TW498079B - Protein having TPO activity - Google Patents

Abstract

The present invention relates to a protein having TPO activity, a method for making the same, DNA fragment with sequence encoding the protein, a recombinant vector containing such DNA fragment, a host cell transformed with said DNA fragment, and a pharmaceutical composition with such protein as effective ingredients. The present invention makes mass production of said protein possible, and is effective in the treatment and diagnosis of platelet defect. The present invention discloses a protein having TPO activity and a DNA sequence encoding the same.

Description

A7 B7 V. Description of the invention ([0001] [Industrial scope of use]-(Please read the precautions on the back before filling out this page) The present invention is related to the ability to promote the proliferation and differentiation of megakaryocyte precursor girls' cells. In vivo, specifically stimulates platelet production or enhances the activity of chopping protein, the DNA encoding this protein, and the manufacturing method of this protein. [0002] [Previous technology], megakaryocytes are mainly in the bone marrow It is observed that it is a multinucleated, large cytoplasm-rich large cell that can produce platelets. The source of megakaryocytes is the pluripotent stem ® 0S in the epiphysis. The pluripotent stem cells differentiate to a certain extent and become only towards the megakaryocytes. The megakaryocyte precursor cells (CFU-MK) re-proliferate and differentiate into megakaryocytes. The megakaryocytes complete nucleus multiplication (ρ 〇1 yp 1 〇idizati ο η) ·, cytoplasmic maturation, and finally Platelets released into the bloodstream as non-nucleated cytoplasmic fragments. An average of 1,000 grade platelets are produced from a mature megakaryocyte. There are many differences in the mechanism of platelet production. It is presumed that the megakaryosphere is located under the endothelium of the venous hole of the bone marrow, and its cytoplasm rewards the endothelium. A cord-like protrusion grows in the venous hole and releases platelets. This type of megakaryocyte hematopoietic and platelet production indicates the existence of a specific regulatory mechanism for production. It is known that healthy and normal animals maintain an effective number of platelets. When anti-platelet substances are administered to normal animals, platelets are only drastically reduced in a short period of time. After that, it began to increase, and temporarily exceeded the normal value. 498079 A7 B7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of the Invention (2) Finally returned to the normal value. It is also known clinically, although red blood cells Counts and white blood cells are normal, but they are also involved in low platelet counts (thrombocytopenia) and thrombocytosis. However, to date, they are responsible for specific regulatory factors of this production mechanism (for example, in the production of red blood cells, such as erythropoietin exists The identification and identification were not successful. [0004] The most important function of platelets is to stop Thrombosis is formed in the institution. Thrombocytopenia, the hemostatic mechanism cannot function normally, showing bleeding tendency. Radiotherapy and chemotherapy for cancer > Thrombocytopenia due to epiphyseal inhibition is a fatal comorbidity, in order to prevent bleeding in patients with this cancer Platelet transfusion is performed. Patients who have received bone marrow transplantation and patients with aplastic anemia also perform platelet transfusion. [0005] For such platelet transfusion, platelets are transfused from the blood of a healthy blood provider by platelet centrifugation. Preparation, platelet storage for blood transfusion has a short life span and the possibility of bacterial infection. It also exposes patients to dangerous viruses such as human immunodeficiency virus (ΗIV) or various hepatitis viruses, allowing patients to derive tissues on the surface of blood transfusion platelets Antibodies to a suitable antigen (HLA) or the risk of graft disease to host disease (GVHD) caused by lymphocytes mixed with platelets for blood transfusion. [0006] Thus, it would be extremely advantageous if the endogenous platelet production could be stimulated in patients with thrombocytopenia while reducing the dependence on platelet transfusion. Also, for patients undergoing radiation therapy and chemotherapy for cancer, if they can correct or prevent platelets (please read the precautions on the back before filling out this page). Binding · Order · Line-5-This paper uses China Standard (CNS) Α4Λ grid (210X297mm) Printed by the Central Standards Bureau of the Ministry of Economic Affairs and Consumer Cooperatives 498079 A7 B7 Fifth, the description of the invention (5) is reduced, making these treatments safer, and the concentration of treatment can be improved. Look forward to a better healing effect. [0007] For these reasons, many studies on the isolation and identification of specific regulatory factors that regulate the production of megakaryocytes and platelets have been performed enthusiastically. Based on the research in the test tube, it is considered that the regulatory factors related to the proliferation and differentiation of megakaryocytes can be roughly divided into the following two types (for example, refer to Williams et al., J, Cell ·. Physiol, 110 vol. '101-104 (1 882)). Megaspheroid community stimulation factor (Mes-CSF) is a regulator that promotes the proliferation and differentiation of CFU-MK. Experimentally, in a semi-solid culture medium, it has the ability to induce the formation of a colony formed by megaspheres (Colony). Of activity. Another regulatory factor is called megakaryocyte expansion factor (MerPot), megakaryocyte stimulus factor, megakaryocyte. Proliferation-promoting activity, platelet generation stimulus factor, etc., mainly make the megakaryocyte function in the differentiation stage under CF11-, and promote differentiation ,mature. In experiments, it is often detected to increase the activity of Meg-CSF. In addition, experimentally, the serum and plasma of the thrombocytopenia of the animal that caused the thrombocytopenia were injected into other normal animals > It is presumed that there is a humoral factor with a platelet increasing effect in the body from the increase of platelets, It is called thrombopoietin (TP0). [0008] In recent years, several selected cytokines have been investigated for the effects of genes in test tubes on megakaryocytes and platelet increase. Human IL-3 stimulates the formation of human megakaryocyte communities (Bruno et al., Exp. Hematol ·, Vol. 16, 37 pp. 1-377, 1 988)) At least it also leads to an increase in monkey platelets. This paper is applicable to Chinese standards. (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page) ---------------- ^ ------ 、 Order 丨 498079

V. Description of the invention (4) (Donahue et al., Science, · 241, 1 820-page (1 988)), however, IL-3 is a factor that can bring about the proliferation and differentiation of all hematopoietic cells and can distinguish megakaryocytes Specific regulators of hematopoietic and platelet production. Human IL-6 does not show Meg-CSF activity, but acts on immature megakaryocytes, and promotes differentiation into mature megakaryocytes (Williams et al., Exp. Hematol., Vol. 18, p. 69 (19 9 0)). In mice and monkeys, it showed platelet increase effect, which increased the size and nucleus of bone marrow megakaryocytes, but it was also considered to have side effects such as weight loss and induction of acute phase proteins (Asano et al. Blood, Volume 75, 1602 -1605 (1990); Stahl et al., Blood, vol. 78, 1467-1475 (1991)). Human IL-11 has also been reported not to show Meg-CSF activity, exert Meg-Pot activity, and have a platelet-increasing effect in mice (Neb eri et al. Blood, Vol. 81, pp. 1-908 (1993)). In addition, human LIFs deliberately increase platelet counts in monkeys (Mayer et al., Blood > Vol. 81, 3226-3323 (1 993)) and have a weak effect on megakaryocytes in test tubes (Burstein et al., J. Cell. Physiol., Vol. 153, pp. 305-312 (1992)). [0009] Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) These cytokines are expected to have the potential for clinical application as platelet increasing factors, but not only for Megakaryocytes have specific effects and are also recognized to have side effects. Therefore, platelet-increasing factors with fewer clinical side effects and specificity for platelet 糸 are expected. [0010] Meg- -7- This paper size applies Chinese national standards (CNS) ) Α4 size (210 × 297 mm) 498079

Printed by the Central Laboratories of the Ministry of Economic Affairs and Consumer Cooperatives. V. Description of the invention (5) CSF, Meg-Pot or TP0 activity 'but this factor K exists alone or in multiples to exert activity' and is similar to the known factors, almost the same Unexplained. [0011] In terms of factors derived from human organisms, Hof f man is equal to that of aplastic anemia after reduction of megakaryocytes in the bone marrow and patients without megakaryotic thrombocytopenic purpura. In addition to thrombocytopenia, healthy patients Compared with intentionally high Meg-CSF activity (Hoffman et al., N. Eng. J. Med., Vol. 305, pp. 533-538 (1981)), Mazur et al. Are more cyanotic and are associated with aplastic anemia Patients' serum showed this Meg-CSF activity to be different from IL-3 or GM-CSF (Mazur et al., Blood, Vol. 76, pp. 290-297 (1 990)). Similar Meg-CSF activity was also detected in the serum of cancer patients undergoing centralized chemotherapy and patients undergoing bone marrow transplantation (Mazur et al., Exp. Hematol., Vol. 12, pp. 624-828 (1984); de A 1 arc ο η and S ch in ieder, Pr og · C 1 i η · B i 〇 · Res ·, Vol. 2 1 5 > pp. 335-340 (1986)). Hoffman et al. Reported the purification of Mes-CFS with an apparent molecular weight of 46,000 from the plasma of patients with thrombocytopenia with low megakaryocyte formation (Hoffman et al., J. Clin. Invest. ≫ 75, 1174-1182 (1985)), In subsequent studies, it was understood that the purity of the purified reference standard was not sufficient to determine the amino acid sequence (Hoffman et Blood, Vol. 76, 1196-1212 (1989)). 75S e-selenomethionine (75S e-selenomethionine) was partially purified from the plasma of patients with similar thrombocytopenia or from the urine of patients with idiopathic thrombocytopenic purpura (I TP). TP0 sample paper size, using China National Standard (CNS) A4 specification (210X297 mm) binding ^^ (please read the precautions on the back before filling this page) 498079 A7 B7 V. Description of the invention (6) Activity, related to plasma-derived activity, has been shown to have an optic molecule of 40,000 (Guangzhou 331, et al., Hematologica, Vol. 72, '291-295' (1987); Vannucchi et al., Leukemia, Vol. 2, pp. 236-240 (1,988) ) [0012] Meg-CSF activity and TP0-like activity were also detected in the urine of patients with aplastic anemia and severe ITP (Kawakita et al., Br. J. Haematol. ≫ Volume 48 > 609 -6 Page 1 5 (1981); Kawakita et al., Blood, Vol. 61, pp. 556-560 (1983)). In addition, Kawakita et al. Report Meg-CSF activity contained in urinary extracts from patients with aplastic anemia under dissociation conditions. Gel filtration 45000 (Kawakita et al., Br > · J · H ae ni at ο 1 ·, Vol. 8 2, 7 1 5-7 2 2 H (1 98 6)) 0 Erikson-M i 1 1 er etc. also reported from the same sample Refined Me es-CSF, but its structure is not explicitly stated (E riks ο n-Μ 1 1 er, etc., B 1 ο od C e 1 1 G rowth Factors · their present and future use in hematology and oncology) Murphy edited 'AlphaMed Press, Dayton, Ohio, pages 204-220 (1992)). Turner et al. Refined megakaryocyte stimulating factor (MSF) with Meg-CSF activity from the urine of patients undergoing bone marrow transplantation and gene Breeding (Turner et al., Blood, printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page), Volume 78, Page 1106, 279a (1991) (abstr ·, suppl · 1)) This MSF is a dimer of a protein having a molecular weight from 28,000 to 35,000. Whether this factor is the same factor as Me g-C S F detected in the serum and plasma of patients with thrombocytopenia so far, and whether it has a platelet increasing effect in vivo is unknown. -9- This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) 498079 A7 __ WJ__ V. Description of the Invention (7) [0013] From the human fetal kidney cell strip (Η El (cell)) The supernatant was cultured to refine the TP0-like activity with a molecular weight of 320,000, and various traits are being investigated, but its structure is still unknown (McDonald et al., J. Lab. Clin, Med., Vol. 106, pp. 162-174 (1985); McDonald ^ Int. J. Cell Cloning, Vol. 7, pp. 139-155 (1989)). On the other hand, other researchers have also reported that EK cells are present in the distribution medium and promote the megakaryocyte maturation in test tubes. The activity is through the production of known cytokines from this cell, namely IL-6 and TP0 (Withy et al., J. Cel 1, Physiol., Vol. 15 "3362-372 (1992)). [0014] Factors, Evatt et al. Reported that the rabbit thrombocytopenic plasma injected with anti-platelet antibodies has a TP0-like activity that promotes the incorporation of rabbit neonatal platelets into 75Se-selenomethionine (Evatt et al., J. Ub Clin. Med., Volume 83 > pages 364-371. (1 974)) Several other similar reports can be seen from the 1960s to the 1970s (for example, Odell et al., Proc · Soc, Biol. Med ·, Vol. 108, pp. 428-431, (19 61); Evatt and Levin, Printed by J. Clin. Invest. Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the notes on the back before filling out this page), Vol. 48, 1615-1626 (1969); Harker, Am. J. Physiol., Volume 218, pages 1376-1380 (1970); Shreiner and Levin, J. Clin · Invest ·, Volume 49 pages 1709-1713 (1970); Penington, Br. Med. J, Volume 1, pages 606-608 (1970) )). Evatt et al., And Hi 1 1 and Lev i η, partially purified TPO-like activity from rabbit plasma during the platelet-reduction phase induced by the administration of antiplatelet antibodies (Evatt et al. Blood, -10- 498079 Ministry of Economic Affairs Printed by A7 B7 of the Consumer Standards Cooperative of the Central Bureau of Standards (5) Invention Description (8) 54 pp. 377-388 (1979); Hill and Levin, Exp. Hematol., Vol. 14, 75 2-759 (1986)) , And then the activity of K to promote the differentiation and maturation of megakaryocytes in the test tube, that is, the activity of M eg-P ot as an indicator, The refinement of this factor was explicitly stated on the gel, and the molecule was found to have a molecular weight of 40,000 ~ 46,000, but it did not reach the full refinement (Keller et al., Exp. Hematol ·, Vol. 16, pp. 262-267 (1 988); Hi 11 et al., Exp, Hematol., Vol. 20, pp. 354-360 (1992)). IL-6 activity was not detected in the plasma of rabbits with severe acute thrombocytopenia induced by anti-platelet antibody, showing that this TPO-like activity and IL-6 are carried by different molecules (Hill et al., Blood, Volume 80, 346 -Page 351, (1992)). [0015] Tayrien and Rosenberg also purified the megakaryocyte-derived rat culture cell strips from the same rabbit plasma or HEK cell culture supernatant to stimulate platelet factor 4 to produce a factor with an apparent molecular weight of 15,000, Its structure is not explicitly stated (371 ^ 611 and 1 ^ 〇501 ^ 61 ^, ".131〇1.〇11 ^ 111., Vol. 26, pp. 3262-3268 (1987)). [0016] In addition, Nakeff found Meg-CSF activity in the serum of mice that reduced platelets by administering anti-platelet antibodies (Nakeff, ,, Experimental Hematology Today, edited by Baum and Ledney, Springer-VeHag, NY 11 Bu P. 123 (1977)). On the other hand, in the same way that K treated with thrombocytopenia in rabbit plasma, megakaryocyte maturation was detected in a test tube (Keller et al., Exp. Hematol., Vol. 16, pp. 262-267 (1988); Hill et al., Exp Hematol ·, Volume 17, (Please read the notes on the back I # before filling and loading-: write this page) Order -1 ^ ^ Paper size applies Chinese National Standard (CNS) A4 specifications (210X297 mm) Printed by the Central Laboratories of the Ministry of Economic Affairs and Consumer Cooperatives 498079 A7 B7 V. Description of the Invention (9) pp. 903-907 (1989)) and the activity of stimulating megakaryocytes to change the shape of platelets (Leven and Yee, Blood, 69 Vol. 1046-1052 (1 987)), but is not considered to have Meg-CSF activity. The reason for such a mistake is unknown. [0017]

Miura et al. Picked out Meg-CSF activity in the platelet-reducing plasma of whole-body-irradiated rats with sublethal doses of radiation (Mi ura et al. Blood, Vol. 83, pp. 1060-1066 (1984)). Platelet transfusion without reduction, the induction of Meg-CSF activity in the living body, not the reduction of thrombocytopenia but the reduction of megakaryocytes is necessary (Miura et al., Exp · Hematol ·, Vol. 16 > pages 139-144 (1988)). Mazur and

South reported that Mes-CSF was detected in the serum of dogs with aplastic anemia by radiation irradiation > found on gel filtration to have a molecular weight of 175,000 (Mazur and South, Exp. Hematol., Volume 13, 1164-11 72 (1 985)). In addition, factors derived from serum, plasma, or urine are also reported by StranevaH (Straneva ^, Exp * Herarat ο 1. ^^ 15, 6 5 7-66 3 pages, (1 987)) and others. [0018] Thus, although human and animal biological samples of thrombocytopenia have been confirmed to have factors that promote megakaryocyte hematopoietic and platelet generation, so far, such factors have been isolated, biochemically and biologically. The above identification and characteristics determine that it is unsuccessful because natural sources such as blood and urine only exist in trace amounts of such factors. [0019] This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) --------- install-(Please read the precautions on the back before filling this page) Order 498079 A7 _B7___ V. Description of the invention (10) [Problems to be solved by the invention] Therefore, the purpose of the present invention is to isolate from a natural source, identify and promote the proliferation and differentiation of megakaryocyte precursor cells, or stimulate or enhance living organisms. A protein (TP0) that specifically produces platelet activity (TP0 activity). Furthermore, the gene encoding this protein is isolated by using the sister DNA technology to provide a means for homogeneous mass production of the protein. If possible, it can replace the current platelet transfusion, reduce the frequency, or be used for the treatment and diagnosis of platelet disorders. [Means for Solving the Problems] According to the present invention, a novel D N A (hereinafter referred to as "D N A of the present invention") is provided which encodes an amino acid sequence of a protein having TPO activity. The D N A of the present invention has an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. [0021] Furthermore, in the DNA of the present invention, the amine group shown in the sequence number 2 •, sequence number 4, or sequence number 6 described above. In the case sequence, as long as TP 0 activity is maintained, one part is changed (Substitutions, deletions, insertions, and additions), that is, DNA encoding the TPO derivative is also included. Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling out this page) [0022] In other words, the DN A of the present invention includes the serial number 2 •, serial number 4, or serial number DNA of the amino acid sequence shown in 6. The words "substantially the amino acid sequence shown in sequence number 2, sequence number 4, or sequence number 6" herein include "sequence number 2, sequence number 4, -13- (CNS) A4 specification (210X297 mm) 498079 A7 B7 V. Amino acid sequence shown in the description of the invention (n) or sequence number 6 "plus" as long as TP0 activity is maintained, in sequence number 2, sequence number 4, or A part of the amino acid sequence shown in SEQ ID NO: 6 includes amino acid sequences such as substitutions, deletions, insertions, and additions. " [0023] Here, the term "DMA encoding an amino acid sequence" means DNA of all base sequences having a degenerative relationship. · [0024] Furthermore, the DNA of the present invention is an amino acid sequence encoding a protein with TP0 activity, and contains 选自 ~ (〇.) Selected from K. [0025] ⑶ sequence number 2, sequence number 4 or sequence number 6 The DNA or their complementary strands shown. (B) Forms a kind of DNA with the DNA of (a) or a fragment thereof (under strict conditions) 〇 (〇If there is no degeneracy of the genetic code, K and ⑻, (B) DHA forms hybrid DNA. [0026] Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page). In other words, the DNA of the present invention also includes the following K DNA represented by any of (b). [0027] DNA DNA encoding an amino acid sequence of a protein having TPO activity incorporated into the vector PEF18S-A2α (trust number FERM BP-4565) carried in E. coli DH5 , Or code with a carrier incorporated into coliform DH5 -14- This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) 498079 Printed by A7 B7, Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (12) ~ pHTI-231 (subject to FERM BP- 45 6 4) amino acid sequence of TP0-active protein. (B) DNA encoding amino acid sequence of TP0-active protein ·, and DNA or fragments thereof (under strict conditions) A hybrid DNA is formed. [0028] The DNA of the present invention contains a base sequence encoding an amino acid sequence from the 1st position to the 16th position in the amino acid sequence shown in SEQ ID NO: 6, and also contains an encoding A protein with TP0 activity. [0029] This type of DNA can include the supply of the site that is cut off by restriction enzymes, and / or to enable the expression of the expression vector to the beginning, the end, or the middle When the non-mammalian host is selected, it can also be incorporated there to express the preferred codons (priority codons). [0030] The DNA of the present invention includes mammals that originate from K humans. After the mRN A of cells is modulated, the cDN A gene library prepared by a known method is used to screen the obtained cDNA. The source of niRN A in this case can be the cell line MdR from rat liver cells 994 8994 cells, H TC cells, Η 4-I -E cells, rat liver, kidney, brain, small intestine, human liver, etc. [0031] The D N A of the present invention may also be derived from mammalian cells starting from humans. The library screens genomic DNA by known methods. The supply of genomic DNA in this case can be chromosomal DNA of humans, rats, mice, etc. (Please read the precautions on the back before filling in this page) -Packing · ^ 1 ▼ Line-15- This paper size is applicable to China National Standard (CNS) A4 (21〇 > < 297mm) 498079 Α7 Β7 V. Description of the invention (15) [0032] In addition, by modifying the c DNA encoding the protein with TP0 activity thus obtained by well-known site-specific mutation method, a part of the amino acid sequence can be changed Or DNA encoding a TP0 derivative. [0033] In addition, the amino acid sequence / base sequence of a protein having TP0 activity disclosed in this detailed description, and DNA encoding a protein that changes a part of the amino acid sequence, can be easily borrowed by the industry. It is obtained by chemical synthesis of part or all of it. [0034] The DNA of the present invention is a useful substance for mass-producing a protein having TPO activity through various genetic recombination techniques. [0035] Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page). Furthermore, the DNA of the present invention is a gene encoding a protein associated with TP0, and other mammalian genes. TPO's CDNA, M and gene DNA are suitable for use as labeled probes. In addition, there are gene therapies for humans and other mammals. The DNA of the present invention can be used as eukaryotic cells for the mass production of TPO Useful for the development of mammalian species that have been transformed with foreign genes used by the host (Palmiter et al., Science, 222, 809-814 (1983)). [0036] According to the present invention, it is provided to isolate and purify the TK with TP0 activity as described above. The protein is the carrier of DNA, the host cell transformed by the vector, and the host cell is cultured to produce a protein with TP0 activity. -16- This paper size applies to Chinese National Standard (CNS) A4 specification (210 × 297) ) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 ____ ^ B7 _ ^ ___ 5. Description of the invention (U) Manufacturing of proteins with TP0 activity [0037] * As the host cells in this case, prokaryotes (such as coliforms), true cough organisms (such as yeast, mimicry, or mammals) cells can be used. Examples of mammalian cells include COS cells, China Hamster ovary (Chinese Hamster Ovary) cells, 0127 0 cells, BHK (young hamster kidney) cells, etc. Examples of yeasts include baker's yeast (Saccharomyces cerevisiae) and methanolized yeast (Pichia pastoris). Examples are silkworm culture cells, etc. [0038] Among the vectors that can be used to transform these host cells, pKC3 0 (Shiniatake H. and M. Rosenberg, Nature, 292, 128-132, 1981), pT "C99A (Amann E. et al., Gene 6, 69, 3 0 1-3 1 5, 1 9 8 8). As mammalian cells, PSV 2-neo (Southern and Berg; J. Mol. AppI. Genet, & gt 1 > 327-341, 1982), pCAGGS (Niv / a et al .; Gene, 108, 193-200, 1991) or pcDL-SR a296 (Takebe et al .; Mol. Cell. Biol. ≫ 8, 466 -472, 1 988), etc. As yeast, pG-1 (Schena M. and Yamamoto KR; Scien ce, 241, 965-967, 1 988). As a transfer vector for producing silkworm cells with heavy sister virus, pAc: 373 (Luckow et al., Bio / Technology, 6 '47-55' 1988) and the like. [0039] These vectors must contain the origin of replication, selection markers, and promoters as required. -1 7-This paper is based on the Chinese National Standard (CNS) A4 specification (210X297 mm) (please read the note on the back first) Please fill in this page for more details)

1T 498079 A7 B7 V. Description of the invention (15) In addition, as a carrier for eukaryotic cells, R N A splicing sites and polyadenylation are added as required. [0040] As a starting point of replication, a vector for mammalian cells can be derived from SV 40, adenovirus, U.S. Babbioma (Eve > Θ ^ 7) virus, and the like. As the carrier for coliform, Co I E1 ·, R factor, F factor and the like can be used. Yeast shake can be used from 2 to in D N A, A R S 1 and so on. As the promoter for gene epitope, vectors for mammalian cells can be derived from retrovirus, polymorphoma virus, adenovirus, SV40, and the like. As the vector for coliform, phage-derived persons, t r p, 1 p p ·, 1 a c, t a c promoters, and the like can be used. For baker's yeast, A D Η ·, P Η 0 5 ·, G P D, P G K _, MAF cx promoters can be used, and for methanol-qualified yeasts, A0X1 promoter can be used. As the carrier for silkworm cells, those derived from nuclear polyhedrosis virus can be used. [0042] As a selectable marker, a neomycin resistance gene, a thymidine kinase (TK) gene, a dihydrofolate zymogenase (DH FR) gene, and a coliform xanthine guanine phosphoribosyl transfer can be used as a mammalian carrier Enzyme (Ecogpt) gene and so on. For the large intestine, a concomycin resistance gene, an ampicillin resistance gene,

X Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) Tetracycline resistance genes, etc. For yeast, Lue2, Trp1, and Ura3 genes can be used. [0043] When using a host vector strip as described above to obtain a protein with τ p 0 activity, 'If the gene is incorporated in an appropriate part of the above recorder Recombinant D Ν △ -1 8-This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) '' 498079 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 _B7_ V. Description of the invention ( 16). After the host cell is transformed, the obtained transformant is cultured, and then the polypeptide can be separated and purified from the intracellular or culture medium. The means and methods used in these methods can be combined with known methods. [0044] In the case of using host expression, in order to more uniformly homogenize the N-terminus of the expression product, the original signal sequence may be changed, or other protein signal sequences may be used. By changing (substituting or adding) the amino acid residues at and near the N-terminus (for example, in the case of coliform bacteria, adding methionine residues, adding lysine, etc.), it is also possible to change N End homogenization. The novel protein with TPO activity of the present invention (hereinafter referred to as "the protein of the present invention") is a protein formed from the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 . In addition, as long as TP 0 activity is maintained, the present invention also includes a part of the amino acid sequence that has been changed (replaced, deleted, inserted, and added), that is, a TP0 derivative. [0046] In other words, the protein of the present invention contains an amino acid sequence substantially as the protein having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. As used herein, "substantially the amino acid sequence shown in sequence number 2, sequence number 4, or sequence number 6" means "sequence number 2 •, sequence number 4, or sequence number 6" "Amino acid sequence" plus "As long as TPO activity is maintained, there are substitutions, deletions, insertions and additions of amines in a part of the amino acid sequence shown in sequence number 2, sequence number 4, or sequence number 6" Amino acid sequence. " (Please read the precautions on the reverse side before filling out this page) -19- ^ The paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 498079 Printed by the Central Standards Bureau of the Ministry of Economic Affairs-Industrial and Consumer Cooperatives A7 B7 Explanation (17) [0047] The protein of the present invention also contains the amino acid sequence shown in SEQ ID NO: 6, including the amino acid sequence from position 1 to 16 and having a TP 0 activity. [0048] As another example of the TP 0 derivative of the present invention, for example, those who attempt to increase stability (substitution, deletion, insertion, and addition) of amino acids to seek stability and increase in the body in a sustained manner , Additional deletion of more than one potential part of the sugar chain or addition that causes changes, deletion of 1 cysteine residue or other amino acid residues (such as alanine or serine residues) ) Those who replace cystine. [0049] Preferably, the protein K of the present invention is isolated from a host cell containing cDNA, genomic DMA, or DNA transformed by chemical synthesis, and purified from host cells. [0050] As a host, when bacteria (such as coliforms) are used for epidermal cell surface expression, a protein having a methionine residue on the H-terminus side of a protein having TP0 activity can be obtained, and this protein is also included in this invention. Depending on the host used, there may be cases where the produced protein with glycosylation is glycosylated, or there may be cases where there is no glycosylation, any of which is included in the present invention. [0051] In addition, the protein of the present invention also contains refined and isolated from natural supply sources (for example, distribution medium containing TP 0 activity or human urine, serum, plasma) -20- This paper size applies Chinese national standards (CNS ) A4g (210X297mm) (Please read the precautions on the back before filling this page) —Package ·

1T 498079 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of invention (π) Natural protein with TP0 activity. According to the present invention, a method for refining from such a natural supply source is included. As such a purification method, there are processes using general purified proteins (ion exchange chromatography, lectin affinity ephemeris, pigment adsorption ephemeris, hydrophobic interaction chromatography, gel percussion, and inverse Phase chromatography, heparin affinity calendar, sulfated gel calendar, hydroxyapatite chromatography, metal chelate calendar, isoelectric point chromatography, fractional electrophoresis, and isoelectric point electrophoresis Method, etc.). In addition, the physicochemical properties of the estimated TPO estimated by K from the examples described later are also included in the present invention. Furthermore, an antibody column capable of recognizing an antibody to TPO can also be used. [0053] The present invention also includes proteins of a kind encoded by a part of DNA complementary to the protein codon of TPO · human cDNA or genomic DNA, ie, such as Tramontano et al. (Nucl · Acids Res ·, 12, 5049-5059 (1 984)) ★ Complementary reverse direction protein 〃. [0054] The present invention also includes a test substance labeled with a reagent that can be used to detect and quantify TPO or cells that retain its receptor in a liquid sample such as solid tissue and blood or urine (for example, K 1 25 I radiolabeled, or biotinylated) protein of the invention. [0055] The biotinylated protein of the present invention is transplanted into the epiphysis of the bone marrow. It is applied from bone marrow-21- this paper A degree applies the Chinese National Standard (CNS) A4Λ grid (210X297 public director) --------- equipment --- (Please read the notes on the back before filling this page), ^ 1 498079 A7 _B7 _ V. Description of the Invention (β) The removal of megakaryoblasts is useful for binding with immobilized chain avidin . In addition, in the case of self or allogeneic bone marrow migration, it is the case of concentrating itself or allogeneic megakaryocytes as cells, and it is sometimes used for binding to the immobilized chain antibiotic protein. Such as ricin, a combination of toxin of diphtheria toxin and TP 0 toxin, and radioisotopes are useful therapies for anti-cancer therapy or callus transplantation. [0056] In addition, the present invention also provides the occasion of labeling with a detectable label (such as a radiolabel or a non-isotopic label such as biotin), and to identify the position of the TPPO gene on the chromosome and / or any related gene system The hybrid method used for the position of the group includes the nucleic acid substance. These are also used to confirm that the previous human TP0 gene disorder in DNA can also be used as a genetic marker to identify adjacent genes and their disorders. [0057] Furthermore, the present invention also includes a peony composition containing and suitable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers together with a therapeutically effective amount of the protein of the present invention. . The term "a therapeutically effective amount" used in this patent specification means an amount that provides a therapeutic effect for a given condition and administration method. This composition is liquid or freeze-dried or dried, and contains various ingredients and ionic strength (for example,

Tris s-hydrochloric acid, acetate, phosphate) Diluents selected · Additives such as albumin or gelatin that do not adsorb on the surface, surfactants (such as Tween 20, Tween 80, P1 uron ί c F68 ·, Bile salts), solubilizers (eg, glycerin, polyethylene glycol), antioxidants (eg, ascorbic acid, -22- This paper size applies Chinese National Standard (CNS) grid (210X297)) ρύ 'V pack— ( (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs -Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Description of the invention (20) Sodium metabisulfite), preservatives (such as thimerosal, benzyl alcohol, parabens), excipients or Isotonic agents (eg lactose, mannitol) blended preparations. In addition, the protein contains a covalent bond with a polymer such as polyethylene glycol, a complex with a metal ion, or a granular preparation of a polymer such as polylactic acid, polyglycolic acid, or hydrogel, or the like. Surfaces, or ribosomes, microemulsions, microcells, mono- or multilayer cells, erythrocytes, or protoplast spheroids are incorporated. This sister compound is affected by the physical state, solubility, stability, release rate in vivo, and in vivo clearance rate of TP0. The choice of sister compound is based on the physical and chemical properties of the protein with TP0 activity. On the characteristics. Furthermore, a polymer (for example, F 今 4 P, &lt; os M y〆) and a tissue-specific receptor, an antigen bound to a ligand or an antibody, or a tissue-specific receptor binding The granular TP0-coated granular compound is also included in the present invention. As other dosage forms of the composition of the present invention, parenteral, transpulmonary, transdermal, and oral routes are used for various administration routes, and granular forms, protective coatings, protease inhibitors, or absorption enhancers are blended. [0058] The peony composition containing the protein of the present invention is usually 0.5 micrograms / kg body weight to 1 mg / kg body weight as an active ingredient, and can be administered several times a day according to the condition, sex and administration route . [0059] In addition, the present invention includes a protein containing the present invention plus, for example, EP 0 ·, G-CSF, G M-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 &gt; IL-12 -23- This paper size is applicable to China National Standards (CNS) Μ Specifications (210X297 mm) I --------- φ— ^ —— (Please read the notes on the back before filling this page) -i'tv line-498079 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Crack A7 B7 V. Description of the Invention (21), IL-13, LIF, SCF, a factor of one K as a composition of additive hematopoietic factors. [0060] The protein of the present invention, alone or in combination with other additive hematopoietic factors, is useful for the treatment of most thrombocytopenia. Other additive hematopoietic factors include EPO, G-CSP, GM-CSF, M-CSF, IL-1, IL-2, 1 ~ 3, IL-4-IL-5 &gt; IL-6, IL -7 ^ IL-8-IL-9 &gt; IL-10, Hi, IL-1 2, IL-1 3, LIF, SCP. Due to platelet disorders such as platelet dysfunction and shortened platelet life (the increase in platelet destruction or the increase in platelet depletion), the number of blood platelets is reduced to a majority of the diseases of special molds, which can be treated with the protein of the present invention. For example &gt; Congenital Fanconi's anemia, aplastic anemia accompanied by chemotherapy and radiation therapy, dysplasia syndrome, acute myelogenous leukemia or thrombocytopenia with bone marrow insufficiency caused by bone marrow transplantation Disease. Can be used to promote platelet recovery in these patients. It is also useful for thrombocytopenia, which is abnormal due to TP0. Thrombocytopenia due to the shortened lifespan of platelets and megakaryocytes, such as idiopathic thrombocytopenic purpura, acquired immune deficiency syndrome (AIDS), disseminated intravascular coagulation syndrome, and thrombotic thrombocytopenia etc. Platelet recovery is useful in these patients. In addition, it is also useful for administering TPO before surgery to increase platelets, and to use the platelets as a platelet for blood transfusion (self platelet transfusion) during self-operation. [0062] -24- This paper size is in accordance with China National Standard (CNS) A4 (21 × 297mm) --------- install-(Please read the precautions on the back before filling This page) Order line 498079 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (22) Furthermore, other uses of the protein of the invention are, for example, due to chemicals or pill drugs, or therapeutic measures. Treatment of obstacles caused by transient platelet defect or injury. TP 0 can be used to promote the release of new + non-invasive platelets in such patients. [0063] The invention further relates to antibodies that specifically bind TPO. The protein of the present invention can be used as an antigen. Such an antibody contains both a single-source and a multi-source anti-antibody, and a chimeric antibody produced by a known method, that is, a recombinant antibody. In contrast to conventional antibody (multi-source antibody) preparations which generally contain various antibodies against various epitopes, single-source antibodies are antibodies each against a single epitope on the antigen. Specific antibodies against TP0 are useful for the selective and specific use of antigen-antibody reaction diagnostics and improved analytical assays to isolate and purify TP0. Moreover, these can be thrown away from the serum to neutralize or remove TP0. The advantage of single-source antibodies is that they can be synthesized by hybridoma cells in a medium without mixing with other immunoglobulins. Monoclonal antibodies were prepared from the culture supernatant of the tumor cells, or by ascites derived from hybridoma cells inoculated into the abdominal cavity of mice. The hybridoma technology originally documented by Kohler and Milstein (Eur. J. Immunol. 6, 51, 5 1 9 (1 976)) is widely used to generate hybrid tumor cells that retain high levels of single-source antibodies to most specific antigens. article. The present invention will be described in more detail below. [0065] (A) Purification of rat TP0 ~ -25 of partial amino acid sequence of rat refined TP0-This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) ------ --- Equipment-(Please read the precautions on the back before filling this page), ^ Τ Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Description of the invention (25) Analysis ~ Rat refined TP0 Analysis of biological characteristics The present inventors first tried to purify a protein having a rat CFU-MK proliferation and differentiation promoting activity (rat TP 0). In this refining, the choice of various natural supply sources, in addition to the choice of calendar carrier, selection of separation means, etc., refined sister match, repeated many trial and error. As a result, the present inventors purified the plasma of thrombocytopenia-derived rats derived from X-ray or 7-ray irradiation, and refined TP0 activity based on rat CFU-MKK as described in the "Reference Example" described later as an index, and purified The protein with TPO activity determines a part of its amino acid sequence, <Examples 1 to 2>. [0066] The biological characteristics of rat TP 0 derived from its plasma were also tested (Example 3). [0067] A summary of the determination of the partial amino acid sequence from the purified to the purified rat TP 0 is as follows. [0068] (1) Collecting about 1 100 parts of plasma of thrombocytopenia rats irradiated with X-rays or 7-rays, and performed Sep hadex G-25 calendar analysis and anion exchange chromatography (Q-Sepharose FF) The step of re-lectin calendar analysis (WGA-Agarose) method to obtain WGA-Agarose adsorbed TP0 active calendar fractions 0 [0069] (2) Second, this WGA-Agarose adsorbed TP0 active chromatography fractions to perform pigment adsorption Affinity Chromatography (TSK AF-BLUE 6 50MH), Hydrophobic Phase-26- This paper size applies to China National Standard (CNS) A4 (210X297 cm) (Please read the precautions on the back before filling out this page) • Equipment. • Line 498079 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Invention Description (24) Interaction Chromatography (Phenyl Sepharose 6 FF / LS), to Sephacryl S-Chromatography 200 HR). Here, Sephacryl S-200 HR is divided into four peaks due to TP0 activity (from the larger molecular weight, F1 ·, F2, F3, F4). The TP0 activity chromatograms F2 and F3 are concentrated separately as a polymer TP0 control standard. P2, low-molecular-weight TP0 control standard F3, was subjected to the following purification steps, respectively. [0070] (3) Take the low molecular TPO control standard F3M reverse phase ephemeris (YMC-Pack PROTE IN-RP), reverse phase ephemeris (YMC-Pack CN-AP) ·, and then the reverse phase layer Analytical method (Capcel 1 Pack C1). When the obtained TP0 activity was analyzed by SDS gel electrophoresis, and the TP0 activity was extracted from the electrophoresis gel, a band with an apparent molecular weight of about 1700 to 190 00 was confirmed under non-progenitor conditions. [0071] (4) Therefore, all TP0 active chromatographic fractions were run on non-transgenic SDS gel electrophoresis and transcribed on PVDF membrane. Second, the fragmentation of the protein on the PVDF membrane by systematic restriction enzymes to perform peptide fragmentation can determine a part of the amino acid sequence of the TP 0 protein in the rat (based on the amino acid sequence data of the two segments, and implement the rat Genetic selection of TP0). [0072] (5)-In terms of the high-molecular TP0 control standard F2 obtained from Sephacryl S-200 HR, it is also purified as the low-molecular TP0 control standard P3, and the final step of reverse phase chromatography (Capcell Pack C1) The TP0 activity chromatogram obtained was run by SDS gel electrophoresis, and TP0 activity oil was taken (please read the precautions on the back before filling this page).

, ^ T line-27- This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) 498079 Printed by A7 B7 of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (25) ° After investigation, The TP0 from F3 is the same. It can be confirmed that there is activity in the band with an apparent molecular weight of about 17,000 to 22,000 under non-prototype. [0073] (B) Rat TPO production specificization of female cells ~ preparation of raRNA ~ structure of rat c DNA bank Since purified rat TPO is derived from plasma, it is necessary to select the supply of mRNA for cDN A colonization Source of organs or cells. Therefore, based on the biochemical and biological properties of TPO from rat plasma, the TPO activity in the culture supernatant of various organs or cells was screened. As a result, the hairpins were derived from rat liver cell-derived McA ~ RH 8994 cells, HTC cells, and -4-I ~ E cells in the culture supernatant, and the rat's primary liver cells were cultured in the supernatant. In addition, it also had about the same TPO activity as that of TPO from rat plasma. Example 4 &gt;. [0074] On the one hand, the c D N A expression vector, PEF18S, and two types of epidermal vectors, PME18S and pEPBOS, will be incorporated in monkey cells (C 0 S 1 cells) <Example 5>. Since the vector has a variety of selection sites that can be used for the incorporation of the insert &gt; c D N A can be easily selected using a vector with high expression efficiency. [0075] In the structure of the c D N A library, in addition to the above-mentioned table 琨 vectors, p u c 糸 and P B R 糸 plastid vectors, human phage vectors, and the like are mainly used. [0076] The inventors, etc., cultured McA-RH8994 cells, and added guanidine thiocyanate solution ~ 28 ~ This paper uses Chinese National Standard (CNS) A4 size (210X297 mm) I ------- Equipment-(Please read the precautions on the back before filling this page) Threading-498079 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7___ V. Description of the invention (26) After homogenization, use the CaCl density gradient centrifugation method A total RNA &lt; Example 6 &gt; was obtained. [0077] The preparation of R N A can also be performed by the thermal phenol method and the acid guanidol chloroform method. C 0078] Since then, poly (A) + RNA was purified from oligo-dT immobilized latex particles of all RMA, and oligo-dT with restriction enzyme Not I cut sequence was used as a primer, and single-stranded cDNA was synthesized by reverse transcriptase, using RNase双 and E · coli DNA polymerase I to obtain double-stranded cDNA. This was ligated with KNoTI and EcoRI to cut off the cDNA epitope vector PEF18S constructed in Example 5 to transform the competent coliform strain DH5 to construct a cDNA library. [0079] (C) Obtaining rat TPO cDNA fragments according to the PCR method (selection) Based on a part of the amino acid sequence of rat TP 0 purified from rat plasma, a DNA sequence estimated to encode the sequence is predicted and synthesized in Degenerate primers used in polymerase chain reaction. The primer used may be an amino acid sequence based on a position other than the primer M used in the present invention. Further, primers having a high degree of degeneracy can be used without inosine. Furthermore, it is possible to design primers that are used frequently in rats to reduce the degree of degeneracy (see Wada et al., Nucleic Acids Res., 1, 8, 2367 ~ 2411, 1 990) 〇 * [0080] Prepared from the front Extraction of plastid DNA from the entire cDNA library &gt; A band of approximately 330 bp was detected when PCR was performed using this DNA as a template &gt; When determining the base sequence, understand the DN A fragment that is part of the gene encoding rat TP0 ( AIM paragraph) &lt; Example 8 &gt;. (Please read the notes on the back Φ— • Items and then fill in-Pack —: Write this page), ιτ 线 -29- This paper size applies to China National Standard (CNS) Α4 size (210 × 297 mm) 498079 Central Ministry of Economic Affairs Printed by Standards Bureau's Consumer Cooperative

A7 ___B7__ V. Description of the invention (27) [0081] (D) Screening of rat TPC cDNA by PCR ~ Sequence of rat TPΌ cDNA ~ Table 琨 Confirm that the cDNA library of the previous sister is divided into about 10,000 each From the aggregates of pure strips, plastid DNA was taken from 100 of them. Using these DNAs as a template and using the newly synthesized primers to perform p c R based on the base sequence of the A 1 fragment, 3 aggregates out of 100 aggregates were detected as specific bands. Therefore, one of these aggregates was selected, and this aggregate was each divided into about 90 pure sub-aggregates. When the same PCR was performed on 100 aggregates among them, three Sub-aggregates picked up the zone. Among these, 1 aggregate was selected and subdivided into 40 pure lines of sub-aggregates. Finally, 1 pure 各 was selected by the same PCR, and successfully isolated the rat with the TP 0 gene. c The plastid p EP 1 8 S _ A 2 α of the DNA fragment is pure &lt; Example 9-9 1 &gt;. When the base sequence of this c D N A fragment was determined, it was understood that it encodes a partially-determined amino acid sequence purified from rat plasma, and was considered to be the rat TPO gene <Example 10 &gt;. [0083] When the purified pure plastid DNA obtained as described above is transfected into COS 1 cells, TP 0 activity can be detected in the culture supernatant. As a result, it was confirmed that the obtained pEF18S-A2a was loaded with a cDNA encoding rat TPO <Example 11>. [E] TPO mRNA detected in various tissues of rats (please read the precautions on the back before filling this page)-binding · lining · -30- This paper size applies Chinese National Standard (CNS) A4 specifications ( 210 X 297 mm) Printed by the Consumers' Cooperative of the China Standards Bureau of the Ministry of Economic Affairs 498079 A7 _____B7 ____ V. Description of the invention (28) The TP 0 niR NA surface tissues analyzed in the rat body by PCR were analyzed in the brain and liver , Small intestine and kidney detection specific table <Example 1 2>. (F) Construction of the human c D N A library From the results of Examples 4 and 12, the liver was selected as the starting tissue for the selection of human TPOcDN A. Therefore, a c D N A library was constructed with m R N A from commercially available normal human livers. In the vector, as in the gene bank of rats, c E F 1 8 S was used to synthesize c D N A in the same manner as in the case of rats, and the restriction gene Not I and the EcoRI site acted as a gene bank with directivity. Introducing large intestine strain 1) Η 5 The number of pure tadpoles in the gene bank was approximately 1.2 million <Example 13>. [0086] (G) Acquisition of human TPO cDNA fragments according to the PCR method (colony) Based on the base sequence encoding the cDNA of rat TPO loaded on pEP18S-A2a, several primers for PCR were synthesized. When mRNA was synthesized from a commercially available normal human liver and PCR was performed using these primers, a band around 6 to 20 bp was observed. When the base sequence was determined, it was understood that it was a D N A fragment having about 86% identity with rat TP 0 and was considered to be a part of a gene encoding human TP 0 &lt; Example 14 &gt;. [0087] (H) Screening of human TPOcDNA by PCR ~ Sequence of human TPOcDNA ~ Table 琨 Confirmation that the previously prepared human cDNA library was expanded and divided into aggregates of approximately 100,000 pure strips each, from 90 aggregates Extract plastids. Using this DNA as a template, -31-· This paper size is applicable to China National Standard (CNS) A4 specification (210X297 male |) —-------- install-(Please read the precautions on the back before (Fill this page), §! Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 498079 A7 ___B7___ V. Description of the invention (29 :) Based on the base sequence of the human TP 0 fragment obtained in Example 14 and the newly synthesized primers are used for P At CR, zones were observed at 3 aggregates. From these, 1 aggregate was selected and divided into K 5000 sub-aggregates that were purely 1 aggregate, and the plastid DNA was purified from 90 of the aggregates. When these same PCRs were performed as horizontal plates, five sub-aggregate detection zones were performed. Therefore, one aggregate was selected from these and then divided into 250 sub-aggregates that were pure and aggregated into one aggregate. From these 90 aggregates, plastid DNA was purified and detected again by PCR. Zones were observed for each aggregate. Therefore, '1 aggregate was selected> and it was divided into K 30 pure sub-aggregates of 1 aggregate, and 90 of these aggregates were used to refine plastid DNA. The results of PCR were applied to 3 aggregates. Zones were observed. From one of these aggregates, 90 colonies were selected to prepare plastid D N A to perform P C R to obtain pure 糸 H L · 3 4 &lt; Example 1 5 &gt;. [0088] When determining the base sequence of the plastid DN A which this pure pupal possessed, it was understood that the load had cDN A &lt; Example 16 &gt; having a similarity to the base sequence of rat TPO of about 84¾. [0089] The purified plastid DNA was purified, and the TPO activity was detected in the culture supernatant when transfected into COS1 cells. It was confirmed that the plastid DNA possessed by the pure peptone was loaded with a gene cDNA fragment encoding human TPO. Example 17 &gt;. [0090] However, this cDN A fragment has no stop codon, but also has an A tail-like sequence held at the 3f end / this is considered to be an artificial product at the time of breeding. Therefore, 'Sister-32-' This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) --------- 31 ^ -pack-(Please read the precautions on the back before filling (This page) Printed by the Consumer Cooperative of the Central Standards Bureau, Ministry of Economic Affairs, 498079 A7 ___B7_ V. Description of the invention (50) Amino acid expression vector encoded before the poly A tail-like sequence, performed on C 0 S 1 cells At the time of the table, p 0 activity was detected in the culture supernatant <Example 18>. [0091] PCR was used to analyze the structure of the full-length CDNA, and human τρ〇3, a DNA fragment on the end side was obtained. [0092] When determining the base sequence of this fragment, it was learned that the cDNAS crocodile of the pure strip HL34 obtained from Example 15> The open reading translation architecture was also extended, and the pre-coding side could be formed by a total of 35 3 amino acids. Protein. That is to say, the human TP0 protein contains 21 signal sequences made of 353 amino acids <Example 19>. [0093] In addition to the colony selection of human CD0A, in addition to the colony method K described above, genes such as the pΡC · line and p BR 糸 plastid vectors, human phage vectors, and other constructs are also used. The library can also be used for community tritification or plaque hybridization using rat TPO cDNA fragments as probes. In the design of the degenerate probe, in order to reduce the degree of degenerate, inosine can also be used. When an assay for specifically and sensitively detecting TP 0 activity can be used, performance breeding can be performed using various expression gene banks that are discarded by the present invention. [0094] However, as described in Example 15, in the normal human liver, considering that the RN A encoding human TPO contains significantly lower content (the content calculated from this example is 1 in 3 million ), Using synthetic oligonucleotide probes and cDN AH segments of rat and human TP0 as probes, by using the hybridization of -33-This paper standard applies to China National Standard (CNS) A4 specification (21 〇 &gt; &lt; 297 mm) --------- ^ 丨 Packing-(Please read the notes on the back before filling this page) Order 498079 A7 B7 V. Description of the invention (51) Screening method The number of treated pure lines and plaques became enlarged, and because the sensitivity and specificity of hybridization were not as good as the PCR method, it was extremely difficult to predict. Actually, the present inventors also have about 2 million pure pupae of the normal human pheasant cDNA gene library prepared in Example 13 and the T rat cDNA fragment of K rat was used as a probe to carry out community dehydration, but no pure human TPO cDNA was obtained. [0095] (I) The restructuring group of the human TPO cDNA library. Since the pure 糸 HL34 obtained in Example 15 is considered to be pure with incomplete cDN A: For the purpose of obtaining full-length human TPO cDNA, a commercially available Poly (A) + RNA from normal human liver was used to reconstruct the human TPO cDNA library (hTP0-F1). The number of clones of a gene bank made by a human large intestine strain was about 1 million &lt; Example 20 &gt;. (J) Rescreening of human TPOcDNA by community hybridization ~ Sequence of human TPOcDNA ~ Performance confirmation Based on the base sequence of human TPOcDNA of part length obtained in Example 14 (SEQ ID NO: 3), and in Example 19 The base sequence of human TP 0 c DNA of the estimated full length (sequence number 6) ... a primer for PCR synthesis. [0096] Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs --------- 0 ^ II (Please read the precautions on the back before filling out this page) The library (hTOP-F1) was divided into three aggregates U1 ~ 3), and plastid DNA was prepared from the respective aggregates. These DNAs were used as templates, and PCR was performed using the synthesized primers. In the case of using the DNA from the aggregate of # 3, in order to enlarge the predicted size of DN A, the aggregate of tf 3 was divided into sub-aggregates of 15 000 each, and PCR was performed using the previously synthesized primers. At the time, 6 of the 90 aggregates had a predicted DN A size of -34- This paper size applies to China National Standard (CNS) A4 specifications (210X: 297 mm) 498079 Α7 Β7 Employees of the Central Standards Bureau of the Ministry of Economic Affairs Cooperative print 5. Explanatory Notes (52) Expanded. One aggregate was selected from these and divided into sub-aggregates with 1,000 pure strips as one aggregate, and plastid DNA was prepared for PCR 'but no enlargement of DNA was observed. This is considered to be because the purity of the pure plutonium required is slower than that of other pure plutonium, so the recovery rate of plastid DNA becomes worse. [0097] Therefore, return to the aggregate of # 3, so that pure mash was planted into a community of 4 100 per 1 LB petri dish, and 1,000 petri dishes and individual replica baking dishes were made. . Regarding the D N A extracted from the colony of the petri dish, when the PCR was performed in the same manner as described above, the expansion of the zone was observed in 1 of the 100 aggregates. From the quasi-cultivation dish of the aggregate, a replicating membrane was prepared, and colonization was performed using a radiolabeled probe (EcoRI / BamHI fragment of plastid PEF18S-HL34). As a result, one positive signal was observed, and the community was picked from the original plate. The plastid DNA was prepared by self-replanting the community obtained from the LB petri dish, and by implementing P C R, pure 糸 p H TF 1 <Example 2 1 &gt; was finally obtained. For the screening of D N A pure strips, the main use is hybridization, PCR, performance breeding, etc., but combining these suitable sisters can increase the efficiency and sensitivity of the screening, or reduce labor. [0098] When the base sequence of the pure PHTF1 obtained here is determined, an open reading framework exists, and it is considered that the amino acid sequence of the protein encoded by this open reading framework is inferred from the human TP of Example 19 The amino acid sequence (sequence number 6) is completely identical. The base sequence differs from the putative one (sequence number 6) in three places and does not cause amino acid conversion. From this, it can be confirmed that the human TP 0 protein is made of 35 3 amino acids with a signal sequence of 21 residues. <-35- This paper size applies to China National Standard (CNS) A4 (210X297 mm) ) (Please read the note on the back 4 • Fill in the items and then fill in:: Write this page)

, TT Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 498079 A7 _B7 __ V. Description of the Invention (is) Example 2 2 &gt;. TP0 activity can be detected in the culture supernatant when transfected into COS1 cells from the purified peptone DNA prepared from the pure 糸 PHTF1 obtained as described above. <Example 23 &gt; 〇 (K) Screening of human TP 0 chromosome DNA ~ Human TP 0 chromosome DNA sequence ~ Performance confirmation The human gene library obtained from Professor Tokuyama Yamato of the Tohoku University Gene Experiment Facility will be implanted into 30,000 NZY M petri dishes Bacteriophages were made into 18 petri dishes, and individual replicating membranes were made. The human TPO cDNA fragment (base sequence number 1 78-1 025 of SEQ ID NO: 7) contained in pure peptone PHTF1 was amplified by PCR and purified, and 32P-labeled was used as a probe to perform plaque hybridization. Result &gt; As 13 positive signals were detected, plaques were picked from the respective original culture dishes, so that one thousand plaques per NZY culture dish were re-planted, and the reproduction from each production was performed. The filter membrane performs plaque hybridization. As a result, since signals were detected in all the organs of the 13 groups, the plaques were isolated and the phage DNA was prepared. 13 of the 13 pure lines related to whether the sum code range of human TPO cDNA was checked by PCR. [0099] Of the 3 pure maggots, there are 5 pure maggots. Since it is considered to contain all the coding regions expected from c DHA, a pure line (λ HGT1) is selected for Southernblot ink dot analysis (the probe used is the same front). In the case of digestion with restriction enzyme H ind 1 [, a band of about 10 kbp was observed, so the restriction enzyme H ind H [was used to digest pure DNA of lambda HGT1 and then run on an agarose gel. Cut and refine the 10kb band and subselect it on the breeding carrier pUC13 -36- This paper size is applicable to the Chinese national standard (〇 Bi) eight 4 ^ grid (210 father 297 mm) ^-(Please read the back first Please pay attention to this page and fill in this page again) Order 498079 Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (54) In the end, pure pHGTIC Example 24 &gt; was obtained. When determining the base sequence of this pure: line, the loaded chromosome D N A contains the coding region of all the proteins of human TP 0 estimated in Example 19, and the base sequences are completely identical in terms of its range. It is also equivalent to the region of the epidermis, and is divided by four non-executives. It has been understood that the base sequence of the full-length human TPO cDNA loaded with the pure Ηρ Η T F 1 obtained in Example 21 is different from three. Finally, when determining the base sequence of the 4 pure strips of the 5 pure stalks selected, 2 positions are the same as those of the pure stalk PHGT1, and the 1 of the stabs and 2 pure spurs are the same as those of the PHGT1. , And then 2 pure 糸 and PHTF1. <Example 25> The EcroRl fragment of the plastid-pure PHGT1 obtained above was ligated to the epidermal vector pEF18S &gt; Human TP0 epidermal plastid pEFHGTE was prepared, and when transfected into COS1 cells, τρ could be detected in the culture supernatant. active. <Example 26 &gt; (L) Preparation of human TP0 deletion derivatives ~ Tables in COS1 cells ~ Activity confirmation From the results of Examples 18 and 22, it is predicted that the human TPO protein does not require a full-length amine in terms of its activity performance Based acid. Therefore, in order to analyze the range necessary for the activity expression of human TP 0 protein, the pure DNA of PHTK31 obtained in Example 18 was used as a horizontal plate, and the primers synthesized by M were subjected to PCR &gt; The expression at the end is plastid, that is, plastid DNA encoding deletion derivatives of amino acids 1-211, 1-1-91, 1-171, and 1-16. When the respective plastid DNAs obtained were transfected into COS1 cells, TPO 'activity was detected in the culture supernatant of any of them. <Example 27 &gt; -37- This paper size is applicable to China National Standard (CNS) Α4 size (210X297mm) --------- install-(Please read the precautions on the back before filling this page ), 1T

Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 ______B7_ V. Description of the Invention (55) Derivatives (deletions, substitutions, insertions, additions, etc.) based on the TDNA thus obtained can be used to bear changes in the original protein activity Protein. As a method used in this case, there are a PCR method, a site-directed mutation generation method, and a chemical synthesis method. [0100] A method for measuring the TP 0 activity (in vitro measurement system) used in the present invention is &lt; Reference Example &gt; &Lt; Reference Example &gt; A, Rat megakaryocyte precursor cell (CFU-MK) assay (rat CFU-MK assay) 糸 (liquid culture 糸) megakaryocytes are energy-dependently incorporated outside of cells 5-Hydroxytryptamine accumulates in densely stained particles (Pedorko, Lab. Invest., Vol. 36, pp. 310-320 (1 977)). This phenomenon has been considered at least in small, single-core acetylcholine-positive girls (Brieker and Euckerraan, Exp. Heniatol. &Gt; 12 volumes) in the differentiation stage between CFU-MK and the identifiable megakaryocytes. '672-675 (1 9 8 4)), followed by an increase in the size of the megaspheres and an increase in serotonin incorporation (Schick and Weinstein, J. Lab. Clin. Med., Vol. 98, 607 -P. 615, (1981)). Furthermore, it is known that only megakaryocytes and systemic cells are specific for bone marrow cells (S c h i c k and Weinstein, J. Lab. Clin. Med. ’98 Vol.’ Pp. 607-615 (1 981)). This assay is performed by cultivating highly concentrated rat c Fu u MK (G p b / M a + CF ϋ-M K; described later) in the presence of the test subject. Proliferation and differentiation, 14C-5-hydroxytryptamine (Hydroxytryptamine-38- This paper size applies Chinese National Standard (CNS) A4 specifications (210X297 cm)) (Please read the precautions on the back before filling this page) ~ ^ Installation ·- Then fill in Taiding 498079 Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7_V. Description of Invention (56) Creatine Sulfate); 5Η T) The measurement method is incorporated as an indicator. [0102] The advantage of this measurement system is that the CFU-M1 (the ratio is extremely high (refer to the "measurement method" described later) contained in the female cells used), on the contrary, the female cells other than the target cells of TP 0 are mixed There are few cells, which can alleviate the indirect influence of mixed cells (for example,> a substance other than the factor is mixed into the cell to induce Meg-CSF activity, and mixed into the cell to produce a point factor that acts synergistically with the factor). Only a small number of whole female cells can be cultured in one hole, and a good culture environment can be maintained for a relatively long period of time. Moreover, during the culture period, many large-sized mature cells generated from CFll-MK by the active control standard The zone nucleus can be observed under a phase-contrast microscope, and it is also advantageous to qualitatively determine the presence and extent of the activity. The result of this qualitative determination corresponds to the quantitative result by the incorporation of 1 4 C-5 -hydroxytryptamine. Therefore, Qualitative judgments can be used in combination to improve the reliability of quantitative results. [0103] "Measurement Method" First, the highly concentrated rat CFU-MK ("Gp I b / I CFU-MK part") used for measurement As reported Methods (Miyazaki et al., Exp. Heraatol., Vol. 20, pages 855 ~ 861 (1992)) were prepared by several improved methods. The outline is as follows. [0104] Wistar rats were extracted, 8-12 weeks old) The femur and tibia were prepared according to the conventional method of preparing bone marrow cell suspension. In the preparation of bone marrow cell suspension, use the media reported by Levine and Fedorko for meganucleus sphere separation ((Please read the note on the back 4-item and then fill-fill-nest page), π -39- This paper size is applicable China National Standard (CNS) A4 specification (210 × 297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Description of the invention (57)

Levine and Fedorko, BloocL, Vol. 50 &gt; pages 713-725 (1977)), several modified media (13.8 dicitrate citrate, glucose @ sugar, 1 nM adenosine, 1 raM mandrine, 10 raM HEPES ( pH 7.25) '0.5¾ Bovine serum albumin (hereinafter abbreviated as (PathHyte 4; Biochemical Industry) and Ca-free: 2 &quot; ^ Mg2 + 2Hanks balanced salt solution (hereinafter referred to as HATCH solution) solution) ° will Bone marrow Iffl cell suspension fluid, Percoll stock solution (manufactured by Pharmacia) is coated with a Per col 1 discontinuous density gradient solution (density; 1.050 g / ml / 1.06 3 g / ml / 1.082) prepared by dilution with MHATC solution. G / ml), centrifugation at 20 t: 400 × g for 20 minutes. After centrifugation, the cells were collected at the interface of density 1.063 g / ml and 1.02 g / ml. Wash the cells After 'the 10% bovine fetal serum (hereinafter abbreviated as PCS) KI sc 〇ve changed 1) u 1 becc 〇 culture solution (hereinafter abbreviated as IMD Μ culture solution) was suspended' into a tissue with a diameter of 1000 mm In a plastic dish, incubate for 1 hour at 3 7 1C in a 5 SK carbon dioxide gas incubator. Then, recover the non-adsorbed cell part 'and put it in a plastic dish with a diameter of 100 mm, and incubate it at 37 t for 1 hour' to collect the non-adsorbed cell part. Resuspend the recovered female cells in HA TC Η solution, put in P55 antibody that was previously adsorbed as a mouse anti-rat platelet G PB 11 b / 111 a antibody (Miyazaki et al., Thorab. Res., Vol. 59, pp. 941-953 (1 990)) For a bacterial examination, a 100-mm diarrhea petri dish (Falconl005; manufactured by Becton-Dickinson) was allowed to stand for 1 hour at M. After that, the non-adsorbing cells were sufficiently washed and removed with a HATCH solution, and then pipetted The cells adsorbed to the solid-phase P 5 5 antibody are removed and recovered. Generally, 3 to 4 X 10 β cells can be obtained from one rat. The fineness of the obtained -40- This paper size applies Chinese National Standards (CNS) Α4 Specifications (210 × 297 mm) --------- ^ Packing -------- Order ----- (Please read the notes on the back before filling out this page) 498079 Central Bureau of Standards, Ministry of Economic Affairs— Printed by the Industrial and Consumer Cooperatives A7 B7 V. Description of the Invention (58) The cellular part of the rat CPU-MK is highly concentrated and is called "GpEb / Ha + C F U-MK part "), by the test in the presence of rat IL-3 which measures the saturation concentration of radon due to the community described later, it is known that it contains a CPU-MK of usually about 5 to 10%. The hybridoma (p55 cell) producing the above-mentioned P55 antibody was deposited on February 14, 994, under the trust number FERM BP-4563, and was deposited in the Institute of Life Engineering and Industrial Technology, Industrial Technology Institute, Ministry of Economic Affairs. [0105] Next * part of the obtained Gp It b / HI CFU-MK was resuspended in IMDM culture medium containing 10¾ PCS, and 10Λ cells were added to each hole to distribute to the flat bottom of 93 holes A petri dish, and then for IMD M culture fluid, add a fully dialyzed standard (described in detail later) and a test subject to make the final culture fluid volume to 200 microliters / hole. The petri dish was placed in a carbon dioxide gas incubator and cultured at 37 for 4 days. 3 hours before the end of the fourth day of culture, 0.1 microcurie (3.71 ^ 9) of 140 serotonin was added to each well, and the culture was continued at 37 ° C. After the end of the culture, centrifuge the Petri dish K 1 0 0 0 “pro for 3 minutes, suck and remove the supernatant, and then add 200 μl of PBS containing 0, 05% EDTA to each well, and centrifuge the Petri dish. The mixer was shaken for 5 to 10 minutes to fully lyse the cells. 150 microliters of the obtained cell lysate was transferred to a commercially available cup-shaped solid scintillator (Ready Cap; manufactured by Beckman) &gt; at 5101. Allow to stand overnight in a desiccator to dry it out the next day, place Ready C ap into a glass vial, and measure the radioactivity of 1 4C with a liquid scintillation counter. Alkaline esterase activity is in accordance with -41-This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page) • Binding and ordering 498079 Staff of Central Bureau of Standards, Ministry of Economic Affairs Printed by Consumer Cooperatives A7 B7 V. Description of Invention (59)

The method of Ishibashi and Burstein (Ishibashi and BUrstein, B Uod, Vol. 67, 151 2-1 514 pages (1 986)), also obtained a result very similar to the quantitative result by the incorporation of 1 4 C ~ 5 ~ serotonin . [0107] Search A product 1 First, the following method was used to prepare the standard plasma of thrombocytopenia rats. [0108] The above-mentioned P55 antibody M was administered at a dose of 0.5 mg once, and was intravenously administered twice to an interval of about 24 hours into 7 to 8-week-old normal Wistar rats ($) * The second thrombocytopenia after about 24 hours of administration, laparotomy under anaesthesia> 3. 8¾ (v / v) sodium citrate solution was used as the coagulant 10 Blood is collected automatically in a milliliter syringe. The blood was transferred to a plastic centrifuge tube and centrifuged at 1 200 × g for 10 minutes to recover the plasma portion. Centrifuge the plasma fraction at 1,200 x g for 10 minutes, and then take care to collect the plasma fraction without aspirating the spherules made of cells and platelets. The plasma is called Γ TRP ") ° [0109] Next, Ca-treated TRP (standard product C) prepared from TRP according to the method described in Example 1, WGA-Sepharose column active part (standard product) W) or Phenyl Sepharose 6 FF / LS column active part (standard product), and thoroughly dialyzed a sufficient amount of IMD M culture medium as a standard product for activity test. -42- This paper size applies Chinese National Standard (CNS) A4 specification (210X297mm) (Please read the precautions on the back before filling out this page)-Binding and binding 498079 A7 B7 Five 'invention description (4〇) [0110 ] In the rat τρ0 purification process shown in Example 1, the standard product c was used initially, the standard product W was started halfway, and the standard product P was used thereafter. The activity of the standard c is arbitrarily defined as 1 'relative activity of the standard iSW and standard P based on jfcb calibration. The method of determining the relative activity of the test subject 'draws the response curve of the amount of the standard and the test subject'. When the test subject 2 'activity is η times the activity of the standard C', the relative activity of the test subject is η. [0111] Β. Community measurement: Bone callus cells were cultured in a semi-solid medium in the presence of a test subject, and cultured by estimating the number of CFU-Ml (the number of megakaryocyte communities formed by proliferation and differentiation). The method for measuring Meg-CSF activity. [0112] "Assay" (a) When non-isolated rat bone marrow cells are used, printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out (This page) Cells containing rat non-isolated bone marrow cells, Gp H b /] H ^ CFU-MK from the previous stages of separation and concentration operations, or Gp H b / HI a + CFU-MK fractions, 10VFCS, 2mM glutamic acid, 1 mM sodium pyruvate, 50 μM 2-heptyl ethanol, 0.3¾ of agar (AGAR NOBLE, manufactured by DIFC0), the final volume of 1 ml of IMDM culture solution is put into a 35 mm diameter Tissue culture plastic blood is solidified at room temperature, and then cultured in a carbon dioxide gas incubator at 37 ° C. Generally, the number of female cells implanted per dish is non-isolated bone marrow cells, Per col 1 Cells in the centrifugation phase and the plastic plate adsorption cell removal phase, and Gplb / lla + CFU -MK parts are 2 ~ -43- This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297 mm) 498079 A7 B7 V. Description of invention (41) 4X105, 2 ~ 5X104, 0.5 ~ 2X103. On the 6th to 7th days, take out the agar gel from the dish, place it on a glass slide (76 mm X 52 mm), and then put a nylon mesh filter paper with an aperture of 50 μm to absorb moisture. After removing them, place them in the chamber. Allow to dry thoroughly at 50 ° C. After heat fixing on a hot plate of 50 υ for 5 minutes, the acetylcholinesterase staining solution prepared according to the method of Jackson (Jackson, Blood, Vol. 42, pp. 413-421 (1973)) was used. After immersion for 2 to 4 hours, confirm that the megakaryocytes are fully stained and then take out water for washing. After drying, stain in Har rvi s hematoxylin for 30 seconds, and stain after washing with water to air dry. K acetylcholinesterase positive megakaryocytes The clusters formed on the three K are one community to estimate the number of megakaryocyte communities. [0113] (b) Cases of using non-isolated epiphyseal cells of mice, K. The number of female cells per 1 petri dish is 2 to 4. X 105 can be carried out in the same manner as described above. [0114] (〇 Using human bone marrow cells In the case of human umbilical cord blood cells, human bone marrow cells and human umbilical cord cells can be used as they are, but the concentrated CF11-MK part can also be used as follows. [0115] Printed by the Consumer Cooperative of the China Standards Bureau of the Ministry of Economic Affairs Read the notes on the back and fill in this page.) First, cover the bone marrow fluid or umbilical cord blood with Lymph prep (manufactured by Daiichi Chemical Co., Ltd.). After centrifugation, collect the white blood cells at the interface. Since then, the female cells have been conjugated with biotinylated antibodies to biotinylated human cell surface antigens (CD 2, CDllc, and CD 19). Remove the magnetic beads. Cells that can be removed by this magnetic bead method -44- This paper size is in accordance with Chinese National Standard (CNS) A4 (210X297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 5. Description of the invention (42) For B cells, T cells, macrophages and part of the pellet. The remaining cells were stained with M FITC-labeled anti-CD 34 antibody and PE-labeled anti-HLA-ljR antibody, and then a cell sorter (for example, ELITE; manufactured by Coulter) was used to recover CD34-positive and HLA-DR-positive cell sections. Serving. In this part, the CFU-MK is concentrated (slightly CD34 + DR + CFU-MK part). The human community measurement was performed using the above-mentioned rat medullary cells, which is about the same as the community measurement, but the number of CD34 + DIT CFU-MK parts implanted in each plastic dish of K is 3 ~ 5X103. A mixture of 12.5¾ human AB plasma and 12.5¾ PCS was used instead of 10¾ PCS. In addition, the culture period until the formation of the megakaryosphere community is from 12 days to 14 days. For the detection of human megakaryocytes, the megakaryocytes were immunostained using the alkaline phosphatase anti-alkaline phosphatase antibody method of human G p I b / melon a mouse monoclonal antibody against the surface antigen of megakaryocytes (for example, T era in ura et al., EX p. H emat vol. 1 · 1 6, 8 4 3-8 4 8 pages U 9 8 8)), it is estimated that the community formed by 3 megaspheres on M is a megasphere community . [0116] C. An assay line using human megakaryocytic penetrating cells (M-07e assay) is an M-07e cell of a human megakaryoblastic cell line, which is known to respond to CM-CSF, IL-3, SCF ·, Proliferating cell lines such as IL-2 (/ ^ "2 丨 etc. &gt; J. Cel 1. Physiol., Vol. 145, 45 8-46 4 (1 990), Kiss et al., Leukeniia, Vol. 7), also understand It is clear that TP0 also responded. * An alternative assay for rat CF11-M K test strips can be used. [0117] "Assay" -45- This paper size applies Chinese National Standard (CNS) M specifications (210 × 297 cm) (Please Read the precautions on the back before filling in this page) Pack. Order 498079 A7 B7_ V. Description of the invention (45) Recover M-07e cells subcultured in the presence of CM-CSF &gt; After washing thoroughly, suspend in 10% FCS in IMD M broth. In a 96-well flat-bottomed petri dish for sister weaving, put K-07 cells into the M-07e cells at a number of 104 per hole, and add standards and test specimens to make the final volume of 200. Microliters / holes. Place the petri dish in a 5% carbon dioxide gas incubator and incubate at 37 for 3 days. 4 hours before the end of the culture on day 3 'Add 1 microcurie (37 KBq) / hole 3Η-thymidine, after the end of the culture' K cell harvester collects the cells on a glass fiber device, K liquid scintillation counter Measurement of radioactivity at 3 ° (e.g., / 3 generic: manufactured by Pharmacia) ° [0118] &lt; Examples &gt; The present invention will be described in detail with reference to the following examples. [0119] &lt; Examples 1-1 &gt; Purified from rat TPO by administration of plasma of thrombocytopenia rats administered with anti-platelet antibodies " "Modulation" Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) with the aforementioned "Reference Example" A. Rat megakaryocyte precursor cells (CFU-MK) measurement In this method, about 1,000 portions of TRP in rats were prepared as a source of purification. [0120] "Refined rat TPO from TRP" -46- This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) 498079 A7 B7 V. Description of the invention (44) At first, based on the content of TPO in TRP At most, it is estimated to be only 1/3 million. About 1,000 parts of TPR of K rats are the result of refining the material, and a slightly refined control standard of rat TP0 is obtained. Next, the partial amino acid sequence analysis of this control standard was tried, and the results of partial amino acid sequences of three kinds were obtained. These are similar to the amino acid sequence of serine protease inhibitor (SP I) produced by the liver and are similar. Therefore, TPO is the possibility of having a structure similar to a serine protease inhibitor, and K and the possibility of a sequence derived from a protein other than TP0 are not negated. Based on these uncertain sequences, TP 0 gene selection was performed from the rat c D N A library &gt; As a result, no candidate genes were obtained. This is thought to be due to the lack of purity and quantity of the reference standard of the analysis &gt; In order to obtain the final purified control standard necessary for analysis of the amino acid sequence, the purification described in Example 1-2 below was performed. In addition, it should be particularly mentioned that from the results of Example 2 and Example 2, the amount of TP0 present in TRP or XRP (described later) is surprisingly estimated to be 1 to 10 parts per 100 million of the total plasma protein. 1 part per billion, since it is extremely difficult to refine this, knowing its content is 虿 0 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) [0122] 〈 Example 1-2 &gt; Purifying TPO from plasma of thrombocytopenia rats irradiated with X-ray or 7-ray "i-47- of this blood plasma of thrombocytopenia rats irradiated with X-ray or 7-ray Standards are applicable to China National Standards for Standards (CNS) A4 (210x297 mm) 498079 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (45) Preparation ”For Wistar 糸 normal rats 7 to 8 weeks old (¾) Whole body irradiation with X-rays or 7-rays with a sub-lethal dose (6 to 7 G y), blood collection on the 14th day of the thrombocytopenia period &gt; Plasma fractions were prepared in the same manner as the above-mentioned TR P (hereinafter, This plasma is called "XRPJ". [0123] Therefore, a total of about 1,100 rat portions of XRP (about 8 liters) were used as the source of purification. [0124] "Refined rat TPO from XRP" is to make the total protein mass of the plasma of about 1,100 rats reach 493,000 ¾ Grams, can not be processed at once. Therefore, among the refining steps described below, (1) to (4) are divided into eleven sisters each of about 100 to carry out. Then, it is implemented in 6 batches from (5) to (7). (8) After K, 1 110 coarse refined products were collected for implementation. .

[0125] The refining example of K in a certain group (XW9) and a certain batch (XB6) is taken as an example for illustration, and the steps related to the refining are described in combination. In addition, in all the steps of purification, the rat CPU-MK measurement described in the aforementioned &lt; Reference Example &gt; was used to measure TPO activity. [0127] Except that the reverse phase chromatography was carried out at room temperature, purification was performed at 4 ° C unless otherwise noted. -48- This paper size applies Chinese National Standard (CNS) A4 specification (210X297mm) --------- ip pack-(Please read the precautions on the back before filling this page) Order 498079 A7 B7 V. Description of the invention (46) [0128] In addition, the quantification of protein uses the Comexy dye binding method (test reagent manufactured by PIERCE company, catalog number 23236 X), or the bicinconic acid (Θ &gt; 7 7 acid) method (PIERCE test reagent, catalog number 23225). [0129] A summary of the purification is shown in Table 1. [0130] [Table 1] ⑴ Calcium chloride treatment, centrifugation treatment, and protease inhibitor treatment of rat plasma Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) Approximately 100 servings of XRP (742 ml, protein concentration 54.8 mg / ml, total protein mass 40 6 8 6 mg) stored in -80 were thawed and transferred to a polypropylene centrifuge tube (manufactured by Narugen Corporation) ). Here, the final concentration of each was 100 μm, calcium chloride powder was added, and the mixture was left to stand at 4 ° C overnight. After centrifugation at 80,000 rpm for 60 minutes, the supernatant was recovered. To this supernatant containing TP 0 activity (742 ml, protein concentration 54.9 mg / ml, total protein mass 40 7 40 mg), add a final concentration of 1 mM P-AP MSP (protease inhibitor) Amino diphenyl methanesulfonium fluoride hydrochloride, Wako Pure Chemical Industries, Catalog No. 010-1 0 393) was carried out by a Sephadex G-25 column to buffer exchange step described below. [0131] / In this way, every 100 parts are concentrated into a group of> Calcium Chloride P-APMSF treatment, a total of 11 sisters, about 1100 parts of XRP (total volume 8184 ml, total protein 493007 mg) ) Processing is carried out separately -49- This paper size is applicable to China National Standards (CNS) A4 (210X297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7_ V. Description of the invention (47)

Sephadex G-25 column separation. 0 Sephadex G-25 <Yuanchong exchange> The calcium chloride obtained in (1) was treated with the supernatant (742 ml ', protein concentration 54.9 mg / ml, total protein mass 40740 mg), M A flow rate of 40 to 70 ml / min was added to a Sephadex G-25M column (prepared by Farumasian Biochemical Co., Ltd., pre-recorded No. 1 7-0033-〇3; diameter 11.3 cm) , Total height of 47 cm), 1300 ¾ litres are discarded until the protein dissolves. Secondly, from the time of the start of UV absorption, the electrical conductivity was collected up to 500 microseconds / cm, and the protein fraction containing TP 0 activity (1 37 7 ml) was recovered and replaced with a 20 mM Tris-HCl P Η 8 solution. , Protein concentration of 27. 5 6 mg / ml). The total protein mass of the TP0 active fraction was 378 82 mg, and the protein yield at this step was 93¾. The relative activity of TP0 was 2.3. '[0133] In this way, the results of Sephadex G-25 column implementation for all sisters, the total volume was 21117 ml, the total protein mass was 48 0 30 6 mg, the average relative activity was 1,8, and the relative activity was 864,600. TPO active portion of Sephadex G-25. ⑶ Q-Sepharose FF &lt; Strong Anion Exchange Chromatography &gt; The TPO active portion of Sephadex G-25M (1 375 ml, protein concentration 27.5 mg / ml, total protein mass 3784 1 mg) obtained in (2) Relative activity 2,3) At a flow rate of 40 ml / min, added to 0-56 卩 3 "〇36 -50- This paper size applies the Chinese National Standard (CNS) A4 specification (210 &gt; &lt; 297 mm) (Please read the precautions on the back before filling out this page)-Packing · 498079 Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (48) FP (made by Farumasia Biochemical Company, self-recorded number 1) 7-0 5 1 0-0 1; diameter 5 cm, filter pad 27 cm high), K20 mM Tris-HCl pH 8 dissolves non-retained F1 portion (3949¾ liters, protein concentration 0.93 mg / ml, total protein The amount is 3870 mg, and the relative activity is 0). [0135] Next, replace it with 20 mM Tris-HCl containing 175raM NaCl in 8 buffer solution to dissolve the TPO active F2 portion (4375 ml, protein concentration 5.36 mg / ml). [0135] 0136] Finally, κ 20 μαΜ Tris-HCl containing 100 μm NaCl and 8 buffers , Dissolve F3 (1221¾ liters, protein concentration 3.9 mg / ml, total protein mass 47 83 mg &gt; relative activity 3. 8). Total protein in the active F2 portion of TP0 is 23440 mg &gt; The protein yield was 61.9¾. Moreover, the relative activity of TP0 increased to 6.8. [0137] In this way, all the sisters of the TP0 active part of Sephadex G-25 ran Q-Sepharose FF, and the total volume was 35842. Ml, total protein mass 31 438 4 mg, average relative activity 8.6, relative activity P2 part of Q-Sepharose FF 虿 2704000. [0138] ⑷ Wheat germ agglutinin (WGA) -Agarose &lt; exogenous lectin Affinity chromatography &gt; The TPO active F2 portion of Q-Sepharose FF obtained in ⑶ was divided into 3 times and added to WGA-Agarose (manufactured by Fengnian Co., Ltd.) at a flow rate of 5 ml / min. -51-This paper is for China National Standard (CNS) A4 (210X297 mm) I -------- Crack-- (Please read the notes on the back before filling this page} Order 498079 A7 _____B7 V. Description of Invention (4θ) Table of Contents No. 800273; diameter 5 cm, _ cushion height 22,5 cm), to Zhang Yuan Chong solution (DPBS), such as Dalbert Coriolis Phosphate, obtained F 1 fraction without retention (9336¾ liters, protein concentration 2.30 mg / ml, total egg mortar mass 21407 mg, relative activity 6.9) 〇 [0139] Next , 20 mM sodium phosphate containing 0.2 mM N-acetamidoglucose (GlcNAc, manufactured by KONE Corporation, catalog number 0 0 5-20), 150 mM NaCl, and 0,02 ¾ plate nitride The aggregates dissolving in 2 buffer solution were concentrated by a super hydrazone unit (manufactured by Felton Corporation, Ω ultra-fixed molecular weight 8000 cutting) to obtain WGA-Agarose adsorption TPO active F2 part (2993 ml, protein concentration 0.376 mg / ml) . The total protein mass of this TPO active F2 fraction was 1125 mg, and the protein yield of F 2 at this step was 4.8%. Also, the relative activity of TP 0 increased to 10 1. The F2 obtained here is stored at -80t :. [0141] In this way, the result of repeated running of WGA-Agarose for the entire group of TPO activity F2 part of Q-Sepharose FF, the total volume was 33094 ml, the total protein mass was 1 50 30 mg, the average relative activity was 132, and the relative activity was 1 987000 WGA-Agarose's TP0 active F2 part printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before completing this page). [0142] (1) TSK-sel AF-BLUE 6 50 MH (pigment adsorption affinity chromatography): A total of 215 parts of XRP starting from XRP, WGA-Agarose adsorbed the active portion of TPO and XGA WGA- Agarose adsorption-52- This paper size is in accordance with Chinese National Standards (CNS) A4 specifications (210X297 gadolinium) 498079 A7 B7 V. Description of the invention (50) TP0 active F2 part is collected as batch XB6 and collected (5974 ml, protein Concentration 0.388 mg / ml, total protein mass 2319 mg, relative activity 150) 〇 [0143] To this volume 5974 ml, add 0.85 mole NaCl (296.76 g) 'to make the final concentration of 0.822M NaCl, 6132 ml of solution , M7 ml / min flow rate was added to M 1M NaCl &gt; 20mM sodium phosphate, 7.2 pre-equilibrated TSK-gel AF-BLUE 6 50 MH column (manufactured by Tosoh Corporation, catalog number 0 8 7 0 5; (5 cm in diameter, with cymbals 23 cm high). [0144] After the end of the addition, collect with 20 mM sodium phosphate, 1M NaCl, and 7.2 dissolve the non-retentive solution (approximately 8470 ml). This K ultra unit (manufactured by Felton Company, Ω ultra fixed molecular weight) 8000 cutting) concentrated to obtain the F1 fraction without retention (543 ml, protein concentration 2.05 mg / ml, total protein mass 111 2 mg, relative activity 31). [0145] Next, the eluent was changed to 2M NaSCN to obtain the dissociated TSK-gel AF-BLUE 6 50 MH adsorbing the TPO active moiety F2 (1 427 ml, protein concentration 0.447 mg / ml). Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) [0146] The total protein mass of this TP0 active F2 fraction is 638 mg, and the protein yield of F2 at this step is 27.5 percent. In addition, the relative activity of TP0 increased to 15 0 0 〇 [0147] -53- This paper size applies the Chinese National Standard (CNS) A4 specification (21〇X297 gong) 498079 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (51) So, for all batches of WGA-Asarose adsorbing TP0 active F2 part, running AF-BLUE 6 50 MH respectively, the total volume was 1 06 55 ml, the total protein mass was 4256 Mg, average relative activity 905, relative activity of TSK-gel AF-BLUE 650MH, TP0 active F2 fraction. [0148] SepPhenyl Sepharose 6 FF / LS <Hydrophobic Interaction Chromatography> For the TPO-active F2 portion of TSK-gel AF-BLUE 650 MH obtained in 毫升 (1 424 ml, protein concentration 0.447 mg / ml, Total protein weight 6 38 mg, relative activity 1 500) volume 1 424 ml, add 1.5 moles of ammonium sulfate (282 · 2 g) powder to a final concentration of 1, 35M ammonium sulfate, 1 5 8 1 Ml of solution. [0149] This M 7 ml / min flow rate was added to a pre-equilibrated Phenyl Sepharose 6 FF (Low Sub) column (Falumasia) in a M 1.5 saddle sulfate, 50 M sodium phosphate pH 7.2. Company-made, catalog number 1 7- 096 5 -0 5; diameter 5 cm, filter pad 10 cm high), after the addition is complete, change the eluent to 0.8 M ammonium sulfate, 38 mM sodium phosphate, the flow rate of the collection 1 〇 Ml / min dissolved part (about 3,160 ml), this was concentrated with an ultrafiltration unit (made by Felton Corporation, Ω ultra-fixed molecular weight 8000 cutting) to obtain Pi (485 ml, protein concentration of 0.194 mg / Ml, total protein mass 94.2 mg, relative activity 0). ‘[0150] Next, the eluent was changed to 20 mM sodium phosphate, pH 7.2, to obtain the TP0 active F2 fraction (about 3,500 ml). Take this as an ultrafiltration unit (Phil-54- this paper size applies to China National Standard (CNS) A4 specifications (210X297 mm) --------- ^ installed ------ order- --- 4 ^ (Please read the precautions on the back before filling in this page) 498079 A7 B7 V. Description of the invention (52) Made by the company, Ω ultra-fixed molecular weight 8 0 0 0 cut) Concentrated, one sampling. 0%。 The protein concentration of the TP0 active F2 part (220 ml) at this stage was 1.45 mg / ml, the total protein mass was 319 mg, and the protein yield of F2 at this step was 50.0%. The relative activity of TPO was 1 230. [0151] In this way, for all batches of the TPO-active F2 portion of TSK-gel AF-BLUE 6 50 MH, Phenyl Sepharose FF / LS was repeatedly run, and the total volume was 1 966 ml, and the total protein mass was 2762 mg. The average relative Activity 847, relative activity of 2339000 Phenyl Sepharose PP / LS, TPO active F2 fraction. [0152] ⑺ Sephacry 1 S-200 0 HR &lt; Gel Filtration Chromatography> TPO active F2 fraction (217 liters, protein concentration 1, 45 mg / L) of Phyne 1 Sepharose 6 FF / LS obtained in ⑹亳, total protein mass 315 mg, relative activity 1 230) &gt; Add 144, 8 ml of 5M NaCl solution to 362 ml of 2M NaCl, and then ultrafiltration 犟 element (manufactured by Amicon Corporation; 3 Membrane, 76 mm diameter) was concentrated to 50 ml. [0153] Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). Add an equal volume (50 ml) of 8M urea to make the final concentration of 1M NaCl &gt; 4M urea. The solution is about 100 ml. Reconcentrate to about 80 ml, and finally make a sample of 88.78 ml, injected into the $ 09 ^ (! 门 13-2〇〇HR column (manufactured by Farumasia Biochemical Company, catalog number 1 7 -0584-01; Diameter 7.5 cm, filter pad height 100 cm). [0154] -55- This paper size applies to China National Standard (CNS) A4 (210X297 mm) 498079 Printed by A7, Consumer Cooperative of the Central Standards Bureau, Ministry of Economic Affairs B7 V. Description of the invention (55) Thereafter, the flow rate of K 3 ml / min K DPBS was developed, and the void volume (120 ml) was collected in 45 ml of 60 polypropylene tubes each. This dissolution pattern is shown in Figure 1. Each 2 tubes were measured, and all 11,000 parts were frozen and stored at -85t under the method of Sephacryl S-200 HR until completion. From the results of the measurement, XB6 sorted out the historical fractions as follows. (Figure 1). [0155] (F1) tube number 1 ~ 15 (molecules near the void volume of 94000 or more (F2) tube number 16 ~ 26 (molecular weight 94000 ~ 33000) (F3) tube number 27 ~ 44 (Molecular weight 33000 ~ 3000) (F4) Tube number 45 ~ 55 (Molecular weight 3000 or less) P heny 1 Sephar 〇se 6 FF / LS for all batches of TP 0 active F2, run Sep h aery 1 200 HR, respectively, to determine the individual calendar analysis, at -8 5 ° C Cryopreservation. After all batches of Sephacryl S-200 were run, immediately before the next reverse phase chromatography (YMC-Pack PROTEIN-RP) was thawed, an ultrafiltration unit (manufactured by Amicon Corporation; YM 3) was used. Membrane, 76 mm in diameter) was concentrated to obtain the two reference standards described below. In W, the concentrated reference standard for the TPO active F2 portion of this Sephacryl S-200 HR is called "Polymer TPO reference standard F2" The concentrated reference standard of the TPO activity F3 portion of Sephacryl S-200 HR is called "low molecular TPO control standard F3". The high molecular TPO control standard F2 and the low molecular TPO control standard F3 described herein are It is convenient to arrange the different parts of the dissolution position of the gel filtration ephemeris method and it is said that * may not indicate the true molecular weight. -56- This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) ) --------- Install-(Please read the precautions on the back first Page write) to provide 498079 A7 __B7_ five described (54) [0156] [Table 2] under Μ invention, a low-molecular TP0 reference standard and the polymer P3 ΤΡ0 respective control standard purification step of F2. The following steps (8) to (11) describe the steps for purifying the low molecular TP0 control standard F3. (8) YMC-Pack PRTEIN-RPC reverse phase chromatography> The low molecular TP0 control standard F3 obtained in (7) (total protein mass 50, 3 mg, protein concentration 0.184 mg / ml, Relative activity 20000, relative activity 1 007000, total volume 274 fluorescent liters plus printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Development (please read the precautions on the back before filling this page) A (0.025¾ trifluoroacetic acid (TFA )) And developing solvent B (containing 0.025¾ TFA of 1-propanol) to prepare a final volume of 508.63 ml, the final propanol concentration is about 20¾, the TFA concentration is 0.012¾, and the protein concentration is 0.0989 mg / ml. This produces insolubles, so this is centrifuged, the supernatant is divided into 25 4 · 3 ml (25.2 mg) twice, and the flow rate of K 2 ml / min is added to the YMC-Pack PROTEIN- K30% B equilibration in advance RP (YMC, catalog number A-PRRP- 33-03-1 5 diameter 3 cm, filter pad height 7.5 cm) column. The precipitate was added with CHAPS (5-[(3- Group) dimethylammonium] -bupropionate; manufactured by Tongren Institute of Chemistry, catalog number 75621 ~ 03_3) 20mM ethyl Sodium, pH 5.5 20 ml can dissolve, merge into the column [0159] -57- applies the Chinese national scale present paper rubbing registration (CNS) A4 size. (210 &gt; &lt; 297 mm) 498 079

B7 V. Description of the invention (55) (Please read the precautions on the back before filling in this page) After adding the sample, pass in about 50 ml of solvent (developing solvent A •• developing solvent B = 3: 1). The stranded part. Next, the program was developed (a linear concentration gradient of 30% 120 to 45% B), and a total of 30 polypropylene tubes were collected and 10 chromatographic fractions each were taken. This operation was repeated and collected in the same tube, the last 20 ml each, and a total of 36 chromatographic fractions. The non-retained part was concentrated to 20 ml with the K__ unit (manufactured by Amicon Corporation; γ W 3 membrane, 76¾ m in diameter). [0160] From the non-retained part and each 20 ml of the tube _ No. 1 to 36, take 0.1 ml, add 20 microliters of 5% BSA and centrifuge> to dry by evaporation, and finally dissolved in 0 · 25 ml of IMDM assay medium for assay to specify the TPO active fraction. As a result, there was TPO activity in tube numbers 17 to 27 (in the range of propanol concentration 38.0 to 43.0%), and this was used as the TPO activity of YCM-Pack PR0TEIN-RP from the low molecular TPO control standard. Part P2. Store at -85 ° C until the next YMC-Pack CN-APM. [0161] YMOPack PR0TEIN-RP from the low molecular TP0 control standard F3 TP0 activity F2 total volume protein concentration total protein relative activity relative activity 220 ml printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs / Ml 2.85 mg 130000 371000 (9) YMOPack CN ~ AP &lt; Reverse Phase Chromatography> -58 This paper size applies to China National Standard (CNS) A4 (210X297 mm) 498079 Employee Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Print A7 B7 V. Description of the invention (56) 214.9 ml of the TP0 active F2 part of the YMC-PackPR0TEIN-RP from the low molecular TP0 control standard F3 obtained from (8) (total protein mass 2.79 Mg, protein concentration of 0.0130 mg / ml, relative activity of 13,000 (relative activity of 3,630), 0.5% of glycerol in 0.6 ml was added, and concentrated to 1.8 ml. The final volume was 5 ml, and the propanol concentration was 20%. The glycerol was about 6%. [0162] This was divided into 5 times (injection of 0.5 5 5 mg of protein, 1 ml in volume). With each use of propofol containing developing solvent A (0.12 TF A) and developing solvent B (0.055 TPA), the flow rate of K 0.6 ml / min is injected into K15% B balanced YMC-Pack CN -AP (manufactured by YMC, catalog number AP-1 5 3; diameter 6 mm, filter pad height 250 mm) in a column. After the end of the injection, the propanol concentration was increased from 15% B to 25¾ B, and then a linear concentration gradient of 65/5 B was developed from 25¾ B to 50¾ B K. The last time, 1 ml of a solution of the same composition containing no protein was injected and developed to recover the TP 0 activity remaining in the column. A total of 6 portions were collected in the same polypropylene tube, and each chromatographic portion contained 7.2 ml of 44 tubes. [0163] Take 30 microliters (one 240th of the chromatographic fraction), add 20 microliters of 5¾ BSA, centrifuge to evaporate to dryness, and finally dissolve in 0.24 ml of IMD Μ assay culture solution, do the measurement , The TPO active part is specified. As a result, due to strong TPO activity in tube number 28 to 33 (in the range of propanol concentration 37.0 to 42. 0¾), this is not the YMOPack CN-AP from the low molecular TPO control standard F3. The main TPO active FA part. -59- This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297mm) --------- install-(Please read the precautions on the back before filling this page) Order • Fee 498079 A7 B7 Total volume protein concentration Total protein mass Relative activity Relative activity amount V. Description of the invention (57) [0164] YMC-Pack CN-AP TPO active FA part from low molecular TPO control standard F3 43.20 ml 0.00 86 3 mg 0.37 3 mg 800 000 298 400 (1 0) C epce 1 1 P ak C 1. 3 0 0 A &lt; Final Reverse Phase Analytical Method> The low molecular TPO control standard obtained in (9) 43.20 ml of TP0 active PA part of product F3 (total protein mass 0, 3 72 mg, protein concentration (K 0 0 8 6 3 mg / ml, relative activity 8 0 0 0 0 0 & gt Relative activity amount 2 9 7 6 0) &gt; Add 0.2 ml of 50% glycerol and concentrate to 0.1 ml of glycerol solution. [0165] Add solvent A (0 · 1% TP) A): 2 ml of a solution of developing solvent B (containing 0.5% TFA in propylene) = 85:15 (15¾ B), and finally prepared into a volume of 2.1 ml, Alcohol concentration of about 1 4¾, glycerin of about 4. 8¾, and protein concentration of 0.177 mg / ml. This was injected into Capcell Pack Cl 300A (made by Shiseido, catalog number Cl TYPE: SG 30 0 A) balanced at 15¾ B. Diameter 4, 8 mm, filter pad height: 250 mm), with a linear flow rate gradient of 27 ¾ B to 38 ¾ B for 65 minutes at a flow rate of 0.4 ml / min, collected in 72 polypropylene tubes 0,6 ml. [0166] 60 This paper size applies to the Chinese national standard (CNS> A4 size (210X297 mm) --------- packing-(Please read the precautions on the back before filling this page ) Order printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Description of the invention (π) Take 3 microliters (1/200 chromatography) from each chromatographic portion, add 20 microliters 5¾ BS A, and finally Substitute in 225 microliters of IMDM assay culture medium, and measure the one diluted 75 times from the original volume. [0167] From each chromatographic fraction, take 1 microliter (1 / 600th of a aliquot) for electrophoresis and centrifuge , Evaporate, add 10 microliters of osmogen-free SDS gel electrophoresis sample buffer, and process at 9 5 ° C for 5 minutes. A gel (prepared by Daiichi Chemical Co., Ltd.) prepared in advance with 15-25% S D S-polypropylene amidine was used for SDS gel electrophoresis, K 21)-silver staining reagent. The "first" silver dyeing kit (manufactured by Daiichi Chemical Co., Ltd., catalog number 1 67997, hereinafter referred to as "silver dyeing kit") was used for dyeing. Use "first" for molecular weight markers. DI low molecular weight mark (manufactured by Daiichi Chemical Co., Ltd., catalog number 181061, K is referred to as "D P C I I I"). [01683 As a result of the above analysis, the tube number 35 to 43 (in the range of the propanol concentration · 30.0 to 32.5¾) apparently has TPO activity. Among them, 36 ~ 42 (in the range of 30.5 ~ 32.0% of propanol concentration) in the tube series are used as the main TPO active part FA. The results on K are shown in Figure 2. [0169] Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page). Estimate the protein quality from the chromatogram. Combined with the measurement results, the total protein cover is 39.6 micrograms. The protein concentration was 9.4 μg / ml, the relative activity was 48 9 0 0 0, and the relative activity was 19 360 0. Investigating the SDS gel electrophoresis images of tube number 36 to 42 of the TPO active part, it is clear that there is a zone where the intensity of the activity is related to the concentration of staining. Moreover, the molecular weight of this zone, in appearance, is -61-This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 498079 A7 __ B7 _ V. Description of the invention (59) That is, The standard molecular weight protein was found to be a powerful zone to be a candidate for TP0 at the positions of 17000 to 1900. (11) Extraction of TPO activity from the electrophoresis gel &lt; 15¾ SDS Polyacrylamide Gel Electrophoresis &gt; IP-D ..... Partial analysis example is obtained from the low molecular weight obtained in (10) TP0 control standard is based on 4,200 microliters of TP0 active PA fraction (total protein mass 39.6 micrograms' protein concentration 9.4 micrograms / ml, relative activity 4890000, relative activity amount 1 93600) within 5.5 microliters (764 points 1 calendar fraction) extraction activity, in order to stain 2.5 microliters (1 / 680th of 1 aliquot) silver staining, respectively into the sample tube, centrifugation, evaporation, and adding SDS without reducing agent 10 microliters of the gel electrophoresis sample, granules, were processed for 1 hour at 37, and then SDS-formed by allowing to stand at room temperature for 18 hours. [0171] Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page). The molecular weight markers use prestine (7.i / 7, 4-7 in Canton). Xiaofan Park mark (BU-Rad company 16 1-0305) and DPCIII mark. These samples were subjected to customary usage (Laeminli, Nature, Vol. 227, pp. 680-685 (1970)) and electrophoresis was performed on a 15T SDS polypropylene amidamine gel using a microplate gel at 4TJ. Immediately after the end of the swim, the k-knife cut off the part handed over to the silver stain, put it into the fixing solution, and made the silver stain with the silver staining kit. [0172] On the one hand, the active part and the entire range of molecular weight should be cut out, and thinly cut with a knife into 34 gels of 1, 5 ~ 2, 5 mm, and the method of Kobayashi-62- China National Standard (CNS) A4 specification (210X297 mm) ου / y A7

V. Description of the invention (60) (Kobayashi Sahiko, Biochemistry, Vol. 59, No. 9 (1 987)) Improved gel breaking. Add 0.3 ml of extraction buffer (20niM T "is-HCl, pH 8, 500mM NaCl, 0.05% BSA) to the gel broken into fine fragments, and extract at 4 ° C for 6 hours with shaking [0173] Next, add 6.8 mK potassium phosphate at a final concentration of 20 mM to produce 6.8 and shake at 410 for 1 hour. To remove the precipitated SDS, move to ultra-free filtration-C 3 GV 0 · 2 2 The filter unit of the micron filter (manufactured by Milipor, model UPC3 OGV OS), centrifuged at 10,000xg (4000RPM) for 15 minutes, and the filtrate was recovered. This was moved to the ultra-free pass. C3-LGC molecular weight 10,000 Filtration element (Milipor company, model [JF (: 3 LGC 0 0) is centrifuged at 3 0 0 X g (7 0 0 0 R PM). When the concentrate reaches about 50 microliters, add 3 0 0 Microliter of 20 mM phosphate was buffered with 7.2, and then subjected to ultrafiltration. [0174] This was repeated twice to remove the remaining SDS. The same operation was repeated for the measurement culture solution, and finally prepared to 300 microliters This is sterilized to determine the activity of TP0. [0175] Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back first) (Fill in this page again.) As a result of this experiment, K silver staining can clearly detect the proteins. For DPC I labeling, the apparent molecular weight is about 1 7000 ~ 1 9000, 1 4000, and 1 1 000. Capce 1 1 The electrophoresis of the TP0 active portion of the Pak C1 column can observe the intensity of the activity that is related to the concentration of the stained zone. The band with an apparent molecular weight of about 1 70 00 ~ 1 90 0 0 is also detected in this experiment. Vision molecule-63- This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) 498079 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 Β7 V. Description of the invention (61) The amount is about 17000 ~ 19000. Above The result is shown in Fig. 3. [0176] The result on M shows that the protein with TP 0 activity can be confirmed in the active part of the Ca P ce Π P ak C 1 3 0 0 Α column, and finally refined to Can be confirmed on an electrophoresis gel. This sample is electrophoresed on a 15% SDS polypropylene with amine gel (not under Takihara), the concentration of the silver-stained band &gt; full TP 0 in the active fraction The amount of TP0 candidate protein with a molecular weight of about 1 70 0 0 to 1 90 00 is about 1.7 micro . [0177] Κ at (12) to (15) to a polymer in each step of the purification ΤΡ0 reference standard F2 of statements. (12) YMC-Pack PRTEIN-RPC reverse phase ephemeris method> The polymer TPO control standard F2 obtained in (7) (total protein mass 257 mg, protein concentration 0. 894 mg / ml) relative activity 7840, relative activity of 20 1 500 0, total volume of 287 ml), adding development solvent A (0. 025% TFA) and 1/3 of the sample volume of 95.8 ml development solvent B (containing 0.025 ¾ TF A), so that the final propanol concentration is about 25¾, the TFA concentration is 0.008, and the protein concentration is 0.671 mg / ml. As a result of insoluble matter, the supernatant after centrifugation was divided into 62.3 ml (42.8 mg) of 6 times each and injected at a flow rate of 2 ml / min into YMC-Pack PROTEIN-RP equilibrated with 30% B in advance. (Manufactured by YMC, catalog number A-PRRP- 33-03 ~ 15; diameter 3 cm, filter pad height 7.5 cm). Precipitate plus 5 mM CHAPS 20raM sodium acetate pH 5 · 5 10ml-64- This paper size applies to China National Standard (CNS) Α4 size (210 × 297 mm) (Please read the precautions on the back before filling this page )-Packing and ordering 498079 A7 B7 V. Description of the invention (62 can be dissolved, so they are combined and sent to [0179] After injecting each of the _ products A: Expanding solvent B = 3: 1), the formula (from 120/3 to 3 (UB propylene tube collection takes a total of 24 pieces and similarly reverses the search for the most retained part and the bud compilation 1, Y Μ 3 film, diameter 7 6 lm [0180] from the tube containing no retentate 10 microliters of 5% β S Α IMD Μ after separation to measure the broth, as a result, tube number 10 to 15 (with TP 0 activity, this is used as YMC-Pack PR0TEIN-RP YMC-Pack CN-AP up to the column. Pass in about 50 ml of solvent (after developing the solvent to collect the non-retained part, start the development process to a linear concentration gradient of 45¾ B) &lt; K poly chromatographic fractions are each 1 5 milliliters. From the first to the subsequent, there are 24 aliquots of 90 milliliters each. The ultrafiltration unit (manufactured by Amican Corporation) is used to concentrate to 90 milliliters without change. 0, 3 ml of the calendar fractions No. 1 to 24 were evaporated to dryness, and finally dissolved in 0.3 ml to determine the TPO active fraction. This propanol concentration was 3 4 · 0 ~ 3 9 · 5 Scope) from the TP 0 active portion F 2 of the polymer TP0 control standard F2. Until the next, save at -8 5 ° C. (Please read the precautions on the back before filling this page)

、 1T _ Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs [0181] YMOPack PR0TEIN-RP from the polymer TPO control standard F2 TPQ activity F2 Total volume protein concentration Total protein mass relative activity 5 4 0 * ml 0. 21 mg / ml 11.4 mg 227000 65- This paper size applies to the Chinese National Standard (CNS) A4 (210X297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 ____B7 V. Description of the invention ( 65) Relative activity 25 8 8 0 0 0 (13) Gel filtration calendar analysis in the presence of Superdex 7 5 pgCCHAPS &gt; YMC-Pack PR0TEIN- from polymer TPO control standard F2 obtained in (12) Add 5 ml of 50% glycerol to 5 38.2 ml of the TPO activity F2 portion of RP (total protein mass 11.3 mg, protein concentration 0.021 mg / ml, relative activity 2270 0 0, relative activity 25 6 5 0 0 0) , Centrifugation, evaporation and concentration. Then add 6 ml of 20 ra M C H A P S. Add 18ml of 20inM CHAPS, mix and move to 4 ° C, and after 41 hours, inject the initial sample into HiLoad 26/60 Superdex 75 ps (manufactured by Falumestia Biochemical Company, catalog number, at a flow rate of 1ml / min). 1 7-1 070 -01; diameter 2 · 6 cm, filter pad 60 cm in height), DPBS with 5niM CHAPS unfolded. -Inject 4 ml of sample (protein concentration 0.466 mg / ml, protein amount 1.86 mg) into the column. [0182] The M column was divided into 6 developments, and the M S u p er d e X 7 5 p g column was divided to collect the entire TPO active portion of YMC-Pack PROTEIN-RP. The six calendar portions are 5 ml each, that is, a total of 30 ml of calendar portions becomes 45. [0183] · Take 1 ml of each chromatographic fraction, add 10 microliters of 5¾ BSA, centrifuge and evaporate to dryness> Finally dissolve in 0.25 ml of IMD M assay culture solution, make the measurement, and The TP 0 active moiety is specified. As a result, the tube number 1 3 to 31 (in the range of molecular weight 78 0 00 to 300 0) has TP0 activity &gt; This is taken as -66- Superde'x 75 ps from polymer TP0 control standard F2-this paper Standards are applicable to China National Standard (CNS) A4 specifications (210 ^ 297 cm) (Please read the precautions on the back before filling this page)

1T 498079 A7 B7 V. Description of the invention (64 The TP 0 active P 2 part is printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. [0184] The TP 0 active F 2 part of the Superdex 75 pg from the polymer TP0 control standard F2 5 40 ml 0.02 16 mg / ml 1.17 mg 1750000 2041000 (14) YMC-Paek CM-AP <Reverse Phase Chromatography &gt; The polymer TPO control standard F2 obtained in (13) Superdex 75 pg TPO active F2 part (molecular weight 78000 ~ 3 0 0 0) 5 5 1 3 · 2 liters (total protein mass 1 · 1 1 荦 g, protein concentration 0.00 2 1 6 mg / Ml, relative activity 17 50 0 0 0, relative activity 1 943000) After adding 1/10 volume of the developing solution B (containing 0.05¾ TPA of bupropion), the injection was injected at a flow rate of 0.6 ml / min. YMC-Pack CN-AP (developed by YMC Co., Ltd.> catalog number AP _ 5 1 3; diameter 6 mm, filter pad height 250 mm) balanced with developing solvent A (0.1% TFA) and developing solution B ^ 15¾ B The column. After the injection, the concentration of propanol is increased from 15% B to 25% B, and then a linear gradient of 65% from 25 to 50% B is developed. [0185] When performing a YMC-Pack C N-AP column, '1/20 of all the input samples were performed in batches to confirm that the activity can be recovered well. Therefore, it was divided into 2 parts and taken off 19/10/20. That is, 3 times of total expansion. Total volume protein concentration Total protein 虿 Relative activity Relative activity amount -67- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) ) (Please read the precautions on the back before filling this page). Packing 'Order 1_ 498079 A7 B7 V. Description of the invention (65) Collect the three chromatographic fractions collected in three separate parts in the same polypropylene 44 A total of 3, 6 ml of each tube. [0186] 5 microliters (1/20 of the chromatographic portion) was taken out, and finally replaced in 0.25 ml of IMDM measurement culture solution. As a result of the measurement, Since tube number 24 ~ 30 (in the range of propanol concentration 36 · 0 ~ 42.0%) has strong TP0 activity, this is taken as the main TP0 of YMO Pack CN-AP from polymer TP0 control standard P2 Active FA part [0187] Superdex 75 pg from polymer TPO control standard F2 POL active FA part (please read the precautions on the back before filling out this page)-Packing · Printed by Zhenong Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 25.20 ml 0 · 0 2 4 6 g / ml 0.6 2 0 mg 700000 434000 (15) Capcell Pak Cl 300AC final inverse phase ephemeris method> YMO? 3〇1 ^^ 4? 1? 0 activity obtained from (14) from the polymer TP0 control standard F2 "Part 4 To 25.20 ml of 24.66 ml (total protein mass 0.606 mg, protein concentration 0.02 46 mg / ml, relative activity 700,000, relative activity amount 424000), add 0.4 ml of 50% glycerol, centrifuge and concentrate by evaporation. [0188] Finally, the concentration of propanol is several%, the concentration of glycerol is 10 ¾, the concentration of protein is the total volume, the concentration of protein, the total protein mass, the relative activity, and the relative activity-6 8-This paper uses the Chinese National Standard (CNS) A4 specification (210X297 mm) Order printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Description of the invention (6β) &quot; A 2 ml sample of 0.303 mg / ml. Capcell Pak ci 30 0 Α (Shiseido catalog number Cl TYPE: SG 3) was developed using a developing solvent a (〇, u TFA) and a developing solvent B (containing 0.05% 1TΑ2 propanol). 0 0 A; diameter 4 · 6 mm, filter mat height 2 500 mm), column with a linear gradient of 27¾ B to 38¾ B 65/65, κ 0.4 ml / min unrolled at 72 polypropylene Collect 0.6 ml in each tube. [0189] Take 0.75 microliters (chromatographic fraction of 1/800) from each Λ fraction, add 20 microliters of 5% BSA, and finally replace with 225 microliters of IMD. The measurement was performed by diluting 300 times from the original volume. [0 1 9 0] Take 2 microliters (1 / 300th of the chromatographic fraction) from each historical analysis for electrophoresis, centrifuge and evaporate, and add 10 microliters of SDS electrophoresis sample without reducing agent. 5 minutes at 9 5 T]. This gel was pre-made with 15 to 25% S D S -polyacrylamide (prepared by Daiichi Chemicals) and run on S D S gel electrophoresis, and stained with a silver staining kit. Molecular weight markers are labeled with DPCM. [0191] The analysis result of K clearly has TP 0 activity in tube numbers 33 to 39 (in the range of a propanol concentration of 29.5 to 31.5%). Among them, tube number 34 ~ 39 (in the range of propanol concentration 33.0 ~ 31 · 5¾) is the main TPO active FA part. The SDS gel electrophoresis image of the main active part of TP0 was clearly found in the molecular weight range of 17000 ~ 2200 from the start of the low molecular TP0 control standard F3 described in (10). The intensity of the activity is related to the staining concentration. -69-This paper size is in accordance with Chinese National Standard (CNS) A4 specification (210 × 297 mm) (Please read the notes on the back before filling out this page) ··, ιτ 498079 Printed by A7, Consumer Cooperative of the Central Standards Bureau, Ministry of Economic Affairs _ ^ B7 V. Explanation of the invention (67) [0192] &lt; Example 2 &gt; Analysis of partial amino acid sequence of rat refined TP 0 By the method of Iwamatsu (Iwamatsu et al., New Basic Biochemical Experiment Method, Section 4 Vol. 3, 3 ~ 8, 4 pages (Nine Goods); Iwamatsu Akiko, Biochemistry, Vol. 63, No. 2, pp. 139-143, · (1991); Akihiro Iwamastu, Electorophoresis, Vol. 13, 142 -147 pages, (1992)), analysis of the amino acid sequence of the rat TPO candidate protein in the TPO FA portion of the Capcell Pak Cl 300A column obtained in (1) of Example 1 was performed. That is, the samples were run on SDS gel electrophoresis, and K was electrotranscribed on a polyvinylidene difluoride (PVD F) membrane. Then, the protein S-alkylated on the PVD membrane was degraded with three kinds of protease restriction enzymes in stages, and the peptide was fragmented. This K reverse phase ephemeris method was used to separate and refine the obtained peptide. Sensitive amino acid sequence determination. K is followed by K for details. &gt; [0193] Capce 1 1 PaJcJLLJLQO A Capce 1 1 PaJcJLLJLQO A clipped from the low-density TP control reference standard analysis example of the TPΠ candidate g white matter (1) Capcell Pak Cl 30 0 / TPO FA section of the column Concentration of aliquots (tube number 36 to 42) The TP0 active FA portion of the Capcell Pak Cl 300 A column from the low-molecular-weight TP0 Cenzhao standard F3 obtained in Example 1 (10) (tube number 36 to 42) 4200 microliters (total protein mass of 39.6 micrograms, protein concentration of 9.4 micrograms / ml, relative activity 4890000, relative activity ί 1 93600) of 4151 microliters (98.8% of the total chromatographic fractions) were subjected to amino acid Sequence (please read the precautions on the back before filling this page) -Installation · 1T. • Line-70- This paper size is applicable to China National Standard (CNS) A4 specification (210 × 297 mm) 498079 A7 B7 V. Description of the invention (68) Analysis, the protein mass estimated from the chromatogram was 39.1 micrograms, and the amount of TP0 candidate protein with a molecular weight of about 1 7000 to 1 9000 on the appearance of silver staining by SDS gel electrophoresis was about 1.6 micrograms. Glycerin was added to this sample, centrifuged, evaporated and concentrated to make 5 microliters of glycerol solution. Add the SDS electrophoresis sample powder without distant source reagent and the adjusted 1M Tr is-HCl, and then make it 8 and finally make it into 200mM TrMs-HCl, pH 8.0, 50mM Tris-HCl, pH 6.8, 1.1¾ Approximately 25 microliters of SDS, 2mM EDTA, 0.002¾ BPB, 30¾ glycerol. [0195] In order to fully convert the sample to DS without excessive heating, the sample was first allowed to stand at room temperature for 14 hours, followed by 60 ° C for 5 minutes. [0196] (2) Preparation of slab gel (4,0% acrylamide concentrated gel, printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs, according to customary usage of electrophoresis (please read the notes on the back before filling this page) 15¾ acrylamide separation gel), SDS gel electrophoresis was performed at room temperature at 12. 5 iuA followed by a constant current of 17.5 mA for 2 hours. The molecular weight markers were prestine small-range markers (B 〇-Ra d 16 1 -030 5) and DPCIII. Immediately after the end of electrophoresis, it was transcribed on the PVDF membrane (item). [0197] In addition, a part of the sample to be delivered for analysis is in a non-exogenous state and is re-naturalized with dithiothreose (DTT). K 15 ~ 25¾ a preliminarily prepared gel of polyacrylamide (first Manufactured by a chemical company; multiple gels (7 / V / ff, \ /) -71-This paper size is printed in China National Standard (CNS) A4 (210X297 mm) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Description of the Invention (69) 15/25 Catalog No. 21 1 072) Electrophoresis. When this silver-stained kit was silver-stained, it was confirmed that the expected TP0 region was a TP0 candidate protein with a molecular weight of about 1900 0 under EBARA and in the TP0 active portion of the Capce 1 1 Rak Cl 300 A column. The purity is about several%. Because of the different mobility of non-prototype and reduction, it is suggested that there is at least one S-S bond in the molecule. 0 [0198] (3) Detection of the region transcribed on the PVDF membrane by the electro-ink dot method Using a semi-dry transcription device (manufactured by Malisso &gt; Weltford transcription device KS-8460), according to customary usage, a constant current of l60raA (11 ~ 17V) was applied for 1 hour to perform transcription on a PVDF membrane ( (Porr B 1 010, manufactured by Abrad Biochemical Systems, catalog number 4 0 094 4). For the anolyte, 0.3 M TrMs, 20¾ methanol &gt; pH 10.4, the transcription membrane solution was 25mM TrMs, 20¾ methanol, out of 10.4, and the catholyte was 25mM! &Gt; is, 40mM aminocaproic acid, 20¾ methanol, Out of 10.4. [0199] Staining the transcribed membrane K-Citriol-S staining solution (0.1 milligrams of Cichlid Red-S and 1 milliliter of acetic acid) in staining &gt; multiple zones were stained, Among them, it can be confirmed that the molecular weight of TPO is about 19,000. This cut is used to fragment the peptide (second term). (4) Fragmentation and peptide gene mapping. Amino acid sequence analysis To systematically fragment the transcript S-alkylated TPO candidate protein transcribed on the PVDF membrane, the following three proteases * are used to perform stepwise defined enzyme degradation. -72- The size of this paper is applicable to China National Standard (CNS) A4 (210X297mm) (Please read the precautions on the back before filling this page) Order 498079 A7 B7 V. Description of the invention (70) [0200] One digestion Amino acid peptide endonuclease (Achroraobacter lyticus ηι497-1, manufactured by Wako Pure Chemical Industries Ltd., Catalog No. 1 29-025 4 1) Secondary digestion endonuclease Asp-N (manufactured by Beringer Mannheim, Catalog No. 1054 589) Secondary digestion trypsin-TPCK (manufactured by Worthington Biochemical Co., Ltd., Catalog No. 3740) The peptide fragments obtained by digestion with each enzyme were collected and used in developing solvent A containing 0 · 05¾ TFA, and contained in developing solvent B 0. 02¾ TFA isopropanol: acetonitrile = 7: 3 mixture, MW ak 〇si 1- II 5 C 1 8 C 1 8 reverse phase column (made by Wako Pure Chemical Industries; diameter 2.0 mm, length 1 50 mm), column temperature 3 (TC, flow rate 0.25 ml / min, linear concentration gradient from 1% B to 50% B over 30% unfolded for gene mapping (Figure 4), and the recovered Peptide fragments. Each peptide fragment was analyzed by a gas phase amino acid sequence analyzer (Shimadzu Corporation) (PPSQ-2), after decomposing by Edelman method, the ΓΓΗamino acid at the N-terminus recovered will be identified by single-solution dissolution method KC 1 8 reverse phase column chromatography. The results are summarized below [0201] Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs --------- ^ equipment-(Please read the precautions on the back before filling out this page) One digestion is carried out in the peptide chain The amino acid sequence of the peptide fragment of the Dicer enzyme is the amino acid sequence. [0202] [Table 3] [0203] [Table 4] -73- The paper size applies to the Chinese National Standard (CNS) A4 specification (21〇297297) 498079 A7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs __ _ B7_ V. Description of the invention (71) [0204] [Table 5] In the above sequence, those indicated by () are deduced from the systemic enzyme digestion [0205] (5) Similarity analysis of the obtained amino acid sequence &lt; homogeneity survey &gt; Whether the obtained amino acid sequence is included in the reported protein, Or if there is a protein with a similar sequence, use a marker vector as a sequence analysis software 7. 、) 7 彳 ”勹 (Kodak International Biotechnologies) analysis. The information of known proteins or known genes uses Entrez Release 6 data base (National Center for Biotechnology Information, National Library of Medicine, National Institutes of H ea 11 h, issued August 15, 1993. The various data bases included in it are as follows: 0, [0206] Entrez Re e ease 6 data base NCBI-GenBank, August 15, 1993 (Release 78.0) EMBL, July 15 &gt; 1993 (Release 35.0 plus updates) DDBJ, July 15, 1993 SWISS-PR0T, April, 1993 (Release 25.0) PIR, June 30, 1993 (Release 37.0) PDB, Apr i 1, 1993 PRF, May, 1993 i -74-This paper standard applies to China National Standard (CNS) A4 (210X297 mm) (please read the notes on the back 1 · • fill in the items on the next page),? Τ 5. Description of the invention (72) Α7 Β7 dbEST, July 15, 1993 (Release 1.10) Ministry of Economic Affairs S. and Europian Patents (— Department) printed by the Consumer Standards Cooperative of the Central Bureau of Standards. As a result, the API 2 sequence (K) corticosteroid-binding globulin (CBG) precursor of DSFUDVK rats [PIR database-based accession number A4006; Smith and Hammond; ss R at corticosteroid-binding Globulin: primary structure and messenger ribonucleic acid levels in the liver under different physiological conditions. 〃 Mol · Endocrinol., (1989), 3, 420-426,] within The sequence KDSFLADVK is exactly the same. C 0207] A more thorough investigation revealed that a sequence of KQY YESE with a high similarity to the AP3 sequence (K) XYYESZ (where X is any of A, S, G ·, M, and Q) and U is E or K), including In the amino acid sequence of rat CBG. These sequences corresponding to AP1 2 ′, AP3 are continuous in rat CBG, and correspond to the internal amino acid sequence called KDSFUDVK QYYESE. [0208] However, no known proteins and genes should be considered for the amino acid sequences' of fragments other than A P 1 2 and A P 3. &Lt; Example 3 &gt; Analysis of biological characteristics of TP 0 from plasma of thrombocytopenia rats (1) CFU-MK measurement in rats: line (liquid culture line) As a representative example, the Thrombocytopenia in rat plasma τρ 0 -75- This paper size is in accordance with Chinese National Standard (CNS) A4 (210X297 mm) (Please read the notes on the back before filling this page) 498079 Employees of the Central Standards Bureau of the Ministry of Economic Affairs Consumption cooperative prints A7 B7 V. Description of invention (75) Partially refined reference standard (YMC Pack Protein-RP column TP0 activity F2 part described in Example 1 (8)) Shown in circle 5. When the culture was observed under the microscope over time, it was considered that the differentiation and maturation of megakaryocytes, that is, the increase in cell size, also considered that an increase in cells might occur. As a particularly significant change, on the 4th day of the last day of culture, it is considered that there are protrusions formed by a large number of giant nucleus balls (which can hardly be recognized on the third day of culture). This protrusion formation is called cyt ο p 1 as ni ic Process formation ( LevenPI Yee »Blood» Volume 69 &gt; pages 1046-1052, (1987)) or proplatelet process formation (Topp et al., Bld., D. 76, 912-924 (1990)), etc. The differentiated platelet precursor structure is considered to be the final morphology of megakaryocyte differentiation that can be observed so far in vitro. Because the TP 0 control standard alone is considered to have a high frequency of change in this form, it is believed that this factor alone can promote the proliferation and differentiation of CPU-M K, make mature megakaryocytes, and finally proceed to platelets. The possibility that arises. [0210] (2) In a community assay, a reference standard was purified for the TP0 fraction from the plasma of thrombocytopenia rats to use rat non-isolated bone marrow cells, isolate and concentrate cells at various stages, or GpEb / l [ a + CFU-MG part of the community measurement: At the time of the test, TPO from the plasma of the rat, so that a giant nucleus community was formed. Compared with the megakaryocyte community formed by TP0 and other known cytokines, namely rat IL-3, mouse GM-CSP, or the megakaryocyte community formed by human EP0, there are megakaryocytes formed by TP0. Among the communities, the number of meganuclei balls that make up each community -76- This paper size is applicable to the Chinese National Standard (CNS) A4 (210 ×: 297 mm) ----------- (Please read the Note for this page, please fill in this page again), ιτ 498079 A7 B7 5. The invention description (74) is small, but the size of each giant nucleus ball is large, which is a feature that enhances maturity. Furthermore, the colonies of other cell systems are scarcely formed, and the Mes-CSP activity shown by TP 0 is considered to be a megasphere-specific activity. From these things, it is clear that DP and other known cytokines that exert Meg-CSF activity measured in this community, namely rat IL-3, mouse GM-CSF, or human EP0, have different biological characteristics and play a role Unique M eg-CSF activity. [0211] For the CD 3 4 + DR + cell fraction derived from human bone marrow cells or human umbilical cord blood cells, the purified reference standard of the TPO fraction from the plasma of thrombocytopenia rats also showed Meg-CSF activity, making it intentional Number of human giant nucleus communities. This matter shows no species specificity in this factor. [0212] &lt; Example 4 &gt; Specificization of rat TP 0 producing cells (1) Exploration of rat TPO producing organs Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before (Fill in this page) First of all, for the purpose of selecting rat TP 0 gene based on part of the amino acid sequence of rat TP 0, or ensuring the supply of mR HA for epitope selection, rat TP0 generates organs. Exploration and specialization. At the beginning, P55 antibody was administered to rats with thrombocytopenia, and bone marrow, lungs, liver, and spleen were removed over time, and culture supernatants of their cells (in the case of lungs and liver, organs were cut) were taken. Solution, rat CFU-MK test strips to evaluate the activity in the supernatant, no clear results can be obtained. Then consider the report suggesting the correlation between rat liver and TP 0 (S i e me n s in a et al., J · L a b.

Cl in · Med., Vol. 86, 8 1 7-8 (33 pages (1975)), with P55 anti-77- This paper is suitable for China National Standards (CNS) A4 size (210X297 mm) A7 A7 Economy Printed by the Ministry of Standards and Technology Bureau's Consumer Cooperatives ~ ^ ---- __ V. Description of the invention) _ is administered to rat livers that cause thrombocytopenia, and hepatocytes prepared by the collagenase reflux method are cultured. In the supernatant, the adsorbed part of the WG A-Asa rose column was developed on a V ydac P heny 1 reverse-phase column in a rat C FI) -assay system to recognize the TP0 activity and Extremely similar activity at the same position. The culture supernatant from normal rat liver cells was also poorly recognized but also active. From these results, the possibility of liver dysfunction as one of the organs of TP0 is strongly suggested. (2) Screening of rat TPO-producing cell lines Based on the above results, screening of rat TPO-producing cell lines was performed. First, the cell lines from 20 types of rat liver were cultured in the respective culture medium for subculture to approximately a confluent culture, and the serum K contained in the culture solution was unified in each of 5¾ FCS. Replacement of other culture liquids> The culture was continued for another 3 days, and the respective culture supernatants were collected. Partially refined the method described in the supernatant K (1), and investigated the presence or absence of TP0. It was confirmed from three types of rat liver parenchymal cell lines, namely, McA-RH8994 cells (ATCC deposit number CRL 1602, Becker et al., Oncodevelopmental Gene Expression &quot; ed by Fishman and Sell,

Academic Press, NY, pp. 259-270 (1976), purchased by Dainippon Pharmaceutical Co., Ltd., H4-E-E cells (ATCC deposit number CRL1548, Pitot et al., Nat * CancerInst * Monogr., 13 #, 229-245H ( 1964), purchased from Dainippon Pharmaceuticals) and Η TC cells (T homps ο η et al. &Gt; Proc. Natl. Acad · Sci. USA, Vol. 56, pp. 296-303, (1 966), purchased from Dainippon Pharmaceuticals) Apparently produces TPO activity. -78- This paper size is in accordance with Chinese National Standard (CNS) A4 specification (210 × 297 mm) (Please read the precautions on the back before filling out this page) Qin 498079 Printed by A7, Consumer Cooperative of the Central Standards Bureau, Ministry of Economic Affairs _— —_B7_ V. Description of the invention (76) [0214] (3) Detailed analysis of TP 0 activity produced by McA-RH8994 cells, H4-HE cells, and HTC cells The activity of τρ 〇 secreted from these three rat cell lines and The TP0 activity from rat plasma, which was purified in parallel, was compared and reviewed in detail from two aspects, biochemical and biological. [0215] McA-RH 8994 cells were suspended in an alpha-MEM (-) broth containing 10¾ FCS, and placed in a broth plastic bottle with a bottom area of 175 cm2 to make 1 X 1 0 β cells / Bottle in a 5% CO 2 gas oven, 37. () Cultured for 3 days' and replaced with IMD M culture solution containing 5¾ FCS, and cultured for another 3 days, and the supernatant was recovered. The Η 4-I-Ε cells were suspended in Du 1 becc containing 10% FCS. Change Eagle culture solution (containing 4.5 g / L of glucose) (hereinafter, referred to as DME M culture solution), put it in a plastic bottle for tissue culture with a bottom area of 175 crni2 to make 5 X 105 cells per bottle in 5¾ carbon dioxide gas After 3 days of incubation at 37 t] in an incubator, the cells were replaced with an IMD M medium containing 5¾ FCS and cultured for another 3 days to recover the supernatant. The HTC cells were suspended in DMEM containing 5% FCS. The culture liquid was put into a plastic bottle for tissue culture with a bottom area of 175 cm2 to make 2,5 X 10δ cells / bottle, and then baked in a 5% carbon dioxide gas incubator at 37 t! For 3 days &gt; It was placed in IMDM culture medium containing 5¾ FCS, and cultured for another 3 days, and the supernatant was recovered. [0216] The culture supernatants of the three cell strains thus obtained were respectively 2 liters, as described in Example 2 From TRP refining method of XRP, cell-derived (please read the precautions on the back before filling out this page) ΙΦ ▼ Fill in again 』Binding · Binding-79- China National Standard for Ladder (CNS) A4 (210X297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 _B7 5. Refinement of part of TP0 of the invention description (77). The following is a summary. [0217] First of all The culture supernatant was concentrated 6 times by ultrafiltration and exchanged with Sephade X G-25 column in T "is-HCl (pH 8.0) buffer. The eluate was added to Q-Sepharose FF tube. The column was washed with M20 iuM Tris-HCl (pH 8 · 0), and the adsorbed part was dissolved with 20 μαM Tris-HCl (pH 8.0.0) containing 175 raM NaCl. This part was added to a WGA-Agarose column. After washing with KPBS, the adsorbed portion was dissolved by 20 mM sodium phosphate (out 7, 2) containing 0.2 MG 1 c N ac and 0.1 15 M N a C 1. This eluate was added to TSK-gel AP-BLUE 6 50 ΜΗ column, 20 ιαΜ o-acid (Na ^) 7.2) containing 1M NaCl, washed and dissolved by 24 ^ 311. This was added to a Phenyl-Sepharose 6 PP / LS column. Μ 50 inM sodium phosphate (pH 7.2) with 1.5 Μ ammonium sulfate (/ Unionium Sulfate) After washing &gt; K then 0.8 mM amine sulfate. 38 mM sodium phosphate after washing ... with 20 mM phosphoric acid Nano (Ex. 7.2) dissolves. After concentrating the adsorbed portion, the M reverse-phase Vy da c Protein C4 column (manufactured by The Separations Group, catalog number 214TP51015; 1 cm in diameter and 15 cm in height of the filter pad) was dissolved into each portion. Used in developing solvent A containing 0.12¾ TFA, and developing solvent B containing 0.055 TFA of propanol, K20 in advance; «B equilibrate the column ... after injecting the sample &gt; at a flow rate of 1 ml / min Dissolve by a linear concentration gradient of 20¾ B to 40¾ B. As a result, the TP 0 activity from any cell line can be dissociated at a concentration of 30¾ to 43¾. [0218] The reference standard of each purification stage was measured in rat CFU-MK: -80-. This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back first Fill out this page again), π Printed by the Consumer Cooperatives of the Central Bureau of Standards, Ministry of Economic Affairs, 498079 A7 ___B7__ V. Description of the Invention (78) The results show TP 0 activity from 3 cell lines, any of which is related to TP 0 from XRP The activity has a very similar behavior (refer to Example 1-2) (the relative specific activity, activity yield, etc. of each stage of the rat CFU-MK measurement system described in Table 6). In addition, the part dissociated from the reverse phase column in the final stage was measured in rat CFU-MK: As a result of measuring the activity, the TP 0 activity from the three cell lines was the same as the TP 0 activity from XRP. The peak position dissolves. K The active part of this reverse-phase column is centered, and the non-adherent cells obtained in the removal stage of the separation and concentration process of rat Gp E b / Itt 、 CPU-MK are used to determine the community of the non-adherent cells &gt; Either one was in rat CFU-Ml (the lysis pattern for measuring the activity of plutonium was approximately the same and was considered to form a megakaryocyte community (Table 7), and, similar to the TP0 activity from XRP, the activity from three cell lines Either one of them specifically formed the megasphere community, while the other communities of the Orthodox system hardly formed. [0219] According to the method described in Example 1-2, the active part of the pooled reverse phase column was provided as SDS Polyacrylamide Gel Electrophoresis》 Protein from the gel oil after electrophoresis. When measuring activity in rat CFU-MK, the molecular weight of TP0 from XRP is 1 7000 ~ 22000, and TP0 from McA-RH8994 cells. The molecular weight is 33000 ~ 39000, the apparent molecular weight of TP0 from Η 4-EE cells is 31000 ~ 38000, the apparent molecular weight of TP0 from HTC cells is 1 700 0 ~ 22000, and the molecular weight is 28000 ~ 35000. [0220] As above, ^ ^ 4-〇8 994 cells, ^ 1 4-1- £ Cells and [11 '(: The activity of TP 0 produced by cells is obviously different from that of TP 0 from XR Ρ -81- This paper is for Chinese national standard (CNS) ) A4 size (210 X 297 mm) I Binding (please read the precautions on the back before filling out this page) 498079 A7 B7 V. Description of the invention (79) The amount of the sub-quantity, some of the biochemical properties are different in biology The properties are equal. In the present invention, it should be specifically mentioned here that molecules that have TP 0 activity present in the blood are specific or non-specific in the molecule, within the cell after secretion or secretion from the production cell. The possibility of things being cut off at specific positions. Also, there is a possibility of existence of things of various lengths in the TP0 gene (mRNA and cDNA). [0221] [Table 6] [0222] [Table 7] &lt; Example 5 &gt; The expression vector (PEF18S) for eDNA library preparation The vector was incorporated into the c DHA fragment to easily create a highly efficient vector for selective DNA clones, and TP 0 c DNA clones were prepared in advance And epiphyseal. Also known as the extension factor of the epigenetic efficient promoter 1 ct (EF 1 α) promoter gene and easy-to-handle expression vector PME18S SR α promoter gene change &gt; Construct epitope vector PEF18S (see Figure 6). The elongation factor 1 α promoter gene will express the vector pEF -BOS (MizushUa et al., Nucleic Acids Res., UL 5 322, 1990) 1 microgram printed with the restriction enzyme Hindi! And the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page)

After EcoR I was partially digested, electrophoresis was performed using a 2¾ agarose gel (manufactured by FMC BioProducts), and a 1 200 bp DNA fragment was separated using bleb (7 ° V, '/ Guangyiyi>) DNA purification kit (manufactured by Bio-Ryder, based on Wi 1 1 is et al., Bio Techniques, 9 „, 92-99 &gt; 1 990, using a porous silicon-based substrate to absorb -8 2-this Paper size applies to Chinese National Standard (CNS) A4 (210X: Z97 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 ___ — _B7 _ 5. Description of the invention (80) Attached, selectively refined DNA sleeve (Group)) Refined. This DNA fragment was inserted into 100 μg of M H ind I [50 nanograms of expression vector pME18S digested with Eco RI (Liu et al., Proc. Natl. Acad · Sci. USA » 2A, 8 9 57-8961, 1993). The host strain uses Compendium &gt; 6 '' 孑> Bu · y, 4) E. col i DH5 (manufactured by Toyo Kaori Corporation; by Hanahan et al., J. Mo 1. Bio1., 1 6 6, 5 5 7-58 0, 1 983 (the competent host bacteria produced by the variation of the method), randomly selected 12 communities Protoplast DNA, by their type of DMA's K-restriction enzyme digestion, select one from 10 pure pupae containing the target plastid (PEF18S) to prepare a large amount of plastid DNA. [0223] Plastid DNA The purification of DNA is substantially rewarded as described in Μ ο 1 ecu 1 ar C 1 ο ning [SambrvookW &gt; Cold Spring Harbor Laboratory Press (1 9 8 9)]. P ME 1 8 S in 50 liters of LB medium containing 5 μg / ml of aspirin (1% B act 0-trypsinized knee, 0.5% B act 0-yeast extract, 0.5% Na C 1) After incubation overnight, the cells obtained by centrifugation were suspended in 4 ml of TEG-lysozyme (25niM Tris, Cl (PH 8), OraM EDTA, 50raM glucose, 0,5¾ lysozyme) solution &gt; Add 8 ml of 0.2N NaOH / 1% SDS solution to completely suspend. Add 6 ml of 3 M potassium / 5 M acetate solution to completely suspend and centrifuge to obtain the supernatant. After the supernatant was treated with phenol-chloroform (1: 1), an equal amount of isopropanol was added and centrifuged to obtain pellets. The pellets were dissolved in a TE solution (10 μM TrMs-hydrochloric acid (pH 7.5) &gt; ImM EDTA), then treated with RNse, treated with phenol-chloroform (1: 1), and then ethanol precipitated. The pellets are redissolved ~ 8 3 _ This paper size adopts Chinese National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling this page) ▼ 装 · ου / y Α7

V. Description of the invention (U) ϋ · — 11_1 — · ϋ — ^ ϋ —mm— ml «ϋ1« s «111 m nn ml ^ (Please read the precautions on the back before filling this page) a C 1 · and polyethylene glycol 3 000 were added to 6.3 3 M, and 7.5% were added to the human and centrifuged. Finally &gt; after the spheroids were dissolved in the TE solution, &gt; ethanol precipitation was performed to obtain about 300 μg of plastid D ΝΑ. This DNA 100 micrograms of M restriction enzyme E (: (RI) and Not I was completely tritiated, and electrophoresed on an agarose gel (manufactured by PMC BioCociucts) of M. 8¾, and the vector fragment was recovered. Κ- 普列布 -A ~ Refined DNA refining kit (made by Biochemical Ryder Co., Ltd.) was refined, and about 55 micrograms of plastid DN a was obtained. This DNA was prepared by using the following c DNA library. [0224] &lt; Example &gt; Refining of mRNA from McA-RH8994 cells As a result of the community measurement in Example 4, McA-RH8 994 cells with higher activity were selected as the material for rat TPOcDMA colony for experiments under M. [0225] The single-printed RMA printed by the Consumer Cooperative of the China Standards Bureau of the Ministry of Economics is essentially implemented as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)]. Me A-RH 8994 cells After completely and quietly proliferating in 15 petri dishes with a diameter of 90 mm, after removing the culture medium from the petri dishes, add 0, 8 ml of a 5M guanidine solution (5M guanidine thionate, 5mM lemon_ Sodium (pH 7 · 0), 0.1 Μ / 3-cyclyl ethanol, 0 ♦ 5¾ inosamine sodium sulfate), After thoroughly suspending, collect the mixed solution in a tube, add guanidine solution to make the total volume 20 ml. This cell is destroyed into a viscous mixture, and use a 20 ml volume syringe filled with 18 G and 2 1 G injection needles Repeatedly about 84- This paper size is applicable to the Chinese National Standard (CNS) A4 (210X297 mm) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 498079 A7 _; ______B7 V. Description of the Invention (82) 20 inhalation and discharge until Almost no viscosity. In a polymer centrifuge tube suitable for SW28 rotator manufactured by Beckman Company, 18 ml of 5.7M C s C BU 0.1 · 1M EDTA (pH 7 · 5) was used as a buffer to make Approximately 20 ml of the above mixture was carefully filled on the tube with a full filling, and the layer was carefully re-applied without damaging the layer. The centrifuge tube thus prepared was centrifuged at 20 to 2500 0 r · p, m, and centrifuged for 20 hours. The obtained pellets were washed twice with a small amount of 80% ethanol. The pellets were dissolved in a TE solution, extracted with phenol-chloroform (1: 1), and added with 1/10 amount of 3 M sodium acetate and 2.5 Ethanol precipitation with twice the amount of ethanol yields total RNA (about 2.5 mg of total RNA from about 10s of female cells) [0226] U) + RNA was purified from full RNA using 01igtexTM-cJT30 (Super) (Nippon Synthetic Rubber / Japan Roche Co., Ltd .; oligo-d TK was covalently fixed on the surface of the latex particles, and poly (A) RNA In the purification, the oligo dT column can be purified in the same way as poly (A) + RNA). From about 500 micrograms of total RNA, 20 micrograms of poly (A) + RNA was obtained. [0227] &lt; Examples &gt; Structure of rat cDNA library 5 micrograms of poly (A) + RNA obtained from Example 5 was used to shake TimeSaverTM cDNA synthesis kit (manufactured by Pharmacia; Okayaraa-Berg method: by Mol. Cell · Biol ·, Z_, 161-170, 1982, and DIRECTIONAL CLONING TOOLBOX (manufactured by Pharmacia; primers for cDNA synthesis with Notl sequence: 5, AACTGG / UG / UTTCGCGGCCGCAG * GAA (T) ia-3 '&amp; EcoRI Preface-8 5-This paper size uses Chinese National Standard (CNS) A4 (210X297 mm) I Binding (Please read the precautions on the back before filling this page ) · 498079 A7 B7 V. Description of the invention (85) Additional attachments: 5'-AATTCGGCACGAG-3f & 5'-CTCGTGCCG -3 'group), with EcoRI at the 5' end and Natl recognition at the 3 'end Of double-stranded cDNA. The synthesized CDNA was ligated with 1.2 micrograms of KEcoRI and Hotl-treated surface card carrier PEF18S (refer to Example 5), and 8,4 ml of Compendium E. col i DH5 (manufactured by Toyobo Corporation) was transformed. . As a result, 5.3 X 105 transformations were obtained. &Lt; Example 8 &gt; Obtaining of rat TPO cDNA fragments by PCR method (breeding) The McA-RH8994 cDNA library prepared in Example 7-530,000 pure 糸 K containing 50 μg / ml of aspirin 5 After cultured overnight in 0 ml LB medium, QIAGEN-tiplOO (manufactured by DIAGEN; a silica-based anion exchange column for DN A purification) was purified from the bacterial cells obtained by centrifugation, and Me AR Η 8994 eDNA library was purified to obtain about 200. Micrograms of plastid DNA. [0229] Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). Synthesis of two counter-inductive nucleosides corresponding to the amino acid sequence of the peptide fragment AP8 described in Example 2. The human elongation factor lc corresponding to the plastid carrier PEF18S shown in FIG. 6 (the first inductive nucleotide primer EFla_l, which does not express gardenia, used for the production of the acid primers-AP8-1R, AP8-2R, and the eDNA library. EFla-2. The 394DNA / RNA synthesizer (based on the / 3-cyanoethylamidad (down &gt; ^ ^) method) manufactured by Abrad Biochemical Co., Ltd. was used for the synthesis. DNA purification using 0PC column (reverse-phase silica-filled column to refine a column with trityl synthetic DNA). The purified synthetic DNA is dissolved in TE solution to 50 μM, to use -86 -This paper uses Chinese standard (CNS) A4 specification (210X297 mm) 498079 A7 B7 5 'invention description (84) and keep it at -20Ί〇. All synthetic oligonucleotides used below are synthesized and purified in the same way [0230] Primer-AP8-1R, AP8-2R are connected by Mixed M primers made of 17 consecutive nucleotides, such as the use of deoxyinosine in the study of Takahashi et al. (Γ ak: ahashi et al. Pr 0 c ♦ Na a 1 · A cad · S ci. (USA) 8 2 · 1 93 1-1 935 (1 985) [0231] The primers-EF 1 α-1 and EF 1 a-2 are based on U etsuki et al., J. Biol. Cheiu., 26 4 &gt; 5791-5798 The gene sequence in (1989) was synthesized from the base sequence of 1 4 9 1-1 5 1 2, 1 5 1 3-1 5 2 2 and was composed of 2 1 and 20 nucleotides of primers. The sequence of the synthetic primers is shown in Table 8. [0232] [Table 8] Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) KMcA-RH8994cDNA library plastid 3 micrograms is horizontal Plate, AP8-1R (· 500 picomoles) ·, EP1 a -1 (100 picomoles) as primers, GeneAmp ™ PCR Reagent Kit with AnipliTaq ™ DNA Polymerase (manufactured by Takara Shuzo Co., Ltd .; heat-resistant TaqI for PCR) Polymerase, reaction buffer, sister of dNTP), GeneArapTM PCR System 96 0 0 (manufactured by PERKIN-ELMER; PCR reactor) at 100 1 liter volume for PCR reaction (after heating at 95 ° C for 2 minutes, at 95 t: denaturing conditions at 1 minute, bonding conditions at 40 ° C for 1 minute, and synthesis conditions at 1 minute at 72 ° C The reaction was performed 3 to 5 times, and then cultured at 7 2 for 7 minutes. In order to improve the specificity of the amplified DNA fragments, the obtained paper size is -87- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 cm) 498079 A7 B7 V. Description of the invention (85) 1 microliter of PCR reaction solution is Template, EF 1 α-2 (100 picomoles), AP 8-2R (500 picomoles) were used as primers, and PCR reaction was performed in the same volume of 100 microliters (heated at 95 ° C). After 2 minutes, 35 times of reaction was performed at 9 5 t: denaturing conditions for 1 minute, 45 ° C for 1 minute of bonding conditions, 72 t! For 1 minute of synthetic conditions, and then at 72 t: 7 Minutes of training) 〇 [0023] Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). The reaction solution obtained here will be run using a 2¾ 瑷 lipose gel (FMC BioProducts (Manufactured by the company) was electrophoresed, and a DNA fragment of about 330 bp, which is the main product of the PCR reaction, was separated, and the bleb refined DNA purification kit (made by Bio-Ryder) was used. T4DNA ligase was used to sub-select this DNA fragment in PCRTMII (Invitrogen; PCR product TA colony vector: because the thermostable polymerase used in P CR has a terminal transferase activity, it is used in DNA amplified by PCR. ^ The property of attaching a deoxyadenylic acid to the end is performed on the vector pCRtmII with a 5 夂 dT overhanging end as the method of sub-selection) vector. For 28 pure lines selected arbitrarily, plastid DNA was purified using QIAGEN_tiplOO (manufactured by DIAGEN), and Ding aq Dye DeoxyTM Terminater Cycle Sequening Kit (manufactured by Abladd Biochemical System Co., Ltd .; using PCR to perform the second deoxygenation method with fluorescent dyes) · Sanger et al., Proc. Natl. Acad. Sci. USA * 7 4. 5 4 6 3 -5 467, 1977) &gt; Using a 37 3A DNA sequence analyzer (fluorescent Optical sequence analyzer) to determine the base sequence of each pure: line. [0234] This paper size applies to China National Standards (CNS) A4 (210X297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 ____B7_ V. Description of the invention (86) In the resulting DNA fragment, select the code When the N-terminal side of the amino acid of AP8 (Ile / Th "/ Se")-Val-Pro is adjacent to the primer of AP8-2R, the full-length sequence contains c DNA with 261 bp. This c The base sequence of 173 to 175 of the DNA fragment is the one encoding methionine, the structure of which is consistent with the structure of the amino acid of AP 8. Furthermore, this sequence is suitable for Kozak's sequence (Kozak, M ·, Cell, ^ _, 283-292, 1 98 6), so it is presumed to be the start of translation, and is considered to be the c DNA fragment encoding the N-terminal portion of the rat TP 0 protein. This c DNA fragment is named A 1. The base sequence of the A 1 fragment excluding the sequence of the vector and the amino acid sequence deduced therefrom are shown in the sequence listing (sequence number 1). [0235] &lt; Example 9 &gt; Screening of mouse TPO cDNA divided the aforementioned c DHA library into aggregates of about 10,000 pure tadpoles each, containing 50% After baking overnight in 1 ml of LB medium of μg / ml of ampicillin, an automatic plastid separation device P 100 (made by Kurashiki Textile Co., VER-3.0; based on the SDS method: Molecular Cloning [Sambrookl ^, Cold Spring Harbor Laboratory,

Press, 1 98 9]: The automatic method for extracting plastid DNA from the method of change) to extract plastids. Using 1/30 of this amount as a template, two synthetic oligonucleotides 5, CGAGGGTGTACCTGGGTCCTG3 '(the induction sequence of sequence 1-17; CGAG is the sequence of the linker) and 5 are designed based on fragment A1, CAGAGTTAGTCTTGCGGTGAG3f (-89 of the serial number 1) This paper size applies to the Chinese National Standard (CNS) A4 size (2l0x: Z97 mm) (Please read the precautions on the back before filling this page)

1T 498079 A7 B7 printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs V. Invention Note (87) 212-232 reverse induction sequence) Synthesized and refined as primers, using GeneAmpTM PCR Reagent Kit with Amf &gt; liTaqTM DHA Polymerase (Manufactured), PCR was performed using GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER) (denaturation condition at 94 10 for 30 seconds, bonding condition at 66 t: 30 seconds for 1 second, 72 minutes at 1 minute 30 reactions under synthetic conditions) As a result, specific bands were detected in 3 of the 100 aggregates. One of the aggregates was divided into about 900 pure lines, and the plastid D N A was extracted. When the same PCR reaction was performed, the bands were detected in 3 of the 100 aggregates, and one of the aggregates was divided into aggregates of 40 pure strips. The same selection was performed. 1 Among the 0 aggregates, 3 aggregates were observed to have a band that was considered specific. One of these aggregates was selected and planted on an LB petri dish (LB medium containing 15¾ agar) containing 50 μg / ml of ampicillin, and one of the colony oil-producing DNA appeared, and the same was performed. As a result of the PCR reaction, .2 of 100 pure lines detected positive bands. &Lt; Example 10 &gt; Sequence of rat TPOcDNA Purification of plastid DNA was performed substantially as described in Molecular Cloning [Sarobrook et al., Cold Spring Harbor Laboratory Press (1 989)]. In Example 9, the two pure mashes finally obtained were cultured overnight in 50 ml of LB medium containing 50 μg / ml of ampicillin, and then purified in the same manner as in Example 5 to obtain about 300 μg of plastids. (Please read the notes on the back before filling in this page) I Binding and ordering. -90- 498079 A7 B7 V. Description of the invention (88) DNA ° [0237] Taq Dye DeoxyTM Terminater C for plastid DNA obtained here yc 1 e S equening K it (Abbr Biochemical: System Co., Ltd.) 'was sequenced as in Example 8 to determine the base sequence of the full length of cDN A. As a result, it was understood that the base sequences of the two pure lines were completely identical to each other. One pure line was selected from among them, and the pure strips carrying the cDN A were named p E F 1 8 S-A 2 α. The base sequence and the amino acid sequence derived from it are not in the ordered list (sequence number 2). [0238] Printed by the Central Standards Bureau of the Ministry of Economic Affairs and Consumer Cooperatives (Please read the notes on the back before filling this page) The sequence features shown in the sequence list (serial number 2) are significant. 5 of 72bp, the sequence after the non-translated range, is presumed to be a signal sequence for secretion of 21 proteins starting from methyl sulfanilic acid, encoding a hydrophobic amino acid. This protein has 126 amino acid residues. After the stop codon (TAA), the 3 f untranslated sequence of 1,022 bases and the majority of the adenine at the polyA tail continue. This protein contains only the sequence corresponding to the amino acid sequence A P 8 resolved in Example 2 (amino acid numbers 1 to 12 in sequence number 2). The glycosylation sequence does not exist. In addition, the base sequence of 1 624 to 1 629 near the end of the 3 'non-translated sequence is different from the inherent sequence, but is considered to be a potential polyadenylation sequence. [0239] 'PEF18S-A2a, a carrier of coliform DH5, was deposited on February 14, 994, under the trust number FERM BP-45 65, at the Institute of Biotechnology and Industrial Technology, Industrial Technology Institute, Ministry of International Trade and Industry. -91-This paper size applies the Chinese National Standard (CNS) A4 (210X297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Description of the Invention (89) [0240] &lt; Example 1 1 &gt; Performance of COS1 cells in rat TPOcDNA ~ Activity confirmed transfection of COS1 cells of plastid PEF18S-A2cx, in accordance with DEAE-daichestrand treated with clokin (// α σ 7) Λ 卜 今 &gt;) (Scrapayraclf, Proc. Natl. Acad. Sci. USA 78 vol., 7575-7578 (1981); Uthman et al. Nucl · Acids Res ·, 1 vol., 1 2 9 5-1 3 Page 0 (1 9 8 3)) plus a few changes to the following methods. COS1 cells (ATCC CRL 1 6 50) suspended in DMEM culture solution containing 10¾ FCS were placed in a 100 mm diameter plastic culture dish for culture, and cultured in a 5% carbon dioxide gas incubator at 37 υ. To about 40% confluent cultures. On the one hand, in DEA E-Daichestrand (Faromaxia), 50 / iM Crokin (Sigma), 8BS (ν / ν) HBS ( 21mM HEPES-145mM NaCl, 7.1) and 9¾ (v / v) of Nu-Seruro (Clabrettiv). In 4 ml of DMEM culture medium, the plastids will be dissolved in 30 microliters of HBS. PEF18S-A2 cx (10 micrograms) was mixed and added to COS1 cells written in DMEM culture medium 2 times before transfection, and then cultured in a 5¾ carbon dioxide gas incubator at 37 ° C for 5 hours. After that, the culture supernatant was removed by suction. After washing the dish twice with K DMEM culture solution, 15¾ liters of DMEM culture solution containing 10¾ FCS was added, and cultured in a 5¾ carbon dioxide gas incubator at 37¾ for 3 to 5 days, and recovered. Culture supernatant. [0241] After the obtained culture supernatant is fully dialyzed against 1 MD M culture solution, the paper size of K rats is in accordance with the Chinese National Standard (CNS) A4 specification (210X297 mm) 1-1 (please read the note on the back first) Please fill out this page again) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 498079 A7 B7 _ V. Description of the invention (9Q.) CFU-MK measurement system evaluation. As a result, a dose-dependent T P 0 activity was observed in the culture supernatant of C 0 S 1 cells of epidermis, which was transfected with pEF18S-A2a plastid (Fig. 7). In addition, as in the case of rat T P 0, most cytoplasmic processes were observed on the fourth day of culture. On the one hand, no TPO activity was detected in the COS1 female cell culture supernatant transfected with the insert-free PEF18S plastid (Figure 7). In addition, in the M-0 7e assay, a dose-dependent M-0 7 e cell proliferation-promoting activity was also observed in the culture supernatant of the plastid PEF18S-A2 c No activity was observed in the culture supernatant of COS1 cells transfected with the insert-free plastid PEF18S. From these results, it was confirmed that PE 卩 18S-A2 cx was loaded with a gene encoding a protein having TP0 activity. [0242]. Next In order to investigate the platelet-increasing effect of TP 0 activity, some refined products were prepared. In the preparation process, TP0 activity was measured using rat CFU-M1 (assay 糸. First, the COS1 cells transfecting plastid PEF18S-A2 α The serum-free medium containing BS A was cultured for 3 days to obtain about 5.8 liters of serum-free culture supernatant. In this serum-free culture supernatant, P-APMSF was added to make the final Concentration ImM, the filtrate was taken through the filter. For this volume 5793 ml (protein concentration 0.229 mg / ml, total protein mass 1 326 mg, relative activity 1,000, relative activity 1 326080) plus 0 per 1,000 ml 0 · 85 mols of NaCl (total 288 grams) to a final concentration of 0.822M NaCl 5 After 849 ml of the solution, K was added at a flow rate of 7 ml / min to a TSK-gel AF-BLUE 6 50 MH column (Tosoh Corporation) equilibrated with 1 μN Na C 1 ·, 20 mM sodium phosphate and 7 · 2 in advance. System, catalog number-93- This paper size applies to China National Standard (CNS) Α4 size (21〇 ×: 297 mm) (Please read the precautions on the back before filling this page) -Packing ·, ιτ ^ «079 ^ «079 A7 printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs —___ V. Description of the invention (91) 0 8 7 0 5; diameter 5 cm, cymbal pad height 6 cm). After the addition, collect 20 mM sodium phosphate, 1 M NaCl, 7.2 dissolved non-retentate (approximately 7900 ml), this was concentrated by an ultrafiltration unit (Filton Company. Ω ultra-fixed molecular 虿 8 0 0 0 cut) to obtain a non-retained portion F 1 (460 Ml, protein concentration 2.11 mg / ml, total protein mass 97 3 mg, relative activity 16.3). Secondly, the dissolving agent was changed to 2M NaSCN, and the dissociated TSK ~ gel AF-BIUE 6 5 0 MH adsorbed TP0 active F2 part (28 40 ml) was concentrated to 6 with an ultra-thin unit (manufactured by Amicon; YM3 membrane, 76 mm in diameter) 81 ml. The total protein mass of this TPO active P2 fraction is 12.5¾ grams, and the protein yield of F2 at this step is 0,62¾. In addition, the relative activity of TP0 is 240. Secondly, it is injected at a flow rate of 1 ml / min. Ί L 〇ad 2 6/6 () Superde X 2 0 0 picogram (manufactured by Farumasia Biochemical Company, catalog number) 17-107, 0 01; 2.6 cm in diameter, 60 cm high filter pad), and the column was developed with 20 mM sodium acetate, 50 mM NaCl, pH 5.5. TPO activity can be confirmed in the part and range of 194 ml to 260 ml dissolved after the initial development. Therefore, this collection was used as the portion of Super &gt; dex 200 picograms of TP0 activity F2 (66 ml &gt; protein concentration 0.112 mg / ml, total protein mass 7.41 mg, relative activity 142860, relative activity 10 58 58). Next, prepare developing solvent A (20 mM sodium acetate, pH 5.5) and developing solvent B (20 mM sodium phosphate containing 500 μαM NaCl, pH 7.2), inject at a flow rate of K 1 ml / min and equilibrate at 100% A RESOURCE S of the strong cation exchange column (manufactured by Farumasia Biochemical Company, catalog number 17-1178-01; company with a diameter of 0.64, and company with a filter pad height of 3). Then the flow rate of K 0, 3 ml / min, with a linear density from 100% A to 100 $ B -94- A degree of this paper applies the Chinese National Standard (CNS) A4 specification (210X297 cm) (please First read the notes on the back, then this page) [Equipment · π 498079 A7 B7 Five 'Invention Note (92) Gradient expansion of 40 minutes. As a result of the measurement, it was understood that the TPO activity was dissociated in a wide range (range from 5¾ B to 32¾ B). The TP0 active fraction was pooled, and the UF ultrafiltration unit (manufactured by Amicon; HM3 membrane, 25 mm in diameter) was concentrated to obtain the TP0 active fraction F2 of RESOURCE S (1.65 ml, protein concentration 4.74 mg / Ml, total protein mass 7.82 mg, relative activity 71400, relative activity 5 5 8 6 0). [0243] The obtained control standard was not a pure TP 0 control standard 'Determination of CFU-MK in rats', because it was confirmed to have very high activity, therefore, an administration experiment on mice was performed, that is, the day before Platelet counts of ICR mice (9 weeks old, $) were administered subcutaneously for 5 days to a portion of TP0 refined products (474 micrograms (100 microliters) / mouse / day), and BSA as a control ( 200 micrograms (100 microliters) / mouse / day), or a buffer solution containing the same composition as that used to dissolve some TP 0 refined products (printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economy ( Please read the precautions on the back before filling in this page) (100 microliters / mouse / day). On the 6th day, blood platelets were collected from the heart and the platelet count was measured (F8 0 0; East Asia). Compared with the pre-administration mice that had been given the TP0 partial refined product, an increase in platelet count of about 2, 14 times was observed on average. In addition, in the comparison between the administration groups, it was observed that the mice administered with the TP0 partial refined product were approximately 1.74 times as compared with those administered with the BSA, or approximately 1 compared with those administered with the buffer only. 90-fold increase in platelet count. This result &gt; It is understood that TP 0 has a platelet increasing effect in the living body. In addition, in mice administered with some refined products of TP 〇, no increase in immunosuppressive acidic protein (IAP), which is one of the acute phase proteins of mice, was observed. Therefore, it is strongly suggested that TP 0 is related to the acute phase. The derivation of protein is different from IL-6 and -95-. This paper size applies the Chinese National Standard (CNS) A4 specification (21 × 297 mm) 498079 A7 _ B7_ 5. Description of the invention (95) IL-11 has different characteristics. [0244] &lt; Example 12 &gt; TPO mRNA was picked up from various rat tissues. To confirm what kind of tissue TPO mRNA expressed in rats, R N A was extracted from various tissues of rats. Various tissues (brain, thymus, lung, liver, heart, spleen, small intestine, kidney · Testis and bone marrow cells) Quickly freeze with liquid nitrogen. The full R N A oil release test R N A single release test reagent ISO GEN (Wako Pure Chemicals). Add the IS0GEN reagent in an amount corresponding to the weight of the frozen sister's weaving &gt; by means of a homogenizer (Siscolon (Ιί / Λ) bu α 7); Riyin Medical Science Manufacturing Co., Ltd .; NS-6 60), After the treatment is completed (about 1 00 00 RPM, 45 ~ 60 seconds), the whole RNA is extracted (based on the method of AcidGuanidium Phenol Chrolo form by Choniczynski and others: refer to Anal. Biochem .., H 1 56 -1 59, 1 987). As a result, 1.1 mg to 5.6 mg of all 0.8 was obtained from each tissue. [0245] Refined from full RNA U) + RHA 01U〇texTM-dT30 (Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page)

Super) (Nippon Synthetic Rubber Co., Ltd., Japan), 20 micrograms of poly (A) + RNA was obtained from about 500 micrograms of total RNA. [0246] One microgram of (A) + R N A was obtained from each of the obtained various tissues, and one chain of cDNA was synthesized by throwing random primers. Dissolve 1 microgram of poly (A) + RNA in 10 -96- This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 498079 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention ( 94) Microliter of sterilized water &gt; at 70 t: heat for 15 minutes and then cool down quickly, add each test reagent to make 75 micro picolar random primers (manufactured by Takara Shuzo Co., Ltd.), 10 U RNase Inhibitor ( (Made by Barringa Mannham), 50inM Tr is-HCl (pH 8.3), 75mM KC1_, 3raM MgC 1 2-20 0 U SuperScr iptTMI I (reverse transcriptase by Life Technology) (20 microliters in total), Incubate at 3 7 10 for 1 hour. The reaction solution was heated at 70 υ for 10 minutes to inactivate the enzyme, and stored at -20 until use. [0247] Based on the cDNA sequence of rat TPO obtained in Example 10, a new synthetic PCR deflector was used. The sequence of the synthetic primers is as follows. [0248] rTP0-I: 5 '-CCTGTCCTGCTGCCTGCTGTG-3' (sequence number 2 of 347 ~ 367) "TP0-N: 5,-TGAAGTTCGTCTCCAACA / \ T03 '(corresponding to 1005 ~ 1025 of the sequence number 2 reaction chain) Use the 0, 1 volume of the synthesized c DNA reaction solution as a template, and perform PCR using the primers used for the synthesis at 1 κ M. GeneArapTM PCR Reagent Kit with AmpliTaqTM DNA Polymerase (manufactured by Takara Shuzo Co., Ltd.) &gt; by GeneAmpTM PCR System 9600 (PERKIN-

(Made by ELMER), PCR reaction (at 9 5 T) for 2 minutes with M 100 microliters volume, denaturing the file at 95 minutes for 1 minute, bonding conditions at 57 minutes for 1 minute, synthesis at 72 minutes for 1 minute The reaction was performed 30 times under the conditions, and then incubated at 72 ° C for 7 minutes). The reaction solution obtained here was run using 2¾ Agaro gel (Qiao F mouth-Λ Y 'in /) (pMC -97- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) ( Please read the notes on the back before filling in this page), 1T if 498079 A7 __ B7 _ V. Electrophoresis of the invention (95) B ioProducts) Electrophoresis, observe the enlarged zone in the brain, liver, small intestine and The kidney observed a zone that was considered specific. From this result, it is impossible to judge the amount of the epidermis, but it can be considered that the expression of TPOmRN A in the rat is the epidermis of these tissues. It is suggested that the same table 琨 pattern is also displayed in humans. If it is consistent with the result of Example 4, it is judged that the liver is suitable as a starting material for obtaining human TPOcDNA. C 0249] &lt; Example 1 3 &gt; ^ From the results of Example 12 from the cDN A library of human normal liver, the liver was selected as the material for human TPO cDNA colony, and commercially available normal human liver was used. Poly (/ 0 + RNA (manufactured by C 1 οntech: also extracted based on the Acid Guanidiuni Phenol Chroloform method of Chmczynski et al.) 5 μg As in Example 7, TimeSaverTM cDNA Synthesis.Kit (Pharmacia System) and DIRECTIONAL CLONING T00LB0X '(printed by the Ministry of Economic Affairs _ Central Standards Bureau employee consumer cooperative (please read the precautions on the back before filling out this page) P harmacia company), synthesized on the 5' end with E co RI, on 3 ^ Double-stranded c DNA with No 11 recognition site at the end. The synthesized c DNA was linked to 1.2 micrograms of pre-treatment vector pEF18S treated with Ec ¢) RI and NotI, and 8.4 ml of Compendium E. col i DH5 (manufactured by Toyo Keiko Co., Ltd.) transformed. As a result, 1.2 x 10 s transformations were obtained. &Lt; Example 1 4 &gt; Human TPO cDNA fragment was obtained by PCR (selection) From commercially available normal human liver poly (A) + RN A (Cion tech Co.-98- This paper size is applicable) China National Standard (CNS) A4 (210X297 mm) 498079 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of Inventions (%)

(Secretary) 1 microgram was used to synthesize the first strand of c D N A using random primers. Dissolve 1 microgram of poly (A) + RNA in 10 microliters of sterilized water, and cool it quickly after heating at 70 t: 15 minutes, add each test reagent to make 75 picomoles random primers (Treasure Wine Manufacturing Co., Ltd.) (Manufactured), 10U RNase Inhibitor ^ BEHRINGER MANHAM CO., M) ·, 50 η m Tr is-HC 1 (pH 8 * 3), 7 5 mM KCl, 3 mM

MsCU, 200U Super ScriptTMII (manufactured by Life Technology Corporation) (20 microliters in total), and incubated at 37 υ for 1 hour. The reaction solution was heated at 70 ° C for 10 minutes to inactivate the enzyme and stored at -20 until use. [0251] A primer for PCR was synthesized from a rat TP0cDNA sequence (sequence number 2). The sequence of the primers is as follows. [0252] rTPO-AIN: 5T- ATGGAGCTGACTGATTTGCTd '(sequence number 2 1 7 3 to 1 9 3) rTPOl: 5'- TGAAGTTCGTCTCCAACAATC'V (corresponding to the reaction chain of 1005 to 1025 of sequence number 2) synthesized c A 0.1 vol volume of DNA reaction solution was used as a template, and PCR was performed using the synthesized primers (TPPO-ΑΙΝ, rTPO-N) 1αM. GeneAmp ™ PCR Reagent Kit with AiupliTaq ™ DNA Polymerase (made by Takara Shuzo Co., Ltd.) The PCR reaction was performed using a 100 microliter volume of GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER) (after heating at 95 TJ for 2 minutes, and then denaturing the sample at 95 ° C for 1 minute at 40. Adhesive conditions for 1 minute, 35 times reaction at 72 ° C for 1 minute, and 7 minutes at 7 2 ° C. 99- This paper size is applicable to China National Standard (CNS) A4M (210X297 mm) (Please read the precautions on the back before filling this page) _pack

1T 498079 A7 B7 V. Description of the invention (97) '[0253] The reaction and liquid run obtained here are separated by 2 electrophoresis gels (manufactured by FMC B i ο P nducts) for this PCR reaction The main product is about 620 bp of DNA {Segment 'refined with Pleb-A-refined DNA purification kit (manufactured by Bio-Ryder Co., Ltd.) ° This refined DNA fragment is Ta q [) ye D eoxy τ M Terininater Cycle Sequening Kit (Ablai, Biochem: System Co., Ltd.) 'The base sequence was determined by direct sequencing by Abladd Biochem: System Co., Ltd. 3 7 3 ADNA sequence analyzer. The base sequence from which the primer is removed and the amino acid sequence deduced therefrom are shown in the Sequence Listing (SEQ ID NO: 3). [0254] The primer sequence without this DNA fragment has a length of 5 8 0 b P, which is the same as that of rat c DNA. As a result of the comparison of the base sequences, it was obviously 86% identical, and was considered as a DHA fragment encoding a part of human TPoc DNA. &Lt; Example 1 5 &gt; Human TPO cDNA was screened by PCR method Based on sequence number 3 of the sequence table, a p C R promoter gene corresponding to human TP 0 was synthesized. The sequence is as follows. [0256] hTPO-I: 5,-TTGTGACCTCCGAGTCCTCAG ~ 3, (sequence number 3 of 60-80) hTPO-J: 5T ~ TGACGCAGAGGGTGGACCCT03, (corresponding to the reaction chain of sequence number 3 of 479-499) This paper Standards are applicable to China National Standard (CNS) A4 specifications (210X297 mm) (Please read the notes on the back '4, and then fill in and fill out —: write this page) Printed by the Consumer Cooperatives of the Central Sample Bureau of the Ministry of Economic Affairs 498079 Economy Printed by the Central Standards Bureau Zhengong Consumer Cooperative A7 B7 V. Invention Description (98) The human cDN A library of Example 13 will be expanded and divided into 1 set of about 100,000 pure 糸 aggregates & gt Incubate overnight in 1 ml of LB culture medium containing 50 μg / ml of aspirin, and use a plastid automatic separation device PI-100 (made by Kurabo Industries, VER_3.0) to extract plastid DNA. The oily D N A was dissolved in a τ E solution. [0257] The extracted DN A 5¾ was used as a template, and the synthetic starter genes (hTPO-I, hTPO-jJ, respectively) were used to perform PCR. Gene / UpTM PCR Reagent Kit with AmpliTaqTM DNA Polymerase (manufactured by Takara Shuzo Co., Ltd.) was used. GeneArapTM PCR System 9600 (manufactured by PERKIH-ELMER Co., Ltd.) was performed under 20 microliters of denaturing conditions for PCR reaction minutes, bonding conditions at 59 ° C for 1 minute, and synthetic samples at 1 minute at 7 2 t 35 reactions and incubate for 7 minutes at 72)) 3 bands in 90 aggregates were detected as specific bands. One of these aggregates was selected and divided into about 5000 each Pure aggregates' plastid DNA was purified from 9.0 aggregates, and then PCR was performed under the same conditions. As a result, bands were detected in 5 aggregates. From these, 1 aggregate was selected, Divided into K 25 0 sub-aggregates of pure maggots as 1 aggregate, plastid DNA was extracted from 90 aggregates. When PCR was performed in the same manner, a band was observed in 3 aggregates. Choose among these 1 aggregate, cultured in LB containing 50 μg / ml of aspirin On the plate, the plastid DH A was similarly extracted from 90 communities, and the result was confirmed by PCR to obtain a pure strip HL34. [0258] -101-This paper size is applicable to the Chinese family standard (CNS) A4 specification ( 210X297 mm) --------- • —install-(Please read the notes on the back before filling this page) Printed by the Central Consumers Bureau of the Ministry of Economic Affairs, Consumer Cooperatives 498079 A7 B7 V. Description of the invention (99) &lt; Example 16 &gt; Purification of the sequence of human TP 0 c DNA plastid DNA was performed substantially as described in Molecular Cloning [Sambrook et al.> Cold Spring Harbor Laboratory Press (1 98 9)]. After pure HL34 was cultured in 50 ml of LB medium containing 50 μg / ml of aspirin for 1 night, the cells obtained by centrifugation were suspended in 4 ml of TEG-lysozyme solution, and 8 ml of 0. 2N NaOH / 1¾ SDS solution was used to suspend the solution thoroughly. Add 6ml 3M potassium / 7 5M acetate solution to completely suspend and centrifuge to obtain the supernatant. After the supernatant was treated with phenol-chloroform (1: 1), add The amount of isopropanol was centrifuged to obtain pellets. After the pellets were dissolved in TE solution, K RNAse was applied. Phenol - chloroform (1: 1) treatment, ethanol precipitation. The pellets were re-dissolved in the TE solution, and NaCl and polyethylene glycol 3,000 were added to make them 0.63 M, 7.5%, and centrifuged. Finally, the pellets were dissolved in the TE solution and then ethanol precipitated. By this, about 300 micrograms of plastid I) NApEF18S-HL34 was obtained. The purified plastid DNA was sequenced using the Taq Dye DeoxyTM Terrainater Cycle Sequening Kit (manufactured by Ablaide Biochemicals), and sequenced by the Abd Biochemical Co., Ltd. 373 ADNA sequence analyzer to determine the full-length bases. sequence. The base sequence and the amino acid sequence deduced therefrom are shown in the Sequence Listing (SEQ ID NO: 4). The primers necessary for determining the base sequence are primers synthesized based on the sequence of sequence number 3 and synthetic primers based on the internal sequence design analyzed by the sequence response of those primers. [0260]-102- This paper applies Chinese national standards (CNS) A4 specification (210X297 mm) --------- 0 ^ ------ 1T ----- (Please read the notes on the back before filling this page) 498079 Central Ministry of Economic Affairs Printed by the Consumer Bureau of Standards Bureau A7 B7____ V. Description of the Invention (100) As a result, the obtained plastid was PEF18S-HL34 containing the cDNA pair of 861 test pair, which was confirmed to be the same as that of rat TP0 analyzed in Example 2. Some amino acid sequences AP8 (sequence number 4: amino acid numbers 1 to 12) and τρ2 / TP3 (sequence number 4: amino acid numbers 157 to 162) are highly identical sequences. This DNA fragment is thought to encode an open_translated framework starting from the 25th base but without a stop codon, and at 3 ends has a tail-like sequence formed from 76 bases. The 253 amino acid sequences just before the poly-A tail-like sequence were compared with rat TPOcDNA (the amino acid sequence portion of 147 residues of SEQ ID NO. 2), which was 84% identical and considered to be A DNA fragment encoding a portion of human cDNA corresponding to rat TPO. Since there is no stop codon, there is a poly-A tail-like sequence at the 3 end, which indicates that this pure and non-reflective substance is not a complete c DNA, but an artificial product when the c DNA library is made. [0261] &lt; Implement Example 17 &gt; Performance of human TPO cDNA in COS1 cells ~ The transfection of the plastid-pure PEF18-HL34 obtained from the confirmation of the activity into COS1 cells was performed according to Example 11. That is, the transfection using the crokin-containing DEAE-Dieszdel method was performed using DNA A 10 micrograms, and the culture supernatant was recovered after 3 to 5 days. After the obtained culture supernatant was sufficiently dialyzed against the IMD M culture solution, the K rat CFU-MK measurement system was evaluated. As a result, the culture supernatant of COS1 cells in epidermis was transfected with PEF18S-HL34, and the amount-dependent TP0 activity was observed (Fig. 8). On the 4th day of culture, protrusions formed by megakaryocytes were observed. One side (please read the precautions on the back before filling this page) -Packing-, 1T line -103- This paper size applies to China National Standard (CNS) Α4 size (210 × 297 mm) ^ 8079

Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the Invention (101) In the culture supernatant of COS1 cells transfected with epidermal bodies without inserts, no activity was observed (Figure 8). In addition, in M-0 07e assay, only PEF18S-HL34 was transfected into the supernatant of the cultured COS1 cells, and M-0 0e cell proliferation-promoting activity was observed. From these results, it is understood that p E F 1 8 S -H L3 4 is loaded with a gene fragment encoding a protein having TPO activity. In addition, it is clear that human TP 0 also acts on rats (no species specificity). [0263] &lt; Example 18 &gt; The epitope of human TP 0 deletion c D Ν Α ~ activity was confirmed from the human TP 0 c DNA of pure HL 3 4 obtained in Example 15 with a polymer on its 3 'side. A tail-like continuous adenine sequence, but this sequence cannot be observed in rat TP 0 c D Ν A, suggesting the possibility of experimental artificial products. Therefore, c D N A with a continuous adenosine sequence lacking the poly-A tail was prepared, and it was examined whether the protein expressing 琨 showed activity as TP0. Restriction enzyme recognition sequences are added to the respective ends (EcoRI is added to hTP05, Notl and 2 stop codons TAATGA are added to hTP03). C 0264] [Table 9] PCR was performed using 1 microgram of the plastid DNA of the plastid-pure PEF18S-HL34 obtained in Example 16 as a horizontal plate, and using synthetic primers (hTP05, hTP03) at 10 / iM each. The GeneAmpTM PCR Reagent Kit with AnipliTac (TM DNA Polymerase (manufactured by Takara Shuzo Co., Ltd.) was used to perform a PCR reaction with a GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMSR) at a volume of 100 μl (denaturation strip for 1 minute at 951-104- This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) --------- ^ Packing -------- Order ----- (Please read the precautions on the back before (Fill in this page) 498079 A7 B7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (102) pieces, bonding conditions under 1 minute at 65 υ, 15 times under 1 minute synthesis conditions Reaction, and incubate at 7 2 t for 7 minutes). The obtained bands of K restriction enzymes E c 〇RI and N 〇11 of about 8000 bp were purified after digestion, and similarly sub-selected by restriction enzyme treatment Table 琨 vector PEF18S. From the obtained transformants, 5 pure 糸 containing DKA fragments of about 800 bp were selected, and plastid DNA was prepared from the big 虿 (method refer to Example 5). Regarding the respective plastids, the related K The base sequence was determined in the entire range of about 8 0 0 bp of the PCR amplification. Confirmation with the analysis in Example 16 The base sequences (SEQ ID No. 4 and 780) are completely identical. [0265] This plastid is named P Η T1- 2 3 1. The vector P Η Τ 1-2 3 1 loaded on coliform · D Η 5 On February 14, 1994, the trust number FER Μ Β Β-4 5 6 4 was deposited in the Institute of Life Science Engineering Technology of the Industrial Technology Institute of the Ministry of International Trade and Industry. [0266] The obtained plastids were in CO S The expression of 1 cell was performed according to Example 11. That is, using 10 micrograms of plastid D N A each, transfection with crokin-containing DEA Ε-Daclides method was performed, from 3 to 5 The culture supernatant was recovered in the future. [0267] The obtained culture supernatant was dialyzed against the IMD Μ culture solution, and evaluated with a rat CPU-M K assay system. As a result, PHT1-231 was transfected to make COS1 in Table VII. TPO activity was observed in the cell culture supernatant (Figure 9), and protrusions formed by a majority of megakaryocytes were observed on the 4th day of culture. On the one hand, epitoplasts transfected with no cDN A insert No activity was observed in the C0S1 cell culture supernatant (Fig. 9). Also, the M- 0 7 e assay was only performed at the transfection-105-this paper is for China National Standard (CNS) Α4 specification (210 × 297 mm) ^ 1—— · ^ ϋ— I mi ml tn HI nn n (Please read the precautions on the back before filling this page)

1T 498079 A7 B7 V. Description of the invention (105) P Η T1-2 3 1 caused M-0 7 e cell proliferation-promoting activity to be observed in the culture supernatant of C 0 S 1 cells of epidermis. From these results, it is apparent that the cDN A fragment contained in pHTI-231 is a gene encoding a protein having TPO activity. &Lt; Example 1 9 &gt; The human TPO C-terminal region of human TPO by PCR was cloned from the pure human HL34 cDNA of HL34 obtained in Example 15 with a poly A tail-like sequence at its 3 'end, hint 3 'Some are incomplete purity. Therefore, an attempt is made to obtain a 3 'region of full length from PCR. Four types of 5 C side primers for PCR were synthesized from the base sequences determined in Example 16. [Table 10] Further, as the 3 'side primer, it is expected to expand from the polymerization of the part A, and a 3' end mixed nucleotide is synthesized to a primer containing 4 bases or less. In addition, fixed primers containing no mixed portion were synthesized. [0270] [Table 11] As in Example 14, printed from the normal human liver (A) + printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) RNA ( (Clontech) 1 microgram of the first strand of synthetic cDNA. However, an oligo-dT primer was used as a daughter (Appendix to TimeSaver ™ cDNA Synthesis Kit manufactured by Pharmacia, 0.5 µg). A 0.1 volume of the reaction solution for cDNA synthesis was used as a template, and the first PQ was performed. GeneAinpTM PCR Reagent Kit with AmpliTaqTM DNA Polymerase (manufactured by Takara Shuzo Co., Ltd.) and -106- used in PCR. This paper size is applicable to China National Standard (CNS) A4 specification (21 × 297 mm) 498079 Employee Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Printing A7 B7 V. Description of the invention (104)

GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER). The primers were reacted using hTPO-H (20 μM) and hTP03mi X (10 μM) with a capacity of K 50 microliters (after 96 minutes: 2 minutes below, 1 minute at 96 ° C / 48 ° C Cycle 1 reaction at 1 minute / 72 ° C for 1 minute, and then, 7 minutes at 7 2). The second PCR was performed using 0.1 volume of the first reaction solution as a template. The reaction was performed in a primer using hTPO-K (20 / i Μ) and hTP03mix (lOuM) with a capacity of 50 μl (96t! After 2 minutes, 96¾ / minute / 63 ° C 1 minute / 72t! 1 minute Cycle the reaction 10 times, and more 7 2 7 minutes). The third PCR was performed using 0.1 volume of the second reaction solution as a horizontal plate. The primers used TP0-0 (20 // Μ) and hTP03mix (10 "M), M50 microliter capacity reaction (96t: after 2 minutes, cycle 10 times 96t! 1 minute / 63 ° C 1 minute / 721 〔1 minute reaction, then 72TJ 7 minutes). Use the 0.1 volume of the 4th reaction solution as the shore plate, and perform the 5th PCR. The primers use TP0-0 (20 v M) and hTP03anchor (20 / iM), the reaction was performed with a capacity of 50 microliters (96t: after 2 minutes, 9 times of 10 cycles of 6 ° C 1 minute / 5 8 ° C 1 minute / 7 2 ° C 1 minute reaction, and then 72 ° C 7 points). The obtained reaction solution was run using a 2¾ Agaro gel (manufactured by FMC BioProducts) electrophoresis &gt; A 600 bp DNA fragment that was the main product of the PCR reaction was separated using Puleb Refined DNA Refining Kit (manufactured by Bio-Ryder). This refined DNA fragment was produced using Taq Dye DeoxyTM Terminater Cycle Segiien ing Kit (manufactured by Ablaide Biochemical Co., Ltd.) and manufactured by Ablaide Biochemical Co. The 373ADAN sequence analyzer directly sorts and determines the base sequence. The base sequence and the amino acid sequence deduced from it are shown in the sequence listing (Sequence-107- this paper) Applicable to China National Standard (CNS) A4 specification (210 X 297 mm) (Please read the notes on the back before filling out this page} • Equipment · τ Printed by the Central Consumers Bureau of the Ministry of Economic Affairs Consumer Cooperatives 498079 A7 _ B7____ 5 2. Description of the invention (105), column number 5) [027 1] This DNA fragment has a base sequence encoding 130 amino acids starting from the primer h TP 0-0, and 180 bases on the 3 'side. The sequence above the amino group continues (cannot be interpreted after the 5th and 7th bases). The amino acid sequence of 30 starting glycines encoded by this DNA fragment and the amino acid number 2 03 described in sequence number 4 The amino acid sequence of ~ 232 is consistent. At the same time, the base sequence from No. 1 to 94 is also consistent with the sequence number of 6 9 2 to 7 8 5. [0272] From the cDN AH · paragraph of Example 17 As a result of analysis and analysis of the pCR amplified fragment in this example, as described in SEQ ID NO: 6, the human TP0 protein is estimated to be formed from 3 5 3 amino acids of a signal sequence containing 21 residues. [0273] & lt Example 2 0 &gt; The reconstituted group of cDN A library from human normal liver was obtained in Example 15 The pure line HL 3 4 'does not have a stop codon in the open translation framework. It has a poly A tail-like sequence at its 3 f-terminus. It is considered to be a cDN / l · synthetic artificial product. Of normal human liver (α-wide RNA (C 1 ο ntech)) 5 ug 'restructured

System for cDNA Synthesis and λ Cloning Kit and SuperScriptTMII RNaseH- (also manufactured by LIFE TECHNOLOGIES). After poly (A) + RNA is thermally denatured, add 20 μl of reaction solution (50mM -1 0 8-attached to the kit containing oligo dT with Notl sequence as primers) Standard (CNS) A4 specification (210x297 mm) -------- · 1 pack ------ order ----- β 丨 line (Please read the precautions on the back before filling this page) Printed by the Employees' Cooperative of the China Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Description of Invention (106)

Tris-HCl &gt; pH8.3 / 75raM KCl / 3mM MgCU / lraM DTT / lmM d N T P / 2 0 0 U Superscript ™ !! RNaseH-), at 37t: incubate for 60 minutes. Synthesizing the second strand of c DNA (containing 25 mM Tr is-HC 1, pH 8.3, 100 μM KCl, 5raM MgCU, ^ 250 α M dNTPs ·, 5mM DTT, 40U E. Coli DNA Polymerase I, 2U E co1i RNaseH, 1 0 UE. c ο 1 ί DNA ligase reaction solution, incubate at 16 t] for 2 hours), add 1 OU T4 DNA polymerase and incubate at 16 ° C for 5 minutes. After heating the reaction solution at 65 ° C for 10 minutes, an equivalent amount of phenol-chloroform was used to remove the low-molecular weight DMA attached to the SizeSepTM 400 spun column (TiiueSaverTM cDNA Syn thesis kit manufactured by Pharmacia). spu η column) to remove c DNA smaller than 400 bp. To this sample, E c 〇RIA dapt 〇r (made by P harmacia and attached to the TimeSaverTM cDNA Synthesis Kit) was added after digestion with K restriction enzyme NO ot I, and S ize S e ρ τ Μ 4 0 was added again. 0 spu η column to remove low molecular weight DN Α. The resulting double-stranded c D Ν Α with the EcoR I at the 5 ′ end and the N τt I recognition sequence at the 3 τ end were 1.3 μg and the table treated with EcoRI and Not I in advance.琨 The carrier pEFISS is connected to transform 9.2 ml of Compendium · Sea E · co 1 i DH5 (manufactured by Toyobo Corporation). As a result, the obtained human liver cDNA library (hTPO-F1) was a product containing 1.0 × 100 transformants. C 0274] &lt; Example 2 1 &gt; Screening of pure TPO cDNA from human liver cDNA library-hTP0-F1 Based on the sequence listing-sequence numbers 3 and 6, the corresponding human TP 0 c D ΝA -109- Paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) -------- install ------ order ----- line (Please read the precautions on the back before filling this page ) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 __ — — — B7 V. Description of the invention (107) Primers for PCR. The sequence is as follows. [0275] hTPO-I: 5, ~ TTGTGACCTCCGAGTCCTCAG-3f (Serial No. 3 of 60-80) hTPO-O: 5'- AGGATGGGTTGGGGAAGGAGA-3 '(corresponding to 90 1-921 counter induction chain of Sequence No. 6) will be The human liver cDNA library constructed in Example 20-hTP0-F1 (1.0 × 10Θ) was divided into 3 aggregates (# 1 ~ 3) and stored frozen. Plastid DNA was prepared from the respective aggregates, and 1% of it was used as a template, and PCR was performed using synthetic primers (hTPO-I and hTP0-KU), respectively. A Gene / UpTM PCR Reagent Kit with AiupliTaq ™ DNA Polymerase (manufactured by W Shusei Co., Ltd.) was used to perform PCR using a GeneAmpTM PCR System 9600 (manufactured by PERKIN-EIMER) at a volume of 100 μl (denaturation conditions at 95 ° C for 1 minute, 5 9 1 minute bonding conditions, 7 2 1 minute synthesis conditions, 3 5 times of reaction, and 7 2 incubation for 7 minutes) · As a result, using the plastid DNA from the aggregate of # 3 'prediction The size of the DNA is enlarged. Therefore, this # 3 aggregate was divided into 15 000 sub-aggregates, and then baked in 1 ml of LB medium containing 50 μg / ml of aspirin for 1 night, and then automatically separated with plastids. The device P 1-1 0 0 'draws the plastid DNA. The extracted DNA was dissolved in TE1 solution 'and 5% was used as a template to perform PCR (primer used, reaction conditions are the same as described above). the result'

The DNA of 6 aggregates in 90 aggregates has a predicted size of DNA. Select one aggregate among these, and divide 1,000 pure: into sub-aggregates of 1 set, and The above-mentioned preparation of plastid DNA is the same, and PCR is performed, but DNA -110- This paper size is applicable to the Chinese National Standard (CNS) A4 specification (21 × 297 cm) ----------- pack ----- -Order ----- line (please read the precautions on the back before filling this page) 498079 Printed by the Consumers' Cooperatives of the China Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Expansion of the invention description (108) was not observed. K. The concentration of bands on a series of PCR-amplified DNA electrophoresis, which becomes lighter as the sub-aggregates become smaller. Therefore &gt; It is thought that due to the poor proliferation of the pure line, the recovery of plastid DNA is not good, and the expansion of P (: R becomes weak, and finally it cannot be expanded. Therefore, return to the aggregate of Lin 3, Screening through the hybridization of the community. [0276] From the aggregate of forest 3, plant 4 100 pure LB petri dishes per 15 cm: line, make 100 petri dishes For individual petri dishes, each made a duplicate petri dish. Among them, half of the petri dishes were baked at 37 ° C for 6 hours to recover the community to extract the plastid DNA. The DNA was subjected to the same PCR as the above, 1 0 0 An expansion of the predicted size band was observed in one of the aggregates. Therefore, two replication filters were made from the polymer petri dish (using BIODNETM A TRANSFER MEMBRANE manufactured by PALL). The denaturation of the filter was 10% SDS for 10 minutes. 0.5 NN a 0 Η / 1.5 MN Na C 1 10 minutes, 0.5 M Tris-HCl (pH8.0) /1.5 M NaCl for 10 minutes, 30 minutes air-drying After that, it is dried in a vacuum oven at 80 TJ for 1 hour. The dried filter membrane M 6 XSSC (2 〇 XSSC is based on 1 liter It contains 1 7 5 · 3 g of Na C 1 and 88.2 g of solution with pH 7.0) / USDS After lightly washing, pre-hybridization is performed. The composition of the reaction solution is 50¾ formamidine, 5 X SSC, 5 X Denhardt's solution (50 × Denhardtfs solution is 500 ml containing 5 grams of F ic ο 1 1 ·, 5 grams of polyvinyl pyrrolidone, 5 grams of bovine serum albumin part V solution), 1¾ SDS, 20 μg / ml salmon The sperm DNA was shaken for 30 minutes at 42 ° C in a 30 ml solution while incubating. After the pre-hybridization and shaking, exchanged with 30 ml of a tritiated solution of the same composition, and added [α -111- this paper Applicable to China National Standard (CNS) Α4 size (210 × 297 mm) Pack-(Please read the precautions on the back before filling out this page}, 11 &gt; Line-Consumer Co-operation, Central Standards Bureau, Ministry of Economic Affairs, printed 498079 A7 __B7_ V. Description of the invention (109) -32P] dCTP (manufactured by Amarium) radiolabeled probe (refined plastid PEF18S-HL34 EcoRI / BaraHI Η segment: sequence number 4-5, end to end 4 5 8 bases, labeled by K random primers; using ana 1. B ioche ηι., 1 3 2, 6-1 3, According to the method described in 193, Ama, Shame Co., Ltd. (M e g a p r i in e D n A (Labeling system) was used), shaking at 4 2 ° C for 20 hours while incubating. The membrane was washed for 30 minutes at 42 ° C in a 2X SSC / 0 · 1¾ SDS solution, and then washed for 30 minutes at 4 2 ° C in a 0.2 x S S C / 0.1% S D S solution. Autoradiography was performed at -7 0 ° C for 16 hours with enhanced fluorescent fluorene and X-OMATTMAR 5 film (manufactured by Eastman Kodak). As a result, a signal that was considered positive was observed. Therefore, the signal Zhou Lin's community was recorded and written from the original petri dish, and re-planted on the 10 cm LB petri dish. Fifty colonies were obtained from the culture, and DNA was prepared by PCR using primers hTPO-I and hTPO-KU. (Conditions are the same as above). As a result, only one zone, as predicted, was expanded. This pure plutonium was named Ph H TF 1. &Lt; Example 2 2 &gt; Sequence of human TPOcDNA pure strip PHTF1 Purification of plastid DNA was performed substantially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (19 8 8)). Pure: p H TF 1 was cultured overnight in 50 ml of LB medium containing 50 μg / ml of ampicillin. The cells obtained by centrifugation were suspended in 4 ml of TEG-lysozyme solution, and 0.2 ml of 8 ml was added. N NaOH / l% SDS solution was thoroughly suspended. Add 6 ml of 3 卩 potassium / 5M acetate solution and suspend thoroughly, then centrifuge to obtain the supernatant. The supernatant is phenol-chloroform-11 2-This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) — · — Packing ------ Order ----- Line (Please read the back first Note: Please fill in this page again.) 498079 Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs A7 _B7_ V. Invention Description (11〇) (1: 1) After processing, add the same amount of isopropanol to centrifuge and get pellets . After the pellets were dissolved in the TE. Solution, they were treated with R N A s e, phenol-chloroform (1: 1), and then ethanol precipitated. The pellets were re-dissolved in TE solution, and NaC 丨 and polyethylene glycol 3 000 were added to make them into 0.63 M and 7.5% centrifugation, respectively. Finally, the pellets were dissolved in a TE solution and then ethanol precipitated. Approximately 300 micrograms of plastid DNApHTFI-[0278] Refined plastid DMA, Taq Dye DeoxyTM Terminater C yc 1 e S equening K it (manufactured by Ablyd Biochemical Systems) The 3 7 3 ADNA sequence analyzer made by De Biochemical System Co., Ltd. sorts and determines the full-length base sequence. The base sequence and the amino acid sequence deduced therefrom are shown in the Sequence Listing (SEQ ID NO: 7). The primers required for the base sequence are determined, and the primers used for the synthesis of the sequence based on the sequence number 6 and the synthesized primers of the internal sequence design analyzed by the sequence reaction of their primers. [0279] As a result, it was confirmed that the obtained pure PH TP 1 was composed of a 172 base pair fragment, and was the amino acid sequence AP 8 (SEQ ID NO: 7, amino acid), which was analyzed in Example 2. Nos. 1 to 1 2) and TP 2 / TP 3 (sequence number 7; amino acid No. 1 5 7 to 1 6 2) have highly identical sequences. This D Ν Α fragment is thought to encode from the 5 'non-reading region to the 101st base, followed by the methionine encoded by the 102nd to 104th base pairs, and to the 1st base. The open reading structure of glycine followed by 3 5 3 amino acids encoded by 5 8 to 1 160 bases followed by a non-coding region after the stop codon (TAA) has 531 bases Base and 30 poly A tail sequences. It is considered to be coded by this open translation architecture (please read the notes on the back before filling out this page). Binding and ordering -113- This paper is A degree applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) Ministry of Economic Affairs Printed by the Consumer Standards Cooperative of the Central Bureau of Standards 498079 A7 B7_ 5. The amino acid sequence of the protein of the invention description (111) is exactly the same as the predicted amino acid sequence of human TP 0 described in Sequence Number 6. The base sequence encodes a cDNA having a length of nucleobases of 77 bases and a length of 347 bases longer than the sequence predicted by SEQ ID NO: 6 with a length of 77 bases and a position before the poly A tail sequence on the 3f side. The base sequence differs from the one predicted by sequence number 6 in three points. The different base sequences are A in sequence number 7 (the 8th base), C in sequence number 6, A (the 74th base) and T, and G (the 1198th base) as A. Only the second base (740th base) is a variation in the coding region of the protein &gt; but it is the third codon of threonine and does not cause amino acid conversion. It is unknown at this point why the substitution of this base sequence started, but from the analysis of the obtained pure 糸 PH TF 1, it can be confirmed that the human TP 0 protein consists of 3 5 3 amines of a signal sequence containing 21 residues Based on acid. The estimated molecular weight of the protein excluding the signal sequence is 3 5 4 6 6 ^ [0280] pHTFI, a carrier of E. coli DH5, was deposited on March 24, 994, under the trust number PER Μ BP-4 6 1 7 Institute of Industrial Technology, Industrial Technology Institute. &Lt; Example 2 3 &gt; Expression of human TPOcDNA pure pHTFI on COS1 cells ~ Confirmation of activity The transfection of COS1 cells with pure purified PHTF1 was performed in accordance with Example 11. That is, the plastids D N A 10 0 g were shaken, transfection was performed using croatin-containing D E A E-Daichestrand method, and the culture supernatant was recovered 3 days later. [0282] -114- This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) -------- install ------ order ----- ®-line (please Read the notes on the back before filling this page) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 ____B7_ V. Description of the invention (112) After dialysis of the obtained culture supernatant on IMDM medium, K rat CFU-MK Evaluation of the measurement system. As a result, a dose-dependent TP 0 activity was observed in the culture supernatant of COS1 cells cultured from PHTF1 epithelium, but the culture supernatant of C 0 S 1 transfected with p-plasmid without K DNA insert was not available. Activity was observed (circle 10a). Similar results were also obtained in the M-07e assay, and intentional M-07e cell proliferation-promoting activity was observed in the COS1 cell culture supernatant of the PHTF1 epitope (Fig. 10b). [0283] From these results, c D N A contained in P H TP 1 is clearly a gene encoding a protein having TPO activity. <Example 2 4 &gt; Selection of human TPO chromosomal DNA K human TPO cDNA was used as a probe to perform selection of human TPO chromosomal DNA. The genomic library used for breeding was obtained from the gene experiment of Tohoku University Professor Shi · Shiben Tokuo (for the gene bank described in Sakai et al., J. Biol. Cheia., 289, 2173 -21 82, 1 9 94, Human chromosomal DNA is partially degraded by restriction enzymes and linked to the gene bank of the BaraHI site of Lambda EMBL3, a bacterial cell vector produced by Stratagene, Inc.). From this gene library, K human TPO cDNA was screened using probes. All the methods used were essentially performed as described in Molecular Cloning [Sarabrook et al., Cold Spring Harbor Laboratory Press (1989)]. Using E. coli LE392 as the host, each NZYM baking dish of 15 cm (1 liter contains 10 grams of NZ amine ·, 5 grams of Na C 1 ·, 5 grams of bact 0 yeast extract -115- paper Standards are applicable to China National Standard (CNS) A4 (21〇 &gt; &lt; 297mm) binding (please read the precautions on the back before filling out this page) 498079 Α7 Β7 Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Description of the invention (115), 2 g of Mg CU 7H20, 15% agar, pH 7.0) were seeded into phages in an amount of 30,000, and 18 petri dishes were made. Two replicated diaphragms were made from each of the baking dishes (using BIODNETM A TRANSFER MEMBRANE manufactured by PALL). ® Denaturation was performed at 0.5N NaOH / 1.5M NaCl for 10 minutes, 0.5M Tris-HCl (pH 8.0) /1.5M Ν a C 1 for 10 minutes, and air-drying for 30 minutes at 8 0 υ vacuum oven for 1 · 5 hours. Pre-hybridization was performed in a solution of 50 ml of 50% formamidine, 5 X S S C ·, 5 X Denhardt, solution, 1¾ SDS, 20 µg / ml salmon sperm D N A at 4 2 C for 1 hour. As a probe, a human TPO cDNA fragment (base sequence number 178- 1 025 of SEQ ID NO: 7) was amplified by PCR and purified, and the primer was labeled with Random Primer DNA (manufactured by Takara Shuzo Co., Ltd .; using Ana 1. Β 〇〇chem · , 1 2 3, 6-1L 1 9 8 3 DNA labeling method of random primers) 32P. The sequence of primers used for PCR is as follows. [0285] hTPO-I: 5f- TTGTGACCTCCGAGTCCTCAG-S '(Serial No. 7 of 178-198) hTP0 ~ N: AGGGAAGAGCGTATACTGTCC-3, (corresponding to the reverse chain of SEQ ID No. 7 00 5-10 25) Hybridization Use 500 ml of a solution with the same composition as the pre-hybridization, and add the radiolabeled probe at 42 ° C for 20 hours. The filter was washed 3 times in 2 XSSC / 0.1% SDS solution for 5 minutes each at room temperature, and then 'cleaned at 68 ° C for 1 hour in 0.1 XSSC / 0.1% SDS solution. Enhanced screen and X-0MATTM AR 5 negative (made by Eastman Kodak) -116- This paper size is applicable to China National Standards (CNS) (210X297mm) (Please read the precautions on the back before filling (This page) ϋ_ϋ— ι__ϋ1 nn ·

Line 1T-♦ 498079 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 __B7_____ V. Description of the Invention (114) -70 t: 16 hours autoradiography. As a result, 13 positive signals were obtained. Pick out 13 plaques around the positive signal from the original culture dish, and re-plant them so that each NZYM dish of 15 cm has 100 plaques. Two replication filters were prepared from each petri dish, and hybridization was performed under the same conditions as described above. As a result, positive signals were detected in all of the filters. From each culture dish, one plaque was isolated, and phage DNA was prepared by the P late Lysate method described in KMolecuUr Cloning. For 13 pure phage DNA, M PCR was used to check whether it contained the coding region of cDNA. The sequence of primers used for inspection is as follows. HTP0-L: 5'- GGCCAGCCAGACACCCCGGC03 '(sequence number 6-21) hTP0-P: ATGGGAGTCACGAAGCAGTTT'V (corresponding to the reaction chain of sequence number 1 2 7-1 4 7) hTP〇-P: 5 *-TGCGTTTCCTGATGCTTGTAG-3 '(sequence number 6 of 5 0 3-5 2 3) hTP〇-V: 5'- AACCTTACCCTTCCTGAGACACA' (should correspond to sequence number 6. 1 0 70-1 0 90 counter-induction strand) When these primers were used to perform PCR, 5 of the 13 pure hydrazones were considered to contain the coding region of the amino acid predicted from cDN A. The length of the chromosomal DNA contained in these five pure lines was about 20 kbp, and the same pattern was also shown in the restriction enzyme analysis of the preliminary review. Therefore, one of these pure lines was selected (pure strip λ H G T1) and S o u t h e r n b 1 o t analysis was performed. That is, 1 μg each of the DNA of the pure λ HGT1 K restriction enzyme EcoRI or (Please read the precautions on the back before filling this page) • Pack.

， 1T line-1 1 7-This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Description of the invention (115) H ind 1 [Complete After digestion and electrophoresis on a 0.8% agar gel, it was transcribed on BIODNETM A TRANSFER MEMBRANE manufactured by PALL. The filter was air-dried for 30 minutes, and then dried in a vacuum oven at 80 ° C for 2 hours. The prehybridization was performed in a 50 ml solution containing 50¾ formamidine, 5XSSC, 5xDenhardtTs solution, 1SS S DS, 20 μg / ml salmon sperm D N A, and 42 ° C for 1 hour. For the probe, a human TPO cDNA fragment (base sequence number 7 78-105 2) of K P C R was expanded, and the refiner made a Random Primer DNA label kit (manufactured by Takara Shuzo Co., Ltd.) to report 32P. For the deuteration, 50 ml of a solution with the same composition as the pre-hybridization was used, and the radiolabeled probe was added at 42 for 20 hours. The filter was washed 3 times in 2 XSSC / 0.1% SDS solution for 5 minutes each at room temperature, and then washed in 0 · 1 XSSC / 0.1% SDS solution at 6 ° C for 1 hour. Autoradiography at 70 ° C for 16 hours was performed with enhanced fluorescent power and X-0MATTMAR 5 negatives (manufactured by Eastman Kodak). As a result, in the case of digestion by the K-restriction enzyme H yn d II &gt; a single band of about 10 k b p was observed. Therefore, the K restriction enzyme H ind M was digested purely: after 10 μg of DNA of λ HG T1, electrophoresis was performed on a 0.8% agarose gel, and a band of 10 kbp was cut out. DN A refined kit (made by Bio-Ryder Co., Ltd.), sub-selected on pure carrier PUC 1 3 (manufactured by Pharmacia Co., Ltd.) also digested with Hind I (the host bacteria uses Toyobo Sangyo Co., Ltd. Sea E. col i DH5). [0287] Among the obtained pure lines, a pure: line containing H yn d m fragment of 10 k b p was selected and named P H G T1. [0288] -118- This paper size applies Chinese National Standard (CNS) (210 × 297 mm) I ------------- Order ----- line (Please read the precautions on the back first Refill this page) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Description of the invention (116) The schematic diagram of the restriction enzyme of phage blunt strip λ HG T1 is shown in Figure 11. &Lt; Example 2 5 &gt; Human TP 0 chromosome purity: ¾ Sequence of P H G Τ 1 A pure line of P H G T 1 was cultured, and plastid D N A was purified. The method is essentially performed as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)]. After culturing pure pHGTI K containing 50 μg / ml of aspirin and 50¾ liters of LB medium overnight, the cells obtained by centrifugation were suspended in 4 ml of TEG-lysozyme solution, and 8. ml of · 2N NaOH / USDS solution for complete suspension. 6 ml of a 3 M potassium / 5 M acetate solution was added, and the suspension was thoroughly suspended and centrifuged to obtain a supernatant. After the supernatant was treated with phenol-chloroform (1: 1), an equal amount of isopropanol was added and centrifuged to obtain pellets. After the pellets were dissolved in the TE solution, RNAse treatment, phenol-chloroform (1: 1) treatment, and ethanol precipitation were performed. The pellets were dissolved again in the TE solution, and Na C 1 · and polyethylene glycol 3 0 0 were added to make them 0 · 6 3 M, 7 · · 5%, and centrifuged. Finally, the pellets were dissolved in a TE solution for ethanol precipitation. Approximately 300 micrograms of plastid D Ν Α ρ H G Τ 1 was obtained. As for the purified plastid DNA, Taq Dye DeoxyTM Terminater Cyc 1 e S equening K it (manufactured by Ablyd Biochemical System Co., Ltd.) was used, and it was determined by the sequence of 373 ADNA sequence analyzer made by Ablyd Biochemical System Co., Ltd. The base sequence surrounding the protein coding region predicted from the base sequence of cDN A. The base sequence and the amino acid sequence deduced therefrom are shown in the Sequence Listing (SEQ ID NO: 8). The primers required for the base sequence determination were used in Example 22. -119- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 cm) (Please read the precautions on the back before filling this page)

498079 A7 B7 V. Description of the invention (117) Two for the analysis of the basic S sequence of cD N A, and the primers designed and synthesized based on the internal sequence analyzed by the sequence reaction of their primers. [0291] Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). As a result, the plastid is pure: 糸 Chromosomal DNA loaded with PHGT 1, all contained in the amine predicted by sequence number 6 The coding region of a base acid> The base sequence of the coding region is completely the same. Also, the region corresponding to the amino acid-expressing protons is divided into four non-extractives, and the length of the non-existing exosome is 5 and the sides are 2 3 1 b p, 286 bp, 1932 bp, and 236 bp. It is different from the cDHA base sequence M described in SEQ ID NO: 7. The different bases are the same as those described in Example 22 (differences from sequence numbers 6 and 7). A (84th base) of sequence number 7 is C, Λ (740th base) is τ, G (Base 1198) is A. In other words, the human TPOcDMA pure: base sequence of pHTF1 obtained in Example 21 is obviously different from the base sequence of the human chromosome by three points. Therefore, in order to analyze whether the variation of this base sequence reflects the original sequence, in order to screen the last five pure chromosomal DNA strips selected, we will determine the sequence of the four pure 下 below the base sequence. . Sequence analysis was performed by a direct base sequence determination method using a phage DNA prepared by the plate lysate method described in Molecular Cloning. In the reaction, the sequence primers synthesized in Example 22 and Taq Dye Deο XyTM Terrainater Cyc 1 e Sequening Kit (Abbr Biochemical: System Co., Ltd.) which can analyze the positions of the base sequence mutation points at the three positions described above were used. Sorted by 3 7 3 ADNA sequence analyzer made by Ablyd Biochemical Company. As a result, in all four pure plutoniums, the 84th base of sequence number 7 is C and the 740th base is T, which is different from cDN A of sequence number 7 and is consistent with that of sequence number 6. Regarding the No. -120- Taiji Paper Cultivation Center, CNS) A4 (210 X 297 males) 498079 A7 B7 V. Description of the invention (118) 11 9 8 bases, G pure line Is 2, and the pure order of A is 2. That is, obviously, there are two kinds of base sequences of the chromosome D N A originally. The difference in this sequence is a gene from the same chromosome or from a plurality of genes, which was unknown at the time. In addition, the purchased poly (A) + RNA manufactured by Clontech was from a Caucasian, while the chromosome D N A was from a Japanese, and it was suggested that the variation in the base sequence may be due to the difference in race. [0292] The vector PHGT1 loaded with coliform DH5 was deposited on March 24, 1994 with the trust number F E R M B P-4 6 1 6 at the Institute of Biotechnology and Industrial Technology, Industrial Technology Institute, Ministry of International Trade and Industry. [0293] --------- 4 ^ · 装 — (Please read the notes on the back before filling in this page) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs &lt; Example 26 &gt; Human TP0 Chromosomal DNA The expression and activity in COS1 cells were confirmed in the purely plastid pH GT1 obtained from the sub-selection. Three positions were inserted on the human body, and the total number of EcoR I recognition sequences was 4 in one place in the vector. In the DNA fragment of about 4.3 kbp between the EcoR I recognition sequence on the 5 f side and the EcoR I recognition sequence in the vector, the coding region of the human TPO protein is all included, and it is clear from the analysis of the base sequence. Therefore, by linking this fragment with the expression vector PEP18S treated by EcoRI, 4 human TP0 tables are obtained. Plastids P E F H G T E # 1-4 (the aforementioned garden 11). These plastids D N A were prepared and subjected to a table test. The purification of plastid DNA was performed substantially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)) to obtain about 250 micrograms of plastid DNA. -121 ^ Paper New York 14 Family Standard (⑽) Α4 threat (21GX297 public dust) Booking fee 498079 A7 B7 V. Description of the invention (119) [0294] The obtained pure line pEPHGTE # 1-4 was transfected into C0S1 cells' Proceed as in Example 11. That is, &gt; 10 micrograms of plastid DNA was used, and transfection was performed using the Crocetin-containing DEA E-Driesland method, and the culture supernatant was recovered after 3 days. [0295] After the obtained cultured supernatant was dialyzed against IMDM culture solution, it was evaluated by rat CFU-MK assay. As a result, a dose-dependent TP 0 activity was observed in the culture supernatant of COS1 cells of the pEFHGTE epitope. Serum, and activity was observed. Any of the pure lines # 1 to 4 gave about the same results. The results of pEFHGTE # 1 as a representative example are shown in FIG. 12 a. In addition, when measured at M-07e, it is also possible to observe the intentional proliferation activity of M-07e cells in a COS1 cell culture supernatant that expresses PEFHGTE. As a representative example, the results at pEFHGTE # 1 are shown in Fig. 12b. [0296] From these results, it is understood that the pure DH A carried by pEFHGTE is the functional chromosome D N A of human TP 0. [0297] Printed by the Consumers' Cooperative of the China Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) &lt; Example 2 7 &gt; Production of human TP 0 missing derivatives and CO S 1 girl cells Performance-Activity confirmation From the results of Example 18, it is understood that even when human TP 0 is not full-length, K can be used to express activity. In order to analyze the area necessary for its activity performance, enter -122-This paper standard applies to China Standard (CNS) A4 specification (210X297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 __ — _B7___ 5. Description of the invention (12) Production and display of missing derivatives. Based on the plastid pure HPP T1-2 3 1 obtained in Example 18, epitoplasts in which 20 amino acids were sequentially deleted from the c-terminus side were produced, and the content was maintained up to TP 2/3 (p.163). No.) epitopes of amino acids. Making use of PCR. The sequence of primers used to make PCR is as follows. HTPO-5: TTTGAATTCGGCCAGCCAGACACCCCGGCC-3, (the E co RI sequence is added to the sequence number 2 of 21; the same as those described in Table 9) hTP0-S: 5, TTTGCGGCCGCTCATTAGCTGGGGACAGCTGTGG-TGGGTGGG-3 '(Sequence number 4 5 5 2-576 reverse induction chain; additional two stop codons TAATG A and Not I sequences; for the production of deletion derivatives encoding amino acid 163) hTP0-4: 5, TTTGCGGCCGCTCATTACAGTGTGAGGACTAGAG -AGGTTCTG-3 '(reverse induction chain of sequence number 4 5 7 6-6 0 0; additional two stop codon sequences TAATGA and Notl; production of deletion derivatives encoding amino acids 1-17_1) hTPO- 3: 5: TTTGCGGCCGCTCATTATCTGGCTGAGGCAGTGA-AGTTTGTC-3 '(reverse induction chain of SEQ ID Nos. 636-660; additional 2 stop codons TAATGA and Hotl sequences; production of deletion derivatives encoding amino acids 1-191) hTPO -2: 5. TTTGCGGCCGCTCATTACAGACCAGGAATCTTGG-CTCTGAAT-3 '(the opposite chain of SEQ ID Nos. 696-720; additional 2 stop codons TAATG A and Not I sequences; a deletion derivative encoding amino acid 211 Function) -123- This paper size is applicable to China National Standard (CNS) Α4 size (210X297) 嫠 --------------- IT ----- (Please read the note on the back first Please fill in this page again) 498079 Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (121) Use 1 μg of plastid DNA of pure 糸 p HT 1-2 3 1 obtained in Example 18 as horizontal Plate, use synthetic primers (use h TP 0-5 as the "side, use h TO ρ-2,-3,-4, and-S as the 3 'side) to perform PCR using 1 0 α Μ respectively. Use GeneAinpTM PCR Reagent Kit with AmpHTaqTM DHA Polymerase (manufactured by Takara Shuzo Co., Ltd.), PCR was performed using a GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER) K 1 0 0 microliters (9 5 t: denaturing conditions of 1 minute, 6 6 t] Adhesive conditions for 1 minute, 72 T] Reaction was performed 20 times for 1 minute, and then reacted at 72t: mint for 7 minutes). After digesting the bands obtained by the respective PCRs with the restriction enzymes EcoRI and Notl, electrophoresis using a 1¾¾ liposugar gel (manufactured by PMC BioProducts) was run. Isolate the main DNA fragments of predictable size for each PCR reaction, purified by K-Puleb-A-refined DNA refining kit (manufactured by Biochemical Ryder), sub-selected on the same restriction enzyme treated PEF18S (As a host strain, Compendium E. col i DH5 manufactured by Toyo Kansai Co., Ltd. was used). Among the obtained transformants, the pure lines containing inserts with predictable lengths were selected respectively from 4 to 5 * to prepare plastid D N A. The method is essentially implemented as described in Μ ο 1 ecu 丨 a "C 1 ο ning [S a in br ο ok, et al., C ο 1 d Spring Harbor Laboratory Press (1989)] &gt; With a series of operations, Deletion derivatives encoding amino acids 1-1 6 3 • (P Η T 1-1 6 3 #Bu 5) ·, deletion derivatives encoding amino acids 1-1 7 1 (P Η Τ1-1 7 1 # 1- 4), deletion derivatives encoding amino acids 1-1 9 1 (ρΗΠ-191 # 卜 4) _, plastid DNA encoding deletion derivatives of amino acids 1-211 (PHT 211tt H) [0299] -124- This paper size is applicable to China National Standard (CNS) A4 (210X297mm) I ------- 0 ^-(Please read the precautions on the back before filling this page)

、 1T 498079 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7_V. Description of the Invention (122) For the refined plastid DNA &gt; Using Taq Dye DeoxyTM Te "minater Cycle Sequening" (Manufacturable) 'Sequenced by Abled Biochemical Co., Ltd.'s 373 ADN A sequence analyzer' to confirm the replacement of the entire full-length abasic sequence, as predicted by the TPO cDNA sequence. [0300] Individual pure line transfection obtained In COs 1 cells, the procedure was performed as in Example 11. That is, 10 micrograms each of plastid DNA was used, and transfection was performed with the Croakin-treated β EAE-Dygistrand method, and the culture supernatant was recovered after 3 days. After the culture supernatant was dialyzed against the IMDM culture medium, it was evaluated with a rat CFU-MK measurement system. As a result, any of the COS1 cells cultured in pHTI-211, pHTI-191, pHTI-171, and pHTI-163 was cultured. A dose-dependent TP0 activity was also observed in the solution. As representative examples, the results of ρ Τ 卜 2 1 1 # 1 ·, ρ Τ Τ 1-1 9 1 # 1 and ρΗΤΙ-171 # 2 are shown in Fig. 13a The results of ρΗΤ 卜 163 # 2 are shown in 圚 1 3b. As a result, in the M-07e measurement system, we can also obtain any of the following: COS1 cell culture supernatants were also observed to use M-07 e cell proliferation-promoting activity. As representative examples, pHTI-211 # 1, pHTI-191 # 1 ·, pHTI-171 2 and pHTI- The results of 163 2 are shown in FIG. 14. [0301] From these results, it is understood that among the 3 5 3 amino acids (including 21 signal sequences) constituting the human TP 0 protein, the 164 position The amino acid deletion after arginine also maintains the activity in vitro. Tip: -12 before the 16-position arginine. 5- This paper size is applicable to China National Standards (CNS) A4 (21〇X297). (%) (Please read the precautions on the back before filling out this page.) Packing. 11 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Zero Economics ^ 8079 A7 37_— V. Description of the invention (123), including table TP [0302] &lt; Example 28 &gt; Preparation of human TPO C-terminal deletion body and expression on COS1 cells ~ Activity confirmed as human TP 0 egg The analysis of the necessary regions of the white matter's active epitopes was performed for the purpose. The missing body obtained in Example 27 was evaluated and a derivative in which the amino acid was deleted at the c-terminus was examined. Based on the plastid-pure PEF 1 8 S-HL 3 4 obtained from Example 16 and the serine of M No. 16 3 as the reference, the epitope of amino acids missing from the C-terminus was sequenced in sequence. body. When constructing, use PCR. The sequence of primers made for PCR is as follows. [0303] hTPO-5: 5. TTTGAATTCGGCCAGCCAG ACACCCCGGCC-3 '(the EcoRI sequence is added to the sequence number 1-2 1; the same as described in Table 9) hTPO-150: 5f- TTTGCGGCCGCTCATTAGAGGGTGGACCCTCCT ACAAGCAT -3 1 (Reverse induction chain of sequence number 4 5 1 4-5 37; sequence with 2 stop codons TAATG A and Not I; deletion mechanism of amino acid 1-150)

hTPO-151: TTTGCGGCCGCTCATTAGCAGAGGGTGGACCCT CCTACAA-3 '(Reverse induction chain of sequence number 4 5 1 8-5 4 0; additional 2 stop codons TAATGA and Not I sequences; encoding deletion of amino acid 1-151 -126-This paper size applies to China National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling out this page) • Packing · 498079 Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (124)) hTPO-153: 5'- TTTGCGGCCGCTCATTATTTGACGCAGAGGGTG GACC (;-3 f (sequence number 4 of 5 2 6-5 4 6); 2 additional stop codons TAATG A and Not I sequence; the role of the deletion system encoding amino acid 153) hTPO-154: TTTGCGGCCGCTCATTATTGCCTGACGCAGAGG GTGGA ~ 3 τ (sequence number 4 5 2 9-5 4 9 anti-induction chain; additional 2 stop codons TAATG Α and Not I sequence; the role of the deletion system encoding amino acids 1-154) hTPO-155: 5τ- TTTGCGGCCGCTCATTAGGCCCGCCTGACGCAG AGGG Τ ~ 3 1 (Sequence number 4-5 5 3 5 5 5 2 counter-induction chain; additional 2 stop code Sub T / UTGA And Notl sequences; the deletion system encoding amino acid 155, and its rejection) hTPO-156: 5, TTTGCGGCCGCTCATTATGGGGCCCGCCTGACG CAGAG-3 f (sequence number 4 of 5 3 5-5 5 5 reaction chain; additional 2 stop code Sub-TAATG A and No t I sequences; production of deletions encoding amino acid 156) hTPO-157: 5'-TTTGCGGCCGCTCATTAGGGTGGGGCCCGCCTG ACGCA-3 'β (reverse induction chain of SEQ ID NO: 5 38-5 5 8; Attach 2 termination codes I -127- (Please read the precautions on the back before filling out this page) • Install. Τ ▼ The size of the paper is applicable to China National Standard (CNS) Α4 specification (210 X 297 mm) 498079 Economy Printed by the Consumer Standards Cooperative of the Ministry of Standards of the People's Republic of China A7 B7 V. Description of Invention (125) Sequences of TAATGA and Not I; the role of the deletion system encoding amino acids 1-157) [0304] Using pure PEF18S- obtained in Example 16 1 microgram of HL34 plastid DNA was used as a template, and synthetic primers (hTPO-5 was used as the 5 f side, hTPO-15 0, ~ 151, -153, -154 &gt; -155 &gt; -156, -157 as 3 * Side-by-side shake) Apply PCR at 10v MH respectively. The PCR reaction was performed using a Gene AmpTM PCR System 9600 (manufactured by PERKIN-ELMER Co., Ltd.) M100 microliters with a Gene A Ni p TMPCRR eagent Kit with AmpliTa〇 (TM DNA Polymerase (manufactured by Shishu Sangyo Co., Ltd.) (95¾ 1 minute denaturation) Pieces, 6 6 t: Adhesive conditions for 1 minute, 7 2 TJ for 1 minute, and reacted 20 times, and then incubated at 7 2 for 7 minutes.) The zone K restriction enzymes EcoRI and Notl obtained from each CR After digestion, electrophoresis using 1¾1lipose gel (manufactured by FMCB i ο P 〇ducts) was performed to separate the main DNA fragments of predictable sizes for each PCR reaction, and purified with plebe-A-refined DNA Refinement (made by Bio-Ryder Co., Ltd.), sub-selected in the same expression vector PEF18S treated with restriction enzyme (as the host bacteria, Compete ♦ Hai E. co 1 ί DH5 manufactured by Toyo Kanji Co., Ltd.). Among the transformants, three to five pure lines containing inserts of respective predictable lengths were selected to prepare plastid DNA. The method used was essentially Molecular Cloning [Sambrook: et al., Cold Spring Harbor Laboratory Press (1989) ]in It is implemented as a record. Through a series of operations, at sequence number 4, the deletion body (ρ Η Π-1 5 0 # 2 1, 2 2, 2 5) encoding the amino acid number 1-150 is obtained. Missing body of amino acid number 1-1 5 1 (ρ Η Τ 卜 1 5 1 # 1 6., 1 7, 1 8) This paper size applies to China National Standard (CNS) Α4 size (210X297) ( Please read the notes on the back before filling this page) • Equipment · Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 ___ 5. Description of the invention (126), missing body encoding amino acid number 1-153 (pHTI -153 # 1-5), the missing body encoding the amino acid number BU 154 (PHTW54 1-5), the missing body encoding the amino acid number: BU 1 5 5 (p Η T 1-1 5 5 # 1- 5). The quality of the deletion body encoding amino acid number 156 (ρΗΤ1_ 156 # 卜 5), the quality of the deletion body encoding amino acid number 1 to 1 5 7 (ρ η T 1-1 5 7 # 1-5) [0305] Purified plastid DNA pHTI-150 # 21, 22, 25 and ρΗΠ-151 # 16, 17, 18, using Taq Dye DeoxyTM Terminater Cycle S eq ii ening 1U t (Abred Biochemical System Corporation), Abu Ryder by biochemical system which is manufactured 3 7 3 A D N A sequence analyzer sort, confirming that no base replacement of the entire sequence of the full-length, having a TP 0 c D N A sequence of such prediction. [0306] Transfection of COS 1 cells with each of the obtained pure pupae was performed according to Example 11. In other words, transfections were performed using the Crocetin-containing DEAE-Davistad method using 10 micrograms each of plastid DNA, and the culture supernatant was recovered 3 days later. After the culture supernatant was sufficiently dialyzed against the IMDM culture medium, the M M-07e was measured and evaluated. As a result, a cysteine containing a limbic acid at the position of 151 was coded by the amino acids 1 to 15 at the 1st position, 1 to 15 at the 3rd position, 151 to the 4th position, and 1 to 1 at the 5th position. The purely transfected C0S1 cell culture supernatant of the C-terminal deletion derivative formed at positions 5, 15, 15 and 15-7 was assayed for dose-dependent TP0 activity. However, the K-coded cysteine-deleted amino acid to the 151st amino acid at the C-terminal deletion at position 150 is pure: transfected C 0 S 1 cell culture supernatant No TPO activity was detected. -129- This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) (Please read the notes on the back before filling this page) One Pack-Order Printed by the Central Consumers Bureau of the Ministry of Economic Affairs Consumer Cooperatives 498079 A7 _____B7___ V. Description of the invention (127). [0307] <Example 2 9 &gt; Preparation of human TPO N-terminal deletions and performance in COS 1 cells ~ Confirmation of activity is required for analysis of activity expression of human TP 0 protein For the purpose &gt; Based on the deletion body obtained in Example 28, a derivative having an N-terminal amino acid deletion was prepared, and it was examined whether TP 0 activity was expressed. Based on the plastid pure PEF18S-HL34 obtained in Example 13 and the pure plastid obtained in Example 27, 糸 Η Η T 1 16 3 &gt; The table 琨 carrier which deleted the N-terminal amino acid of the contiguous signal sequence. P C R is used in the construction. The sequence of the primers produced for PCR is as follows: 0 [0308] hTP0 ~ 5: 5f- TTTGAATTCGGCCAGCCAGACACCCCGGCC-3 1 (EcoRI is added to the sequence number 4 1-21; the same as described in Table 9) hTP03 ·· 5 '-TTTGCGGCCGCTCATTATTCGTGTATCCTGTTCAGG TATCC-31 (reverse induction chain of sequence number 757-780; added two stop codons TAATGA and Notl sequence; same as the one synthesized for the deletion mechanism encoding amino acid 231) hTP. -S: 5 T- TTTGCGGCCGCTCATTATTCAGTGTCAGGACTAGA GAGGTTCT-3 f (Reverse induction chain of sequence number 4 5 7 7-6 02; addition of 2 stop codons TAATGA and Notl sequence; production of deletion body encoding amino acid 1-163 Yong-130- This paper size applies to China National Standard (CNS) Α4 size (210 × 297 mm) (Please read the precautions on the back before filling this page).

1T 498079 A7 ___B7 V. Description of the invention (128)

hTPO-13: AGTAAACTGCTTCGTGACTCCCATGTCCTTCACA GCAGACTGAGCCAGTG-3 f (SEQ ID NO: 124-173; for the production of derivatives with missing amino acid number 1-1 12) hTP0-13R: 5, -CATGGGAGTCACGAAGCAGTTTACTGGACAGCGT TAGCCTTGCAGTTAG-3 f (SEQ ID 4) 64-87 and 124-148 counter-induction strands; missing derivatives of amino acid numbers 1-1 2) hTPO-7: 5'-TGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGA CTCCCATGTCCTTC-3 f (Serial Number 4 of 1 0 6-1 5 4; Production of derivatives with missing amino acid numbers 1-6) hTP0-7R: 5'-TTTACTGAGGACTCGGAGGTCACAGGACAGCGTT AGCCTTGCAGTTAG-3? (Serial number 4 of 6 4-8 7 and 1 0 6-1 2 9 reaction chain ; For the production of derivatives with lost amino acid numbers 1-6) hTPO-8: 5'- GACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTC CCATGTCCTTCACA-3 f. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) ) (Sequence number 1 0 9-1 5 7; for the production of derivatives lacking amino acid number 7) hTPO-8R · 5. CAGTTTACTGAGGACTCGGAGGTCGGACAGCGTT AGCCTTGCAGTTAG-3 f ( Column number 4 of 6 4-8 7 and the reverse induction chain of 1 0 9 ~ 1 3 2; missing amine group -m- This paper size applies to China National Standard YcNS) A4 size (210X297 mm) — 498079 A7 B7 5 Explanation of the invention (129) For the production of the derivatives of acid numbers 1 to 7 ° [0309] (1) Production of the derivatives of the missing amino acid numbers 1-12 (PHT13-231) using the pure PEF obtained from the implementation of the tooth 18 1 8 S-HL 3 4 plastid DNA 1 · 4 μg as template, using synthetic primers (combination of h TP 0-1 3 and h TP 0 3: use hTPO-5 and hTP0-13R together) 5 V Μ, PCR was performed using Gene / UpTM PCR Reagent Kit with AnipliTac (TM DNA Poly in erase (manufactured by Takara Shuzo Co., Ltd.), Gene Amp TMPCR System 96 0 0 (manufactured by PERKIN-ELMER) at 100 micron Litre capacity for PCR reaction (95T) After 5 minutes denaturation, 95 ° C 1 minute denaturation conditions, 65 1] 1 minute bonding conditions, 7210 1 minute synthesis conditions

30 reactions, and then cultivate at 72 ° C for 7 minutes). The individual PCR products were run on a 2¾ agarose gel (manufactured by FM.C BioProducts), and the major fragments of each predictable size were separated. NA-fragments were purified with plebe-A-refined DNA. (Made by Ryder) Refining &gt; TE buffering agent dissolved in 15 microliters. K One microliter of each solution was used as a horizontal plate, and the second PCR was performed. 〇 [0310] Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) hTPO-5 and hTP03) PCR was performed at 5 μM, respectively. ° PCR was performed using GeneAnipTM PCR Reagent Kit with AmpliTaqTM DNA Polymerase (manufactured by Takara Shuzo Co., Ltd.) with a capacity of K100 microliters of GeneAnipTM PCR System 9600 (manufactured by PERKIN-ELMER). After 5 minutes of denaturation at ° C, the denatured pieces at 95 ° C for 1 minute, the bonding conditions at 65 ° C for 1 minute, and the synthesis conditions at 7 2 ° C for 1 minute -132- This paper size applies Chinese national standards ( CNS) A4 size (210 X 297 mm) 498079 A7 B7__ printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (15o) 30 reactions, and then incubate at 7 2 t! For 7 minutes). The PCR products were run by electrophoresis using a 1.22⁄2 wax gel (manufactured by FMC BioProducts) to separate the major DNA fragments of each predictable size. The K-Pribe-A-refined DNA purification kit (Bio-Ryder) Refined), dissolved in 15 microliters of TE Yuan granules. After digestion with the restriction enzymes EcoR I and Not I, the same amount of phenol / chloroform was used for extraction and ethanol precipitation was performed. The precipitate obtained by centrifugation was dissolved in 15 microliters of TE buffer, and then sub-selected on the epitope carrier PEF 1 8 S, which was also treated with restriction enzymes. ο 1 i D Η 5). Among the obtained transformants, there were 4 5 pure puppets with predictable lengths inserted into the human body to prepare plastids D Ν Α. The method was essentially performed as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Dress (1 9 8 9)] ° in sequence number 4 to obtain the protein encoding amino acid number 1-2 31 The plastid DN A of the deletion derivative (p Η T 1 3-2 3 1) of 1-12 is deleted. For these, PCR was performed using the primers of hTPO-5 and hTP03, and 8 of the 45 pure pupae were pure: inserts with a predictable size were confirmed. Among them, three pure tadpoles were sequenced using the Taq Dye DeoxyTM Terrainater Cycle Sequening Kit (Ablad Bio: Systems, Inc.) &gt; 373 A DNA sequence analyzer made by Ablade Biosystems, and occurred in one pure tadpole. If the predicted deletion, and other parts also include no substitution of the base sequence across the full length, a pure strip pHT13-231 # 3 with TPOcDNA as designed can be obtained. [0311]

(2) Production of a derivative with amino acid number 1-6 (pH T7-163) produced from the above-mentioned deletion derivative (1), designing C-133 of TP 0 protein- This paper is applicable to Chinese countries. Standard (CNS) A4 (210X297 mm) ^ Binding (please read the precautions on the back before filling this page) 498079 A7 ______ B7____ V. Description of the invention (151) The amino acid at the end is No. 231, but because It was confirmed that the TP 0 activity was exhibited even when the C-terminal side was deleted. Therefore, in this case, the C-terminus was designed to be an amino acid of No. 16 to 3. In the following (3), the amino acid No. 163 is similarly designed as a C-terminus. The plastids (^ 1.4 micrograms) of the pure line 1 ^ 1-163 obtained in Example 27 were used as templates, and the synthesized primers (hTPO-7 and hTP〇-S) were combined to make two: hTPO-5 and hTP0-7R (Sisters use) 5 / iM PCR. GeneAmpTM PCR Reagent Kit with ArapIiTaqTM DNA Polymerase (manufactured by Takara Shuzo Co., Ltd.) was used to perform a PCR reaction with a GeneAinpTM PCR System 9 600 (manufactured by PERKIN-ELMER) at a volume of 100 microliters (9 5 ° C after 5 minutes denaturation, 9 5 ΐ! Denaturing conditions for 1 minute, 6 5 t: Adhesive conditions for 1 minute, 72. () 30 times of reaction for 1 minute of synthesis conditions, and then incubate at 7 2 ° C for 7 minutes). The PCR products were run using a 1 · 2 agarose gel (manufactured by PMC BioProducts), and the major DNA fragments of each predictable size were separated. The DNA was purified with Blebe refined DNA (made by Bio-Ryder) and dissolved. 15 microliters of TE buffer. Use 1 microliter of each solution as a template to perform the second PCR. [0312] (Please read the precautions on the back before filling this page)

, 1T front-line Printed synthetic primers (using hTPO-5 and hTP0-S) from the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs, respectively, performed PCR using 5 μM. The GeneAmpTM PCR Reagent Kit with AmpliTaqTM DNA Polymerase (manufactured by Takara Shuzo Co., Ltd.) was used to perform PCR (95t) with a volume of 100 microliters using GeneAmpTM PCR System 9600 (manufactured by PERKIH-ELMER). After 5 minutes of denaturation, 95 ° C for 1 minute Denaturation conditions, 65 ^ 1 minute bonding pieces, 72 ° C 1 minute synthesis conditions 30 times-134- This paper size applies to China National Standard (CNS) Α4 size (210 × 297 cm) 498079 Central standard of the Ministry of Economic Affairs Bureau employee consumer cooperative printed A7 ____ B7 Fifth, the response of invention description (152) 'cultivated for another 7 minutes at 72T]. The PCR products were electrophoresed using a 1.2% loose sugar gel (FMCB i ο P 〇ducts) electrophoresis, the main DNA fragments of predictable size were separated, and purified by plebe-A-refined j) n A The kit (made by Bio-Ryder) was refined and dissolved in 15 µl of τ E buffer. After digestion with M restriction enzymes EcoR I and No11, the same amount of phenol / chloroform was used for extraction twice, followed by ethanol precipitation. The precipitate obtained by centrifugation was dissolved in 5 microliters of τ Eyuan granules, and sub-selected on the epitope vector p E 卩 丨 8 S which was also treated with restriction enzymes (as a host strain, Combiden manufactured by Toyobo Corporation) was used. Hai E. c 〇1 i D Η 5). Among the obtained transformants, 30 pure lines with a predictable length of the human body were selected to prepare plastid DNA. The method was essentially performed as described in Moiecuiar Cloningt Sambrook ^, Cold Spring Harbor Laboratory Pres s (1 898). In SEQ ID NO: 4, the plastid D N A of the deletion derivative (P Η T 7-1 6 3) of BU 6 among the proteins encoding amino acid BU 163 was obtained. For these pure lines, h C P 0-5 and h T P 0-S were used as primers to implement P C R. In all pure lines, confirm that the human body has a predictable size. From these pure: three were selected, and Taq Dye Deox yTM Termina Ter Cycle Sequening Kit (manufactured by Ablaide Biochemical System Co., Ltd.) was sequenced by 373A DNA sequence analyzer made by Ablaide Biochemical Co., Ltd., and occurred in two pure lines. If the predicted deletion also includes other partial substitutions across the full-length abasic sequence, two pure TP 0 c DNA sequences can be obtained as designed: ρ Η T7-1 6 3 # 4, and # 2 9 . [0313] (3) Preparation of Derivatives with Missing Amino Acid Numbers 1 to 7 (ρ Η Τ8-1 6 3) Method of Making Derivatives with Missing Amino Acid Numbers 1-7 (PTT 8-163) (please Please read the precautions on the back before filling this page) Binding and binding * 135- This paper size is applicable to China National Standard (CNS) A4 specifications (210X: 297 gigabytes) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Description of the invention (155) The quality is the same as the above-mentioned production of the derivative (ρ Η T7-16 3) of amino acid number B6 missing. The same method is used. Using the purified plasmid DNA of pHTl-163 1 · 4 micrograms obtained in Example 27 as a template, the synthetic primers (hTPO-8 and hTPO-S were used together: hTPO-5 and hTP0-8R were used in combination ) Each

ArapHTaqTM DNA Polymerase (manufactured by Takara Shuzo Co., Ltd.) was used to perform a PCR reaction using a GeneAmpTM PCR System 9600 (manufactured by PERKIN-ELMER) in a capacity of K 100 microliters (95 after 5 minutes of denaturation &gt; 9 5 ° C for 1 minute) Denaturation conditions, 6 5 t bonding conditions for 1 minute, 7 2 t] synthesis conditions for 1 minute for 30 reactions, and 7 2 T] cultivating mint for 7 minutes) '. The respective PCR products were run on a 1.22⁄2 liposugar gel (PMC Bi P οducts), and the major DNA fragments of each predictable size were separated. K-Pleb-A-Sperm DNA Refined kit (made by Bio-Ryder), dissolved in 15 microliters of TE buffer. One microliter of each solution was used as a template, and the second PCR was performed. [0314] Synthetic primers (using hTPO -5 and hTP0_S) PCR was performed using 5 μM each. GeneAmpTM PCR Reagent Kit with AmpliTaciTM DMA Polymerase (manufactured by Takara Shuzo Co., Ltd.) was used. Gene / UpTM PCR System 9600 (manufactured by PERKIN-ELMER Co., Ltd.) had a capacity of K 100 microliters. Perform PCR reaction (denaturation condition at 95 ° C for 5 minutes, denaturation condition at 95 ° C for 1 minute, 6 5 t) bonding condition for 1 minute, synthesis condition at 7 2 ° C for 1 minute, and then react 30 times. 7 2 lG incubation for 7 minutes). Run the PCR product using a 1.2¾ agarose gel (FMC BioProducts -136-) This paper size applies to China National Standard (CNS) A4 (210X297 mm) Binding ^^ (please (Please read the notes on the back before filling out this page) 498079 A7 B7 printed by Standards Bureau Consumer Cooperative V. Electrophoresis of Invention (154)). Predictable size of major DNA fragments. Prebleb ~ A-refined DNA refining kit (made by Bio-Ryder). Refined, dissolved in 15 microliters of TE Yuan granules. After being cured with the restriction enzymes EcoRI and Notl, it was extracted once with an equal amount of phenol / chloroform and then ethanol precipitated. The precipitate obtained by centrifugation was dissolved in 15 microliters of TE After the buffer, it was sub-selected on the expression vector p EF 1 8 S treated with the same K-restriction enzyme (as a host strain, Compete · hai E. c ο 1 i D Η 5 manufactured by Toyobo Corporation) was used. Among the shapes, 30 pure cymbals containing inserts of predictable length were selected to prepare plastid DMA. The method was essentially performed as described in Molecular Cloning [Sarabrook: et al., Cold Spring Harbor Laboratory Press (1989)]. No. 4 ·, the plastid D Ν Α of the deletion derivative (P Η Τ8-1 6 3) of the protein encoding amino acid number 1-1 6 3 was obtained. For these pure lines, hTPO-5 was implemented. PCR with hTP〇-S as primers Of the insert. Three of these pure lines were selected and sorted by Ta q Dye DeoxyTM Terminater Cycle Sequening Kit (manufactured by Able Biotech Co., Ltd.) &gt; sequenced by 373 AD ΝΑ sequence analyzer made by Able Biochemical Co., Ltd., in 2 Two pure lines with a TPO cDNA sequence as designed, such as the predicted deletion, and the replacement of other parts across the full length without a test sequence, can be obtained. [0315] (4) Confirmation of epitope and TPO activity of COS1 cells with deletion derivatives. The transfection of each of the obtained pure bodies of the deletion bodies was performed in COSlffl cells according to Example 11. That is, 10 micrograms each of plastid DNA was used, and transfection was carried out by using the DEAE ~ Daiche Sderland method containing crorkin. 3 days later, the culture supernatant was recovered. -137- This paper size is applicable to Chinese National Standard (CNS) Α4 specification (210 × 297 mm) --------- install-(Please read the precautions on the back before filling this page)

Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, 1T 498079 A7 B7 V. Description of the invention (155) After the culture supernatant has been fully dialyzed against the IMDM broth, it is determined by M-07e: Department Evaluation. [0316] As a result, a purely transfected COS1 cell culture supernatant that encodes a deletion derivative at position 7 to 16 of the amino acid was found to have a weak but dose-dependent TP0 activity. On the one hand, TPO activity was not detected in purely transfected COS 1 cell culture supernatants encoding derivatives missing at positions 8-16 and 13-23 at amino acids 1 to 23. [0317] <Example 3 0 &gt; Preparation of human TPO full-length cDNA plastid (ρΗΤΡΙ) The composition of the animal cell expression vector with the entire amino acid coding region of human TP Q c DNA predicted as sequence number 6 was performed . [0319] The base sequence of the primers used is as follows: [0320] hTPO-1: 5, TTGTGACCTCCGAGTCCTCAG-3 '(SEQ ID NO: 105 to 125) hTP0-S: 5, -CAGGTATCCGGGGATTTGGTC-3, (corresponding to the sequence number 6 of 7 45 ~ 76 5 counter-induction chain) hTP〇-P ·· 5T- TGCGTTTCCTG ATGCTTGTAG-3 f (Serial No. 6 50 3 ~ 523) hTPO-K0: 5 f- GAGAGAGCGGCCGCTTACCCTTCCTGAGACAGAT T_3, -138- This paper Applicable to China National Standard (CNS) A4 specification (210 × 297 mm) (Please read the notes on the back before filling out this page) Equipment, 1T Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Invention Explanation (1%) (The recognition sequence of the restriction enzyme N 〇11 and the GAGAGA sequence are added to the reverse induction strand sequence corresponding to the sequence number 6 from 106 to 108.) [0321] The first PCR was performed using 300 nanograms of the pure p E F 1 8 S-H L 3 4-3 obtained in Example 16 as a template. Primer-hTPO-1 and hTP〇-S 5.uM each, using Vent RT M 1) NA polymerase (N ew E ng 1 and B i OLabs) 1 unit, the reaction was performed (9 6 t : 1 minute, 6 2 T] 1 minute, 7 2 1 minutes of reaction after 30 cycles, at 7 2 C for 7 minutes). The composition of the reaction solution is as follows. The final concentration is 10 mM KC1, 10 iuM (fUU) 2S04, 20 mM Tris-HCl (ρΗ8 · 8), 2niM MgS ", 0.1% Triton X-100, 200yM dNTP ία ix-[0322] will come from the market Of human normal liver (A) + RMA (C 1 ο ntech) 1 μg heated at 7 (Π) for 10 minutes and quenched on ice, add 10 mM D ΓΓ, 5 0 0 α M d NTP ra i X, 25 nanogram random primers (manufactured by Takara Shuzo Co., Ltd.) · 10 units of RN ase I nhibit 0 r (Bringa Mannham Corporation) · 2, 0 units of Super S cript τ MIIRN ase Η -(LIFE has been (: _01001 £ 3 company), incubate at 37 ° () for 1 hour to synthesize 0 ^. Use 1 / 20th of the synthesized cDN A reaction solution as a template and perform the second PCR The reaction was performed using 2.5 μM of each of primers-hTP0-P and hTP0-K0, 25 units of AmpliTaqTM DNA polymerase (manufactured by Takara Shuzo Co., Ltd.) for the reaction (95 ° C for 1 minute, 5 8 ° C for 1 minute, 7 2 ° C 1 minute reaction for 30 cycles) ° [0323] This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) (Please read the (Please fill in this page for the matters needing attention). Pack-order 498079 Printed by A7 B7, Consumer Cooperatives of the China Standards Bureau of the Ministry of Economic Affairs. 5. Description of the invention (137) Run the 2nd PCR solution on the U agarose gel. Cloth-A-Refined DNA refining kit (made by Bio-Ryder Co., Ltd.) Refined major zones of predictable size. Refined amounts of 1 in 20 of each amount are horizontal plates, and the third PCR is performed. Vent RTM DNA polymerase (New Eng 1 and Bi 0 L abs) was used to carry out the reaction (after heating at 9 6 ° C for 2 minutes, 9 6 2 minutes, 7 2 ° C for 2 minutes) After 3 cycles at 72 ° C for 7 minutes), hTPO-I and hTPO-KO were added to the reaction solution to make 1 α M, and then heated at 96 ° C for 2 minutes. After that, 91 minutes , 62t] 1 minute, 72t! After reacting for 25 cycles in a 1 minute reaction, it was reacted for 7 minutes at 72. The reaction solution was extracted once with the same amount of water-saturated phenol-chloroform, and then extracted with the same amount of chloroform. After 1 time, ethanol precipitation was performed (0.3M sodium acetate, 0.5 microliters of Beringa Manham Co., Ltd., hepatose, 2.5 times the amount of ethanol), and DNA was recovered. The recovered DNA was digested with the restriction enzymes BaraHI and Not]: After digestion, run U agarose gel electrophoresis, and use Pulib-A-refined DNA purification kit (made by Bio-Ryder) to refine the predicted size. After banding> Pre-ligated to M restriction enzyme B a mH I and Η 〇11 digested pBluescriptll SK + vector (Stratagene), then Compomed E. col i DH 5 (manufactured by Toyobo Co., Ltd.) was transformed . Four pure lines were selected from the obtained community to prepare plastid D N A. For the purified plastid D Μ A, the Ta q Dye DeoxyTM Terminater Cycle Sequening kit (Abbr Biochemical: System Co., Ltd.) was used, and sorted by Ablade Biochemical System Co., Ltd. 373A DNA sequence analyzer, from BamHI to Not] : The substitution of the base-free sequence in the region can be confirmed to be a pure pBLTP with a predicted TP 0 c DNA sequence. 〇-140-This paper size applies the Chinese National Standard (CNS) A4 specification (21〇 &gt; &lt; 297)嫠) • m · (ϋ · I nm · ml In ml i (Please read the precautions on the back before filling this page)

1T line 498079 A7 ___ —__ B7 _ V. Description of the invention (138) [0324] pBLTP digested with restriction enzyme Ecoll & Bamin, run 1% agarose gel electrophoresis &gt; dn 纟 Refined Kit (made by Biochemical Ryder) Refined high molecular weight zone. Similarly, pEF18S-HL34 was treated with a restriction enzyme and a band of 45 0 bp was purified. The respective DNAs were ligated, and Compendium-Sea E. Co 1 i DH5 (manufactured by Toyobo Co., Ltd.) was transformed. The plastid DNA was prepared from the obtained community, and the pure line pBLTEN containing human TP 0 c D Μ A insert was obtained. [0325] After digestion with the obtained pBLTENM restriction enzymes EcoRI and Notl, run 1¾ agarose electrophoresis, and use pribe- -Refined DNA refining kit (made by Biochemical Ryder Co., Ltd.) After refining a band of 1 200 bp, it was connected to the expression vector PEP18S treated with the same K restriction enzyme, and Compendium-Sea E, coli DH5 (Manufactured by Toyobo Corporation). A plastid DNA was prepared from the obtained community, and a pure line p H TP 1 containing all coding regions of human TPOcDNA was obtained. This pure plastid D N A is abundant. It is prepared for the following experiments. The preparation of plastid DNA is essentially carried out as described in Molecular Cloning (Sambrook: et al., Cold Spring H.S.borab.:/Press, 198, 9). [0326] Consumption by employees of the Central Bureau of Standards, Ministry of Economic Affairs Printed by the cooperative (please read the precautions on the back before filling this page) <Example 3 1 &gt; The CH0 cell sister vector, pDEF202-hTP0-Pl, will consist of pDH MGFR pMG MG 1 After 1 μg of the K restriction enzymes EcoRI and BamHI, run agarose gel electrophoresis to recover a fragment containing the mouse D Η PR small gene (about 2 * 5 kbp). The recovered。 section was dissolved in -14 ^ paper size Applicable to Chinese National Standard (CNS) A4 specification (210X297 gong) 498079 A7 B7__ V. Description of the invention (159) 50niM Tris-HCl (pH7,5), 7raM MgCl2, ImM Tetonyl alcohol, 0.2mM dNTP reaction solution 25 microliters, add 2 units of Klenow fragments, and react at room temperature for 30 minutes to smooth the ends of the DNA. Then, treat with phenol / chloroform and precipitate with ethanol, and then dissolve in IOiuM Tri-HC1 (pH 8. 0) ,

10 microliters of a solution of 1 mM EDTA. [0327] Next, the obtained mouse DHFR-containing small gene fragment was treated with the animal epitope vector PEF18SK restriction enzyme Sma I, and K alkaline phosphatase (manufactured by Takara Shuzo) was dephosphorylated to the selected vector DNAKT 4 DNA ligase (manufactured by Takara Shuzo) was combined to obtain expression vector pDEF 202. [0328] Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) Secondly, this carrier PDEF 2 0 2 is treated with restriction enzymes E c RI and S pe I to After liposugar gel electrophoresis recovered a large vector fragment, the plastid pHTPl Μ restriction enzyme EcoRI containing this fragment and human TPO cDNA (P1 pure: line) was ligated with MT 4DNA of human TPO cDNA (P1 pure line) obtained by Spel treatment. Enzyme (made by distiller's liquor) was combined to obtain the epithelial carrier PDEF 202-hTPO-Pl. This plastid contains the replication initiation region of SV 40, the human elongation factor-α promoter region, the initial polyadenylation site of SV40, the mouse DHFR minigene, the replication initiation region of pUC ι8, and / 3-lactamase Gene (Smp), downstream of the human TPO cDNA downstream of the human elongation factor-l-α Kaixun region. [0329] &lt; Example 3 2 &gt; · Human TPO on the surface of CH0 cells: CC 0 cells (dhfr- Strains, U r 1 aub and C hasi η; ρ r 0 c · -142- This paper size is applicable to China National Standards (CNS) A4 (210X297 male) 498079 A7 B7 V. Description of the invention (U〇)

Natl. Acad. ScM. USA; Vol. 77, p. 4216, 1 980) in a 6 cm diameter broth containing 10 ¾ bovine fetal serum with minimal essential medium (with addition of α-Μ E Μ (-), thoracic spine, hypoxanthine) The culture was propagated in a Petri dish (F a 1 c ο η company), and this was transformed by the calcium phosphate method (Ce 1 1 Phect, manufactured by Farumasia Company). [0330] That is, 10 micrograms of pDEP202-hTP0-Pl plastid prepared in Example 31. Zhongjiayuan Granule-A: 120 microliters and H20: 120 microliters were mixed and then 'left at room temperature for 10 minutes. Secondly, add buffer ~ B to this solution: 120 μl, mix again, and let stand at room temperature for 30 minutes. This DNA solution was dropped into a petri dish, and then baked and cultured in a C 0 2 incubator for 6 hours. Remove the culture medium from the petri dish, and after adding K cx -MEM (-) 冼 twice, add a -MEM (-) containing 10¾ dimethylarene 飒 and print it at the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please first (Please read the notes on the back and fill in this page.) 2 minutes at room temperature. Next, add non-selective medium containing 10% dialyzed bovine fetal serum (add α-MEM (-), hypoxanthine, thymidine) and culture for 2 days. Add a-ME Μ (-), hypoxanthine, thymidine) option. Selection was performed by trypsinizing the cells and dividing each 6-cm diameter petri dish into 5 10-cm diameter petri dishes or 20 24-well petri dishes, and then selecting the culture medium every 2 days. The medium was exchanged while the culture was continued. In the culture dish or hole where the cells have been proliferated, the human TPO activity in the culture supernatant is measured, and the human TPO activity in the culture supernatant is determined in a new culture dish or hole. The ηM methotrexate medium divides the cells into 1: 15 and continues to grow to proliferate fef cells that are resistant to methotrexate for colonization. -143- This paper is again applicable to Chinese National Standard (CNS) A4 (210X: 297 Gong)> 498079 A7 B7 V. Description of the Invention (U1) [0 33 1] Also, C Η 0 Niu Cell's transformation is also It is possible to simultaneously transform ρ Η TP 1 and oral payment 0 1 to c Η 0 cells ((: 0 called: "3 ^ (^ (: 1: 丨 (^). [0332] &lt; Example 3 3 &gt; \ 63.6.5.3. Cells were treated with the elder sister vector, 8 ^ (003 ^ 〇 ^? 0- 丨) 1. Animal cells were treated with epidermal vector BMCGSneo 1 microgram with restriction enzymes Xhol and No. I. Then, agarose gel electrophoresis was performed to recover the d NA portion of the carrier. Then the DNA fragment containing the obtained DNA and human TP 0 c D Η A plastid p BLTENK restriction enzymes X h ο I and N 〇11 treatment, KT 4 The DNA ligase was combined with the obtained human TPO cDNA (Pure pure 糸) to obtain epidermal plastid BMCGSneo-hTPO-Pl. This epidermal plastid contains the initial cytomegalovirus start region and is derived from the rabbit / 3 globulin gene. Part of the human / 3 globulin gene of epidermal ganglia and polyadenylation base, 69% of bovine papilloma virus 1 gene, thymidine kinase promoter and polyadenylation site, phosphotransferase ( new [W gene]), ρ BR 3 22 replication initiation region and / 3 -lactamase gene (Ampr), downstream of the cytomegalovirus initiation region is followed by human TP 0 c D NA. [0333] &lt; Example 3 4 &gt; Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) at X 6 3, 6.5.3, the cell performance will be 63.6.5.3. The culture was propagated in 〇11156〇! (: 0, minimally necessary (DME) medium containing 10% bovine fetal serum, and this was transformed by electroporation. [0334] -144- This paper size is suitable for Chinese countries.) Standard (CNS) A4 (210X297 mm) 498079 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 ______B7 V. Description of the invention (142) That is, the BMCGSneo-hTPO-Pl plastid prepared in Example 33 was added with 20 μg 750 microliters of DME medium containing 107 cells was fixed in a small cup with a 4 mm lid for electroporation, and left at 40 t: 10 minutes. This micro cup was fixed to the electroporation device (BTX 600, BTX company), the gene was introduced under the conditions of 380V, 25mF, 24 Angstrom (A). After 15 minutes at the cup, the cells were washed once with D ME containing 10% bovine fetal serum. Next, the cells were suspended in 50 ml of d μE containing 10% bovine fetal serum, divided into 96-well culture dishes, and then cultured in a CO 2 incubator for 2 days. Thereafter, the broth was exchanged for DME containing 1 mg / ml of G 4 18 (GIBCC Co.) and 10% bovine fetal serum, and the medium was exchanged every 3 days thereafter. With respect to the virus that has proliferated cells, human TPO activity in the culture supernatant was measured. Those who were identified as human TP 0 active in the culture supernatant were allowed to undergo proliferative tolerance cells for 2 times to establish human TP 0 to produce a female cell line. Example 3 5 &gt; Massive expression of human TPO on COS1 cells ~ Refined transfection of COS1 cells was performed using the Crookin-containing DEAE-Dygistrand method described in Example 11 . COS1 cells (ATCC CRL1650) were placed in a 175-cm square culture flask treated with collagen, and IMDM containing 1 (U (v / v) FCS was used in a 5¾ carbon dioxide gas incubator at 37 ° C. Cultivate until it becomes a 1005S confluent culture. The collagen coating of the culture bottle is processed to prepare K ImM HC1 into a 0.3 mg / ml collagen solution (Cellmatrix typel-C manufactured by Iwaki Co., Ltd.) each-175 square centimeters. Add 25 ml to the culture bottle, and leave it at room temperature for 1 hour to recover the collagen solution. The content is 20 ~ (Please read the precautions on the back before filling this page).

, tT line-145- This paper size is applicable to China National Standard (CNS) A4 (210X297mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Description of the invention (145) 50 ml of PBS washing Come once. Transfection was performed at a depth of 175 cm 2 in each culture flask, and 250 micrograms / ml of DEAE-Dagestrand (Faromaxia) _, 60 y crokin (Sigma Company) ) And 20% IMDM solution containing 10% (ν / ν) Nu-Serum (Clabrettiv), mixed and dissolved in 50 microliters of HBS plastid pHTPl (40 mg), just turned Before the staining, M IMD was added and the COS1 cells described above were washed once, and then cultured in a 37% 5% carbon dioxide gas incubator for 3 hours. Thereafter, the culture supernatant was removed by suction. After washing once with I MD, 50 ml of a serum-free medium was added, and the culture supernatant was baked in a 37% 5% carbon dioxide gas incubator. The culture supernatant was recovered after 5 days. . In one operation, 750 to 260 baking bottles of 175 cm 2 were used, and 5 to 131 culture supernatants were recovered. The elder sister of serum-free medium became 5 μg / ml I nsu 1 i η (made by Sigma Co., Ltd.), 5 μg / ml Transferrin (made by Sigma Co., Ltd.), 10 // Μ monoethanolamine (Wako Pure Chemical Industries, Ltd.) , 2 5 η MS 〇diumse 1 enite (manufactured by Sigma), IMDM of 200 micrograms / ml BSA (fatty acid-free high-purity bovine albumin manufactured by Nichi Corp.). [0336] Purification and activity of human full-length TPO on the surface of COS1 cells (from expressing plastid pTOP1 &gt; P1 pure: line) confirmed human full-length TPO on the surface of COS1 cells (from epiplast pTP) Examples of the activity and purification are described below. To investigate the platelet-increasing effect of TP0, a portion of the refined product was first prepared and refined to obtain a highly purified TP0 control standard. In the following preparation process, the TP 0 activity was measured. The main test was M-07e, and the rat CFU-MK test strip also showed the same. -146- This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X: 297). (Please read the notes on the back before filling out this page). Equipment-Line 498079 Printed A7 B7 by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 5. TPO activity of the invention description (U4). For these measurements, human serum albumin (HSA) was added to the control standard at a final concentration of 0.02 to 0.05%. [0337] First, the method of Bridge and Sudo (Hideya Ohashi and Tadashi Sudo, Biosci. Biotech. Biochem ,, 58 (4), 7 5 8-7 5 9, 1 994) was used to transfect COS1 cells of plastid pHTPI. In serum-free IMD Μ culture solution containing 0.2 g of BSA per 1000 ml, 5 mg of bovine insulin, 5 mg of human lysoferrin and 0.02 inM ethanolamine, 25 nM of sodium selenite, Incubate at 37 ° C for 5 days in a CO2 gas incubator to obtain approximately 7 liters of serum-free culture supernatant. In the serum-free culture supernatant, P-APMSF and Refabloc SC ΐ 2-aminoethyl)-benzylsulfonium hydrochloride (manufactured by Merk) catalogue number 2 48 39) are proteolytic enzyme inhibitors. Add the final concentration of about lmM, and take the filtrate of 0.22 // m filter. The K ultrafiltration unit (made by Felton Company, Yamika ultra-combination (彳 X square, 'spun / V 歹 歹, / /)) molecular weight 8 0 0 0 Jiashi (Fang ,, Changbu), Or manufactured by Jingli Boer Company ♦ PLGC Peli Kanga Jie He (1, J &gt; Nai ,, / /) molecular weight 1 000 when the best) concentrated about 10 times, for a volume of 723 ml (protein concentration 3. 38 mg / ml 'total protein 4 2 4 4 5 mg, relative activity 43000, relative activity 105 1 0 0 0 0 0 0) plus 1.6 moles of ammonium sulfate per 1 000 ml (total of 28 8 g), so as to contain Finally, 804 ml of a 1.5M ammonium sulfate solution was concentrated in a Macr 〇-P rep M ethy 1 Η IC tube equilibrated with a 20 mM sodium citrate buffer (pH 5 · 2) containing a saddle of 1.25M sulfate. Columns (manufactured by Bio-Rad, catalog number 1 56-0 0 8 0; diameter 5 cm, filter pad height 9 cm) were added at a flow rate of 10 ml / min. After adding, it must be included (please read the precautions on the back before filling in this page)-Binding. Thread -147- This paper is suitable for the New Zealand Banking Standard (CNS) A4 (210 ^ 297 mm) Ministry of Economic Affairs Printed by the Consumer Standards Cooperative of the Central Bureau of Standards 498079 A7 _____B7_ V. Description of the invention (U5) 1.25 shirts of ammonium sulfate 20 — citrate Nayuan solution (out 5.2) dissolves without retention F 1 (238 4 ml, protein concentration 0 86.4 mg / ml, total protein mass of 601 mg, relative activity of 6 0 0). [0338] Next, the dissolving agent was changed to 20 iuM citrate to dissolve 5.8, and the dissociated fraction F2 (1092 ml, protein concentration 0.7776 mg / ml, total protein mass 8 47 g, relative activity 1 5 0 0 0 0). [0339] In addition, Macro-Prep Methyl HIC column F2 (1081¾ liters) was added at a flow rate of 10 ml / min, and SP Sepharose Fast Flow (Farlu) was equilibrated in advance with 20 citrate Nayuan solution (out 5.8). Made by Marcia Biochemical Technology Co., Ltd., Catalog No. 17- 0729-0 1; diameter 3 cm, S pad height 10 cm). After the addition is complete, it is necessary to pool K containing 1 1 0 ιηΜ 1 (: 1 of 20 — sodium citrate buffer solution (out 5.6) part of the dissociated 卩 '1 (2262¾ liters, protein concentration 0.270mg / ml, total protein The amount is 610 mg and the relative activity is 30000). Secondly, the eluent is changed to a 20 mM sodium citrate buffer solution (out of 5.4) containing 400 niM NaCl, and the dissolved fraction F2 (856 ml, protein concentration) is collected. 0.189 mg / ml, total protein 1 162 mg, relative activity 300,000). Secondly, the dissolving agent was replaced with 20 μαM NaCl citrate solution (pH 5.2) containing 1 000 mM NaCl, and the dissolved fraction F 3 (3 70 ml, protein concentration 0.034 mg / ml &gt; total protein mass 12.6 mg, relative activity 15 0 0 0 0) [0340] In addition, in SPS ephar 〇se F ast F 1 〇 w The main TP 0-148- in the column is applicable to the Chinese National Standard (CNS) A4 size (21 OX297 mm) (Please read the precautions on the back before filling this page) —I ml ml ml _1 ϋϋ · Ϋ_ι— ml m · m HI n nm— ^ mamn 11 mi in —-— ϋ v: V 11_11 Hal In ml 燊 i cable 498079 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7__ 5. Description of the invention (Η6) Add 1-propanol with a final concentration of about 10¾ to F2 (85 4 5 ml), and add a flow rate of 3 ml / min. LA ~ WGA columns (manufactured by Toyona Corporation, catalog number WG-〇07; 2 cm in diameter, filtered in advance with 2 0 111 M Na 2 1 0 111M sodium citrate buffer solution (out 5.4)) The height is 14 cm). After the addition is complete, a portion of Pl (64.4 ml) of the dissociated Pl (64.4 ml) containing 400 Mltt NaC1 20 μαM sodium citrate buffer solution (out 5.4) and propofol is dissolved. , Protein concentration of 0,01 · 78 mg / ml 'total protein mass 1. 15 mg &gt; relative activity 1 7220). Second &gt; the eluent was changed to 20 mM sodium citrate containing 0.4M GlcNac, 10 ¾ propanol Buffer (pH 6.1) &gt; Collect the dissolved fraction F2 (45 ml, protein concentration 0.0104¾ g / ml, total protein mass 0.47 mg, relative activity 6 7 5 0 0 0). [0 3 4 1】 Therefore, after adding the final concentration of about 0.005% TFA to the main TPO active part "2 (340 liters) in the LA-WGA column, YMOPack CN-AP (manufactured by YMC, catalog number AP-513; diameter 6 mm, filter mat height 250 mm) unfolded. That is, using 1-propanine containing developing solvent A (containing 0. 15Ϊ TF A) and concentrated solvent B (containing 0,05¾ TFA) at a flow rate of 0.6 ml / dispensing person K 1 5% Β equilibrated Y MC-P ack CM-AP Column. After the injection was completed, the propanol concentration was increased from 15¾ B to 25¾ B, and then developed with a linear concentration gradient of 25¾ B to 50¾ B for 65 minutes, and each 1.5 ml (2.5 minutes) was collected in a polypropylene tube. [0342] Separately take 0.5 microliters (chromatographic fraction of 1/3000), add HSA, concentrate by ultrafiltration, and use 0. 05% HSA as the final ♦ 25 ml IMDM culture medium -149 -This paper size is in accordance with the Chinese Standard (CNS) A4 (210X297 mm) (Please read the notes on the back before filling out this page} • Sang Ding _ Printed by the Central Consumers Bureau of the Ministry of Economic Affairs 498079 A7 _B7 _ V. Description of the invention (U7) solution, this is determined, the TP0 active part is specified by adding K. This result is due to tube number 24 ~ 30 (in the propanol concentration of 35. 0 ~ 42 · 0¾% range ) Is there a strong activity? 〇 Activity (relative activity is about 6 3 0 0 0 ~ 48 00000), as the active part of Ding P0 (13 · 5 ml) ° [0343] So again Μ YMC-Rack CN- The FA obtained by AP is: Capcell Pak Cl 3 0 0 A (manufactured by Shiseido, catalog number Cl TYPE) using 1 M acetone containing solvent (including 0.1% TFA) and 1-acetone containing developing solvent B (including 0.05 ίκ TFA). : SG 3 0 0 A; diameter 4 · 6mm, minus 150mm height plus 4.6mm diameter in succession, before «pad height 3.5mm The column is expanded. That is, a part of F A obtained from YMC-Pack CN ~ AP is taken (8 ♦ 9 ml) &gt; After 0.3 ml of glycerol is added, it is concentrated by centrifugation and evaporation ' Add approximately 2.5 ml of 10% B solution &gt; K 0.4 ml / min into the M 20¾ B equilibrated Capcell Pak Cl 3 0 0 A column. After injection, M 20¾ B for 5 minutes After dissolution, a linear concentration gradient of κ 20% Β to 40% Β 50 was spread out, and 1 ml (2.5 points) of each was collected in a polypropylene tube. [0344] Take 1 microliter (1 1 / 200th percentile) HSA was added, concentrated by ultrafiltration, and finally prepared with 0.05% [^ 4 of .0.25ml of 1 ^ {〇 ^ {Measurement of the culture solution, this was measured, The active part of TP 0 was specified. As a result, strong TPO activity (relative activity of about 3000 000 to 225 0 00 00) was obtained in tube numbers 20 to 23 (in the range of propanol concentration 28.5 to 32.5%). [0345] -1 5 0-This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) -------- · Package ------ Order ----- line (please read the back first Please pay attention to this page, please fill in this page) Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7 V. Description of the invention (148) &lt; Example 37 &gt; The molecular weight of human TP0 in COSlffl cell was measured in C (3 S The molecular weight of human full-length TP 0 (derived from epidermal pH TF 1, F1 pure line) of 1 cell epidermis, and the addition of sugar chains are considered to be larger than the molecular weight estimated from κ obtained from the size of the peptide chain. Therefore, first, from a culture medium containing human COS1 female cells and epithelial cells, a fully grown TPO (from epidermal plastids ρ H TF 1, F 1 pure) culture supernatant, the purified TPO part was prepared as follows. That is, using a developing solvent / U. 1¾ trifluoroacetic acid (TFA)) and a developing solvent B (containing 1-propanol of 0.055 TFA), the culture supernatant was 0.3 liters K 0 · 4 ml / The flow rate was divided into a YMC-Pack PROTEIN-RP (YMC company, catalog number A-PRRP- 3 3-46 -25; diameter 0.46 cm, pad height 15 cm) equilibrated at 25¾ B in advance. After the injection was completed, after 20 minutes of dissolution at 20¾ B, a linear concentration gradient of K 20¾ B to 40% B was developed at 50 minutes, and 1 ml (2.5 points) of each was collected in a polypropylene tube. [0344] Take 1 microliter (chromatographic fraction of 1/1000) and add HSA 'ultrafiltration to concentrate, and finally make 0.25 ml of IMSA measurement culture solution containing 0.055 of HSA, and make this measurement. The TP0 active part is specified. This result is' in the tube number 20 ~ 23 (in the range of propanol concentration 28. 5 ~ 32 · 5¾), it can be obtained that shows strong TP0 activity (relative activity is about 3000 00 0 ~ 2500000) High activity control standard. [0345] &lt; Example 37 &gt; Molecular weight measurement of human TP0 expressed by COS1 cells -151- The size of this paper applies to China National Standard (CNS) A4 (210X297 mm) --- ------ φ--install ------ order ----- line (please read the precautions on the back before filling this page) 498079 Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7_F 、 Explanation of the invention (U9) The molecular weight of human full-length TPO (derived from epidermal jHTFl, P 1 pure line) in K C0S1 cells, with sugar chains attached As a result, it is considered to be larger than the molecular weight estimated from the size of the peptide chain. Therefore, firstly, the culture supernatant of humans that express KC 0 S 1-containing cells completely long TP0 (derived from epidermal plastid pHTF1, P1 pure line) was cultured. The partially purified TP 0 portion was prepared as follows. That is, a developing solvent A (containing 0 · 1¾ trifluoroacetic acid (TFA)) and a developing solvent B (containing 0 · 05% TPA) · -propanol were used in advance. 25% B Balanced Υ Μ Ο Pack PR 0 TEIN ~ RP (YMC, catalog number A-PRRP-3 3-4 6-2 5; diameter 0.4 6 cm, filter pad height 15 cm) tube The column was filled with culture supernatant 0.3 ml, K flow rate 0.4 ml / min, 25% Β was passed for 5 minutes, and linear concentration gradient chromatography from 25¾ Β to 50% Β was performed for 50 minutes at KK. TP 0 activity Dissolve at 3 4 · 5% ~ 4 3 · 5 (named 1-propanol range), evaporate this by centrifugation and dry to obtain a partially refined control standard. [0346] Next, from the After reducing the gel of SDS-PAG E and extracting the protein, the K M-07e assay strip was used to investigate the activity of TPO, or by the Western analysis described in Example 45. , Was moved to the original processing of D P C I [Determination of the molecular weight marker promoter. As a result, TPO activity exists in a wide range of apparent molecular weight of about 69,000 to 94000, and heterogeneity of molecular weight can be confirmed. In the same way, we investigated the DPCH markers treated with exogenous TP0 by the H-linked sugar chain cut of N-glycanase (manufactured by Jansheim, catalog number 1 472-00). Depending on the molecular weight, it is 36 000 ~ 4 0 0 0, which is larger than the estimated molecular weight of about 3 5 0 0 from the size of the peptide chain, which strongly suggests that it also contains a 0-linked sugar chain. -152- This paper size is in Chinese National Standard (CNS) Α4 size (210 × 297 mm) (Please read the precautions on the back before filling this page)-Packing ·

, TT line 498079 A7 B7 V. Description of the invention (Qiao 〇) [0347] Same as S @, when the apparent molecular weight of the CpS1 cell epitope τρ〇 (from epiphyseal II ρΗΤΡί, P1 pure line) is investigated, [0348] &lt; Example 38 &gt; Biological characteristics of human TP0 A culture supernatant containing TP0 activity mainly obtained by transfecting epidermal pHTF1 to COS1 cells was used. Liquid to investigate the effects of human TP0 on human and rat blood cells. [0349] Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) to use the CD 3 4 +, DR 'cell fraction community test system prepared from human cord blood A deliberate number of megakaryocyte communities have been formed from human TPO. For example, under the condition of adding 10% of epithelial COS1 cell culture supernatant after transfection of epidermal pH Τ1-231, the average formation of CD 3 4 + and DR + cells from 1 0 is 1 1.5 megamegasphere community. From the white blood cell portion obtained by using the Pic 〇 丨 1- Paque specific gravity centrifugation method from human to remove blood, remove the plastic adherent cells, and then add the cells with affinity to SBA (soy lectin) to make AIS Micro selector-CD 34 (Asahi Kasei Co., Ltd.) Pan Lin (eight ° &gt; &gt; method removed, and finally AIS micro selector-CD34 (Asahi Kasei Co., Ltd.) Pan Lin method was used to obtain CD 34+ cells In this part of the cell, human TP0 was added (transfected with epitoplasts PHTF1 to make the cultured supernatant of COS1 cells in epitope), and Gp I b / HI a + The selective proliferation of cells, and here G p I [b / M a + cells, the Ploidy of the nucleus (Ploidy) increased. Since then, it is strongly suggested that human TP0 pair -153-this paper size applies to the Chinese country Standard (CNS) A4 (210X297 mm) 498079 A7 __ —_B7 V. Description of the invention (151) Human megakaryocytes precursor cells act specifically to promote their proliferation and differentiation. 0 [0 3 50] Μ Uses obtained from G pb / jj a + cell fraction and G isolated from rat bone marrow cells The community determination of the plastic non-adherent cell part at the previous stage of p I b / I a + cell part was performed at the time of detection. An intentional number of megakaryocyte communities were formed from human T p 0. For example, 20% of Transfection of plastid ρ η TF 1 under the conditions of the culture supernatant of C 0 S 1 cells of epidermis, on the 5th day of culture, an average of 3 GP I b / HI cells were formed on the 3rd day. 4.5. 5 megakaryospheres from the 20000 plastic non-adherent female cells formed an average of 28.5 meganuclei communities. Moreover, megakaryocytes exist in the plastic non-adherent cell part. Various lines other than K are precursors to female cells, but the addition of human τρο only forms a megakaryocyte community, which strongly suggests that human TP 〇 has a specific effect on megakaryocyte precursor cells. Also, it will be derived from rat bone marrow. G p it b / melon cell portion in the presence of human τ P 0 (transfection of plastids P Η TF 1 to the surface of C 0 S 1 Nie cell culture supernatant added 20%), liquid culture 3-5 days, when investigating the nuclear polyploidy (PU i dy), the polyploidy obviously increased on the fifth day of cultivation. [0351] Ministry of Economic Affairs Printed by the Consumer Bureau of Standards Bureau (please read the notes on the back before filling out this page) &lt; Example 3 9 &gt; Glutathione-S-transferase (GST) and human TP0 (amino acid W74) Fusion protein (hereinafter referred to as the fusion protein "GST-TP0 (1-174)", the structure of the vector for expression of coliform bacteria. In order to facilitate the expression of human TP 0 in coliform bacteria, a human-154- Applicable to China National Standard (CNS) A4 specification (210X297 mm) Printed by the Consumers' Cooperative of the China Standards Bureau of the Ministry of Economic Affairs 498079 A7 ___B7 _ V. Description of the invention (152) TP 0 The artificial gene that contains the codons of the coliform priority. The D N A base sequence has an amino terminal methionine codon (ATC) at the 1 position for the start of translation of coliform bacteria. [0352] The synthetic nucleotides 1-12 shown in Table 12 were prepared, and a synthetic oligonucleotide of 2- 1 1 was added to 1 ATP and 1 0 by T4 kinase (manufactured by Farumasia Corporation). Phosphorylation in a solution of ra M T ris-acetate, 10 in M magnesium acetate, 50 ro M potassium acetate. In order to combine these synthetic oligonucleotides into six strands of DNA, 1 and 2; 3 and 4; 5 and 6; 7 and 3; 9 and 10; 11 and 12 respectively One strand of DNA was bonded in a solution of 10 μM Tris / HCl (7.5 μL), 10 in M M CU, and 50 m N Na C 1. Secondly, the two strands of the three sisters of 1 and 2; 3 and 4; 5 and 6 and the two strands of three of 7 and 8; 9 and 10 11 and 12 were used T4 ligase (Life Technology (Manufactured by the company), and the two reaction solutions were similarly treated with T4 ligase reaction. The DNA obtained by this ligation reaction was digested with BamHI (manufactured by Brigham Mannham Company), and electrophoresed on a 2% agarose gel to recover a fragment of about 390-400 bp in size. Lieb-A-refined DNA purification kit refined, sub-selected in M Xbal ·, BamHI 潸 PUC18 (host uses E. c ο 1 i D Η 5 cx). The obtained pure line: In the analysis of the base sequence, a pure line having an artificial gene encoding the human TP 0 shown in Table 13 and containing the coliform preferential codon was selected. This is PUC 1 8 (X Β) ( 1-1 2 3). The D N A sequence of the coding chain in Table 13 represents a sequence listing (sequence number 9). [0353] [Table 12] ~ 1 5 5-This paper A degree is applicable to the Chinese National Standard (CNS) M size (210 ^ 297 mm) (Please read the precautions on the back before filling this page). Assembly and thread 498079 Printed by A7 __B7_ of the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the invention (155) [0354] [Table 13] Synthesized from the normal human liver (A) + 1 microgram of RNA, using oligo dT primers as primers For the first strand of c DNA, PCR was performed with the 0.1 volume of the c DNA synthesis reaction solution as a horizontal plate. The primers were reacted using hTP〇-C and hTPO-B (EcoRI), K 100 microliters (96 t: 2 minutes, after 9 5 t) 1 minute / 5 8 υ 1 minute / 72 ° C 1 minute The reaction was performed for 30 cycles, and then at 7: TC for 7 minutes). The human TP 0 c DNA fragment M Ba roH I thus obtained was digested with EcoRI, and then agarose gel electrophoresis of M 2¾ was used. A fragment of about 600 b ρ size was recovered, and pleub-A-Sperm 1) Ν Α refined kit sister refined, sub-selected in K EcoRI, BamHI digested pUC18 (the host uses E.col i D Η 5 α). A pure line encoding a human TP 0 base sequence was selected by analyzing the base sequence as Pl) C 1 8 (Β Ε) (1 2 4-3 3 2). The sequences of the PCR primers used herein are as follows. [0355] hTP0-C: 5! -GGAGGAGACCAAGGCACAGGA'V (pHTFI Pure 329-349) hTP〇-Z (EcoRI): CCGGA ATTCTTACCCTTCCTGAGACAGA TT-3 '(corresponding to 11 4 3 of PH TF 1 pure 糸-11 6 3 on the other hand should attach E co RI sequence). [0356] After digesting pUC18 (BE) (124-332) with BamHI and EcoRI, electrophoresis on an agarose gel with 2¾ and recovering humans with a size of approximately 600 bp ) • Binding and binding b—line-156- This paper size is applicable to the Chinese National Standard (CNS) grid (210X297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 _ _B7 _ V. Description of the invention (彳 54) The C-terminal side fragment of TPOcDNA was purified with plebe DNA refined kits and sub-selected from PUC18 (XB) (W23) digested with EcoRI and BaroHI (E, coli DH5a was used as the host).糸 is p UC 1 8 (XE) (Bu 3 3 2). [0357] Preparation of various deletion structures on the C-terminal side of human TP 0 c DNA to facilitate the implementation of the in vitro measurement of human TPO activity using its surface For example, since it is apparent that human TPO amino acid 163 retains human TPO activity, the epitope vector containing this peptide fragment is constructed. Here, the epitope of GST-TP0 (1-174) is performed. [0358] Equivalent The base sequence of PHTF1 pure line 68 1 -68 6 (amino acid 1 73-1 74) is due to the restriction enzyme Sa c I recognition, using this restriction enzyme recognition site, the stop codon was introduced by two synthetic oligonucleotides. Specifically, the two synthetic oligonucleotides SSE1 and SSE2 were put in 10M Tr is / HCl (pH 7. 5.), 10 mM MgCU, 50 iuM NaCl for binding, the two strands of DNA obtained here were digested with SacI, EcoRI, and about 480 bp of pUC18 (XE) at the C-terminal side of human TPOcDNA was removed (Bu 332 ) Was introduced using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.) to obtain pure 糸 PUC18 (XS) (1-174) (the host used E. C 〇1 i D Η 5 α). The synthetic oligonucleosides used here The sequence of the acid is shown in Table 1. [0359] [Table 1 4] After MXbal and EcoRI digested pl) C18 (XS) (Bu 174), M 2¾-157-This paper is for Chinese country (CNS) Α4 specification (210X297 mm) an ·· —, —-m ml I 111 Βϋ · Μ— mu m (Please read the precautions on the back before filling this page) Line 498079 Staff consumption of the Central Standards Bureau of the Ministry of Economic Affairs Cooperative printed A7 _B7___ V. Description of the invention (155) Lipose gel gel swimming, recovering the size of about 60 0 bp encoding human TP 0 amino acid 1-1 7 4 Section &gt; Preble-A-refined DNA Refined Kit Refined 'Sub-selected in KX ba I, E c 〇RI Digested p B 1 uescript Π SK + (Tara-Dajing (X 卜) 7 (Manufactured by the company) (E · col i DH5 a was used as the host), and the pure fluorene obtained therefrom was PBL (XS) (1-1 7 4). After the same, the PBL (XS) (1-1 7 4) Μ X ba I, Η nd nd II was digested to recover the fragment of about 5 50 bp encoding human TPO amino acid 174. 'Puleb-A-refined DNA purification kit refined, sub-selected colonies in KX ba I, Hind I [digestive PCFM 5 36 (European Patent Application No. 1 36 490 specification) (host makes E. coli DH5ct) OM pure obtained here: pCPM5 36 / hT (1-1 7 4). [0360] In order to express GST-TP0 (1-174) by using PGEX-2T (manufactured by Farumasia Corporation) as a carrier protein for fusion protein with glutathione transferase (GST), proceed as follows. M p CF Μ 5 3 6 / h T (1-1 7 4) is a horizontal plate, and PCR is performed with two PCR primers-GEX1 and GEX3 (96t) 2 minutes later> 95 t] 1 minute / 4 1 t : 1 minute / 72 1 minute reaction 22 cycles "and 72 ° C for 7 minutes). The resulting human-encoded TP0 fragment was digested with K Nael and EcoRI and electrophoresed on a 2% agarose gel to recover a 550 bp fragment, which was purified by K-Pleb-A-refined DNA kit, Select PGEX-2T cloned from KEcoRI and SmaI (host uses E. coli i) Η 5), and analyze the base sequence to select the correct pure PGEX-2T / hT encoding human TP 0 base sequence (l-174), which is used as a transformation for the expression of GST-TP0 (1-174). The primer sequences for PCR used here are as follows (please read the precautions on the back before filling in this page). Binding-158- This paper size is applicable to China National Standard (CNS) A4B (210X297 mm) Central Standard of the Ministry of Economic Affairs Bureau Consumer Consumption Cooperative Mark 498079 A7 B7 V. Invention Description (156) [0361] GEX1: 5, ATCGCCGGCTCCGCCAGCTTGTGAC'V (with Nael sequence attached to serial number 10-21-39) GEX3: 51- GCCGAATTCTCATTAGAGCTCGTTCAGTGT-3f (serial number 1 0 of 5 2 3-5 4 9). This epitope is connected to a GST protein, which contains a numbered thrombin recognition sequence and a sequence of human TIP (amino acid 1-174). The sequence encoding the thrombin recognition sequence and human TP 0 (amino acid 174) is shown in the sequence table (sequence number 10) ° [0363] &lt; Example_0 4 &gt; GST-TPO (W74) in E. coli Performance The transformed strain obtained in Example 39 was cultured in 60 ml of L Β medium containing 50 μg / ml of ampicillin, and cultured overnight with shaking at 37, and 25 ml of this culture solution was added to Picillin was 50 μg / μL of 1000 mL of L B medium, and cultured at 37 1 with shaking to 0D / U. . Until 0.7 ~ 0.8. IPTG was then added to bring the final concentration to 0 · 1, followed by shaking culture for 3 hours to induce the expression of GST-TP0 (1-174). <Example 4 1 &gt; Purification and activity confirmation of GST-TP0 (1-174) from a pure strip pGEX-2T / hT (1-174) encoding human TPO base sequence expressed as coliform-159 -This paper size is applicable to China National Standard (CNS) A4 (210X297 cm) (Please read the precautions on the back before filling this page)

498079 A7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs _____B7_ V. Description of the invention (157) In the GST-TP0 (pure GEX-2 T / h T (1 -174) from the pure line encoding human τP 0 base sequence ( 174) Production of 5.9 g of frozen bacteria was added to 10 ml of water to suspend, and the bacteria were broken by a high-pressure crusher. GST-TP0 (1-174) was recovered by centrifugation to remove most of the protein and bacteria components mixed in it. Next, add 5 ml of water to the precipitate containing the recovered G s T-τ p 〇-17 4) to suspend, then add 丄 T Trisyuan flushing solution while stirring to produce 8.56 ml, 10 mM 120 ml of urea and 16 ml of water. After stirring at room temperature for 5 minutes to dissolve, the solution was divided into 4 equal portions, and each of the following four operations (1) to (4) was performed. [0365] (1) KK 20 mM Tris buffer solution 8.5 diluted 10-fold. The prototype glutathione and oxidized glutathione were added thereto to the final concentration of 5 niM and 0.5 μm, respectively, and allowed to stand overnight at 4: C. By centrifugation, G S T-T P 0 (1-1 7 4) was recovered from the supernatant. Here, it was diluted 2 times with 5.5 μm of 20 μM sodium citrate sodium diluent, and adjusted to 5.5 with acetic acid. GST-TPO (1-174) was adsorbed on K 2 0 in Μ sodium citrate buffer to produce 5.5-equilibrium SPS epha "〇se F ast Flow (manufactured by Biochem Technology Co., Ltd., Catalog No. 17 — 0729) -01) on a cation exchange column. The resin K 20 mM sodium citrate is washed out of the liquid 5 · 5, and then washed with 20 mM sodium citrate buffer 含 5 · 5 containing 500 M sodium chloride. GST-TP 0 (1-1 7 4) dissolve. Add 2.6 ml of 1 M Tris buffer to 8.5 to 2.9 ml of the eluent, add 8.5 to 8.1, and add to glutathione. Peptide agarose 4B (manufactured by Farumasia Biosciences, catalog number 17-0 7 5 6 -01) in a column to adsorb GST-TP0U-174), after washing with MPBS &gt; 1 ^ Prototype glutathione 20mM Tris buffer solution (please read the precautions on the back before filling this page) -pack, tr _line-160- This paper size applies to China National Standard (CNS) A4 specification (210X297 Mm) 498079

8.5 Dissolve GST ~ TP0 {P174). Thrombin 37 NIH® was added to the eluate. After standing at room temperature for 4 hours, it was diluted with PBS 0 times and added to a glutathione cyclolipose 4 Β column, so that the adsorption was cut off GS T, and non-adsorption Partially recovered TP 0 (Bu 174). Dilute the recovered non-adsorbed K20 iuM sodium citrate buffer solution 3,5 times and add it to the balance of the solution: SPS epha "〇se F ast P 1 〇w cation exchange column, The buffer solution was used for dissolution according to a linear concentration gradient method of sodium chloride concentration from 0M to 500 mM. [0366] (2) The operation of (1) was all performed in 0.1% polyoxyethylene sorbitan ester [0367] (3) Dilute 8.5 times with 20 μM Tris solution to dilute 10 times. Here, add the prototype glutathione and oxidized glutathione to the final concentration of 5 respectively. mM and 0.5 m Μ, left to stand overnight at 4 T). By centrifugation, 0 3? -1 '卩 0 (1-174) was recovered from the supernatant, and here 20—citrate Nayuan rinse After diluting 5.5 times, adjust the pH to 5.5 with acetic acid to make GST-TP0 (1-174) adsorbed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs --------- ^ w— ^! ( Please read the precautions on the back before filling this page.) For the thread, use 2011 ^ sodium citrate solution to produce 5.5 balanced 5? 309113 ″ 〇3 6 ″ 33 seven Flow cation exchange resin. The resin M 20 raM lemon Soda The pH of the washing solution was 5, 5, and the solution was dissolved in 20 mM sodium citrate containing 500 μm sodium chloride to release 5.5 to dissolve 031 ^ 0 (1-174). Add 1> to the dissolution solution. 3 Buffer solution 8.5 to make about 8, add Thrombin 320 HI Η unit and let stand in the room for 4 hours, then dilute 5 times by κ 20 mM sodium citrate buffer pH 5.5, add to the buffer with the buffer SP Sepharose Fast Flow Cation Exchange Columns' With a linear concentration ladder of sodium chloride concentration from 0M to 50 0 mM in this buffer 161 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 _______ __B7__ 5. Description of the invention (159) Degree of dissolution. [0368] (4) All operations of (3) are performed in the presence of 0.1% polyoxyethylene sorbitan ester 80. [0369] (1) ~ (4 The SP Sepharose Past Flow cation exchange calorimetry obtained from the operation) was dissolved in sodium chloride at a concentration of about 200 mM to 400 ηM, and the IMDM culture medium was fully dialyzed. W rat CFU-MK: the result of evaluation, TP0 activity was identified as capacity-dependent. Results of SDS-PAGE analysis of the fraction in the presence of the original agent For this purpose the main portion of the tape detection region of one protein, molecules move: 19 K channel zone of ear Throughput (purity BU 20¾). With regard to this band, the method described in Example 1, after SDS-PAGE, was transcribed on a PVDF membrane, and the results of N-terminal sequence analysis were performed. As a fusion protein from this pGEX-2T / hT (1-1 7 4), Confirmation contains TP 0 sequence that can be represented. [0370] <Example 4 2 &gt; An epitope carrier for coliform of human TPO (amino acid 163) converted to amino acid 1 (Ala), amino acid 3 (Ala-Va) After digestion of PUC18 (XE) (Bu332) M Xbal _, EcoRI produced in Example 39, K 2% agarose gel electrophoresis was performed to recover about 1 0 0 encoding human TP 0 amino acid 1-3 3 2 0 b P size fragments, refined with plebe ~ A-refined DNA refining kit, sub-selected to colonize biuescrip digested with X ba I _, E c 〇RI .tjiang SK + (manufactured by Stella Rada ) (Host uses E ♦ co 1 i DH5 α). The pure 糸 obtained here is p BL (X Ε) (1-3 3 2) ° -162- Shi Congji to do Perthian oa containing from A / guang vrc, a &gt; sister Cong ,, 1 Λ ν ιοί Eight arrived, --------- install-(Please read the precautions on the back before filling this page) Order 1 498079 Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (16) [0 37 1] Second, the B a mH I recognition sequence from the pure PBL (XE) (1-32) is changed to the amino acid 16 (36-6-489) and the coliform is preferred. a. The synthetic oligonucleotides 13-20 shown in the table were prepared, and the synthetic oligonucleotides of 13 and 14; 15 and 16; 17 and 18 were used in the same tube, and T4 kinase (manufactured by Farumasia Corporation) was used. ) Phosphorylated in a solution of 0.1raMATP, 10mM ^ &gt; is-acetate, 10! UM magnesium acetate, 50mM potassium acetate. Add 1 / 10th volume of a solution of 100mM TrMs / HC1 (pH 7.5), 100mM MgCU, and 500 μM NaCl, and boil in a water bath for 3 minutes, and then place to form a double-stranded D fU. Next, the three sets of double-stranded DNAs of 13 and 1 4; 15 and 16; 17 and 18 were ligated using a DNA ligation kit (made by the channel brewing company), using this as a template, K 19 and 20 The synthetic oligonucleotide was used as a primer for PCR reaction. After the PCR products K BaniH I and H d thus obtained were lysed, K 2¾. Agarose gel electrophoresis was performed, and a fragment of 130 b p was recovered by using a pleib-A- refined D N A purification kit. This was digested with B a m Η I and Η d Μ, and sub-selected for p B L (X E) (Bu 3 3 2) (E · c ο 1 i D Η 5 was used as a host). The obtained pure: the selected sequence has the base sequence shown in Table 15 and M is p B L (X Η) (1-1 6 3). [0372] [Table 1 5] Furthermore, in order to increase the amount of human TPO (amino acid 163) and prevent K-terminal protease from decomposing, the purpose is to make the human TPO (amino acid [163] The amino acid at position 1 converts Ser—AU, the amino acid at position 3—Ala—Val, and then converts the 1 position to Lys and the -2 position to Met: a variant human TP 0 (amino acid 1 · &Quot; 1 6 3) (κ will refer to such protein as-163- this paper;? Jl 逋 use Chinese National Standard (CNS) A4 specification (210X297)) --------- ( (Please read the notes on the back before filling out this page)

498079 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7 V. Description of invention (161) "h 6 T (1-1 6 3)" The performance of the carrier of the carrier. The four types of synthetic oligonucleotides shown in the table were prepared, and the synthetic oligonucleotides of 2-9 and 3-3 were subjected to T4 kinase (manufactured by Falumasia Corporation) in lmM ATP, 10mM TrMs-acetate, Phosphorylation in a solution of 10 mm magnesium acetate and 50 mM potassium acetate. In order to turn these synthetic oligonucleotides into two double-stranded DNAs, combine single-stranded DNA of 1-9 and 2-9; 3-3 and 4-3 respectively in 10mM Tris / HCl (pH 7.5), 10raM MsCU, 50mM NaCl solution. Next, the double-stranded DNA of 1-9 and 2-9; 3-3 and 4-3 of the second sister were ligated with DNA (made by Takara Shuzo Co., Ltd.). The DNA obtained by this ligation reaction was sub-selected into p BL (X Η) digested by KX ba I and N ru I (Bu 16 3). Based on the analysis of the base sequence, synthesis was performed from Table 16 Oligonucleotides were selected as the pure 糸 (the host used ε · c ο 1 i D Η 5 α), which was ρ BL (X Η) Η 6 Τ (1-1 6 3). [0373] [Table 16] After digesting p BL (X X) h 6 T (1-1 6 3) with Xba I and Hind El, about 50% of the mutant human TP0 amino acid 163 was recovered and encoded. Η Η Η ，, Μ 普里布 -A-精 D Ν Α refined repertoire refined, selected from κ X ba I, Hind digestion 0 [: [^ 5 36 (European Patent Application No. 1 3649 (Instruction No. 0) (KpMWI UTCC No. 39933 pre-transformed E. coli JM109 was used as the host). The pure gallium obtained here is pCMF5 36 / h6TU-163), and the coliform strain with this epitope is a mutant human TPO, that is, a transformant for h6T (1-1 63) expression. This expression plastid contains the D N A sequence shown in the sequence listing (sequence number Π). [0374] (Please read the precautions on the back before filling this page) Binding-Line-164- This paper size applies to China National Standard (CNS) A4 ^ i (210X297 mm) Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Preparation 498079 A7 B7 V. Description of the invention (1S2) &lt; Example 4 3 &gt; h 6 T (1 6 3) is expressed in the table of coliform 琨 The expression of plastid PCFM536 is controlled by the XPL activation gene, which is Under the control of I 8 5 7 repressor gene. The transformed strain obtained in <Example 4 2 &gt; was cultured in 30 ml of 60 ml of LB medium containing 50 μg / ml of ampicillin and 12.5 μg / ml of tetracycline at 30 t! With shaking overnight. 25 milliliters of this culture solution was added to 1 000 liters of LB medium containing 50 μg / ml of apicillin, and shaken at 30 ° C. to 0D to Ae000.1-1.2. Next, the final temperature was set to 42T], and about 3 30 ml of LB medium at 65 ° C was added, followed by shaking and culture at 42 ° C for 3 hours to induce the epithelium of h 6 (1-16 3). [0375] &lt; Trade Example 4 4 &gt; The purification and activity of h6T (l-163), which is pure human P0 base sequence encoding the E. coli epitope from PTO (: PM536 / h6TU-163), was confirmed at h 6 T (1- 1 6 3) produces heavy cells and freezes the cells. 3.6 grams of water is added to 10 ¾ liters to suspend the cells &gt; By centrifugation, the precipitate is recovered to remove most of the protein and bacteria components mixed in it. After adding 7 ml of water to the precipitate containing the recovered h 6 T (W 6 3) and suspending, add 1 M Tris buffer while stirring to produce 8. 5 3 ml, and then add urea (final concentration 8M), hydrochloric acid Guanidine (final concentration 6M), N-lauryl sarcosinate (final concentration 2%), stirring at room temperature for 5-20 minutes to help dissolve. This K 20 mM is &gt; is buffer pH 8.5 was diluted 10 times, and the protein was rolled back (refolded) in 4 overnight. At this time, in addition to air oxidation, glutathione and copper sulfate were added as additives, and the h6T (-165-) in the supernatant was recovered by centrifugation. This paper size applies to China National Standard (CNS) A4 (210X297).嫠) --------- Installation ------ Order ----- line (please read the precautions on the back before filling this page) 498079 Printed by the Consumer Standards Cooperative of the Central Standard Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (165) 1-1 6 3). The results of s DS-PAGE analysis of each recovered part in the presence of the regenerant 'can be detected in any method as the main protein band of this part with a molecular weight of about 18K channels (purity 30) -40¾). About this band, according to the method described in Example 1, after SDS-PGAE, it was transcribed on a PVDF membrane, and the result of the end sequence analysis was performed.> From this p CF Μ 5 3 6 / h 6 T (1-1 6 3) The variant τρ 〇 can be confirmed to contain the TP 0 sequence that can be expressed. After each part was fully dialyzed against the IMDM culture medium, K rat CFU-MK: the result of the evaluation 'was determined as a volume-dependent TP 0 activity in all parts. [0376] &lt; Example 4 5 &gt; Production of anti-TPO peptide antibody and detection of TPO by SDS-PAGE-Weiss analysis If an anti-TPO antibody can be produced, it can be detected by immunological methods TPO protein. Therefore, among the amino acid sequences of the rat TPO originally identified, 'selection was considered to be more suitable as the three regions of the antigen (shown in Table 17), by Tara (Proc. Natl. Acad. Sci. USA, 85, 5 4 0 9-5 4 1 3, 1 988) method to synthesize 4-chain multi-antigen peptide (MAP) type peptides, each 100 micrograms, continued 8 times, respectively in 2 groups of rabbits As a result, these antisera can be obtained. [0377] Contains an amine sequence in a synthetic cystamine [0378] [Table 17] Next, in these antisera, first the anti-RT1 peptide and anti-RT2 peptide antibodies-1 6 6-this paper is applicable China National Standard (CNS) A4 specification (210 X 297 mm) --------- install-(Please read the precautions on the back before filling out this page) Order-line 498079 Employees of the Central Standards Bureau of the Ministry of Economic Affairs Printed by Consumer Cooperative Association A7 B7 V. Description of Invention (164), using protein A column (PR 0 SEP-A: BU pr 〇cessing company, catalog number 8 4 2 7), get I g G part (respectively 2 3 4 4 mg; [9 2 0 mg), 54 mg of these were taken respectively, and 32 mg were made by coupling with active biotin (NHS-LOBiotin II, manufactured by PIERCE, catalog number 21336) Into. As in the method described in Example 1, the control standard containing recombinant TP 0 was run by SDS-PAGE, followed by PVDF, or electroblQtting on a nitrocellulose membrane. These biochemical antibodies were used as primary antibodies for Wei-Yang analysis. That is, the film after the ink dot method was washed with 20 mM Tr is-HC 1 _, 0.5 m N a C 1 (pH 7, 5) (TBS) for 5 minutes, and M plus 0.1¾ Tween. TBS (TTBS) was washed twice for 5 minutes, and then treated with a blocking agent (B 1 ο Ace, manufactured by Dainippon Pharmaceutical Co., Ltd., catalog number uk-B 2 3) for 60 minutes. Secondly, after treatment with a TTBS solution containing a concentration of 10 micrograms / ml of bioanti-TP 0 peptide antibody, 0.05: ¾ BSA, 10 $ B 丨 ck Ace for 60 minutes, K TTBS was washed for 5 minutes 2 times. Next, • Alkaline phosphatase-labeled avidin (manufactured by Leinco Technologies, catalog number Λ108) Μ containing 10¾ BlockAce, a solution of TTBS solution diluted 50000 times for 30 minutes, 5 times 2 times After washing with TTBS and TBS for 5 minutes, color development was performed with an alkaline phosphatase substrate (Cat. No. 1 Catalogue No. 170 ~ 6 4 3 2). The Wei's analysis on M was performed at room temperature. [0380] As a result, not only all kinds of heavy sister rats TP0 expressed in COS1 cells, but also various recombinant human TP0 expressed in COS1 cells and coliforms (specifically, '-167- This paper standard applies to China National Standards (CNS) Α4 specification (210 X297 mm) -------- install ------ order ----- 41 ^ line (please read the precautions on the back before filling this page) Central Bureau of Standards, Ministry of Economic Affairs Printed by the employee consumer cooperative 498079 A7 B7 V. Description of the invention (165) As described in the embodiments so far: () s 1 human cell epitope from human pT TP 1 or human TP of pTF TF 1 〇TP 0 _ after its N-linked sugar chain cleavage, expressed in coliform (: ST-TP0 (1-174) and its TP0 after thrombin dysfunction, is the variant TP 0 expressed in coliform h 6 T (1-1 6 3) can also be identified. Therefore, it can be used for the analysis of these various recombinant human TP0. [038 1] Same as the above method, suggesting that it is possible to make anti-human TP0-like antibodies. By K this Antibodies obtained by other methods, not only Weiss analysis, starting with TP0 purification through antibody columns, can be applied to the use is usually considered All of the immunological antibody method [0382] [Sequence Listing] SEQ ID NO: 1 - Sequence length: 261 Sequence type: nucleic acid Number of strand: double-stranded linear geometry ··

Sequence type: cDNA to mRNA Source · Rat (R a 11 us η 〇rvegicus) Cell line: McA-RH8994 Sequence GGTGTACCTG GGTCCIGAAG CCCITCTTCA CCIGGATAGA TTCCTTGGCC CACCTGTCCC GAGCCAGCCCAGGCCCAGGCCCAGGCCACGGGGACCCGGAGCCGGAGCCAGGG CTG ACT GAT TTG CTC CTG GTG GCC ATA CTT CTC CTC ACC GCA 220 -168-^ Paper money is applicable to China National Standards (CNS) Α4 · (21QX297) ------------- Order-- --- 41 ^ line (please read the precautions on the back before filling this page) 498079 Printed by the Consumers' Cooperative of the China Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Invention Description (166) Met Glu Leu Thr Asp Leu Leu Leu Val Ala lie Lou Lou Leu Thr Ala 1 5 10 15 AGA CTA ACT CTG TCC AGC CCA 〇TT CCT CCC OCC TQT OAC CC 281 Arg Leu Thr Leu Ser Ser Pro V &amp; l Pro Pro Ma Cya Asp 20 25 Sequence length: 2 Sequence length: 1 6 6 3 Sequence type: Number of nucleic acid strands: Double-stranded Geometry: Linear sequence Type: cDNA to mRNA Source: Rat (R a 11 us η 〇rvegic us). Cell line: McA-RH8994 * SEQUENCE: GGTGTACCTG GCTCCTGAAG CCCTTCTTCA CCTGGATACA TTCCTTGGCC CACCTGTCCC 60 CACCCCACTC TGTGCAGAGG TACAAAAGCT CAAGCCGTCT CCATGGCCCC CTC GTC CTC ATC GCCAGCC ACCC Met Glu Leu Thr Asp Leu Leu Leu Val Ala lie Leu Leu Leu Thr Ala -20 -15 -10 AGA CTA ACT CTG ICC AGC CCA GTT CCT CCC GCC TGT GAC CCC AGA CTC 268 Arg Leu Thr Leu Ser Ser Pro Val Pro Pro Ala Cys Asp Pro ArK Leu -5 1 5 l〇 (Please read the precautions on the back --- items before filling out this page), 1T -16 9- This paper size applies Chinese National Standard (CNS) Α4 specifications (210 × 297) Epidemic) 498079 A7 B7 V. Description of Invention (167) CTA AAT ^ AAA CTG CTT CGT GAC TCC TAC CTC CTT CAC AGC CGA CTG AGT 316 Leu Asn Lya Leu Leu Human rg Asp Ser Tyr Leu Leu His Ser Arg Leu Ser 15 20 25 CAG TGT CCT GAC GTC AAC CCT TTG TCT ATC CCT GTC CTG CTG CCT GCT 384 Gin Cys Pro Asp Val Asn Pro Leu Ser lie Pro Val Leu Leu Pro Ala 30 35 40 GTG GAG TTT AGC CTG GGA GAA TGG AAA ACC CAG ACG GAA CAG AGC AAG 412 Val Asp Phe Ser Leu Gly Glu Trp Ly a Thr Gin Thr Glu Gin So r Lys 50 66 GCA CAG GAC ATT CTA GGG GCA GTG TCC CTT CTA CTG GAG GGG GTG ATG 4Θ0 Α1λ Gin Asp lie Leu Gly Ala Val Ser Leu Leu Leu Glu Gly Val Met 80 65 70 75 GCA CCA CGA GGA CAGA TTG GAA CCC TCC TGC CTC TCA TCC CTC CTG GGA 508 Ala Ala Human rg Gly Gin Leu Glu Pro Ser Cys Leu Ser Ser Leu Leu Gly 80 85 90 CAG CTT TCI GGT CAG GTT CGC CTC CTC TTC GTG GGA CCC CAG GGC CTC .558 Gin Leu Ser Gly Gin Yal Arg Leu Leu Lou Gly Ala Leu Gin Gly Leu 95 100 105 CTA GGA ACC CAG GTA AGT ccc CAG ACC TAT kGk AAC TAC CCT CTT ACT 604 Leu Gly Thr Glh Val Ser Pro Gin Thr Tyr Arg Asn Tyr Pro Leu Thr 110 115 120 CAG TIC CTC 813 Gin Phe Leu 125 Economy Printed by the Ministry of Standards and Staff's Consumer Cooperatives (please read the notes on the back first) Complete this page) TAAGCACCTG GGAAAAGACA AGGGATTCTA GATTCTAGGT GTCTTCAGTG TATGAAAGCT 673 GGTCTATACG GAGTGATGCT TCTCAGCCAC AATACCTGGG TGCTCGCACT AAATCTTTCC 733 ACCTTAGTGA GAAGAGGCCT GATATGTGGG CCAACTCACT GGCCTCAGGC CCATCCTCTG 703 CCTTCAGCTI CCTCCACACG GCAGGACCAC AGCTCACAAG GACCCCAGTG CCCTCTTCTT 853 -170- This paper; O complex applicable Chinese National Standard (CNS) A4 size (210X297 Kimiyoshi) Ministry of economic Affairs Bureau of standards employees consumer cooperatives printed 498079 A7 _B7_ V. description of the invention (168) CAGCTTCCAA CAACTGCTTC GGGGAAAGGT GCGCTTCCTG CTGCTGGTAG AAGCTCCCGC 013 CCTCTGTGTC AGACGGACCC TTCCCACCAC AGCTGTCCCA AGCAGAACCT CTCAACTCCT 973 CACACTAAAC AAGTTCCCAA ACAGACTTCT GGATTGTTGG AGACGAACTT CAGTGTTGTA 1033 GCCAGAACTG CTGGCCCTGG ACTTCTGAAC AGGCTTCAAG GATTCAGAGC CAAGATTATT 1093 CCTGGTCAGC TAAATCAAAC CTCCGGGTCC TTACACCAAA TCCCTGGATA CCTGAACGGG 1153 ACACACGAAC CTGTGAATGG AACTCATGGG CTCTTTGCTG GGACCTCACT ACAGACCCTG 1213 GAAGCCCCG ACGTGCTCC AACAGGGTCC CCAC TGATGGAT ACACACTTTT CCCTCCTTCA 1333 CCTACCTTCC CCACCCCTGG GTCTCCACCC CAGCTCCCCC CCGTTTCCTG ACCCCTCCAC 1393 CACCATACCT AACTCTACCA ACCCTCATCC AGGACTTGGT CTCAGTAAGC GTCCCGTGCA 1453 CTGGCACGGA GCGCGATCGT CTGCAACATC TCTCAGGGGC AAGCTTCCTC AGGAAGGCTC 1513 TGAGGCACCT CACTACACAT CCTGCTCTCG CCTAACGGGC CCTGGGAAAC GGATACACAG 1573 GCCAGGACAC TGTACAACCT TAGGAGCGAT TTTTTTCTTA ACCTATCAAC AATATTCATC 1Θ33 AGAGC the aqueous UA (2 5 Salt-yl) 1Θ83 sequence Number: 3. Sequence length: 8 8 Sequence type: Nucleic acid · Number of chains: Double-stranded Geometry: Linear

Sequence type: cDNA to niRNA source · ♦ Synapsabine (Η ο m 〇 S a p i n s) Tissue type: liver Sequence: CIC GTG GTC ATG CTI CTC CTA ACT GCA AGG CTA ACG CTG TCC AGC CCG 48

Leu Val Val Met Leu Leu Leu Thr Ala Arg Leu Thr Lgu Sor Ser Pro 15 10 15 -171-This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) ---- ^ ---- -Install ------- Order ----- line (please read the notes on the back before filling out this page) Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 _ B7__ V. Description of Invention (169) GCT CCT CCT GCT TGT GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT GAC 96

Ala Pro Pro Ala Cya Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp 20 25 30 TCC CAT ^ GTC CTT CAC AGC AGA CTG AGC CAG TGC CCA GAG GTT CAC CCT 144

Ser His Val Leu His Ser Ar ^ Leu Ser Gin Cya Pro Giu Val His Pro 35 40 45 TTG CCT ACA CCT GTC CTG CTG CCT GCT GTG GAC TTT AGC TTG GGA GAA 192

Leu Pro Thr Pro Val Leu Leu Pro Ala VbI Asp Phe Ser Leu Giy Glu 50 6B 60 TCG ΑΑΛ ACC CAG ATG GAG GAG ACC AAG GCA CAG GAC ATT CTG GGA GCA 240

Trp Lys Thr Gin Met Glu Glu Thr Lys Ala G】 d Αλρ Leu Gly Ala 85 70 75 80 GTG ACC CTT CTG CTG GAG GGA GTG ATG GCA GCA CGG GGA CAA CTG GGA 288

Vai Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly 85 90 95 CCC ACT TGC CTC TCA TCC CXC CTO GGO CAC CTT TCT GGA CAG GTC CGT 336

Pro Thr Cys Leu Ser Ser Lau Leu Gly Gin Leu Ser Gly Gin Val Arg 100 106 110 CTC CTC CTT GGG GCC CTG CAG AGC CTC CTT GGA ACC CAG NNN NNN NNN 384

Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Giy Thr Gin Xaa Xaa Zaa 115 120 125 NNN NNN NHN NCC ACA GCT CAC AAG CAT CCC ΛΑΤ CCC ATC TTC CTG AGC 432

Xaa Xaa Xaa Xaa Ihr Ala His Lys Asp Pro Asn Ala lie Phe Leu'Ser l3〇 135 140 TTC CAA CAC CTG CTC CCA CGA AAG CTG CGT TTC CIG ATG CTT GIA GGA 480

Phe Gin His leu Leu Arg Gly Lys Ve.1 Arg Pho Leu Met Leu Vai Gly 146 150 155 160. -172- The A degree of this paper applies the Chinese National Standard (CNS) A4 specification (21〇X 297 mm) I-- ----- Install-(Please read the notes on the back before filling this page) Order 498079 A7 B7 V. Description of the invention (17〇) GGG TCC ACC CTC TGC GTC AGG CGG GCC CCA CCC ACC ACA GCT GTC CCC Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala Yal Pro 165 170 175 AGC AGA ACC TCT CTA GTC CTC ACA CTG AAC CAG CTC CCA AAC AGG ACT Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn Arg Thr 528 576 180 185 190 TCT G Ser 580 sequence number: 4 sequence length: 8 6 1 sequence type: number of nucleic acid strands: double-stranded geometry: linear sequence type: cDN A to mRNA source: homosabin (Η ο η ι o S apins) Tissue type: liver sequence 歹 u: I GGCCAGCCAG ACACCCCCCC CAGA ATG GAG CTG ACT GAA TTG CTC CTC GIG GTC ATG CTT CTC CTA ACT CCA Het Glu Leu Thr Glu Leu Leu Leu Val Yal Met Leu Leu Leu Thr Ala 24 72 I ------- Handle-out ------ Order ----- ϋ 线 (Please read first Note on the back, please fill in this page) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs -15 -10 AGC CTA ACG CTG TCC AGC CCG CCT CCT CCT GCT TGT GAC CTC CGA GTC Arg Leu Tbr Leu Ser Ser Pro Ala Pro Pro Ala Cya Asp Leu Arg Val -5 1 5 10 CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC AGC AGA CTG Human GC Leu Ser lys Leu Leu Arg Asp Ser His Val Leu His Sor Ar | Leu Ser 15 20 25 -173 -120 168 This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 B7___ V. Description of the invention (171) CAG TGC CCA GAG GTT CAC CCT TTG CCT AGA CCT GTC CTG CTG CCT GCT 210

Gin Cys Pro Glu Val Hia Pro Leu Pro Thr Pro Vai Leu Leu Pro Ala 30 35 40 GTG GAC ITT AGC TTC GGA GAA TGG AAA ACC CAG ATG GAG GAG ACC AAG 264

Val Asp Phe Ser Leu Gly Glu Γγρ lys Thr Gin Met Glu Glu Thr Lys 45 60 55 GCA CAG GAC Human CTG GGA GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG 312

Ala Gin Asp lie Leu Gly Ala Val TI \ r Leu Leu Leu Glu Gly Val Met 80 85 1 '70 75 GCA GCA COG GGA CAA CTG GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG 360

Ala Ala Arg Gly Gin Leu Giy Pro Thr Cys Leu Ser Ser Leu Leu Gly 80 85 90 CAG CTT TCT CGA CAG CTC CGT CTC CTC CTT GGG GCC CTG CAG AGC CTC 408

Gin Leu Ser Gly Gin VaI Arg Leu Leu L «u Gly Ala Leu Gin Sor Lou Θ5 100 105 GTT GGA ACC CAG CTT CCT CCA GAG GGC AGG ACC ACA GCT GAG AAG GAT 450

Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp 110 115 120 CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG 504

Pro Asn Ala lie Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val 125 130 136 CGT TTC CTG ATG CTT GTA GGA CGG TCC ACC CTC TGC CTC AGG CGG GCC 552 people rg Phe Leu Met Uu Val Gly Gly Sor Thr Leu Cys Val Arg Arg AIb 140 145 150 166 CCA CCC ACC ACA GCT GTC CTC AGC AGA ACC TCT CTA GTC CTC ACA CTG 600

Pro Pro Thr Thr Ala Vtl Pro Ser Arg Thr Ser Leu Yai Ιβυ Thr Leu 180 165 170 -174- This paper size is applicable to China National Standard (CNS) A4 specifications (210X: Z97 Public Manager Γ (Please read the precautions on the back before (Fill in this page). Binding-Order 498079 A7 B7 V. Invention Description (172) AAC GAG CTC CCA AAC AGG AsnGlu Leu Pro Asn Arg 175 GCC TCA GCC AGA ACT ACT Ala Ser Ala Arg Thr Thr .180 TTC AGA GCC AAG ATT CCT Phe Arg Ala Lys lie Pro 205 GAC CAA ATC CCC GGA TAC Asp Gin lie Pro Gly Tyr 220 225 Below water UA (7 6¾ base) ACT TCT GGA Thr Ser Gly 180 GGC TCT GGG Gly Ser Gly 195 GGT CTG CTG Gly Leu Leu 210 CTG AAC AGG Leu Asn Ar Name TTG TTG Leu Leu CTT CTC Leu Leu AAC CAA Asn Gin ATA CAC Ilo His 230 GAG ACA AAC TTC ACT Glu Thr Asn Phe Thr 185 AAG TGG CAG CAG GGA L ”Trp Gin Gin Gly 200 ACC TCC AGG TCC CTG. Thr Ser Arg Ser Leu 215 GAA CTC IT Glu Leu 696 744 785 881 I ------- install-- (Please read the precautions on the back before filling this page) Central Ministry of Economic Affairs% Consumption of Employees Cooperative society ¾. Serial number: 5 Sequence length: 5 7 6 Sequence type: Nucleic acid strand number: Double-stranded geometry: Linear sequence type · cDNAg mRNA source: homoplastic sabin (Η 〇mo S apins) Tissue type: Liver sequence: AG CCA TTC AGA GCC AAG ATT CCT GGT CTG CTG AAC CAA ACC TCC AGG Arg Ala LyS] ΐθ pr〇〇iy Leu Leu Aen Gin Thr Sor hrs 1 5 10 15 47 Order k -175- This paper is applicable to f Ugj home turn s) M specifications (: 297 mm ) 498079 A7 B7 V. Description of invention (175) TCC CTG GAC CAA ATC CCC GGA TAC CTG AAC AGG ATA CAC GAA CTC TTG 95 Ser Leu Asp Gin lie Pro Giy Tyr Leu Asn Ar $ lie His Glu Leu Leu 20 25 30 AAT GGA ACT CGI GGA CTC ITT CCT GGA CCC TCA CGC AGG ACC CTA GGA 143 Asn Gly Thr Arg Gly leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly 35 40 45 GCC CCC GAC ATT TCC TCA GGA ACA TCA GAC ACA GGC TCC CTG CCA CCC 19i Ala Pro Asp lie Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro SO 55 60 AAC CTC CAG CCT GGA TAT TCT CCT TCC CCA ACC CAT CCT CCT ACT GGA 239 Asn Leu Gin Pro Gly Tyr Ser Pro Se r Pro Thr His Pro Pro Thr Gly 65 70 75 CAG TAT ACG CTC TTC CCT CTT CCA CCC ACC TTG CCC ACC CCT GTG GTC 287 Gin Tyr Thr Lou Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val g85 85 95 CAG CTC CAC CCC CTG CTT CCT GAC CCT TCT GCT CCA ACG CCC ACC CCT 335 Gin Leu Hia Pro Leu Lou Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro 100 105 110 ACC AGC CCT CTT CTA AAC ACA TCC TAC ACC CAC XCC CAG AAT CTG TCT 383 Thr Sor Pro Lou Leu Asn Thr Sor Tyr Thi His Ser Gin Asn Leu Ser 115 120 125 CAG GAA GGG XAAGGTTCTC AGACACTGCC GACATCAGCA TTGTCTCATG 432 Cln Glu Gly 130-TACAGCTCCC TACCA AAA GCACATG GCAGATG ACTGTACATT ATAAACCTTC AGAA 570 -176- This paper size applies to Chinese National Standard (CNS) A4 (210X297). Please read the precautions on the back before binding line 498079 A7 _B7 V. Description of the invention (174) Central Bureau of Standards, Ministry of Economic Affairs Employee Consumption Cooperative Printed Serial Number: 6 Sequence Length: 1 2 6 7 Sequence type: Nucleic acid strand number: Double-stranded Geometry: Linear sequence Type: cDNA to mRM A Source: Homo Sap Us Type: Liver sequence: GGCCAGCCAG ACACCCCGGC CAGA Human TG GAG C Ding G ACT GAA TTG CTC CTC GTTC GTG ATG CTT CTC CTA ACT GU Met Glu Leu Thr Glu Leu Leu Leu Val VrI Met Leu. Leu Leu Thr Ala -20 -15 '-10 AGO CTA ACG CIG ICC AGG GCG GCT CCT CCT GCT TGT GAC CTC CGA GTC Arg Leu Thr leu Ser Ser Pro Ala Pro Pro Ala Cya Asp Leu Arg Val -5 1 5 10 CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC Leu Ser .Lys Leu Lou Arg Asp Sor His Val Lou His Sor Arg Lou Sor Dagger 15 20 26 CAG TGC CCA GAG GTT CAC CCT TTG CCT ACA CCI GTC CTG CTG CCT GCT Gin Cya Pro Glu Yel Hia Pro Leu Pro Thr Pro Val Leu Leu Pro Ala 80 3S 40 GTG GAC TIT AGC TTG GGA GAA TGG AAA ACC CAG ATG GAG GAG ACC AAG 264 Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin H ^ t Glu Glu Thr Lys 45 50 55 -177- 4 This paper size applies the Chinese National Standard (CHS) A4 specification (210X297 Director &quot; Γ 1 ~ 24 72 120 188 210 (Please read the precautions on the back before filling out this page) • Binding and ordering printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs $-498079 A7 _B7__ V. Invention Description (175) GCA CAC GAC ATT CTG GGA GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG 312

Ala Gin Aap lie Leu Gly Ala Vel Thr Leu Leu Leu Glu Gly Yai Met SO 85 70 75 GCA GCA CGG GGA CAA CTG GGA CCC ACT TGC CTC TCA ICC CTC CTG GGG 3Θ0

Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Sar Ser Leu Leu Gly80 85 90 CAG CIT TCT GGA CAG GTC CGT CTC CTC CTT GGG GCC CTG CAG AGC CTC 408

Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu 95 100 105 CTT GGA ACC CAC CTT CCT CCA CAG GGC AGG ACC ACA GCT CAC AAG GAT 458

Leu Gly Thr Gin Leu Pro Pro Gin Giy Arg Thr Thr Ala His Lys Asp 110 115 120 CCC AAT GCC ATC TIC CTG AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG 504

Pro Asn Ala lie Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val 126 130 135 CGT TTC CTG AT (j CH GTA GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC 652

Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys VbI Arg Arg Ala 140 145 150 155 CCA CCC ACC ACA GCT GTC CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG 600

Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Sor Leu Val Leu Thr Lou 160 105 170 AAC GAG CTC CCA AAC ACG ACT TCT GGA TIG TIG GAG ACA AAC TTC ACT 048

Aan Glu Leu Pro Aan Arg Thr Ser Gly Leu Leu Glu Thr Asa Phe Thr 175 180 185 GCC TCA GCC AGA ACT ACT GGC TCT GGG CTT CTG AAG TGG GAG CAG GGA 090

Ala Sei Ala Ar 《Thr Thr Gly Sor G】 y Lou Lou Lys Trj &gt; Gin Gin Gly 1Θ0 195 200 TTC AGA CCC AAG ATT CCT GGT CTG CTG AAC CAA ACC TCC AGG TCC CTG 744

Phe Arg Ala Lys llo Pro Gly Leu Leu Asn Gin Thr Ser Arg Ser Leu 206 210 215 -178-. This paper size applies to China National Standard (CNS) A4 (210X297 mm) --------- ^ -Install—r (Please read the notes on the back before filling this page) Order 498079 A7 B7 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs V. Invention Description (176) GAC CAA ATC CCC GGA TAC CTG AAC AGG ATA CAC GAA CTC TTG AAT GGA 7Θ2 Human sp Gin lie Pro Gly Tyr Leu Asn Arg lie His Glu Leu Leu Asn Gly 220 225 230 235 ACT CGT CGA CTC TIT CCT GGA CCC TCA CGC AGC ACC CTA GGA GCC CCG 840

Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Tbr Leu Gly Ala Pro t G L L (7 G P S S Tian Ding Yi _ 240 245 250 ... GAC AIT TCC TCA GGA ACA TCA GAC ACA GGC TCC GTG CCA CCC AAC CTC 888

Asp lie Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu 255 280 2Θ5 CAG CCT GGA TAT TCT CCT TCC CCA ACC CAT CCT CCT ACT GGA CAG TAT 938

Gin Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Tbr Gly Gin Tyr 270 275 280 ACG CTC TTC CCT CTT CCA CCC ACC TTG CCC ACC CCT GTG GTC CAG CTC Θ84

Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gin Leu 285 290 295 CAC CCC CTG CTT CCT GAC CCT TCT GCT CCA ACG CCC ACC CCT ACC AGC 1032

His Pro Leu Leu Pro Ajjp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser 300 305 310 315 CCT CTT CIA AAC ACA TCC TAC ACC CAC TCC CAG AAT CTG ICT CAG GAA 1080

Pro Leu Leu Asn Thr Ser Tyr Thr His Sor Gin Asn Leu Ser Gin Glu 320 325 330 GGG TAAGGTTCTC AGACACTGCC CACATCAGCA TTGTCTCATG TACAGCTCCC 1133

Gly TTCCCTGCAG GGCGCCCCTG GGAGACAACT GGACAAGATT TCCTACTTTC TCCTGAAACC 11Θ3 CAAAGCCCTG GTAAAAGGGA TACACAGGAC TGAAAAGGGA ATCATTTTTC ACTGTACATT 1253 Serial number: 7 -179- This paper size applies to China National Standards (CNS) A4 specifications I 210 (Please read the notes on the back before filling out this page) Order ¾ Printed by the Consumers' Cooperative of the Ministry of Economic Affairs of the Ministry of Economic Affairs 498079 A7 ___—_ B7____ 5. Description of the invention (177) Sequence length: 1 7 2 1 Sequence type: Nucleic acid Number of chains: double-stranded geometry: linear sequence type: c DNA to m R Μ A source: homosabin (Η 0 1110 sa Pins) tissue type: liver sequence: GCGGCACGAG GGGGGTGTCT GGCTGGCGTG GCTCCCTGTT TGGGGCCTCT CCCCTGAAIC Θ0 CTTCCTGGGG CCATGGAGGC CAGACA CCCCGGCCAG A 101 ATG GAG CTG ACT GAA TTG CTC CTC GTG GTC ATG CTT CTC CTA ACT GCA 149

Met Glu Leu Dinghr Glu Leu Leu Lsu Yal Val Met Leu Leu Leu Thr Ala-20 -15- -10 AGG CTA ACG CTG TCC AGC CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC 193

Arg Leu Thr Leu Ser Sor Pro Ala Pro Pro Ala Cys Asp Lou Arg Val -5 1 5 10 CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CI1 CAC AGC AGA CTG AGC 245

Leu Ser Lys Leu Lou Arg Asp Ser Ilis Yal Leu His Ser Arg Leu Ser] B 20, 25 CAG TGC CCA GAG GTT CAC CCT TTG CCT ACA CCT GTC CTG CTG CCT GCT 2Θ3

Gin Cys Pro Glu Val Ilis Pro Leu Pro Thr Pro Val Leu Leu Pro Ala 30 35 40 GTG GAC ITT AGC TTG GGA GAA TGG AAA ACC CAG AIG GAG GAG ACC AAG 341

Val Asp Pho Sor Lou Gly Glu Trp Lys Thr G3n Mot Glu Glu Thr Lys

45 50 BB GCA CAC.GAC ATT CTG GGA GCA GTG ACC CTT CTG CTC GAG GCA CTC ATG 389 -180- This paper is again applicable to China National Standard (CNS) A4 specifications (210X297 mm) (Please read the notes on the back first (Fill in this page again)

498079 A7 B7 V. Description of the invention (port 8)

Ala Gin Asp lie 00 GCA GCA CGG GGA Ala Ala Arg Gly Printed by CAG CTT Gin Leu CTT GGA Leu Gly CCC AAT Pro Asn 125 CGT TTC Arg Phe 140 CCA CCC Pro Pro AAC GAG Asn Glu GCC TCA Λ] β Sor TTC AGA PhG Arg 205 TCT GGA Ser Gly 95 ACC CAG Thr Gin 110 GCC ATC Ala He CIG ATG Leu Met ACC ACA Thr Thr CTC CCA Leu Pro .175 GCC AGA Λ15 Arg 190 GCC AAG Ala Lys

Leu Gly 65 CAA CTG Gin Leu 80 CAG GTC Gin Vai CTT CCT Leu Pro TTC CTG Phe Leu CXT GTA Leu Val 146 GCT GTC Ala Yal 160 AAC AGG Asn Arg ACA ACT Thr Thr ATT CCT Ilo Pro

Ala Val Thr GGA CCC ACT Gly Pro Thr CGT CTC CTC Arg Leu Leu 100 CCA CAG GGC Pro Gin Gly 115 AGC TTC CAA Ser Phe Gin 130 GGA GGG ICC Gly Gly Ser CCC AGC AGA Pro Sor Ar ^ ACT TCT GGA Thr Ser.Gly 180 GGC TCT GGG Gly Ser Gly 106 GGT CTG CTG Gly Lou Leu 210

Leu Leu Leu Glu 70 TGC CTC TCA TCC Cys Leu Ser Ser 85 CTT GGG GCC CTG Leu Gly Ala Leu AGG ACC Arg Thr CAC CTG His Leu ACC CTC Thr Lou 160 ACC TCT Thr Ser 185 TTG TTG Leu Leu ACA GCT Thr Ala 120 CTC CGA Leu Arg 135 TGC GTC Cys Vai CTA GTC Leu Val GAG ACA Glu Thr

CTT CTG AAG TGG Leu Leu Lys Trp 200 AAC CAA ACC TCC Asn Gin Thr Ser 21B

Gly Val Hot? 6 CTC CTG GGG Leu Leu Gly 90 CAG AGC CTC Gin Ser Leu 105 CAC AAG GAT His Lye Asp GGA AAG GTG Cly Lys Val AGG CGG GCC Arg Arg Ala 155 CTC ACA CTC Leu Thr Leu 370 AAC TTC ACT Aen Phe Thr 186 CAG CAG GGA Gin Gin Gly AGG TCC CTG Arg Ser Leu 437 485 533 581 629 877 725 773 (Please read the precautions on the back before filling out this page)-Install and order P! Line 181 This paper size applies to Chinese national standards (CNS) A4 specification (210X297 mm) 821 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 498079 A7 ____B7__ V. Description of the invention (179) GAC CAA ATC CCC GGA TAC CTG AAC AGG ATΛ CAC GA into CTC HG AAT GGA 860

Asp Gin lie Pro Gly Tyr Leu Asn Arg Ilo His Glu Leu Leu Asn Gly 220 225 · 230 235 ACT CGT GGA CTC ITT CCT GGA CCC TCA CGC AGG ACC CTA GGA GCC CCG 817

Thr Arg Gly Leu Phe Pro Cly Pro Ser Arg Arg Thr Leu Gly Ala Pro 240 245 250 GAC ATT TCC TCA GGA ACA TCA GAC ACA GGC TCC CTG CCA CCG AAC CIC 965

Asp lie Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu 255 260 265 CAG CCT GGA TAT TCT CCT ICC CCA ACC CAT CCT CCT ACT GGA CAG-TAT 1013

Gin Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gin Tyr 270 275 280 ACG CTC TIC CCT CTI CCA CCC ACC TTG CCC ACC CCT GTG GTC CAG CTC 10Θ1

Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gin Leu 285 290 205 CAC CCC CTG CTT CCT CAC CCT TCT GCT CCA ACG CCC ACC CCT ACC AGC 1109

His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser 300 305 310 815 · CCT CIT CTA AAC ACA TCC TAC ACC CAC TCC CAG AAT CTG TCT CAG GAA 1157

Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gin Asn Lou Ser Gin Glu 320 326 330 GGG TAAGGTTCTC AGACACTGCC GACATCAGCA TTGTCTCCTG TACAGCTCCC 1210

Gly TTCCCTGCAG GGCGCCCCTG GGAGACAACT GGACAAGATT TCCTACTTTC TCCTGAAACC 1270 CAAAGCCCTG GTAAAAGGGA TACACAGGAC TGAAAAGGGA ATCATTTTTC ACTGTACATT 1330 ATAAACCTTC AGAAGCTATT TTTTTAAGCT ATCAGCAATA CTCATCAGAG CAGCTAGCTC 1390 TTTGGTCTAT TTTCTGCAGA AATTTGCAAC TCACTGATTC TCTACATGCT CTTTTTCTGT 14S0 GATAACTCTG CAAACGCCTG GGCTGGCCTG GCACTTGAAC AGAGGGAGAG ACTAACCTTG 1510 AGTCAGAAAA CAGAGAAAGG GTAATTTCCT TTGCTTCAAA TTCAAGCCCT ICCAACGCCC 1570 CCATCCCCTT TACTATCATT CTCAGTGGCA CTCTGATCCC ATATTCTTAA CAGATCTTTA 1630 -182- The size of this paper is applicable to Chinese National Standard (CNS) A4 (210X297mm) I ---- r-- ·· 1 Pack ------ Order ----- ^ Line (Please read first Note on the back, please fill in this page again) 1721 Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 498079 A7 __B7 V. Description of the invention (18〇) CTCTTGAGAA AIGAATAAGC TTTCTCTCAG AAATCCTGTC CCTATACACT AGACAAAACT 1Θ90 Tail of water UA below G3 3 0¾¾ Serial number: 8 Sequence length: 4 5 0 6 Sequence type: Nucleic acid strand number: Double-strand geometry: Linear

Sequence type: the cDNA to roRNA Source: isotype Sabin (Η ο ία 〇S apins) type tissue: Liver sequence:. GAATTCAGGG CTTTGGCAGT TCCAGGCTGG TCAGCATCTC AAGCCCTCCC CAGCAXCTGT Θ0 TCACCCTGCC AGGCAGTCTC TTCCTAGAAA CTTGGTTAAA TGTTCACTCT TCTTGCTACT 120 TTCAGGATAG ATTCCTCACC CTTGGCCCGC CTTTGCCCCA CCCTACTCTG CCCAGAAGTG 180 CAAGAGCCTA AGCCGCCTCC ATCCCCCCAG GAAGGATTCA GGGGAGAGGC CCCAAACAG.G 240 GAGCCACCCC AGCCAGACAC CCCGGCCAGA ATG GAG CTG ACT G GTGAGAACAC 293

Met Glu Lou Tbr G -20 ACCTGAGGGG CTAGGGCCAT ATGGAAACAT GACAGAAGGG GAGAGAGAAA GGAGACACGC 353 TGCAGGGGGC AGGAAGCTGG GGGAACCCAT TCTCCCAAAA ATAAGGGGTC TGAGGGGTGG 413 ATICCCTGGG TTTCAGGTCT GGGTCCTGAA TGGGAATTCC TGGAATACCA GCTGACAAIG 473 ATTTCCTCCT CATCTTTCAA CCTCACCICT CCTCATCTAA G AA TTG CTC CTC GTG 528 lu Leu Leu Leu Val -15 GTC AIG CTT CIC CTA ACT GCA AGG CTA ACG CTG TCC ACC CCG GCT CCT 576

Vai Met Leu Leu Leu Thr person la Arg Lou Thr Lou Ser Ser Pro Ala Pro -JO -5 1 CCT GCT TOT GAC CTC CG person GTC CTC AGT ΑΛΛ CTG CTT CGT GAC TCG CAT 624 -183- This paper size applies to Chinese national standards (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page)

498079 A7 _ B7 V. Description of the invention (181)

Pro Ala Cys Asp Leu Ar 豸 Val Leu Ser Lys Leu Leu Arg Asjp Ser His 5 10 15 20 GTC CTT CAC AGC AGA CTG GTGAGAACTC CCAACATTAT CCCCTTTATC CGCGTAACTG Θ82 Yal Leu His Ser Arg Leu 25 GTAAGACACC CATACTCCCA GGAAGACACCCTCCCTCTCTCTCCTCTCCT CTGACAACGA TTATTCTTCA 802 CAATACAGCC CGCAITTAAA AGCTCTCGTC TAGAGATAGT ACTCATGGAG GACTAGCCTG 8Θ2 CITATTAGGC TACCATAGCT CTCTCTATTT CAGCTCCCTT CTCCCCCCAC CAATCTTThr Leu ProCCT CUC CCT CTCT CCT 30 35 40 CCT GCT GTG GAC TTT AGC TTG GGA GAA TGG AAA ACC CAG ATG CTAAGAAAGC 1025 Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met 45 50 55 CATCCCTAAC CTTGGCTTCC CTAACCCCTACCTCACCCCTACCATTACCCTACCATTACCCTCAGCC TCTACATTCA CCTATGAIGA TAGCCTGTGG ATAACATGAT GGCTTGCAGG TCCAATATGf 1205 GAATAGATTT GAAGCTGAAC ACCATGAAAA CCIGCAGAGA AATCCCT CAT CCCCATGCCT 1265 TTGACCTAIT CCTGTTCAGT CTTCTTAAAT TGGCATGAAG AAGCAAGACT CATATGTCAT 1325 CCACAGATGA CACAAAGCTG GGAAGTACCA CTAAAATAAC AAAAGACTGA ATCAAGATTC 1385 AAATCACTGA AAGACTAGGT CAAAAACAAG CTGAAACAAC AGAGATATAA ACTTC1ACAT H45 GTGGGCCGGG GGCTCACGCC TGTAATCCCA GCACTTTGGG AGGCCGAGGC AGGCAGATCA 1505 Ministry of Economic Affairs Bureau of Standards Employees Co-op ------- printed dress - - (please read the back of the precautions to fill out this page) line CCTGAGGGCA GGAGTTTGAG AGCAGCCTGG CCAACATGGC GAAACCCCGT CTCTACTAAG 1565 AATACAAAAT TAGCCGGGCA TGGTAGTGCA TGCCTGTAAT CCCAGCTACT TGGAAGGCTG 1625 AAGCAGGACA ATCCCTTGAA CCCAGGAGGT GGAGCTTCIA GTGAGCTGAG ATCATGCCAA 1885 TGCACTCCAG CCTGGGTGAC AAGACCAAAA CTCCGTCTCA ΑΑΛΛΟΛΑΑΑΑ AAAATTCTAC 1745 ATGTGTAAAT TAATGAGTAA AGTCCTATTG CAGCTITCAG GCCACAATGC CCTGCTTCGA 1805 TCATTTAAGC CTCTGGCCCT AGCACTTCCT ACGAAAAGGA TCTGAGACAA TTAAATTGCC 1885 -184- This paper size applies to China National Standard (CNS) Α4 size (210X297 mm) 498079 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 _B7 _; _ V. invention is described in (182) CCCAAACTTA CCATGTAACA TTACTGAAGC TGCTATTCTT AAAGCTAGTA ATTCTTGTQT 1925 GTTTGATGTT TAGCATCCCC ATTCTGGAAA TGCTCGTACA GAACTCTATT CCGAGTGGAC 1Θ85 TACACTTAAA IATACTGGCC TGAACACCGG ACATCCCGCT GAAGACATAT GCtAATTTAT 2045 TAAGAGGGAC CATATTAAAC TAACATGTGT CTAGAAAGCA GCAGCCTGAA CAGAAAGAGA 2105 CTAGAAGCAT GTTTTATGGG CAATAGTTTA AAAAACIAAA ATCTATCCTC AAGAACCCTA 2185 GCGTCCCTTC TTCCTTCAGG ACTGAGTCAG GGAAGAAGGG CAGTTCCTAT CCGTCCCTTC 2225 TAGTCCITTC TTTTCATCCT TATCATCATT ATGGTAGAGT CTCATACCTA CATTTAGTTT 22B5 ΑΤΤΤΑΠΑΤΤ ATTATTTGAG ACGGAGTCTC ACTCTATCCC CCAGGCTGGA GTGCAGTGGC 2345 ATGATCTCAA CTCACTGCAA CCTCAGCCTC CCGGATTCAA GCGATTCTCC TGCCTCAGTC 2405 TCCCAAGTAG CTGGGATTAC AGGTGCCCAC CGCCATGCCC AGCTAATTTT TGTATTTTTG 24Θ5 GTAGAGATGG CGTTTCACCA TGTTGGCCAG GCTGATCTTG AACTCCTGAC CTCAGGTGAT 2525 CCACCTGCCT CAGCCTCCCA AAGTGCTGGG ATTACAGGCG TGAGCCACTG CACCCAGCCT 2585 TCATTCAGTT TAAAAATCAA ATGATCCTAA GGTTTTGCAG CACAAAGAGT AAATTTGCAG 2845 CAGTAGAACC AAGAGGTAAA AGCTGIAA CA GGGCAGATTT CAGCAACGTA AGAAAAAAGG 2705 AGCTCTTCTC ACTGAAACCA AGTGTAAGAC CAGGCTGGAC TAGAGGACAC GGGAGTTTTT 2785 GAAGCAGAGG CTGATGACCA GCTGTCGGGA GACTGTGAAG GAATTCCTGC CCTGGGTGGG 2825 ACCTTGGTCC TGTCCAGTTC TCAGCCTGTA TGATTCACTC TGCTGGCTAC TCCTAAGGCT 2885 CCCCACCCGC TTTTAGTGTC CCCTTTGAGG CAGTGCGCTT CTCTCTTCCA TCTCTTTCTC 2945 AG GAG GAG ACC AAG GCA CAG GAC ATT CTG GGA GCA GTG ACC CTT CTG CTG 2995 Glu Glu Thr Lys Ala Gin Asp lie Lou Gly Ala Val Thr Leu Levi Leu 80 85 70 GAG GGA GTG ATG GCA GCA CGG GGA CAA CTG GGA CCC ACT TGC CTC TCA 3043 Glu Gly Vftl Met Ala Ala Arg Gly Gin Lou Gly Pro Ding br Cys Lou Sor 75 80 85 TCC CTC CTG GGG CAG CTT TCT GGA CAG GTC CGT CTC CTC CTT GGG GCC 3091 Sor Leu Leu Gly Gin Lsu Ser Giy Gin Val Arg Leu Leu Leu Gly Ala β〇95 100 CTG CAG AGC CTC CTT GGA ACC CAG GTAAGTCCCC AGTCAAGGGA TCTGTAGAAA 3H5 Leu Gin Ser Leu Leu Gly Thr Gin -185-(Please read the precautions on the back before filling out this page) Packing-Order-Line paper is also applicable to Chinese National Standard (CNS) A4 specification( 210X297 mm> 498079 A7

V. Description of the invention (185) 105 Π0 CTGITCTTTT CTCACTCAGT CCCCCTAGAA GACCTGAGCG AAAGAGGGCT CTTCCAGCGA 3205 CCTCAACCGC AGAAGAGCTG ATCTACTAAG AGTGCTCCCT GCCAGCCACA ATGCCTGGGT CTCCT CCT CCT CCT GCC CCCC GCC CCCC

Leu Pro Pro Gin Gly Arg Thr Thr Ala 115 120 CAC AAG GAT CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC CIG CTC CGA '3428

Hia Lys Asp Pro Asn Ala lie Phe Leu Ser Phe Gin His Leu Leu Arg 125 130 135. GGA AAG GIG CGT TTC CTG ATC CTT GTA GGA CGG TCC ACC CTC TGC GTC 3474

Gly Lys Val Ar ^ Phe Leu Mot Leu V ^ l Gly Gly Ser Thr Leu Cya Val 140 145 150 AGC CGG GCC CCA CCC ACC ACA GCT GTC CCC AGC AGA ACC TCT CTA GTC 3522 Please read the note on the back I, J Printed by the Consumers' Cooperatives of the Ministry of Economic Affairs

Ari Arg Ala Pro Pro Thr Thr Ala Val Pro Sc r Arg Thr Ser Leu Val 155 160 185 CIC ACA CTG AAC GAG CTC CCA AAC AGG ACT TCT GGA TTG TTG GAG ACA 3570 Leu Thr Leu Asn Glu Leu Pro Asn Arg Thr Sor Gly Leu Leu Glu Thr 170 175 180 AAC TTC ACT GCC TCA GCC AGA ACT ACT GCC TCT GGC CTT CTG AAG TCG 3318 Asn Pho Thr Ala Ser Ala Arg Thr Thr Giy Scr Gly Lou Lou Lys Trp 185 190 195 200 CAG CAG GGA TTC ACA GCC AAG ATT CCT GOT CTG CTG AAC CAA ACC TCC 3666 Gin Gin Gly Phe into AH Lys Ilo Pro Gly Leu Leu Asn Gin Thr Ser 205 210 215 AGG TCC CTG GAC CAA ATC CCC GCA TAC CTG AAC AGG ATA CAC GAA CTC 3714 Ror Sor Leu into ap Gin lie Pro Gly Tyr Lou Asn Arg lie Hia Glu Lou 220 225 230 Alignment This paper size applies to China National Standard (CNS) A4 (210X297 mm) 498079 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7_____ V. Description of the Invention (184) TTG AAT GGA ACT CGT GGA CTC TTT CCT GGA CCC TCA CGC AGG ACC CTA 3702 Leu Asu Gly Tiir Arg Gly Lisu Phe Pro Gly Pro Sor Arg Arg Thr Lou 235 240 245 GGA GCC CCG GAC ATT TCC TCA GGA ACA TCA GAC ACA GGC TCC CTG CCA 3810 Gly Ala Pro Asp lie Ser Ser'Gly Thr Ser Asp Thr Gly Ser Leu Pro 250 255 J 260 CCC AAC CTC CAG CCT GGA TAT TCT CCT TCC CCA ACC CAT CCT CCT ACT 3858 Pro Asn Leu Gin Pro Gly Tyr Ser Pro Ser Thr His Pro Pro Thr 285 270 275 280 GGA CAG TAT ACG CTC TTC CCT CTT CCA CCC ACC TTG CCG ACC CCT GTG 3908 Gly Gin Tyr Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Yal 285 2Θ0 295 GTC CAG CTC CAC CCC CIG CTT CCT GAC CCT TCT GCT CCA ACG CCC ACC 3Θ54 Val Gin Leu Hia Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr 300, 305 310 CCT ACC AGC CCT CTT CTA AAC ACA ICC TAC ACC CAC TCC CAG AAT CTG 4002 Pro Thr Ser Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gin Asn Leu 315 320 325 TCT CAC GAA GGG TAAGGTICTG AGACACTGCC GACATCAGCA TTGTCTCATG 4064 Ser Gin Glu Gly 330 TACAGCTCCC TTCCCTCCAC GCCGCCCCTG CGAGACAACT GGACAACATT TCCTACTTTC 4114 TCCTGAAACC CAAAGCCCTG GTAAAAGCGA TACACAGGAC TCAAAACGGA ATCATTTTTC 4174 ACTGTACATT ATAAACCTTC AGAAGCTAIT TTTTTAAGCT ATCAGCAATA CTCATCAGAG 4234 CACCIACCTC TTTGGTCTAT TTTCTGCAGA AATTTGCAAC TCACTGATTC TCTACATGCT 4294 CTTTTTCTGT GATAACICTC CAAAGGCCTG GGCTGCCCTG GCAGTTGAAC AGAGGGACAC 4354 ACTAACCTTG AGTCAOAAAA CAGAGAAAGG GIAATTTCCT TTGCTTCAAA TICAACGCCT 4414 TCCAACGCCC1 CCATCCCCTT TACTATCATT CTCAGTGGGA CTCTGATCCC ATATTCTTAA 4474 CAGAICITTA CTCTTGAGAA ATCAATAACC TT 4508 -187- (please Read the notes on the back and fill out this page) ---------- ^, order ---------

•----I This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 498079 A7 B7 V. Description of the invention (185) Sequence scam: 9 Sequence length: 4 1 6 Sequence type: Number of nucleic acid strands ·· Double-stranded Geometry: Linear Sequence Type: Synthesis I) NA Sequence:

CTAGAAAAAA TGTTCTGTCT GGAAGTTCAC ATGGAAAACC TCTGGAAGGC TGGCCAGCTG CCAGCTGCCG

CCAAGGAGGT AAACTGCTTC CCGCTGCCGA CAGATGGAAG GTTATCGCTG TCTGGCCAGC CCAGAGGGCC

AAIAAATAAT GCGACTCTCA CCCCGGTTCT AGACCAAAGC CACGTGGCCA TTCGTCTGCT GTACCACTGC

GAGCCCGGCT CGTGCTGCAC GCTTCCGGCT TCAGGACATC GCTTGGCCCG GCTCGGCGCT TCACAAGGAT

CCGCCAGCTT TCTCGTCTGT GTCGACTTCT CTGGGTGCAG ACCTGCCTGT CTGCAGTCTC CCGAACGCTA GTGACCTTCG 60 CCCAGTGCCC 120 CCCTGGGTGA ISO TAACTCTGCT 240 CTTCCCTGCI 300 TGCTTGGCAC n. Ministry of Economic Affairs, Tsinghua Bureau, Consumer Consumption Cooperative, printed serial number: 10 sequence length: 5 4 9 sequence type: number of nucleic acid chains: double-stranded geometry: linear sequence type: synthetic DHA sequence: -188- paper Standards apply to China National Standard (CNS) A4 (210X297 mm)

Printed by the Jm line Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 498079 A7 B7___ V. Description of Invention (186) CTG GTT CCG CGT GGA ICC CCG GCT CCG CCA GCT TGT GAC CTT CGT GTT 48

Leu Val Pro Ar ^ Gly Ser Pro Ala Pro Pro Ala Cys Asp Leu Ar ^ Val -5 + i 5 10 CTG TCT AAA CTG CTT CGC GAC TCI CAC GTG CTG CAC TCT CGT CTG TCC 90

Leu Ser Lys Leu Leu Arg Human 8p Ser His Val Levi Hia Ser Arg Leu Ser 15 20 ^ 25 CAG TGC CCG GAA GTT CAC CCG CTG CCG ACC CCG GTT CTG CTI CCG GCT 144

Gin Cys Pro Glut Val His Pro Leu Pro Thr Pro Yal Leu Leu Pro Ala 30 35 40 GTC GAC TTC TCC CTG GGT GAA TGG AAA ACC CAO ATG GAA GAG ACC AAA 192

Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys 45 50 55 GCT CAG GAC ATG CTG GGT GCA GTA ACT CTG CTT CTG GAA GGC GTT ATG 240

Ala Gin Asp lie Leu Gly Ala Val Thr Leu Leu Leu Giu Gly Val Met 00 65 70 75 GCT CCA CGT GGC CAG CTT GGC CCG ACC TCC CTG TCT TCC CTG CTT GGC 288

Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Sor Leu Leu Gly 80 85 90 CAG CTG TCT GGC CAC GTT CGT CTG CTG CTC GCC GCT CTG CAG TCT CTG 336

Gin Leu Ser Gly GId Yal Arg Leu Leu Leu Giy Ala Leu Gin Ser Leu Θ5 100 105 CTT GGC ACC CAG CTG CCG CCA CAG GGC CGT ACG ACT GCT CAC AAG GAT 384

Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His lys Asp 110 Π5 120 CCC ΑΛΤ GCC ATC TTC CTG AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG 4S2

Pro Asn Ala lie Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lya Val 125 130 135 CGT TTC CTG ATG CTT GTA GGA GGG TCC ACC CTC TGC G1X AGG CGG GCC 480 into rg Pbo Leu Met Lou Val Gly Gly Ser Thr Lou Cys Val Arg Arg Ala 140 145 160 155 -189- — This paper New Zealand Financial Standard for Family Care (CNS) A4 ^ (21GX297 mm) I ------— HI ---- Order-- (Please read the back first Note for re-filling this page) Line 498079 Printed by A7 _B7_______ of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs V. Invention Description (187) CCA CCC ACC ACA GCT GTC CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG 528 Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu 160 165 170 AAC GAG CTC TAATGAGAAT TC 649 Asn Glu Leu Sequence Number: 11 Sequence Length: 5 3 5 Sequence Type: Nucleic Acid Chain Number: Double-stranded Geometry: Linear Sequence Species: Synthetic DNA Source: Human (isotype sabin). CTAGAAAAAA CCAAGGAGGT ΑΛΤΑΑΑΤΑ 28 ATG AAA GCA CCT GTA CCA CCT GCA TGT GAT TTA CGG GTC CTG TCT AAA 70 Met Lys Ala Pro Val Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys +1 5 10 CTG CTG CGC GAC TCT CAC GTG CTG CAC TCT CGI CTG TCC CAG TGC CCG 124 Leu Leu Arg Asp Ser His Yal Leu His Scr kr $ Leu Ser Gin Cys Pro 15 20 25 30 GAA GTT CAC CCG CTG CCG -ACC CCG GTT CTG CTT CCG GCT GTC GAC TTC 172 Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala V &amp; l Asp Phe 35 40 45 TCC CTC GGT GAA TGG AAA ACC CAG ATC GAA GAG ACC AAA CCT CAG GAC 220 Sor Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lye Ala Gin Asp 50 55 60 ATC CTG 'GGT GCA GTA ACT CTG CTT CTG GAA GGC GTT ATG GCT GCA CGT 208 -1 9 0 ~ (Please read the notes on the back before filling this page)- Loading ·

1. The paper size of the 1T line is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 498079 A7 B7 V. Description of the invention (188) lie Leu Gly Ala 85 GCC CAG CTT GGC Gly Gin Leu Gly 80 GGC CAG GTT CGT Gly Glu Val Arg 95 CAG CTG CCG CCA Gin Leu Pro Pro ATC TTC CTG TCT lie Pie Leu Ser 130 ATG CIG GTT GGC Met Leu Val Gly 146 ACT GCT GTT CCG Thr Ala Val Pro 1Θ0

Yal Thr Leu Lou Leu 70 CCG ACC TGC CTG TCT Pro Xhr Cys Leu Ser 85 CTG CTG CTC GGC GCT Leu Leu Leu Gly Ala 100 CAG GGC CCT ACC ACT Gin Gly Arg Thr Thr 115 TTC CAG CAC CTG CTG Phe Gin Ria Leu Leu 135 GGT TCT Human CC CTG TGC Gly Ser Thr Lou Cys 150 TCT TAATGAAAGC TT Sot

Glu Gly Val Met A1r Ala Arg 75 TCC CTG CTT GGC CAG CTG TCT Ser Leu Lsu Giy Gin Leu Ser 90 CTG CAG TCT CTG CTT GGC ACC Lqu Gin Ser Leu Leu Gly Thr 105 110 GCT CAC AAG GAT CCG kkC GCT Ala His Lys Asp Pro Asn Ala 120 126 CGT GGC AAA GTT CGT TTC CTG Arg Gly L ”Val Arg Phe Leu 140 'GTT CGT CGG GCG CCG CCA Human CC Val Arg Arg Ala Pro Pro Thr 165 318 3Θ4 412 4Θ0 508 535 (Please read the back first Note # 1 Refill and install — nest page) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs [Simplified description of the figure] [Figure 1] Represented in TP 0 from XRP rat, refined on gel Analytical chart of filtration chromatography (Sephacryl s ~ 200 HR column) and set in rat CFU-MKM-191-This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) 498079 A7 ___B7 5 Figure of TPO activity of the invention description (189). [Figure 2] Reverse phase ephemeris method (C apce Η P ak C 1 3 0 0 A column) purified from XRP rat low molecular TP0 control standard ) Calendar analysis [Li, and graph of TPO activity measured in rat CFU-MK [Circle 3] Reverse phase ephemeris method (C apce 1 1 P ak C 1 3 0 0 Α column) purified from rat low molecular TP 0 control standard derived from XR PF FA (Tube number 3 6 ~ 4 2) Photographs separated by SDS-polyacrylamide gel electrophoresis, and the TP0 activity measured in rat CFU-MK. [Circle 4] Shown in XRP Peptide circle diagram of the TP 0 active portion of the TP 0 active fraction FA purified by reverse-phase chromatography (C ace Π Pak C 1 3 0 0 A column) purified from a mouse low-molecular TP0 control standard. [With 5] Shows TP0 activity from rat blood paddles measured in rat CFU-MK. [Figure 6] A conceptual diagram showing the composition of the expression vector PEF18S. Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs --- ----- Pack-(Please read the precautions on the back before filling this page) The line [Figure 7] is shown in the rat CFU-MK assay guide PEF18S-A2 cx to make the culture supernatant expressing C0S1 cells Figure of TP 0 activity. [Fig. 8] The CFU-MK measurement of rats was introduced to PEF18S-HL34. The table is C0S1 -192- This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) Central Bureau of Standards, Ministry of Economic Affairs Printed by Employee Consumption Cooperative 498079 A7 _________B7 _ 5. Description of the invention (19) A diagram of TP 0 activity in cell supernatant. [Circle 9] FIG. F shows the activity of the culture supernatant of COS1 cells of epidermis by measuring pHTI-231 in rat CFU-MK. [圜 10a] shows the activity of TP0 in the supernatant of COS1 cells introduced into pETFI as measured by rat CFU-MK. [Fig. 1 Ob] A graph showing the measurement of TP0 activity in the culture supernatant of cultured COS1 cells induced by pHTFI at M ()-7e. [011] Fir strains are pure λ HG T1 restriction enzymes (E ′: EcORI, H: Hindi, S: Sail in the garden) [Fig. 12a] It is shown in rat CFU-MK assay that pEFHGTE leads to COS1. TP 0 of cell culture supernatant [Fig. 12b] Fig. 12b shows the measurement of TP 0 activity in COS1_ cell culture supernatant of pEFHGTE introduced into M0-7e to measure the epithelium. [Fig. 13a] The measurement and introduction of pH CTI-MK in rats -211 # 1 and pHTI-191 1 and P 1 T 1 ~ 1 7 1 # 2 are graphs showing the activity of TP 0 in the C 0 S 1 cell culture supernatant [Figure 13b] -193- Paper size applies Chinese National Standard (CNS) Α4 specification (210 × 297 mm) ------- ||| ρ · Packing-(Please read the precautions on the back before filling this page) Thread 498079 A7 B7_ 5 The description of the invention (191) is shown in the rat CPU-MK measurement guide ρ Η T 1-1 6 3 # 2 to show the expression of TP0 activity in c 0 S 1 cell culture supernatant. '[Figure 14 ] In M 0 -7 e Assay ρ Ε 丁 1-2 1 1 # 1, ρ Ε 丁 1-1 9 1 it 1 _, pETl-171 # 2 and pETl-163 # 2 to express the expressed COS1 cell culture supernatant ΤΡ0 active warm. (Please read the precautions on the back before filling out this page)-Pack

, 1T Line Ministry of Economics, Central Bureau of Standards, Consumer Cooperation Du printed -194 A paper size is applicable to Chinese National Standard (CNS) A4 specifications (210 × 297 mm) 498079 A7 B7 V. Description of the invention (192) Written details of the chemical formula etc. Instructions I-Please [-Table 1] 1-Table 1 | f Rat plasma Ding P0 made of branches • · — 1 step '纮 ® white world phase talk sex talk sex fi talk sex recovery insects ^ (mg) ( %) | Same blood acetylene 493000--% '4 Ca-treated blood rose / G25 4803CO 2 864600 100 | &lt;# Subexchange &gt; Q-Scpharosc FF 314400 9 2704000 • 313,' &lt; Xion 丨: source lectin &gt;WGA-Aga; 〇3c 15030 132 198700D 230 &lt; Pigment Adsorption: &gt; TSK.gdAF-B 丨 ue 650MH 4236 905 383 True 443 &lt; Hydrophobic Phcnyl-Scpharo. ^ 6 FF / LS 2762 S47 2339000 271, ' -&lt; Gel Filtration &gt; S-200HRF3 (β: Molecular TPO) 51 200CO 1020000 '118 S.200HR?! (¾Molecular DPO) 262 783S 20550CO 238 Low-Molecular DPO (S-2COH31 F3l ^) 'Step phase phase activity phase phase activity S activity recovery abundant (mg)' (% ) &lt; coagulation-filtration &gt; S.2COHRF3 (low molecular TPO) 50.3300 20000 _0C0 116 &lt; inverse buckle &gt; y MC Protein.RP prep 2.8540 130000 371000 43 &lt; inverse phase &gt; YMCCN-AP 0.3730 8CO000 298400 35 Capccll PakCl 0.0396 4890000 1936CO 22 &lt; Swim &gt; U% SDS-PAGE (Unreduced) 0.0317 149000000 250300 29 β-molecule tpo (from SS-20: 0HR F2) Step Secret White noble phase live a phase live ttft activity Ugly f (mg) • f (%) sound. Rrf SJOOKRn (¾molecular TPO) 257.0000 7840 2013000 233 &lt; turn &gt; YMC Proicin-RP prep 11.4000 227000 25S8000 300 &lt; gel filtration &gt; 75pg / CHAPS \ A660 1750000 2011000 236 隹 &lt; Back buckle &gt; VMCCN-ΛP 0.6200 7CXXXX3 43 ^ 000 50 Ψ- Capctll? Bk Cl 0.0630 20000000 1260000 146 members &lt; Hungry swimming &gt; 15% SDS.PAGE (Unselected treatment) 0.0030 330CXXXXX) 990000 117 Printed by Industrial and Consumer Cooperatives 195-498079 A7 B7 printed by Employees' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (195) [Table 2] Molecular weight of TP0 active part in Sephacryl S-200HR Total volume Total volume (after concentration) Total protein mass Average relative activity Relative activity amount [Table 3] Born from Fh amine-like microscope, a large cleavage enzyme-like segment of amine, an ether sequence, fragment name, amino acid sequence AP3 (κ ) XYYESZ (X is any of A, S, G, M, Q.) (B is E or! () ΑΡ6 (K) XRAAZ (X is E or 0) (B is E or N) AP7 (K ) AGXCSG (but the wholeness cannot be determined) AP8 (K) XPVPPACDPRLL (X is any of I., T, S)

API 2 (K) DSFLADVK AP5 (Unable to determine) AP 2 0 (Unable to determine) AP 2 1 (Unable to determine) AP23 (Unable to determine)-196-This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) I ------ ~ Pack ------ Order ----- AWI 0 (Please read the precautions on the back before filling this page) Low-molecular ΤΓΡ0 Control standard F3 33000 ~ 3000 6 4 8 ( ) 280 ¾ liter 5 1 · 3 g 20 0 0 0 1020000 Polymer TP0 reference standard F2 94000 ~ 33000 3420 ¾ liter 2 9 3 ml 2 6 2 g 7 8 3 8 2055000

α or R) TLPTXAVP (K or R) TLPTXAVP 498079 A7 B7 V. Description of the invention (19 [Table 4] 'The second digestion cuts the cystic fragment of _ A sp-N by the egg crust. Amino acid sequence AS P 1 (Unable to determine) ASP 2 (Unable to determine) ASP6. (Unable to determine) AS Pl 1 1 (Unable to determine) [Table 5] Three digestions by trypsin-TPC1 (Amino group of peptide fragment Acid sequence Η paragraph name ___________________________ amine_ 醅 sequence _________________________________________________________________________________ TP2 TP3 I ------...-Install ------ order ------ line (Please read the precautions on the back before filling in this Page) Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs The paper size applies to the Chinese National Standard (CNS) A4 (210 X 297 mm) 498079 A7 B7 V. Description of the invention (195) [Table 6]

Bi ^ odlw learn g 袅 _ + &lt; Chu 97¾. W1P'5

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs ¾ Zhen 15. .Wns,, ero5c * 50 £ wpx &gt; 061 0 卜 1 &gt; .u 8AZ ra olnp π ·· ΤΓιco ^ t 9 §Ι ^ Θ- μ5έ §00§ 3 §ss 00s 82 ο, νηi§ Ja-dv P30IW1 OOP 06 0.08 §ΓΊΓηονηίΝ0ΓΝ2009 ^ 2 ονοει 0.9 ^^^^-γοΛν spcs2 ISI 0 ^ 997 ^. &lt; Νπε 09seors. P61 fe ^ a 〇vn003 ζ HO inch oovo / i. Ϋ́ S3ocolp 91 saB-s4d3s036 ^ g £? Igz ^ Qgse 8 _l ^ fel-H 迚 Ώ 迚 Ώε) ΏΕ) ΏΕ} -198- This paper size applies Chinese National Standard (CNS) A4 Specifications (210X297 mm) (Please read the precautions on the back before filling out this page) 498079 A7 B7 V. Description of the invention (196) Form II ------ · 丨 install ------ order ----- line (Please read the precautions on the back before filling this page) Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economy -199- Dissolved propanol concentration XRP McA-RH8994 HTC Η 4-II-E Number of communities Number of communities Number of communities 3 0 · 5% ~ 3 2. 6% 0 33 4 18 32 .6¾ ~ 34.7% 0 1 3 2 19 5 0 34. 7% ~ 36.7% 67 290 291 2 30 3 6 · 7% ~ 3 8 · 8% 70 214 183 103 38, 8¾ ~ 40. 9¾, 2 15 5 28 40.9¾ ~ 43.0¾ 0 4 0 4 This paper size is applicable China National Standard (CNS) A4 specification (210X: 297 mm) 498079 A7 B7 V. Description of the invention (197) [Table 8] AP8 P [. Va | pr. Pr. Ah c "Asp pr" r "&quot; ^

Thr Se r

APHR -ACG CTG GGC CCI GA [GA-5,

APi-ZR

TC • GGG GCG CGG ACC CTC GG-5 # AA People AA (Please read the notes on the back before filling out this page) [Table 9] hTP05: 51 HTP0 3: 5 'EFI printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs S ^ GGA TCT TGG TTC ATT CTC AAG-3 ^ EF1 d S ^ CCT CAG ACA GTG GTT CAA AC-3f TTT GAA TTC GGC CAG CCA GAC ACCCCGGCC-3 f (Sequence_ 4 of 1-2 1)

TTT GCG GCC GCT CAT TAT TCG

TGT ATC CTG TTC AGG TAT CC-3T (corresponding to the anti-induction chain of serial number 4 75 7-78 0) -200 This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 498079 A7 B7 Description of the invention (198) [Table 10] 5 ′ side primer hTP〇-H: AGCAGAACCTCTCTAGTCCTC-3! (Sequence number 4 of 5 7 4-5 9 4) hTPO-K: 5, ACACTGAACGAGCTCCCAAAC-3, (sequence number 4 of 5 9 5-6 1 5) hTPO-N: 5 f- AAC TA CTGGC TC TG GGC TTC T- 3 τ (serial number 4 of 6 6 0-6 8 0) hTPO-O: 5 τ- AGGGATTCAGAGCCAAGATTC-3 f (Sequence number 4 of 6 9 2-7 1 2) [Table 11] ^ 3 f-side primer h TP 0 3 mix: 5,-TAG [: GGCCGC (T) 7 GGGG-3 'AAA A CTTT CCC hTP03anchor: 5f ~ TAGCGGCCGC (T) ii-3f (Please read the precautions on the back before filling out this page) -Printing and Threading Printed by the Employees' Cooperatives of the China Standards Bureau of the Ministry of Economics-201-This paper size applies to Chinese national standards ( CNS) A4 size (2IOX297 mm) 498079

GCCG-3 '-ccccgtccccaccccctgaacttccgcgcactggcacacacgacactccagcacctcacagtcgc 5 * .CTACAAAAAACCAACGACCTAATAAATAATCACCCCGGCTCCCCCACCTTCTGACCTTCCTGTTC TCTCT-S * 5 · .CACTTTACACACAACACGAAGGTCACAACCTCCCCCACCCCGCCTCATTATTTATTACCTCCTTC CTTTTTT-3 * 5 * -aaactccttccccactctcacgtgctgcactctcgtctctcccactgccccgaagttcaccccct CAAC-3f 5 · • ACCCCGGTTCTGCnCCGCCTGTCGACnCTCCCKGGTGAATGUAiUCCCAGATCGAAGAGAC CAAA'3 · 5 * -CTCACCTTTCGTCTCTTCCATCTCGCTTTTCCATTCACCCAGCCAGAACTCCACACCCCCAACCA CAAC-3f 5, · GCTCACCAUTCCTGCCTGaCTAACTCTGCTTCTCCAAGCCGTUTCGCTGCACGTGGCCAGCT TCGC- 3f 5 '-gctcgggccaagctccccacctccacccataaccccttccacaaccacacttactgcacccacca TCTC-3 · 5f -CCGACCTGCCTGTCTTCCCTCCTTOGCCAGCTGTCTGCCCAGGTTCGTCTCCTCCTCGCCCCTC TGCAC-3 * • 5 * -cacagactccacaccgccgagcagcagaccaacctccccacacacctcgccaaccacccaaca CACGCA-3.' '· 5 * -TCTCTGCTTGGCACCCACCTCCCGCCACACCGCCCTACCACTCCTCACAAGCATCCCAACCCT ATCTTCC-3 * 2: 5 * -aacacaggaacataccgttcggatccttgtgaccactgctaccgccctgtccccccaoctccc TCCCAAC-3 * Ministry of economy winning Falcon Industrial and consumer cooperatives -202- This paper size applies to China's National Standard (CNS) A4 size (210X297 mm). Hudu 79 Five printed by the Central Bureau of Standards of the Ministry of Economic Affairs and Consumer Cooperatives printed A7 B7, invention description (200) tg 1 3 ] 10 i 20 30 40 50 80

CTAGAAAAAA CCAAGGACCT AATAAATAAT GAGCCCCGCT CCGCCACCTT CTCACCTTCG

TTTTTT CGTTCCTCCA ΤΤΑΤΤΤΛΤΑ CTCCCGCCGA CCCCCTCGAA CACTGCAAGC

Xba I 2-One 70 80 3 90 1 00 1 1 0 120 KTTCTGTCT AAACTGCnC GCOACTCTCA CGTGCTGCAC TCTCGTCTGT CCCAGTGCCC acaacacaca'tttgacgaag ccctcacact gcaccacctc acagcacaca ccgtca.cgcc 4 130 HO 150 5 160! 70 180

GG, UGTTCAC CCGCTGCCGA CCCCGGTTCT GCTTCCGG.CT GTCGACTTCT CCCTGGGTGA CCTTCAACTC CCCGACGCCT CCGCCCAAGA CCAACCCCCA CACCTCAACA CCCACCCACT 6 190 200 210 220 7 230 240

ATCGAAAACC CACATGCAAC AGACCAAACC TCACCACATC CTCGCTCCAC TAACTCTCCT

TACCTTTTGC CTCTACCTTC TCTGCTTTCG AGTCCTGTAG GACCCACCTC ATTGACACCA 8 a 250 260 270 280 290 £ 300

TCTGCAACGC CTTATGGCTG CACGTGCCCA CCTTCGCCCC ACCTCCCTCT CTTCCCTCCT ACACCTTCCC CAATACCGAC CTCCACCCCT CCAACCCGCC TGCACGGACA CAACGGACCA

JJ '3 1 0 320 S30 3-iO 350 360

TGCCCAGCTG TCTCGCCAGG TTCGTCTGCT CCTCGGCGCT CTGCAGTCTC TGCTTGGCAC

ACCCGTCCAC AGACCGCTCC AACCACACCA CGACCCCCGA CACCTCACAG ACCAACCGTG Π 370 S80 390 400 4! 0 420

CCACCTCCCC CCACACCCCC GTACCACTCC TCACAACGAT CCGAACCCTA TCTTCC CCTCCACCGC CGTGTCCCCG CATGGTGACG ACTCTTCCTA CGCTTCCCAT ACAACCACAC Αλ 12 ΒβώΙΠ 203 (Please read the precautions on the back before filling this page)

$ ^ 张 纽 it Financial Standards (CNS) (210X297mm) 498079 A7 B7 V. Description of Invention (2〇1) C «1 4]

SSE1: CTAATCAG

SSE2: AATTCTCATTAGAGCT

SacI SSE1 EcoRI 5 (-CTAATCAG-3 '3 * -TCGAGATTACTCTTAA-S · SS.E2 (Please read the precautions on the back before filling out this page) Binding and Ordering Printed by the Consumers' Cooperatives of the China Standard Associate Bureau of the Ministry of Economic Affairs 1 5] 1 3: 51-GATCCGAACGCTATCTTCCTGTCTTfCCACCACCTGCTGCGT-3, 1 4: 5 * -TTTGCCACGCACCAGGTGCTGGAAAGACACGAAGATAGCCTTCG-3, 1 5: 5 * -GCCAAAGTTCGTTTCCCCTGGGCCCCAGGACCA-GCATCACGCA-TCAGC-ATC 3, 1 8: 5 * -AGCTTTCATTAAGACGGAACACCAGTGGTTGGCGGCGCCCGACGA-3, 1 9: 5 * -AAGGATCCGAACGCTATCTTCCTC-3, 2 〇: 5 * -AGAAGCTTTCATTAAGACGGAACA-3, line-2 0 4-This paper is in accordance with the Chinese National Standard (CNS) A4 specifications (210X297 mm) V. Description of the invention (202) [Table 16] 2- 9: 51-CACCTGGTACAGGTGCTTTCATTATTTATTACCTCCTTCGTTTTTT-S, 3- 3: 5 * -CCTGCATGTCATTTACGGGTCCTGTCTAAACTGCTGCG-3, 4- 3: 5 * -CGCAGCAGTTTAGACAGGACCCGTAAATC , 10 Μ 20 30 40 50 60

CTAGAAAAAA CCAAGCAGCT AATAAATAAT GAAAGCACCT CTACCACCTC CATGTGATTT

TTTTTT CGTTCCTCCA TTATTTATTA CTTTCCTGCA CATGCTGCAC GTACACTAAA 2J. 3 ^ 3 70 80 acgggtcctg tctaaactcc tccc

TGCCCAGCAC AGATTTGACC ACCC tl Window of the Central Standards Bureau of the Ministry of Economic Affairs (printed by the Industrial and Consumer Cooperatives-20 5-498079 A7 B7 V. Description of the invention (205) [Table 17] Contains a certain amine sequence in the synthetic cysteine rat TP0 ( 9-28) (fcRTl area):

PRL LNK.LLRDSYLLHOLSQ (but the L of No. 24 is derived from a genetically determined amino acid residue of S)-rat T P 0 (4 8-8 β) (suck P R 2 region:

FSLGEWKTQTEQSKAQDILGA taiwan mouse TP0 (163-1 80): (like RT4 area): SRTSQLLTLNKFPNRLUl. (But Ul after K 178 is derived from genetically determined amino group_residue is TSG) (Please read the note on the back first) Please fill in this page for further information.) • Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs-20 6-This paper size applies to China National Standard (CNS) A4 (210X297 mm)

Claims (1)

498079 No. 083105113 Patent Application Benefit Chinese Patent Application Amendment (June 91). Sixth, application: i | ψρ. Wai I j —91, 6 · m Ο 14 Amendment ^ JJ: Ψ Λ 1 Supplement 1 — 1 • A protein with thrombopoietin (TPO) activity, the amino acid sequence of which is composed of 1-332 amino acid residues shown in SEQ ID NO &gt;: 6: [SEQ ID NO: 6] GGCCAGCCAG ACACCCCGGC CAGA 24 ATG GAG CTG ACT GAA TTG CTC CTC GTG GTC ATG CTT CTC CTA ACT GCA 72 Met Glu Leu Thr Glu Leu Leu Leu Val 'Val Met Leu Leu Leu Thr Ala -20 -15 -10 AGG CTA ACG CTG TCC AGC CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC 120 Arg Leu Thr Leu Ser • Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val -5 1 5 10 CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC 168! Leu Ser Lys Leu Leu Arg Asp Ser His • Val Leu His Ser Arg Leu Ser 15 20 25 CAG TGC CCA GAG GTT CAC CCT TT G CCT ACA CCT GTC CTG CTG CCT GCT 216 Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala 30 35 40 GTG GAC TTT AGC TTG GGA GAA TGG AAA ACC CAG ATG GAG GAG ACC AAG 264 Val Asp • Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys 45 50 55 GCA CAG GAC ATT CTG GGA GCA GTG ACC CTT CTG .CTG GAG GGA GTG ATG 312 Ala Gin Asp lie Leu Gly Ala Val Thr Leu Leu Leu Glu Gly. Val Met 60 65 70 75 GCA GCA CGG GGA CAA CTG GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG 360 Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly 80 85 90 CAG CTT TCT GGA CAG GTC CGT CTC CTC CTT GGG GCC CTG CAG AGC CTC 408 Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu 95 1.00: 105 CTT GGA ACC CAG CTT CCT CCA CAG GGC AGG ACC ACA GCT CAC AAG GAT 456 Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp 110 115 120 CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC CTG CT C CGA GGA AAG GTG 504 Pro Asn Ala lie Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val 125 130 135 CGT TTC CTG ATG CTT GTA GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC 552 Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala. 140 145 -1-150 155 This paper size is applicable to Chinese National Standard (CNS) A4 size (210X297 mm) A8 B8 C8 D8 Six. Patent application scope CCA ccc ACC ACA GCT GTC CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG 600 Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser. Leu Val Leu Thr Leu 160 165 170 AAC GAG CTC CCA AAC AGG ACT TCT GGA TTG TTG GAG ACA AAC TTC ACT 〇 4 8 η Glu Leu Pro Asn Arg Thr Ser Gly -Leu Leu Glu • Thr Asn Phe Thr 175 180 185 t GCC TCA GCC AGA ACT ACT GGC TCT GGG CTT CTG AAG TGG CAG CAG GGA 696 Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gin Gin Gly 190 195 200 TTC AGA GCC AAG ATT CCT GGT CTG CTG AAC CAA ACC TCC AGG TCC CTG 744 Phe Arg Al a Lys lie Pro Gly Leu Leu Asn Gin Tin Ser Arg Ser Leu 丨 205 210 2.15 GAC CAA ATC CCC GGA TAG CTG AAC AGG ATA CAC GAA CTC TTG AAT GGA 792 Asp Gin lie Pro Gly Tyr Leu Asn Arg lie His Glu Leu Leu Asn Gly 220 225 230 235 ACT CGT GGA CTC TTT CCT GGA CCC TCA CGC AGG ACC CTA GGA GCC CCG 840 Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro 240 245 250 GAC ATT TCC TCA GGA ACA TCA GAC ACA GGC TCC CTG CCA CCC AAC CTC 888 Asp lie Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu 255 2 60 265 CAG CCT GGA TAT TCT CCT TCC CCA ACC CAT CCT CCT ACT GGA CAG TAT. 936 'Gin Pro Gly Tyr Ser Pro Ser Pro Thr • His Pro Pro Thr Gl.y Gin Tyr 270 275 280 ACG CTC TTC CCT CTT CCA CCC ACC TTG CCC ACC CCT GTG GTC CAG CTC 984! Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gin Leu 285 290 295 CAC CCC CTG CTT CCT GAC CCT TCT GCT CCA ACG CCC ACC CCT ACC AGC 1032 His Pro L eu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser 300 305 310 315 CCT CTT CTA AAC ACA TCC TAC ACC CAC TCC CAG AAT CTG TCT CAG GAA 1080 Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gin Glu 320 325 330 | GGG TAAGGTTCTC AGACACTGCC GACATCAGCA TTGTCTCATG TACAGCTCCC1 1133 Gly TTCCCTGCAG GGCGCCCCTG GGAGACAACT GGACAAGATT TCCTACTTTC TCCTGAAACC 1193 CAAAGC2A standard China 12 standard AGATCAC ATCAC AGATCAC 1 (Mm) 498079 A8 B8 C8 D8 VI. Patent Application Fanyuan 2. A protein with TPO activity 'whose amino acid sequence is any of the amines shown in the amino acid sequence shown in SEQ ID NO: 6 below Composition of amino acid residues: Amino acid 1-151, Amino acid 1-153; Amino acid 1-154; Amino acid 1-155; Amino acid 1-156; Amino acid 1-157; Amine Amino acid 1-163; amino acid 1-171; amino acid 1-191; amino acid 1-211; Acids 1-231, amino acids 1-232; 7-163 or amino acids. 3. The protein according to item 2 of the scope of the patent application, which is a protein consisting of amino acid residues 1 to 63. 4. A protein with TPO activity, whose amino acid sequence is shown in SEQ ID NO: 2: C SEQ ID NO: 2] 60 120 17 2 220 GGTGTACCTG GGTCCTGAAG CCCTTCTTCA CCTGGATAGA TTCCTTGGCC CACCTGTCCC CACCCCACTC TGTGCAGAGG TACAA ^ AAGCT CAAGCCGTCT CCATGGCCCC AGG GCCCCACACA GGGAGCCACT GCAGTCAGAC ACCCTGGGCA GA ATG GAG CTG ACT GAT TTG CTC CTG GTG GCC ATA CTT CTC CTC ACC GCA _ -3 -___ This paper size applies to China National Standards (CNS) A4 specifications (210x 297 mm) A8 B8 C8 D8 Six The scope of patent application Met Glu Leu Thr Asp Leu Leu Leu Val Ala He Leu Leu Leu Thr Ala -20 • -15 -10 AGA CTA ACT CTG TCC AGC CCA GTT CCT CCC GCC TCC GAC CCC AGA CTC 268 Arg Leu Thr Leu Ser Ser Pro Val Pro Pro Ala Cys Asp Pro Arg Leu • -5 1 5 10 CTA AAT AAA CTG CTT CGT GAC TCC TAC CTC CTC CTT CAC AGC CGA CTG AGT 316 Leu Asn Lys Leu Leu Arg Asp Ser Tyr Leu Leu His Ser Arg Leu Ser 15 20 25 j CAG TGT CCT GAC GTC AAC CCT TTG TCT ATC CCT GTC CTG CTG CCT GCT 364 Gin Cys Pro Asp Val Asn Pro Leu Ser lie Pro Val Leu Leu Pro Ala j 30 35 40 GTG GAC TTT AGC CTG GGA GAA TGG AAA ACC CAG * ACG GAA CAG AGC AAG 412 Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Thr Glu Gin Ser Lys 45 50 55 GCA CAG GAC ATT CTA GGG GCA GTG TCC CTT CTA CTG GAG GGG GTG ATG 460 Ala Gin Asp lie Lea Gly Ala Val Ser Leu Leu Leu Glu Gly Val Met 60 65 70 75 GCA GCA CGA GGA CAG TTG GAA CCC TCC TGC CTC TCA TCC CTC CTG GGA 503 Ala Ala Arg Gly Gin Leu Glu Pro Ser Cys Leu Ser Ser Leu Leu Gly 80 • 85 90 CAG CTT TCT GCT CAGT GG CTT CGC CTC CTC TTG GGA GCC CTG CAG GGC CTC 556 Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Gly Leu 95 100 105 CTA GGA ACC CAG GTA AGT ccc CAG ACC TAT AGA AAC TAC CCT CTT ACT 604, Leu Gly Thr Gin Val Ser Pro Gin Thr Tyr Arg Asn Tyr Pro Leu Thr 110 115 120 CAG TTC C.TC 613 Gin Phe Leu 125 TAAGGAC CTG GGAAAAGACA AGGGATTCTA .GATTCTAGGT GTCTTCAGTG TATGAAAGCT 673 GGTCTATACG GAGTGATGCT TCTCAGCCAC 'AATACCTGGG TGCTGGCAGT AAATCTTTCC 733 j ACCTTAGTGA GAAGAGGCCT GATATGTGGG CCAACTCACT GGCCTCAGGC CCATCCTCTG 793 J CCTTGAGCTT CCTCCACAGG GCAGGACCAC AGCTCACAAG • GACCCCAGTG CCCTCTTCTT 853 I GAGCTTGCAA CAACTGCTTC GGGGAAAGGT GCGCTTCCTG CTGCTGGTAG AAGGTCCCGC 913 j CCTCTGTGTC AGACGGACCC TTCCCACCAC AGCTGTCCCA AGCAGAACCT CTCAACTCCT 973 CACACTAAAC AAGTTCCCAA ACAGACTTCT GGATTGTTGG AGACGAACTT CAGTGTTGTA 1033 GCCAGAACTG CTGGCCCTGG ACTTCTGAAC AGGCTTCAAG GATTCAGAGC CAAGATTATT 1093 CCTGGTCAGC TAAATCAAAC CTCCGGGTCC TTAGACCAAA TCCCTGGATA CCTGAACGGG 1153 ACACACGAAC CTGTGAATGG AACTCATGGG CTCTTTGCTG GGACCTCACT ACAGACCCTG 1213 GAAGCCCCAG ACGTTGTGCC AGGAGCTTTC AACAAAGGCT CG.TTGCCACT CAACCTCCAG 1273 -4- this paper scale applicable Chinese national standard (CNS) A4 Specifications (210X297 mm) A8 B8 C8 D8 VI. Patent Application Range AGTGGACTTC CTCCTATCCC AAGCCTTGCT GCTGATGGAT ACACACTTTT CCCTCCTTCA 1333 CCTACCTTCC CCACCCCTGG GTCTCCACCC CAGCTCCCCC CCGTTTCCTG ACCCCTCCAC 1393 CACCATACCT AACTCTACCA ACCCTCATCC AGGACTTGGT CTCAGTAAGC GTCCCGTGCA 1453 CTGGCACGGA GCGCGATCGT CTGCAACATC TCTCAGGGGC AAGCTTCCTC AGGAAGGCTC 1513 TGAGGCAGCT CACTAGACAT CCTGCTCTCG CCTAACGGGC CCTGGGAAAG GGATACACAG 1573 GCCAGGACAC TGTACAACCT TAGGAGCGAT TTTTTTCTTA ACCTATCAAC AATATTCATC 1633 AGAGCAAAAA A AAAA AAAA A AA AAAA AAAA 1663 5. The scope of the patent The protein of item 2, whose amino acid sequence is 1-232 amino acid residues as shown in SEQ ID NO: 4: [SEQ ID NO: 4] GGCCAGCCAG ACACCCCGGC CAGA 24 ATG GAG CTG ACT GAA TTG CTC CTC GTG GTC ATG CTT CTC CTA ACT GCA 72 Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala -20 -15 -10 AGG CTA ACG CTG TCC AGC CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC 120 Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Va 丄 -5 1 5 10 CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC 168 Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser 15 20 25 CAG TGC CCA GAG GTT CAC CCT TTG CCT ACA CCT GTC CTG CTG CCT GCT 216 Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala 30 35 40 GTG GAC TTT AGC TTG GGA GAA TGG AAA ACC CAG ATG GAG GAG ACC AAG 264 Val Asp Phe Ser Leu Gly Glu Trp Lvs Thr Gin Met Glu Glu Thr Lys 45 50 55 GCA CAG GAC ATT CTG GGA GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG 312 Ala Gin Asp lie Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met 60 65 70 75 GCA GCA CGG GGA CAA CTG GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG 360 Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly 80.: 85 90 · CAG CTT TCT GGA CAG GTC CGT CTC CTC CTT GGG GCC CTG CAG AGC CTC 408 Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu 95 1.00 105 -5- This paper size applies to China National Standard (CNS) A4 (210 x 297 mm) 498079 A 8 B8 C8 D8 785 845 861, patent application scope CTT GGA ACC CAG CTT CCT CCA CAG GGC AGG ACC ACA GCT CAC AAG GAT 45β Leu Gly Thr Gin Leu Pro Prp Gin Gly Arg, Thr Thr Ala His Lys Asp 1,10 115 120 CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC CTG CTC CGA GGA AAG CTG 504: ^,% 1 Pro Asn Ala lie Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val | 125 130 135-I CGT TTC CTG ATG CTT GTA GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC 552 | _] Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala | 140 '145 •' 150 155 j CCA CCC -ACC ACA GCT GTC CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG 600 丨 Pro Pro'Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu 160 165 1 * 70 AAC GAG CTC CCA AAC AGG ACT TCT GGA TTG TTG GAG ACA AAC.TTC ACT 648 Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr 175 180 · 185 GCC TCA GCC AGA ACT ACT GGC TCT GGG CTT CTG AAG TGG CAG CAG GGA 696 Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gin Gin Gly 190 · 195 200 TTC AGA GCC AAG ATT CCT GGT CTG CTG AAC CAA ACC TCC AGG TCC CTG 744 Phe Arg Ala Lys lie Pro Gly Leu Leu Asn Gin Thr Ser Arg Ser Leu 205 210 215 GAC CAA ATC CCC GGA TAC CTG AAC AGG ATA CAC GAA CTC TT Asp Gin lie Pro Gly Tyr Leu Asn Arg lie His Glu Leu 220 225 230 NO: 7 shows: [SEQ ID NO: 7] GCGGCACGAG GGGGGTGTCT GGCTGGCGTG GCTCCCTGTT TGGGGCCTCT CCCCTGAATC 60 CTTCCTGGGG CCATGGAGGC CAGACAGACA CCCCGGCCAG A 101 ATG GAG CTG ACT GAA TTG CTC CTC GTG GTC ATG. Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Thr Ala -20 -15 '-10 AGG CTA ACG CTG TCC AGC CCG GGT CCT CCT GCT TGT GAC CTC CGA GTC 193 Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val -5 1 5 10 -6-This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 498079 A8 B8 C8 D8 VI. Patent application scope CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC 245 Leu Ser Lys Leu Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser 15 20 25 CAG TGC CCA GAG GTT CAC CCT TTG CCT ACA CCT GTC CTG CTG CCT GCT 293 Gin Cys Pro Glu Val His Pro Leu P · χΌ Thr Pro Val Leu Leu Pro Ala 30 .. 35 40; GTG GAC TTT AGC TTG GGA GAA TGG AAA ACC CAG ATG GAG GAG ACC AAG 34lj Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys f! 45 50 55 GCA CAG GAC ATT CTG GGA GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG 389: Ala Gin Asp lie Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met j 60 65 70 75 ί GCA GCA CGG GGA CAA CTG GGA CCC • ACT TGC CTC TCA TCC CTC CTG GGG 437! Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly i 80 85 90 CAG CTT TCT GGA CAG GTC CGT CTC CTC CTT GGG GCC CTG CAG AGC CTC 485 Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu 95 100 105 CTT GGA ACC CAG CTT CCT CCA CAG GGC AGG ACC ACA GCT CAC AAG GAT 533 Leu Gly Thr Gin Leu Pro Pro Gin Gly-.Arg Thr ThrT Ala His Lys Asp .110 115 120 --- ---- CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG 581 Pro Asn Ala lie Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val 125 130 135 CGT TTC CTG ATG CTT GTA GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC 629 Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala 140 145 150 155 CCA CCC ACC ACA GCT GTC CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG 677 Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Lhuiu 160 165}. 170 AAC GAG CTC CCA AAC AGG ACT TCT GGA TTG TTG GAG ACA AAC TTC ACT 725 Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr 175 180 185 GCC TCA GCC AGA ACA ACT GGC TCT GGG CTT CTG AAG TGG CAG CAG GGA 773 丨 Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gin Gin Gly! F 190 195 200 i TTC AGA GCC AAG ATT CCT GGT CTG CTG AAC CAA ACC TCC AGG TCC CTG 821 Phe Arg Ala Lys lie Pro Gly Leu Leu: Asn Gin Thr Ser Arg Ser Leu 205 · 210 215 i GAC CAA ATC CCC GGA TAC CTG AAC AGG ATA CAC GAA CTC TTG AAT GGA 869 Asp Gin lie Pro Gly Tyr Leu, As'n A * rg lie His Glu Leu Leu Asn Gly 220 225 ..... * 230 235 235 -7 - The paper size of this paper applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) 498079 A8 B8 C8 _______D8 VI. Application scope of patent ACT CGT GGA CTC TTT CCT GGA CCC TCA CGC AGG ACC CTA GGA GCC CCG Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro 240 245 250 GAC ATT TCC TCA GGA ACA TCA GAC ACA GGC TCC CTG CCA CCC AAC CTC Asp He Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu 255. 260 265 CAG CCT GGA TAT TCT CCT TCC CCA ACC-CAT CCT CCT ACT GGA CAG.TAT Gin Pro G.ly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gin Tyr 270 275 280 ACG CTC TTC CCT CTT CCA CCC ACC TTG CCC ACC CCT GTG GTC CAG CTC Thr Leu Phe Pro Leu Pro Pro Thr-Leu Pro Thr Pro Val Val Gin Leu 285 290 295 CAC CCC CTG CTT CCT GAC CCT TCT GCT CCA ACG CCC ACC CCT 'ACC AGC ·: ^ His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser 300 305 310 315 CCT CTT CTA AAC ACA TCC TAC ACC CAC TCC CAG AATf CTG TCT CAG GAA Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gin Asn Leu Ser Gin Glu 320 325 330 GGG TAAGGTTCTC AGA CACTGCC GACATCAGCA TTGTCTCGTG TACAGCTCCC Gly TTCCCTGCAG GGCGCCCCTG GGAGACAACT GGACAAGATT TCCTACTTTC TCCTGAAACC 1270 CAAAGCCCTG GTAAAAGGGA TACACAGGAC TGPlAAAGGGA ATCATTTTTC ACTGTACATT 1330 ATAAACCTTC AGAAGCTATT TTTTTAAGCT ATCAGCAATA.CTCATCAGAG CAGCTAGCTC 1390;:. TTTGGTCTAT TTTCTGCAGA AATTTGCAAC TCACTGATTC TCTACATGCT CTTTTTCTGT 1450 GATAACTCTG CAAAGGCCTG GGCTGGCCTG GCAGTTGAAC AGAGGGAGAG ACTAACCTTG 1510 AGTCAGAAAA CAGAGAAAGG GTAATTTCCT TTGCTTCAAA TTCAAGGCCT TCCAACGCCC 1570 CCATCCCCTT TACTATCATT CTCAGTGGGA CTCTGATCCC ATATTCTTAA CAGATCTTTA 1630 CTCTTGAGAA ATGAATAAGC TTTCTCTCAG AAATGCTGTC CCTATACACT AGACAAAACT 1690 GAAAAAAAAA AAAAAAAAAA AAAAAAAAAA A &gt; 1721 a 7. A protein with a TPO acid sequence of ID-1. Its amino group is 1 Composition of 174 amino acid residues: [SEQ ID NO: 10] CTG GTT CCG CGT GGA TCC CCG GCT CCG CCA GCT TGT GAC CTT CGT CTT 48 Leu Val Pro Arg Gly Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val +1 5 10 CTG TCT AAA CTG CTT CGC GAC TCT CAC GTG CTG CAC TCT CGT. CTG TCC 96, Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser ^ 15 20. 25 -8- This paper size applies to China National Standard (CNS) A4 (210 x 297 mm) 917 965 1013 1061 1109 1157 1210: equipment A8 B8 C8 D8___ VI. Patent Application Range CAG TGC CCG GAA GTT CAC CCG.CTG CCG ACC CCG GTT CTQ CTT CCG GCT 144 Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Ceu Pro Ala 30 35 40 GTC GAC TTC TCC CTG GGT GAA TGG AAA ACC CAG ATG GAA GAG ACC AAA 192 Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys 45 50 55 GCT CAG GAC ATC CTG GGT GCA GTA ACT CTG CTT CTG GAA GGC GTT ATG 240 Ala Gin Asp lie Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met 60 65 70 75 GCT GCA CGT GGC CAG CTT GGC CCG ACC TGC CTG TCT TCC CTG CTT GGC 288 Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly 80: 85, 90 CAG CTG TCT GGC CAG GTT CGT CTG CTG CTC GGC GCT CTG CAG TCT CTG 336 Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu 95. 100 105 CTT GGC ACC CAG CTG CCG CCA CAG GGC CGT ACC ACT GCT CAC AAG GAT 384 Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp 110 115 120 CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG 432? Ro AsnAJLa lie Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val 125 130 135 CGT TTC CTG ATG CTT GTA GGA GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC 490 Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala 140 145 150 155 CCA CCC ACC ACA GCT GTC CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG 528 Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu 160-165 170 AAC GAG CTC TAATGAGAAT TC 549 Asn Glu Leu 〇8. A protein with TPO activity, the amino acid sequence of which is shown in SEQ ID NO: 1 1 [SEQ ID NO: 1 1] CTAGAAAAAA CCAAGGAGGT AATAAATA 28 ATG AAA GCA CCT GTA CCA CCT GCA TGT GAT TTA CGG GTC CTG TCT AAA 7 〇Met Lys Ala Pro Val Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys + 1 5 10 CTG CTG CGC CGC GAC TCT CAC GTG CTG CAC TCT CGT CTG TCC CAG TGC CCG 124 Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro 15 20 25. 30 -9- This paper size applies to China National Standard (CNS) A4 (210X297 mm) 498079 A8 B8 C8 D8 VII. Application Patent scope GAA GTT CAC CCG CTG CCG ACC CCG GTT CTG CTT CCG GCT ^ GTC GAC TTC 172 Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe 35:40 45 TCC CTG GGT GAA TGG AAA ACC CAG ATG GAA GAG ACC AAA GCT CAG GAC 220 Ser Leu Giy Glu · Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp • 50 55. 60 ATC CTG GGT GCA GTA ACT CTG CTT CT0 'GAA GGC GTT ATG GCT GCA CGT 268 lie Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg β5 70.: Ί 5 GGC CAG CTT GGC CCG ACC TGC CTG TCT TCC CTG CTT GGC CAG CTG TCT 316 Gly Gin Leu Gly Pro Thr Cys Leu Ser Leu Leu Gly Gin Leu Ser 80 85 90 GGC CAG GTT CGT CTG CTG CTC GGC GCT CTG CAG TCT CTG CTT GGC ACC 364 Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr 95 100 105 110 CAG CTG CCG CCA CAG GGC CGT ACC ACT GCT CAC AAG GAT CCG AAC GCT 412 Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asa Ala 115 120 125 ATC TTC CTG TCT TTC CAG CAC CTG * CTG CGT GGC ΛΑΑ GTT CGT TTC CTG 4 60 lie Phe Leu Ser Phe Gin His L eu Leu Arg Gly Lys Val Arg Phe Leu 130 135 ..... 140 ATG CTG GTT GGC GGT TCT ACC CTG TGC GTT CGT CGG GCG CCG CCA ACC 508 Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr 145 150 155 ACT GCT GTT CCG TCT TAATGAAAGC TT 535 Thr Ala Val Pro Ser 160 〇9 · According to the first patent application, the protein has additional Met residues at the -1 position of the protein. 10 · The protein according to item 2 of the patent application scope, which has additional Met residues at the · 1 position of the protein. 1 1 · The protein according to item 1 of the scope of the patent application, which has additional residues of Met and Lys at the -1 and -2 positions of the protein, respectively. -10-This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) A8 B8 C8 12. The protein according to item 2 of the patent application scope, which has additional residues of Met and Lys at the "" and _2 positions of the protein, respectively. 13.-A protein with TPO activity, its amino The acid sequence is composed of 1-163 amino acid residues shown in SEQ ID NO: 6, but it contains the following substitutions: • S r1 and A1 a3 residues are replaced by a 1 a and V a 1 residues, respectively. 1 4. A pharmaceutical composition for increasing platelets, which contains a protein according to any one of claims 1 to 5 and 7 to 13 of the scope of patent application. 1 5-a pharmaceutical composition for treating platelet disorders, It contains the protein according to any one of claims 1 to 5 and 7 to 13 in the scope of the patent application. 1 6 · —A pharmaceutical composition for treating thrombocytopenia, which contains the claims 1 to 5 in the scope of patent application And the protein of any one of items 7 to 13. 17. The pharmaceutical composition according to item 16 of the scope of patent application, wherein the thrombocytopenia is caused by chemotherapy, radiation therapy or bone marrow transplantation. ϊϋStandard Secretary) Domain _X297 mm) Binding Line -11-