WO1996003434A1 - Megakaryocyte differentiation/proliferation factor - Google Patents

Abstract

A novel megakaryocyte differentiation/proliferation factor (NTPO protein) usable as a remedy for thrombocytopenia. A DNA coding for this protein can be applied in the production of the protein by genetic engineering techniques and the analysis of genes coding for the protein.

Description

 Description of damage Megakaryocyte differentiation and growth factor Background of the invention

 Field of the invention

 The present invention relates to a novel megakaryocyte differentiation growth factor. The present invention can be applied to the field of thrombocytopenia treatment.

 Description of related technology

 Conventionally, oat platelet transfusion has been used to treat thrombocytopenic patients. However, the treatment of concentrated platelet transfusion requires the provision of a blood donor to supply the OAT platelet product, the inability to store platelets for a long time, the matching of major histocompatibility antigens, and virus infection. However, there are problems such as the need to consider the dangers of traffic, and this is not always a problem. Instead of transfusion, if patients could improve their own platelet hematopoietic ability to treat thrombocytopenia, such problems could be overcome. In other words, if the administration of a therapeutic agent can specifically stimulate the differentiation and proliferation of megakaryocytes, which are progenitors of platelets, and promote the increase of platelets, it is a highly useful alternative to vague platelet transfusion It can be a treatment for thrombocytopenia.

It has been suggested that the plasma of animals that have been experimentally rendered thrombocytopenic has an activity that promotes megakaryocytic-platelet hematopoiesis, and the substance responsible for this activity is called thrombopoetin. . More recently, a substance having thrombopoietin activity (hereinafter abbreviated as TP0. TP0 is a substance that is also referred to as c-Mp1 ligand or megakaryocyte proliferation / differentiation factor.で Ρ Ρ Ρ 統一 Ρ 構造 と 共 に 構造 構造 構造 構造 構造 構造 構造 構造 構造 構造 方法 方法 方法 方法 方法 方法 構造 方法 方法 方法 方法 方法 方法 方法 方法 方法 方法 方法 方法 方法. , ature, Vol.369.p.533 (1994), S. Lok et al., ature.Vol.369.p.565 (1994) K. Kaushansky et al..Nature.Vol.369.p.568 ( 1994) and TD Bartley et al. Cell, Vol. 77, p. 1117 (1994)]. As a treatment for thrombocytopenia that replaces platelet transfusion, a treatment that uses a more effective therapeutic agent with fewer side effects is desired, and the development of a therapeutic agent for that purpose is urgently needed. However, TPO has not yet been tested in clinical trials, and its effectiveness is uncertain. Under these circumstances, if a plurality of novel therapeutic agents having the effect of promoting the increase of platelets are provided, it is possible to expect a further excellent therapeutic effect on thrombocytopenia by using them. Disclosure of the invention

 Summary of the Invention

 The present invention provides a novel protein which can replace TPO and has a more useful protein and a gene encoding the protein, and a method for mass-producing such a protein using gene recombination technology. Aim.

 The present inventors have screened cDNA libraries derived from various human tissues to achieve the above object. As a result, the present inventors have discovered a cDNA encoding a novel protein having a different amino acid sequence from TPO. The present inventors have also succeeded in expressing a novel protein having megakaryocyte and platelet increasing activity (hereinafter abbreviated as NTPO protein) by genetic recombination technology. The present invention has been completed in this manner.

 That is, the present invention

 (1) E: a protein having an amino acid sequence consisting of all or a part of the amino acid sequence described in Column No. 1 and having megakaryocyte differentiation / proliferation activity; and a protein substantially equivalent to the protein. A protein,

 (2) a peptide having an amino acid sequence consisting of all or part of the amino acid sequence at position 160-286 in the amino acid sequence of SEQ ID NO: 1; A peptide that is substantially equivalent to a peptide,

 (3) a DNA encoding the protein having the amino acid sequence of SEQ ID NO: 1, a DNA of SEQ ID NO: 2, and a DNA substantially equivalent thereto;

 (4) a vector containing the DNA,

(5) a transformant carrying the vector, (6) a method for producing a protein having megakaryocyte differentiation / proliferation activity, comprising culturing the transformant and collecting an expression product thereof;

 (7) a polyclonal antibody which has an anchorage to at least a part of the amino acid sequence at position 160 to 286 in the amino acid sequence of SEQ ID NO: 1. Or a monoclonal antibody,

 (8) DNA having a nucleotide sequence consisting of all or a part of the nucleotide sequence shown in SEQ ID NO: 2 is used as a probe or a primer, and is hybridized with a test DNA. A method for analyzing an arrester of a protein having a megakaryocyte differentiation / proliferation activity, and

 (9) A platelet proliferation promoter or platelet comprising, as an active ingredient, a protein having an amino acid sequence consisting of all or a part of the amino acid sequence of SEQ ID NO: 1 and having megakaryocyte differentiation / proliferation activity. A pharmaceutical composition comprising a prophylactic agent or a therapeutically effective amount of the protein and an excipient;

About.

 When the present inventors compared the NTP0 protein of the present invention with TP0 reported by Sauvage et al. [See Nature. Vol. 369, p. 533 (1994)], the NTPO protein was found to be TP0 It was found that the intermediate cDNA was deleted due to further splicing inside the cDNA. That is, based on the numbers in the nucleotide sequence of TPO published in Nature, vol. 369 [see Fig. 3a in the report], the NTPO protein is found in the nucleotide sequence of TP0. As a result of the deletion of positions 693-808 of the TPO, the positions after position 139 in the amino acid sequence of TPO were deleted and changed. This base deletion causes a shift in the amino acid-encoding triplet. As a result, in the NTPO protein, the amino acid sequence starting at position 160 in SEQ ID NO. It is completely different. That is, the NTPO protein has the same amino acid sequence at position 11-159 in SEQ ID NO: 1 as TP0; the amino acid sequence at positions 160-286 is novel and The number of amino acids is 67 less than TPO. The amino acid sequence described in SEQ ID NO: 1? Kooi-, positions 1 to 21 indicate signal peptides.

DNA containing all or part of the DNA encoding the NTP0 protein of the present invention The NTP0 protein gene can be analyzed by using it as a probe or a primer and hybridizing it with a test DNA. For example, since the nucleotide sequence coding around position 160 of the amino acid sequence of the NTP0 protein (SEQ ID NO: 1) is a nucleotide sequence characteristic of the NTP0 protein, At least a part of the nucleotide sequence of the probe or primer may be appropriately selected so that this position can be detected.

 A portion of the DNA that encodes the NTP0 protein of the DNA used as a probe or primer consists of at least six consecutive nucleotides (nucleotides). It is preferably at least 8 bases which are preferably contiguous, more preferably at least 10 bases which are contiguous and particularly preferably at least 15 bases which are contiguous — Consists of 25 bases.

 Examples of the DNA encoding the NTP0 protein include DNA of SEQ ID NO: 2S, but the DNA is not limited thereto, and each amino acid of the NTP0 protein is encoded by The present invention also encompasses DNAs composed of various types of triplets to be loaded.

 The NTPO protein can be used as an element for producing antibodies or as a component of research or diagnostic reagents using the antibodies, by using all or part of the NTPO protein as a vector. Can be. “Ebitotop” refers to the antigenic determinant of a polypeptide and is generally composed of at least six amino acids. It is known that a polypeptide composed of six amino acids binds to an antibody [WO 84/03564 (published September 13, 1984, assignee: COMMONWEALTH SERUM LABS). AND GEYSEN, HM) and the corresponding published patent publication 60-500684].

 A portion of an NTPO protein refers to at least six consecutive, preferably at least six consecutive amino acids based on the amino acid sequence of the NTPO protein of the present invention. At least 10 amino acids, more preferably at least 10 amino acids, particularly preferably at least about 15-25 It means a polypeptide consisting of two amino acids.

The characteristic structure of the NTP0 protein is an amino acid sequence at positions 160 to 286 in SEQ ID NO: 1. Therefore, it is preferable to select the web from this site. In addition, the anti-NTP0 antibody should recognize the position around position 160 in the amino acid sequence. Is preferred. By using such an antibody, the NTPO protein can be distinguished from the TPO, and the NTP0 protein can be quantified.

 A protein having megakaryocyte differentiation / proliferation activity, which comprises all or a part of an amino acid sequence obtained by deleting one or more amino acids from the amino acid sequence of an NTPO protein; A protein having megakaryocyte differentiation / proliferation activity, comprising all or a part of an amino acid sequence obtained by substituting one or more amino acids in the amino acid sequence of the protein; and Megakaryocyte differentiation-proliferating activity, including all or part of an amino acid sequence in which one or more amino acids are added or inserted into the amino acid sequence of the NTP0 protein Are also included in the NTP0 protein of the present invention. Among such NTPO proteins of the present invention, those having a sequence similar to the amino acid sequence at positions 160 to 286 in SEQ ID NO: 1, which is a characteristic structure, are preferred. Similarity means that the homology is generally 70% or more, preferably 80% or more.

 Detailed description of the invention

 (1) Isolation of human NTP0cDNA

 Since human NTPO protein is synthesized in various tissues, its cDNA can be isolated from commercially available human tissue-derived cDNA (for example, Quick Clon cDNA, Clontech). May be used. Cloning is performed by a known method, for example, the PCR method.

[M. A. Innis et al., PCR Protocols, Academic Press (1990)]. The resulting clone can be subcloned to a plasmid such as M13mp18 if necessary. The nucleotide sequence of the cDNA thus obtained is determined by the dideoxy method [Sanger, F., Science, Vol. 214, p. 1205 (1981)] using a commercially available kit. be able to.

 It has been confirmed that the human NTPO cDNA obtained by the present inventors has the novel nucleotide sequence shown in SEQ ID NO: 2, and the amino acid of the novel protein encoded by the nucleotide sequence is The sequence was deduced as in E column number 1. The present inventors named the protein having the amino acid sequence represented by SEQ ID NO: 1 as NTPO protein, and hereinafter, it will be referred to as NTPO protein.

 (2) Recombinant expression vector and its transformant

The DNA encoding human NTPO protein obtained by the above method Alternatively, a transformant can be obtained by incorporating the fragment into an appropriate vector and transferring the obtained vector to an appropriate host cell. If this is cultured by a conventional method, a large amount of human NTPO protein can be obtained from the culture.

 Specifically, it is performed as follows. First, a DNA encoding human NTPO protein or a fragment thereof is ligated downstream of the promoter of a vector suitable for its expression by a known method using restriction enzymes and DNA ligase, and the recombinant expression vector is obtained. Create a chart. Examples of vectors that can be used include plasmids pBR322 and pUC18 derived from Escherichia coli, plasmid BUB110 derived from Bacillus subtilis, and plasmids derived from yeast. Smid p YE, vectors derived from the bacterium offage, Agt10 and Lgt11, and pSV2, but are vectors that can be replicated and amplified in the host. If it is, there is no particular limitation. The promoter and terminator are not particularly limited as long as they correspond to the host used to express the nucleotide sequence of the DNA encoding the human NTPO protein, and they are appropriate for the host. Can be combined. The DNA used for preparing the transformant may be any DNA encoding human NTPO protein, and is limited to the DNA having the nucleotide sequence of SEQ ID NO: 2. Not something. It may be a sequence in which a part of the nucleotide sequence of SEQ ID NO: 2 is substituted or deleted, whether intentional or not, a sequence in which insertion or addition occurs in the sequence, and It may be a DNA having a sequence generated by combining these. Further, DNA may be synthesized by chemical synthesis.

The recombinant expression vector thus obtained can be used for the recombinant cell method [see J. Mol. Biol.. Vol. 53, p. 154 (1970)], the protoplast method [ Natl. Acad. Sci. USA, vol. 75, p. 1929 U 978)], calcium phosphate method [Science. Vol. 221. p. 551 (1983)], invitro package Natl, Acad. Sci. USA, Vol. 72, p. 581 (1975)], Wilsberger method [Cel, Vol. 37, p. 1053 (1984)], etc. According to the above method, a transformant is introduced into a host to prepare a transformant. As a host, for example, Escherichia coli, Bacillus subtilis, yeast, insect cells or animal cells are used. The obtained transformant is cultured in an appropriate medium according to the host. Cultivation is usually performed under conditions of 20 to 45 and pH 5 to 8, with aeration and ventilation as necessary. Stirring is performed. The separation and purification of the NTPO protein from the culture may be carried out by appropriately combining known separation and purification methods. Examples of such known methods include salting out, solvent precipitation, dialysis, gel filtration, electrophoresis, ion exchange chromatography, and affinity chromatography. Examples include mattography and reversed-phase high-speed liquid inlet mattography.

 (3) Preparation of antibody

 Antibodies can be prepared by an ordinary method using all or part of the NTP0 protein as an antigen.

 For the production of polyclonal antibodies, for example, multiple injections of the antigen subcutaneously, intramuscularly, intraperitoneally or intravenously into animals such as mice, guinea pigs, and egrets , And then blood is collected from such animals and serum is separated. When immunizing, a commercially available adjuvant can also be used.

 • Monoclonal antibodies are prepared, for example, as follows. First, mouse spleen cells immunized with a peptide containing the NTPO protein or a part thereof are fused with commercially available mouse mice cells to produce a hybridoma. Thereafter, the hybridoma is cultured, and a monoclonal antibody is prepared from the culture supernatant. Alternatively, the hybridoma is administered to a mouse, and a monoclonal antibody is prepared from the ascites of the mouse.

 What is used as the antigen does not necessarily need to be an NTP0 protein having an entire amino acid structure. It may be a peptide having a partial structure of the protein, a mutant or derivative thereof, or a fusion peptide of the peptide with another peptide. The method for preparing the antigen may be any of a biological technique and a chemical synthesis technique.

 These antibodies enable identification and quantification of the NTP0 protein in a human biological sample, and can be applied to diagnostic reagents and the like. The immunological measurement of the NTP0 protein using these antibodies may be performed according to a known method, and for example, can be performed by a fluorescent antibody method, a passive agglutination reaction method, or an enzyme antibody method.

 BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the growth promoting activity of NTP0 on human leukemia cell line UT7-GM. FIG. 2 is a graph showing megakaryocyte differentiation / proliferation activity of NTP0 in a mouse bone I cell culture system.

 Example

Hereinafter, the present invention will be described in detail and specifically with reference to examples. However, the present invention is not limited to only these examples. General Experimental methods Experiments harm [e.g. J. Sambrook et al., Molecular Cloning , a laboratory manual. Cold Spring Harbor Laboratory Press (1989)] is himself截₀

 (Example 1) Cloning of DNA sequence encoding human NTPO protein

 A commercially available human fetal liver cDNA library (Quick Clone cDNA, manufactured by Clontech) was used as a raw material, and four synthetic oligonucleins were synthesized according to known methods. PCR was performed using the nucleotide primers (A, B, CsD). The sequence of the synthetic oligonucleotide was selected based on the report of de Sauvage [see Nature. Vol. 369, p. 533 (1994)].

 A: 5 'GCC AGA ATG GAG CTG ACT GAA TTG CTC CTC 3'

 B: 5 'AGA GCC TTA CCC TTC CTG AGA CAG ATT CTG 3'

 C: 5 'ATG GAG CTG ACT GAA TTG CTC CTC GT 3'

 D: 5 'CCT TAC CCT TCC TGA GAC AGA TTC TG 3 "

 Taisei oligonucleotide was synthesized using an automatic DNA synthesizer (manufactured by Alabio Biosystems), and then purified by polyacrylamide gel electrophoresis. PCR was performed with a combination of primers A and B and a combination of primers C and D.

For the first PCR, 200 bicograms of the above cDNA were used. The PCR reaction was performed using a commercially available kit (TaKaRa Taq or TaKaRa Ex Taq, manufactured by Takara Shuzo) in a reaction volume of 0.05 ml. As the buffer, the one attached to the kit was used, and the final concentration of dNTP was 0.25 mM. As a reaction device, a DNA thermal cycler (manufactured by Parkin Elmer Theta) was used. The PCR reaction is first performed with 95% heat denaturation for 5 minutes, followed by heat denaturation at 95 ° C for 1 minute, annealing at 68 ° C for 1 minute, and extension reaction at 72 ° C for 1 minute. 40 cycles were repeated with 5 minutes as one cycle. Thereafter, 72 extension reactions were performed for 5 minutes to complete the PCR. 0.005 from the PCR reaction solution The second PCR was performed using the obtained ml. The cycle setting conditions were the same as those for the first PCR, except that the annealing temperature was changed to 65 ° C. In the second PCR reaction, the following two types of synthetic oligonucleotides (E, F) were used.

 E: 5 'GATTAAGCTTATGGAGCTGACTGAATTGCTCCTCGT 3'

 F: 5 'GATTAGATCTTTACCCTTCCTGAGACAGATTCTGGGA 3'

 After the completion of the second PCR reaction, 0.005 ml of the reaction solution was taken out, and the progress of the reaction was confirmed by agarose gel electrophoresis. As a result, a major reaction product of about 1050 bp and, in addition, a DNA reaction product encoding about 950 bp of NTP0 protein could be confirmed.

 The remaining 0.045 l of the reaction mixture was subjected to ethanol precipitation to recover the DNA. Thereafter, the DNA was digested with restriction enzymes Hind 111 and Bgl II. The obtained DNA ′ fragment was subjected to agarose gel electrophoresis, and the desired DNA fragment of about 950 bp was recovered from the gel together with the DNA fragment of about 1050 bp. About one-fifth of the recovered DNA fragment was further digested with the restriction enzyme Bara HI, and the resulting DNA fragment was separated by agarose gel electrophoresis. The separated DNA fragments of about 430 bp and about 530 bp were derived from a novel gene encoding NTPO protein, and were recovered from the gel.

 The DNA fragment of 430b was ligated in the presence of T4DNA ligase to the M13mp18 vector, which had been digested with the restriction enzymes BaraHI and HindIII beforehand. On the other hand, the 530 bp DNA fragment was ligated to the M13mp18 vector that had been digested with the restriction enzyme BamHI in the presence of T4DNA ligase. Thereafter, E. coli JM109 was separately transformed using each reaction solution. The resulting black was isolated and cultured, and RFDNA and single-stranded DNA were prepared from the culture.

 The second PCR was also performed using Deep Vent DNA polymerase (New England Biolabs). At that time, the buffer attached to the kit was used.

After performing PCR using Deep Vent DNA polymerase, DNA fragments were recovered. The DNA fragment was M13 which had been digested with the restriction enzyme Hinc11 in advance. The lig9 vector was ligated in the presence of T4 DNA ligase. Thereafter, Escherichia coli JM109 was transformed using the vector. The obtained plaque was isolated and cultured, and RFDNA and single-strand DNA were prepared from the culture solution.

 A portion of RFDNA was digested with restriction enzymes Eco R1 and Hind II], and the digest was subjected to agarose gel electrophoresis. Thus, a clone containing the desired DNA fragment of about 950 bp was selected.

 The PCR proliferation reaction using TaKaRa Ex Taq was performed as follows. That is, the reaction was carried out at a reaction volume of 0.1 ml using 200 pg of kidney cDNA and oligonucleotide (:, D. Use the buffer supplied with the kit. The final reaction temperature of dNTP was set to 0.25 mM.1 The reaction was performed by heat denaturation at 94 ° C for 30 seconds, annealing and extension at 68 ° C for 5 minutes. The cycle was repeated 40 cycles, and the extension reaction was completed at 72 ° C for 5 minutes to complete the PCR.The desired 950 bp reaction product was separated by agarose gel electrophoresis, and the gel was separated. The purified DNA was used in the experiments of Examples 3c and 4b.

 The target clone can also be selected using the human kidney cDNA or the human aorta cDNA library.

 (Example 2) Determination of the nucleotide sequence of DNA encoding human NTPO protein

 The base (nucleotide) sequence of the single-stranded DNA of M13mp18 and M13mp19 containing the DNA fragment derived from the NTP Oif gene obtained in Example 1 was converted to a commercially available DNA. It was determined using a nucleotide sequence determination kit (Sequenase Version 2.0 T7 DNA Polymerase Sequencing Kit, USB). At that time, if necessary, the following synthetic oligonucleotides G and H were used as primers for DNA sequencing.

 G: 5 'GTC TGA TGT TCC TGA GGA AA 3'

 H: 5 "TCT GGC TGA GGC ACT GAA GT 3 '

The reaction reagent used was the one attached to the kit. The reaction procedure followed the protocol attached to the kit. The nucleotide (nucleotide) sequence of the gene encoding the NTPO protein thus determined is shown in SEQ ID NO: 2. Based on this nucleotide sequence, the amino acid sequence of NTP0 was deduced as described in SEQ ID NO: 1. (Example 3) Preparation of NTP0 gene regulated by signal peptide of human plasma gen-activator

 It is well known that the expression of a protein secreted extracellularly is defined by the amino acid sequence of a signal peptide that is processed after its biosynthesis. The expression of human plasma activator (tPA) in animal cells has been well studied so far, and examples of its expression at high levels have been reported.

 The present inventors, for the purpose of regulating the expression of NTP0 in animal cells, to convert the signal peptide of the original NTP0 gene into a human brassinogen activator (tPA) gene-derived gene. Replaced with something.

 a) Isolation of tPAcDNA

 For the isolation of tPA cDNA, the nucleotide sequence of tPA DNA published by Pennica et al. was used based on Pennica et al. Nature. Vol. 301, pp214-221 (1983). The following oligonucleotides I and J were synthesized. Using these, from a human placenta cDNA library (Quick Clone cDNA, Clontech), tPA cDNA is amplified by the PCR method, and the PCR product is subjected to agarose gel electrophoresis. Analyzed by electrophoresis. As a result, the target tPADNA of about 1700 bp was confirmed and isolated.

 I: 5 "GCA ATC ATG GAT GCA ATG AAG AGA GGG 3 '

 J: 5 "TGG TCA CGG TCG CAT GTT GTC ACG AAT 3 '

 ) Preparation of tPA gene expression cassette

 Based on its structural characteristics, tPA follows a signal peptide and, in order from the N-terminus, includes a finger region (F), a growth factor-like region (G), a king 1 region (K1), and a It is distinguished into a ring 2 region (K 2) and a serine mouth thease region (S Ρ).

 The present inventors introduced a restriction enzyme cleavage site into the tPA gene for the purpose of facilitating genetic manipulation for expressing the NTPO protein controlled by the tPA signal peptide. That is, the following synthetic oligonucleotides K and L were prepared in order to introduce a Hindlll site on the 5 'side of tPA cDNA and an Xbal site on the 3' side of K1 and obtained in a). PCR was carried out targeting about OObp of tPA cDNA.

K: 5 "AATTAAGCTTGCAATCATGGATGCAATGAAGAGAGG 3 ' L: 5 'AATTTCTAGATTAGTCACTCTTTCCCTCAGACCACGCAGGGG 3'

The PCR product was treated with the restriction enzymes Hind I and Xba I. Next, the treated product was separated by agarose electrophoresis, and a target DNA fragment of about 660 bp was recovered. This DNA was designated as HTPAFGK1-XH. Use this DNA, the pre-Hind II I Contact and have been cut with Xba I click the bra scan Mi Dobeku data one p UC l 8 b Ichiyu ring the _c obtained bra scan Mi de, after the genetic manipulation Was. The plasmid containing this tPA gene fragment was named pUCHTPAFGKl.

c) Preparation of NTP0 gene controlled by tPA signal sequence

 At the boundary between the signal peptide sequence of the tPA gene and the mature tPA sequence, the only restriction site (BglII) is present. Therefore, if this site is used to place an NTP0 gene on the 3 ′ side of the signal peptide sequence of the tP gene, a gene for expressing NTP0 protein regulated by the tPA signal sequence can be used. A child can be made. For this purpose, a BglII cleavage site may be introduced at the 5 · side of the NTP0 gene DNA, and an Xba1 cleavage site may be introduced at the 3 · side.

 The present inventors first synthesized the following oligonucleotides M and N. Next, using these, a PCR reaction was performed using the NTPO gene described in Example 1 as a target DNA.

 M: 5 'ATTAAGATCTCCGGCTCCTCCTGCTTGTGACCTCC 3'

 N: 5 'TTAATCTAGATTAGGAAGCAGGGGGTGGAGCTGGACCAC 3'

The PCR product was digested with restriction enzymes Bgl II and Xba 1, and the digest was subjected to agarose gel electrophoresis. Thus, the desired DNA of about 800 bp was separated and recovered. This DNA fragment was designated as NTP 0—HX. The previous pUCHTPAFGKl-XH was treated with the restriction enzymes Bgl11 and XbaI, and the processed product was subjected to agar-mouth gel electrophoresis. From the gel, a pUC vector containing the tPA signal sequence was purified and collected. Next, the NTPO-XH gene DNA was cloned into this tPA signal pUC vector by a conventional method. The thus obtained plasmid was named pUCHTPAsig-NTPO-XH. pUCHTPAsig-NTPO-XH plasmid DNA was treated with restriction enzymes Hind111 and Xba1, and the treated product was subjected to agarose electrophoresis. The gel contains the tPA signal peptide sequence and the NTP0 gene. DNA was purified and recovered. This was named DNA fragment HTPAsig-NTPO-XH. (Example 4) Preparation of human brass minogen activator-NTP0 fusion gene From the nucleotide sequence of the NTP0 gene described in Example 2, the amino acid sequence was Asparagine-Kingdai type sugar is not added to the predicted NTP0 protein. It has been observed that glycoproteins are often involved in the extracellular secretion of proteins, so if sugars can be added to the NTP0 protein in some way, extracellular secretion of that protein will occur. Highly promising. Since tPA is a glycoprotein, this object can be achieved by preparing a fusion protein of tPA and NTP0. The NTPO protein can be recovered by recovering the secreted protein and treating it by an appropriate method to remove the tPA portion.

 a) Preparation of cassette for expression of tPA N-terminal region

 In order to prepare a gene encoding a protein obtained by fusing an NTP0 protein to the C-terminal side of the K1 region of tPA, the following ligand nucleotide 0 was synthesized.

 0: 5 'TTTATCTAGAAGATCTGGGGAAGTCACTGTTTCCCTCAGAGCAGCCAGGGGTGCT 3' Using the oligonucleotide 0 and the oligonucleotide I, target about 1700 bpt PAc DNA described in Example 3a)). A PCR reaction was performed as DNA.

 By using oligonucleotide 0, the amino acid sequence of the C-terminal region of the K1 region of tPA becomes Asp Cys Tyr Phe Gly Asn Ser Asp Phe Pro Arg Ser.

The NTPO protein is linked immediately after this. In addition, the Phe ProArg sequence in this amino acid sequence is a specific recognition sequence for limited proteolytic thrombin, and thus is obtained by treating the fusion protein with thrombin. The tPA portion can be removed from the fusion protein of tPA and NTPO.

 The PCR product was treated with the restriction enzymes Hind1Π and Xba1, and the treated product was subjected to agarose-sgel electrophoresis to separate and collect the desired DNA of about SSObp. This DNA fragment was named HTPAFGK1.

 ) Creation of NTP0 gene that is compatible with HTPPAFGK1 gene

HTPAFGK 1 In order to ligate the Ρ Τ Ρ 0 gene from which the signal sequence was removed to the 3 ′ end of the iti gene, the following oligonucleotide was synthesized, and then the oligonucleotide was synthesized. PCR using the NTP0 gene DNA described in Example 1 was carried out by using the nucleotide P in combination with the above-mentioned oligonucleotide N.

 P: 5 'ATTATCTAGACCCGCTCCTCCTGCTTGTGACCTCCGA 3'

 The PCR product was treated with the restriction enzyme Xba1, and the treated product was subjected to agarose gel electrophoresis, whereby a target DNA fragment of about 800 bp was separated and collected. This DNA was named NTP0—XX.

 (Example 5) Expression of NTP0 gene in mammalian cells

 a) Construction of expression vector pK4KMCS34

 A method for producing the expression vector pK4K has been reported by Kato et al. [See Japanese Patent Application Laid-Open No. 5-33992]. The exogenous gene expressed using the present vector can be introduced into the restriction site (HindII!) And the restriction enzyme (BamHI) cleavage site of the present vector. However, since the NTP0 gene has an internal BamHI cleavage site, it is difficult to use the expression vector pK4K as it is. To circumvent this difficulty, Taisei oligonucleotide Q, which contains the following restriction enzyme cleavage sites between the Hind111 cleavage site and the BamHI cleavage site of the expression vector ρρ4Κ, R was inserted to produce a modified pK4K vector, which was used as a vector for expression of the {0} gene. Increasing the number of restriction sites that can be used will expand the range of use of this vector.

 Q: 5 "AGCTTAGTCGATATCAGCGGCCGCATCTAGAAATG 3 '

 R: 5 'GATCCATTTCTAGATGCGGCCGCTGATATCGACTA 3 ·

 The 5th side of the above-mentioned oligonucleotides Q and R was phosphorylated according to a conventional method. Thereafter, the obtained phosphorylation reaction solution was heated to 100 ° C., cooled, and oligonucleotide Q was allowed to anneal with oligonucleotide R. The double-stranded DNA obtained in this manner is digested with Hindi 11 and Bam HI in advance and cloned into a pK4K vector that has been subjected to a phosphorylation treatment. Expression vector PK4KMCS34 obtained c

It was confirmed by agarose gel electrophoresis that PK4K CS34 DNA was cleaved at one site by each of the restriction enzymes Hind111, EcoRV, Not1, XbaI, and BamHI.

b) Expression vector pl Production of KMCSHTPAsig-NTPO-XH The DNA fragment HTPAsig-NTPO-XH obtained in Example 3c) was digested with the restriction enzymes Hind 111 and ADE31 in advance and dephosphorylated to a plasmid PK4KMCS34, which was obtained by a conventional method. Joined. E. coli TB1 was transformed with the N thus obtained. A plasmid DNA was prepared from the transformed Escherichia coli according to a conventional method, and this was named pK4KMCSHTPAsig-NTP0-; (H.

 c) Expression vector p! KMCSHTPAFGKl-NTPO-XX

 The DNA fragment HTPAFGK1 obtained in Example 4a> was ligated to a plasmid pK4KMCS34, which had been digested with the restriction enzymes Hindill and Xba1 and dephosphorylated beforehand, in a conventional manner. Escherichia coli TBI was transformed with the DNA obtained in this manner. A plasmid DNA was prepared from the transformed E. coli according to a conventional method. By digesting this plasmid DNA with a restriction enzyme, it was confirmed that it contained the DNA fragment HTPAFGK1. This plasmid was named pJKMCSHTPAFGKl. The DNA fragment NTPO-XX obtained in Example 4b) was ligated to a plasmid pK4KMCSHTPAFGKl which had been previously digested with the restriction enzyme Xba1 and dephosphorylated, in a conventional manner. E. coli TBI was transformed with the DNA obtained in this manner. A plasmid DNA was prepared from the transformed Escherichia coli according to a conventional method. The plasmid DNA was digested with the restriction enzyme BamHI, and based on the results, clones in which the NTPO-XX was correctly oriented in the vector were selected. N T P 0 — XX force ブ ラ The correctly oriented brassmid was named pl KMCSH-PAFGKINTPO-Π.

d) Preparation of expression vector PK4KMCSHTPAFGK1-XH

 The DNA fragment HTPAFGK1-XH obtained in Example 3b) was ligated to pK4KMCS34, a vector previously digested with Hind111 and XbaI and dephosphorylated, in a conventional manner. Thereafter, the same operation as that described in Example 5b> was performed to obtain the desired brassmid pK4KMCSHTPAFGKl-XH.

e) Expression by BHK cells

 There is a detailed report by Kato et al. On the expression of foreign genes using PK4K vector and BHK cells (see Japanese Patent Application Laid-Open No. 5-339292). With reference to the method, the present inventors conducted experiments as follows.

First, BHK cells [tk-tsl3 strain, Waechter, DE and Baserga, R. Proc. Natl. Acad. Sci. USA. Vol. 79, p. 1106 (1982)] was inoculated in a 5 × 10 ⁵ eel 1 s / ml / 5 ml / 25 cm ² culture flask and cultured. Hereinafter, all cultures were performed in a carbon dioxide incubator adjusted to 5% C0 _} . After the culture 1, the plasmid p! UKMCSHTPAFGKl-XH3.5 described in b) was transfused by the calcium phosphate method using cellphect (a kit manufactured by Pharmacia). Yuxion. The medium used was a 1MDM medium supplemented with fetal calf serum so that its concentration was 5%. Door lance off _Λ click shea Yo down after two days, the cells were collected by re-trypsin treatment, it, the 250ηΜ ΜΤΧ including including media, were passaged in culture off La scan U-75cm '. The medium was changed every 2-3 days, and the culture was performed for 10 days. The cells were then passaged into 225 cm ² culture flasks. ^Four days later, the medium was changed with the cells growing confluent. Thereafter, the medium was further changed 3 to 5 times every day, and 800 ml of the culture supernatant was collected.

 The amount of the human plasminogen-activating factor-NTPO fusion protein contained in the culture supernatant was measured by the ELISA method described in Example 7. As a result, the amount of the protein was 40 ng / ml in terms of the amount of tPA antigen.

 Example 6 Purification of Human Brassica-Genogen Activator-NTPO Fusion Protein and Release of NTPO from the Protein

 An antibody that binds to FGK1 was selected from the anti-human tPA mouse monoclonal antibodies purchased as reagents. The antibody was conjugated to a formylcell mouth fin (manufactured by Seikagaku Corporation) to prepare an anti-tPA column.

 Purification of the human plasminogen activator-NTP0 fusion protein was performed by affinity chromatography using this antibody force ram. That is, 750 ml of the above-mentioned culture supernatant was provided to an antibody column previously equilibrated with 50πΜ-tris-hydrochloric acid (ρΗ7.5) -0.5Μ NaCl buffer, and then the column was added with the above buffer. Washed. Thereafter, elution was performed using a 0.2 M glycine / hydrochloric acid buffer (pH 2.5). The eluted fraction was immediately neutralized with 1/10 volume of 1 M Tris-HCl buffer (pH 12.0). The neutralized eluted fraction obtained in this way was dialyzed against Dulbecco's PBS (-). After that, the dialyzed solution was subjected to ultrafiltration using Centrifuge 10 (manufactured by Amicon) and subjected to τιδ.

The human plasminogen activator thus obtained is obtained as follows. The amount of quality was measured by the EL1SA method shown in Example 7. As a result, the amount of the protein was 52 gZnil x 3 ml in terms of the amount of tPA antigen.

 Next, this protein was treated with thrombin to prepare free NTPO. That is, 1 mg of bovine thrombin (manufactured by Sigma) was added per 1 absorbance (measured at 280 nm) of the obtained human plus plasminogen activator-NTPO fusion protein. The mixture was incubated at 37 ° C for 1 hour. Thereafter, 0.5 mM DFP was added to the mixture to inactivate thrombin. The release of NTPO was confirmed by SDS polyacrylamide gel gel electrophoresis.

 (Example 7) Determination of the protein amount of the human brass minogen activator-NTPO fusion protein

The measurement of the amount of the human plus actinogen-NTP0 fusion protein was performed by ELISA using an antibody against tPA. The same antibody as that used in Example 6 was used as the primary antibody, and the anti-human tPA ゥ sagi polyclonal antibody biotin-labeled was used as the secondary antibody. . The primary antibody was dissolved in Dulbecco's PBS (-) to a concentration of 5 g / ml. This primary antibody solution was added to a round bottom 96-well plate at 100 n1 / well, and incubated at room temperature for 1 hour. Then, wash each well of the plate [200 1 washing solution (wash 4 times with 20 mM phosphate buffer (pH 7.5) containing 0.93 iNaCl and 0.055 iT * een 20, etc.)] Then, add a block of 4 times diluted with deionized water (manufactured by Dainippon Pharmaceutical Co., Ltd.) in 200 wells, incubate at room temperature for 1 hour, and perform the blocking. After washing each hole of the plate, human t-PA as a sample or a standard was added to each well, each of which was 100. Each of the sample and the standard tPA solution was 20 mM Tris-HCl. It was diluted to an appropriate concentration using a block ace (hereinafter referred to as 10- ^l BA) diluted 10-fold with a buffer solution (pH 7.5, containing 0.05¾ Tween 20) and used. After washing each well, add 10-solution of secondary antibody containing appropriate concentration of secondary antibody to each well and incubate for 1 hour at room temperature After washing each well of the plate, 100 / well of 1- (H-ΒΑ) solution of HRP-labeled avidin (Vector Lab) diluted 1/2000 (Γ-ΒΑ) was used. in addition, for 30 minutes at room temperature I Nki Interview base - sheet and Yo down the blur -. after washing each well of bets, the substrate solution [1.5nig / _m l O walk We two les emissions Jia Mi emissions' 2 hydrochloride And 50 mM monocitrate containing 0.03% hydrogen peroxide [Phosphate buffer (pH 5.0)] was added at 100 1 / well to develop color, and then 1N hydrochloric acid was added at 100 / 1Z well to stop the reaction. The absorbance at 450 nm of the solution in each well was measured, and the amount of antigen of each sample, which was equivalent to the amount of antigen of standard tPA, was determined.

 (Example 8) Measurement of proliferation promoting activity of megakaryocyte cells

 It was evaluated whether or not the sample (NTPO) obtained in Example 6 had a growth promoting activity on a human leukemia cell line UT7-GM in which the expression of a TPO receptor (c-npl) was confirmed.

 That is, BSA was added to the sample to a final concentration of 0.1%, and the obtained sample containing BSA was dialyzed against 1MDM medium. The solution obtained in this way was sterilized by filtration with a 0.22 m filter (Millipore). The solution after filtration sterilization was serially diluted in a 1 MDM medium containing 10% fetal bovine serum to a 50/1/1 / well on a 96-well plate.

On the other hand, UT7-GM was subcultured in advance in IMDM medium containing 10% fetal calf serum and 1 ng / ral GM-CSF (R & D). At the time of use, this was collected by centrifugation at 1,000 rpm for 5 minutes and suspended in a medium containing 10% fetal calf serum without GM-CSF. Perform this centrifugation twice more to sufficiently remove GM-CSF, and then transfer UT7-GM to a medium containing 10% fetal bovine serum without GM-CSF for 1 x 10 ^sec 1 s. / ml. Add this suspension at 50 1 / well to the 96-well plate described above, then add UT7-GM for 37 days in a 5% C02 adjusted CO ₂ incubator for 3 days. Cultured.

 Proliferation of UT7-GM was measured by colorimetric assay using MTT. That is, MTT was dissolved at 5 mg / ml in Dulbecco's PBS (-), and the obtained solution was added to the above-mentioned 96-well plate containing UT7-GM with a hole. After that, UT7-GM was cultured for 4 hours, and then a 10% SDS solution containing lmM NH ^ OH was added at 100 μl / well. 96-hole plate, 37 more. The cells were incubated overnight at C to thaw the cells. Thereafter, the absorbance at 590 nm of each well was measured with a 96-well plate absorbance meter.

Figure 1 shows the results of the assay for the human plus plasminogen activator-NTPO fusion protein and the sample obtained by treating it with trombin. All samples clearly showed a growth-promoting activity against UT7-GM in a dose-dependent manner. In Fig. 1, the horizontal axis is the amount of protein in terms of tPA antigen of the added sample, and the vertical axis is measured by the MTT method. OD ,,. , Which reflects the number of cells. OD _s , where no sample is added. Was 0.3 to 0.35.

 For comparison, the human plasmid-gen activator-TP0 fusion protein and only the tPA portion of the fusion protein were prepared in the same manner as described in Examples 4 to 7. did. These were also evaluated for their growth promoting activity on UT7-GM. As a result, the human plus plasminogen activator-TPO fusion protein and the sample obtained by subjecting it to thrombin treatment were the human plus plasminogen activator-NTPO fusion protein and the same, respectively. It showed almost the same growth promoting activity as the sample obtained by treating trombin with the amount of protein converted to tPA antigen.

 In addition, no growth promoting activity was observed in the tPA portion alone or in a sample obtained by treating the tPA portion with trombin.

 (Example 9)

 It was evaluated whether the sample (NTPO) obtained in Example 6 had megakaryocyte differentiation / proliferation activity in a mouse bone ft! Cell culture system.

 That is, BSA was added to the sample to a final concentration of 0.1%, and the obtained sample containing BSA was dialyzed against 1MDM medium. The solution obtained in this manner was sterilized in a port with a 0.22 μm filter (Millipore). The solution after port sterilization was serialized in 1MDM medium containing 0.1% BSA and 1% Nutridoma (manufactured by Behringer Mannheim) so that 150 1 / well on a 9S well plate. Diluted.

—Mouse (BDF1, female, 6 weeks old) Suspend the femoral cells in a medium containing 1% fetal calf serum and place them in a 25 ° (1 ^ 1) culture flask at 37 ° C. The cells were allowed to stand for 1 hour at C. Thereafter, the cells not attached to the flask were collected together with the medium, and the medium containing the cells was centrifuged at 1500 rpm for 10 minutes, and the supernatant was collected. The cells were resuspended in a medium containing 0.5 mM DFP and 0.1% BSA, and the resulting suspension was left at room temperature for 20 minutes. After the rounds were performed, the cells were sedimented by centrifugation, and the cells were resuspended in the IMDM medium at a concentration of 2 × 10 ^e cells / ml.

This suspension was added to the 96-well plate at 1 / well, and the cells were then added to the plate. (:., 5% C 0 2 carbon dioxide was prepared in Lee Nki Interview and cultured in a beta 6 days their Then, add 1 M Tris-HCl buffer ( _PH 8.0) containing 1% Triton X at 50 1 / well and 1% quencher containing 1.5 mg / ral DTNB (Sigma). An aqueous sodium phosphate solution was added to each well at 151 / well. Further, 10 mM Yowi-Dai-Acetylthiocholin was added to each well at 151 / well, and immediately thereafter, the absorbance at 405 of each well was measured with a 96-well plate absorbance meter. Let this set value be (A).

 The 96-well plate was incubated at 37 ° C. for 1 hour, and then the absorbance at 405 nra of each well was measured again with the 96-well plate absorbance stab. This measured value is defined as (B).

Measurement from the measurement value (B) a value obtained by subtracting the _{(A), A 0 D 4} . _s . This indicates the activity value of acetylcholinesterase.

Fig. 2 shows the results of the assay for the human plasmin-gen activator-NTP0 fusion protein and the sample obtained by treating it with trombin. All specimens clearly showed megakaryocyte differentiation / proliferation activity in a dose-dependent manner. 2, the horizontal SaiwaiYukari is added protein amount in terms of t PA antigen of the specimen was, and the vertical axis, AOD ₄ showing the Asechiruko re N'e scan Te la chromatography peptidase activity. ,, Which reflects megakaryocyte cell number and differentiation maturity. Δ 0 D when no sample is added. And were almost 0.0.

For comparison, the acetyl cholesterol protein of human plasminogen-activating factor-1 TP0-modified protein and the sample obtained by subjecting it to thrombin treatment were used in the same manner as described above. Thease activity was determined. As a result, the human plasminogen activator-TPO fusion protein and the sample obtained by subjecting it to thrombin treatment were the human plasminogen activator-NTPO fusion protein and the NTPO fusion protein, respectively. It showed almost the same activity of promoting megakaryocyte differentiation and proliferation in terms of the amount of protein in terms of tPA antigen as that of a sample obtained by treating this with thrombin. Sequence Listing SEQ ID NO: 1

Array length: 2 8 6

Sequence type: amino acid

Sequence type: Protein

 Topology: Direct letter

Origin

 Organism name: Homo sapiens

Immediate origin

 One library: fetal human liver c DN A library

Array

 Met Glu u Leu Thr Glu u Leu Leu Leu Val Val et Leu Leu Leu Thr Ala

1 5 10 15

Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val

 20 25 30

 Leu Ser Lys Leu Leu Arg Asp Ser His Va 1 leu His Ser Arg Leu Ser

 35 40 45

 Gin Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala

50 55 60

 Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys 65 70 75 80

Ala Gin Asp lie Leu Gly Ala Val Thr Leu ten Leu Glu Gly Val Met

 85 90 95

Ala Ala Arg Gly Gin Leu Gly Pro Thr C s Leu Ser Ser Leu Leu Gly

 100 105 110

 Gin Leu Ser Gly Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu

 115 120 125

Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp 130 135 140

 Pro Asn A 1 a lie Phe Leu Ser Phe Gin His Leu Leu Arg Gl Lys Asp 145 150 155 160

Phe Trp lie Val Gly Asp Lys Leu His Cys Leu Ser Gin Asn Tyr Trp

 165 170 175

Leu Trp Ala Ser Glu Val Ala Ala Cly lie Gin Ser Gin Asp Ser Trp

 180 185 190

 Ser Ala Glu Pro Asn Leu Gin Val Pro Gly Pro Asn Pro Arglie Pro

 195 200 205

 Glu Gin Asp Thr Arg Thr Leu Glu Trp Asn Ser Trp Thr Leu Ser Trp

210 215 220

 Thr Leu Thr Gin Asp Pro Arg Ser Pro Gly His Phe Leu Arg Asn lie 225 230 235 240

Arg His Arg Leu Pro Ala Thr Gin Pro Pro Ala Trp He Phe Ser Phe

 245 250 255

Pro Asn Pro Ser Ser Tyr Trp Thr Val Val Tyr Ala Leu Pro Ser Ser Thr

 260 265 270

 His Leu Ala His Pro C s Gly Pro Ala Pro Pro Pro Ala Ser

 275 280 285 SEQ ID NO: 2

Array length: 8 6 1

Sequence type: nucleic acid

Number of 鑌: double strand

 Topology: Direct letter

Sequence type: cDNA to mRNA

Origin

 Organism name: Homo sapiens

Immediate origin One library: Human fetal liver cDNA library

Array features

 Characteristic symbol: CDS

 Location: 1.. 8 6 1

 How the feature was determined: E

 Characteristic symbol: sig peptide

 Location: 1.. 6 3

 How the feature was determined: S

 Characters that express the characteristics: mat peptide

 Location: 6 3.. 8 6 1

 ^ Method that determined the feature: S

Array

 ATG GAG CTG ACT GAA TTG CTC CTC GTG GTC ATG CTT CTC CTA ACT GCA 48 Met Glu u Leu Thr Glu u Leu Leu Leu Val Va 1 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 96 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 144 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 192 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 TGC AAA ACC CAG ATG GAG GAG ACC AAC 240 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 288 Ala Gin Asplie 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 366

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 384

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 432

Leu G】 y Thr Gin Leu Pro Pro G】 n 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 GAC 480

Pro Asn Ala lie Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Asp

 125 130 135

 TTC TGG ATT GTT GGA GAC AAA CTT CAC TGC CTC AGC CAG AAC TAC TGG 528

Phe Trp lie Val Gly Asp Lys Leu His Cys Leu Ser Gin Asn Tyr Trp

140 145 150 155

CTC TGG GCT TCT GAA GTG GCA GCA GGG ATT CAG AGC CAA GAT TCC TGG 576

Leu Trp Ala Ser Glu Val Ala Ala Gly lie Gin Ser Gin Asp Ser Trp

 160 165 170

 TCT GCT GAA CCA AAC CTC CAG GTC CCT GGA CCA AAT CCC CGG ATA CCT 624

Ser Ala Glu Pro Asn Leu Gin Val Pro Gly Pro Asn Pro Arglie Pro

 175 180 185

 GAA CAG GAT ACA CGA ACT CTT GAA TGG AAC TCG TGG ACT CTT TCC TGG 672

Glu Gin Asp Thr Arg Thr Leu Glu Trp Asn Ser Trp Thr Leu Ser Trp

 190 195 200

 ACC CTC ACG CAG GAC CCT AGG ACC CCC CGA CAT TTC CTC ACG AAC ATC 720

Thr Leu Thr Gin Asp Pro Arg Ser Pro Gl His Phe Leu Arg Asn lie

 205 210 215

AGA CAC AGG CTC CCT GCC ACC CAA CCT CCA GCC TGG ATA TTC TCC TTC 768 Arg His Arg Leu Pro Ala Thr Gin Pro Pro Ala Trp He Phe Ser Phe

220 225 230 235

CCC AAC CCA TCC TCC TAC TGG ACA GTA TAC GCT CTT CCC TCT TCC ACC 816

Pro Asn Pro Ser Ser Tyr Trp Thr Val Val Tyr Ala Leu Pro Ser Ser Thr

 240 245 250

 CAC CTT GCC CAC CCC TGT GGT CCA GCT CCA CCC CCT GCT TCC TGA 861 His Leu Ala His Pro Cys Gly Pro Ala Pro Pro Pro Ala Ser

 255 260 265 SEQ ID NO: 3

Array length: 30.

Sequence type: nucleic acid

Number of towns: One town

 Topology: linear

Sequence type: Other nucleic acids (synthetic DNA)

Array

 GCCAGAATGG AGCTGACTGA ATTGCTCCTC 30 SEQ ID NO: 4

Array length: 30

Sequence type: nucleic acid

Number of chains: single strand

 Topology: Direct letter

Sequence type: Other nucleic acids (synthetic DNA)

Array

 AGAGCCTTAC CCTTCCTGAG ACAGATTCTG 30 SEQ ID NO: 5

Array length: 2 6 Sequence type: nucleic acid

Number of chains: single

 Topology: Direct letter

Sequence type: Other nucleic acids (synthetic DNA)

Array

 ATGGAGCTGA CTGAATTGCT CCTCGT 26 SEQ ID NO: 6

Array length: 2 6

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acids (synthetic DNA)

Array

 CCTTACCCTT CCTGAGACAC ATTCTG 26 SEQ ID NO: 7

Array length: 3 6

Sequence type: nucleic acid

Number of chains: single strand

 Topology: Direct letter

Sequence type: Other nucleic acids (Taishi DNA)

Array

 GATTAAGCTT ATGGAGCTGA CTGAATTGCT CCTCGT 36 SEQ ID NO: 8

Array length: 3 7

Sequence type: nucleic acid

Number of towns: One bottle Topology: linear

Sequence type: Other nucleic acids (synthetic DNA)

Array

 GATTAGATCT TTACCCTTCC TGAGACAGAT TCTGGGA 37

E column number: 9

Array length: 20

Sequence type: nucleic acid

Number of chains: single strand

 Topology: Direct letter

Sequence type: Other nucleic acids (Taishi DNA)

Array

 GTCTGATGTT CCTGAGGAAA 20 SEQ ID NO: 10

Array length: 20

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acids (synthetic DNA)

Array

 TCTGGCTGAG G CAGTGAAGT 20 SEQ ID NO: 1 1

Array length: 2 7

Sequence type: nucleic acid

Number of reins: single strand

 Topology: Direct letter

Sequence type: Other nucleic acids (synthetic DNA) Array

 GCAATCATGG ATGCAATGAA GAGAGGG 27 SEQ ID NO: 1 2

Array length: 2 7

Sequence type: nucleic acid

Number of towns: One town

 Topology: Direct letter

Sequence type: Other nucleic acids (synthetic DNA)

Array

 TGGTCACGGT CGCATGTTGT CACGAAT 27 SEQ ID NO: 1 3

Array length: 3 6

Sequence type: nucleic acid

Number of chains: single strand

 Topology: Straight

Sequence type: Other nucleic acids (synthetic DNA)

Array

 AATTAAGCTT GCAATCATGG ATGCAATGAA GACAGG 36 SEQ ID NO: 14

Array length: 4 2

Column E type: Nucleic acid

Number of chains: single

 Topology: linear

Sequence type: Other nucleic acids (Taishi DNA)

 Array

AATTTCTAGA TTAGTCACTG TTTCCCTCAG AGCAGGCAGG GG 42 SEQ ID NO: 1 5

Array length: 3 5

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: Other nucleic acids (synthetic DNA)

Array

 ATTAAGATCT CCGGCTCCTC CTGCTTGTGA CCTCC 35 SEQ ID NO: 16

Array length: 3 9

Sequence type: nucleic acid

Number of reins: single strand

 Topology: linear

Sequence type: Other nucleic acids (synthetic DNA)

Array

 TTAATCTAGA TTAGCAAGCA GGGGGTGGAG CTGGACCAC 39 SEQ ID NO: 17

Array length: 5 5

Sequence type: nucleic acid

Number of chains: single strand

 Tobo D J: Direct letter

Sequence type: Other nucleic acids (Taishi DNA)

Array

 TTTATCTAGA AGATCTGGGG AAGTCACTGT TTCCCTCAGA GCAGGCAGGG GTGCT 55 SEQ ID NO: 18

Array length: 3 7 Sequence type: nucleic acid

Number of reins: single strand

 Topology: linear

Sequence type: Other nucleic acids (synthetic DNA)

Array

 ATTATCTAGA CCGCCTCCTC CTGCTTGTGA CCTCCGA 37 SEQ ID NO: 1 9

Array length: 3 5

Sequence type: nucleic acid

Number of chains: single strand.

 Topology: Direct letter

Sequence type: Other nucleic acids (synthetic DNA)

Array

 AGCTTAGTCG ATATCAGCGG CCGCATCTAG AAATG 35 SEQ ID NO: 20

Array length: 3 5

Sequence type: nucleic acid

Number of towns: One town

 Topology: linear

Sequence type: Other nucleic acids (synthetic DNA)

Array

 GATCCATTTC TAGATGCGGC CGCTGATATC GACTA 35

Claims

The scope of the claims

1. A protein having an activity of megakaryocyte differentiation and proliferation, having an amino acid sequence consisting of all or part of the amino acid sequence of SEQ ID NO: 1.

 2. One or more amino acids are added, deleted or inserted into the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1 Amino acid consisting of all or part of the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 1 except that one or more amino acids in the amino acid sequence are substituted. A protein having a megakaryocyte differentiation proliferating activity having a sequence.

 3. The force resulting from addition, deletion or insertion of one or more amino acids at position 160-286 in the amino acid sequence of SEQ ID NO: 1, or 16 The amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1 is the same as that of the amino acid sequence described in SEQ ID NO: 1 except that at least one of the amino acids at 0 to 286 is substituted. A protein having a megakaryocyte differentiation / proliferation activity having an amino acid sequence consisting of all or a part thereof.

 4. A peptide having an amino acid sequence consisting of all or a part of the amino acid sequence at position 160 to 286 in the amino acid sequence of SEQ ID NO: 1.

 5.16 — 2-8 In the amino acid sequence at the 6-position, the amino acid sequence having at least eight continuous amino acids is contained, so-called claim 4 is described. Petit.

 6. A DNA encoding a protein having the amino acid sequence of SEQ ID NO: 1.

7. The DNA of claim 6, which is the DNA of SEQ ID NO: 2.

 8. A DNA encoding a protein having a megakaryocyte differentiation / proliferation activity having an amino acid sequence consisting of all or a part of the amino acid sequence of SEQ ID NO: 1.

 9. One or more amino acids are added, deleted or inserted into the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1 Amino acid consisting of all or part of the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 1 except that at least one amino acid in the amino acid sequence is substituted. DNA encoding a protein having a megakaryocyte differentiation / proliferation activity having a sequence.

10. A protein comprising one or more amino acids added, deleted or inserted at positions 160 to 286 in the amino acid sequence of SEQ ID NO: 1 or 1 6 0 — 2 8 6th place Consists of all or part of the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 1, except that at least one of the amino acid sequences is substituted. DNA encoding a protein having an amino acid sequence and having megakaryocyte differentiation / proliferation activity.

 11. A vector containing DNA encoding a protein having the amino acid sequence of SEQ ID NO: 1.

12. SEQ ID NO: 1 § Mi Roh whole or base click te containing DNA encoding a protein having a megakaryocyte differentiation and proliferation activity with § Mi Roh Sanhai train consisting of a portion of the acid sequence ₀ according to

 13. One or more amino acids are added, deleted or inserted into the amino acid sequence of SEQ ID NO: 1, or the amino acid of SEQ ID NO: 1 An amino acid comprising all or part of the amino acid sequence identical to the amino acid sequence shown in SEQ ID NO: 1 except that one or more amino acids in the sequence are substituted. A vector containing a DNA encoding a protein having a sequence and having megakaryocyte differentiation and proliferation activity.

 14. At least one amino acid added, deleted or inserted at position 160-286 in the amino acid sequence of SEQ ID NO: 1 or 1 6 0 — 2 8 The same amino acid sequence as the amino acid sequence described in SEQ ID NO: 1 except that at least one of the amino acids at position 6 is substituted. A vector containing a DNA encoding a protein having a megakaryocyte differentiation / proliferation activity, which has an amino acid sequence consisting of all or a part thereof.

 15. A transformant having a vector containing a DNA encoding a protein having the amino acid sequence of SEQ ID NO: 1 inserted therein.

 16. It should contain a DNA encoding a protein having megakaryocyte differentiation / proliferation activity having an amino acid sequence consisting of all or a part of the amino acid sequence of SEQ ID NO: 1 inserted therein. A transformant retaining a cluster.

17. One or more amino acids added, deleted or inserted into the amino acid sequence of SEQ ID NO: 1 inserted therein, or described in SEQ ID NO: 1. Consists of all or part of the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 1, except that at least one amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted. A vector containing a DNA encoding a protein having an amino acid sequence and having megakaryocyte differentiation / proliferation activity A transformant retaining the protein.

 18. One or more amino acids are added, deleted, or inserted at positions 160 to 2886 in the amino acid sequence of SEQ ID NO: 1 inserted therein. Or or

16 0 — 2 8 Amino acid sequence identical to the amino acid sequence described in SEQ ID NO: 1 except that at least one of the amino acids at position 6 is substituted. A transformant having a vector containing a DNA encoding a protein having megakaryocyte differentiation / proliferation activity, which has an amino acid sequence consisting of all or a part thereof.

 1 9. Cultivate a transformant carrying the vector containing the DNA encoding the protein having the amino acid sequence of SEQ ID NO: 1 inserted therein, and recovering the expression product A method for producing a protein having megakaryocyte differentiation / proliferation activity.

 20. A vector containing a DNA encoding a protein having a megakaryocyte differentiation / proliferation activity having an amino acid sequence consisting of all or a part of the amino acid sequence of SEQ ID NO: 1 inserted therein. A method for producing a protein having a megakaryocyte differentiation / proliferation activity, which comprises culturing a transformant retaining the promoter and collecting the expression product.

 21. One or more amino acids added, deleted or inserted into the amino acid sequence of SEQ ID NO: 1 inserted therein, or described in SEQ ID NO: 1. Consists of all or part of the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 1, except that at least one amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted. Transforming a transformant containing DNA encoding a protein having an amino acid sequence and having a megakaryocyte differentiation / proliferation activity, and recovering the expression product. A method for producing a protein having a nucleocyte differentiation / proliferation activity.

 22. Is one or more amino acids added, deleted, or inserted at position 160-286 in the amino acid sequence of SEQ ID NO: 1 inserted therein? , Or,

16 0-2 8 The same amino acid sequence as the amino acid sequence described in SEQ ID NO: 1 except that one or more amino acids in the amino acid at position 6 are substituted. Culturing a transformant having a vector containing a DNA encoding a protein having megakaryocyte differentiation / proliferation activity having an amino acid sequence consisting of all or a part thereof, and expressing the expression product A method for producing a protein having a megakaryocyte differentiation / proliferation activity, comprising recovering the protein.

23. Amino acid sequence at position 160-28 in the amino acid sequence of SEQ ID NO: 1 Poly- or monoclonal antibodies that have $$ affinity for at least some of the columns.

 24. 16 0 — 28 8 It has a binding property to an amino acid sequence consisting of at least 8 continuous amino acids in the amino acid sequence at position 6; The clonal antibody or the monoclonal antibody according to claim 23.

 25. A DNA having a nucleotide sequence consisting of all or a part of the nucleotide sequence shown in SEQ ID NO: 2 is used as a probe or primer, and is used as a test DNA. A method for analyzing a gene of a protein having megakaryocyte differentiation / proliferation activity, which comprises hybridizing the gene.

 26. A DNA having a nucleotide sequence consisting of at least 10 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 2 is used as a probe or primer. The method for analyzing a protein having megakaryocyte differentiation / proliferation activity according to claim 25.

 27. A platelet growth promoter or platelet comprising, as an active ingredient, a protein having an amino acid sequence consisting of all or a part of the amino acid sequence of SEQ ID NO: 1 and having megakaryocyte differentiation / proliferation activity减 Small prophylactic agent.

 28. One or more amino acids added, deleted or inserted into the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1 Amino acid consisting of all or part of the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 1 except that one or more amino acids in the sequence are substituted. An agent for promoting platelet proliferation or preventing thrombocytopenia, comprising a protein having an acid sequence and having megakaryocyte differentiation / proliferation activity as an active ingredient.

 29. The force at which one or more amino acids are added, deleted or inserted at positions 160-286 in the amino acid sequence of SEQ ID NO: 1, Or the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 1 except that at least one of the amino acids at position 160 to 288 is substituted. A platelet growth-promoting agent or a thrombocytopenia-preventing agent comprising, as an active ingredient, a protein having megakaryocyte differentiation / proliferation activity having an amino acid sequence consisting entirely or partially of a noic acid sequence.