KR19990081421A - Method for producing human platelet promoter (TPO) using animal cells - Google Patents

Abstract

The present invention relates to an animal cell expression vector comprising a human platelet promoter (Thrombopoietin; hereinafter abbreviated as "TPO") gene and a method for mass production of TPO in animal cells using the same. The present invention relates to a method for producing a large amount of TPO protein by transforming animal cells with a recombinant expression vector comprising a TPO gene substituted with a tPA signal sequence and then culturing the animal cells. In the present invention, by replacing the signal sequence of the TPO with the signal sequence of the human tissue plasminogen activator (hereinafter abbreviated as "tPA") to allow the TPO expressed in the cell to be secreted efficiently extracellularly. It is a useful method to express the type TPO protein in animal cells with high efficiency.

Description

Method for producing human platelet promoter (TPO) using animal cells

The present invention relates to a method for mass expression of human platelet production promoter (Thrombopoietin (hereinafter abbreviated as "TPO") in animal cells. More specifically, the signal sequence of TPO is a human tissue plasminogen activator (tissue) The present invention relates to a method for mass-producing a high-efficiency native TPO protein by substituting with a signal sequence of Plasminogen Activator (hereinafter abbreviated as "tPA") and expressing in animal cells.

TPO is a glycoprotein consisting of 353 amino acids and is a hematopoietic protein that is produced and secreted in tissues such as human liver or kidney and acts on the bone marrow to increase the number of platelets, which are hematopoietic cells in the bone marrow, and promote differentiation. .

Since Odel, et al., Proc. Soc. Exp. Biol. Med., 108, 428, 1961, for the first time, in 1961, Odel et al. Reported the presence of factors that regulate the number of platelets in rat blood. By 1994, the existence of TPO was revealed, and studies of its structure and action were made (Sauvage, et al., Nature, 369, 533-538, 1994; Bartley, et al., Cell, 77, 1117-). 1124, 1994). TPO is a glycoprotein that acts on megakaryocyte colony-forming progenitor cells, which are platelet progenitor cells present in bone marrow cells, to proliferate platelet progenitor cells and ultimately increase the number of platelets and promote their differentiation. It is losing promising protein.

Several hematopoietic growth factors are involved in the differentiation of myocytes from the bone marrow into megakaryocytes via megakaryoblasts. In other words, myocytes initially contain megakaryocyte colony-stimulating factors ("MK-CSFs") and interleukin-3 (IL-3), a type of nonspecific differentiation cytokine, and granulocyte colony stimulating factors (GM). -CSF) and myocyte growth factor (SCF) have been known to undergo maturation by TPO, a type of cytokine that is differentiated into megakaryocytes and later belongs to a specific differentiation family [Gordon, MS et al., Blood 80: 302 (1992).

Since the 1960s, many efforts have been made to purify TPO in the plasma of thrombocytopenic animals, but there are numerous other proteins in the plasma and there is no established method for measuring the activity of TPO during purification. However, the c-mpl gene, a protooncogene, was cloned from v-mpl, an oncogene of myeloproliferative leukemia virus (hereinafter referred to as "MPLV"), which causes blood cancer. It was found to contain four well-conserved cysteine (Cys) residues at the N-terminal site and tryptophan (Trp) -serine (Ser) -X-tryptophan (Trp) -serine (Ser) motifs in the vicinity of the biofilm It is known that the c-mpl gene belongs to the family of hematopoietic receptors [Soryri, M. et al., Cell 63: 1137 (1996); Vignon, I. et al., Proc. Natl. Acad. Sci. USA 89: 5640 (1992); Skoda, R.C. et al., EMBO J. 12: 2645 (1993); Bazan, J. F., Proc. Natl. Acad. Sci. USA 87: 6934 (1990). Later, Metia et al. Reported that c-mpl was a cytokine receptor that regulates megakaryocyte formation, that is, c-mpl mRNA was specifically observed only in cells in which megakaryocyte formation occurred. It has been reported that the introduction of oligonucleotides into hematopoietic myocytes inhibited the expression of this gene, which selectively blocked the process of megakaryocyte formation (Methia, N. et al., Blood 82: 1395 (1993)). The function of c-mpl has become clearer recently by studying the characteristics of transgenic mice made by injecting the c-mpl gene into mice (Gurney, A. L. et al., Science 265: 1445 (1994)).

In addition to these reports, a combination of recombinant c-mpl expressing only the water-soluble portion of c-mpl and serum of irradiated animals loses TPO activity [Wendling, F. et al. Nature 371: 571 (1994) suggests the possibility that the ligand of c-mpl may be TPO. Based on this, Genentech and Amgen are soluble in c-mpl genes. It was successful to isolate and purify ligands from thrombocytopenia using affinity chromatography method, which only expressed a part and fixed it. After confirming the 5 'terminal amino acid sequence of the isolated and purified ligand protein, oligonucleotides prepared using the oligonucleotides were used as probes. (1994), de Sauvage, FJ et al., Nature 369: 533 (1994).

TPO is composed of 353 amino acids in humans and 352 and 356 amino acids in dogs and rats, respectively, and the amino acid sequences between them are known to be very similar. The TPO amino acid sequence is divided into two regions in terms of similarity with other growth factors. The N-terminal region consists of 162 or 153 amino acids and has approximately 25% amino acid similarity with EPO, whereas the C-terminal region has not found similarity with any known polypeptide and has six N-glycosylation sites. exist. Comparing the activity of the truncated TPO obtained after truncation of the C-terminal region with that of the native TPO, the C-terminal region is independent of TPO activity. Bartley, TD et al., Cell 77: 1117 (1994).

Injection of recombinant TPO into mice reported a four-fold increase in megakaryocyte formation and platelet levels in bone marrow and spleen [Kaushansky., Lok S. et al., Nature 369: 568 (1994)]. Has been found to be a humoral factor with the function of promoting platelet production.

As such, TPO is a humoral factor that is specifically involved in the regulation of platelet production, and may be usefully used as a therapeutic agent for promoting recovery of platelet amount when administered to thrombocytopenia patients.

Recently, methods for expressing human TPO in animal cells have been developed [Bartley, T. D. et al., Cell, 77: 1117 (1994); De Sauvage, FJ et al., Nature, 369: 533 (1994)], when human TPO is expressed in animal cells, it is expressed in the form of natural TPO in the human body, so it can be used as a medicine, but its expression is remarkably low. There was no.

The inventors found out that the expression rate of TPO was low because the signal sequence of TPO was not effective and TPO expressed in cells could not be secreted efficiently extracellularly. Since the amount is significantly higher than that of the native TPO, the TPA signal sequence can effectively secrete the TPO protein extracellularly than the TPO signal sequence. Therefore, the TPO signal sequence is substituted with the signal sequence of tPA, thereby replacing the native TPO protein in animal cells. The present invention was completed by expressing with efficiency.

It is an object of the present invention to provide a method for mass expression of human platelet production promoters (TPO) in animal cells.

Figure 1a shows the nucleotide sequence of the human platelet production promoter,

Figure 1b shows the amino acid sequence of the human platelet production promoter,

Figure 2 shows the nucleotide sequence and amino acid sequence corresponding to the signal sequence of the human tissue plasminogen activator,

3 shows a procedure for cloning the platelet generation promoter gene derived from human fetal hepatocytes and replacing the signal sequence of the human platelet production promoter with a human tissue plasminogen signal sequence.

Figure 4 is an autoradiography of SDS-polyacrylamide gel electrophoresis after immunoprecipitation of the cell lysate and cell culture obtained by transfection of the expression vector of the present invention to COS-7 cells with an antibody of human platelet production promoter The results are shown.

Column 1: Cell disruption solution of COS-7 cells not containing expression vectors

Second column: Cell disruption solution of COS-7 cells containing expression vector pLCED4 TPO

Row 3: Cell disruption solution of COS-7 cells containing expression vector pLCED4 tPAL TPO

Row 4: Cell culture medium of COS-7 cells not containing expression vector

Row 5: Cell culture medium of COS-7 cells containing expression vector pLCED4 TPO

Row 6: Cell culture medium of COS-7 cells containing expression vector pLCED4 tPAL TPO

Row M: Standard Molecular Weight Proteins

In order to achieve the above object, the present invention provides an E. coli expression vector comprising a TPO gene whose signal sequence is substituted with a tPA signal sequence.

In another aspect, the present invention provides E. coli transformants for obtaining the expression vector.

In another aspect, the present invention provides a method for producing a large amount of TPO using the expression vector. Specifically, the present invention provides a method for mass-producing a high-efficiency natural-type TPO protein in animal cells, in which the expression vector is transformed into animal cells and then cultured so that the TPO expressed in the cells is efficiently secreted to the cells.

Hereinafter, the present invention will be described in detail.

The present invention uses human fetal liver cells to isolate genes encoding TPO.

Ribonucleic acid (RNA) is extracted from human fetal liver tissue to synthesize cDNA, and a polymerase chain reaction (hereinafter, abbreviated as 'PCR') is performed as a template. Meanwhile, primers for PCR (oligonucleotides having the nucleotide sequences of SEQ ID NOS: 1 and 2) corresponding to the TPO gene are synthesized and used. Specifically, it was designed to include a base sequence corresponding to the amino terminus of the primer TPO of SEQ ID NO: 1 and a restriction enzyme Bam I recognition site, and the primer of SEQ ID NO: 2 was a stop codon, a restriction enzyme Xho I recognition site and the carboxy terminus of the TPO. Has a sequence As a result, a TPO gene of about 1100 base pairs was isolated.

The gene of TPO obtained by the above procedure was inserted into the plasmid vector pLCED4 gDs to prepare an expression vector pLCED4 TPO containing the TPO gene.

In order to clone the tPA signal sequence, PCR was performed using the expression vector pLCED4sf tPA as a template. At this time, a primer for PCR (an oligonucleotide having the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6) corresponding to the tPA signal sequence is synthesized and used. Specifically, the primer of SEQ ID NO: 5 includes a nucleotide sequence corresponding to the amino terminus of tPA and a restriction enzyme BamH I recognition site, and the primer of SEQ ID NO: 6 represents a restriction enzyme Nar I recognition site and the nucleotide sequence of the carboxy terminus of the tPA signal sequence. Include. As a result, a nucleic acid fragment of about 110 base pairs was obtained.

Meanwhile, PCR is performed using the expression vector pLCED4 TPO as a template to obtain a TPO gene from which the signal sequence has been removed. At this time, a primer for PCR (an oligonucleotide having the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4) corresponding to the TPO signal sequence is synthesized and used. Specifically, the primer of SEQ ID NO: 3 comprises a base sequence corresponding to the carboxy terminus of the TPO signal and a restriction enzyme Nar I recognition site, and the primer of SEQ ID NO: 4 is a restriction enzyme Xho I recognition site and the nucleotide sequence of the carboxy terminus of the TPO gene. It includes. As a result, nucleic acid fragments of about 1000 base pairs were obtained.

The expression sequence pLCED4 tPAL TPO of the present invention containing the TPO gene substituted with the tPA signal sequence was prepared by conjugating the TPO gene and the tPA gene signal sequence from which the signal sequence was removed and then transformed into E. coli.

The expression vector pLCED4 tPAL TPO of the present invention was transformed into Escherichia coli HB101 strain (ATCC 33694) and the Escherichia coli transformant HB101 / pLCED4 tPAL TPO was deposited on 8 April 1998 at the Korea Institute of Science and Technology. (Accession No .: KCTC 0452BP).

The present invention also provides a method for producing TPO. Specifically, the expression vector pLCED4 tPAL TPO of the present invention is transformed into animal cells and then cultured to produce a large amount of the native type TPO protein in animal cells with high efficiency.

In the present invention, the expression vector is first transformed into animal cells and then cultured sufficiently at a temperature of 35 to 40 ° C. to induce the expression of the TPO gene. Since the expression vector of the present invention includes a TPO gene in which a signal sequence is substituted with a tPA signal sequence, TPO expressed in a cell can be efficiently secreted extracellularly, and thus a large-scale production of a natural type TPO protein can be performed.

In the present invention, autoradiography was performed to confirm the expression state of TPO. As a result, the expression amount of TPO substituted with the tPA signal sequence of the present invention was about 3 times higher than that of the native TPO.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples are only for exemplifying the present invention, and the present invention is not limited by the examples.

Example 1 Cloning of the TPO Gene

(Step 1) cDNA synthesis from human fetal liver cells

RNA extraction from human fetal liver cells was carried out by Kologinski et al., Cholozynski, P., et al., Anal. Biochem., 162: 156 (1987)]. 5 × 1 ml of RNA extract [4 M Guaniidinium thiocyanate, 25 mM Sodium Citrate, pH 7.0, 0.5% Sarcosyl, 0.1 M 2-mercapto in 106 cells Ethanol (Mercaptoethanol)] was added to shake the cell membrane and then 0.8 ml of 2 M sodium acetate (pH 4.0), 0.8 ml of phenol, 1.6 ml of chloroform-isoamyl alcohol (49: 1, v / v) After mixing well, centrifuge for 15 minutes at 12,000 G and 4 ° C, transfer the aqueous solution of the upper layer to a new container with the same volume of isopropanol, leave for about 1 hour at -20 ° C, and centrifuge for 20 minutes at 12,000 G, 4 ° C. Isolation gave RNA precipitate. The precipitate was washed with 75% ethanol and then dried in vacuo and dissolved in 20 μl of TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). To synthesize cDNA breaks, 10 μl of the RNA solution obtained above was added, 2 μl of 0.1 M methylmercury (CH ₃ HgOH) was added, and the mixture was left at room temperature for 10 minutes to release the secondary structure of RNA, followed by 2 μl of 1 M 2. Mercaptoethanol was added and reacted for 5 minutes. 5 μl of 10-fold concentrated reverse transcriptase solution [500 mM Tris-HCl, pH 8.3, 750 mM KCl, 30 mM MgC1 ₂ , 10 mM Dithionthreitol], 2.5 μl of 10 mM dNTP mixture (dGTP, dATP, dTTP, dCTP 10 mM each, distilled water treated with 24 μl of diethylpyocarbonate (DEPC), 1.0 μl of RNase inhibitor (RNasin, 1U / μl, Promega, USA), 1 μl of oligo (dT ) _12-18 primer (oligo (dT) _12-18 primer, GibcoBRL, USA) was added and allowed to stand at room temperature for 10 minutes to allow RNA template and primer to adhere well, followed by 2.5 μl reverse transcriptase (18U / μl, Superscript RNase H). -Reverse transcriptase, BRL, USA) was added to react the cDNA strand at 37 ℃ for 1 hour.

(Step 2) Synthesis of the primer

To obtain the TPO gene from the cDNA obtained in step 1 using a polymerase chain reaction, the following primers were synthesized using a DNA synthesizer (DNA Synthesizer, Applied Biosystems lnc., U.S.A.).

That is, a primer for PCR (an oligonucleotide having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2) corresponding to the TPO gene is synthesized from the TPO nucleotide sequence information. Specifically, the primer of SEQ ID NO: 1 is a base corresponding to the amino terminal of the TPO. And a primer of SEQ ID NO: 2 comprising a stop codon, a restriction enzyme Xho I recognition site, and a nucleotide sequence of the carboxy terminus of the TPO.

(Step 3) Isolation of TPO Gene

In order to amplify the TP0 gene, a polymerase chain reaction was performed using a primer having SEQ ID NO: 1 and SEQ ID NO: 2. 2 μg of each primer obtained in step 2 was added to the reaction tube, and 1 ng of human fetal liver cell cDNA obtained in step 1 was used as a template, and 10 μl of 10-fold polymerization buffer solution (500 mM KCl, 100 mM Tris-HCl, pH 9.0, 1% Triton X-100, 2.5 mM MgC1 ₂ ), 10 μl of 2 mM dNTP (2 mM each of dGTP, dATP, dTTP, dCTP), 2.5 monomer Taq polymerase and distilled water were added to make the total volume 100 μl. Then, PCR was performed 40 times under conditions of 1 minute (modification step) at 95 ° C, 40 seconds (immersion step) at 55 ° C, and 2 minutes (polymerization step) at 72 ° C. As a result, a TPO gene was obtained, hereinafter referred to as 'fragment TPO'. 2 μg of the fragment 'TPO' obtained above was completely purified by restriction enzymes BamH I and Xho I under conditions of NEB buffer solution 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl _2, 1 mM dithiothreitol, pH 7.9). After cleavage, electrophoresis separation was performed with 7% acrylamide gel to obtain nucleic acid fragments of about 1100 base pairs. This fragment is hereinafter referred to as 'fragment TPO-B / X'.

Example 2 Preparation of TPO Gene Expression Vector

First, 2 µg of plasmid pLCED4 gDs (Korean Patent Application No. 95-22864) was added to the NEB buffer solution 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl _2, 1 mM dithiothitol, After complete cleavage under the condition of pH 7.9), and separated by 1% agarose gel, about 5.5 kb nucleic acid fragments were purified.

100 ng of fragment TPO-B / X and 100 ng of fragment pLCED4-B / X obtained in step 3 of Example 1 were placed in a conjugation reaction tube, followed by 2 μl of 10-fold conjugation solution [50 mM Tris-HCl. (pH 7.8), 10 mM MgCl ₂ , 1 mM dithiothreitol, 1 mM ATP, 25 μg / ml bovine serum albumin], 10 units of T4 DNA ligase and a total volume of 20 μl. Distilled water was added and then reacted at 16 ° C. for 12 hours. After the reaction, E. coli HB101 (ATCC 33694) was transformed to obtain pLCED4 TPO containing the TPO gene. The sequencing of the cDNA fragment cloned into the recombinant plasmid pLCED4 TPO obtained above was performed according to the method of Sanger et al., Sanger, F., et al., PANS USA 74: 5463 (1997). [Dye Cycle Sequencing] System (Applied Biosystems, USA)] and analyzed by the ABI 377 DNA sequence of the company.

Example 3 Signal Sequence Cloning of tPA Gene

The following primers were synthesized using a DNA synthesizer (DNA Synthesizer, Applied Biosystems Inc., U.S.A.) to obtain a signal sequence gene of the tPA gene using a polymerase chain reaction.

A primer for PCR (an oligonucleotide having the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6) corresponding to the tPA gene is synthesized from the nucleotide sequence information of tPA. Specifically, the primer of SEQ ID NO: 5 comprises a nucleotide sequence corresponding to the amino terminus of tPA and a restriction enzyme BamH I recognition site, and the primer of SEQ ID NO: 6 is a nucleotide sequence of the restriction enzyme Nar l recognition site and the carboxy terminus of the signal sequence of tPA It includes.

To obtain the signal sequence of the tPA gene, the expression vector pLCED4sf tPA of Korean Patent Application No. 97-10763, filed on March 27, 1997, was used as a template, and the sequence 5 and the sequence 6 primer were used as in Example 3 The polymerase chain reaction was carried out by the method. As a result, a signal sequence of the tPA gene was obtained and this fragment is hereinafter referred to as 'fragment tPA'. 2 μg of the fragment “tPAL” obtained above was subjected to restriction enzymes BamH I and Nar I under conditions of NEB buffer solution 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , lmM dithiothreitol, pH 7.9). After complete cleavage, electrophoresis separation was performed using 7% acrylamide gel to obtain a nucleic acid fragment of about 110 base pairs. This fragment is hereinafter referred to as 'fragment tPAL-B / N'.

<Example 4> Preparation of the expression vector in which the signal sequence of the TPO gene is substituted with the signal sequence of the gene

TPO genes removed from the signal sequence using the polymerase chain reaction were synthesized using the following primers (DNA Synthesizer, Applied Biosystems Inc., U.S.A.).

A primer for PCR (an oligonucleotide having the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3) corresponding to the TPO gene is synthesized from the nucleotide sequence information of the TPO. Specifically, the primer of SEQ ID NO: 3 comprises a nucleotide sequence corresponding to the carboxy terminus of the TPO signal sequence and a restriction enzyme Nar I recognition site, and the primer of SEQ ID NO: 2 is a nucleotide sequence of the restriction enzyme Xho I recognition site and the carboxy terminus of the TPO gene. It includes.

In order to obtain the TPO gene from which the signal sequence was removed, the polymerase chain reaction was performed in the same manner as in Step 3 of Example 1, using the expression vector pLCED4 TPO of Example 2 as a template and the primers of SEQ ID NO: 3 and SEQ ID NO: 2 . As a result, the TPO gene from which the signal sequence was removed was obtained, which is hereinafter referred to as 'fragment ΔTPO'. 2 μg of the fragment ΔTPO obtained above was subjected to the conditions of NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH 7.9) using restriction enzymes Nar I and Xho I. After complete cleavage, nucleic acid fragments of about 1000 base pairs were obtained by electrophoresis separation with 7% acrylamide gel. This fragment is hereinafter referred to as 'fragment ΔTPO-N / X'.

100 ng of fragment tPAL-B / N obtained in Example 3, 100 ng of fragment ΔTPO-N / X obtained in Example 3, and 100 ng of fragment pLCED4-B obtained in Step 3 of Example 1 above in a conjugation reaction tube / X and then 2 μl of 10-fold conjugation solution [50 mM Tris-HCl (pH 7.8), 10 mM MgCl ₂ , 1 mM dithiothreitol, 1 mM ATP, 25 μg / ml bovine serum albumin], Ten units of T4 DNA ligase and distilled water were added so that the total volume was 20 μl, followed by reaction at 16 ° C. for 12 hours. After the reaction, E. coli HB101 (ATCC 33694) was transformed to obtain pLED4 tPAL TPO containing the TPO gene substituted with tPA signal sequence.

The sequencing of TPO substituted with the tPA signal sequence cloned in the recombinant plasmid pLCED4 tPAL TPO obtained above was performed according to the method of Sanger et al. [Sanger, F., et al., PNAS USA, 74: 5463 (1997)]. It was performed using a cycle sequencing system (Applied Biosystems, USA) and analyzed by the company's ABI 377 DNA a nucleotide sequencer.

Example 5 Expression of TPO Substituted by Signal Sequence of COS-7 tPA Gene in Mammalian Cells

Mammal in order to observe the expression level of the substituted TPO from animal cells as a signal sequence in COS-7 tPA gene COS-7 cells (ATCC CRL1651) for 37 ℃ after a 15-fold dilution pre-sale in 35 mm plates, in a CO ₂ incubator Incubated. After confirming that the cells had grown to about 70-80% on the plate, plasmid pLCED4 TPO and plasmid pLECD4 tPALTPO were transfected into the cells by lipofectamine method (Gibco BRL Cat. No. 18324-012), respectively. The medium used at this time was D-MEM medium containing 10% calf serum [Dulbecco's Modified Eagle Medium; 1 g / L D-glucose, L-glutamine, 110 mg / L sodium pyruvate, Gibco BRL Cat. No. 11885-035]. Cultured cells and cultures were harvested 48 hours after transfection. The cell lysate and culture medium obtained by crushing the cultured cells were mixed with the TPO antibody solution and reacted, followed by immunoprecipitation. This was autoradiography by electrophoresis on 12% SDS-polyacrylamide gel according to the method of Lambley [Leammli, Nature, 227, 680 (1970)]. FIG. 4 shows human platelets of cell lysates and cell cultures obtained by transfecting COS-7 cells with expression vector pLCED4 tPAL TPO containing expression vector pLCED4 TPO and tPA signal sequence comprising a native TPO gene. It is the result of autoradiography by immunoprecipitation with antibody of production promoter and electrophoresis of SDS-polyacrylamide gel.

Here, the fifth and sixth columns show that the expression level of TPO substituted by the tPA signal sequence of the present invention is about three times higher than that of the TPO secreted into the cell culture.

In the present invention, by replacing the signal sequence of TPO with the signal sequence of tPA to efficiently secrete the TPO expressed in the cell extracellularly, the signal sequence of the TPO is not effective, the intracellular expressed TPO is not secreted extracellularly By solving the conventional problem of low expression amount, the natural TPO protein can be expressed in animal cells with high efficiency.

(Sequence list)

SEQ ID NO: 1

Sequence length: 28

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 2

Sequence length: 32

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 3

Sequence length: 37

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 4

Sequence length: 28

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 5

Sequence length: 32

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

Claims (3)

Mammalian cell expression vector pLCED4 tPAL TPO comprising a TPO gene in which the signal sequence of human Thrombopoietin (TPO) gene is substituted with a signal sequence of human tissue plasminogen activator (tPA) Escherichia coli transformant transformed with the expression vector of claim 1 HB101 / pLCED4 tPAL TPO (Accession No .: KCTC 0452BP) A method for producing human platelet promoter (TPO), characterized in that the expression vector pLCED4 tPAL TPO is transformed into animal cells and then cultured.