyethylene glycol 3000 were then added to their final concentrations of 0.63 M and 7.5%, respectively. After centrifugation, the precipitate was dissolved in a TE solution and precipitated with ethanol. Approximately 300 μg of plasmid DNA pHGT1 was obtained in this way.

Using the Taq Dye Deoxy ^TM Terminater Cycle Sequening Kit (kit manufactured by Applied Biosystems), the plasmid DNA thus purified was introduced into a 373A DNA sequencer (Applied Biosystems) to determine the nucleotide sequence of the protein coding region predicted from the cDNA nucleotide sequence. The nucleotide sequence thus determined and the amino acid sequence derived from it are shown in the sequence list (SEQ 1D 8). In this case, the oligonucleotides used in example 22 for nucleotide analysis of cDNA and synthetic oligonucleotides constructed on the basis of the internal sequence obtained by analysis from the sequence were used as primers in determining the nucleotide sequence.

As a result, it was found that the chromosomal DNA carried by the plasmid clone pHGT1 contains the complete coding region of the amino acid sequence derived from SEQ 1D 6, and the nucleotide sequence of this coding region completely coincided with SEQ 1D 6. In addition, the region corresponding to the amino acid coding exon, contained 4 introns with lengths of 231 bp, 286 bp, 1932 bp and 236 bp in this order, from the 5'-side (Fig. 11). Also, this nucleotide sequence was different from the cDNA nucleotide sequence (SEQ 1D 7) at 3 positions, the same as those described in example 22 (different positions between SEQ 1D 6 and 7). Those. A (position 84), A (position 740) and G (position 1198) in SEQ 1D 7 were respectively C, T and A in SEQ 1D 8. Thus, it was found that the nucleotide sequence of the human TPO cDNA clone pHTG1 obtained in Example 21, differs from the nucleotide sequence of the pHFT1 clone of human chromosomal DNA at 3 positions. Therefore, to analyze whether these mutations in the sequence of the pHFT1 cDNA clone reflect the chromosomal DNA sequence, the nucleotide sequences of the other 4 clones were determined among 5 chromosomal DNA clones independently selected by this screening. Sequence analysis was carried out by a direct method for determining the nucleotide sequence using a phage DNA sample obtained according to the plate lysis method described in Molecular Cloning. Each clone was sequenced using a 373A DNA sequencer (Applied Biosystems) using the primers used in Example 22, which were synthesized based on such parts of the sequence that the above 3 positions of the mutations in the nucleotide sequence could be analyzed. Used ^{Taq Dye Deoxy TM Terminater Cycle Sequening Kit} (Applied Biosystems). It was found that the nucleotide sequences of all 4 clones were identical to SEQ 1D 6. Positions 84 and 740 corresponding to SEQ 1D 7 were replaced by C and T, respectively, in these 4 clones. However, position 1198 was G in 2 clones and A in two other clones. In other words, it was found that there are two types of nucleotide sequences inherent in the original chromosomal DNA. It is currently unclear whether such differences in the nullotide sequence are obtained from a homologous chromosome or from multiple genes. In addition, it was hypothesized that differences in the nucleotide sequence may be due to racial differences, as poly (A) ⁺ RNA acquired from Clontech is of Caucasian origin, while chromosomal DNA is of Japanese origin.

 The pH T1 vector carried by E. coli strain DH5 was deposited by the inventors on March 24, 1994 FERM BP-4616 at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan.

Example 26. Expression of chromosomal DNA of human TPO in COS 1 cells and confirmation of TPO activity
The plasmid clone pHGT1 obtained by subcloning contained only 4 EcoRl recognition sequences, 3 in the insertion part and 1 in the vector. Analysis of the nucleotide sequence revealed that the entire region encoding the human TPO protein was included in the DNA fragment of approximately 4.3 kb, which was inserted between the EcoRl recognition sequence closest to the 5'-side in this insertion and the recognition sequence EcoRl in vector. Therefore, this fragment was ligated with the pEF18S-treated EcoRl expression vector and 4 human TPO expressing plasmids 1 to 4 were obtained (see FIG. 11). Having obtained these plasmid DNAs, an experiment was performed on their expression. Purification of plasmid DNA was carried out mainly according to the method described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989), yielding approximately 250 μg of plasmid DNA.

Transfection of COS 1 cells with pEFHGTE 1-4 clones thus obtained was carried out according to the method of Example 11. That is, transfection was carried out with 10 μg of plasmid DNA by DEAE-dextran method, involving treatment with chloroquine. Transfected COS 1 cells were incubated for 3 days at 37 ^{° C.} and then culture supernatants were collected.

 Cultural supernatants were extensively dialyzed against IMDM culture medium and evaluated in rat CFU-MK assay system. TPO activity was detected in a dose-dependent manner in the supernatant of COS 1 cells in which each of the four clones was expressed (pEFHGTE 1-4). In contrast, TPO activity was not detected in the supernatant of COS 1 cells in which only the plasmid pEF18S was expressed. Representative results with pEFHGTE 1 are shown in FIG. 12a. Similar data were obtained in the M-07e test system. The supernatant of COS 1 cells in which each of the clones was expressed (pEFHGTE 1-4) significantly increased the growth of M-07e cells in a dose-dependent manner. Representative data with pEFHGTE 1 is shown in FIG. 12.

 These results showed that plasmid clones pEFHGTE 1-4 carry functional human TPO chromosomal DNA.

Example 27. Obtaining DNA deletion type human TPO and its expression and confirmation of TPO activity
The results in Example 18 showed that human TPO could exhibit its biological activity, even after removing its carboxy third. Therefore, to further evaluate the biologically active parts, experiments were conducted with its deletion derivatives. In this example, deletion derivatives of TPO, with 29, 40, 60, or 68 amino acids removed from the carboxyl-terminal end of the TPO protein (amino acids 1-231) encoded by pHT1-231, were examined for their ability to influence the biological activity of TPO in vitro. The shortest derivative (Amino acids 1-163) still contained the amino acid sequences corresponding to TP2 / 3 TPO of the rat plasma described in Example 2. Deletion plasmids were obtained by PCR using the plasmid clone pHT1-231 DNA obtained in Example 18 as a matrix and synthesized oligonucleotides as primers. The primer sequences used for PCR were as follows.

 hTPO-5: 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3 '(SEQ 1D 56) (obtained by adding the EcoRl recognition sequence to the 1-21 sequence in SEQ 1D 4, this is the identical sequence described in FIG. 9).

hTPO-S: 5'-TTT GCG GCC GCT CAT TAG CTG GGG ACA GCT GTG GTG GGT-3 '(SEQ 1D 57) (antisense primer corresponding to the sequence of positions 555-576 in SEQ 1D 4, obtained by adding two termination codons TAA and TGA and Notl recognition sequences for use in preparing a deletion derivative encoding the positions of 1-163 amino acid residues,
hTRO-4: 5'-TTT GCG GCC GCT CAT TAC AGT GTG AGG ACT AGA GAG GTT CTG-3 '(SEQ 1D 58) (antisense primer corresponding to the sequence of positions 576-600 in SEQ 1D 4 obtained by adding two TAA termination codons and TGA and Notl recognition sequences, for use in obtaining a deletion derivative encoding the positions of 1-171 amino acid residues),
hTPO-30: 5'-TTT GCG GCC GCT CAT TAT CTG GCT GAG GCA GTG AAG TTT GTC-3 '(SEQ 1D 59) (antisense primer corresponding to the sequence of positions 636-660 in SEQ 1D 4 obtained by adding two TAA termination codons and TGA and Notl recognition sequences for use in preparing a deletion derivative encoding the positions of 1-191 amino acid residues), and
. hTPO-2: 5'-TTT GCG GCC GCT CAT TAC AGA CCA GGA ATC TTG GCT CTG AAT-3 '(SEQ 1D 60) (antisense primer corresponding to the sequence of positions 696-720 in SEQ 1D 4 obtained by adding two TAA termination codons and TGA and Notl recognition sequences for use in preparing a deletion derivative encoding the positions of 1-211 amino acid residues).

Using 1 μg of plasmid DNA of the clone pHT1-231 obtained in Example 18 as a template, 10 μM of each of the synthesized hTPO-5 (for the 5 ^' side) and hTPO-2, -3, -4 and S (for 3 'sides) as primers and a kit of reagents for PCR GeneAmp ^™ with AmpliTaq ^™ DNA polymerase (Takara Shuzo), PCR was performed in a volume of 100 μl using PCR system 9600 GeneAmp ^™ (PERKIN-ELMER) with a repetition of a total of 20 cycles, each of which consisted of denaturation at 95 ^° C for 1 minute, hybridization at 65 ^° C for 1 minute and synthesis at 72 ^° C for 1 minute, followed by final incubation of ri 72 ^o C for 7 minutes. The target band obtained from each PCR was digested with restriction enzymes EcoRl and Notl and the resulting cleavage product was subjected to electrophoresis on 1% agarose gel (FMS BioProducts) to isolate the main DNA fragment having the expected size amplified by each PCR. The DNA fragment thus isolated was purified using Prep-A-Gene DNA Purification Kit (Bio-Rad) and then subcloned into the pEF18S expression vector, previously digested with the same restriction enzymes. In this case, the Highly competent E. coli DH5 strain manufactured by TOUOVO was used as the host strain. From the obtained transformants, each derived from its own PCR, 4-5 clones containing the insertion of the expected size were selected to obtain plasmid DNA samples, mainly by the method described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press , 1989). In a number of such operations, samples of plasmid DNA were obtained from a deletion derivative encoding the positions of 1-163 amino acid residues (pHT1-163 1-5), a deletion derivative encoding the positions of 1-171 amino acid residues (pHT1-163 1-4), a deletion derivative encoding the positions of 1-191 amino acid residues (pHT1-191 1-4) and a deletion derivative encoding the positions of 1-211 amino acid residues (pHT1-211 1-4). The full length of the amplified regions of each plasmid DNA was subjected to nucleotide sequence analysis to find its complete identity with the nucleotide sequence shown in SEQ 1D 4.

 Transfection into COS 1 cells of the clones thus obtained was carried out according to the method of Example 11. That is, transfection was performed with 10 μg of each of the plasmid DNA samples according to the DEAE-dextran method, which involves treatment with chloroquine. Transfected COS 1 cells were cultured for 3 days and then culture supernatants were harvested.

 These culture supernatants were extensively dialyzed against IMDM culture medium and evaluated in rat CFU-MK test / system. TPO activity was detected (dose-dependent) in the supernatant of COS 1 cells in which each of pHT1-211, pHT1-191, pHT1-171 or pHT1-163 was expressed. Representative data with pHT1-211 1, pHT1-191 1 and pHT1-171 2 are shown in FIG. 13a and with pHT1-163 2 in FIG. 13. Similar results were obtained in the M-07e test system. The supernatant of COS 1 cells in which each of pHT1-211, pHT1-191, pHT1-171 or pHT1-163 was expressed significantly increased the proliferation of M-07e cells. Representative data with pET1-211 1, pET1-191 1, pHT1-171 2 or pHT1-163 2 are shown in Fig. 14.

 These results showed that human TPO still retains its in vitro biological activity even after deletion of its carboxyl-terminal half at Ser (position 163), and gave a strong basis for the assumption that the biologically active parts of human TPO are located inside the amino-terminal half, ending at Ser (position 163).

Example 28. Obtaining human TPO with a deletion at the C-end and its expression and activity in COS 1 cells
To analyze the region necessary for the human TPO protein to be active, the C-terminal amino acids were deleted from the deletion derivatives obtained in Example 27 and the TPO activity expressed by the derivatives obtained was tested in COS 1 cells. Using the plasmid clone pEF18S-HL34 obtained in Example 16, expression plasmids were obtained by deletion of nucleotide sequences encoding the C-terminal amino acid residues starting at serine at position 163. The construction of these deletion plasmids was performed using PCR. The following primers were used:
. hTPO-5: 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3 '(SEQ 1D 61) (obtained by adding the EcoRl recognition sequence to the sequence of positions 1-21 in SEQ 1D 4, the same sequence is shown in example 18 ),
. hTPO-150: 5'-TTT GCG GCC GCT CAT TAG AGG GTG GAC CCT CCT ASA AGC AT-3 '(SEQ 1D 62) (antisense primer corresponding to the sequence of positions 514-537 in SEQ 1D 4 obtained by adding two TAA termination codons and TGA and Notl recognition sequences for use in obtaining a deletion mutant encoding the positions of 1-150 amino acid residues),
. hTPO-151: 5'-TTT GCG GCC GCT CAT TAG CAG AGG GTG GAC CCT CCT ACA A-3 '(SEQ 1D 63) (antisense primer corresponding to the sequence of positions 518-540 in SEQ 1D 4, obtained by adding two TAA termination codons and TGA and Notl recognition sequences for use in obtaining a deletion derivative encoding the positions of 1-151 amino acid residues),
.hTRO-153: 5'-TTT GCG GCC GCT CAT TAC CTG ACG CAG AGG GTG GAC CC-3 '(SEQ 1D 64) (antisense primer corresponding to the sequence of positions 526-546 in SEQ 1D 4 obtained by adding two TAA termination codons and TGA and Notl recognition sequences, for use in obtaining a deletion derivative encoding the positions of 1-153 amino acid residues),
.hTPO-154: 5'-TTT GCG GCC GCT CAT TAC CGC CTG ACG CAG AGG GTG GA-3 '(SEQ 1D 65) (antisense primer corresponding to the sequence of positions 529-549 in SEQ 1D 4 obtained by adding two TAA termination codons and TGA and Notl recognition sequences for use in obtaining a deletion derivative encoding the positions of 1-154 amino acid residues),
.hTRO-155: 5'-TTT GCG GCC GCT CAT TAG GCC CGC CTG ACG CAG AGG GT-3 '(SEQ 1D 66) (antisense primer corresponding to the sequence of positions 532-552 in SEQ 1D 4 obtained by adding two TAA termination codons and TGA and Notl recognition sequences for use in preparing a deletion derivative encoding the positions of 1-155 amino acid residues),
.hTPO-156: 5'-TTT GCG GCC GCT CAT TAT GGG GCC CGC CTG ACG CAG AG-3 '(SEQ 1D 67) (antisense primer corresponding to the sequence of positions 535-555 in SEQ 1D 4, obtained by adding two TAA termination codons and TGA and Notl recognition sequences for use in preparing a deletion derivative encoding the positions of 1-156 amino acid residues), and
.hTPO-157: 5'-TTT GCG GCC GCT CAT TAG GGT GGG GCC CGC CTG ACG CA-3 '(SEQ 1D 68) (antisense primer corresponding to the sequence of positions 538-558 in SEQ 1D 4 obtained by adding two TAA termination codons and TGA and Notl recognition sequences for use in obtaining a deletion derivative encoding the positions of 1-157 amino acid residues).

PCR was performed using 1 μg of plasmid DNA of clone pEF18S-HL34 obtained in Example 16 as a template and 10 μM of each of the oligonucleotides thus synthesized (hTPO-5 for the 5'-side and hTPO-150 '-151, -153, -154, -155, -156, -157 for the 3 ^' side) as primers. Using a PCR reagent kit GeneAmp ^™ with AmpliTaq ^™ DNA polymerase (manufactured by Takara Shuzo), PCR was performed in 100 μl volume, using a PCR system 9600 GeneAmp ^™ (PERKIN-ELMER) and repeating a total of 20 cycles, each of which consisted of denaturation at 95 ^o C for 1 minute, hybridization at 66 ^o C for 1 minute and synthesis at 72 ^o C for 1 minute, followed by final incubation at 72 ^o C for 7 minutes. The target band obtained in each PCR was digested with restriction enzymes EcoRl and Notl, and the resulting cleavage product was electrophoresed on 1% agarose gel (FMC BioProducts) to isolate the main DNA fragment having the expected size for each PCR. The DNA fragment thus isolated was purified using a Prep-A-Gene DNA Purification Kit (Bio-Rad) and then subcloned into the pEF18S expression vector treated previously with the same restriction enzymes. In this case, the Highly competent strain of E-coli DH5 (TOUOVO) was used as the host strain. 3-5 clones containing the insertion of the expected size were selected from the obtained transformants originating from each PCR to obtain plasmid DNA samples essentially in accordance with the method described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) . Samples of plasmid DNA were obtained from a deletion derivative encoding the positions of 1-150 amino acid residues (pHT1-150 21, 22 and 25), a deletion derivative encoding the positions of 1-151 amino acid residues (pHT1-151 16, 17 and 18) were obtained through a number of such operations. , deletion derivative encoding the positions of 1-153 amino acid residues (pHT1-153 1-5), deletion derivative encoding the positions of 1-155 amino acid residues (pHT1-155 1-5), deletion derivative encoding the positions of 1-156 amino acid residues ( pHT1-156 1-5), and a deletion derivative encoding the position 1-157 amino acid residues (pHT1-157 1-5).

Of these purified plasmid DNA samples, the sequence pHT1-150 21, 22 and 25 and pHT1-151 16, 17 and 18 were checked using a 373A DNA sequencer (Applied Biosystems) using a Taq Dye Deoxy ^TM Terminater Cycle Sequening Kit (Applied Biosystems), confirming that the expected TPO cDNA sequences had no substitutions in all nucleotide sequences.

 Transfection of COS 1 cells with each of the clones thus obtained was carried out in accordance with the method of Example 11. That is, transfection was performed with 10 μg of each of the samples of plasmid DNA with a DEAE-dextran method involving chloroquine treatment, and the culture supernatant was recovered after 3 days of cultivation. The obtained culture supernatants were dialyzed against the IMDM culture medium described previously and evaluated using a M-07e test system. TPO activity was detected in the culture supernatant of COS 1 cells transfected with corresponding clones encoding C-terminal deletion derivatives consisting of amino acid positions 1-151, amino acid positions 1-153, amino acid positions 1-154, amino acid positions 1-155, amino acid positions 1 -156 or amino acid positions 1-157. All these derivatives contain a cysteine residue at position 151. However, TPO activity was not found in the COS 1 culture supernatant of cells transfected with a clone encoding a C-terminal deletion derivative consisting of amino acid positions 1-150, in which the cysteine residue at position 151 was also deleted.

Example 29. Obtaining human TPO with a deletion at the N-end and its expression and activity in COS 1 cells
To analyze the region necessary for the TPO protein to exhibit its biological activity, deletion derivatives with deletion of amino acids at the N-terminus obtained in Example 28 were further deleted and the activity of TPO in the resulting deletion derivatives was determined. Using the plasmid clone pEF18S-HL34 obtained in Example 16 and the plasmid clone pHT1-163 obtained in Example 27, expression plasmids were obtained by deletion of the nucleotide sequences encoding the N-terminal amino acid residues following the signal sequence. The construction of these deletion plasmids was performed using PCR. The sequence of primers made for use in PCR are as follows:
h TPO-5: 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3 '(SEQ ID 69) (obtained by adding the EcoRl recognition sequence to the sequence of positions 1-21 in SEQ ID 4, the same sequence is shown in example 18 ),
hTPO-3: 5'-TTT GCG GCC GCT CAT TAT TCG TGT ATC CTG TTC AGG TFT CC-3 '(SEQ ID 70) (antisense primer corresponding to the sequence of positions 757-780 in SEQ ID 4, obtained by adding two TAA termination codons and TGA and Notl recognition sequences; the same sequence has been synthesized for use in the preparation of a deletion derivative encoding the positions of 1-231 amino acid residues),
. hTPO-S: 5'-TTT GCG GCC GCT CAT TAG CTG GGG AGA GCT GTG GTG GGT-3 '(SEQ ID 71) (antisense primer corresponding to the sequence of positions 555-576 in SEQ ID 4 obtained for use in obtaining a deletion derivative, coding position 1-163 amino acid residues),
.hTPO-13: 5'-AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC CAG TG-3 '(SEQ ID 72) (positions 124-173 in SEQ ID 4 obtained for use in the preparation of a derivative, in which the amino acid residues of positions 1-12) are deleted,
. hTPO-13R: 5'-CAT GGG AGT CAC GAA GCA GTT TAC TGG ACA GCG TTA GCC TTG CAG TTA G-3 '(SEQ ID 73) (antisense primer corresponding to the sequence of positions 64-87 and 124-148 in SEQ ID 4 obtained for use in obtaining a derivative in which the amino acid residues of positions 1-12 are deleted),
hTPO-7: 5'-TGT GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT C-3 '(SEQ ID 74) (positions 106-154 in SEQ IG 4, obtained for use in the preparation of a derivative, in which deleted the amino acid residues of positions 1-6),
.hTPO-7R: 5'-TTT ACT GAG GAC TCG GAG GTC ACA GGA CAG CGT TAG CCT TGC AGT TAG -3 '(SEQ ID 75) (antisense primer corresponding to the sequence of positions 64-87 and 106-129 in SEQ ID 4 , prepared for use in obtaining a derivative in which the amino acid residues of positions 1-6) are deleted,
. hTPO-8: 5'-GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC A-3 '(SEQ ID 76) (positions 109-157 in SEQ ID 4, obtained for use in the preparation of a derivative, in which deleted the amino acid residues of positions 1-7),
.hTPO-8R: 5'-CAG TTT ACT GAG GAC TCG GAG GTC GGA CAG CGT TAG CCT TGC AGT TAG-3 '(SEQ ID 77) (antisense primer corresponding to the sequence of positions 64-87 and 109-132 in SEQ ID 4 obtained for use in obtaining a derivative in which the amino acid residues of positions 1-7) are deleted.

(1) Obtaining a derivative (pHT13-231) in which the amino acid residues of positions 1-12 are deleted
PCR was performed using 1.4 μg of plasmid DNA of the clone pEF18S-HL34 obtained in Example 18 as a template and 5 μM of each of the oligonucleotides thus synthesized (hTPO-13 and hTPO-3 in one combination of both hTPO-5 and hTPO- 13 in another combination) as primers. Using a GeneRmp ^™ PCR reagent kit with AmpliTaq ^™ DNA polymerase (Takara Shuzo), PCR was performed in 100 μl volume using a PCR 9600 GeneAmp ^{™ system} (PERKIN-ELMER) and, after 5 minutes of denaturation at 95 ^{° C.} , was repeated as a whole 30 cycles, each of which consisted of denaturation at 95 ^° C for 1 minute, hybridization at 65 ^° C for 1 minute and synthesis at 72 ^° C for 1 minute, followed by final incubation at 72 ^° C for 7 minutes . Each of the PCR products was electrophoresed on a 1.2% agarose gel (manufactured by FMC BioProducts) to isolate the main DNA product of the expected size, which was then purified using Prep-A-Gene DNA Purification Kit (Bio-Rad) and dissolved in 15 μl of TE buffer. After this, a second PCR was performed using 1 μl of each of the solutions thus prepared as a matrix.

A second PCR was performed using 5 μM of each of the synthesized primers (hTRO-5 and hTPO-3). Using a PCR reagent kit, GeneAmp ^™ with AmpliTaq ^™ DNA polymerase (Takara Shuzo), PCR was performed in 100 μl volume using a 9600 Gene-Amp ^™ PCR system (PERKIN-ELMER), and after 5 minutes of denaturation at 95 ^° C, a total of 30 cycles were repeated, each of which consisted of denaturation at 95 ^° C for 1 minute, hybridization at 60 ^° C for 1 minute and synthesis at 72 ^° C for 1 minute, followed by final incubation at 72 ^° C for 1 minutes Each of the PCR products was subjected to 1.2% agarose gel electrophoresis (FMC BioProducts) to isolate the main DNA fragment of the expected size, which was then purified using Prep-A-Gene DNA Purification Kit (Bio-Rad) and dissolved in 15 μl of TE buffer. After digestion with restriction enzymes EcoRl and Notl, the resulting solution was extracted with the same volume of a phenol / chloroform mixture, followed by precipitation with ethanol. After centrifugation, the resulting pellet was dissolved in 15 μl of TE buffer and then subcloned into the pEF18S expression vector, previously treated with the same restriction enzymes. The highly competent E. coli DH5 (TOUOVO) was used as the host strain. 45 clones containing the insertion of the expected size to obtain plasmid DNA samples were selected from the obtained transformants, mainly in accordance with the method described in Molecular Cloning (Sambrook et al. Cold Spring Harbor Laboratory Press, 1989). In this way, it was possible to obtain plasmid DNA of a deletion derivative (pHT13-231) encoding a protein molecule in which the amino acid residues of positions 1-12 were deleted from the original amino acid sequence 1-231. When conducting PCR of these 45 clones using hTPO-5 and hTPO-3 primers, insertions having approximately the expected sizes were confirmed in 8 clones. Of these, the sequence of 3 clones was verified in a 373A DNA sequencer (Applied Biosystems) using the Taq Dye Deoxy ^TM Terminater Cycle Sequening Kit (Applied Biosystems) to obtain a pHT13-231 clone having the cDNA sequence that they wanted to construct and a deletion which was expected, without substitutions or other changes in the entire nucleotide sequence.

(2) Obtaining a derivative (pHT7-163) in which the amino acid residues of positions 1-6 are deleted
In this case, the amino acid of position 163 was used as the C-terminus to obtain deletion derivatives, although in the previous step, the amino acid of position 231 was used as the C-terminus of TPO protein to obtain the deletion derivative. It was found that TPO activity could be expressed, even with further deletion of C-terminal amino acids. For this reason, the position of amino acid 163 was used as the C-terminus in the next step (3). PCR was performed using 1.4 μg of the plasmid DNA of clone pHT1-163 obtained in Example 27 as a template and 5 μM of each of the synthesized oligonucleotides (hTPO-7 and hTPO-S) in one combination of both hTPO-5 and hTPO-7R in another combination) as primers. Using a GeneRmp ^™ PCR reagent kit with AmpliTaq ^™ DNA polymerase (manufactured by Takara Shuzo), PCR was performed in 100 μl volume using a 9600 GeneAmp ^™ PCR system (PERKIN-ELMER) and, after 5 minutes of denaturation at 95 ^{° C.} , was repeated a total of 30 cycles, each of which consisted of denaturation at 95 ^° C for 1 minute, renaturation at 65 ^° C for 1 minute and synthesis at 72 ^° C for 1 minute, with final incubation at 72 ^° C for 7 minutes . Each of the PCR products was electrophoresed on a 1.2% agarose gel (manufactured by FMC BioProducts) to isolate the main DNA fragment of the expected size, which was then purified using Prep-A-Gene DNA Purification Kit (Bio-Rad) and dissolved in 15 μl of TE buffer. After this, a second PCR was performed using 1 μl of each of the solutions thus obtained as a matrix.

The second PCR was performed using 5 μM of each of the synthesized primers (hTPO-5 and hTPO-S). Using a GeneRmp ^™ PCR reagent kit with AmpliTaq ^™ DNA polymerase (Takara Shuzo), PCR reactions were carried out in 100 μl volume using a 9600 GeneAmp ^™ PCR system (PERKIN-ELMER) and, after 5 minutes of denaturation at 95 ^{° C.} , was repeated a total of 30 cycles, each of which consisted of denaturation at 95 ^° C for 1 minute, renaturation at 60 ^° C for 1 minute and synthesis at 72 ^° C for 1 minute, followed by final incubation at 72 ^° C for 7 minutes. Each of the PCR products was electrophoresed on a 1.2% agarose gel (manufactured by FMC BioRpoducts) to isolate the main DNA fragment of the expected size, which was then purified using Prep-A-Gene DNA Purification Kit (Bio-Rad) and dissolved in 15 μl of TE buffer. After digestion with restriction enzymes EcoRl and Notl, the resulting solution was extracted with the same volume of a phenol / chloroform mixture, followed by precipitation with ethanol. After centrifugation, the resulting pellet was dissolved in 15 μl of TE buffer and then subcloned into the pEF18S expression vector, which was pretreated with the same restriction enzymes. In this case, the Highly competent E. coli strain DH5 (TOUOVO) was used as the host strain. 30 clones containing the insertion of the expected size were selected from the obtained transformants to obtain plasmid DNA samples mainly according to the method described in Molecular Cloning (Sambrook et al., Cold Sprining Harbor Laboratory Press, 1989). In this way, it was possible to obtain plasmid DNA of a deletion derivative (pHT7-163) encoding a protein molecule in which the amino acid residues of positions 1-6 were deleted from the original sequence of amino acid residues 1-163 in SEQ ID 4. When carrying out PCR with these clones using hTPO-5 primers and hTPO-S insertions, having approximately the expected sizes, were confirmed in all clones. Of these, the sequences of 3 clones were checked using a 373A DNA sequencer (Applied Biosystems) using the Taq Dey Deoxy ^TM Terminater Cycle Sequening Kit (Applied Biosystems) to obtain 2 clones, pHT7-163 4 and 29, each of which has a TPO cDNA sequence, which was constructed, and the deletion, which was expected, without substitutions and any other changes in the entire nucleotide sequence.

(3) Obtaining a derivative (pHT8-163) in which the amino acid residues of positions 1-7 are deleted
A derivative (pHT8-163) with a deletion of amino acid residues of positions 1-7 was prepared basically in the same way as in the above case of obtaining a derivative (pHT7-163) with a deletion of amino acid residues of positions 1-6. PCR was performed using 1.4 μg of plasmid DNA of clone pHT1-163 obtained in Example 27 as a template and 5 μM of each of the synthesized oligonucleotides (hTPO-8 and hTPO-S in one combination and hTPO-5 and hTPO-8R in another combination) as primers. Using a GeneRmp ^™ PCR reagent kit with AmpliTaq ^™ DNA polymerase (Takara Shuzo), PCR was performed in 100 μl volume using a 9600 GeneAmp ^™ PCR system (PERKIN-ELMER) and, after 5 minutes of denaturation at 95 ^{° C.} , was repeated a total of 30 cycles, each of which consisted of denaturation at 95 ^° C for 1 minute, renaturation at 65 ^° C for 1 minute and synthesis at 72 ^° C for 1 minute, followed by final incubation at 72 ^° C for 7 minutes. Each of the PCR products was subjected to 1.2% agarose gel electrophoresis (FMC BioProducts) to isolate the main DNA fragment of the expected size, which was then purified using Prep-A-Gene DNA purification kit (Bio-Rad) and dissolved in 15 μl of TE buffer. After that, a second PCR was performed using 1 μl of each of the resulting solutions as a matrix.

The second PCR was performed using 5 μM of each of the synthesized primers (hTPO-5 and hTPO-S). Using kit for PCR GeneAmp ^TM reagents AnipliTaq ^TM DNA polymerase (manufactured by Takara Shuzo) was carried out PCR in a 100 .mu.l volume using a PCR system 9600 GeneAmp ^TM (PERKING-ELMER) and, after 5 minutes of denaturation at 95 ^o C, was repeated in the a total of 30 cycles, each of which consisted of denaturation at 95 ^° C for 1 minute, renaturation at 60 ^° C for 1 minute and synthesis at 72 ^° C for 1 minute, followed by final incubation for 7 minutes. Each of the PCR products was electrophoresed on a 1.2% agarose gel (FMC Bio-Products) to isolate the main DNA fragment of the expected size, which was then purified using Prep-A-Gene DNA Purification Kit (Bio-Rad) and dissolved in 15 μl of TE buffer. After digestion with restriction enzymes EcoRl and Notl, the resulting solution was subjected to extraction with the same volume of a phenol / chloroform mixture and subsequent precipitation with ethanol. After centrifugation, the resulting pellet was dissolved in 15 μl of TE buffer and then subcloned into the pEF18S expression vector, which was previously digested with the same restriction enzymes. In this case, the Highly competent strain of E.coli DH5 (TOYOBO)
used as a host strain. 30 clones containing the insertion of the expected size to obtain plasmid DNA samples were selected from the obtained transformants, mainly according to the method described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). In this way, it was possible to obtain plasmid DNA of a deletion derivative (pHT8-163) encoding a protein molecule in which the amino acid residues of positions 1-7 were deleted from the original amino acid residues of positions 1-163 in SEQ ID No 4. When carrying out PCR with these clones Using primers hTPO-5 and hTPO-S, insertions of approximately the expected sizes were confirmed in all clones. Of these, the sequence of 3 clones was verified using a 373A DNA sequencer (Applied Biosystems) using the Taq Dye Deoxy ^TM Terminater Cycle Sequening Kit (Applied Biosystems) to obtain 2 clones, pHT8-163 33 and 48, each of which has a TPO cDNA sequence which was constructed, and the deletion that was expected, without substitutions or similar changes in the entire nucleotide sequence.

(4) Expression of a deletion derivative in COS 1 cells and confirmation of TPO activity
Transfection of COS 1 cells with each of the deletion clones thus obtained was carried out according to the method of Example 11. That is, transfection was performed using 10 μg of each of the plasmid DNA samples with a DEAE-dextran method involving chloroquine treatment, and the culture supernatants were recovered after 3 days of cultivation. The culture supernatant thus obtained was thoroughly dialyzed against the IMDM culture medium described above and evaluated using a test system in M-07e.

 As a result, weak but significant TPO activity was detected in the culture supernatant of COS 1 cells transfected with a clone encoding a deletion derivative consisting of amino acids of positions 7-163. However, TPO activity was not detected in the culture supernatant of COS 1 cells transfected with a clone encoding a deletion derivative consisting of amino acids of positions 8-163 or amino acids of positions 13-231.

Example 30. Obtaining plasmids cDNA (pHTP1) TPO full length of a person
An expression vector having all of the amino acid encoding regions of human TPO cDNA as contemplated in SEQ ID 6 was designed for expression in mammalian cells.

 PCR was performed as follows to obtain a DNA fragment encompassing all coding regions of human TPO cDNA.

The nucleotide sequences of the primers used were as follows:
. hTPO-1: 5 'TTG TGA CCT CCG CGT CCT CAG-3' (SEQ ID No 78) (positions 105-125 in SEQ ID No 6);
.SA: 5'-CAG GTA TCC GGG GAT TTG GTC GTC-3 '(SEQ ID No 79) (antisense primer corresponding to the sequence of positions 745-765 in SEQ ID No 6);
. hTPO-P: 5'-TGC GTT TCC TGA TGC TTG TAG-3 '(SEQ ID No. 80) (positions 503-523 in SEQ ID 6);
. hTPO-KO: 5'-GAG AGA GCG GCC GCT TAC CCT TCC TGA GAC AGA TT-3 '(SEQ ID No. 81) (the sequence obtained by adding the Notl recognition sequence and the GAGAGA sequence (SEQ ID No. 82) to the antisense sequence corresponding to sequence of positions 1066-1086 in SEQ ID No 6).

The first PCR was performed using 300 ng of the clone pEF18S-HL34 obtained in Example 16 as a template. Using 0.5 μM of each of the hTPO-1 primers to SA IE Vent R ^TM DNA polymerase (manufactured by New England Biolabs), PCR was performed (a total of 30 cycles were repeated, each of which consisted of incubation at 96 ^° C for 1 minute incubation at 62 ^° C for 1 minute and incubation at 72 ^° C for 1 minute, followed by final incubation at 72 ^° C for 7 minutes). The composition of the reaction solution in final concentrations was as follows: 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 20 mM Tris-HC1 (pH 8.8), 2 mM MgSO ₄ , 0.1% Triton X-100 and 200 μm mixture of dNTP.

1 μg of a commercially available preparation of poly (A) ⁺ RNA obtained from normal human liver (manufactured by Clontech) was heated at 70 ^{° C.} for 10 minutes, rapidly cooled on ice, and then mixed with 10 mM DTT, 500 μM dNTP mixture, 25 ng of an arbitrary primer (manufactured by Boehringer-Mannheim) and 200 units of Superscript ^TM 11 RNase K ^- (manufactured by LIFE TECHNOLOGIES), followed by incubation for 1 hour at 37 ^{° C.} to carry out cDNA synthesis. After that, a second PCR was carried out (repeating a total of 30 cycles, each cycle consisted of incubations at 96 ^° C for 1 min, at 58 ^° C for 1 min and at 72 ^° C for 1 min) using 1/20 of the reaction solution of synthesized cDNA as matrices and 2.5 μM of each of the hTPO-P and hTPO-KO primers and 2.5 units of AmpliTaq ^TM DNA polymerase (manufactured by Takara Shuzo).

Each of the obtained solutions of the first and second PCR was subjected to electrophoresis on a 1% agarose gel to isolate the corresponding main DNA fragments of the expected size, which were then purified using Prep-A-Gene DNA purification kit (manufactured by Bio-Rad). After that, a third PCR was performed using 1/20 of the volume of each of the solutions thus obtained as a matrix. This reaction (heating at 96 ^° C for 2 minutes, followed by a total of 3 cycles, each of which consisted of incubations at 96 ^° C for 2 minutes and at 72 ^° C for 2 minutes, and subsequent incubation at 72 ^° C for 7 minutes) using 1 unit of Vent R ^TM DNA polymerase (manufactured by New England Biolabs). The resulting reaction solution was mixed with 1 μM of each of the hTPO-1 and TPO-KO primers, heated at 96 ^{° C. for} 2 minutes, subjected to 25 PCR cycles, each of which consisted of incubation at 96 ^{° C. for} 1 minute, at 62 ^{° C. for} 1 minute and at 72 ^{° C. for} 1 minute, followed by incubation at 72 ^{° C.} for 7 minutes. The resulting reaction solution was extracted with the same volume of a phenol / chloroform mixture saturated with water, followed by ethanol precipitation (2.5 volumes of ethanol in the presence of 0.3 M sodium acetate and 0.5 μl of glycogen produced by Boehringer-Mannheim to extract DNA. Thus obtained DNA was digested with restriction enzymes BamH1 and Notl and subjected to electrophoresis on a 1% agarose gel and the main band having the expected size thus isolated was purified using Prep-A-Gene DNA Purification Kit (Bio-Rad), ligated with pBLUESCRIPT 11 SK vector ⁺ (Stratagene), which was was digested with restriction enzymes BamH1 and Notl, and then transformed into the Highly competent E. coli strain DH5 (TOYOBO). 4 clones were selected from the obtained colonies to obtain plasmid DNA samples. The sequence of purified plasmid DNA samples was checked using a 373A DNA sequencer manufactured by Applied Biosystems using Taq Dye Deoxy ^TM Terminater Cycle Sequening Kit (Applied Biosystems), resulting in a clone, pBLTP, having a TPO cDNA sequence constructed without substitution in the nucleotide sequence within the region between BamH1 and Notl.

 This pBLTP clone was digested with restriction enzymes EcoR1 and BamH1 and subjected to electrophoresis on a 1% agarose gel and the high molecular weight band thus isolated was purified using Prep-A-Gene DNA Purification Kit (Bio-Rad). In the same way, PEF18S-HL34 was treated with restriction enzymes to purify a DNA fragment of approximately 450 bp. Each of the DNA samples thus obtained was ligated and transformed into the Competent E. coli strain DH5 (TOUOVO). Plasmid DNA samples were obtained from the resulting colonies to obtain a clone, pBLTEN, containing the insertion of human TPO cDNA. The pBLTEN clone thus obtained was digested with restriction enzymes EcoR1 and Notl and electrophoresed on a 1% agarose gel, and the approximately 1200 bp DNA fragment thus isolated was purified using Prep-A-Gene DNA Purification Kit (Bio-Rad) , were ligated with the expression vector pEF18S, which was previously cleaved with the same restriction enzymes, and then transformed into the High Competent E. coli DH5 strain (TOUOVO). Plasmid DNA samples were obtained from the obtained colonies; a clone, PHT1, containing the cDNA coding region of the entire human TPO was obtained. Plasmid DNA was obtained from this clone in large quantities and used in further experiments. Obtaining plasmid DNA was carried out mainly in accordance with the method described in Molecular Cloning (Sambrok et al., Cold Spring Harbor Laboratory Press, 1989).

Example 31. Construction of a mammalian expression plasmid, pDEF202-hTPO-P1, for the expression of human TPO in Chinese hamster ovary cells (CHO)
1 μg of plasmid pMG1 containing mouse dihydrofolate reductase minigene (DHFR) was digested with restriction enzymes EcoR1 and BamH1 and subjected to agarose gel electrophoresis to isolate a fragment (approximately 2.5 kb) containing the minigene with mouse DHFR. The isolated DNA fragment was dissolved in 25 ml of a reaction solution consisting of 50 mM Tris-HCl (pH 7.5), 7 mM MgCl ₂ , 1 mM 2-mercaptoethanol and 0.2 mM dNTP mixture, mixed with 2 units of a Klenov fragment and then incubated at room temperature for 30 minutes to form blunt ends at both ends of this DNA fragment. After treatment with a phenol / chloroform mixture and ethanol precipitation, the resulting fragment was dissolved in 10 ml of a TE solution (10 mM Tris-HCl (pH 8.0) / 1 mM EDTA). The mammalian expression vector pEF18S was digested with Sma1 restriction enzyme, dephosphorylated with alkaline phosphatase (manufactured by Takara Shuzo), and then ligated with a DNA fragment containing the DHFR mouse minigene using T4 DNA ligase (Takara Shuzo) to obtain pDEF202 expression vector.

The constructed expression vector pDEF202 was then digested with restriction enzymes EcoR1 and Spel and then subjected to agarose gel electrophoresis to isolate a large DNA fragment. human TPO cDNA, which was obtained by digestion of a pHP1 plasmid containing human TPO cDNA (clone p1) with restriction enzymes EcoR1 and Spel, were ligated with this linearized vector pDEF202 using T4 DNA ligase (Takara Shuzo) to obtain plasmid pEF202-h TPO-PTP expression of human TPO cDNA. This constructed plasmid contains the start of SV40 replication, the 1-α human elongation factor promoter, and the early SV40 polyadenylation signal for transcription of human TPO cDNA, mouse DHFR minigene, and the start of replication pUC18 and the β-lactamase gene (Amp ^r ).

Example 32. Expression of human TPO in CHO cells
CHO cells (DHFR strain, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, vol. 77, p. 4216, 1980) were maintained in α-minimal stock medium (α-MEM (-), supplemented with hypoxanthine and thymidine) containing fetal calf serum (FCS) using a tissue culture dish 6 cm in diameter (Falcon). The transformation of CHO cells with plasmid pDEF202-hTPO-P1 was carried out by the calcium phosphate method (CellPhect, Pharmacia), as described below. 10 μg of the plasmid pDEF202-hTPO-P1 obtained in Example 31 was mixed with 120 ml of buffer A and 120 ml of H ₂ O and the resulting mixture was incubated at room temperature for 10 minutes. This solution was then mixed with 120 ml of buffer B and allowed to stand at room temperature for another 30 minutes. This DNA mixture solution was then added to the culture and incubated for 6 hours in a CO ₂ incubator. After 6 hours, the culture was washed twice with serum-free α-MEM (-), treated with α-MEM (-) containing 10% dimethyl sulfoxide for 2 minutes and then incubated for 2 days in non-selective medium (α-MEM (-) with the addition of hypoxanthine and thymidine) containing 10% dialyzed FCS. After 2 days of cultivation, cells were selected in selective medium (α-MEM (-)) containing 10% dialyzed FCS. To select transformed cells with a DHFR - positive phenotype, these cells were treated with a tipsin / EDTA solution and resuspended in a selective medium. Cells in a tissue culture dish (6 cm) were divided into 5 plates (10 cm) or 12-24 well culture plates and then cultured in this selective medium. The culture medium was replaced at intervals of 2 days. The activity of human TPO in the culture medium of CHO cells with a DHFR-positive phenotype was measured by CFU-MK test, M-07e test or Ba / P3 test. Cells secreting human TPO into the culture medium were treated with 25 nM methotrexate to isolate a cell clone producing a high amount of human TPO.

 In this case, transfection of CHO cells can also be performed by transfection of SKO cells with plasmids pHTP1 and pMG1.

 CHO strain (CHO-DUKXB11) transfected with plasmid pDEF202-hTPO-P1 was deposited by the inventor on January 31, 1995, under FERM BP-4988 National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan.

Example 33. Construction of a recombinant vector, BMCGSneo-hTPO-P1, for use in X 63.6.5.3. cells
One microgram of the mammalian expression vector pBMCGSneo was digested with restriction enzymes Xhol and Notl and subjected to agarose gel electrophoresis to isolate a portion of the vector DNA. This DNA and human TPO cDNA fragment (P1 clone), which was obtained by digesting the pBLTEN plasmid containing human TPO cDNA, with Xhol and Notl restriction enzymes, was ligated with T4 DNA ligase to obtain the target expression vector, pBMCGSneo-hTPO-P1. This expressing plasmid contains an early cytomegalovirus promoter, portions of an intron derived from rabbit β-globin gene and a polyadenylated region for transcription of human TPO cDNA, human β-globin gene, 69% of bovine papilloma virus 1 gene, thymidine kinase promoter and polyadenylated region of thymidine kinase 1, (neomycin-resistant gene) and the origin (origin) of pBR322 replication and the β-lactamase gene (Amp ^r ) and human TPO cDNA were ligated at a site located 5'-3 'from the cytomegalovirus promoter.

Example 34. Expression in X 63.6.5.3. cells
X 63.6.5.3 cells were cultured in Dulbecco's minimal stock medium (DME) containing 10% fetal bovine serum. Transfection X.63.6.5.3. plasmid cells BMCGSneo-hTPO was performed by electroporation, as described below.

20 μg of the plasmid BMCGSneo-hTPO-P1 obtained in Example 33 was added to 750 μl of DME medium containing 10 ⁷ cells, and the mixture was placed in an electroporation cuvette having a 4 mm gap and allowed to stand for 10 minutes at 4 ^{° C.} The cuvette was then placed in an electroporation device (BTX 600 manufactured by BTX) for gene transfer under conditions of 380 V, 25 mF and 24 angstroms. After gene transfer, the cuvette was again allowed to stand for 15 minutes at 4 ^° C. Then the cells were washed once with DEM containing 10% fetal bovine serum. Transfected cells were suspended in 50 ml of DEM containing 10 FCS, poured into the wells of a 96-well culture plate, and then cultured for 2 days in a CO ₂ incubator. After 2 days of cultivation, the culture medium was replaced with DEM containing 10% FCS and 1 mg / ml G418 (produced by GIBCO) and a change of medium was then performed every 3 days. The activity of human TPO in the culture medium of the transformants was measured by tests with CFU-MK, M-07e or Ba / F3. Transformants secreting human TPO into the culture medium were cloned twice by limiting dilution to establish human TPO-producing cell lines.

Example 35. Large-scale expression of human TPO in COS 1 cells
Transfection of COS 1 cells was carried out according to the DEAE-dextran method, which included chloroquine treatment as described in Example 11. COS 1 cells (ATCC CRL 1650) were cultured in IMDM containing 10% (v / v) FCS using 175 cm collagen-coated ² culture flasks at 37 ^{° C.} in a 5% CO ₂ incubator until the cells merged 100%. To cover the culture flask with collagen, 25 ml of a collagen solution (Cellmatrix type 1-C manufactured by Iwaki), the concentration of which was adjusted to 0.3 mg / ml with 1 mM HC1, was added to each 175 cm ² culture flask, the collagen solution was removed after 1 standing at room temperature and then the flask thus treated was washed once with 20-50 ml of PBS. Transfection was performed by mixing 20 ml of IMDM solution containing 250 μg / ml DEAE-dextran (Pharmacia), 60 μM chloroquine (Sigma) and 10% (vol./about.) Nu-serum (Collaborative) with pHTP1 (40 μg), which was dissolved in 500 μl of HBS by adding the resulting mixture to the COS 1 cells described above contained in one 175 cm ² culture flask, which was washed once with IMDM immediately before transfection. Then these cells were cultured for 3 hours at 37 ^{° C.} in a 5% CO ₂ incubator. After this, the culture supernatant was removed by suction and the cells were washed once with IMDM, mixed in 50 ml of serum-free medium and then cultured for 5 days at 36 ^° C in a 5% CO ₂ incubator to recover the culture supernatant. In one operation, 100-260 culture flasks were used and 5-13 L of culture supernatant was recovered. A serum-free medium was prepared by adding 5 μg / ml insulin (manufactured by Sigma), 5 μg / ml transferrin (Sigma), 10 μM monoethanolamine (Wako Pure Chemicai Industries), 25 nM selenite (Sigma) and 200 μg / ml BSA (free of BSA) to IMDM fatty acids highly purified bovine albumin manufactured by Nichirei).

Example 36. Purification of full-sized human TPO (obtained from the expression plasmid pHTP1, P1 clone) expressed in COS 1 cells, and its biological activity
The following are examples of the activity and purification of full-sized human TPO (obtained from the expression plasmid pHTP1) expressed in COS1 cells. A partially purified product was obtained first, to test its platelet-increasing activity, and then purified to obtain a high purity product. To measure TPO activity in the subsequent stages, the M-07e test system was mainly used. Similarly, TPO activity was determined in a rat CFU-MK test system. During these tests, human serum albumin was added to each sample to a final concentration of 0.02-0.05%.

First, COS 1 cells transfected with plasmid pHTP1 according to the Ohashi and Sudo method (Hideya Ohashi and Tadashi Sudo, Biosci. Biotech. Biochem., 58 (4), 758-759, 1994) were cultured for 5 days at 37 ^{° C.} in 5% CO ₂ using a serum-free 1MDM culture medium containing 0.2 g BSA, 5 mg bovine insulin, 5 mg human transferrin, 0.02 mM monoethanolamine and 25 nM sodium selenite in 1000 ml, resulting in approximately 7 l of serum-free culture supernatant. To it was added p-APMSF Pefabloc SC proteolytic enzyme inhibitors (4- (2-aminoethyl) -benzenesulfonyl fluoride hydrochloride manufactured by Merk Cat. 24839) to a final concentration of approximately 1 mM, followed by filtration using a 0.2 μm filter to obtain a supernatant. The supernatant was concentrated approximately 10-fold using an ultrafiltration apparatus (Omega Ultrasette, with a cut-off of a nominal molar mass of 8,000 manufactured by Filtron; or PLGC Pellicon Cassette, a cut-off of a molar mass of 10,000, Millipore). 723 ml of this concentrate (protein concentration 3.38 mg / ml, total protein 2445 mg, relative activity 43000, total activity 105100000) was mixed with 1.6 moles per 1000 ml of ammonium sulfate (288 g in total), obtaining 804 ml of solution, containing 1.5 M ammonium sulfate (in final concentration). Then, the solution thus obtained was applied at a flow rate of 10 ml / min onto a Macro-Prep Methyl HIC column (diameter 5 cm and height 9 cm) Bio-Rad, cat. 156-0080), which was previously balanced with 20 mM sodium citrate (buffer) (pH 5.2) containing 1.5 M ammonium sulfate. After loading, the samples were eluted with 20 mM sodium citrate buffer (pH 5.2) containing 1.25 M ammonium sulfate to obtain the F1 fraction passing through the column (2384 ml, protein concentration 0.864 mg / ml, total protein 2061 mg, relative activity 6000).

 Then, the elution solution was replaced with 20 mM sodium citrate, pH 5.8, to obtain the F2 fraction (1092 ml, protein concentration 0.776 mg / ml, total protein 847 mg, relative activity 150,000). The thus obtained Macro-Prep Methyl HIC F2 fraction (1081 ml) was applied at a flow rate of 10 ml / min to a SP Sepharose Fast Flow column (diameter 3 cm and height 10 cm) manufactured by Pharmacia Biotech Cat. 17-0729-01), which was previously equilibrated with 20 mM sodium citrate buffer (pH 5.8). After loading the samples, elution was performed with 20 mM sodium citrate buffer (pH 5.6) containing 110 mM NaCl to obtain the F1 fraction (2262 ml, protein concentration 0.270 mg / ml, total protein 610 mg, relative activity 30,000). Then, the eluting solution was replaced with 20 mM sodium citrate (pH 5.4) containing 400 mM NaCl, collecting the F2 fraction (856 ml, protein concentration 0.189 mg / ml, total protein 162 mg, relative activity 300,000). Then, the elution solution was replaced with 20 mM sodium citrate buffer (pH 5.2) containing 1000 mM NaCl, collecting the eluted F3 fraction (370 ml, protein concentration 0.034 mg / ml, total protein 12.6 mg, relative activity 150,000).

 The main TPO-active fraction F2 (845 ml) of the column stage of the SP Sepharose Fast Flow column was mixed with approximately 10% at a final concentration of 1-propanol and applied at a flow rate of 3 ml / min on an LA-WGA column (diameter 2 cm, height 14 cm, Honen Cat. WG-007), pre-equilibrated with 20 mM sodium citrate buffer (pH 5.4) containing 400 mM NaCl. After loading the column, elution was performed with a mixture of 20 mM sodium citrate buffer (pH 5.4) containing 400 mM NaCl with 1-propanol (9: 1) to obtain the F1 fraction (64.4 ml, protein concentration 0.0178 mg / ml, total protein 1.15 mg, relative activity 17220). Then, the elution solution was replaced with 20 mM sodium citrate buffer (pH 6.1) containing 0.4 M GlcNAc and 10% 1-propanol, collecting the eluted F2 fraction (45 ml, protein concentration 0.0104 mg / ml, total protein 0.470 mg, relative activity 675000).

 The main TPO-active fraction F2 (340 ml) of the LA-WGA column operation was mixed with approximately 0.005% at the final concentration of TFA and subjected to column chromatography on YMC-Pack CN-AP (6 mm in diameter and 250 mm in height) manufactured by UMS, cat . AR-513. Using 0.1% TFA as a developing solvent A and 1-propanol containing 0.05% TFA as a developing solvent, a sample was applied at a flow rate of 0.6 ml / min to a YMC-Pack CN-AP column equilibrated previously 15% B. After loading the sample, the concentration of propanol was increased from 15% B to 25% B and elution was performed by a linear gradient from 25% B to 50% B for 65 minutes, collecting eluates in 1.5 ml polypropylene tubes (2, 5 minutes).

 0.5 μl (1/3000 fractions) of the eluate in each tube was mixed with HSA and concentrated by ultrafiltration to obtain 0.25 ml of IMDM test culture solution containing 0.05% HSA for use in identifying TPO-active fractions using this test. As a result, high TPO activity (relative activity of approximately 630000-4800000) was found in tubes with numbers 24-30 (in the range of propanol concentrations of 35.0 and 42.0%), which were combined as a TPO-active fraction of FA (13.5 ml).

 The FA fraction obtained from the YMC-Pack CN-AP column stage was subjected to column chromatography on a Cepcell Pak C1 300A (4.6 mm diameter, 150 mm height manufactured by Shiseido, Cat. C1 TYPE: SG 300A) using 0.1% TFA as a developing solvent A and 1-propanol containing 0.05% TFA as a developing solvent B. A portion (8.9 ml) of the FA fraction obtained in a column operation with YMC-Pack CN-AP was mixed with 0, 3 ml of glycerol was concentrated by centrifugal evaporation and then mixed with approximately 2.5 ml of 10% B. The resulting solution was then applied at a flow rate 0.4 ml / min to a Capcell Pak C1 300A column, pre-equilibrated with 20% B. After loading the column, elution was performed first with 20% B for 15 minutes and then with a linear gradient from 20% B to 40% B for 50 minutes, collecting eluates in polypropylene tubes 1 ml (2.5 min) in each tube.

 A portion of 1 μl (1/1000 fraction) of the eluate in each tube was mixed with HSA and concentrated by ultrafiltration to obtain 0.25 ml of IMDM test culture solution containing 0.05% HSA for use in identifying TPO-active fractions with this test. As a result, a high TPO activity (relative activity of approximately 3000000-22500000) was detected in test tubes with numbers 20-23 (within the range of 28.5 and 32.5% propanol concentration), which were combined as a highly active fraction.

Example 37. The measurement mol. mass of human TPO expressed in COS 1 cells
It was suggested that they say. the mass of full-sized human TPO (obtained from expressing plasmid pHTP1, clone F1) expressed in COS 1 cells should be greater than mol. mass estimated based on the size of the peptide chain due to the addition of the sugar chain. Therefore, as a first step, a partially purified TPO fraction was obtained as follows from a culture supernatant containing a full-sized human TPO (obtained from the expression plasmid pHTP1, F1 clone) expressed in COS 1 cells. That is, using developing solvent A (0.1% trifluoroacetic acid (TFA) and developing solvent B (1-propanol containing 0.05% TFA), a portion of 0.3 ml of the culture supernatant was applied to a YMC-Pack PROTEIN-column RP (diameter 0.46 cm, bed height 15 cm, UMS, Cat. A-RRR-33-46-25), pre-equilibrated previously 25% B. 25% B was passed through the column for 5 minutes at a flow rate of 0.4 ml / min and then fractionation was performed using a linear density gradient from 25% to 50% B for 50 minutes, TPO activity was detected in the eluates in the concentration range radios propanol 34.5% and 43.5%. These eluates were evaporated to dryness by evaporation and centrifugation to yield partially purified sample.

 Then the TPO activity was determined by the M-07e test system after protein extraction from SDS-PAGE gel electrophoresis, carried out under non-reducing conditions, as described in example 1, or mol. the mass was measured based on the recovered DPC111 markers by Western analysis, which will be described below in example 45. As a result of TPO activity was found in a wide range of average mol. masses from about 69000 to 94000, indicating the variation in mol. mass. It was also measured that the average mol. the mass of SRW based on the reduced DRS111 markers after hydrolysis of the sugar chain of the N-glycosidic linkage type using N-glycanase (Genzyme, cat. 1472-00) was 36000-40000, i.e. more than mol. a weight of approximately 35,000, assumed from the size of the peptide chain, which is a strong indication of the presence of also a sugar chain of the O-glycosidic linkage type.

 In the same way it was found that the average mol. the mass of full-sized human TPO (obtained from expressing plasmid pHTP1, P1 clone) expressed in COS 1 cells is 63000-83000.

Example 38. Biological characteristics of human SRW
The effects of human TPO on hematopoietic cells in humans and rats were investigated mainly using a culture supernatant from COS 1 cells transfected with pHTF1.

In a colony-counting test using the CD34 ⁺ DR ⁺ cell fraction obtained from human spinal cord blood, human TPO stimulated the formation of a significant number of megakaryocyte colonies. For example, 11.5 megakaryocyte colonies formed 6,000 CD34 ⁺ DR ⁺ cells in the presence of 10% (v / v) culture supernatant from COS 1 cells transfected with pHT1-231. To test the effect of human TPO on progenitor cells of human megakaryocytes, CD34 ⁺ cells were obtained from human peripheral blood as follows. The leukocyte fraction obtained from human peripheral blood using a Ficoll-Paque density gradient was depleted from cells adhering to plastic cups. Unattached cells were then purified from SBA (soy agglutinin) -positive cells by panning using AIS Microselecter SBA (Asahi Medical). The obtained cells were incubated on AIS Microselecter CD 34 and adsorbed cells, CD34 ⁺ cells, were collected after culturing CD34 ⁺ cells for 10 days in the presence of a culture supernatant from transfected COS 1 cells in a liquid medium, ^and large proliferation of Gp11b / 111a ⁺ cells was observed ^and large found an increase in ploidy in these Gp11b / 111a ⁺ cells. These results provide a strong basis for the assumption that human TPO has a selective effect on progenitor cells of human megakaryocytes and stimulates their proliferation and differentiation.

In the colony counting test using Gp11b / 111a ⁺ cells highly enriched with CFU-MK from rat bone marrow cells and cells that did not adhere to the plastic obtained in the previous stage of Gp11b / 111a ⁺ cells, human TPO induced the formation of a significant number of megakaryocyte colonies. For example, 34.5 megakaryocyte colonies were formed from 1000 Gp11b / 111a ⁺ cells and 28.5 megakaryocyte colonies were formed by 20,000 cells that did not adhere to the plastic in the presence of 20% (v / v) culture supernatant from transfected COS 1 cells. In addition, although the fraction of cells not attached to plastic contains various hematopoietic progenitor cells other than CFU-MK, only colonies of megakaryocytes were formed in the presence of human TPO, which suggests that human TPO exhibits its selective effect on progenitor cells of megakaryocytic origin. When culturing Gp11b / 111a ⁺ cells obtained from rat bone marrow for 3-5 days in the presence of 20% (v / v) culture supernatant from transfected COS 1 cells in liquid culture, an increase was observed on the 5th day of cultivation cell ploidy.

Example 39. Construction of a vector for use in expression of a fusion protein (hereinafter referred to as “GST-TPO (1-174)” glutathione S-transferase (GST) and human TPO (amino acid residues 1-174) in E. coli
To facilitate the expression of human TPO in E. coli, an artificial gene was obtained that encodes human TPO and contains the E. coli preference codon. The nucleotide sequence of this DNA contains the amino terminal codon of methionine (ATG) at position -1 for use in translation initiation in E. coli.

The synthetic oligonucleotides 1-12 shown in Table IV were prepared and each of the synthetic oligonucleotides 2-11 was phosphorylated with T4 kinase (manufactured by Pharmacia) in a solution consisting of 1 mM ATP, 10 mM Tris-acetate, 10 mM Mg-acetate and 50 mM K-acetate. To convert these synthetic oligonucleotides into 6 double-stranded DNA fragments, they were combined in combinations of 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10 and 11 and 12, and each combination was annealed in a solution of 10 mM Tris -CH1 (pH 7.5), 10 mM MgCl ₂ and 50 mM NaCl. Then 3 double-stranded DNA fragments 1 and 2, 3 and 4 and 5 and 6 and 3 other double-stranded DNA fragments 7 and 8, 9 and 10 and 11 and 12, respectively, were treated with T4 ligase (manufactured by Life Technology) and these 2 reaction solutions again treated with T4 ligase in the same manner. The DNA fragment obtained by the ligation reaction was digested with BamH1 (Boehringer-Mannheim) and electrophoresed on a 2% agarose gel, extracting a fragment of approximately 390-400 bp by size, which was then purified using Prep-A-Gene DNA purification kit and subcloned into pUC18, pre-digested with Xbal and BamH1 (E. coli DH5 was used as the host). Of the clones thus obtained, a clone having an artificial gene encoding human TPO and containing the following preferred E. coli codons was selected by nucleotide sequence analysis and named pUC18 (XB) (1-123). The DNA sequence of the coding chain is shown in the sequence list (SEQ ID No 9) (see Table V).

Single-stranded cDNA was synthesized from 1 μg obtained from normal human liver poly (A) ⁺ RNA using an oligo dT primer, and PCR was performed using 0.1 volume of the cDNA synthesis reaction solution as a template. PCR (incubation at 96 ^o C for 2 minutes, repetition and a total of 30 cycles, each of which consisted of incubations at 96 ^o C for 1 minute, at 58 ^o C for 1 minute and at 72 ^o C for 1 minutes, and the final incubation at 72 ^° C for 7 minutes) was carried out with a volume of 100 μl using hTPO-C and hTPO-Z (EcoR1) as primers. The human TPO cDNA fragment thus obtained was digested with BamHI and EcoR1 and subjected to electrophoresis on a 2% agarose gel, extracting a fragment of approximately 600 bp, which was then purified using Prep-A-Gene DNA purification kit and subcloned into pUC18 pre-digested EcoR1 and BamH1 (E. coli DH5α used as the host). The clone encoding the correct nucleotide sequence of human TPO was selected by nucleotide sequence analysis and it was named pUC18 (BE) (124-332). The oligonucleotide sequences of the primers used in this PCR were as follows:
. hTPO-C: 5'-GGA GGA GAC CAA GGC ACA GGA-3 '(SEQ ID No 95) (positions 329-349 of the clone hHTF1); and
. hTPO-Z (EcoR1): 5'-CCG GAA TTC TTA CCC TTC CTG AGA CAG ATT-3 '(SEQ ID 96) (obtained by adding the recognition sequence to the antisense primer corresponding to positions 1143-1163 of the pHTF1 clone).

 Clone pUC18 (BE) (124-332) was digested with BamHl and EcoR1 and electrophoresed on a 2% agarose gel to extract a approximately 600 bp human TPO cDNA fragment that was then purified using Prep DNA Purification Kit -A-Gene and subcloned into pUC18 (XB) (1-123), pre-digested with restriction enzymes EcoR1 and BamHl (E. coli DH5α was used as the host). The clone thus obtained was named pUC18 (XE) (1-332).

 Since the presence of human TPO activity in the peptide fragment of human TPO from positions 1-163 of amino acid residues was confirmed by an example in which various C-terminal deletion constructs of human TPO cDNA were obtained and expressed to measure in vitro TPO activity, an expression vector was constructed containing this peptide fragment. In this case, the expression of the DNA sequence encoding GST-TPO (1-174) was performed.

. 5'-CTAATGAG-3' (SEQ ID No 99)
3'-TCGAGATTACTCTTAA-5' (SEQ ID No 100)
.

- Клон pUC18 (XS) (1-174), полученный выше, расщепляли Xbal и EcoR1 и подвергали электрофорезу в 2% агарозном геле для извлечения фрагмента размером приблизительно 600 п.н., кодирующего аминокислотные остатки 1-174 ТРО человека. Затем этот фрагмент очищали при помощи набора для очистки ДНК Prep-A-Gene и субклонировали в pBluescript 11SK⁺ (изготовляемый Stratagene), предварительно расщепленный Xbal и EcoR1 (E.coli DH5α использовали в качестве хозяина). Клон, полученный таким образом, был назван pBL(XS)(1-174). Таким же образом клон pBL(XS) (1-174) расщепляли Xbal и Hind111, извлекая фрагмент размером приблизительно 550 п.н., кодирующий аминокислотные остатки 1-174 ТРО человека, и затем этот фрагмент очищали при помощи набора для очистки ДНК Prep-A-Gene и субклонировали в рСFМ536 (EР-А-136490), расщепленный предварительно Xbal и Hind111 (E.coli DH5α использовали в качестве хозяина). Клон, полученный таким образом, был назван рСFM536/hТ (1-174).Since the nucleotide sequence corresponding to positions 681-686 (amino acid residues 173 and 174) of the pHTF1 clone is recognized by the restriction enzyme Sac1, two terminal codons were introduced using 2 synthetic oligonucleotides using the recognition site of this restrictase. Those. two synthetic oligonucleotides SSE1 and SSE2 were annealed in a solution consisting of 10 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ and 50 mM NaCl, and using the DNA ligation kit (Takara Shuzo), the double-stranded fragment thus obtained was introduced in pUC18 (XE) (1-332), of which a C-terminal fragment of approximately 480 bp The human TPO cDNA was removed by cleaving it with Sacl and EcoR1 to give clone pUC18 (XS) (1-174) (E. coli DH5α was used as the host). The sequence of synthetic oligonucleotides used in this case were as follows:
SSE1: CTAATGAG (SEQ ID No 97)
SSE2: AATTCTCATTAGAGCT (SEQ ID No 98);

. 5'-CTAATGAG-3 '(SEQ ID No 99)
3'-TCGAGATTACTCTTAA-5 '(SEQ ID No 100)
.

- Clone pUC18 (XS) (1-174), obtained above, was digested with Xbal and EcoR1 and subjected to electrophoresis on a 2% agarose gel to extract a fragment of approximately 600 bp encoding amino acid residues 1-174 human TPO. This fragment was then purified using Prep-A-Gene DNA Purification Kit and subcloned into pBluescript 11SK ⁺ (manufactured by Stratagene), pre-digested with Xbal and EcoR1 (E. coli DH5α was used as the host). The clone thus obtained was named pBL (XS) (1-174). In the same way, clone pBL (XS) (1-174) was digested with Xbal and Hind111, extracting a fragment of approximately 550 bp encoding the amino acid residues 1-174 human TPO, and then this fragment was purified using Prep- DNA purification kit A-Gene and subcloned into pCFM536 (EP-A-136490), pre-digested with Xbal and Hind111 (E. coli DH5α was used as the host). The clone thus obtained was named pCFM536 / hT (1-174).

The following experiment was performed to express GST-TPO (1-174) using pGEX-2T (Pharmacia), which is an expression vector for a glutathione S-transferase (GST) fusion protein. PCR (incubation at 96 ^o C for 2 minutes, repeating a total of 22 cycles, each of which consisted of incubations at 96 ^o C for 1 minute, at 41 ^o C for 1 minute and at 72 ^o C for 1 minute , and the final incubation at 72 ^° C for 7 minutes) was performed using pCFM536 / hT (1-174) as a template and two PCR primers GEZ1 and GEX3. The human TPO coding fragment thus obtained was digested with Nael and EcoR1 and subjected to electrophoresis on a 2% agarose gel, extracting a fragment of approximately 550 bp, this fragment was then purified using Prep-A-Gene DNA purification kit and cloned into pGEX-2T, pre-digested with EcoR1 and Smal (E. coli DH5 used as the host) and then a clone named pGEX-2T / hT (1-174) was selected by nucleotide sequence analysis to obtain a transformant for use in GST-TPO expression ( 1-174). The oligonucleotide sequences of the primers used in the PCR were as follows:
GEX1: 5'-ATS GCC GGC TCC GCC AGC TTG TGA C-3 '- (SEQ ID No. 101) (obtained by adding the Nael recognition sequence to the sequence of positions 21-39 in SEQ ID 10) and
. GEX3: 5'-GCC GAA TTC TCA TTA GAG CTC GTT CAG TGT-3 '(SEC ID 102) (antisense primer corresponding to the sequence of positions 523-549 in SEQ ID 10).

 This expression plasmid contains a DNA sequence encoding a GST protein, followed by a peptide for the recognition of thrombin and human TPO (amino acid residues 1-174). The DNA sequence encoding the peptide recognition of thrombin and human TPO (amino acid residues 1-174), shown in the list of sequences (SEQ ID 10).

Example 40. Expression of GST-TPO (1-174) in E. coli
The transformant obtained in example 39 was cultured overnight at 37 ^{° C.} on a shaker using 60 ml of LB medium containing 50 μg / ml ampicillin, and 25 ml of the resulting culture broth was added to 1000 ml of LV medium containing 50 μg / ml ampicillin , and cultivated at 37 ^{° C.} on a shaker until the CD at A ₆₀₀ reached 0.7-0.8. Thereafter, 1PTG was added to the culture broth to a final concentration of 0.1 mM and culture was continued for another 3 hours to induce the expression of GST-TPO (1-174).

Example 41. Purification of GST-TPO (1-174) expressed in E. coli and confirmation of its biological activity
5.9 g of frozen cells of a recombinant strain producing GST-TPO (1-174) obtained from clone pGEX-2T / hT (1-174) encoding the nucleotide sequence of human TPO was suspended in 10 ml of water and destroyed by a high-pressure disintegrator. After centrifugation of the resulting suspension, GST-TPO (1-174) was recovered in the precipitated fraction, most of the impurity proteins, cellular components, etc. removed. Then, the precipitated fraction thus obtained containing GST-TPO (1-174) was suspended in 5 ml of water, to which 6 ml of 1M Tris buffer (pH 8.5), 120 ml of 10 M urea and 16 ml were then added with stirring. water. After 5 minutes of stirring to dissolve the contents, the resulting solution was evenly divided into 4 parts for the following steps (1) to (4).

(1) One of the samples was diluted 10 times with 20 mM Tris buffer (pH 8.5). Then, reduced-type glutathione and oxidized-type glutathione were added to the corresponding final concentrations of 5 mM and 0.5 mM, followed by incubation overnight at 4 ^° C. The resulting mixture was centrifuged to extract GST-TPO (1-174) as a supernatant liquid, which then diluted 2 times with 20 mM sodium citrate buffer (pH 5.5) and then adjusted to pH 5.5 with acetic acid. GST-TPO (1-174) in solution was applied to SP Sepharose Fast Flow (Pharmacia Biotech, Cat. 17-0729-01) a cation exchange column, previously equilibrated with 20 mM sodium citrate buffer (pH 5.5). After washing the column with 20 mM sodium citrate pH 5.5, GST-TPO (1-174) was eluted with 20 mM buffer containing sodium citrate, pH 5.5, containing 500 mM sodium chloride. A portion of 129 ml of this eluate was mixed with 2.6 ml of 1 M Tris buffer, pH 8.5, and the resulting mixture with pH 8.1 was applied to a Glutathione Sepharose 4B column (Pharmacia Biotech, Cat. 17-0756-01) for GST adsorption -TPO (1-174). After washing the PBS column, GST-TPO (1-174) was eluted with Tris buffer pH 8.5 containing 10 mM of reduced type glutathione. The resulting eluate was mixed with 37 NlH thrombin units, allowed to stand for 4 hours at room temperature, diluted 10 times with PBS and then applied to a Glutathione Sepharose 4B column to adsorb cleaved GST and extract TPO (1-174) in the non-adsorbed fraction. The recovered non-adsorbed fraction was diluted 3 times with 20 mM pH 5.5 containing sodium citrate buffer and applied to a SP Sepharose Fast Flow cation exchange column, previously equilibrated with the same buffer, and the target compound was eluted with a linear density gradient of 0-500 mM sodium chloride dissolved in the same buffer.

 (2) All operations of step (1) were performed in the presence of 0.1% polysorbate 80.

(3) One of the samples was diluted 10 times with 20 mM Tri-buffer with a pH of 8.5. Reduced-type glutathione and oxidized-type glutathione were added to it to the corresponding final concentrations of 5 mM and 0.5 mM, followed by standing overnight at 4 ^° C. The mixture thus obtained was centrifuged to extract GST-TPO (1-174) as supernatant liquid, which was then diluted 2 times with 20 mM containing sodium citrate buffer with a pH of 5.5 and then adjusted to pH 5.5 with acetic acid. GST-TPO (1-174) in this solution was applied onto a SP Sepharose Fast Flow cation exchange resin pre-suspended with 20 mM sodium citrate buffer at pH 5.5. After washing the resin with 20 mM sodium citrate-containing pH 5.5 buffer, elution was performed with GST-TPO (1-174) 20 mM sodium citrate-containing buffer with pH 5.5 containing 500 mM sodium chloride. The eluate was adjusted to pH 8 with 1 M Tris-buffer with pH 8.5, mixed with 320 NlH thrombin units to remove GST polypeptide sequences by cleavage with a peptide linker recognition thrombin and allowed to stand for 4 hours at room temperature, diluted 5 times 20 mM buffer containing sodium citrate, pH 5.5, and then applied to SP Sepharose Fast Flow cation exchange resin, equilibrated in advance with the same buffer, and the target compound was then eluted with a linear density gradient of 0-500 mM sodium chloride dissolved in the same buffer.

 (4) All operations of step (3) were performed in the presence of 0.1% polysorbate 80.

 Each of the fractions eluted at sodium chloride densities of approximately 200-400 mM by column chromatography on a SP Sepharose Fast Flow cation exchange column was extensively dialyzed against IMDM culture medium and then evaluated in rat CFU-MK test system. When analyzing the fraction by SDS-PAGE electrophoresis in the presence of a reducing agent, a band corresponding to mol. weight of 19 kD (purity 1-20%) as one of the main protein bands of this fraction. When analyzing the N-terminal sequence of this band in SDS-PAGE and transferring to a PVDF membrane according to the method described in Example 1, it was confirmed that this band contains the TPO sequence expressed as obtained from pGEX-2T / hT (1-174) fusion protein. The deduced (deduced) amino acid sequence of this obtained TPO was [Gly-1] TPO due to the sequence of the peptide linker for thrombin recognition and the known thrombin activity on this linker.

Example 42. Construction of a vector for use in expression in E. coli human TPO (amino acid residues 1-163) having substitutions at position 1 (Ser -> Ala) and position 3 (Ala -> Val)
Clone pUC18 (XE) (1-332) obtained in Example 39 was digested with Xbal and EcoR1 and electrophoresed on a 2% agarose gel, isolating a fragment of approximately 1000 bp encoding amino acid residues 1-332 of human TPO, and this the fragment was then purified using Prep-A-Gene DNA Purification Kit and subcloned into pBluescript 11SK ⁺ (manufactured by Stratagene), which was digested with restriction enzymes Xbal and EcoR1 (E. coli DH5α was used as the host). The clone obtained in this way was named pBL (XE) (1-332). Then, the region of this pBL clone (XE) (1-332) from its BamH1 recognition sequence to amino acid position 163 (positions 366-489) was replaced by dominant E. coli codons. The synthetic oligonucleotides 13-20 shown below were prepared and each pair of synthetic oligonucleotides 13 and 14, 15 and 16 and 17 and 18 were phosphorylated in the same tube containing a solution consisting of 0.1 mM ATP, 10 mm Tris acetate , 10 mM Mg-acetate and 50 mM K-acetate. After further adding 1/10 of the volume of the solution, consisting of 100 mm Tris / Hcl (pH 7.5), 100 mm MgCl ₂ and 500 mm NaCl, the resulting solution was heated in a boiling bath for 3 minutes and then allowed to cool to room temperature, getting a double-stranded DNA fragment. Then, the three pairs of double-stranded fragments thus obtained consisting of 13 and 14, 15 and 16 and 17 and 18 were ligated using a DNA ligation kit (Takara Shuzo) and PCR was performed using the ligation product as a template and synthetic oligonucleotides 19 and 20 as primers. The resulting PCR product was digested with BamH1 and Hind111 and electrophoresed on a 2% agarose gel, extracting a fragment of approximately 130 bp. using the DNA-A-Gene DNA purification kit. This fragment was subcloned into pBL (XE) (1-332), previously digested with BamH1 and Hind111 (E. coli DN5 was used as the host). A clone having the nucleotide sequences shown in table. VI, was selected from the obtained clones by nucleotide sequence analysis and named pBL (XH) (1-163).

In order to increase the amount of expressed human TPO (position 1-163) and to prevent hydrolysis of the N-terminus with a protease, an expression vector was designed for use in the expression of the mutated type of human TPO (position 1-163) (hereinafter referred to as "h 6T (1-163)" ) having substitutions at position 1 (Ser on Ala) and position 3 (Ala on Val) of human TPO (position 1-163) ([Ala ¹ , Val ³ ] TPO (1-163)) and, at the same time encoding Lys at -1 position and Met at -2 position ([Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ ] TPO (1-163)). Four synthetic oligonucleotides shown below were prepared, and synthetic oligonucleotides 2-9 and 3-3 were phosphorylated with T4 kinase (manufactured by Pharmacia) in a solution consisting of 1 mM ATP, 10 mM Tris-acetate, 10 mM Mg-acetate and 50 mM K- acetate. In order to convert these synthetic oligonucleotides into double-stranded DNA fragments, each pair of single-stranded DNA fragments 1-9 and 2-9 and 3-3 and 4-3 was annealed in a solution consisting of 10 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ and 50 mM NaCl. Then, pairs of double-stranded DNA fragments thus obtained, consisting of 1-9 and 2-9 and 3-3 and 4-3, were processed using a DNA ligation kit (Takara Shuzo). The DNA fragment obtained in the ligation reaction was subcloned into pBL (XH) (1-163), pre-digested with Xba1 and Nru1, and the clone correctly replaced with the following synthetic oligonucleotide, shown in table. VII was selected by nucleotide sequence analysis (E. coli DN5α was used as the host) and named pBL (XH) h6T (1-163).

 Clone pBL (XH) (1-163) was digested with Xba1 and Hind111, extracting a fragment of approximately 500 bp encoding the amino acid residues 1-163 of human mutated TPO, and this fragment was then purified using Prep-A DNA purification kit -Gene and cloned into pCFM536 (EP-A-136490), pre-digested with Xba1 and Hind111 (E. coli JM109, which was previously transformed with pMW1 (ATCC 39933), used as the host). The clone obtained in this way was named pCFM536 / h6T (1-163), and the E. coli strain having this expression vector was used as a transformant for use in the expression of human TPO of the mutated type, namely h6T (1-163). This expression plasmid contains the DNA sequence shown in the sequence list (SEQ ID 11).

Example 43. Expression of h6T (1-163) in E. coli
Expression of the expression plasmid pCFM536 is controlled by the λPL promoter, which itself is controlled by the c1857 repressor gene. The transformant obtained in Example 42 was cultured overnight at 30 ^{° C.} on a shaker using 60 ml of LB medium containing 50 μg / ml ampicillin and 12.5 μg / ml tetracycline, and a portion of 25 μl of the obtained culture broth was added to 1000 ml L in a medium containing 50 μg / ml ampicillin, and cultured at 37 ^{° C.} on a shaker until the OD at A ₆₀₀ reaches 1.0-1.2. After that, approximately 330 ml of LB medium heated to 65 ^° C was added to the culture broth, bringing the final temperature of the medium to 42 ^° C, and the culture was pumped for 3 hours at 42 ^° C to induce h6T expression (1-163).

Example 44. Purification of h6T (1-163) expressed in E. coli and confirmation of its biological activity
A portion of 3.6 g of frozen h6T cells (1-163) of the producing recombinant strain was suspended in 10 ml of water and destroyed using a high-pressure disintegrator. By centrifuging this suspension, a precipitated fraction was obtained, and most of the impurity proteins, cellular components, etc., were removed. The recovered precipitated fraction containing h6T (1-163) was suspended in 7 ml of water and 3 ml of Tris buffer (pH 8.5) was added to the suspension with stirring, followed by the addition of urea (final concentration 8 M), guanidine chloride (final concentration 6 M) and N-lauroyl sarcosinate (final concentration 2%). After 5-20 minutes of stirring at room temperature to dissolve the components, the resulting solution was diluted 10 times with 20 mM Tris buffer at pH 8.5 and subjected to repeated protein folding of the chain by incubation overnight at 4 ^° C. In this case, glutathione was used and copper sulfate as additives other than air oxidation. By centrifugation, 6T (1-163) was recovered from the supernatant fluid. When analyzing each of the extracted fractions using SDS-PAGE in the presence of a reducing agent, a band corresponding to mol. weight 18 kD (purity 30-40%) as the main protein band of each fraction. When analyzing the N-terminal sequence using SDS-PAGE and transferring to a PVDF membrane according to the method described in Example 1, it was confirmed that this band contains the expressed TPO sequence in the form of a mutated type obtained from pCMF536 / h6T (1-163) TPO . With careful dialysis of each fraction against 1MDM culture medium and evaluation using a rat test system with rat CFU-MK TPO, activity was detected in all fractions and it was dose-dependent.

Example 45. Obtaining antibodies against TPO peptide and detection of TPO by SDS-PAGE and Western analysis
Success in producing antibodies against TPO peptide will make it possible to detect the TPO protein by immunological means. In this regard, three regions that seem to be relatively applicable as antigens (see below) were selected from the proven TPO amino acid sequences and a peptide of the type of quadrupole chain peptide with multiple antigens (MAP) was synthesized by the Ta method ^m (Proc.Natl Acad.Sci.USA, 85, 5409-5413, 1988) using each of the selected areas and then each of the 2 rabbits was immunized 8 times with 100 μg of each of the peptides thus synthesized to obtain the corresponding antiserum samples.

(

при положении 24 представляет собой исходно S в аминокислотных остатках, определенных на основании гена)
b) Крысиный ТРО (46-66) (Пептидный район RT2)
FSLGЕWKTQTEQSKAQDILGA (SEQ 1D 117)
с) Крысиный ТРО (163-180) (Пептидный район RT4)

(

в положениях 178-180 исходно представляет собой TSG в аминокислотных остатках, определенных на основании этого гена)
Затем эти пробы антисывороток, анти-RT1 пептид и анти-RT2 пептид подвергали сначала колоночной хроматографии на Protein A (PROSEP-A, Bioprocessinq Ltd. , кат. 8427) для получения 1gG фракций (2344 мг и 1920 мг, соответственно). 54 мг и 32 мг из этих соответствующих фракций биотинилировали связыванием их с активным биотином (NHS -LC - Biotin 11; изготовляемым P1ER CE, кат. 21336). Рекомбинантный ТРО-содержащую пробу подвергали SDS - PAGE и затем электроблоттингу на PVDF или нитроцеллюлозную мембрану, как описано в Примере 1. Вестерн-анализ проводили обычным способом с применением этих биотинилироваиных антител в качестве первых антител.Amino Acid Sequences Contained in Synthesized Peptide Antigens
a) Rat SRW (9-28) (Peptide region R T1)

(

at position 24 represents the initial S in amino acid residues determined on the basis of the gene)
b) Rat SRW (46-66) (Peptide region RT2)
FSLGEWKTQTEQSKAQDILGA (SEQ 1D 117)
c) Rat SRW (163-180) (Peptide region RT4)

(

at positions 178-180 initially represents TSG in amino acid residues determined based on this gene)
Then, these antiserum samples, anti-RT1 peptide and anti-RT2 peptide were first subjected to Protein A column chromatography (PROSEP-A, Bioprocessinq Ltd., cat. 8427) to obtain 1gG fractions (2344 mg and 1920 mg, respectively). 54 mg and 32 mg of these respective fractions were biotinylated by binding to active biotin (NHS -LC - Biotin 11; manufactured by P1ER CE, cat. 21336). The recombinant TPO-containing sample was subjected to SDS-PAGE and then electro-blotted onto a PVDF or nitrocellulose membrane as described in Example 1. Western analysis was carried out in the usual manner using these biotinylated antibodies as the first antibodies.

 That is, the membrane after blotting was washed with a solution consisting of 20 mM Tris-HC1 and 0.5 NaCl (pH 7.5) (TPS) for 5 minutes and twice with 0.1% Tween 20-containing TPS (TPS) for 5 minutes each time and then treated with a blocking agent (Block Ace; manufactured by Dainippon Pharmaceutical, Cat. UK-B25) for 60 minutes. Then, the membrane was treated for 60 minutes with a TTBS solution containing 10 μg / ml biotinylated anti-TPO peptide antibodies, 0.05% BSA and 10% BlockAce, and then washed twice with TTBS each time for 5 minutes. After this, the membrane was treated for 30 minutes with a solution obtained by diluting alkaline phosphatase-labeled Avidin (Leinco Technoloqies, Cat. A108) with 5000 times a solution of TBPS containing 10% BlockAce and washed twice with TTBS and TBS for 5 minutes for 5 minutes . After that, color development was performed using an alkaline phosphatase substrate (manufactured by Bio-Rad, cat. 170-6432). The described Western analysis was performed at room temperature.

 As a result, it was possible to recognize not only various recombinant rat TPO proteins expressed in COS 1 cells, but also recombinant human TPO proteins expressed in COS 1 cells and E. coli cells (for example, human TPO expressed from plasmids pHTP1 or pHTF1 expressed in COS 1 cells, and its product obtained by cleavage of the N-glycosidic bond, GST-TPO (1-174), expressed in E. coli, and its thrombin cleaved product and h6T (1-163), which is a mutated type TPO expressed in E. coli, which were described in the Examples). In addition, these results made it possible to analyze these various types of recombinant human SRW.

 In addition to the above, the possibility of producing antibodies against the human TPO peptide was confirmed. Antibodies obtained by these methods can be used not only for western analysis, but also for purification of TPO using antibody columns and any conventional immunological methods using antibodies.

 The 6 regions in the human TPO amino acid sequence shown in SEQ 1D 7, which were thought to have relatively suitable antigenicity, were selected (see Table 4). Peptides such as a tetrameric peptide with multiple antigens (MAPs) corresponding to these regions have been synthesized. Each peptide was immunized with 2 rabbits 8 times in an amount of 100 mg for each immunization.

 A monomeric peptide was also separately synthesized in which the cysteine residue was linked to the C-terminus of each peptide region shown in Table 4. This peptide was then used as a test antigen to determine antibody titer using enzyme-linked immunosorbent assay. As a result, an increase in antibody titer was shown for the prepared sera, so that these sera were used as antisera.

 Antibodies were obtained by immunization with each antigenic peptide of two rabbits to produce antisera directed against such a peptide. The resulting two antibodies against the HT1 peptide for discrimination are called the antibody against the HT1-1 peptide and the antibody against the HT1-2 peptide, as obtained in two separate rabbits.

 An example of purification of antibodies against the peptide HT1 1 will be shown below.

 First, 30 mg of the monomeric peptide HT1 coupled to a cysteine residue was coupled to 12 ml of Sulfo-Link binding gel (Pierce, cat. 44895). Briefly, a peptide solution containing antigen was bound for 15 minutes to a gel equilibrated with binding buffer (50 mM Tris, 5 mM EDTA-Na pH 8.5) in an amount of 6 volumes to the volume of the gel. Then, after incubation for 30 minutes, the gel was washed with binding buffer in an amount of 3 volumes per gel volume. A binding buffer containing 0.05 ML cysteine-HCl was added to the gel in an amount of 1 ml / ml gel to block unreacted radicals for 15 minutes. After incubation for 30 minutes, the gel was washed with binding buffer (8 volumes per 1 volume of gel). All binding reactions were carried out at room temperature. Thus, the peptide was covalently bound to the gel with a binding efficiency of 28.3%, and an antigen column was prepared containing 0.8 mg of the peptide per 1 ml of gel.

76.7 ml (3620 mg protein content) of an antiserum containing an antibody against the HT1-1 peptide, which was obtained from one of the rabbits in a total of 78.4 ml, was applied to a column with antigen previously equilibrated with 50 mM phosphate buffer (pH 8) containing 150 mM NaCl and 0.05% sodium azide, and then washed with the same buffer to obtain 105.9 ml of a fraction passing through the column (protein content 3680 mg). Then the adsorbed fraction was eluted with 0.1 M citrate buffer (pH 3.0) and immediately neutralized with 21.1 ml of 0.1 M carbonate buffer (pH 9.9), followed by concentration by ultrafiltration (using an Amicon UM 30 membrane) receiving a purified antibody against the peptide HT1-1 in a solution of 50 mm phosphate buffer (pH 8) containing 150 mm NaCl and 0.05% NaN ₃ .

 The following antibodies were similarly prepared: anti-peptide HT1-2 antibody (60.0 mg); anti-HT2-1 peptide antibody (18.8 mg); anti-HT2-2 peptide antibody (8.2 mg); etc. You can also get antibodies against peptides HT3-HT6.

 3 mg of affinity-purified antibodies against the HT1-1 peptide, against the HT1-2 peptide, against the HT2-1 peptide or against the HT2-2 peptide were biotinylated by binding to activated biotin (Pierce, NHS-LC-Biotin 11, cat. 21336).

 It was found that all of the above purified antibodies recognize and detect human TPO when analyzed by Western blotting (on a nitrocellulose filter or on PVDF) after SDS-PAGE of a recombinant standard human TPO partially purified from the culture supernatant of CHO cells into which the gene was introduced coding for the amino acid sequence shown in Table 4, and in which this gene was expressed.

Table 4
Amino Acid Sequences of Human TPO Included in a Tetrameric MAP Synthetic Peptide Antigen
Peptide region HT1 human TPO (amino acid numbers 8-28): DLRVLSKLLRDSHVLHSRLSQ (SEQ 1D 119)
Peptide NT2 region of human TPO (amino acid numbers 47-62): SLGEWKTQMEETKAQD (SEQ 1D 120)
Peptide region of NT3 TPO, human (amino acid numbers 108-126): LGTQLPPQGRTTAHKDPNA (SEQ 1D 121)
Peptide NT4 region of human TPO (amino acid numbers 172-190): NELPNRTSGLLETNFTASA (SEQ 1D 122)
Peptide region HT5 human TPO (amino acid numbers 262-284): SLPPNLQPGYSPSPTHPPTGQYT (SEQ 1D 123)
Peptide NT6 region of human TPO (amino acid numbers 306-332): PSAPTPTPTSPLLNTSYTHSQNLSQEG (SEQ 1D 124)
Example 46 Preparation of Anti-TPO Peptide Antibody Column
Since the anti-rat TPO peptide antibody obtained in Example 45 was able to recognize rat and human TPO molecules, the immunoglobulin fractions (1gG) of the anti-RT1 peptide and anti-RT2 antibodies were coupled to a chemically activated gel carrier to prepare columns with anti-TPO peptides in the following way. Each of the two antibodies used as such materials was obtained from the sera of two rabbits separately. That is, anti-RT1 peptide antibodies obtained from two rabbits were respectively called anti-RT1-1 peptide antibody and anti-RT1-2 peptide, and anti-RT2 peptide antibodies obtained from two other rabbits were respectively called anti-peptide antibody RT2-1 and anti-peptide antibody RT2-2. Since these antibodies were used separately for the preparation of antibody columns, only 5 columns were obtained, namely, two columns with antibodies against the RT1 peptide (hereinafter referred to as the column with anti-RT1-1 antibodies and the column with anti-RT1-2 antibodies), two columns with antibodies against the peptide RT2 (hereinafter referred to as a column with anti-RT2-1 antibodies and a column with anti-RT2-2 antibodies) and one column (column with a mixture of antibodies anti-RT1-2 + 2-1), which was prepared by mixing anti -RT1-2 antibodies and anti-RT2-1 antibodies and binding of this mixture to a gel.

Each antibody was dissolved in a solution of 50 mM sodium phosphate and 0.15 M NaCl (pH 8.0) to a final concentration of 5 mg / ml or 2.5 mg / ml of each of the uniformly mixed anti-RT1-2 antibodies and anti-RT2- 1 antibody in the case of a column with a mixture of anti-RT1-1 + 2 antibodies) and 2.31 ml of the resulting solution were mixed with 1.54 ml in a volume of swollen formyl-activated gel (Formyl-Cellulofine, Chisso) and subjected to a 2-hour binding reaction at 4 ^o C. Then was added 1.1 ml of 10 mg / ml of a reducing agent (trimetilaminborana (TMAV); manufactured by Seikagaku Kogyo, cat 680246.), followed by 6 hours to olnitelnoy binding reaction and followed by centrifugation to extract a gel. 10 ml of purified water was added to this portion of the gel, centrifuged again, and unreacted antibody molecules were removed by repeating this step 4 times. Then, the resulting gel was mixed with 4.6 ml of a blocking solution (0.2 M sodium phosphate and 1 M ethanolamine, pH 7.0) and 1.1 ml of a solution of a reducing agent and treated at 4 ^{° C.} for at least 2 hours to blocking the active groups of unreacted gel. After that, the gel was washed with purified water and DPBS using a centrifuge, packed into a small column tube, washed with 3 M potassium thiocyanate solution and 0.1 M glycine-HC1 solution (pH 2.5), balanced with DPBS and then stored.

 In 5 gels with antibodies against TPO peptides, columns with anti-RT1-1 antibodies, columns with anti-RT1-2 antibodies, columns with anti-RT2-1 antibodies, columns with anti-RT2-2 antibodies and columns with anti RT1- mixture 2 + 2-1 antibodies, the binding efficiency of the respective IgG fractions reached 97.4%, 95.4%, 98.4%, 98.3% and 99.4%. In addition, the number of IgG fractions associated with the gel volume was 5.6 mg / ml gel, 5.8 mg / ml gel, 5.7 mg / ml gel, 5.7 mg / ml gel and 2.9 mg / ml of gel, respectively. Therefore, since it was confirmed that the binding efficiency and the amount of binding did not change significantly depending on the origin of the antibodies and differences in antigens, the experiments shown in the following Example 47 were carried out using these antibody columns.

Example 47. Purification of TPO from the culture supernatant obtained by transfection of COS 1 cells with a pHTP1 expression vector using an anti-TPO antibody column followed by reverse phase column chromatography and confirmation of its biological activity
To evaluate the following purified samples, the M-07e test system was used as an in vitro test. Partially purified TPO samples from the culture supernatant obtained in Example 35 using COS 1 cells transfected with the pHTP1 expression vector (fractions other than the main TPO fraction, namely the passing fraction F1 and the F3 fraction eluted after F2 by washing with 20 mM sodium citrate, pH 5.8, Masgo-Rp Methyl H1C columns and F1 and F3 fractions of SP Sepharose Fast Flow columns) were combined to produce the TPO sub-pool fraction (5453.79 ml; protein concentration 0.490 mg / ml; total protein 2676 mg; relative activity 12100 and total activity 32380000) and concentrated with approx By using an ultrafiltration device (Omega Ultraset, nominal molecular weight cut-off of 8000 manufactured by Filtron), the solvent of the concentrated fraction was replaced with DPBS, and then the obtained small-volume sample was processed on an ultrafiltration device with a UM-10 membrane (Amicon), eventually obtaining a sample of 120 , 2 ml of a DPBS solution containing 0.05% sodium azide. Then all 5 gels with antibodies against TPO peptides obtained in Example 46 were mixed and a single antibody column was prepared (diameter 1.6 cm and height 4.8 cm, called a column of anti-RT1 + 2 antibody mixture). The TPO fraction of the subpool was applied to this column at a flow rate of 0.033 ml / min. After the loading of the column was completed, DPBS was eluted to collect the eluates until the UV absorption was sufficiently low. The eluates collected in this way were concentrated by ultrafiltration with a UM-10 membrane (Amicon) to obtain the F1 fraction passing through the column (86.62 ml; protein concentration 14.3 mg / ml; total protein 1184 mg; relative activity 67500; total activity 79900000) . Then, the F2 fraction adsorbed by the column (92.97 ml; protein concentration 0.12 mg / ml; total protein 11.1 mg; relative activity 257000 and total activity 2860000) was eluted with an acid eluting solution (0.1 M glycine-HC1, pH 2 ,5). Although the F1 fraction passing through the column still contained TPO activity, the TPO adsorbed column with antibodies was further purified, because he found an increase in relative activity of about 20 times, i.e. Using 0.1% TFA as developing solvent A and 1-propanol containing 0.05% TFA as developing solvent B, column chromatography was performed on Capcell Pak Cl 300A (Shiseido, Cat. C1 TURPE: SG 300A; diameter 4, 6 mm and a height of 150 mm, connected to a pre-column with a diameter of 4.6 mm and a height of 35 mm) by mixing the F2 fraction obtained from the antibody column with 1/10 of the volume of developing solvent B and applying the mixture at a flow rate of 0, 4 ml / min per Capcell Pak C1 300A column, balanced in advance with 20% B. After completion of loading, the columns were electrically tion for 5 minutes, 20% B and then within 50 minutes a linear gradient from 20% of the density B to 40% B and eluates were collected into polypropylene tubes in portions of 1 ml (2.5 min).

 Portions of 2 μl (1/500 fractions) of the eluate in each tube were mixed with HSA and concentrated by ultrafiltration to obtain 0.25 ml of 1MDM test culture solution containing 0.02% HSA for use in identifying TPO-active fractions by this method. As a result, high TPO activity was detected in samples of tubes with numbers 20-23 (in the range of propanol concentrations of 28.5 and 32.5%). In addition, these samples were subjected to Western analysis according to the method described in Example 45, and the presence of TPO, which had an average mol. mass of 60,000-70000 when measured under reducing conditions based on DLS111 markers mol. masses, as well as the presence of other molecules having the corresponding mol. masses 32000-43000 and 20000-30000.

Example 48 Confirmation of the biological activity of TPO from the culture supernatant obtained by transfection of COS 1 cells with the expression vector pHTP1 and purified to the column stage of Sappel Pak C1 300A
The TPO-active fractions purified in Example 36, namely tubes 20-23 (in the range of 28.5 and 32.5% propanol concentration) of the Capcell Pak C1 300A column, were combined, mixed with 0.21 ml of glycerol and then concentrated centrifugal evaporation. The concentrate was mixed with 0.21 ml of a 6 M guanidine hydrochloride solution, diluted to a volume of 1 ml DPPS, the buffer was replaced with DPPS containing 0.01% HSA using a Sephadex G-25 column (NAP-10, Pharmacia Biotech, Cat. 17-0854- 01) and then mixed with 1.1 ml of a DPBS solution containing 0.01% HSA, resulting in a TPO-active fraction of FA (2.6 ml). An in vivo test was performed to test the biological activity of this sample. This active fraction was injected subcutaneously with 1CR male mice (8 weeks old), each group including 4 animals, daily for 5 days at a dose of 100 μl (having a total activity of 110,000, measured by a test system with M-07e). As a control, 100 μl of DPBS containing 0.01% NSA was also injected subcutaneously. Immediately prior to administration and the day after the last administration, blood was collected from the fundus to measure platelet counts using a microcytometer (F800, Toa 1yo Denshi). In the group treated with TPO, platelet numbers increased on average by about 1.42 times after completion of administration compared with the value before administration with a significant difference between the values (p <0.05, Student t-test). Based on these results, confirmation was obtained that the aforementioned human TPO produced by mammalian cells is able to increase platelet counts in vivo.

Example 49. Confirmation of the biological activity of the crude TPO fraction obtained from 33 L of culture supernatant obtained by transfection of COS 1 cells with a pHTP1 expression vector, and a partially purified cation exchange column
1) The M-07e test system was used as an in vitro test to evaluate the following purified samples. As described in Example 36, a total of 33 L of serum-free culture supernatant was obtained by transfection of COS 1 cells with a pHTP1 expression vector and filtered through a 0.22 μm filter to obtain the supernatant. It was concentrated approximately 10-fold using an ultrafiltration device (PLGC pellicon Cassette, nominal cut-off molar mass 10000 manufactured by Millipore) and mixed with 1 mM final concentration of p-APMSF protease inhibitor to obtain a 2018 ml sample by volume (protein concentration 3, 22 mg / ml; total protein 6502 mg; relative activity 66000 and total activity 429100000). Then this volume was re-concentrated using an ultrafiltration device (Omega Ultraset, molecular weight cut-off 30,000; Filtron), resulting in a fraction with mol. weighing 30,000 or more (volume 1190 ml; protein concentration 2.54 mg / ml; total protein 3020 mg; relative activity 82,500 and total activity 249,000,000) and a fraction with a mol. weighing 30,000 or less (volume 2947 ml; protein concentration 0.471 mg / ml; total protein 1402 mg; relative activity 4500 and total activity 6310000). The presence of TPO activity in the fraction with mol. weighing 30,000 or less indicates the possibility that this culture supernatant contains TPO, mol. the mass of which decreased during its expression in animal cells or during its secretion from animal cells. On the other hand, a fraction of 30,000 or more after replacing the buffer with 20 mM Na-citrate buffer (6.1) was applied at a flow rate of 5 ml / min to a SP Sepharose Fast Flow column (diameter 2.6 cm and height 29 cm) manufactured Pharmacia Biotech Cat. 17-0729-01), balanced in advance with 20 mM sodium citrate buffer (pH 6.1). After loading the sample, the column was washed and eluted with 20 mM nitrate buffer (pH 6.1). Then, using 20 mM sodium citrate buffer (pH 6.1) as developing solvent A and a solution consisting of developing solvent A and 1 M NaCl, as developing solvent B, elution was performed at a flow rate of 3 ml / min with a linear gradient from 0% B to 50% B for 215 minutes and then from 50% B to 100% B for 20 minutes. Eluates were collected in 30 ml (10 min) in polypropylene tubes. When checking portions of each of these eluates in the M-07e test system to determine the distribution of activity, TPO elution was detected over a wide range. Therefore, the fractions eluted with a NaCl concentration of 50 mM or lower, including the fraction passing through the column, were combined as the F1 fraction, and the main TPO fractions eluted with 50-1000 mM NaCl were combined as the F2 fraction. The volume of the F1 fraction was 1951 ml (protein concentration 2.05 mg / ml; total protein 3994 mg; relative activity 13500 and total activity 53900000). The volume of the F2 fraction was 649.8 ml (protein concentration 1.11 mg / ml; total protein 721 mg; relative activity 268000 and total activity 193000000).

2) Confirmation of the biological activity of the F2 SP Sepharose Fast Flow fraction
A 2.5 ml portion of the F2 SP Sepharose Fast Flow fraction separated in step (1) after exchange of the buffer with 3.5 ml of DPBS solution containing 0.01% HS A using a Sephadex G-25 column (NAP-25, Pharmacia Biotech, cat. 17-0852-01) was tested for the biological activity of TPO in an in vivo test. Those. this active fraction was injected subcutaneously with 1CR male mice (8 weeks old), each group containing 4 animals daily for 5 consecutive days at a dose of 100 μl (having a total activity of 123,000, as measured in the M-07e test system). As a control, 100 μl of DPBS containing 0.01% HSA was injected subcutaneously in the same manner. Immediately prior to administration and the day after the last administration, blood was collected from the fundus to measure platelet count using a microcytometer (F800, Toa 1 yo Denshi). In the group with the introduction of TPO, platelet numbers increased approximately 1.29 times on average after completion of administration compared with the value before administration with a significant difference (p <0.05, Student t-test) between them. These results showed that the aforementioned human TPO produced by mammalian cells is able to increase platelet count.

Example 50. Construction of a recombinant vector, pDEF202-gh TPO, for use in the expression of human TPO chromosomal DNA in CHO cells
The pDEF 202 vector was digested with restriction enzymes Kpn1 and Sp 1 and then subjected to agarose gel electrophoresis to isolate a fragment containing murine DHFR minigen and SV40 polyadenylation signal, and the pDEF 202-gh TPO expression vector was obtained by ligating with T4 DNA ligase (Takara Shuzo) thus, a DNA vector fragment that was obtained by removing the SV40 region containing polyadenylation signal from the pEFHGTE plasmid by treatment with the restriction enzymes Kpn1 and Sp1. This plasmid contains the origin (origin) of SV40 replication, the 1-α human elongation factor promoter, the SV40 early polyadenylation region for transcription of the human TPO gene, murine DHFR minigen, the beginning of pUC 18 replication and the β-lactamase gene (Amp ^r ). The human TPO chromosomal DNA is connected to the site in the direction 5'-3 'from the 1-α promoter of the human elongation factor.

Example 51. Expression of human TPO chromosomal DNA in CHO cells
CHO cells (dhfr ^- strain; Urlaub and Chasin, Proc. Natl. Acad.Sci.USA, vol. 77, p. 4216, 1980) were grown by culturing them in α-minimal stock medium (α-MEM (-) added by thymidine and hypoxanthine) containing 10% fetal bovine serum using a 6 cm diameter dish (manufactured by Falcon) and the resulting cells were transfected with a calcium phosphate method (CellPhect, Pharmacia).

That is, 10 μg of the plasmid pDEF202-gh TPO obtained in Example 50 was mixed with 120 μl of buffer A and 120 μl of H ₂ O and the resulting mixture was incubated at room temperature for 10 minutes. This solution was then mixed with 120 μl of buffer B and incubated at room temperature for another 30 minutes. The DNA solution thus obtained was placed in a dish and cultured for 6 hours in a CO ₂ incubator. After removing the medium and washing the resulting plate, α-MEM (-) was added twice with α-MEM (-) containing 10% dimethyl sulfoxide to the plate and treated for 20 minutes at room temperature. Then a non-selective medium (α-MEM (-) supplemented with hypoxanthine and thymidine) containing 10% dialyzed FCS was added and cultured for 2 days and then selection was made using selective medium (α-MEM (-) lacking hypoxanthine and thymidine) containing 10% dialyzed FCS. The selection was carried out by treating the cells with trypsin, distributing the treated cells in one cup (with a diameter of 6 cm) into 5 cups with a diameter of 10 cm or 20 cups such as 24-well plates and continuing to cultivate these cells with a change in the selective medium every 2 days. The presence of human TPO activity was confirmed in the culture supernatant in these plates or wells in which cell proliferation was observed when measuring the human TPO activity using a Ba / F3 test.

 In this case, transfection of CHO cells can also be carried out by cotransfection of CHO cells with plasmids pEFHGTE and pMG1.

Example 52. Construction of a vector for use in the expression in E. coli of a human TPO protein (amino acid residues of positions 1-163) (hereinafter referred to as "hMKT (1-163)", in which the Lus residue and Met residue were respectively added to positions -1 and -2, and confirmation of its expression
For expression of a protein whose amino acid residues at positions 1 and 3 are the same as in the human TPO protein (amino acid residues 1-163), the pCFM536 / hMKT vector (1-163), also called [Met ^-2 , Lys ^{- 1} ] TPO (1-163), for use in the expression of hMKT (1-163) in E. coli in the same manner as described in Example 42, using the following synthetic oligonucleotides 1-13, 2-13, 3-3 and 4-3 presented in table VIII.

 This expression plasmid contains the DNA sequence shown in the sequence list (SEQ 1D 12). After that, the expression of hMKT (1-163) was induced as described in Example 43.

 By processing the expressed SDS-PAGE protein obtained in this way, transferring it to a PVDF membrane and analyzing the N-terminal amino acid sequence, it was confirmed that this protein contains the hMKT amino acid sequence (1-163) that was to be expressed.

Example 53. Laying the chain of human TPO, h6T (1-163) obtained from human TPO expressed in E. coli using guanidine hydrochloride and glutathione and purification and confirmation of its biological activity
Portions of 1.2 g of frozen cells producing h6T (1-163) recombinant strain obtained in Example 43 were suspended in 3 ml of water and destroyed using a high-pressure disintegrator. By centrifuging this suspension, the precipitated fraction was recovered and most of the impurity proteins, cellular components, etc. removed. The thus obtained precipitated fraction containing h6T (1-163) was suspended in 4 ml of a final volume of water, 1 ml of 1 M Tris buffer (pH 8.5) was added to this suspension with stirring, followed by 20 ml of 8 M guanidine chloride with followed by stirring for 5 minutes at room temperature to dissolve the contents. The resulting solution was diluted 10 times with 20 mM Tris buffer (pH 8.5), 5 mM reduced type glutathione and 0.5 mM oxidized glutathione were dissolved in this diluted solution with stirring, and then the solution thus prepared was incubated overnight at 4 ^o C. By centrifugation, h6T (1-163) was recovered in supernatant fluid. A portion of 160 ml of the obtained supernatant liquid was concentrated using a UM10 ultrafiltration membrane (Amicon) and the buffer was exchanged for DPBS to obtain 3.4 ml in the final volume fraction (protein concentration 2.18 mg / ml). A careful dialysis of this fraction against 1 MDM culture medium and evaluation using a rat CFU-MK test system revealed high TPO activity (relative activity 58,000).

 The resulting TPO-active fraction was subjected to in vivo testing. This fraction was injected subcutaneously with 1CR male mice (7 weeks old), each group consisting of 4 animals daily for 5 consecutive days at a dose of 170 μl (having a total activity of 22,000, as measured in a rat CFU-MK test system) on 1 animal. As a control, 170 μl of DPBS was subcutaneously administered to each of the 6 animals of the control group in the same manner. Immediately prior to administration and the day after the last administration, blood was collected from the fundus to measure platelet counts using a microcytometer (F800 manufactured by Toa Iyo Denshi). In the group with administration of TPO, platelet counts increased on average by about 2.15 times the day after administration was completed compared with the value before administration, and this effect remained unchanged even after 3 days after completion of administration, showing a 2.13 times higher value. The platelet counts on the next day and on the 3rd day after the completion of administration were 1.73 and 1.80 times higher in the group treated with TPO than in the control group, with a significant difference between them (p <0.001, Student t- test). These results showed that human TPO produced by E. coli and obtained as described above is able to increase platelet counts in vivo.

 Example 54. Laying the human TPO chain, h6T (1-163), expressed in E. coli, using sodium N-lauroyl sarcosinate and copper sulfate and purification and confirmation of its biological activity.

 Portions of 0.6 g of frozen cells producing h6T (1-163) recombinant strain obtained in Example 43 were suspended in 3 ml of water, destroyed by a high-pressure disintegrator and then centrifuged to recover the precipitated fraction. After suspending the precipitated fraction in 3.1 ml of water, 0.19 ml of 1 M Tris buffer (pH 9.2), 11.25 μl of 1 M DTT, 38 μl of 0.5 M EDTA and 0.38 ml of 10% solution deoxycholate was added to the suspension with stirring, and the resulting mixture was stirred at room temperature for 40 minutes. The mixture was then centrifuged to recover the precipitated fraction and remove most of the impurity proteins, cellular components, and the like. The resulting precipitate was suspended in 4 ml of water and centrifuged to recover a precipitated fraction containing h6T (1-163). The precipitated fraction thus obtained was suspended in 3.8 ml of water, and 0.2 ml of 1 M Tris buffer (pH 8) and 1 ml of 10% N-lauroyl sarcosinate were added to this suspension, followed by stirring for 20 minutes at room temperature. to dissolve the contents. The resulting solution was mixed with 5 μl of 1% copper sulfate and stirred overnight (about 20 hours) at room temperature. The resulting solution was centrifuged to extract a supernatant fraction containing h6T (1-163) and a portion of 5 ml of this supernatant was mixed with 5 ml of water and 10 ml of 20 mM Tris buffer (pH 7.7) and then with 2.6 g of ion exchange resin 20 -50 mesh Dowex 1-x4 chloride form, followed by stirring for 90 minutes. This ion exchange resin was removed using a glass filter to recover a fraction of the non-adsorbed resin, which was then centrifuged to recover the supernatant liquid. It was applied to a SP Sepharose Fast Flow cation exchange column, previously equilibrated with 20 mM Tris buffer (pH 7.7), and eluted with the same buffer using a linear gradient of 0-500 mM NaC1. After carefully dialyzing each of the eluted fractions from this column against 1MDM culture medium and evaluating them in a rat CFU-MK test system, high TPO activity (relative activity of 19,000,000) was detected in the fraction eluted with a NaCl concentration of approximately 100 mM. This fraction was concentrated 1.6 times (2.5 ml; protein concentration 25 μg / ml) using an ultrafiltration device (Ultra Free CL, nominal cut-off, molar mass 5000, manufactured by Millipore, Cat. UFC4LCC25). When analyzing the concentrated fraction using SDS-PAGE in the presence of a reducing agent, the band corresponding to TPO was detected as the main band (purity 70-80%).

 The TPO active fraction obtained by the method described above was tested in an in vivo test, i.e. this active fraction was injected subcutaneously with 1CR male mice (8 weeks old), each group consisting of 4 animals, daily for 5 consecutive days at a dose of 100 μl (having a total activity of 47500, as measured in a rat CFU-MK test system ) for 1 animal. As a control, 100 μl of 20 mM Tris buffer (pH 7.7) containing 100 mM NaCl was injected subcutaneously in the same manner. Immediately prior to administration and the day after completion of administration, blood samples were taken from the fundus to measure platelet counts using a microcytometer (F800, Toa Iyo Denshi). In the group treated with TPO, platelet counts increased on average by about 1.73 times after completion of administration compared with the value before administration and were 1.59 times higher than in the control group with a significant difference (p <0.01, Student t- test) between them. These results showed that human TPO produced by E. coli and obtained as described above has the ability to increase platelet counts in vivo.

Example 55. Large-scale culture of CHO cells
A large-scale cultivation of human TPO-producing CHO cell line (CHO 28-30 cells resistant to 25 nM MTX), which was obtained by transfection of human TPO-expressing plasmid pDEF202-hTRO-P1 in CHO cells in Example 32, was carried out as follows. CHO cell line was cultured and propagated in DMEM / F-12 culture medium (G1BCO) containing 25 nm MTX and 10% FCS. After separation of the cells with trypsin solution, 1 × 10 ⁷ cells were inoculated into a roller bottle (ex Falcon, Falcon 3000) containing 200 ml of the same medium, and then cultured at 37 ^{° C.} at a rotation speed of 1 rpm for 3 days. After 3 days of cultivation, the culture supernatant was removed by suction and adherent CHO cells were then rinsed with 100 ml PBS. 200 ml of DEMEM / F-12 medium (G1BCO) containing neither 25 nM MTX nor 10% FCS was added to the vial and the cells were cultured at 37 ^{° C.} at a rotation speed of 1 rpm for 7 days. After 7 days of cultivation, the culture supernatant was recovered as starting material for the subsequent purification procedure. The procedures described above were carried out in 500 roller bottles, receiving 100 l of culture supernatant.

Example 56. Purification of human TPO from a human TPO producing CHO cell line
1) Approximately 100 L of the serum-free culture supernatant obtained in Example 55 was filtered through a 0.22 μm filter to obtain a filtrate, which was then concentrated on an ultrafiltration device (PLTK Pellicon cassette, nominal cut-off molar weight 30000, ex Millipore). After replacing the solvent of this concentrate with pure water, pAPM SF (Wako Pure Chemicals) and Pefabloc SC (Merck), which are proteinase inhibitors, were added to the resulting aqueous solution at final concentrations of 1 mM and 0.35 mM, respectively, to obtain 3628 ml of a mole fraction. masses of 30,000 or more (protein concentration of 1.60 mg / ml; total protein of 5805 mg; relative activity of 1230000 and total activity of 7149000000). The fraction was then subjected to Western blotting, as described in Example 45. As a result, the presence of TPO protein was detected at mol. weight between 66000 and 100000. In a more detailed study, it was found that lower molecular weight types of SRW than this SRW were contained in the fraction passing through the column.

 Ultrafiltrate containing SRW with mol. weighing no more than 30,000, concentrated separately using an ultrafiltration device (PLGC Pellicon cassette, nominal cut-off molar mass of 10,000, Millipore), obtaining 1901 ml of a fraction of low molecular weight TPO (protein concentration 0.36 mg / ml; total protein 684 mg; relative activity 245,500 ; total activity 167900000). This shows that during the cultivation, these low molecular weight types of SRW were formed.

 Then to 3614 ml of the fraction containing TPO with mol. weighing 30,000 or more, 764 g of ammonium sulfate and 144.5 ml of 0.5 M sodium citrate-containing buffer (pH 5.5) were added to obtain 4089 ml of a solution containing 1.41 M (final concentration) of ammonium sulfate and 17.7 mm (final concentration) containing sodium citrate buffer. After removing insoluble matter from this solution, a clear solution was obtained by centrifugation.

 This clear solution was then applied to a Masro-Prep Methyl H1C column (Bio-Rad, cat. 156-0080; diameter 5 cm, layer height 24.5 cm), previously equilibrated with 20 mM citrate buffer (pH 5.5) containing 1 , 2 M ammonium sulfate, at 25 ml / min flow rate. After loading was completed, elution was performed with 20 mM sodium citrate-containing buffer (pH 5.5) containing 1.2 M ammonium sulfate. The eluted fraction was then concentrated on an ultrafiltration device (ex Filtron, Omega Ultrasette, nominal molecular weight cut-off of 8000), to obtain the F1 fraction passing through the column (4455 ml; protein concentration 0.400 mg / ml; total protein 1780 mg; relative activity 1831000).

 Then, 20 mM Na citrate (pH 6.0) was passed through a column as an elution buffer to obtain an eluate, which was then concentrated on the same Omega Ultrasette ultrafiltration unit, nominal cut-off mol. mass 8000), collecting the F2 fraction (1457 ml; protein concentration 0.969 mg / ml; total protein 1411 mg; relative activity 1715000). In the F2 fraction, the presence of a protein having a mol. weight from 66000 to 100000, was confirmed on SDS-PAGE. Western analysis showed that this protein was TPO.

 Then F2, eluted from the Macro-PP Methyl H1C column (1443 ml), was applied to an SP Sepharose Fast Flow column (Parmacia Biotech, cat. 17-0729-01; diameter 5 cm, layer height 12 cm), previously balanced with 20 mM Na citrate buffer (pH 6.0) at a flow rate of 15 ml / min. After completion of the application, elution was performed with 20 mM Na-citrate buffer (pH 6.0) containing 50 mM NaCl. The eluate was collected and it was named the F1 fraction (3007 ml; protein concentration 0.226 mg / ml; total protein 679 mg; relative activity 88830). After that, the elution buffer was replaced with 20 mM Na-citrate buffer (pH 5.4) containing 750 mM NaCl to collect the eluted F2 fraction (931 ml; protein concentration 0.753 mg / ml; total protein 710 mg; relative activity 5558000), which then concentrated to 202 ml using an ultrafiltration device (Filtron, Omega Ultrasette, nominal cut-off mol. mass 8000; and Omicon UM 3 membrane).

 Then, the concentrated TPO activity containing F2 fraction (197 ml) was applied to a Sepacryl S -200H gel filtration column (Parmacia Biotech, cat. 17-0584-05; diameter 7.5 cm, layer height 199 cm) at a flow rate of 3 ml / min The eluted volumes of 1200-1785 ml, 1785-2010 ml, 2010-2280 ml and 2280-3000 ml were collected separately to obtain F1 fractions (585 ml; protein concentration 1.00 mg / ml; total protein 589 mg; relative activity 4118000), F2 (225 ml; protein concentration 0.263 mg / ml; total protein 59.2 mg; relative activity 2509000), F3 (270 ml; protein concentration 0.119 mg / ml; total protein 32.1 mg; relative activity 2535000) and F4 ( 720 ml; protein concentration 0.0467 mg / ml; total protein 33.6 mg; relative activity 1,155,000), respectively. Thus, fractionated TPO activity had a wide range of mol. masses as determined by gel filtration. After the SDS-PAGE fraction, followed by Western blot analysis, it was found that F1 had TPO molecules with a mol. weight 66000-100000 (mainly); F2 contained TPO molecules with mol. mass 32000-60000; and F3 contained TPO molecules with mol. weighing 32000-42000. All types of TPO molecules possessed TPO activity.

 Based on the result of sequencing of the N-terminal amino acids of the TPO molecule with a mol. Weights of 66000-100000, we confirmed that the TPO molecule had the amino acid sequence of the protein encoded by the human TPO gene.

 In addition, the TPO molecule having a mol. weight 66000-100000, was subjected to experiments with enzymatic digestion using glycosidase enzymes consisting of N-glycanase (ex Genzyme, cat. 1472-00), neuraminidase (Nakarai-Tesqu, cat. 242-29 P), endo-a- N-acetylgalactoaminididases (Seikagaku Kogyo, cat. 100453) and O-glycosidases (Boehriger Mannheim Biochemica, cat. 1347101), alone or in combination, and then SDS-PAGE assay. As a result, it was found that this polypeptide part of the SRW has a mol. mass of approximately 36,000, as expected based on its theoretical mol. mass, and is a glycoprotein with both N-linked and O-linked sugar chains.

Example 57. Purification of human TPO from producing human TPO CHO cell line
1) To 1 l of serum-free culture supernatant of CHO cells obtained as described in Example 55, 211.4 g of ammonium sulfate was added, after which the mixture was filtered through a 0.2 μm filter (ex Yelman Science, Cat. 12992). The obtained filtrate was applied to a Macro-PP Methyl H1C column (Bio-Rad, cat. 156-0081, diameter 50 mm, layer height 90 mm), previously balanced with 20 mM Na-acetate buffer (pH 5.6), containing 1.2 ammonium sulfate, at a flow rate of 15 ml / min. After application was complete, elution was performed with 450 ml of 20 mM Na-acetate buffer (pH 5.6) containing 1.2 M ammonium sulfate. Then, the eluted fraction was applied to a reverse phase Vydac C4 column (The Separation Grouh, Cat. 214BTP54, diameter 4.6 mm, height 250 mm) at a flow rate of 0.75 ml / min. After washing the column with 10 mM Tris buffer (pH 6.4) containing 5% ethanol (called developing solvent A) for 15 minutes, elution was performed for 66 minutes with a linear gradient from developing solvent A to 10 mM Tris buffer (pH 6 , 4) containing 94% ethanol (called developing solvent B). The obtained chromatogram is shown in Fig. 15. As a result of SDS-PAGE analysis, molecules having mol. mass 65000-100000, i.e. putative TPO molecules, corresponding fractions with a retention time of 68-72 minutes after addition of the sample, appeared on the SDS-PAGE gel as a single band (see Fig. 16). Subsequent Western analysis showed that the resulting protein is TPO.

 Analysis of the N-terminal amino acids of this protein sample revealed that the protein had the amino acid sequence of a protein encoded by the human TPO gene.

Example 58. Obtaining a recombinant virus for the expression of human TPO in insect cells
The plasmid pHTP1 obtained in Example 30 was digested with restriction enzymes EcoR1 and Notl and then subjected to electrophoresis on a 1% agarose gel to give a 1200 bp band, which was then purified using Prep-A-Gen DNA Purification Kit. The purified DNA was then ligated with the transport vector pVL 1393 (1nvitrogen), pretreated with the same restriction enzymes, followed by transformation into Highly competent cells of the E. coli strain (Toyo Boseki). From the obtained colonies, a clone pVL 1393 / h of TPO was selected containing the full length coding region of human TPO cDNA. Then plasmid DNA was obtained from this clone by the method described in Molecular Cloning (Sambrook et al., Cold Spring Habor Laboratory Press, 1989), followed by transfection into insect cells Sf 21 1nvitrogen) using the BaculoGold ^™ transfection kit (Farmingen). Transfected cells were cultured in Sf-900 medium (Lifetechnology) at 27 ^{° C.} for 4 days, collecting the virus-containing supernatant. The supernatant was diluted to 1:10 ⁹ -1: 10 ⁵ and approximately 7 • 10 ⁵ Sf 21 cells were infected with 1 ml of diluted supernatant at 27 ^{° C.} for 1 hour in a dish with a diameter of 35 mm. After removal of the supernatant, 1% hot agarose in an Sf-900 medium was placed in this culture and allowed to harden, after which it was cultured for 6 days at 27 ^{° C.} in a humid atmosphere. The only plaque that was formed was scraped out and the virus was isolated in 200 μl of Sf-900 medium, Sf 21 cells were infected with the obtained viral clone in a 24-well plate for virus propagation. A portion of the virus-containing fluid obtained from a single plaque was treated with a phenol / chloroform mixture, followed by ethanol precipitation to isolate viral DNA, which was then used as a template for PCR using primers specific for human TPO cDNA. Recombinant virus containing human TPO cDNA was selected based on amplification of a specific DNA fragment. Then, Sf 21 cells were infected with a supernatant containing this recombinant virus carrying human TPO cDNA to propagate the virus.

Example 59. Expression of human TPO in Sf 21 insect cells and identification of TPO activity
Sf 21 cells were cultured in 175 cm ² culture flask until 80% fusion was achieved, followed by infection with a recombinant virus carrying human TPO cDNA obtained in Example 58 at 27 ^° C for 1 hour, after which the obtained cells were cultured in Sf-900 medium at 27 ^o C for 4 days to obtain a culture supernatant. The resulting culture supernatant was subjected to medium change to 1MDM culture medium on a NAP ^TM -5 column (Pharmacia). Significant TPO activity in the obtained supernatants was found in a dose-dependent manner both in the test with rat CFU-MK and in the M-07e test. Recombinant human TPO expressed in Sf 21 cells was also identified by Western analysis described in Example 45.

Example 60. Laying of a variant human TPO chain, h6T (1-163), obtained from clone pCFM536 / h6T (1-163) carrying the human TPO base sequence, by expression in E. coli, using sodium N-lauroyl sarcosinate and sulfate copper and h6T purification (1-163)
30 g of the frozen h6T-producing (1-163) recombinant microorganism obtained in Example 43 was suspended in 300 ml of water, destroyed in a high pressure disintegrator (10,000 psi, Rannie High Pressure Laboratory) and then centrifuged to recover the precipitated fraction. This precipitate fraction was suspended in 90 ml of water and water was added with stirring to a total of 150 ml. Then, 9 ml of 1 M Tris buffer (pH 9.2), 540 μl of 1 M DTT, 1.8 ml of 0.5 M EDTA and 18 ml of 10% sodium deoxycholate were added to this mixture with stirring, followed by stirring for 30 minutes at room temperature. The precipitated fraction was recovered by centrifugation and most of the impurity proteins and components of the microorganisms were removed. 180 ml of 5 mM DDT was added to this precipitate to give a suspension, which was then centrifuged to collect a precipitate fraction containing h6T (1-163). To the resulting precipitate was added 300 ml of water, obtaining a suspension, to which water was then added until the amount of liquid 570 ml with stirring. 30 ml of 1 M Tris buffer (pH 8), 150 ml of 10% N-lauroylsarcosine were added to this mixture at room temperature with 20 min stirring to dissolve h6T (1-163). 750 μl of 1% copper sulfate was added to the solution, and the mixture was stirred overnight (approximately 20 hours) at room temperature. After removing the supernatant fraction containing h6T (1-163), 750 ml of water and 1500 ml of 20 mM Tris buffer (pH 7.7) were added to 750 ml of this supernatant by centrifugation, followed by 3 ml of Polysorbate 80 (Nikko Chemicals) with stirring . To the resulting solution was added 600 g of Dowex 1-X4 ion exchange resin (20-50 mesh, chloride form) and stirred for 90 minutes at room temperature. After removing the non-adsorbed fraction using a glass filter, the ion-exchange resin was washed with 750 ml of 20 mM Tris buffer (pH 7.7). The combined solution of the non-adsorbed and wash fractions was adjusted to pH 9.2 with 2N sodium hydroxide, after which it was applied to a Q Sepharose Fast Flow anion exchange column (1D 5 cm x 10 cm), equilibrated with 20 mM Tris buffer (pH 9.2) containing 0.1% polysorbate 80, to extract the non-adsorbed fraction. The pH of the obtained non-adsorbed fraction was adjusted to 7.2 with hydrochloric acid, applied to a Sp Sepharose Fast Flow cation exchange column (1D 5 cm x 10 cm), balanced with 20 mM Tris buffer (pH 7.2) containing 0.1% polysorbate 80, and then eluted with a linear gradient from 0 to 500 M NaCl in the same buffer. The eluted fractions were subjected to SDS-PAGE analysis to collect the TPO fraction, to which trifluoroacetic acid was then added to a concentration of 0.1%. After applying the resulting solution to a Capcell Pak 5 μm 300A Cl column (1D 2.1 cm x 5 cm x 2, Shiseido), reverse phase HPLC was performed by a linear gradient elution method with an increasing concentration of 1-propanol in 0.1% trifluoroacetic acid. The eluate was analyzed on SDS-PAGE in the absence of a reducing agent. As a result, three fractions were fractionated based on the position of the elution in reverse phase HPLC and each fraction was diluted 3 times with 0.1% trifluoroacetic acid. Each diluted fraction was applied to a reverse phase HPLC column as described. The resulting three TPO fractions were designated Fr. S-a, Fr. Sb and Fr. Sc, depending on the elution order on the reverse phase HPLC (see FIG. 17). They were analyzed for SDS-PAGE in the absence of a reducing agent. As a result, one strip was discovered at the pier. a mass of approximately 18 kDa (Fr. S-a), approximately 19 kDa (Fr. sb) or 18 kDa (Fr. S-c) (see FIG. 18). After their amino acid analysis, each of the amino acid compounds almost coincided with the corresponding value determined from the sequence information. Additionally, the results of the N-terminal amino acid analysis showed that the expected sequences were obtained. The amount of protein of each fraction, determined by amino acid analysis, was 0.64 g (Fr.S-a), 1.81 mg (Fr. Sb) and 3.49 mg (Fr. S-c). After complete dialysis of each of the fractions against 1MDM medium, the M-07e test was performed. As a result, the relative activity of TPO in each fraction was approximately 1620000 (Fr. Sa), 23500000 (Fr. Sb) and 746000000 (Fr. S-c).

Example 61. Laying the chain of variant human TPO, h6T (1-163), obtained from clone pCFM536 / / h6T carrying the base sequence of human TPO, through its expression in E. coli, with guanidine chloride and cysteine-cystine and purification of h6T (1 -163)
50 g of the h6T-producing (1-163) frozen recombinant microorganism obtained in Example 43 was added to 500 ml of water and suspended, disrupted by a high pressure device (10,000 psi, Rannie High Pressure Laboratory) and then centrifuged to recover the precipitate fraction. The precipitate fraction was suspended in water and the amount of liquid was then adjusted to 250 ml with stirring by addition of water. 15 ml of 1 M Tris buffer (pH 9.2), 900 μl of 1 M DTT, 3 ml of 0.5 M EDTA and 30 ml of 10% sodium deoxycholate were added to the suspension and stirred at room temperature for 30 minutes. After centrifugation, a precipitate fraction was recovered, leaving most of the impurity proteins, components of the microorganism, etc. in the supernatant. 300 ml of 5 mM DTT was added to the precipitate and suspended, after which the precipitated fraction containing h6T (1-163) was collected by centrifugation. The resulting precipitated fraction containing h6T (1-163) was suspended in water in a total volume of 104 ml. To the suspension was added 20 ml of 1 M Tris buffer (pH 8.5) and then 376 ml of 8 M guanidine chloride with stirring and then stirred at room temperature for 10 minutes to dissolve h6T (1-163). To the solution was added 2500 ml of 20 mM Tris buffer (pH 8.5) containing 0.1% polysorbate 80 and then 2000 ml of 20 mM Tris buffer (pH 8.5) containing 1 M guanidine chloride, with stirring, followed by addition 5 mM cysteine and 0.5 mM cystine. The solution thus obtained was incubated overnight at 4 ^{° C.} and then centrifuged to obtain h6T (1-163) in the supernatant, which was then concentrated using a Prep Scale UF cartridge PLDC (Millipore) ultrafiltration membrane. The buffer of this concentrate was replaced with 20 mM Tris buffer (pH 9.2) containing 0.1% polysorbate 80, until a final volume of 1000 ml was reached. The resulting solution was applied to a Q Sepharose Fast Flow anion exchange column (1D 5 cm x 10 cm) balanced with 20 mM Tris buffer (pH 9.2) containing 0.1% Polysorbate 80, and then eluted with a linear gradient from 0 to 500 M NaCl in the same buffer, obtaining a fraction (240 ml) eluted at approximately 20-150 mM NaCl. The resulting fraction was diluted 4 times with 20 mM Tris buffer (pH 7.2), bringing the final volume to 960 ml, and then the pH was adjusted to 7.2 with acetic acid, applied to a SP Sepharose Fast Flow cation exchange column (1D 5 cm x 10 cm) balanced with 20 mM Tris buffer (pH 7.2) containing 0.1% polysorbate 80, and then eluted with a linear gradient from 0 to 500 M NaCl in the same buffer. The eluate was analyzed on SDS-PAGE collecting the TPO fraction. Trifluoroacetic acid was added to the TPO fraction to a final concentration of 0.1%. The resulting solution was applied to a Capce11 Pak 5 μm 300A C1 column (1D 2.1 cm x 5 cm x 2, Shiseido), then reverse phase HPLC was performed using a linear gradient elution method with an increasing concentration of 1-propanol in 0.1% trifluoroacetic acid. The eluate was analyzed by SDS-PAGE in the absence of a reducing agent and fractionated into two fractions depending on the position when eluting in reverse phase HPLC. Two fractions of SRW were designated as Fr. G-and Fr. Gd based on the order of their elution in reverse phase HPLC (see FIG. 17). Each fraction was then analyzed for SDS-PAGE in the absence of a reducing agent. As a result, we found one strip at the pier. a mass of approximately 18 kDa (Fr. G-a) or approximately 32 kDa (Fr. Gd) (see Fig. 18). Further, SDS-PAGE analysis of these fractions in the presence of a reducing agent showed that a band at mol. mass of approximately 20 kDa. The result of the amino acid analysis of these fractions showed that each of the amino acid compositions almost coincided with the corresponding theoretical value determined from the sequence information. Additionally, the results of the N-terminal amino acid analysis showed that these fractions contained the expected sequences. The amount of protein of each fraction, determined from the results of amino acid analysis, was 2.56 mg (Fr. G-a) or 1.16 mg (Fr. Gd). After complete dialysis of each fraction against the 1MDM medium, an M-07e test was performed. As a result, this fraction had a relative TPO activity of approximately 3960000 (Fr. G-a) or approximately 7760000 (Fr. Gd).

Example 62. Construction of recombinant vector pDEF202-hTPO163 for the expression of human TPO partial length (amino acids 1-163) (hereinafter referred to as hTPO163) inside CHO cells
The vector pDEF202 constructed in Example 31 was digested with restriction enzymes EcoR1 and Sp1, followed by extraction of a large vector fragment using agarose gel electrophoresis. This fragment was ligated using T4 DNA ligase (Takara-Shuso) with hTPO163 cDNA, which was obtained by treating plasmid pEF18S-hTPO163 containing cTPNA ATPO163 encoding amino acids -21-163 (SEQ 1D 13), restriction enzymes EcoR1 and Spe1, obtaining expression vector pDEF202-hTPO163. This plasmid contained the origin of replication (origin) of SV 40, the 1-α promoter of the human elongation factor, the site of early polyadenylation of SV 40, murine DHFR minigen, the beginning of replication of pUC18 and the β-lactamase gene (Amp ^r ), in which hTPO163 cDNA is connected at the site in the direction of 5'-3 'from the 1-α-promoter of the human elongation factor.

Example 63. Expression of hTPO163 in CHO cells
CHO cell line (dhfr ^-..... Strain, Urlaub and Chasin, Proc Natl Acad Sci USA, 77, p 4216, 1980) was grown in α-minimum essential medium (α-MEM (-) with thymidine and hypoxanthine) containing 10% fetal calf serum in a 6 cm dish (Faleon) and then transformed with plasmid pDEF202-hTPO163 according to the transfectum method (Seikaga Ku Kogyo K.K.).

Briefly, 20 μg of the plasmid pDEF202-hTPO163 obtained in Example 62 was mixed with 240 μl of 0.3 M NaCl, then with a mixture of 20 μl of transfectum and 220 μl of water. This DNA solution was added dropwise to the plate and cultured for 6 hours in a CO ₂ incubator. The medium was removed from the plate, which was then washed twice with α-MEM (-) and 10% DMS O α-MEM (-) was added before incubation for 2 minutes at room temperature. After that, 10% dialyzed FCS-containing non-selective medium (α-MEM (-) with hypoxanthine and thymidine) was added to the plate for 2 more days, then selection was performed in 10% dialyzed FCS-containing selective medium (α-MEM (-) without hypoxanthine and thymidine). The selection was performed by trypsinization of the cells, their distribution into 5 cups of 10 cm or 20 24-well cups from 1 cup (6 cm) and cultivation was continued with medium change at 2-day intervals. Supernatants from plates or wells were tested for TPO activity using a Ba / F3 test, observing TPO activity. Cells in which TPO activity was observed in the culture supernatants were transferred to fresh plates or to fresh wells after separation to a cell concentration of 1:15 with selective medium containing 25 nM methotrexate. Cells were then recultivated to grow and clone methotrexate resistant cells.

 Alternatively, the transformation of CHO cells can be performed by cotransfection of pEF18S-hTPO163 and pMG1 into CHO cells.

 CHO strain (CHO-DUKHB11) transfected with pDEF202-hTPO163 plasmid was deposited by the author of this application on January 31, 1995 under accession FERM BP-4989 at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan.

Example 64. Large-scale culture of CHO cells
Large-scale cultivation of the hTPO163 CHO producing cell line (CHO 109 cells obtained by selection as described above in 10% dialyzed FCS selective medium [α-MEM (-) without hypoxanthine and thymidine]), which was obtained by transfection of the hTPO163 expressing plasmid pDEF202-hTPO163 in the cells in Example 63 were performed as follows. CHO 109 cells were grown in DMEM / F-12 medium (G1BCO) with 10% FCS. After cell collection (or trypsinization) using a trypsin solution, 10 million cells were inoculated into a Falcon roller bottle (FALCON 3000) containing 200 ml of the same medium and cultured at a rotation speed of 1 rpm, at 37 ^{° C.} for 3 days . The culture medium was removed by suction and the surface of the cell culture was rinsed with 100 ml of PBS. Then, 200 ml of DMEM / F-12 (G1BCO) without 10% FCS was added to the cell culture and cultured for 7 days at 37 ^{° C.} at 1 rpm. The culture supernatant was collected and used as starting material for the subsequent purification step. Similar procedures were carried out in 300 roller bottles, receiving 60 l of serum-free culture supernatant.

Example 65. Purification of KTPO163 from hTPO163 CHO-producing cell line
1) 60 l of the serum-free culture supernatant obtained in Example 64 was filtered, collecting the filtrate, which was then concentrated using an ultrafiltration device (Filtron, molecular weight cut-off 10000), obtaining a concentrated fraction (600 ml, 11.2 mg / ml, total protein 6430 mg). Using Western analysis of this fraction using antibodies against the HT1 peptide, the presence of the hTPO163 protein expressed in the range of mol. masses from 20,000 to 26,000.

 The obtained concentrated culture supernatant (537 ml) was treated on a Sephadex G-25 Fine column (Pharmacia Biotech, Cat. 17-0032-02; diameter 10 cm and height 30 cm), pre-equilibrated with 10 mM Na-phosphate buffer (pH 6.8) obtaining the F1 protein fraction (938 ml, protein concentration 4.9 mg / ml, total protein 4594 mg) as a solution in 10 mM Na-phosphate buffer (pH 6.8).

 This protein fraction (929 ml) from a Sephadex G-25 Fine column was applied at a flow rate of 15 ml / min to an SP Sepharose Fast Flow column (Pharmacia-Biotech, Cat. 17-0729-01; diameter 5 cm and height 12 cm), pre-equilibrated with 10 mM Na-phosphate buffer (pH 6.8), followed by elution with 10 mm Na-phosphate buffer (pH 6.8), then with the same buffer containing 10% ethanol. The eluates were combined as an F1 fraction (1608 ml, protein concentration 2.13 mg / ml, total protein 3426 mg). Then, a second elution was performed with 10 mM Na-phosphate buffer (pH 6.8) containing 750 mM NaCl and 25% ethanol, collecting the main eluate of hTPO163 fraction F2 (651 ml, protein concentration 1.67 mg / ml, total protein 1087 mg) .

 Ethanol and purified water were added to the TPO-containing activity of the F2 fraction (200 ml) from the Sepharose Fast Flow SP column to obtain 300 ml of a 45% (final conc.) Ethanol solution. Insoluble substances formed by the addition of ethanol were removed by centrifugation. The supernatant was introduced into a SOUR CE 15RPC column (Pharmacia-Biotech, Cat. 17-0727-02; diameter 2 cm, height 20 cm), pre-equilibrated with 50% solvent A (10 mM Na-acetate buffer, pH 6.7) plus 50 % solvent B (10 mM Na-acetate buffer, pH 6.7, containing 90% ethanol) at a flow rate of 2 ml / min. The column was then washed with 55% solvent A plus 45% solvent B until the non-adsorbed substances were completely removed. After that, elution was performed at a flow rate of 1.5 ml / min in accordance with the following elution profile; 50% B for 5 minutes; a linear gradient of 50% B to 100% B for 140 minutes and then 100% B for 35 minutes. Fractions were collected at intervals of 5 minutes (corresponding to 7.5 ml of volume). All fractions were subjected to SDS-PAGE and then Western analysis to test the elution range of hTPO163. As a result, it was found that highly purified hTPO163 with an average mol. weighing approximately 20,000 to 26,000 elutes in the range of 66-87% ethanol. Among these proteins hTPO 163, hTPO163 protein with mol. of a higher mass was previously eluted from the reverse phase column, suggesting that the glycosylated hTPO163 molecule has increased hydrophilicity.

 To 88.8 ml of the eluted hTPO163 fraction (i.e., the hTPO163 fraction (90 ml) eluted at a concentration range of 68-86.5% ethanol) from a SOUR CE 15RPC column, CHAPS was added, and this mixture was concentrated and washed to obtain 2, 5 ml of a concentrate containing approximately 5% ethanol and 4 mM CHAPS. This concentrate was then applied to a Superdex 75 pg column (Pharmacia-Biotech, Cat. 17-1070-01; diameter 2.6 cm and height 60 cm), balanced in advance with 10 mM Na-phosphate buffer (pH 6.8) containing 10 % ethanol, at a flow rate of 1.5 ml / min. Starting from the time 60 minutes after application, the eluted fractions were collected 6 ml each (i.e. every 4 minutes). As a result, hTPO163 was eluted in fractions with tube numbers 16-31, as determined by SDS-PAGE. This elution position corresponds to a molecular weight range of from about 44,000 to about 6,000, as determined by gel chromatography using standard molar markers. masses (mixture of Bio-Rad, Gel Filtration Standard, cat. 151-1901; and Calbiochem insulin, cat. 407696). Additionally, these hTPO163 molecules appear to be eluted in order to decrease the degree of glycosylation. All types of hTPO163 tubes 16-31 (elution volume 180-276) were collected as fraction FA. In addition, the fractions of tubes with numbers 16-18 (eluting volume 180-198 ml), 19-24 (eluting volume 198-234 ml) and 25-31 (eluting volume 234-276 ml) were collected separately and designated as fractions FH, FM and FL, respectively. The FA fraction was also obtained by combining portions of the fractions FH, FM and FL. This is shown in FIG.

 2) Then the fractions of hTPO163 FH, FM, FL and FA from the Superdex 75 pg column described in (1) will be illustrated in more detail, the N-terminal amino acid sequences of the TPO163 types obtained above were determined by the same method as described in Example 1, but most of the N-terminal residues of Ser could not be identified. This suggests that O-linked sugar is added to the N-terminal Ser. Further, it was confirmed that the sequence after the N-terminal Ser is the amino acid sequence expected based on the sequence of the hTPO gene. The concentrations of hTPO163 proteins in FH, FM, FL and FA were 10.2 ng / ml, 6.2 ng / ml, 0.84 ng / ml and 3.2 ng / ml, as determined by amino acid analysis (Acq. TAQ method, Wasters), respectively, provided that these concentrations were concentrations of the peptide moieties of the sugar chain-free molecules. These fractions (100 ng each) were subjected to SDS-PAGE under non-reducing conditions using a 15/25 multigel (Dai-ichi Kagaku Jakuhin, 15-25% preformed polyacrylamide gel) or under reducing conditions using DTT followed by silver staining (Daiichi Pure Chemicals). As a result, it was found that each of these fractions contains highly purified hTPO163. The average mol. masses of hTPO163 in FН, FM, FL and FA, as calculated using DPC111 markers mol. the masses (Daiichi Pure Chemicals) as a standard under reducing conditions were 24000-21500, 23000-21000, 23000-20500 and 23500-20500, respectively (see FIG. 20).

 In addition, the average mol. hTRO masses in FH, FM, FL and FA calculated using the biotin-labeled standard for SDS-PAGE (Bio-Rad, Biotynylated SDS-PAGE Standards, Broad Range: Cat. 161-0319) under reducing conditions in Western analysis, were 26000-22000, 25500-22000, 26000-21000 and 26000-21000, respectively. Heterogeneity among the pier. masses of FH, FM, FL and FA may be due to the heterogeneity of O-linked sugar chains. Therefore, each fraction of FH, FM, FL, and FA was digested with neuraminidase (Neuraminidase, Nacala tesque, Cat. 242-29 P), DTT was reduced, and then analyzed for SDS-PAGE. As a result, in all fractions the average mol. the mass was about 19,000, indicating that the heterogeneity of the mol. The mass of hTPO is mainly due to the heterogeneity of the amount of sialic acid in the sugar chains associated with the hTPO163 protein, and that the hTPO163 thus obtained is expressed as a glycoprotein in CHO cells.

 3) Fractions FN, FM, FL and FA obtained in (2) were analyzed for their in vitro activity using the M-07e test system. As a result, the relative specific activities were 511000000, 775000000, 1150000000 and 715000000 / mg of hTPO163 protein per weight of the peptide part that does not contain sugar mass).

Example 66. Construction of E. coli vector for the expression of human TPO (amino acids 1-332), which added Lys at position -1 and Met at position -2, respectively (hereinafter referred to as hMKT (1-332), and expression of hMKT (l -332)
To express the full-length amino acid sequence of human TPO in E. coli, codons from amino acid 164 to amino acid 332 have been replaced with codons preferred for E. coli, as described below.

Synthetic oligonucleotides: 21 and 22; 23 and 24; 25 and 26; 27 and 28; 29 and 30; 31 and 32; 33 and 34; 35 and 36; 37 and 38, or 39 and 40, were phosphorylated in a solution of 0.1 mM ATP, 10 mM Tris acetate, 10 mM Mg acetate, 50 mM K acetate using T4 kinase (Pharmacia) in the same tube. Then, 1/10 volumes of a solution of 100 mM Tris-HCl (pH 7.5), 100 mM MgCl ₂ , 500 mM NaCl were added to the reaction mixture, boiled for 3 minutes in a water bath and allowed to cool, obtaining double-stranded DNA. 4 sets of double-stranded DNA, i.e. oligonucleotides 21 and 22/23 and 24 (combination-A); oligonucleotides 25 and 26/27 and 28 (combination-B); oligonucleotides 31 and 32/33 and 34 (combination-C) and oligonucleotides 35 and 36/37 and 38/39 and 40 (combination-D) were separately ligated using a DNA ligation kit (Takara-Shuso) and then combination-A was ligated together with combination-B and also combination-2 with combination-D, obtaining respectively ligation-1 and ligation-2. Using ligation products 1 and 2, PCR was performed as matrices, in which oligonucleotides 41 and 42 for ligation product 1 and oligonucleotides -43 and -44 for ligation product 2 were used as primers. PCR products 1 and 2 were digested with Sac1 and EcoRV and EcoRV and Hind 111, respectively, followed by electrophoresis on a 2% agarose gel and purification with the help of a DNA purification kit Rp-A-Gene, obtaining fragments of approximately 240 bp and 250 bp, respectively. The two fragments thus obtained were subcloned into pBluescript 11 Sk + (Stratagene), previously cleaved with Sac1 and Hind 111 (E. coli DN5 was used as the host). From the obtained colonies, a clone was selected having the base sequence shown in table. 6, by sequencing, and was named pBL (SH) (174-332) (see SEQ 1D 154).

 Next, pBL (XH) h 6T (1-163) obtained in Example 42 was subjected to PCR as a matrix in the presence of the following synthetic oligonucleotides 45 and 46 as primers, and the PCR product was decoupled by BamH1 and Sac1 followed by electrophoresis on a 6% polyacrylamide gel receiving a fragment of approximately 160 bp from the gel.

45: 5'-AAGGATCCGAACGAASTGSTATSTTSSTG-3 '(SEQ 1D 155)
46: 5'-GGGAGGSTSGTTSAGGGGTSAGAASSAGA
GAGGTACGAGACGGAACAGCAGTGGTTGG-3 '(SEQ 1D 156)
Also, pBL (SH) (174-332) was digested with Sac1 and Hind 111, and the cleavage product was purified using a Ppper-A-Gene DNA purification kit to obtain a fragment of approximately 480 bp. The resulting two fragments were subcloned into pBluescript 11SK + (Stratagene), pre-digested with BamH1 and Hind 111 (E. coli, DH5 was used as the host).

 Of the obtained clones, a clone was selected by sequencing having the base sequence shown in Table 1. 7 and named pBL (BH) (123-332) (see SEQ 1D 157).

 The pBL (XH) h6T (l-163) obtained in Example 42 was digested with BamH1 and Hind 111 and obtained as described above, pBL (BH) (123-332) was also decoupled to give a fragment of approximately 640 bp after whereby these two fragments are ligated together to give the clone pBL (XH) h6T (1-334). Next, pCFM536 / h MKT (1-163) obtained in Example 52 was digested with Xba1 and Sfi1 to obtain a fragment of approximately 270 bp which was then ligated with pBL (XH) -h6T (1-334) cleaved with the same restriction enzymes to give a pBL (XH) hMKT clone (1-334). After cleavage of pBL (CH) hMKT (1-334) Xba1 and Hind 111, a fragment of approximately 1040 bp was extracted and purified that encodes a human TPO of the mutated type (amino acids 1-332). It was cloned into pCFM536 (EP-A-136490), previously decoupled by Xba1 and Hind 111 (E. coli jM109, previously transformed by pMW1 (ATCC 39933), used as the host). The resulting clone was named pCFM536 / hMKT (1-332) and the E. coli strain carrying this expression vector was used as a transformant for expression of the mutated type hMKT (1-332) protein. This expression plasmid pCFM536 / h MKT (1-332) contains the DNA sequence shown in SEQ 1D 14.

This transformant was cultured in 60 ml of LB medium containing 50 μg / ml ampicillin and 12.5 μg / ml tetracycline overnight at 30 ^{° C.} and this culture (25 ml) was then added to 1000 ml of LB medium containing 50 μg / ml of ampicillin, and the culture was pumped at 36 ^° C until the OD ₆₀₀ reached 1.0-1.2. Then, after adding to the culture approximately 330 ml of LB medium at 65 ^° C, so that the final temperature of the culture became 42 ^° C, the culture was pumped for another 3 hours at 42 ^° C to induce the expression of the variant human TPO, hMKT protein (1-332) also called [Met ^-2 , Lys ^-1 ] TPO (1-332).

 The resulting culture was immediately subjected to SDS-PAGE analysis. It used Multigel 15/25 Dai-ichi Chemical Co.). After electrophoresis and Coomassie blue staining, a protein specific for the induction of expression was detected at mol. a mass of approximately 35 kDa on the gel in relation to the transformant. This specific protein confirmed the expression of the hMKT protein (1-332). In addition, after SDS-PAGE electrophoresis and electroblotting (using an introcellulose membrane), the expressed protein reacted with antibodies against the HT1 peptide obtained in Example 45. After staining, a band was detected at mol. weight of approximately 35 kD, indicating that hMKT was expressed (1-332).

Example 67. Obtaining substituted derivatives of human TPO, their expression in COS 7 cells and identification of their activity
In accordance with the following procedures, it was investigated whether TPO activity had some derivatives in which the amino acids of human TPO were partially replaced by other amino acids.

 The pSMT201 vector for expression in animal cells, for use in this Example, was constructed as follows.

 First, the expression plasmid pXM-mEPORn containing the mouse erythropoietin receptor cDNA (obtained from Dr. D'Andrea at the Dana Farbor Cancer Institute; Cell: 57, 277-285 (1989)) was digested with restriction enzymes Kpn1 and EcoR1 and subjected to agarose gel electrophoresis to obtain rXM vector fragment in which the mouse erythropoietin receptor cDNA was removed. Then, two oligonucleotides were synthesized using DNA synthesizer (AB1) to introduce different restriction sites into the expression vector. The synthesized oligonucleotides have the following base sequences, presented in table IX.

These two oligonucleotides were mixed and annealed to obtain a double-stranded oligonucleotide that was ligated with the pXM vector obtained as described above in the presence of T4 DNA ligase (Takara-Shuso) to obtain the pDMT201 expression vector. To remove mouse DHFR to the DNA contained in pDMT201 from pDMT201, the pDMT201 and pEF18S vectors were separately digested with Not 1 and Hpa1, followed by agarose gel electrophoresis, to obtain a large fragment from the pDMT201 vector DNA and a smaller fragment from the pEF18S vector DNA. These fragments were then ligated together using T4 DNA ligase (Takara-Shuso) to obtain the pDMT201 expression vector. This vector, as shown in FIG. 21, has an SV40 origin (start) of replication, an enhancer sequence, a three-part adenovirus main late promoter sequence, an adenovirus leader sequence, a splicing signal sequence, an SV40 early polyadenylation site sequence, an adenovirus VA RNA gene sequence , pUC18 replication start and β-lactamase gene (Amp ^r ), together with the following restriction sites for ligation of the desired gene: Bgl 11, Pst1, Kpn 1, Xho1, EcoR1, Sma1, Spe1 and Not1. The pHTP illustrated in Example 30, carrying the full length human TPO cDNA, was digested with EcoR1 and Sp1 to obtain a full length human TPO cDNA fragment, which was then subcloned into the pSMT201 vector, also cleaved in advance to obtain the plasmid pSMT201-hTPO.

 Using the plasmid pSMT201-hTPO thus obtained as a matrix, the βGL-TPO / pBLue plasmid was first constructed, which was used as a matrix to obtain the target derivatives, as follows.

 In βGL-TPO / pBLue, an attempt was made to add the leader sequence of rabbit β-globin to the 5'-untranslated hTPO site by the method of Annweiler (Annweiler et al., Nucleic Acids Research, 19: 3750, 1991). For this purpose, two DNA strands are chemically synthesized as shown in Table X.

 SEQ 1D 160 and 161 were ligated with a fragment that was obtained by digesting the Bluescript 11SK + (Toyo-boseki) EcoR1 and Sma1 vector, yielding the βGL / pBLue vector into which the leader sequence was inserted.

PCR was performed using the following primer sequences:
Sma-ATG: 5'-GGCCCGGGATGGAGGSTGASTGAATTGSTC-3 '(SEQ 1D 162) (i.e., 102-122 of the SEQ 1D 7 sequence to which the Sma1 sequence is connected); and (end): 5'-TCAAGSTSTASTGTSSSTTSSTGAGASAGATTSTG-3 ^' (SEQ 1D 163) (i.e., the antisense sequence 1140-1160 of SEQ 1D 7 to which the Sre1 and Hind 111 sequences were connected).

PCR was performed using 1 ng DNA of plasmid pSMT201-hTPO as a template with 10 μM of these synthetic primers. Using the Takara amplification PCR kit (Takara-Shuzo) and the Programmable Thermal Controller (MJ Research), PCR was performed in a volume of 100 μl under the following conditions: 3 cycles of 94 ^° C for 1 minute, 55 ^° C for 2 minutes and 72 ^° C for 2 minutes ( cycle); then 20 cycles: 94 ^{° C. for} 45 s, 55 ^{° C. for} 1 minute and 72 ^{° C. for} 1 minute (cycle). The PCR product was extracted with chloroform and then precipitated with ethanol twice, followed by suspension in 100 μl of TE buffer.

The PCR product was then digested with restriction enzymes Sma1 and Hind 111, extracted with phenol / chloroform and precipitated with ethanol. The resulting pellet was dissolved in 10 μl of TE buffer, after which it was subcloned into the βGL / pBlue vector, previously digested with the same restriction enzymes (Competent E. coli strain JM109 (Toyo-boseki) was used as the host). From the obtained transformed cells, 20 clones were selected, from which plasmid DNA was obtained, mainly as described in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989). Purified plasmid DNA was identified by sequencing using a Taq Due Deoxy ^TM Terminater Cycle sequencing kit (Applied Biosystems, Inc.) and a 373A DNA sequencer (Applied Biosystems, Inc.). As a result, it was confirmed that the plasmid encoding βGL-TPO / pBLue had the expected TPO cDNA sequence without any substitutions along its entire length. Then, after cleavage of βGL-TPO / pBLue Bg111 and Spe1, the cleavage product was extracted with a phenol / chloroform mixture and precipitated with ethanol. The resulting pellet was dissolved in 10 μl of TE buffer and subcloned into pSMT201 vector, which was digested with the same enzymes (Competent-High E. coli JM109 (Toyo-boseki) was used as the host). In accordance with the method described in Molecular Cloning (Sambrook et al., Supra), plasmid DNA was obtained from transformant cells. The resulting pSMT / βGL-TPO expression vector had a structure in which the human β-globin leader sequence and human TPO cDNA were connected at the Bg111 / Spe1 restriction site in the 5′-3 ′ direction from the signal splicing sequence of the pSMT201 vector, as shown in FIG. 21. This pSMT / βGL-TPO vector was used to transfect into C0S 7 cells.

To obtain substituted human TPO derivatives, PCR was used in which the Ito method was used (Ito et al., Gene, 102: 67-70, 1991). In particular, two human TPO derivatives were obtained in which Arg-25 and His-33 human TPO were replaced by ASn and Thr, respectively. These derivatives can be called, respectively, [ASn ²⁵ ] TPO and [Thr ³³ ] TPO. The following primers were used in PCR:
T7: 5'-TAATASGASTASASTATGGGGCG-3 '(SEQ 1D 164) (corresponding to the T7 region of the BLuescript 11 S K + promoter); ΔBgl11: 5'-AATTSSAAGATSACASASTASTGC-3 '(SEQ 1D 165) (to replace the recognition sequence of Bgl 11); (end): 5'-TCAAGSTSTASTGTSSSTTSSTGAGASAGATTSTG-3 '(SEQ 1D 166) (antisense sequence 1140-1160 SEQ 1D 7, to which the sequences Sl and Hind 111 are connected); 3: 5'-TGGGACASTGGSTCAGGTTGSTGTGAAGGACATGGGG-3 '(SEQ 1D 167) (Arg 25 -> Asn SEQ 1D 7); and 09: 5'-TGTAGGCAAAGGGGGTAAASSTSTGGGCA-3 '(SEQ 1D 168) (His - 33 -> Thr SEQ 1D 7).

The first stage of PCR was performed using 1 ng of plasmid DNA βGL-TPO / pBLue as a template together with 10 μg of each of the synthetic primers. The combinations of primers were as follows: [1] Δ Bgl11 and end primers; [2] T7 and N3; and [3] T7 and 09. Using a PCR kit, amplification (Takara Shuso) and Programmable Temperature Controller (My Research), PCR was performed in a volume of 100 μl under the following conditions: 3 cycles 94 ^° C for 1 minute, 55 ^° C for 2 minutes and 72 ^{° C. for} 2 minutes (1 cycle); then 17 cycles of 94 ^{° C. for} 45 s, 55 ^{° C. for} 1 minute and 72 ^{° C. for} 2 minutes (1 cycle). Each of the PCR products was extracted with chloroform and precipitated twice with ethanol and the precipitate was dissolved in 100 μl of TE buffer. The resulting PCR products (1 μl each) were then subjected to a second PCR. Matrix combinations were as follows: PCR products [1] / [2] and PCR products [1] / [3]. In both cases, a combination of T7 and "end" were used as primers. After incubation at 94 ^° C for 10 minutes and the addition of Takara Taq, PCR was carried out under the following conditions: 3 cycles of 94 ^° C for 1 min, 55 ^° C for 2 minutes and 72 ^° C for 2 minutes (cycle); then 9 cycles of 94 ^{° C. for} 45 s, 55 ^{° C. for} 1 minute and 72 ^{° C. for} 1 minute (cycle). To each of the obtained PCR products were added 1 μl of proteinase K (5 mg / ml), 2 μl of 0.5 M EDTA and 2 μl of 20% SDS, incubated at 37 ^° C for 30 minutes to inactivate Taq, and extracted with phenol / chloroform and precipitated with ethanol. After this, the precipitate was dissolved in 20 μl of sterile water and digested with Bgl11 and Sp1, followed by extraction with a phenol / chloroform mixture and precipitation with ethanol. The pellet thus obtained was dissolved in 10 μl of TE buffer and then subcloned into the pSMT201 expression vector, previously digested with the same restriction enzymes, and treated with calf intestinal alkaline phosphatase (Boehringer-Mannheim) (Competent-High E. coli JM109 was used as the host). In each case, two clones were selected from the obtained transformed cells, from which plasmid DNAs were obtained mainly as described in Molecular Cloning (Sambrook et al., Supra). Purified plasmid DNA was sequenced using the 373A DNA sequencer and Taq sequencing kit Dye Deoxy ^TM Terminate Cycle (two Applied Biosystems, Inc.).

The plasmid obtained by PCR using these primers [1] and [3] contained cDNA encoding a substituted TPO derivative (09 / TPO). Replacing the amino acid His 33 (CAC) with Thr (ACC) and lengthening the initial C-terminus (Gly 332) by adding the amino acid sequence TSIGYPYDVPDYAGVHHHHHH (SEQ 1D 169) were confirmed. This derivative is called [Thr ³³ , Thr ³³³ , Ser ³³⁴ , Ile ³³⁵ , Gly ³³⁶ , Tyr ³³⁷ , Pro ³³⁸ , Tyr ³³⁹ , Asp ³⁴⁰ , Val ³⁴¹ , Pro ³⁴² , Asp ³⁴³ , Tyr ³⁴⁴ , Ala ³⁴⁵ , Gly ³⁴⁶ , Val ³⁴⁷ , His ³⁴⁸ , His ³⁴⁹ , His ³⁵⁰ , His ³⁵¹ , His ³⁵² , His ³⁵³ ] TPO.

On the other hand, a plasmid obtained by PCR using primers [1] and [2] contained cDNA encoding a substituted TPO derivative (N3 / TPO). The replacement of the amino acids Arg 25 (AGA) with Asn (AAC) and Glu 231 (GAA) with Lys (AAA) and the extension of the initial C-terminus (Gly 332) by the addition of the amino acid sequence TS1GУРУDVРD YАGVННННННН were confirmed. This derivative is named [Asn ²⁵ , Lys ²³¹ , Thr ³³³ , Ser ³³⁴ , Ile ³³⁵ , Gly ³³⁶ , Tyr ³³⁷ , Pro ³³⁸ , Tyr ³³⁹ , Asp ³⁴⁰ , Val ³⁴¹ , Pro ³⁴² , Asp ³⁴³ , Tyr ³⁴⁴ , Ala ³⁴⁵ , Gly ³⁴⁶ , Val ³⁴⁷ , His ³⁴⁸ , His ³⁴⁹ , His ³⁵⁰ , His ³⁵¹ , His ³⁵² , His ³⁵³ ] TPO.

 Transfection into COS 7 cells of each obtained clone was carried out by the DEAE-dextran method, which was treated with chloroquine as described in Example 35. Briefly, 40 μg of plasmid DNA was used in transfection and after 5 days the culture supernatant was recovered, which was then concentrated to a volume of 1/20 using Centricon-30 (Amicon) to evaluate their TPO activity using the M-07e test. As a result, in the culture supernatants of COS 7 cells, into which plasmids containing cDNA encoding human TPO derivatives N3 / TPO and 09 / TPO were respectively transfected, dose-dependent TPO activity was detected (see FIG. 22).

Example 68. Obtaining insertion or deletion derivatives of human TPO, their expression in COS 7 cells and identification of TPO activity
This Example shows whether TPO activity has the insertion and deletion derivatives of hTPO163.

The following derivatives were prepared: His 33-deletion derivative ([ΔHis ³³ ] TPO (1-163), dH33); Gly116-deletion derivative ([ΔGly ¹¹⁶ ] TPO (1-163), dG 116); Arg 117 is a deletion derivative ([ΔArg ¹¹⁷ ] TPO (1-163), dR 116); Thr insertion derivative ([His ³³ , Thr ³³ , Pro ³⁴ ] TPO (1-163), T33 '); Ala insertion derivative ([His ³³ , Ala ^{33 '} , Pro ³⁴ ] TPO (1-163), A33') and Gly insertion derivative ([His ³³ , Gly ^{33 '} , Pro ³⁴ , Ser ³⁸ ] TPO (1- 163), G33 '), in which Thr, Ala and Gly were independently integrated between His 33 and Pro 34; and an Asn insertion derivative ([Gly ¹¹⁶ , Asn ¹¹⁶ ', Arg ¹¹⁷ ] TPO (1-163), N116'). Ala-insertion derivative ([Gly ¹¹⁶ , Ala ^{116 '} , Arg ¹¹⁷ ] TPO (1-163), A116') and Gly-insertion derivative ([Gly ¹¹⁶ , Ala ^{116 '} , Arg ¹¹⁷ ] TPO (1-163, G116 '), in which Asn, Ala and Gly were independently embedded between Gly 116 and Arg 117, all sequences are based on SEQ 1D 13.

 Among these derivatives, T33 ′ and N116 are intended for the introduction of mucin type chains and sugar chains of the Asn link chain type, respectively.

 The above derivatives were obtained using PCR according to the method described by Jto et al. (Jene, 102: 67-70, 1991).

The following primers were used in PCR:
dH33: 5'-TGTAGGCAAAGGGAASSTSTGGGCA-3 '(SEQ 1D 172) (to obtain His 33-deletion derivative based on SEQ 1D 7);
T33 ': 5'-TGTAGGCAAAGGAGTGTGAASSTSTGG-3' (SEQ 1D 173) (to obtain a Thr insertion derivative with Thr embedded between His 33 and Pro 34 in SEQ 1D 7);
A33 ': 5'-TGTAGGCAAAGGGAGGGGTAASSTSTGG-3' (SЕQ 1D 174) (to obtain an Ala-insertion derivative with Ala embedded between His 33 and Pro 34 in SEQ 1D 7);
G33 ': 5'-TGTAGGCAAAGGTSSGTGAAASSTSTGG-3' (SEQ 1D 175) (to obtain a Gly insertion derivative with Gly embedded between His 33 and Pro 34 in SEQ 1D 7);
dG116: 5'-AGSTGTGGTSSTSTGTGGAGGGAAG-3 '(SEQ 1D 176) (to obtain a Gly116-deletion derivative based on the sequence shown in SEQ 1D 7);
dR117: 5'-GTGAGGSTGTGGTGCCCTGTGGAGGG-3 '(SEQ 1D 177) (to obtain an Arg 117-deletion derivative based on the sequence shown in SEQ 1D 7);
N116: 5'-AGSTGTGGTSSTGTTGSSSTGTGGAGGG-3 '(SEQ 1D 178) (to obtain an Asn insertion derivative with Asn embedded between Gly116 and Arg117 in SEQ 1D 7);
A116 ': 5'-AGSTGTGGTSSTAGGСGSSSTGTGGAGGG-3' (SEQ 1D 179) (to obtain an Ala insertion derivative with Ala embedded between Gly116 and Arg 117 in SEQ 1D 7);
G116 ': 5'-AGSTGTGGTSSTTSSGSSSSTGTGGAGGG-3' (SEQ 1D 180) (to obtain a Gly insertion derivative with Gly embedded between Gly116 and Arg117 in SEQ 1D 7);
dR1: 5'-CGGGSTGCAGGATATSSAAGATSTSA-3 (SEQ 1D 181) (to replace EcoR1 restriction enzyme recognition sequence);
T7: 5'-TAATASGASTASASTATGGGGСG-3 ^' (SEQ 1D 182) (corresponding to the T7 region of the Bluescript 11S K + promoter); and end Not: 5'-GGGCGGSSGSTCAGSTGGGGACACGSTGTGGTGGG-3 '(SEQ 1D 183) (antisense nucleotide sequence 633-653 of SEQ 1D 7, to which the termination codon and Not1 recognition sequence are added).

The first stage of PCR was performed using 1 ng DNA of plasmid βGL-TPO / pBlue obtained in Example 67 and 10 μM synthetic primers as a template. The combination of primers was: [1] dR1 and end Not1 or [2] T7 and mutated primers (dH33, A33 ', G33', T33 ', dG116, dR117, A116', G116 'and N116'). PCR amplification kit (Takara Shugo) was used, and Programmable Temperature Controller (MJ Rescarch) and PCR were performed in a final volume of 100 μl. In the first PCR of reaction [1], one cycle was 94 ^° C for 1 min, 45 ^° C for 2 minutes and 72 ^° C for 2 minutes and 3 cycles were carried out followed by 17 cycles in which each cycle was 94 ^° C for 45 s, 45 ^° C 1 min and 72 ^o C 1 min. On the other hand, the first reaction [2] was carried out under the conditions: 94 ^{° C. for} 1 minute, 55 ^{° C. for} 2 minutes and 72 ^{° C. for} 2 minutes. For 3 cycles, followed by 17 cycles, each of which consisted of 94 ^{° C. for} 45 s, 55 ^{° C. for} 1 minute and 72 ^{° C. for} 1 minute. Each of the PCR products was extracted with chloroform before precipitation twice with ethanol, and then the resulting precipitate was dissolved in 100 μl of TE buffer.

Then, the second stage of PCR was performed using 1 μl of each of the PCR products [1] and [2]. Used a combination of T7 and end Not1 as primers. After incubation at 94 ^{° C.} for 10 minutes, Takara Taq was added to each PC reaction mixture. The second PCR was carried out under the conditions: 94 ^° C for 1 minute, 55 ^° C for 2 minutes and 72 ^° C for 2 minutes for 3 cycles, followed by 94 ^° C for 45 s, 55 ^° C for 1 minute and 72 ^° C for 1 minute for 9 cycles. To each of the PCR products thus obtained, 1 μl of 5 mg / ml proteinase K, 2 μl of 0.5 M EDTA and 2 μl of 20% SDS were added and the mixture was kept at 37 ^{° C.} for 30 minutes to inactivate Taq. The PCR product was extracted with phenol / chloroform, followed by ethanol precipitation, and the resulting precipitate was dissolved in 20 μl of sterile water. After digestion with EcoR1 and Not1, the solution was extracted with phenol / chloroform and then precipitated with ethanol. The resulting pellet was dissolved in 10 μl of TE buffer and subcloned into the pEF18S expression vector, previously digested with the same restriction enzymes, and treated with alkaline phosphatase (from calf intestine, Boehringer-Mannheim). The host cells were Competent-High E.coli M109 (Toyo-Boseki). Plasmid DNA was obtained from the obtained transformant according to the method described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press (1989)). Thereafter, the nucleotide sequence of the purified plasmid DNA was confirmed by Taq Dye Deoxy ^TM Terminater Cycle kit sequenced (Applied Biosystems) and DNA sequencer 373A from the same company. As a result, it was confirmed that all these plasmids encoding dH33, A33 ', T33', dG116, dR117, A116 ', G116' and N116 'had the expected TPO cDNA sequence without any nucleotide sequence substitutions at sites other than those intended for replacement sites. Moreover, in G33, a base substitution [Pr38 (CCT) -> Ser (TCT)] was observed other than a substitution at the intended site.

 Transfection of each clone obtained in COS 7 cells was carried out according to the method in Example 11. Briefly, transfection was performed using 10 μg of each plasmid DNA with a DEAE-dextran method involving chloroquine treatment, and after 4-5 days the culture supernatant was recovered, which was evaluated using M -07e-test systems. As a result, dose-dependent TPO activity was detected in each supernatant of COS 7 cell cultures transfected with plasmids encoding amino acid insertion or deletion derivatives (see FIG. 23). In contrast, TPO activity was not detected in the supernatant of the COS 7 cell culture, the expression plasmid of which pEF18S, transfected therein and expressed, did not contain cDNA encoding the TPO derivative.

Example 69. Obtaining and purification of antibodies against human TPO peptide
1) Six regions (shown in Table 8) that are considered relatively suitable as antigens were selected from the amino acid sequence of human TPO SEQ 1D 191, from which a 4-chain peptide of the type of peptide with multiple antigens (MAP) was synthesized by the Tam method (Proc. Natl. Acad. Sci. USA, 85, 5409-5413, 1988). Two rabbits were immunized 8 times with 100 μg of this peptide.

 Single chain peptides were obtained separately by linking the cysteine residue to the C-terminus of each of the peptide regions shown by SEQ 1D 119-124 (called peptide regions of HT 1-6, respectively, each of the partial peptides corresponds to 8-28, 47-52, 108-126 , 172-190, 262-284 and 306-332 positions, respectively, of the sequence SEQ 1D 191). Using them as test antigens, the amounts of antibodies to them in the sera obtained from rabbits thus immunized were measured by enzyme immunoassay, and an increase in the number of antibodies in all tested sera was confirmed. Thus, these sera are hereinafter referred to as antisera.

 In addition, the aforementioned single-chain synthetic peptide having a cysteine residue linked to its C-terminus was also used as an antigen for affinity purification of antisera in the next step (2).

 2) Each of the two rabbits was immunized with one of the aforementioned antigenic peptides, and antibodies obtained from each rabbit were obtained separately. Specifically, for antibodies against the HT1 peptide, antibodies against the HT1-1 peptide and antibodies against the HT1-2 peptide from two immunized rabbits were obtained. Further, as one example, purification of an anti-HT1-1 peptide antibody is illustrated.

 First, 30 mg of the HT1 single chain peptide having a cysteine residue associated with it was combined with 12 ml of Sulfo Link Coupling GE1 (manufactured by Pierce Co., cat. 44895). More specifically, the antigen-containing peptide solution was coupled to a gel equilibrated with binding buffer (50 mM Tris, 5 mM EDTA-Na, pH 8.5) in an amount 6 times larger than the gel volume over a period of 15 minutes. Then, after standing for 30 minutes, the gel was washed with binding buffer in an amount of 3 volumes of gel. Then, a binding buffer containing 0.05 M L-cysteine-HCl was added to the gel at a ratio of 1 ml / ml gel, by which the untreated groups were blocked for 15 minutes. After standing for 30 minutes, the gel was washed with binding buffer 8 times in an amount equal to the volume of the gel. The binding operation was carried out at room temperature. In this way, a peptide containing an antigenic region is attached to the gel by covalent binding at a binding efficiency of 28.3% to form a gel with an antigenic peptide having 0.8 mg of peptide bound per ml of gel in an antigenic column. After taking all the blood from each immunized rabbit, 78.4 ml of antiserum containing antibodies against NP-1 peptide was obtained. 76.7 ml of this antiserum (protein content 3620 mg) was added to an antigenic column, previously equilibrated with 50 mM phosphate buffer (pH 8) containing 150 mM NaCl and 0.05% sodium azide, then the column was washed with the same buffer. In this way, 105.9 ml of a fraction passing through the column was obtained (protein content 3680 mg). Then, the adsorbed fraction was eluted with 0.1 M nitrate buffer (pH 3.0), which was then immediately neutralized by adding 21.1 ml of 0.1 M carbonate buffer (pH 9.9), concentrated by ultrafiltration (using an UMZ membrane produced Amicon Co.) and purified in 50 mM phosphate buffer (pH 8) containing 150 mM NaCl and 0.05% sodium azide. Thus, 11.2 ml of purified antibodies against the HT1-1 peptide (protein content 77.7 mg) were obtained in the buffer. Antibodies against the HT1-2 peptide (60.0 mg), antibodies against the HT2-1 peptide (18.8 mg), antibodies against the HT2-2 peptide (8.2 mg), etc. were obtained in the same way.

 Antibodies against HT3-HT6 peptides are prepared as described above.

Example 70. Detection of SRW using Western analysis
1) To evaluate the antisera obtained in Example 69, they were tested as described below.

 A standard sample of recombinant human TPO partially purified from the supernatant of the CHO cell culture, into which the gene encoding amino acids -21-332 SEQ 1D 6 was introduced, and in which it was expressed, was subjected to SDS-polyacrylamide (SDS-PAGE) electrophoresis according to with the usual method and then electroblotted onto a PVDF or nitrocellulose membrane. After blotting, the membrane was washed with 20 mM Tris-HCl and 0.5 M NaCl (pH 7.5) (TBS) for 5 minutes, then washed twice with 0.1% Tween 20-containing TBS (TTSS) for 5 minutes and treated with a blocking agent (Block Ac; manufactured by Dai Nippon Pharmaceutical Co., Cat. UK-B25) for 60 minutes. Antisera containing (each) one of the antibodies; antibody against the HT1-1 peptide, antibody against the HT2-1 peptide, antibody against the HT3-2 peptide, antibody against the HT4-1 peptide, antibody against the HT5-2 peptide and antibody against the HT6-1 peptide, diluted with a solution of TTBS containing 0.05 % BSA and 10% Bloc Ace before 1/1000 dilutions. Antibodies thus diluted were used as a primary antibody for Western analysis. In particular, a recombinant human TPO sample processed as described above was treated with a TTBS solution containing an anti-TPO peptide antibody, 0.05% BSA and 10% Block Aca, for 60 minutes and then washed with TTBS for 5 minutes. It was then treated with a TTBS solution containing anti-rabbit (ab ') goat biotinylated antibodies (Saltag Co., Cat. 43015) as a secondary antibody, together with 0.05% BSA and 10% Block Ace, for 60 minutes and then washed twice TTBS for 5 minutes. It was then treated with 1/5000 dilution of alkaline phosphatase-labeled avidin (manufactured by Leinco Technologies Co., Cat. A108) diluted with 10% Block ACE TTBS solution for 30 minutes and then washed twice with TTBS for 5 minutes and then TBS in within 5 minutes. The membrane was then developed using an alkaline phosphatase substrate (manufactured by Bio-Rad Co., cat. 170-6432). Western analysis was performed at room temperature. He confirmed that human TPO is recognized and detected by these antisera.

 2) 3 mg of each of the antibodies against the HT1-1 peptide, against the HT1-2 peptide, against the HT2-1 peptide and against the HT2-2 peptide, which were purified by affinity chromatography in Example 69, were weighed and biotinylated by binding to activated biotin (NHS -LC-Biotin 11 manufactured by Pierce Co., Cat. 21336). The same standard recombinant human TPO sample used in (1) was subjected to SDS-PAGE, then electrically blotted onto a PVDF or nitrocellulose membrane and subjected to routine Western analysis using each of these biotinylated antibodies as primary antibodies. Immediately after blotting, the membrane was washed with TBS for 5 minutes and then washed twice with TTBS for 5 minutes, and then treated with a blocking agent (Blos Ace) for 60 minutes. Then it was treated with a TTBS solution containing 1 μg / ml of biotinylated anti-TPO peptide antibody, 0.05% BSA and 10% Block Ace for 60 minutes and then washed twice with TTBS for 5 minutes. The membrane was then treated with 1/5000 dilution of alkaline phosphatase-labeled avidin (Leinco Technologies Co., Cat. A108) diluted with a 10% Block ACE TTBS solution for 30 minutes and then washed twice with TTBS for 5 minutes and then TBS in within 5 minutes. The membrane was then developed using an alkaline phosphatase substrate (Bio-Rad Co., Cat. 70-6432). The described Western analysis was performed at room temperature. He confirmed that human TPO is recognized and detected by these purified antibodies.

Example 71. Obtaining columns with antibodies against TPO peptide and detection of human TPO
Antibodies against human TPO peptide obtained in Example 69 were tested for recognition of human TPO. These antibodies were individually coupled on a chromatographic carrier to obtain anti-TPO peptide antibody columns. As one example, the following illustrates the preparation of an anti-HT1-2 peptide antibody column.

1) A solution was prepared containing 50 mM sodium phosphate and 0.15 M NaCl (pH 8.0) and containing 5 mg / ml antibodies, 0.8 ml of this solution was combined and bound to 1.54 ml of swollen formyl-activated gel ( Formyl-Gellulofine manufactured by Chisso Co.) at 4 ^{° C.} for 2 hours. Then, 1.1 ml of a solution containing 10 mg / L of reducing agent (trimethylamine borane (TMAB) manufactured by Seikagaku (Kogyo K.K., cat. 680246) was added and the binding continued for another 4 hours. Then, only part This gel was taken from the reaction mixture by centrifugation. 10 ml of pure water was added to it and only part of the gel was removed from this mixture by centrifugation. This process was repeated 4 times to remove unreacted antibodies. Then, the gel thus obtained was treated with 2.1 ml of blocking buffer ( 0.2 M Na-phosphate, 1 M e tanolamine, pH 7.0) and 1.1 ml of a solution of a reducing agent added to it at 4 ^{° C.} for 2 hours, as a result of which the active groups in the unreacted gel were blocked. Finally, the resulting gel was washed with water, 20 mM Tris -HCl and 0.15 M NaCl (pH 8.0) using a centrifuge, filled with a small column tube, which was washed with 3 M sodium thiocyanate solution and 0.1 M glycine-HC1 solution (pH 2.5) and then balanced again 20 mM Tris-HCl and 0.15 M NaCl (pH 8.0). The column thus balanced was stored.

 The anti-TPO peptide antibody gel in the anti-HT1-2 antibody column, in particular, had a binding efficiency of up to 94.2% for each 1gG fraction. The amount of 1 gG fraction bound on the gel per unit volume of this gel was 1.9 mg / ml gel. In the same manner as described above, a gel was prepared on which 21.8 mg per ml of 1 gG gel lacking TPO as antigen was bound. This gel was used as a pre-column (hereinafter referred to as an antibody pre-column) to remove non-specifically binding molecules from anti-TPO antibody columns, as mentioned in (2).

 2) The following is another example of the preparation of a column with antibodies against HT1-2.

 A standard sample of recombinant human TPO, which was partially purified from the supernatant of the CHO cell culture, into which a gene encoding the amino acid sequence of any of SEQ 1D 119-124, which was expressed in these cells, was introduced, was added to the antibody pre-column (containing 1 ml of gel) prepared in (1), and a volume of 20 mM Tris-HCl (pH 8.0) and 0.15 M NaCl, equal to the gel volume, was passed through the column 10 times, collecting the fraction passing through the column. Then, the fraction adsorbed on the pre-column was eluted 10 times in the volume of the gel with an acidic eluent (0.1 M glycine-HCl, pH 2.5). Then, the fraction passing through the pre-column was added to the anti-HT1-2 antibody column (containing 2 ml of gel) prepared in (1), and the column was washed 10 times in the gel volume of 20 mM Tris-HC1 (pH 8.0) and 0 , 15 M NaCl, collecting the fraction passing through the anti-HT1-2 antibody column. Then, the fraction adsorbed on the column was eluted 8 times in the volume of the gel with an acidic element (0.1 M glycine-HCl, pH 2.6). These fractions were analyzed by SDS-PAGE, which confirmed that TPO is not adsorbed on the antibody pre-column, but specifically binds to the anti-HT1-2 antibody column, and that almost all TPO in the sample added to this column is bound to the last column. Based on this experiment, it was confirmed that at least 200-300 μg / ml TPO gel was bound to the gel in the last column.

Example 72. The effect of TPO on increasing platelet count
20 normal male 1CR strain mice, eight weeks old (blood was already taken from their orbital veins and platelet counts were measured using an F-800 microcytometer manufactured by Toa Iyo Denshi) were divided into 4 groups by a non-selective method. One of the 4 groups (control-iv-group) was injected with 100 μl of PBS intravenously (iv) once a day for 5 consecutive days. One of the remaining groups (TPO-iv group) was injected with 100 μl of a solution (prepared by replacing a concentrated solution of the TPO-active fraction F2 SP Sepharose Fast Flow obtained in Example 56 with PBS buffer using a NAP-25 column [Pharmacia Biotech; cat. 17- 0852-02] followed by dilution of PBS; containing the relative activity of 211900 in the M-07e test) intravenously once a day for 5 consecutive days. Another group (control-SC group) was injected with 100 μl PBS subcutaneously (SC) 100 μl PBS 1 time per day for 5 consecutive days, while the last group (TPO-SC group) was injected 100 μl 1 time per day for 5 consecutive days, as in the TPO-iv group, but subcutaneously (Sс). After 6, 8, 10, 13, and 15 days from the start of the injection, blood from the orbital vein was taken from each of the mice and platelet counts were counted using a microcytometer (F-800; Toa Iyo Denshi). Changes in platelet numbers are given in FIG. Thus, in the control group -iv, the platelet count increased by 44% compared with the number before administration even on the 6th day (day 0 is the day the injections started), when the platelet counts were highest, and in the control group, the increase was only 47 % was noted even on day 8, when platelet counts were highest. On the other hand, in the TPO-iv group, an increase of 177% was observed on the 6th day compared with the amounts before administration, and in the TPO-Sc group, an increase of 347% was observed already on the 6th day and the highest increase was observed on the 8th day 493%.

 These results confirmed that human TPO increases platelet count regardless of differences in type, as well as in the route of administration.

Example 73. The effect of TPO on the increase in platelet count
16 normal male mice of C3H / HeJ strain of 11 weeks of age (blood was already taken from their orbital veins and platelet counts were measured using a F-800 type microcytometer manufactured by Toa Iyo Denshi) were divided into 4 groups by a non-selective method. One of the four groups (control group) was injected with 100 μl of PBS subcutaneously 1 time per day for 5 consecutive days. Another group (TPO-1 group) was injected subcutaneously with 100 μl of a solution (prepared by substituting the TPO-active Sephacryl S-200H fraction obtained in Example 56 with PBS using a NAP-25 column, followed by dilution of PBS; containing a relative activity of 83,000 in M -07e-test) 1 time per day for 5 consecutive days. Another group (TPO-2 group) was injected with 100 μl of a solution (obtained by diluting the TPO-active fraction used in the TPO-1 group, PBS 2 times; containing the relative activity of 41,500 in the M-07e test) subcutaneously 1 time per day for 5 consecutive days. The last group (TPO-3 group) was injected with 100 μl of a solution (obtained by diluting the TPO-active fraction used in the TPO-2 group 2 more times; containing the relative activity of 20750 in the M-07e test) subcutaneously 1 time per day for 5 consecutive days.

 Blood was drawn from the orbital veins of mice on the 6th, 8th, 10th and 12th days after injection and platelet counts were counted using a microcytometer (type F-800; Toa Iyo Denshi).

 Changes in platelet numbers are given in FIG. Thus, in the control group there was almost no change in platelet counts, whereas in the groups that were injected with TPO, there was an increase in platelet counts in all groups, and the highest platelet counts were on the 8th day after administration. In addition, in these groups, differences were observed in the dose-response relationship. So, in the group TRO-1, approximately 200% increase was noted already on the 6th day, the increase increased to about 270% on the 8th day, and then, however, a decrease was observed, even on the 12th day 65% was still observed an increase, meanwhile, a significant difference was observed from the control group (p <0.01; Dunnet multiple comparison test). The same increase was found in the TPO-2 group, and although the increasing effect was lower than in the TPO-1 group, increases of 140% and 160% were noted on the 6th and 8th days, respectively. On the 12th day, platelet counts decreased to approximately the same level as in the control group. The increasing effect in the TPO-3 group was weaker than in the TPO-2 group, and on the 8th day, when the platelet counts were the highest, a 110% increase was noted and there was also a significant difference (p <0.01) compared to the control group.

 The results described above showed that human TPO increases platelet counts regardless of the strain of mice and that its effect has a dose-response characteristic.

Example 74. The inhibitory effect of TPO on thrombocytopenia caused by the introduction of an anticancer agent
30 male eight-week old 1CR mice were injected 200 mg / kg of 5 FU intravenously and randomly divided into 2 groups (each group consisted of 15 mice). From the next day (day 1), one of the groups (control group) was injected with 100 μl of PBS subcutaneously 1 time per day for 5 consecutive days, while the other group (group of TPO) was injected with 100 μl of TPO-active fraction used in Example 72 (containing the relative activity of 211900 in the M-07e test) subcutaneously 1 time per day for 5 consecutive days. On the 4th, 6th and 8th days, 5 mice were randomly selected from each of the two groups, then blood was taken from the orbital veins and platelet counts were counted using a microcytometer (type E-2 500; Toa Iyo Denshi). Changes in platelet counts are given in FIG. So, in the control group, platelet counts after administration of 5-FU decreased over time and on the 6th day this value was the lowest (approximately 28% of the value before 5-FU administration), although approximately 1 was noted on the 8th day. 3x increase as a reaction to this decrease. Even in the case of the TPO group, platelet counts after administration of 5-FU decreased over time and on day 6, this value was the lowest (approximately 52% of the value before administration of 5-FU). However, the difference between platelet counts on days 4 and 6 was very low and on day 6, a significantly higher (p <0.05: Dunnet test) value remained compared to the control group. On day 8, an increase of approximately 200% of the value was observed before administration of 5-FU.

 The result confirmed that human TPO inhibits the decrease in platelet count caused by the anticancer agent.

Example 75. The therapeutic effect of TPO for thrombocytopenia
Nimustine hydrochloride (ACNU) (50 mg / kg) was injected intravenously to each of 36 male seven-week old 1CR mice, and then these mice were randomly divided into two groups (each group consisted of 18 mice). From the next day (day 1), one of the groups (control group) was injected with 200 μl of PBS subcutaneously twice a day for the next days, while the other group (group of TPO) was injected with 200 μl of the TPO-active fraction used in Example 73 ( without diluting PBS), to which 0.04% Tween 80 (relative activity 380,000 in the M-07e test) was added, subcutaneously twice a day for subsequent days.

 After injection, mice in each of the groups were randomly divided into three groups: from one group, blood was taken on the 5th and 12th days; from another group on the 8th and 14th days and from the third group on the 10th day. Blood was taken from the orbital veins and platelet counts were counted using a microcytometer (type F-800; manufactured by Toa Iyo Denshi). Changes in platelet counts are given in FIG. 27. So, in the control group, platelet counts after administration of ACNU decreased over time and on days 8-10, this value was the lowest (approximately 29% of the value before administration of ACNU) and the recovery was only approximately 49% and 74% by 12th and 14th days, respectively. On the other hand, in the TPO group, a decrease to approximately 38% of the value was noted before the administration of ACNU on the 5th day, and then the situation changed to increase. So, on the 8th day, the value was restored to approximately 63% of the value before the introduction of ACNU and on the 10th day and after it the platelet counts were greater than before the introduction of ACNU. So, on the 12th and 14th days, increases were up to about 300% and 400%, respectively.

 As noted above, on days 8–10, when a decrease was observed in the control group, an increase was already observed in the TPO group, and on the 10th day platelet counts were higher than the numbers before the administration of ACNU. Therefore, it was confirmed that human TPO accelerates a return to normal with thrombocytopenia caused by an anticancer agent.

 Considering also the results of Example 74, it can be expected that human TPO inhibits a decrease in platelet counts and accelerates a return to normal in case of thrombocytopenia, regardless of the type of anticancer agent.

Example 76. The therapeutic effect of TPO on thrombocytopenia after bone marrow transplantation (BMT)
48 male mice of C3H / HeN strain of seven weeks of age were irradiated with 10 Gy of radiation exposure (whole body) and immediately thereafter they were injected intravenously with 1 x 10 ⁶ bone marrow cells from mice of the same strain. Then, the mice were randomly divided into two groups from the next day (day 1), one of the groups (control group) was injected with PBS, while the other group (TPO group) was injected with the TPO-active fraction used in Example 73 (relative activity 44000 in M -07e-test) with a dose of 100 μl subcutaneously 1 time per day for the next 20 days.

 After the start of injection, every 6 mice were randomly selected on the 5th, 10th, 14th and 21st days, blood was taken from their ophthalmic veins and platelet counts were counted in a microcytometer (type E-2500, Toa Iyo Denshi).

 Changes in platelet counts are given in FIG. So, in all groups, platelet counts decreased over time after the injection of bone marrow cells. On the 10th day, the amounts were the lowest (approximately 3% of the value before using TDC). After this, a gradual recovery was noted and on the 4th day platelet counts were 11 and 13% in the control group and in the SRW group, respectively. On day 21, only 37% recovery was observed in the control group, while in the TPO group, recovery was approximately 65%, after which a significant effect of accelerated recovery was observed. The described results confirmed that human TPO, obtained as in Example 56, enhances recovery to normal platelet counts after TDC.

Example 77. The therapeutic effect of TPO on thrombocytopenia after exposure to radiation
36 male mice of an 8-week-old 1CR strain were irradiated with 5Gy x-rays over the whole body and randomly divided into two groups. From the next day (day 1), one of these groups (control group) was injected with PBS, while the other group (TPO group) was injected with the TPO-active fraction used in Example 73 (relative activity 1440000 in the M-07e test) one once a day subcutaneously for 3 consecutive days and from the next day, the relative activity of 360,000 (according to the same test) was injected subcutaneously once a day for 7 consecutive days. After the start of injections, each of the groups was divided into three groups: the group from which blood was taken on the 4th, 11th and 21st days; another group from which blood was taken on the 7th and 13th days, and a third group from which blood was taken on the 9th and 15th days. Blood was taken from the orbital veins and platelet counts were counted using a microcytometer (type F-800, Toa Iuo Denshi). Changes in platelet counts are given in FIG. So, in the control group, platelet counts decreased over time after irradiation with X-rays and on the 9th day the numbers were the lowest (approximately 25% of the value before irradiation with X-rays). On the 11th and 13th days, platelet counts recovered to approximately 38 and 67%, respectively, and on the 15th day they recovered to values before exposure to X-rays. In the SRW group, these amounts decreased to approximately 24% of the values before x-ray irradiation on day 7. After this, an increase was noted and on the 9th day platelet counts were restored to approximately 82%. On the 11th day and after it, the quantities were larger than before x-ray irradiation, and this trend continued until the 21st day. As mentioned above, on the 9th day, when there was still a decrease in the control group, the platelet counts in the TPO group were already changing upwards and on the 11th day the platelet counts were higher than before the X-ray exposure, remaining high afterwards. On the other hand, in the control group, it took 15 days to restore platelet counts to values before exposure to x-rays. These results showed that the human SRW obtained in Example 56 reduces the time needed to recover to normal after platelet counts are reduced after x-ray irradiation, and that it exhibits a therapeutic effect on thrombocytopenia after x-ray irradiation.

Example 78. The effect of hTO163 on increasing platelet count
15 healthy male mice of strain C3H / HeN, from which blood was already taken from the orbital veins and counted the number of platelets using a microcytometer (type F-800, Toa Iyo Denshi), were randomly divided into 4 groups: the control group and groups A, B and C. The first group (control group)
were injected subcutaneously with PBS containing 0.1% serum from mice, 1 time per day for 5 consecutive days. Groups A, B, and C were injected with the TPO-active Sepharose Fast Flow SP fraction obtained in Example 65, followed by dilution of PBS containing 0.1% mouse serum at doses of approximately 40,000,000, approximately 8,000,000 and approximately 1,600,000 / kg body weight, respectively , according to the relative activity in the M-07e-test HTPO163, subcutaneously for 5 consecutive days.

 On the 6th, 8th, 10th and 12th days after injection, blood was taken from the orbital vein of each mouse and platelet counts were counted using a microcytometer (type F-800 manufactured by Toa Iyo Denshi). Changes in platelet counts are shown in FIG.

 So, in the control group there was almost no change in platelet counts, while in the received TPO groups, increases in platelet counts were observed in all cases and the highest values were on the 8th day after injections. Among each of the obtained TPO groups, a dose-response relationship was noted. For example, in group A, increases of approximately 88% and approximately 100% were observed on the 6th and 8th days, respectively, and even on the 10th day, approximately 97% increase remained, after which all the results showed a significant difference from the control group ( p <0.01 according to the Dunnet test). Similar increases were also noted in group B and, although the degree of increase was lower than in group A, increases of about 65% and about 84% were noted on days 6 and 8, respectively, with a significant difference compared with the control group ( p <0.01, Dunnet test). The enhancing effect in group C was lower than in group B. An approximately 31% increase was still observed on day 8 when the highest platelet counts were observed. The described results confirmed that the hTPO163 obtained in Example 65 increased platelet counts and that its effect showed a dose-response response.

Example 79. The therapeutic effect of hTPO163 on thrombocytopenia
100 male mice of an eight-week old C3H / HeN strain were injected intravenously with 50 mg / kg nimustine hydrochloride (ACANU) and then randomly divided into 4 groups: a control group and groups A, B and C, each of which consisted of 25 mice. From the next day (day 1), the control group was injected subcutaneously with 5 ml / kg PBS once a day for the following days, while groups A, B and C were injected subcutaneously with the TPO-active SP Sepharose Fast Flow fraction obtained in Example 65 diluted with PBS at doses of 16000000, approximately 48000000 and approximately 160,000,000 / kg body weight, respectively, in the values of the relative activity of hTPO163 in the M-07e test, once a day for the next days.

 After the start of injection, mice in each group were randomly divided into 5 groups and blood was taken on the 5th, 8th, 10th, 12th and 14th days, respectively. Blood was taken from the orbital vein and platelet counts were counted using a microcytometer (type E-2500, manufactured by Toa Iyo Denshi). Changes in platelet counts are shown in FIG. So, in the control group, platelet counts decreased over time after administration of ACNU, giving the lowest values (approximately 15-16% of the value before administration of ACNU) on days 8-10, and the degree of recovery was only about 28% and approximately 51% on the 12th and 14th days, respectively. On the other hand, in group A, these amounts decreased to approximately 25% of the values before the administration of ACNU on day 8. After that, however, they changed upward and recovered to approximately 34% and approximately 89% on the 10th and 12th days, respectively. On the 14th day, the platelet count was greater than before the administration of ACNU.

 As mentioned above, the lowest platelet counts were observed on day 8 in all groups, although in the groups receiving human TPO, the decrease was slower than in the control group giving the lowest platelet count. In the control group, there was almost no recovery in platelet counts even on the 10th day, while in groups receiving human TPO, platelet count recovery was noted in all of these groups, although their levels varied. In the 50 μg / kg group, recovery was observed on day 12, when platelet counts were higher than the values before ACNU administration. If we compare this with the fact that in the control group the recovery was only approximately 51%, compared with the value before the administration of ACNU, even on the 14th day, it becomes clear that hTPO163 obtained as described in Example 65 enhances the recovery of the amount platelets after their decline. Thus, it was confirmed that hTPO163 has a therapeutic effect on thrombocytopenia caused by anticancer agents.

 The following are examples of pharmaceutical preparations.

Example 80
The human TPO fraction obtained in Example 56 was concentrated, subjected to aseptic treatment, and frozen at -20 ^{° C.} to obtain a frozen product for use as an injection.

Example 81
The human TPO fraction obtained in Example 56 was concentrated, 5 ml was placed in 10 ml vials under aseptic conditions, dried by lyophilization at -20 ^° C and the vial was closed with a rubber stopper to obtain a lyophilized substance for use as an injectable preparation.

Example 82
The human TPO fraction obtained in Example 56 was concentrated, subjected to aseptic filtration, and placed in a 10 ml vial to obtain a product for use as an injectable preparation.

Example 83
The hTPO163 fraction obtained in Example 65 was concentrated, subjected to aseptic treatment, and lyophilized at -20 ^{° C.} to obtain a frozen product for use as an injection.

Example 84
The hTPO163 fraction obtained in Example 65 was concentrated, 5 ml was placed in a 10 ml vial by aseptic operation, lyophilized at -20 ^° C and the vial was sealed with a rubber stopper to obtain a lyophilized product for use as an injection.

Example 85
The hTPO163 fraction obtained in Example 65 was concentrated, sterile filtered, and placed in a 10 ml vial to obtain the product for use as an injection.

Example 86
The human TPO fraction obtained in Example 57 was concentrated, subjected to aseptic processing, and frozen at -20 ^{° C.} to obtain a frozen product for use as an injection.

Example 87
The human TPO fraction obtained in Example 57 was concentrated, 5 ml was placed in a 10 ml vial under sterile conditions, lyophilized at -20 ^° C and the vial was sealed with a rubber stopper to obtain a lyophilized product for use as an injection.

Example 88
The human TPO fraction obtained in Example 57 was concentrated, sterile filtered and placed in a 10 ml vial to obtain the product for use as an injection.

Example 89. Aplastic plasma of dogs
Heparinized aplastic dog plasma ("APK9") or normal dog plasma ("NK9") was prepared as described [Mazur, E. and South, K. Exp, Hematol. 13: 1164-1172 (1985); Arriaga, M., South K., Cohen. JL and Mazur, EM Blood 69: 486-492 (1987); Mazur, E., Basilico, D., Newton, JL, Cohen, JL, Charland, C., Sohl, PA, and Narendran, A. Blood 76: 1771-1782 (1990)], except that 450 rad (4.5 J / kg) whole body irradiation was used for recipients.

Example 90. Test for the determination of human megakaryocytes
Standard methods for many of the procedures described in the following examples or other suitable procedures are given in well-known widely described molecular biology methods, such as, for example, Sambrook, et al., (Eds), Molecular Cloning, Second. Edition, Cold Spring Harbor Laboratory Press (1987) ub Ausubel, et al., (Eds), Current Protocols in Molecular Biology, Green associates / Wiley Interscience, New York (1990).

APK9 and fractionated ARP9 were analyzed for their ability to stimulate the development of human megakaryocytes from GD34 ⁺ progenitor cells, CD34 - selected cells were obtained from peripheral blood cells as described (Hokom, M.N., Choi, E., Nichol, Hornkohl, A., Arakawa, T., and Hunt, P. Molecular Biology of Haematopoiesis 3: 15-31, 1994), and incubated in the following culture medium: Dulbecco medium modified by the Claims method (1MDM; G1BCO, Grand Island, NY) with the addition of 1 % Glutamine Pen-Strep (Irvine Scientific, Santa Ana, CA) and 10% heparinized, low platelet AB human plasma. Also included in environment 2-mercaptoethanol (10 ^- 4 M), pyruvic acid (110 .mu.g / ml), cholesterol (7.8 ug / ml), adenosine, guanine, cytidine, uridine, thymidine, 2-dezoksitsitozin, 2-deoxyadenosine 2-deoxyguanosine (10 μg / ml each, Sigma); human recombinant insulin (10 μg / ml), human transferrin (300 μg / ml), soy lipids (1%, Boehringer - Mannheim, Indianapolis, 1N); Recombinant major human fibroblast growth factor (2 ng / ml), Genzyme, Cambridge, MA); recombinant human epidermal growth factor (15 ng / ml) derived from platelet growth factor (10 ng / ml, Amgen, Inc., Thousand Oaks, CA). CD34 selected cells were seeded at 2 x 10 ⁵ cells / ml of culture medium, with a final volume of 15 μl, into the wells of Tepacaku microtiter plates (Vanguard, Inc., Neptune, NJ). Cells were incubated at 37 ^{° C.} for 8 days in humidified boxes in 5% CO ₂ in air, fixed directly in the culture wells with 1% glutaraldehyde and incubated with a mixture of monoclonal antibodies (anti-GPIb, anti-GP11B, (Biodesign) and anti- GP1b (Dako, Carpinteria, CA). An immune response was exhibited by a streptavidin-β-galactosidase detection system (HistoMark, Kirgegaard and Perry). Megakaryocytes identified by blue color were counted using an inverted microscope at 100X magnification. The results were presented as average numbers. megakaryocytes per lu ku ± standard error of the mean (SEM). In some cases, the data are presented in "megakaryocyte units / ml", where the degree to which this sample induced the development of megakaryocytes is normalized to a positive ARK9 control for this experiment. One unit is defined as the amount of material that leads to the same number of megakaryocytes as 1 μl of the ARK-9 standards.The activity was considered to be associated with the MpL ligand, if it could be blocked with 5-10 μg / ml MPL-X (soluble Mpl receptor). It has been shown that ARK9 contains factor (s) that stimulate the development of human megakaryocytes in this system. CD34 selected cells incubated with 10% NK9 for 8 days showed a small number of blue stained megakaryocytes, while CD34 selected cells incubated with 10% NK9 for 8 days found a very large number of blue stained megakaryocytes .

 Fig. 32 shows that increasing concentrations of Mpl-X added to the human megakaryocyte culture system block the development of megakaryocytes in an increasing manner. At MPl-X concentrations greater than 5 μg / ml, inhibition was complete. In this experiment, CD34-selected cells were stimulated with 5% ARP9. This shows that activity that interacts with Mpl-X (preferably the MPl ligand) is necessary for the development of human megakaryocytes, and implies that the Mpl ligand is present in the APC itself.

 It was further shown that the MPl ligand activity necessary for the development of human megakaryocytes is present in APC9. APC9 (135 ml) was diluted 6 times in Isc media and applied to an MPl-X affinity column. Unbound material (fraction passing through the column) was collected and concentrated to the original volume before analysis. The bound material was eluted in 10 ml of 1 M NaCl and a 20% pool was diafiltered and concentrated 4 times before analysis. CD34-selected cells, incubated only in the medium, did not develop into megakaryocytes. Cells incubated in 5% ARP9 (the same pool that is applied to the column) developed at 48 ± 8 megakaryocytes per well. Cells incubated in 10% unbound material did not develop into megakaryocytes. Cells incubated in 10% of the eluted pool developed at 120 ± 44 megakaryocytes per well. Both column loading activity and eluted pool activity were substantially completely inhibited by 5 μg / ml Mpl-X in this test.

Example 91. Transfection of a mouse or human Mpl receptor into a murine cell line A. Mouse Mpl receptor
The full length mouse Ml receptor cDNA was subcloned into an expression vector containing a transcription promoter derived from the LTR of Moloney mouse sarcoma virus. 5 μg of this construct and 1 μg of selectable marker plasmid pWLneo (Stratagene) were co-electroporated into a 1L-3-dependent mouse cell line (32D, clone 23; Greenberger et al., PNAS 80: 2931-2936 (1983)). Cells were cultured for 5 days, recovered and then incubated in selective media containing 800 μg / ml Geneticin (G418, Sigma) and 1 ng / ml mouse 1L-3. The surviving cells were then divided into pools of 2 x 10 ⁵ cells and cultured until a cell population was grown, which was then analyzed. 6 populations were tested for surface expression of the Mpl receptor using FACS analysis using polyclonal rabbit antipeptide serum. One population was selected for FACS sorting (using a cell sorter with excitation of fluorescence) using the same antipeptide serum. Unicellular clones of the parent cell line were selected for growth in 10% APC9 and Genetic. After selection in ARK9 for 35 days, the cells were kept in 1 ng / ml mouse 1L-3. One of the subclones, 1A6.1, was used for this part of the work.

B. Human Ml receptor
The full length human MPl receptor cDNA sequence (Vigon, 1., et al., PNAS 89: 5640-5644 (1992)) was subcloned into an expression vector containing the mouse sarcoma Maloney virus transcription promoter (the same vector as in the case of the mouse receptor ) 6 μg of this construct and 6 μg of the amphotrophic retroviral packaging construct (Landau, NR, Littman, DR, J. Virology 66: 5110-5113 (1992)) were transfected into 3 x 10 ⁶ 293 cells using a Stratagene transfection kit with CaPO ₄ . The same cells were re-transfected after 2 days and again after 4 days. The day after the last transfection, 293 cells were cultured together with a 1L-3-dependent mouse cell line (32D, clone 23; Greenberger et al., PNAS 80: 2931-2936 (1983). After 24 hours, 32D cells were isolated and obtained as BSA gradient bands (Path-o-cyte; mills 1nc.). The cells were increased in volume by 1 ng / ml of mouse 1L-3 and then selected for growth in 20% APC. Cells were sorted for expression on their receptor surface using FACS using polyclonal rabbit anti-peptide serum, which cells were then used in tests.

Example 92. 1A6.1 analysis to determine MPl ligand
1A6.1 cells were washed from 1L-3 culture and placed on Terasaki microtiter plates (1000 cells / 15 μl total count / well) in α-MEM (Gibco) supplemented with 10% fetal calf serum (FCS), Geneticin (800 μg / ml) and 10% foam / strep (Gibco) in serial dilutions of 1: 1 test samples. After 48 hours, the number of viable cells per well was determined under a microscope. For one unit of activity, the amount of activity that led to 200 viable cells per well was taken. Activity was considered to be associated with the MPl ligand, if it could be completely blocked by adding 5-10 μg / ml MPl-X to the reaction medium. The activity of MPl ligand in APC9 was on average 4400 ± 539 units / ml aplastic plasma. Unless otherwise indicated, units of MPl ligand activity were determined in the 1A6.1 test.

 Tests with cells transfected with the human Mpl receptor gene were performed essentially the same as with 1A6.1 cells.

Example 93. Demonstration that the MPl ligand is present in aplastic plasma or serum from sources such as mouse, dog, pig and human
MPL ligand is present in aplastic plasma or serum from mice, dogs, pigs and humans (table 9). Plasma was collected from BDF1 mice with pre-irradiation and 12 days post-irradiation (500 rad). Plasma was tested in a 1A6.1 test in which it showed 2000 units / ml of activity, which was essentially completely inhibited by Mpl-X (10 μg / ml). Irradiated murine plasma was also positive in the text for human megakaryocytes, in which it found an activity of 1833 units / ml. Plasma was collected from dogs with pre-irradiation and 10 days of post-irradiation (450 rad). Plasma was tested both in the 1A6.1 test and in tests with the development of human megakaryocytes. Activity was detected and completely inhibited by Mpl-X (10 μg / ml) in both texts. Plasma was collected from pigs pre-irradiated with 10 days post-irradiation (650 rad). Plasma was tested both in the 1A6.1 test and in the test with the development of human megakaryocytes. In both tests, she found the activity of Mpl-ligand (inhibited by 10 μg / ml Mpl-X), comparable to the activity found in dog aplastic plasma. Human serum was obtained. This material was collected from bone marrow transplant patients. Sera from 6 patients were tested in the 1A6.1 test, where they showed activity of 903 units / ml, 88% of which was associated with the Mpl ligand (10 μg / ml Mpl-X was inhibited). Sera from 14 aplastic patients (with developmental anomaly) were tested in a test with the development of human megakaryocytes. As a group, they found a major activity, 941 megakaryocyte units / ml, which was completely inhibited by 10 μg / ml Mpl-X. Data are given with mouse 1L-3 to show the specificity of the 1A6.1 test. Although this recombinant cytokine induces the growth of this cell line, it is not blocked by 10 μg / ml Mpl-X.

Example 94. Obtaining derivatives of human TPO expressed in the E. coli system
In an attempt to improve biological activity, stability, and solubility, some TPO derivatives were engineered and expressed in E. coli. These derivatives generated using the amino acid sequence shown in SEQ 1D 11 included: [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹²⁹ ] TPO (1-163), in which arginine replaces leucine at the amino acid position 129 (also called L129), [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹³³ ] TPO (1-163), in which arginine replaces histidine at amino acid position 133 (also called H133R), [Met ^{- 1} , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹⁴³ ] TPO (1-163), in which arginine replaces methionine at amino acid position 143 (also called M143R), [Met ^-2 , Lys ^-1 , Ala ¹ , Val ^3, Leu ^82] TPO (1-163), wherein the leucine replaces glycine at polo enii 82 amino acids (also referred to as ^{G82L), [Met -2, Lys} -1, Ala ^1, Val ^3, Leu ^146] TPO (1-163), wherein the leucine replaces glycine at amino acid position 146 (also called G146L), [ Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Pro ¹⁴⁸ ] TPO (1-163), in which proline replaces the series at amino acid position 148 (also called S148P), [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ⁵⁹ ] - TPO (1-163), in which arginine replaces lysine at amino acid position 59 (also called K59R), [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹¹⁵ ] TPO ( 1-163), in which arginine replaces glutamic acid at amino acid position 115 (also called Q115R).

Upon receipt of these TPO derivatives, h6T (1-163) described in Example 42 was used as a matrix, and the following synthesized oligonucleotides were used as primers:
Ll29R: 5'-ATSTTSSGTTSTTTSSACGCACST-3 '(to obtain an Arg-substituted derivative with Leu-129 substituted by Arg in the sequence shown in SEQ 1D 11);
H133R: 5'-TSTTTSSACGCGTSTGSTGCGT-3 '(to obtain an Arg-substituted derivative with His-133 substituted by Arg in the sequence shown in SEQ 1D 11);
M143R: 5'-CGTTTSSTGCGTSTGGTTGGC-3 '(to obtain an Arg-substituted derivative with Met-143 substituted by Arg in the sequence represented by SEQ 1D 11);
G82L: 5'-GGCCACGSTTSTGSSGACSTGCST-3 '(to obtain a Leu-substituted derivative of Gly-82 substituted by Leu in the sequence shown in SEQ 1D 11);
G146L: 5'-ATGCTGGTTCTGGGTTCTACCCT-3 '(to obtain a Leu-substituted derivative with Gly-146 substituted by Leu in the sequence shown in SEQ 1D 11);
S148P: 5'-GTTGGСGGТССGАСССТGТGСG-3 '(to obtain a Pro-substituted derivative with Ser-148 substituted by Pro in the sequence shown in SEQ 1BD 11);
K59R: 5 ^' -GAAGAGASSCGCGSTCAGGACATCC-3 ^' (to obtain an Arg-substituted derivative with Lys-59 substituted by Arg in the sequence shown in SEQ 1D 11) and
Q115R: 5'-CTGCCGCCACGTGGCCGTACCAC-3 '(to obtain an Arg-substituted derivative with Gln-115 substituted by Arg in the sequence shown in SEQ 1D 11).

 Mutant plasmids were constructed using the Sculptor in vitro mutagenesis kit (Amersham). Fragment Xba 1 - Hind 111, obtained from pCFM536 / h6T (1-163) described in Example 42, was introduced into the phasal vector Bluescript 11SK (-) (Stratagene, California, USA) after cleavage of Xba 1 and Hind 111 to obtain plasmid pSKTO which was then transformed into E. coli JM109. Single-stranded DNA for mutagenesis was obtained from pSKTO using the helper phage M13KO7 (Takara-Shuzo, Japan). Mutations were introduced into the TPO gene in accordance with the supplier's protocols using the oligonucleotides listed above. Sequencing was performed with a DNA sequencing kit (Applied Biosystems, USA) according to the protocols of the supplier and TPO mutations were confirmed.

 Fragments of Xba 1 - Hind 111, obtained from mutated pSKTRO, were introduced into plasmid pCEM536 cleaved with Xba 1 and Hind 111. Plasmids pCFM536 carrying the mutated genes were transformed into E. coli 261 to obtain a transformant expressing genes derived from TPO.

 Cultivation of E. coli 261 transformed with mutated pCFM536 was carried out in accordance with Example 43, as described previously. The transformant cell pellet was collected and stored in the freezer for at least 1 day. A frozen cell pellet (3 g) was suspended in 30 ml of 20 mM Tris buffer (pH 8.5) containing 10 mM EDTA, 10 mM DTT and 1 mM PMSF. The suspension was stored on ice and sonicated 5 times with an interval of 1 min (at least). The sonicated cells were collected by centrifugation at 15,000 rpm for 10 minutes.

The precipitate was suspended in 30 ml of 10 mM Tris buffer (pH 8.7) containing 8 M guanidine chloride, 5 mM EDTA, 1 mM PMSF and recovered by adding 50 mg DTT. After stirring for 1-1.5 hours, the pH of the solution was adjusted to 5.0 with dilute HC1 and then the solution was cooled to 4 ^{° C.} before dilution.

 The sample solution was gradually diluted in 1.5 L of 10 mM CAPS buffer (pH 10.5) containing 30% glycerol, 3 M urea, 3 mM cystamine and 1 mM L-cysteine overnight. After mixing the sample for at least 2 days, the precipitate was removed by centrifugation at 8000 rpm for 45 minutes. The pH of the solution was adjusted to 6.8 with 6M phosphoric acid and the solution was diluted 2 times. The solution was filtered through 2 layers of filter paper 2 (diameter 90 mm, Toyo filter paper, Japan) and then applied to a column (2.6 x 10 cm) of CM-Sepharose Fast Flow (Pharmacia). The column was washed with 400 ml of 10 mM phosphate buffer (pH 6.8) containing 15% glycerol and 1 M urea, and 10 mM phosphate buffer (pH 7.2) containing 15% glycerol was balanced. Protein was eluted from the column using a linear gradient system from 10 mM phosphate buffer (pH 7.2) containing 15% glycerol to the same buffer containing 0.5 M NaCl at a flow rate of 1.0 ml / min. Protein elution was observed by absorbance at 280 nm and the fraction containing the TPO derivative was confirmed by SDS-PAGE and collected.

After concentrating the TPO fraction (approximately 30 ml) to 2 ml using Centriprep 10 (Omicon), the mutant TPO was further purified using HPLC with reverse phase (Waters) with an α Bondasphere C4 column (3.9 x 150 mm). Protein was eluted using a linear gradient from 0.05% TFA in H ₂ O to 2 propanol containing 30% CH ₃ CN and 0.02% TFA at a flow rate of 0.5 ml / min for 40 minutes. Under these conditions, the TPO derivatives eluted after approximately 30 minutes.

 For biological analysis, the TPO sample thus purified was dialyzed twice against 1 L of 10 mM phosphate buffer for 2 days. After concentration with Centriprep 10 (Omicon) to approximately 0.1 mg / ml, biological activity was evaluated in the M-07e test system as previously described. It was found that all mutants have almost the same activity compared to the activity of h6T (1-163), a standard TPO breakdown (see Figs. 33 and 34).

List of deposits
Escherichia coli (pHT1-231 / DH5):
FERM BP-4564 and SSTSS-M95001
Escherichia coli (pEF18S-A2μ / DH5):
FERM BP-4565 and SSTSS-M95002
Escherichia coli (pHGT1 / DH5):
FERM BP-4616 and CCTCC-M95003
Escherichia coli (pHTF1 / DH5):
FERM BP-4617 and ССТСС-М95004
Mouse-Mouse-Hybridoma P55:
FERM BP-4563 and SSTSS-S95001
CHO28 / 1/1/3-C6:
FERM BP-4988 and ССТСС-С95004 СНO163Т-63-79-С1:
FERM BP-4989 and ССТСС-С95005.

Claims (34)

1. The thrombopoietin polypeptide (TPO) having a biological activity of specific stimulation or increased platelet formation and containing the amino acid sequence of SEQ ID NO 6, or a derivative thereof, and which may additionally contain amino acids Met ^-2 -Lys ^-1 , Met ^-1 or Gly ^-1 .  2. TPO polypeptide according to claim 1, containing the amino acid sequence 1-332 of SEQ ID NO 6.  3. The TPO polypeptide according to claim 2, consisting of the amino acid sequence 1-163 of SEQ ID NO 6.  4. The TPO polypeptide according to claim 2, consisting of the amino acid sequence 1-232 of SEQ ID NO 6.  5. The TPO polypeptide according to claim 2, consisting of the amino acid sequence 1-151 of SEQ ID NO 6.  6. TPO polypeptide according to any one of paragraphs. 2-4, having from 1 to 6 amino acids deleted. 7. TPO polypeptide according to claim 3, selected from the group consisting of [ΔHis ³³ ] TPO (1-163), [ΔArg ¹¹⁷ ] TPO (1-163) and [ΔGly ¹¹⁶ ] TPO (1-163). 8. TPO polypeptide according to claim 3, selected from the group consisting of [His ³³ , Thi ^{33 '} , Pro ³⁴ ] TPO (1-163), [His ³³ , Ala ^33' , Pro ³⁴ ] TPO (1-163) , [His ^{33 '} , Gly ^33' , Pro ³⁴ , Ser ³⁸ ] TPO (1-163), [Gly ¹¹⁶ , Asn ^{116 '} , Arg ¹¹⁷ ] TPO (1-163), [Gly ¹¹⁶ , Ala ^116' , Arg ¹¹⁷ ] TPO (1-163), [Gly ¹¹⁶ , Gly ^{116 '} , Arg ¹¹⁷ ] TPO (1-163) and [Ala ¹ , Val ³ ] TPO (1-163). 9. The TPO polypeptide of claim 2, selected from the group consisting of [Thr ³³ , Thr ³³³ , Ser ³³⁴ , Ile ³³⁵ , Gly ³³⁶ , Tyr ³³⁷ , Pro ³³⁸ , Tyr ³³⁹ , Asp ³⁴⁰ , Val ³⁴¹ , Pro ³⁴² , Asp ³⁴³ , Tyr ³⁴⁴ , Ala ³⁴⁵ , Gly ³⁴⁶ , Val ³⁴⁷ , His ³⁴⁸ , His ³⁴⁹ , His ³⁵⁰ , His ³⁵¹ , His ³⁵² , His ³⁵³ ] TPO [Asn ²⁵ , Lys ²³¹ , Thr ³³³ , Ser ³³⁴ , Ile ³³⁵ , Gly ³³⁶ , Tyr ³³⁷ , Pro ³³⁸ , Tyr ³³⁹ , Asp ³⁴⁰ , Val ³⁴¹ , Pro ³⁴² , Asp ³⁴³ , Tyr ³⁴⁴ , Ala ³⁴⁵ , Gly ³⁴⁶ , Val ³⁴⁷ , His ³⁴⁸ , His ³⁴⁹ , His ³⁵⁰ , His ³⁵¹ , His ³⁵² , His ³⁵³ ] TPO. 10. The TPO polypeptide according to claim 2, selected from the group consisting of [Asn ²⁵ ] TPO and [Thr ³³ ] TPO. 11. TPO polypeptide according to claim 3, selected from the group consisting of [Ala ¹ , Val ³ , Arg ¹²⁹ ] TPO (1-163), [Ala ¹ , Val ³ , Arg ¹³³ ] TPO (1-163), [ Ala ¹ , Val ³ , Arg ¹⁴³ ] TPO (1-163), [Ala ¹ , Val ³ , Leu ⁸² ] TPO (1-163), [Ala ¹ , Val ³ , Leu ¹⁴⁶ ] TPO (1-163), [Ala ¹ , Val ³ , Pro ¹⁴⁸ ] TPO (1-163), [Ala ¹ , Val ³ , Arg ⁵⁹ ] TPO (1-163) and [Ala ¹ , Val ³ , Arg ¹¹⁵ ] TPO (1-163) . 12. TPO polypeptide according to claim 3, selected from the group consisting of [Arg ¹²⁹ ] TPO (1-163), [Arg ¹³³ ] TPO (1-163), [Arg ¹⁴³ ] TPO (1-163), [Leu ⁸² ] TPO (1-163), [Leu ¹⁴⁶ ] TPO (1-163), [Pro ¹⁴⁸ ] TPO (1-163), [Arg ⁵⁹ ] TPO (1-163) and [Arg ¹¹⁵ ] TPO (1- 163).  13. The TPO polypeptide of claim 2, covalently linked to a polymer.  14. TPO polypeptide according to claim 13, characterized in that said polymer is polyethylene glycol.  15. TPO polypeptide according to any one of paragraphs. 1-7, characterized in that it is obtained by the method according to p. 24.  16. TPO polypeptide according to any one of paragraphs. 1-7, characterized in that it is obtained by the method according to p. 25.  17. TPO polypeptide according to any one of paragraphs. 1-7, characterized in that it is obtained by the method according to p. 26.  18. DNA encoding a TPO polypeptide according to any one of paragraphs. 1-14.  19. The DNA sequence encoding the TPO polypeptide according to any one of paragraphs. 1-14, having a biological activity of specific stimulation or increase in platelet formation, selected from the group of: a) DNA sequences shown in SEQ ID NO 196, or complementary to it; (b) DNA sequences encoding a polypeptide according to any one of claims. 7-12 and hybridizing under stringent conditions with the DNA sequence described in (a), or fragments thereof.  20. The DNA sequence according to p. 19, characterized in that it is a cDNA.  21. The DNA sequence according to claim 19, characterized in that it is genomic DNA.  22. The DNA sequence according to claim 19, characterized in that it is a synthetic DNA.  23. A DNA sequence encoding a TPO polypeptide having biological activity specific stimulating or increasing platelet formation, including in the 5 ′ -> 3 ′ direction: a) DNA encoding glutathione S-transferase (GST), b) DNA encoding a peptide recognition of thrombin; and c) DNA encoding the TPO polypeptide.  24. A method of producing a TPO polypeptide according to claims. 1-14, comprising the steps of expressing the polypeptide encoded by the DNA of claim 18 in a suitable host and isolating the TPO polypeptide. 25. The method according to p. 24, wherein the TPO polypeptide is a polypeptide according to claim 1, and the method further includes the removal of Met ^-2 -Lys ^-1 from the selected TPO polypeptide.  26. A method of producing a TPO polypeptide having a biological activity of specific stimulation or an increase in platelet formation, comprising the steps of expressing a polypeptide encoded by DNA according to any one of claims. 19-23, in a suitable host and isolation of the TPO polypeptide. 27. A method of producing a TPO polypeptide having biological activity of specific stimulation or increased platelet formation, comprising the steps of: a) expressing the polypeptide encoded by the DNA of claim 23 in a suitable host; and b) isolating said GST (thrombin recognition peptide) of the TPO polypeptide; c) treating said GST (thrombin recognition peptide) of the TPO polypeptide with thrombin; and d) isolating the Gly ^-1 TPO polypeptide.  28. A pharmaceutical composition for treating platelet-related disorders, thrombocytopenia in general, and thrombocytopenia induced by chemotherapy, radiation therapy, or bone marrow transplantation, comprising an effective amount of a polypeptide according to any one of claims. 1-17 in combination with a pharmaceutically acceptable carrier.  29. A method of treating platelet-related disorders, comprising administering an effective amount of a pharmaceutical composition according to claim 28 to a subject having such a disorder.  30. A method of treating thrombocytopenia, comprising administering an effective amount of a pharmaceutical composition according to claim 28 to a subject having thrombocytopenia.  31. A method for treating thrombocytopenia induced by chemotherapy, radiation therapy, or a bone marrow transplant in a subject, comprising administering to him an effective amount of a pharmaceutical composition according to claim 28.  32. Antibody to TPO polypeptide obtained by a method comprising the steps of immunizing an animal with a TPO polypeptide according to any one of claims. 1-14 and isolation of antibodies to the TPO polypeptide.  33. The antibody of claim 32 for isolating a polypeptide according to any one of claims. 1-14.  34. The antibody according to p. 32 for the quantitative determination of the polypeptide according to any one of paragraphs. 1-14.

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