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WO1996003434A1 - Facteur de differenciation/proliferation de megacaryocytes - Google Patents

Facteur de differenciation/proliferation de megacaryocytes Download PDF

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Publication number
WO1996003434A1
WO1996003434A1 PCT/JP1995/001476 JP9501476W WO9603434A1 WO 1996003434 A1 WO1996003434 A1 WO 1996003434A1 JP 9501476 W JP9501476 W JP 9501476W WO 9603434 A1 WO9603434 A1 WO 9603434A1
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WIPO (PCT)
Prior art keywords
amino acid
acid sequence
seq
protein
megakaryocyte differentiation
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PCT/JP1995/001476
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English (en)
Japanese (ja)
Inventor
Shinji Yoshitake
Masayuki Okada
Norio Komatsu
Original Assignee
Eisai Co., Ltd.
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Publication date
Application filed by Eisai Co., Ltd. filed Critical Eisai Co., Ltd.
Priority to AU29914/95A priority Critical patent/AU2991495A/en
Publication of WO1996003434A1 publication Critical patent/WO1996003434A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel megakaryocyte differentiation growth factor.
  • the present invention can be applied to the field of thrombocytopenia treatment.
  • oat platelet transfusion has been used to treat thrombocytopenic patients.
  • 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.
  • problems such as the need to consider the dangers of traffic, and this is not always a problem.
  • transfusion if patients could improve their own platelet hematopoietic ability to treat thrombocytopenia, such problems could be overcome.
  • 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.
  • thrombopoetin 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.
  • 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.
  • NTPO protein megakaryocyte and platelet increasing activity
  • 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 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;
  • 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.
  • a monoclonal antibody 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.
  • 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 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;
  • 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.
  • 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.
  • the NTP0 protein gene can be analyzed by using it as a probe or a primer and hybridizing it with a test DNA.
  • a probe or a primer 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.
  • DNA encoding the NTP0 protein examples 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.
  • “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.
  • 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;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a transformant is introduced into a host to prepare a transformant.
  • Escherichia coli, Bacillus subtilis, yeast, insect cells or animal cells are used as a host.
  • 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.
  • Antibodies can be prepared by an ordinary method using all or part of the NTP0 protein as an antigen.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)].
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 1 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
  • tPA human plasma activator
  • 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.
  • tPA human brassinogen activator
  • tPA cDNA 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.
  • 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 ⁇ ).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • pUCHTPAsig-NTPO-XH 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.
  • 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.
  • 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.
  • 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 Hind1 ⁇ 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.
  • HTPAFGK 1 In order to ligate the ⁇ ⁇ ⁇ 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.
  • 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.
  • 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.
  • HinndII restriction site
  • BamHI restriction enzyme
  • 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 ⁇ 4 ⁇ , 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.
  • 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
  • PK4K CS34 DNA was cleaved at one site by each of the restriction enzymes Hind111, EcoRV, Not1, XbaI, and BamHI.
  • 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.
  • the correctly oriented brassmid was named pl KMCSH-PAFGKINTPO- ⁇ .
  • 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.
  • 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%.
  • the cells were collected by re-trypsin treatment, it, the 250 ⁇ ⁇ 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.
  • 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 50 ⁇ -tris-hydrochloric acid ( ⁇ 7.5) -0.5 ⁇ 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 ⁇ .
  • the human plasminogen activator thus obtained is obtained as follows.
  • the amount of quality was measured by the EL1SA method shown in Example 7.
  • the amount of the protein was 52 gZnil x 3 ml in terms of the amount of tPA antigen.
  • 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.
  • 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 ⁇ 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.
  • 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 6 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.
  • 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.
  • 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 ⁇ l / 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.
  • 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.
  • 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.
  • 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.
  • Example 6 It was evaluated whether the sample (NTPO) obtained in Example 6 had megakaryocyte differentiation / proliferation activity in a mouse bone ft! Cell culture system.
  • 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 ⁇ m 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.
  • 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.
  • 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. ⁇ 0 D when no sample is added. And were almost 0.0.
  • 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.
  • 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.
  • Organism name Homo sapiens
  • 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
  • Sequence type nucleic acid
  • Organism name Homo sapiens
  • Phe Trp lie Val Gly Asp Lys Leu His Cys Leu Ser Gin Asn Tyr Trp
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (Taishi DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (Taishi DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA) Array
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type Other nucleic acids (Taishi DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (Taishi DNA)
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention porte sur un nouveau facteur de différenciation/prolifération de mégacaryocytes (protéine NTPO) servant de remède contre la thrombocytopénie. On peut utiliser un ADN codant pour ladite protéine pour la produire par génie génétique et pour l'analyse des gènes codant pour elle.
PCT/JP1995/001476 1994-07-25 1995-07-25 Facteur de differenciation/proliferation de megacaryocytes WO1996003434A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU29914/95A AU2991495A (en) 1994-07-25 1995-07-25 Megakaryocyte differentiation/proliferation factor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP6/172497 1994-07-25
JP17249794 1994-07-25

Publications (1)

Publication Number Publication Date
WO1996003434A1 true WO1996003434A1 (fr) 1996-02-08

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ID=15943076

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1995/001476 WO1996003434A1 (fr) 1994-07-25 1995-07-25 Facteur de differenciation/proliferation de megacaryocytes

Country Status (2)

Country Link
AU (1) AU2991495A (fr)
WO (1) WO1996003434A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999018716A1 (fr) 1997-10-06 1999-04-15 Netoffice Solutions Llc Procede et dispositif de gestion de messages
EP0968223A4 (fr) * 1997-01-08 2007-11-21 Proligo L L C Bioconjugaison de macromolecules

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BLOOD, (15 Feb. 1995), Vol. 85, No. 4, GURNEY A.L. et al., "Genomic Structure, Chromosomal Localization and Conserved Alternative Splice Forms of Thrombopoietin", pages 981-8. *
FEBS LETT., (10 Oct. 1994), Vol. 353, No. 1, SOHMA Y. et al., "Molecular Cloning and Chromosomal Localization of the Human Thrombopoietin Gene", pages 57-61. *
J. BIOL. CHEM., (13 Jan. 1995), Vol. 270, No. 2, CHANG M.-S. et al., "Cloning and Characterization of the Human Megakaryocyte Growth and Development Factor (MGDF) Gene", pages 511-514. *
PROC. NATL. ACAD. SCI. U.S.A., (20 Dec. 1994), Vol. 91, No. 26, FOSTER D.C. et al., "Human Thrombopoietin: Gene Structure, cDNA Sequence, Expression and Chromosomal Localization", pages 13023-7. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0968223A4 (fr) * 1997-01-08 2007-11-21 Proligo L L C Bioconjugaison de macromolecules
WO1999018716A1 (fr) 1997-10-06 1999-04-15 Netoffice Solutions Llc Procede et dispositif de gestion de messages

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