JPH06225767A - Gene coding magakaryocyte amplifier - Google Patents

Abstract

PURPOSE:To obtain a new DNA useful for the production of a megakaryocyte amplifier known as an agent for the treatment of diseases relating to the thrombocytopenia or platelet hypoactivity. CONSTITUTION:A DNA containing a base sequence coding the amino acid sequence of formula, a base sequence obtained by partly substituting or depleting the above base sequence or adding a fragment to the sequence or a base sequence hybridizing to the above sequence. It can be produced by preparing an mRNA from a cell capable of producing megakaryocyte amplifier, e.g. established cell HPC-Y5 (FERM BP-3703) originated from human pancreatic carcinoma cell and converting the mRNA to double stranded cDNA by conventional method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to genes, and more specifically, it acts on interleukin-3 (IL-3) which acts on Megakaryocyte Colony-Forming Units differentiated from pluripotent blood stem cells. Megakaryocyte Colony-Stimulating Fac
tor: abbreviated as Meg-CSF) A human-derived megakaryocyte amplification factor (hereinafter referred to as Meg-PO) that promotes maturation of megakaryocytes in the presence of a substance having activity.
Abbreviated as T])) A gene encoding a polypeptide having activity, a recombinant vector containing the gene,
The present invention relates to a transformant using the vector and a method for producing a megakaryocyte amplification factor using the gene.

[0002] The gene of the present invention has IL in vitro.
In the presence of -3, it encodes a megakaryocyte amplification factor having an action of amplifying megakaryocyte colonies in a dose-dependent manner. By inserting the gene of the present invention into an appropriate vector and transforming a conventional host cell, a large amount of uniform megakaryocyte amplification factor can be produced. From this, the present invention can provide a therapeutic agent expected to be clinically useful for diseases associated with, for example, thrombocytopenia or decreased platelet function.

[0003]

2. Description of the Related Art Platelets are one of the blood-forming components that are important for hemostasis and thrombus formation in the living body. Platelets are converted from hematopoietic stem cells in the bone marrow to megakaryocyte progenitor cells to megakaryocyte blasts, and then released into blood from mature megakaryocytes.

It is considered that two types of factors having different actions are necessary for the formation of megakaryocyte colonies from bone marrow cells (Williams, N et al. "J. Cell Physiol.
110. 101 (1982)). That is, Meg-CSF which forms a megakaryocyte colony only by adding it and Meg-CSF which has no activity for forming a megakaryocyte colony by itself,
Meg-POT showing the action of increasing the number of megakaryocyte colonies and promoting its maturation when added together with eg-CSF
Is.

In humans, IL-3 (Teramura, M et al. “Exp. Hematol.” 1 ) is considered to have Meg-CSF activity.
6 , 843 (1988)) and granulocyte / macrophage colony stimulating factor (Teramura, M et al. “Exp. Hematol.” 17 , 1011
(1989)) are known. In humans, Meg-PO
Interleukin 6 (Ter) has T activity.
amura, M and Mizoguchi, H "Int. J. Cell Cloning" 8, 2
45 (1990)), Interleukin 11 (Teramura, M et al.
"Blood" 79 , 327 (1992)), erythropoietin (Brun
O, E et al. “Blood” 73, 671 (1989)) and the like are known.

However, it is known that these are not factors specific to the megakaryocyte / platelet system, but rather have an action on other blood cells and cells other than blood cells. Therefore, when these substances are administered as pharmaceuticals in the expectation of their action on the megakaryocyte / platelet system, it is feared that they may exert an action other than that. From this, a physiologically active substance that acts specifically on the megakaryocyte / platelet system and is highly useful as a drug is desired.

[0007]

Therefore, at the same time as finding a new megakaryocyte growth factor that acts on the megakaryocyte-platelet system,
In order to use the megakaryocyte amplification factor for medical use, it was necessary to obtain it in a large amount. In the method of isolating the megakaryocyte amplification factor from the culture supernatant of the producing cells, the megakaryocyte amplification factor concentration in the culture supernatant is low, and a complicated purification step is required to obtain a uniform megakaryocyte amplification factor, and There were some difficulties, such as obtaining only a very small amount. Therefore, for the above purpose of use, recombinant DN
It has been desired to produce a large amount of a novel megakaryocyte amplification factor that acts on the megakaryocyte / platelet system using the A technique.

[0008]

In such a situation, the present inventors have established a human pancreatic cancer cell-derived cell line "HPC-".
The megakaryocyte-amplifying activity was found in the culture supernatant of "Y5", and a novel megakaryocyte-amplifying factor having the objective megakaryocyte-amplifying activity as an index was purified from the culture supernatant to clarify its properties (international application No .: PCT / JP92 / 01689, International Publication Number: WO93 / 13132).

Further, an oligonucleotide primer was synthesized based on the information on the amino acid sequence, and "HPC-Y
A DNA fragment encoding a megakaryocyte amplification factor was obtained from the cDNA library prepared from the mRNA prepared from "5" by the polymerase chain reaction (hereinafter abbreviated as PCR) using the above synthetic primers. Then this D
A cDNA library was screened using the NA fragment as a probe, and a gene encoding a novel novel megakaryocyte amplification factor was successfully isolated, and its entire nucleotide sequence was clarified.

Further, a novel megakaryocyte is obtained by inserting this gene into an appropriate vector, culturing a transformant transformed with this expression vector, and then separating and purifying the produced target protein. It was also revealed that the amplification factor can be produced in large quantities.

Accordingly, the present invention provides a gene encoding a polypeptide having human megakaryocyte amplification factor activity.

The present invention further provides a recombinant vector containing a gene encoding a polypeptide having human megakaryocyte amplification factor activity.

The present invention also provides a prokaryotic or eukaryotic host cell transformed with a recombinant vector containing a gene encoding a polypeptide having human megakaryocytic amplification factor activity.

The present invention further comprises culturing a transformant obtained by transforming with a recombinant vector containing a gene encoding a polypeptide having human megakaryocyte amplification factor activity, and producing the desired protein, preferably the target protein. Provided is a method for producing a protein having human megakaryocyte amplification factor activity.

[0015]

[Specific Description] The megakaryocyte amplification factor gene can be obtained, for example, by preparing mRNA from cells producing a megakaryocyte amplification factor, and then converting it into a double-stranded cDNA by a known method. In the present invention, the cell serving as the source of this mRNA is a human pancreatic cancer tumor cell-derived cell line “H”.
PC-Y5 "(Nozomi Yamaguchi et al. CANCER RESEA
RCH 50 7008 1990) [December 27, 1991 International Institute for Microbial Technology, Institute of Industrial Science and Technology, Microdevelopment Article No. 3703 (FERM BP-3703), an international deposit based on the Budapest Treaty] Not only the cell line, but also cells that can be separated from mammals or other established cell lines may be used.

In the present invention, mRNA is treated with guanidine thiocyanate and then subjected to cesium chloride density gradient centrifugation (Chirgwin et al. Biochemistry 18 5294).
1979) Total RNA was obtained, but the method already used for cloning genes of other physiologically active proteins, for example, the treatment with a surfactant and the treatment with phenol in the presence of a ribonuclease inhibitor such as vanadium complex (Berger & B
irkenmeier, Biochemistry, 18 5143 1979) method can be used.

Preparation of poly (A) ⁺ RNA from total RNA can be carried out by affinity column chromatography using an oligo (dT) -bound carrier such as sepharose or cellulose, or by a batch method. Also,
The poly (A) ⁺ RNA can be further purified by a sucrose density gradient centrifugation method or the like. In addition, there is a method of directly obtaining poly (A) ⁺ RNA without once preparing RNA.

From the mRNA obtained as described above, double-stranded c
To obtain DNA, for example, using mRNA as a template, 3 '
DNA complementary to mRNA by treating with reverse transcriptase the oligo (dT) complementary to the polyA-chain at the end or a random primer or a synthetic oligonucleotide corresponding to a part of the amino acid sequence of the megakaryocyte amplification factor as a primer
(CDNA) is synthesized.

After decomposing / removing mRNA by alkaline treatment, the obtained single-stranded cDNA is used as a template for reverse transcriptase or DNA polymerase (for example, Klenow fragment).
After treatment, treat with S1 nuclease or directly RN
Double-stranded cDNA can also be obtained by treating with asase H and DNA polymerase (Maniati
s et al. Molecular Cloning, Cold Spring Harbor Lab
oratory 1982 and Gubler & Hoffman, Gene 25 263 1
983).

The cDNA encoding the megakaryocyte amplification factor can be isolated by a method such as direct expression cloning using the megakaryocyte amplification activity as an index or an antibody.

[0021] The megakaryocyte amplification activity can be measured by applying a soft agar culture method using bone marrow cells in the presence of IL-3.

That is, horse serum (56 ° C. for 30 minutes, B
iocell) 0.2 ml, mouse (C57BL / 6N male, 6-12 weeks old) femur bone marrow cell suspension 0.1 ml (2
× 10 ⁵ nucleated cells), recombinant mouse IL-3 at 5 ng /
Iscove's Modified Dulbecco's culture solution containing ml (IMD
M) Modified McCoy's 5A containing 0.2 ml and agar of 0.75%
0.4 ml of the culture solution and 0.1 ml of the test sample (diluted with IMDM containing 10% horse serum) were mixed and put in a tissue culture plastic dish with a diameter of 35 mm to solidify, and then 37 ° C, 5 % Carbon dioxide / 95% air, 10
Culture is performed under the condition of 0% humidity.

On the 6th day of culture, the whole agar layer was taken out on a slide glass and dried, and a film-shaped sample was fixed with 5% glutaraldehyde, followed by the method of Nakeff et al. (Proc. So.
c.Exp.Biol.Med. 151 587 1976) and stained with acetylcholinesterase, and the number of megakaryocyte colonies is calculated. At this time, an aggregate containing 4 or more cells positive for acetylcholinesterase staining is designated as a megakaryocyte colony. The magnification of the speculum is 40 times. The Meg-POT activity was generated by the recombinant IL-3 alone without the number of megakaryocyte colonies generated by adding the test sample and the test sample (adding only IMDM containing 10% horse serum). The difference from the number of megakaryocyte colonies is used as an index.

The present inventors isolated and purified a megakaryocyte amplification factor from the culture supernatant of a cell line that produces a megakaryocyte amplification factor,
A primer is synthesized based on the information of the amino acid sequence,
Using PCR, a gene fragment encoding a polypeptide having human-derived megakaryocyte amplification activity was cloned.
CDNA using the DNA as a probe by a known method
The library was screened for clones containing the full-length cDNA encoding the novel novel megakaryocyte amplification factor.

These cDNAs were designated as pBluescript S
PKPO27 inserted between the EcoRI and XhoI cleavage sites of K (-)
E. coli strain JM109 containing pK, and pK
E. coli JM109 strain containing PO21 is
1st year in 1992, respectively, at the Institute of Microbial Engineering, Institute of Industrial Technology
On 12th of January, Micro Engineering Research Article No. 4029 (FERM BP
-4029), and on November 10, 1992
No. 071 (FERM BP-4071) has been deposited internationally under the Budapest Treaty.

Further, by repeating the PCR used in the present invention, full-length cDNA can be obtained. Alternatively, the desired cDNA can be obtained by directly synthesizing a probe from the information of the amino acid sequence and directly screening a cDNA library without using PCR.

Thus, the cloned gene encoding the megakaryocyte amplification factor can be transformed into another prokaryotic or eukaryotic host cell by incorporating it into an appropriate vector DNA.

Furthermore, it is possible to express the gene in each host cell by introducing an appropriate promoter and a sequence for expression into these vectors. In addition, by linking a gene encoding another polypeptide to the gene of interest and expressing it as a fusion protein, facilitating purification or increasing the expression level, and subjecting it to an appropriate treatment in the purification step, It is also possible to cut out the target protein. In addition, the gene to be linked is a fusion protein using that of another bioactive factor,
Attempts to enhance the megakaryocyte amplification activity are also possible.

Generally, eukaryotic genes are considered to exhibit polymorphism as known in human interferon genes and the like (for example, Nishi et al., J. Biochem. 97 153).
1985), one or more amino acids may be substituted by this polymorphism, or the amino acids may not be changed at all even if the base sequence changes.

Also, a polypeptide lacking or adding one or more amino acids in the amino acid sequence of SEQ ID NO: 12 or 13 or a polypeptide in which an amino acid is substituted with one or more amino acids is a megakaryocyte. It may have amplification activity. For example, it is already known that a polypeptide obtained by converting a nucleotide sequence corresponding to cysteine of human interleukin 2 (IL-2) gene into a sequence corresponding to serine retains IL-2 activity. (Wang et al., Science, 224 1431 1984).

When expressed in eukaryotic cells, most of them are added with sugar chains, but sugar chain addition can be regulated by converting one or more amino acids. In this case as well, megakaryocytes are used. It may have amplification activity. Therefore, all genes encoding a polypeptide obtained by artificially modifying the megakaryocyte amplification factor gene of the present invention are included in the present invention. At that time, a gene encoding a polypeptide in which amino acids are randomly changed can be prepared, but for example, the amino acid of a protein having megakaryocyte amplification factor activity of an animal species other than human (mouse, rat, monkey, etc.) It is also possible to produce genes encoding various modified forms by substituting or deleting the nucleotide sequence with reference to the sequence.

Furthermore, the present invention also includes a gene in which the obtained polypeptide has megakaryocyte proliferating activity and hybridizes with a gene encoding a polypeptide containing the amino acid sequence shown in SEQ ID NO: 12 or 13. Incidentally, as the hybridization conditions, it is possible to apply the conditions of probe hybridization that is usually performed (for example, Molecular Cloning: A Laboratory Manual,
Sambrook et al., Cold spring Habor Laboratory Press,
1989).

The expression vector of the present invention contains an origin of replication, a selection marker, a promoter located in front of the gene to be expressed, an RNA splice site, a polyadenylation signal and the like.

As promoters for gene expression in mammalian cells, retroviruses, polyomaviruses,
Adenovirus, simian virus 40 (SV40) and other viral promoters and human polypeptide chain elongation factor 1α (HEF-1)
A cell-derived promoter such as α) may be used. For example, when using the SV40 promoter, Mullig
It can be easily implemented according to a method such as an (Nature 277 108 (1979)).

Origins of replication include SV40, polyoma virus, adenovirus, bovine papilloma virus (B
PV) and the like can be used, and the selection marker is phosphotransferase APH (3 ′).
II or I (neo) gene, thymidine kinase (TK)
A gene, an Escherichia coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene, a dihydrofolate reductase (DHFR) gene, etc. can be used.

Further, in the case of a prokaryotic host cell such as Escherichia coli, transformation can be carried out using pBR322 which is a vector using the host (Boliver et al .: Gene 2).
95 (1975)). pBR322 contains genes for ampicillin and tetracycline resistance, and transformed cells can be identified by utilizing either resistance. As a promoter required for gene expression in a prokaryotic host, a β-lactamase gene promoter is used.
(Chang et al .: Nature 275 615 (1978)), lactose promoter (Goeddel et al .: Nature 281 544 (1979)) and tryptophan promoter (Goeddel et al .: Nucleic Aci
d Res. 8 4057 (1980)), tac promoter and the like, and any promoter can be used for expression of the human megakaryocyte amplification factor of the present invention.

Among the hosts used in the expression system of the present invention, examples of prokaryotic host cells include Escherichia coli.
coli ), Bacillus subtilis
, Bacillus thermophilu
s ) and the like. Among eukaryotes, examples of eukaryotic microbial host cells include Saccharomyces cerevisiae , and examples of mammalian-derived host cells include COS cells, Chinese hamster ovary (CHO) cells, C127
Cells, 3T3 cells, Hela cells, BHK cells, Nabarva cells and the like. The culture of the transformant of the present invention may be carried out by appropriately selecting culture conditions suitable for the host cell.

As described above, the transformant transformed with the gene encoding the desired megakaryocyte amplification factor is cultured, and the produced megakaryocyte amplification factor is isolated from inside or outside the cell and purified to homogeneity. can do.

The human megakaryocyte amplification factor, which is the target protein of the present invention, can be separated / purified by using the separation / purification method used for ordinary proteins, without any limitation. For example, various chromatography, ultrafiltration,
By appropriately selecting and combining salt folding, dialysis and the like, the human megakaryocyte amplification factor can be separated and purified.

[0040]

EXAMPLE A method for obtaining a gene encoding a megakaryocyte amplification factor of the present invention, a recombinant vector having the gene, a transformant containing the same, a protein of interest obtained by culturing the transformant, and About the manufacturing method of
The present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Meg-POT producing cell line HP
A tumor obtained from a lymph node of an established pancreatic cancer patient of CY5 was used in RPMI1640 medium containing 10% fetal bovine serum (FBS).
Carbon dioxide incubator (carbon dioxide concentration 5%, humidity 1
It was established by culturing in (00%). This cell line was acclimated to Ham's F10 medium containing 1% FBS, and then the FBS concentration was gradually reduced until finally the protein-free Ha
Acclimation was allowed to grow in m's F10 medium. This cell line grew in a monolayer on a plastic dish and had a doubling time of about 33 hours (Nozomi Yamaguchi et a
l. CANCER RESEARCH 50 7008 1990). This cell line
It is called PC-Y5 cells.

Example 2 Subculture of HPC-Y5 and
Mass culture with roller bottle Subculture of the HPC-Y5 cells described in Example 1 was performed as follows. Plastic culture flask (150 cm
² (manufactured by Corning), 10 ^-8 M sodium selenite, 100 U / ml penicillin G potassium and 100 μm
g / ml) Ham's Nutrient Mixt containing kanamycin sulfate
HPC-Y5 cells were cultured in 50 ml of ure F12 medium,
The culture medium was exchanged every 4 days.

The culture medium was removed at the time of cell passage, and 0.125% trypsin (GIBCO) preheated to 37 ° C was used.
), 0.01% ethylenediaminetetraacetic acid (EDT
A) Calcium- and magnesium-free Dulbecco's Phosphate-buffered saline (PBS) containing (manufactured by Wako Pure Chemical Industries, Ltd.)
The solution was added and heated at 37 ° C. for 5 minutes. The cells were detached by pipetting operation, transferred to a 15 ml plastic centrifuge tube, and centrifuged at 1500 rpm for 5 minutes to collect the cells. The cells were suspended in the above medium and subcultured in 4 to 5 new flasks. After standing overnight, the culture medium was removed together with the non-adherent cells, and the above medium was newly added to continue the culture. After that, the culture medium was exchanged every 4 days.

Large-scale culture of HPC-Y5 cells in a roller bottle for purification of Meg-POT described in Example 3 was carried out as follows.

The HPC-Y5 cells subcultured as described above were collected from a 150 cm ² plastic culture flask in which they were completely densely grown, using trypsin-EDTA as described above, and the cells were collected in 0.2% fetal bovine. Serum (Hyclone)
Suspended in 250 ml of the above medium containing 1700 cm ²
Was transferred to a plastic roller bottle (made by Corning) and subjected to rotary culture at a speed of 0.5 rpm. After 7 days, the culture medium was replaced with the above-mentioned medium containing no serum, and the serum-free medium was replaced every 4 days thereafter to collect the serum-free culture supernatant for purification.

Example 3 From the culture supernatant of HPC-Y5 strain
Purification of Meg-POT Tween 20 was added to the culture supernatant (27.3 liters) of HPC-Y5 cells obtained according to the method described in Example 2 so that the final concentration was 0.01%, and then artificial kidney PAN12 was used.
00 (Asahi Medica) was used to concentrate about 200 times.
The concentrate was dialyzed against 10 mM acetate buffer (pH 5.0) containing 0.01% Tween 20 at 4 ° C. overnight. The dialyzed solution was centrifuged (10000 × g, 60 minutes) to remove insoluble matter, and the supernatant was used for the following purification.

(Step-1) S-Sepharose ion
Exchange Chromatography S-Sepharose Fast obtained by equilibrating the above-mentioned centrifugation supernatant with 20 mM acetate buffer (pH 5.0) containing 0.01% Tween 20.
It was added to a Flow (Pharmacia) column (5 × 10 cm). After washing the column with the same buffer,
The adsorbed protein was eluted by sequentially increasing the concentration of Cl to 0.15M, 0.5M and 1.0M. As a result of measuring the activity of the flow-through fraction, the wash fraction, and the elution fraction at each salt concentration according to the method described above, Meg-POT activity was observed in the 0.15 M-NaCl elution fraction.

(Step-2) DEAE-Sepharose
The active fraction obtained in the ion exchange chromatography step-1 was added to 10 mM Tris-hydrochloric acid buffer (pH 7.4) containing 0.01% Tween 20 at 4 ° C.
Dialyzed overnight. DE in which dialysis solution is equilibrated with the same buffer
The mixture was added to an AE-Sepharose Fast Flow (Pharmacia) column (2.2 × 13 cm), and the column was washed with the same buffer solution. The concentration of NaCl in the buffer is 0.15M, 0.5
The adsorbed protein was eluted by successively increasing M and 1.0M.
As a result of measuring the activity of the flow-through fraction, the wash fraction and the elution fraction at each salt concentration according to the above-mentioned method, 0.
Meg-POT activity was observed in the 15M-NaCl elution fraction.

(Step-3) Reversed-phase HPLC (I) To the active fraction obtained in Step-2, 5% trifluoroacetic acid (TFA) was added to adjust the pH to about 2, and 0.1% TFA was added.
Was added to a reversed-phase HPLC column (Protein C4, 10 × 250 mm, manufactured by Vydac) equilibrated with 5% acetonitrile containing 1.0 g / min. Adsorbed protein is a linear concentration gradient of acetonitrile (5% → 65%, 120 minutes, 0.5
% Acetonitrile / min) at a flow rate of 1.0 ml / min. The eluted protein was detected by following the absorbance at 220 nm and 280 nm, and fractionated in 1 ml portions. As a result of activity measurement of each fraction, Meg-POT activity was observed in the fraction having an acetonitrile concentration of 40-45%.

(Step-4) Reversed-phase HPLC (II) The active fraction obtained in Step-3 was diluted 2-fold with 0.1% TFA, and then reversed by equilibration with 35% acetonitrile containing 0.1% TFA. Phase HPLC column (Protein C4, 4.6x
250 mm, manufactured by Vydac) at a flow rate of 1.0 ml / min. The adsorbed protein was a linear concentration gradient of acetonitrile (35
% → 50%, 75 minutes, 0.2% acetonitrile / minute) at a flow rate of 1.0 ml / minute. The eluted protein was detected by following the absorbance at 220 nm and 280 nm and fractionated in 1 ml portions. As a result of measuring the activity of each fraction, M was found to be in the fraction of acetonitrile concentration 40-45%.
Egg-POT activity was observed.

(Step-5)DEAE / ion exchange HPLC  After freeze-drying the active fraction obtained in step-4, 0.0
10 mM Tris-HCl buffer containing 1% Tween 20 (pH
Protein Pak dissolved in 8.0) and equilibrated with the same buffer
G-DEAE column (Waters, 8.2 x 75 mm)
The flow rate was 0.7 ml / min. Adsorbed protein is NaCl
Linear concentration gradient (0.0M → 0.2M, 40 minutes, 5 mM N
aCl / min) at a flow rate of 0.7 ml / min. Elution
The protein was detected at 220 nm and fractionated by 0.7 ml. each
As a result of measuring the activity of the fractions, the NaCl concentration was 75 mM.
Meg-POT activity was observed in the following fractions.

(Step-6) TSKgel G30
00SW gel filtration step-5, the active fraction obtained in 0.01% Tween 20
And TSKgel G3000 equilibrated with 50 ml Tris-HCl buffer (pH 7.4) containing 0.15M NaCl.
The SW column (21.5 × 600 nm, manufactured by Tosoh Corporation, guard column 21.5 × 75 nm) was passed through the column at a flow rate of 3 ml / min, and the eluted protein was detected at 220 nm. The activity of each of the 3 ml fractions was measured, and the result was Meg-POT.
Since the activity was observed in the fraction with an elution time of 49 to 54 minutes,
The fraction was collected.

(Step-7) Reversed-Phase HPLC (III) The active fraction obtained in Step-6 was adjusted to pH about 2 by adding 5% TFA, and the same as in Reversed-phase HPLC (II) of Step-4. Chromatography was performed under the conditions. As a result of measuring the activity of each fraction, Meg-POT activity was observed in the main peak (acetonitrile concentration 40% -45%).
The result is shown in FIG. Fractions indicated by horizontal bars in FIG. 1 were collected as purified Meg-POT.

Example 4 Megakaryocyte amplification factor (Meg-PO
T) amino acid sequence (i) Determination of N-terminal amino acid sequence The purified Meg-POT sample obtained in Example 3 was obtained by Edman degradation using a gas phase protein sequencer type 470A (manufactured by Applied Biosystems). Phenylthiohydantoin (PTH) -amino acid was identified using PTH Analyzer Model 120 (manufactured by Applied Biosystems). As a result, the amino acid sequence near the N-terminal (sequence-1
3), the following three types were recognized.

[0055]

[Table 1] Sequence-1 (SEQ ID NO: 1) Leu Ala Gly Glu Xaa Gly Gln Glu Ala Ala Pro Leu Asp Gly Val Leu 1 5 10 15 (where Xaa represents an unidentified amino acid, the same applies hereinafter) Sequence -2 (SEQ ID NO: 2) Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu Asp Gly Val Leu Ala 1 5 10 15 Sequence-3 (SEQ ID NO: 3) Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu Asp Gly Val Leu Ala Asn 1 5 10 15 (The number below the sequence is the number of cycles at the time of Edman degradation.) The following amino acid sequences were found to be common in the amino acid sequences of sequence-1 to sequence-3 above. .

Gly Glu Thr Gly Gln Glu Ala Ala Pro Le
u Asp Gly Val Leu (SEQ ID NO: 4).

(Ii)Endoproteinase Glu-C digestion  The sample was added with 0.1 M ammonium hydrogen carbonate buffer (pH 7.
8) dissolved in urea and dithiothreitol (DT
The final concentrations of T) are 7.8 M and 50 mM, respectively.
And incubated at 37 ° C. for 2 hours. There
And Endo Glu-C (Boehringer-Mannheim)
After adding 0.5 μg and reacting at 37 ° C. for 2 hours,
The same amount of enzyme was added and the reaction was carried out again at 37 ° C. for 18 hours.
Add 10% trifluoroacetic acid (TFA) to the reaction mixture and add
After setting to 2, C18 color equilibrated with 0.1% TFA
(4.6 × 25 cm, Vydac).

The peptide was eluted by linearly increasing the concentration of acetonitrile in 0.1% TFA from 0 to 64% in 128 minutes and then 80% in 16 minutes. Peptides were detected at two wavelengths, 220 nm and 280 nm. The obtained partially digested peptide fragments were sequentially analyzed by the gas phase protein sequencer 473A (Applied Biosystems).
The amino acid sequence was analyzed using the product manufactured by K.K.

As a result, one of the partially digested fragments was found to have the sequence shown below.

Leu-Ala-Val-Ala-Leu-Ala-Gln-Lys-Asn-Va
l-Lys-Leu-Ser-Thr-Glu-Gln-Leu-Arg-Xaa-Leu-Ala-His-
Arg-Leu-Ser-Glu-Pro-Pro-Glu-Asp-Leu-Asp-Ala-Leu-Pr
o- --- (SEQ ID NO: 5).

Example 5 Poly from HPC-Y5 cells
(A) ⁺ RNA Chirgwin like from Preparation HPC-Y5 cell (Biochemistry, 18 5
294 (1979)) according to the method described above.
A was prepared. That is, about 1 × 10 ⁹ HPC-Y
5 cells containing 49 ml of 31.25 mM sodium citrate and 0.625% sodium lauryl sarcosine 5M
It was completely homogenized in a guanidine thiocyanate (Fulka) solution. Homogenate 0.1M in centrifuge tube
The solution was overlaid on a 5.3 M cesium chloride solution layer containing EDTA, and this was then covered with a SW40 rotor (Beckman) for 3 times.
RNA was precipitated by centrifugation at 1,000 rpm at 20 ° C. for 17 hours.

The RNA precipitate was washed with 80% ethanol and washed with 10 mM Tris-HCl buffer (pH: 1 mM EDTA and 0.5% sodium dodecyl sulfate (SDS)).
7.5) Dissolved in 2.4 ml of pronase (Boehringer
Mannheim) was added at 0.5 mg / ml, and the mixture was incubated at 37 ° C for 20 minutes. Proteins were removed by treating the RNA solution with phenol. After extracting residual phenol in the solution with chloroform, RNA was precipitated with ethanol.

From the total RNA, poly (A) ⁺ RNA is oligo
(dT) mRNA Separat consisting of cellulose spun column
It was purified using or (Clontech laboratories). Purification was increased by repeating purification twice according to the prescription attached to the kit.

The above operation was repeated twice to obtain 37 μg and 190 μg of poly (A) ⁺ RNA, respectively.

Example 6 Construction of cDNA library for PCR Using 10 μg of the above poly (A) ⁺ RNA as material, cDNA
A synthesis kit c-CLONE (Clontech laboratories)
Was used to synthesize a double-stranded cDNA, and an EcoRI linker was attached to the end. The free EcoRI linker was separated by agarose gel electrophoresis, and the cDNA having a length of 600 base pairs or more was recovered by electroelution using Geneluter (manufactured by Invitrogen). This linker-added double-stranded cDNA and λZAPII vector (Strata
(manufactured by gene) and 7 mM magnesium chloride, 1 mM DT
T, 1 mM ATP and 2 units of T4 DNA ligase (manufactured by Takara Shuzo), 50 mM Tris-HCl buffer (pH 7.
5) Incubated at 4 ° C. for 24 hours in ligation.

This is Gigapack Gold II packaging extra
HP using ct (Stratagene) packaging
A cDNA library of CY5 cells was constructed. Further divide this library into 7 pools (AG),
Amplified and recovered in 50 mM Tris-HCl buffer (pH 7.5) containing 5.8 g / l sodium chloride, 2 g / l magnesium sulfate heptahydrate and 0.01% gelatin.

Example 7 Cloning by PCR Primer T7 of the T7 promoter sequence near the EcoRI site of λZAPII, primer T3-2 of the sequence near the T3 promoter, and the partial amino acid sequence Gly-Glu-Thr-Gly-Gln-Glu-Ala near the N-terminal of this factor 381A DNA Synthesizer (Applied) was used as a mixed primer N1 containing all possible codons as a coding gene.
Biosystems).

Further, as shown in Example 4 (ii), Ala-G was added to the Endo Glu-C fragment (SEQ ID NO: 5) of this factor.
ln-Lys-Asn-Val-Lys-Leu -Ser-Thr-Glu-Gln-Leu-Arg-Xaa
-Leu-Ala-His-Arg-Leu-Ser- Glu-Pro-Pro-Glu-Asp-Leu-
Since Asp-Ala was found to have a relatively low codon usage sequence (underlined part), mix primers K4S and K4-2A containing all possible nucleotide sequences as genes encoding these were found based on this amino acid sequence. Was similarly synthesized. The base sequences of these primers are shown below.

T7: 5'-TAATACGACTCACTATAGGG-3 'T3-2: 5'-CATGATTACGCCAAGCTCGAA -3' N1: 5'-GG (GATC) GA (GA) AC (GATC) GG (GATC) CA (GA) GA
(GA) GC-3 '(sense) K4S: 5'-GC (GATC) CA (AG) AA (AG) AA (TC) GT (GATC) AA
(AG) (TC) T -3 '(sense) K4-2A: 5'-GC (GA) TC [(GATC) AG or (TC) AA] (A
G) TC (TC) TC (GATC) GG (GATC) GG (TC) TC-3 ′ (antisense) Pronase, EDTA, and SDS were added to a phage suspension of 7 pools of the amplified cDNA library.
The final concentrations were 0.5 mg / ml, 20 mM and 0.5%, respectively, and the mixture was incubated at 37 ° C for 1 hour. Protein was removed from this solution by treatment with phenol, and then phenol was extracted with chloroform, and then DNA was precipitated with ethanol. The first-stage PCR was performed using the DNA thus purified as a template and a DNA Thermal Cycler (manufactured by Perkin Elmer Cetus).

500 ng of library DNA was added to 10 mM Tris-HCl buffer (pH 8.3), 50 mM potassium chloride,
1.5 mM magnesium chloride, 0.01% gelatin, 10
Amplification was carried out in a 50 μl PCR reaction solution containing 0 μM deoxynucleotide triphosphate (dNTP) and 1 μM each of primers T7 and N1. First, after denaturing at 95 ° C for 7 minutes, the mixture was cooled to 85 ° C and 1.25 units of AmpliTaq DNA polymerase (manufactured by Perkin Elmer Cetus) was added. afterwards,
Denaturation 94 ° C for 1 minute, annealing 60 ° C for 1 minute, extension 7
PCR was performed for 40 cycles of 2 minutes at 2 ° C. Further, a second stage PCR was carried out using the primers K4S and K4-2A for 1/20 amount of these PCR reaction solutions under the same conditions as above.

The PCR reaction solution in the second step was analyzed by 12% polyacrylamide gel electrophoresis, and the gel portion containing the amplified DNA fragment of 84 bp was cut out to obtain 0.1% SDS, 1%.
50 including 0 mM magnesium acetate and 0.1 mM EDTA
It was crushed in 0 mM sodium acetate and kept at 37 ° C. for 16 hours to extract DNA. DNA precipitated with ethanol
20 mM containing 1 mM svermidine and 0.1 mM EDTA
Dissolve in 75 μl of Tris-HCl buffer (pH 9.5), 90 ℃
After heating for 2 minutes at 10 ° C., it is rapidly cooled and 10 μl of 500 mM Tris-HCl buffer (pH 9.5) containing 100 mM 2-mercaptoethanol and 100 mM magnesium chloride is added to 10 mM ATP.
And 13 units of polynucleotide kinase (manufactured by Toyobo Co., Ltd.) were added and reacted at 37 ° C. for 1 hour to phosphorylate the 5 ′ end.

Protein was removed by treating the DNA solution with phenol. After extracting residual phenol in the solution with chloroform, the DNA was precipitated with ethanol.
Vector pSP73 treated with restriction enzymes SmaI (Takara Shuzo) and alkaline phosphatase (Takara Shuzo)
(Promega) and this DNA sample were treated with 7 mM magnesium chloride, 1 mM DTT, 1 mM ATP and 2 units of T4 D.
The ligation was carried out in 20 μl of 50 mM Tris-HCl buffer (pH 7.5) containing NA ligase (Takara Shuzo Co., Ltd.) by incubating at 16 ° C. for 16 hours.

Then, 10 μl of the above ligation mixture was added to 100 μl of competent cells of Escherichia coli JM109, and the mixture was allowed to stand on ice for 30 minutes, at 42 ° C. for 1 minute, and again on ice for 1 minute. Then 400 μl of SOC medium (Molecula
r Cloning: A Laboratory Manual, Sambrook et al., Cold S
pring Harbor Laboratory Press, 1989) was added, and the temperature was 37 ° C.
After incubating for 30 minutes in LB agar medium containing 50 μg / ml ampicillin (Molecular Cloning: A Laboratory
Manual, Sambrook et al., Cold Spring Harbor Laboratory
This Escherichia coli was spread on Press, 1989) and kept at 37 ° C. for 16 hours to obtain an E. coli transformant.

LB containing 50 μg / ml of ampicillin was used for each of 5 clones among the transformants that appeared.
After culturing in 5 ml of the medium at 37 ° C for 16 hours, the culture was subjected to the alkaline method (Molecular Cloning: A Laboratory Manu
al, Sambrook et al., Cold Spring Harbor Laboratory Pres
S., 1989) to prepare plasmid DNA. Sequenase Version 2.0 (United States
Biochemical) was used to determine by the dideoxy sequencing method, and a 40 base pair sequence sandwiched between the primers K4S and K4-2A was revealed as shown in SEQ ID NO: 6.

Sense primers 7D-1S: 5'-CTCTCAACAGAGCAGCTGCG-3 'and 7 based on this nucleotide sequence
D-3S: 5'-CTGGCTCACCGGCTCTCTGA-3 'and antisense primer 7D-1A: 5'-AGAGCCGGTGAGCC
AGACAG-3 'and 7D-2A: 5'-GCGCAGCTGCTCTGTT
GAGA-3 'was synthesized. Under the same conditions as above, using library DNA as a template, T7 and 7D-1S and T3-2 and 7D
The first step PCR was performed with the combination of -1A. In addition, the second step PCR was performed using primers T7 and 7D-3, respectively.
Performed with a combination of S and T3-2 and 7D-2A.

The PCR product was analyzed by 1% agarose gel electrophoresis, the gel portion containing the amplified DNA fragment was cut out, and the DNA was extracted using Sephaglas BandPrep Kit (Pharmacia). About these DNAs fmol DNA S
When the base sequence was directly determined using the Sequencing System (Promega), a part of the gene encoding this factor was confirmed as shown in SEQ ID NOS: 7 and 8.

Based on this nucleotide sequence, sense primer 3AS1: 5'-AACTCCTTGGCTTCCCGTGTG -3 'and antisense primer 7SA1-5'-CGCATCTGGGTTGA
GGAATAG-3 'was synthesized, and PCR was performed on pool D under the above conditions. As a result, 197 base pairs (SEQ ID NO: 9)
Was confirmed to be amplified, and the amino acid sequence encoded by this DNA fragment was the purified Meg-PO obtained in Example 3.
It matched the amino acid sequence determined using T. This DN
The A fragment (Q197A) was used as a probe in the following screening.

Example 8 cDNA library probe
Screening with Q197A Double-stranded cDNA was synthesized using 5 μg of poly (A) ⁺ RNA obtained in Example 5 as a material and using ZAP-cDNA SYNTHESIS KIT (manufactured by Stratagene) to prepare a λZAPII vector (manufactured by Stratagene). Connected to the arm. This is Gi
gapack Gold II packaging extract (Stratagene)
Was used to construct a new cDNA library of HPC-Y5 cells.

The 197 bp DNA amplified by PCR in Example 7 was recovered from the gel after acrylamide gel electrophoresis, and this DNA fragment Q197A was used as a random primer D.
It was labeled with ³² P using NA labeling kit (manufactured by Takara Shuzo). However, instead of the attached random primer, primers 3AS1 and 7SA1 were used at a concentration of 400 nM for labeling. Free dNTPs can be isolated from NICK-Column (Pharmacia
(Made by the company). Next, Benton and Davis method
Plaque hybridization was performed according to (Science 196 , 180, 1977).

Hy was put on the agar medium on which the phage plaque was formed.
The phage was transferred on a bond-N + filter (manufactured by Amersham), and the filter was treated in the following order. 1.5
The DNA was denatured for 5 minutes with 0.5N sodium hydroxide aqueous solution containing M sodium chloride, and then with 0.1N sodium hydroxide aqueous solution containing 1.5M sodium chloride for 1 minute, followed by 0.5M sodium chloride containing 0.5M. Tris-HCl buffer (pH 7.4) twice for 1 minute and finally 2 x SSCP
(240 mM NaCl, 30 mM Na ₃ citrate, 26 mM
KH ₂ PO ₄ , 2 mM EDTA) for 1 minute.

The filter is then dried and treated with 0.4N aqueous sodium hydroxide solution for 20 minutes at 5 × SSPE (Molecu
lar Cloning: A Laboratory Manual, Sambrook et al. Cold
Spring Harbor Laboratory Press, 1989) treated twice for 1 minute. 50% formamide, 5 x SSPE, 5 x De
nhardt's Solution (Molecular Cloning: A Laboratory
Manual, Sambrook et al., Cold Spring Harbor Laboratory
Press, 1989), 0.1% SDS and 0.1 mg / ml modified D
Incubation was carried out at 42 ° C. for 4 hours in a prehybridization solution containing NA (salmon testis DNA, manufactured by Boehringer Mannheim).

A hybridization solution containing the Q197A probe labeled as described above (50% formamide, 5 × SSPE, 5 × Denhardt's Solution, 0.
1 mg / ml denatured DNA (salmon testis DNA), 0.1% SD
Hybridization was carried out in S) for 16 hours at 42 ° C. The filters were washed twice at room temperature with 2 × SSC containing 0.05% SDS for 1 hour, then at 68 ° C. for 0.
1x SSC with 1% SDS twice for 1 hour, then 68
After washing with 0.2 × SSC containing 0.1% SDS at 1 ° C. for 1 hour, the cells were detected by autoradiography.

As a result, 23 positive phage clones were obtained. Full length c of the obtained phage clones
The clones suspected of containing DNA were excised into plasmids using helper phage R408 ZAP-.
cDNA SYNTHESIS KIT (manufactured by Stratagene) was performed according to the attached recipe. The resulting plasmid pKPO27
(Fig. 2) Sequen the nucleotide sequence of the cloned DNA
When examined by the dideoxy sequencing method using ase Version 2.0 (manufactured by United States Biochemical), the nucleotide sequence shown in SEQ ID NO: 10 was obtained. In addition, when the nucleotide sequence of the cloned DNA of the other plasmid pKPO21 was examined by the same method, the 1873th G (guanine) in the nucleotide sequence shown in SEQ ID NO: 10 was A.
Has changed to (adenine) (hence the 593th Va
1 (valine) was changed to Met (methionine)), the rest of the sequence was identical to that of pKPO27.

When these nucleotide sequences and the amino acid sequences encoded by them were examined by Gen Bank Re 1.71, it was confirmed that both the nucleotide sequences and the amino acid sequences were novel.

Escherichia coli containing the plasmid pKPO27 was Escherichia coli JM109 (pKPO27), and E. coli containing the plasmid pKPO21 was Escherichia coli JM109 (pKPO27).
As a hiacoli JM109 (pKPO21), the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology (1-3, Higashi 1-3, Tsukuba, Ibaraki), respectively, on October 12, 1992, Micro Engineering Research Article No. 4029 (FERM BP- 4029), and on Nov. 10, 1992, Mikken Kenjoyori No. 4071 (FERM BP-4
071) was internationally deposited under the Budapest Treaty.

Example 9 pRVHKPO27 vector
Construction (for animal cells) pKPO27 obtained in Example 8 was added to 10 mM MgCl 2
Restriction enzyme Ec in 20 mM Tris-HCl buffer (pH 7.5) containing ₂ , 1 mM DTT, 100 mM NaCl at 37 ° C
Treated with oRI (Takara Shuzo Co., Ltd.) for 2 hours to recover DNA as ethanol precipitate, and further 10 mM MgCl ₂ , 1 mM
After treatment with a restriction enzyme BamHI (manufactured by Takara Shuzo Co., Ltd.) for 2 hours at 37 ° C. in a 20 mM Tris-hydrochloric acid buffer solution (pH 8.5) containing DTT and 100 mM KCl, agarose gel electrophoresis was performed to recover a 1.8 kb DNA fragment. did.

The vector HEF-12h-gγ1 was treated with the restriction enzymes EcoRI and BamHI in the same manner as above, and then alkaline phosphatase (Takara Shuzo) was added to give 6
The 8.7 kbp DNA was dephosphorylated by incubating at 5 ° C. for 2 hours and subjected to agarose gel electrophoresis. The recovered 8.7 kbp DNA was mixed with the above 1.8 kbp DNA fragment and then 6.6 mM MgC was added.
T4 DNA ligase was reacted overnight at 66 ° C. in 66 mM Tris-HCl buffer (pH 7.5) containing 1, ₂ , 5 mM DTT, 1 mM ATP, and this was introduced into Escherichia coli JM109 strain to obtain pRVHKPO27 (FIG. 3). ).

As shown in FIG. 3, this plasmid is E
F1α promoter, human immunoglobulin H chain constant region gene, SV40 enhancer promoter, pBR
322 including the replication initiation region and β-lactamase gene (Amp ^r ), and between the EF1α promoter and the human immunoglobulin heavy chain constant region gene, human Meg-P
A part of the OT cDNA is connected.

The vector HEF-12h-gγ
l was prepared as follows.

The 2.5 kbp HEF-1α promoter-enhancer region, which is a component of the vector HEF-12h-gγl, is located at about 1'to the 5'-end of the gene.
5 kbp DNA, 33 bp in the first exon, 943 bp in the first intron, and 1 in the first part of the second exon.
It consists of 0 bp. This 2.5 kbp HindIII-Eco
The RI fragment was transformed into the plasmid pEF321-CAT (DWKim
Et al., Gene 91 , 217 (1990); and T. Uetsuki et al., J. Bio.
l. Chem. 264 , 5791 (1989)) and pd
KCR vector (M. Tsuchiya et al., Embo J. 6 , 611 (198
7)), K. O'Hara et al., Proc. Natl, Acad. Sci. USA 78 ,
1527 (1981), (R. Fukunaga et al., Proc. Natl. Acad. Sc
i. USA. 81 , 5086 (1984)) and cloned into SV4
About 300 containing 0 early promoter-enhancer
Replaced by HindIII-EcoRI fragment of bp
F-1 was obtained.

PTEF-1 was digested with EcoRI and Kl
Fill in with enow polymerase and Hin
It was linked to a dIII linker. Next, this modified pT
About 1.6 kbp HindIII from EF-1 vector DNA
-The SmaI fragment was excised.

HCMV-12h-gγ1 (Maeda et al., Huma
n Antibodies and Hybridomas 2 , 124 (1991); CA
Kettleborough et al., Protein Engeneering 4 , 773 (199
1)) was partially digested with EcoRI, filled in with Klenow polymerase, and self-ligated to obtain plasmid H from HCMV-12h-gγ1.
CMV-12h-gγ1 (ΔE2) was prepared.

Plasmid HCMV-12h-gγ1 (Δ
E2) was digested with EcoRI, filled in with Klenow polymerase and digested with HindIII.
An approximately 7 kbp fragment containing the DNA sequence encoding the human α-IC region was transformed into the above-mentioned 1.6 kbp HindIII-Sm containing the HEF-1α promoter-enhancer.
Ligation into the aI fragment gave HEF-12h-gγ1. The HEF-1α promoter enhancer region in this vector was identical to that in pTEF-1 except for the 380 bp DNA flanking the 5'-region.

Example 10 Megakaryocyte amplification factor in COS cells
Expression of offspring (Meg-POT) gene I COS cells were suspended in PBS at 1 × 10 ⁷ cells / ml, and pRVHKPO27 was added to 0.8 ml of this cell suspension.
10 μg was added. 1900V, 25μF, 0.4msec
Electroporation method using GenePulsar (BioRad) under the conditions
The plasmid was introduced into COS cells by (electroporation). After a recovery period of 10 minutes at room temperature, the electroporated cells were Dulbec containing 1% fetal bovine serum.
co's Minimum Essential Medium (DMEM) (GIBC
(Manufactured by O company) 25 ml. After culturing for 72 hours, the culture supernatant was collected.

A part of the culture supernatant of the COS cells thus obtained was treated with Centriprep 10 (Amicon) to about 10 parts.
It was concentrated twice and analyzed by SDS / PAGE. Similarly, as a control, the culture supernatants of untreated COS cells and vector-introduced COS cells were also about 1 each.
The 0-fold concentrated product was also run side by side. Gel concentration is 12%, Laemmli method Nature, 227 , 680 (197
The sample was electrophoresed according to 0), and one plate was stained with 2D-silver staining reagent II "Daiichi" (manufactured by Daiichi Pure Chemicals Co., Ltd.) for protein staining.

The molecular weight markers are low molecular weight markers manufactured by Bio-Rad [Phosphorylase B (92.5 kd), bovine serum albumin (66.2 kd), ovalbumin (4
5.0 kd), carbonic anhydrase (31.0 kd), soybean trypsin inhibitor (21.5 kd), lysozyme (1
4.4 kd)] was used.

On the other sheet, a megakaryocyte amplification factor was detected by Western blotting.

The molecular weight markers used at this time were prestained molecular weight markers (Phosphorylase B (106.0 kd), bovine serum albumin (80.0 kD) manufactured by Bio-Rad.
d), ovalbumin (49.5 kd), carbonic anhydrase (32.5 kd), soybean trypsin inhibitor (27.
5 kd) and lysozyme (18.5 kd)] were used. The primary antibody was obtained by immunizing rabbits with an 18-residue peptide synthesized as an antigen based on the sequence obtained by N-terminal sequence analysis of megakaryocyte amplification factor purified from the culture supernatant of HPC-Y5. A polyclonal antibody was used.

As a result, when the bands stained by the silver staining method were compared among the three, the band found at a molecular weight of about 33,000 was the CO into which the gene for megakaryocyte amplification factor had been introduced.
It was found only in the culture supernatant of S cells. Further, even in the Western blotting method, only this band having a molecular weight of about 33,000 was strongly colored, so that this band was considered to be a recombinant megakaryocyte amplification factor.

Example 11 Giant in COS cell culture supernatant
Measurement of karyocytic amplification factor (Meg-POT) activity I The megakaryocyte amplification factor activity of the culture supernatant of COS cells obtained in Example 10 was measured according to the method described above. However, C
Upon stimulation of gene transfer into OS cells, I was added to the culture supernatant.
Since L-6 is known to be induced, the measurement was carried out in a system to which an antibody against mouse IL-6 receptor was added.

As a result, as shown in Table 2, the culture supernatant of untreated COS cells into which no gene was introduced was also Meg.
-The culture supernatant of control COS cells introduced with a vector containing no POT cDNA also did not show megakaryocyte amplification factor activity, whereas the culture supernatant of COS cells introduced with a vector containing Meg-POT cDNA was clearly It showed megakaryocyte amplification factor activity.

[0102]

[Table 2] Megakaryocyte amplification factor activity in culture supernatant of COS cells COS cells Megakaryocyte amplification factor activity ^a) Culture supernatant concentration (%) 50 0.5 0.005 Untreated COS 1.5 3 ND ^b) Control COS 0 2.5 ND Meg-POT cDNA-introduced COS 8.5 7 3.5 a) Formed colonies A value obtained by subtracting the number of colonies formed by IL-3 alone (26.5) from the number.

B) Not measured.

Example 12 From the culture supernatant of COS cells
Purification of recombinant megakaryocyte amplification factor (Meg-POT) I To 350 ml of COS cell culture supernatant obtained in Example 10
Add Tween 20 to a final concentration of 0.01%.
It was concentrated about 10 times by the ultrafiltration method using micon PM-10 (manufactured by Amicon). A recombinant megakaryocyte amplification factor (Meg-POT) was purified from this concentrated solution by the following procedure.

(I) 10 mM containing 0.01% Tween 20
The concentrate was dialyzed against 10 L of Tris-HCl buffer (pH 8.4) overnight at 4 ° C., and then equilibrated with the same DEAE-Sepharose fast flow column (2.2 × 18).
cm, manufactured by Pharmacia). After washing the column with the same buffer, the NaCl concentration was gradually increased to 0.
The protein adsorbed on the column was eluted by increasing the concentration to 1, 0.15, 0.2, 0.5M. The obtained fraction is SDS / PA
As a result of GE analysis, the molecular weight found in Example 10 was about 3.
The 3,000 band was detected only in the first half of the fraction with a NaCl concentration of 0.1M. This fraction was collected and subjected to subsequent purification on reverse phase HPLC.

(Ii) After adding 10% TFA to the above fractions to adjust the pH to 3 or less, a Vydac C4 column (4.6 × 250) equilibrated with 24% acetonitrile containing 0.1% TFA.
mm) and washed the column with the same eluent, then 0.1%
Acetonitrile concentration in TFA of 24-64 in 80 minutes
%, And linearly increased to 80% in 10 minutes to elute the protein adsorbed on the column. Flow rate is about 1 ml / min
The protein detection was carried out at two wavelengths of 220 nm and 280 nm. As a result of SDS / PAGE analysis of the obtained peak, a band having a molecular weight of about 33,000 was observed at a concentration of acetonitrile of about 41%. This fraction was
After diluting with 1% TFA, reverse phase HPLC was performed again under the same conditions to collect the main peak.

Example 13 Recombinant megakaryocyte amplification factor (M
Amino acid sequence analysis of Egg-POT) Regarding the recombinant Meg-POT obtained in Example 12, a gas phase protein sequencer type 473A (Appl
Ned terminal amino acid sequence analysis was performed using ied Biosystems). As a result, the following three types of N-terminal amino acid sequences (a) to (c) were recognized, and the band having a molecular weight of about 33,000 confirmed in Example 10 was confirmed to be a recombinant megakaryocyte amplification factor. Was done.

(A) Ser-Arg-Thr-Leu-Ala-Gly-Glu-Thr-
Gly-Gln-Glu-Ala-Ala-Pro-Leu-Asp- ……… (SEQ ID NO: 1
1) (b) Leu-Ala-Gly-Glu-Thr-Gly-Gln-Glu-Ala-Ala-Pro-
Leu-Asp-Gly-Val-Leu-Ala-Asn-Pro-Pro-Xaa-Ile-Ser-Xa
a-Leu-Xaa-Pro-Arg -... …… (c) Gly-Glu-Thr-Gly-Gln-Glu-Ala-Ala-Pro-Leu-Asp-
Gly-Val-Leu-Ala-Asn-Pro-Pro-Xaa-Ile-Ser-Xaa-Leu-Xa
a-Pro-Arg-Gln-Leu -......... Among these, (b) and (c) are the sequence-1 of the polypeptide from the culture supernatant of HPC-Y5 cells in Example 4 (i) -1 ( It corresponds to SEQ ID NO: 1) and sequence-3 (SEQ ID NO: 3), respectively.

In addition, recombinant Meg-POT contains 10 mg /
After adding 100 μl of 70% formic acid solution containing cyanogen bromide and decomposing cyanogen bromide at room temperature for 24 hours, excess reagents were removed by a centrifugal concentrator. The residue is 0.1% TFA
Vydac C dissolved in 1 ml and equilibrated with 0.1% TFA
Add to 4 columns (4.6 x 250 mm), 0.1% TF
In A, the concentration of acetonitrile was linearly increased to 80% in 40 minutes to elute the cyanogen bromide fragment adsorbed on the column. Amino acid sequence analysis was performed on the C-terminal side fragment of the obtained fragments using a gas phase protein sequencer type 473A, and the following sequences were obtained. This sequence is a partial sequence of a cyanogen bromide fragment obtained by similarly decomposing a megakaryocyte amplification factor (Meg-POT) obtained from the culture supernatant of HPC-Y5 in the same manner as described below. Matched

[0110]

[Table 3]

The amino acid sequence represented by "cDNA" in the above table is an amino acid sequence deduced from the nucleotide sequence of the cDNA encoding Meg-POT, and the amino acid sequence represented by "COS" is a cDNA encoding Meg-POT. Is a partial sequence obtained by amino acid sequence analysis of the protein expressed in COS cells, and the amino acid sequence represented by "HPC-Y5" is a human pancreatic cancer cell-derived cell line "H
It is a partial sequence obtained by amino acid sequence analysis of Meg-POT obtained from the culture supernatant of "PC-Y5".

Example 14 pRVHKPO27f vector
(For animal cells) pKPO27 obtained in Example 8 was added to 10 mM MgCl 2
_2, 50 containing 1 mM DTT and 100 mM NaCl
The DNA was recovered as an ethanol precipitate by treating with restriction enzyme XhoI (manufactured by Toyobo Co., Ltd.) for 2 hours in mM Tris-HCl buffer (pH 7.5) at 37 ° C. Next, 5 mM MgCl ₂ , 10
mM DTT, 1 mM dATP, dCTP, dGTP, d
1 in 20 mM Tris-HCl buffer (pH 7.4) containing TTP
The ends were blunted by treatment with Klenow fragment of DNA polymerase for 1 hour at 0 ° C. This DNA as ethanol precipitate
Was recovered, then 10 mM MgCl ₂ , 1 mM DTT,
50 mM Tris-HCl buffer containing 100 mM NaCl (pH
7.5) Restriction enzyme SalI (Toyobo Co., Ltd.) at 37 ° C
And then agarose gel electrophoresis for 2 hours.
A 3 Kbp DNA fragment was recovered.

PRVHKPO27 obtained in Example 9 was treated with 10 mM MgCl ₂ , 1 mM DTT, 100 mM KCl.
After treatment with a restriction enzyme BamHI (manufactured by Takara Shuzo Co., Ltd.) for 2 hours in a 20 mM Tris-hydrochloric acid buffer solution (pH 8.5) containing the above, the DNA was recovered by ethanol precipitation. Next 5 mM
MgCl ₂ , 10 mM DTT, 1 mM dATP, dCT
DNA polymerase Klenow in 20 mM Tris-HCl buffer (pH 7.4) containing P, dGTP and dTTP at 10 ° C.
The fragment was treated for 1 hour to blunt the ends.

The DNA was recovered by ethanol precipitation, then 10 mM MgCl ₂ , 1 mM DTT, 100 mM.
50 mM Tris-HCl buffer containing NaCl (pH 7.5)
It was dephosphorylated by treating with restriction enzyme SalI (Toyobo Co., Ltd.) for 2 hours at 37 ° C. for 2 hours, adding alkaline phosphatase (Takara Shuzo Co., Ltd.) and incubating at 65 ° C. for 2 hours to perform agarose gel electrophoresis. A 8.5 Kbp DNA fragment was recovered. This 8.5 Kbp DNA fragment and the above 1.
After mixing with a 3 Kbp DNA fragment, 6.6 mM MgCl 2 was added.
_2, 5 mM DTT, and reacted overnight T4DNA ligase in 66mM Tris-HCl buffer (pH 7.5) in 16 ° C. containing 1 mM ATP, which was obtained pRVHKPO27f was introduced into E. coli JM109 strain (Fig. 4).

As shown in FIG. 4, pRVHKPO27f contains EF1α promoter, human immunoglobulin H chain constant region gene, SV40 enhancer promoter, pBR322-derived replication initiation region and β-lactamase gene (Amp ^r ), and contains human Meg. -P
The OT cDNA is connected between the EF1α promoter and the human immunoglobulin heavy chain constant region gene.

Example 15 Regeneration of megakaryocytes in COS cells
Gene (Meg-POT) gene expression II COS cells were suspended in PBS at 1 × 10 ⁷ cells / ml, and 0.8 ml of this cell suspension was added to pRVHKPO27f.
10 μg was added. 1900V, 25uFD 0.4msec
The plasmid was introduced into COS cells by electroporation (Generation) using GenePulsar (manufactured by BioRad) under the above conditions. After a recovery period of 10 minutes at room temperature, the electroporated cells were Dulbec containing 1% fetal bovine serum.
co's Minimum Essential Medium (DMEM) (GIBCO) 2
Added to 5 ml. After culturing for 72 hours, the culture supernatant was collected.

A part of the COS cell culture supernatant thus obtained was treated with Centriprep-10 (Amicon) to about 10 parts.
It was concentrated twice and analyzed by SDS / PAGE. As a control, the culture supernatant of COS cells into which only the vector had been introduced was similarly subjected to 10-fold concentration, and then the samples were run side by side. 12% gel concentration, Laemmli's method Natu
Re, 227 , 680 (1970), and one sheet was stained with 2D-silver staining reagent II "Daiichi" (manufactured by Daiichi Pure Chemicals Co., Ltd.) for protein staining.

The molecular weight markers are low molecular weight markers (Phosphorylase B (92.5 kd), bovine serum albumin (66.2 kd), ovalbumin (4
5.0 kd), carbonic anhydrase (31.0 kd), soybean trypsin inhibitor (21.5 kd), lysozyme (1
4.4 kd)] was used.

On the other sheet, a megakaryocyte amplification factor was detected by Western blotting.

The molecular weight markers used at this time were Prestained molecular weight markers (Phosphorylase B (106.0 kd), bovine serum albumin (80.0 kD) manufactured by Bio-Rad.
d), ovalbumin (49.5 kd), carbonic anhydrase (32.5 kd), soybean trypsin inhibitor (27.
5 kd) and lysozyme (18.5 kd)] were used. The primary antibody was obtained by immunizing rabbits with an 18-residue peptide synthesized as an antigen based on the sequence obtained by N-terminal sequence analysis of megakaryocyte amplification factor purified from the culture supernatant of HPC-Y5. A polyclonal antibody was used.

As a result, when the bands stained by the silver staining method were compared with each other, it was found that the band with a molecular weight of about 33,000 was CO introduced with the megakaryocyte amplification factor gene.
It was found only in the culture supernatant of S cells. Further, even in the Western blotting method, only this band having a molecular weight of about 33,000 was strongly colored, so that this band was considered to be a recombinant megakaryocyte amplification factor.

Example 16 Giant in COS cell culture supernatant
Measurement of lymphocyte amplification factor (Meg-POT) activity II The megakaryocyte amplification factor activity of the COS cell culture supernatant obtained in Example 15 was measured according to the method described above. However, C
Upon stimulation of gene transfer into OS cells, I was added to the culture supernatant.
Since L-6 is known to be induced, the measurement was carried out in a system to which an antibody against mouse IL-6 receptor was added.

As a result, as shown in Table 4, the culture supernatant of untreated COS cells into which no gene was introduced was also Meg.
-The culture supernatant of control COS cells introduced with a vector containing no POT cDNA also did not show megakaryocyte amplification factor activity, whereas the culture supernatant of COS cells introduced with a vector containing Meg-POT cDNA was clearly It showed megakaryocyte amplification factor activity.

[0124]

[Table 4] Megakaryocyte amplification factor activity in culture supernatant of COS cells COS cells Megakaryocyte amplification factor activity ^a) Culture supernatant concentration (%) 2 0.2 0.02 0.002 untreated COS 0 ND ^b) ND ND control COS 2 ND ND ND Meg-POT cDNA-introduced COS 7 4 7 1 a) From the number of formed colonies A value obtained by subtracting the number of colonies formed by IL-3 alone (10 colonies).

B) Not measured.

Example 17 From the culture supernatant of COS cells
Purification of recombinant megakaryocyte amplification factor (Meg-POT) II To 350 ml of COS cell culture supernatant obtained in Example 15
Add Tween 20 to a final concentration of 0.01%.
It was concentrated about 10 times by the ultrafiltration method using micon PM-10 (manufactured by Amicon). A recombinant megakaryocyte amplification factor (Meg-POT) was purified from this concentrated solution by the following procedure.

(I) 10 mM containing 0.01% Tween 20
The concentrate was dialyzed against 10 L of Tris-HCl buffer (pH 8.4) overnight at 4 ° C., and then equilibrated with the same DEAE-Sepharose fast flow column (2.2 × 18).
cm, manufactured by Pharmacia). After washing the column with the same buffer, the NaCl concentration was gradually increased to 0.
The protein adsorbed on the column was eluted by increasing the concentration to 1, 0.15, 0.2, 0.5M. The obtained fraction is SDS / PA
As a result of GE analysis, the molecular weight found in Example 15 was about 3.
A 3,000 band was detected in the fraction with a NaCl concentration of 0.1M and this fraction was subjected to subsequent purification by reverse phase HPLC.

(Ii) After adding 10% TFA to the above fractions to adjust the pH to 3 or less, a Vydac C4 column (4.6 × 250) equilibrated with 24% acetonitrile containing 0.1% TFA.
mm) and washed the column with the same eluent, then 0.1%
Acetonitrile concentration in TFA of 24-64 in 80 minutes
%, And linearly increased to 80% in 10 minutes to elute the protein adsorbed on the column. Flow rate is about 1 ml / min
The protein detection was carried out at two wavelengths of 220 nm and 280 nm. As a result of SDS / PAGE analysis of the obtained peak, a band having a molecular weight of about 33,000 was observed at a concentration of acetonitrile of about 41%. This fraction was
After diluting with 1% TFA, reverse phase HPLC was performed again under the same conditions to collect the main peak.

Example 18 Purified recombinant Meg-POT
Recombinant megakaryocyte potentiator purified in megakaryocyte potentiator activity measured Example 17 (Me
g-POT) containing 10% horse serum in Iscove's Modifie
d Dulbecco's culture solution (IMDM) at a predetermined concentration (50.
Test sample 0.1 diluted to 1, 3.1, 0.2 ng / ml)
ml, horse serum (56 ° C for 30 minutes, Biocell)
0.2 ml, mouse (C57BL / 6N male, 6-12)
IM) containing 0.1 ml of femoral bone marrow cell suspension (2 × 10 ⁵ nucleated cells) and 5 ng / ml of recombinant mouse IL-3
Modified McCo containing 0.2 ml DM and 0.75% agar
0.4 ml of y's 5A culture solution was mixed. Then, after putting them in a tissue culture plastic dish having a diameter of 35 mm to harden them, 37 ° C, 5% carbon dioxide gas / 95% air,
The culture was performed under the condition of 100% humidity.

On the 6th day of culture, the whole agar layer was taken out on a slide glass and dried, and a film-like specimen was fixed with 5% glutaraldehyde, followed by the method of Nakeff et al. (Proc. So.
c. Exp. Biol. Med. 151 587 1976) and stained with acetylcholinesterase, and the number of megakaryocyte colonies was calculated. At this time, an aggregate containing 4 or more acetylcholinesterase-staining positive cells was used as a megakaryocyte colony (the colony was inspected at a magnification of 40 times).

The activity of megakaryocyte amplification factor is
The number of megakaryocyte colonies generated by adding egg-POT and no recombinant Meg-POT (IM containing 10% horse serum)
The difference from the number of megakaryocyte colonies generated by recombinant IL-3 alone in the case of adding only DM) was used as an index.

As a result, as shown in Table 5, the purified recombinant Meg-POT exhibited megakaryocyte amplification factor activity.

[0133]

Table 5 Megakaryocyte amplification factor activity of purified recombinant Meg-POT Meg-POT concentration Megakaryocyte amplification factor activity ^b) (ng / ml) ^a) 50.1 9.5 3.1 8.5 0.2 3 a) Measured by amino acid analysis.

B) A value obtained by subtracting the number of colonies formed by IL-3 alone (19.5) from the number of colonies formed.

Example 19 Megakaryocyte amplification factor (Meg-P
In vitro transcription / translation of gene encoding OT) pKPO27 obtained in Example 8 was replaced with 10 mM MgCl 2.
Restriction enzyme Xho in 20 mM Tris-HCl buffer (pH 8.5) containing ₂ , 1 mM DTT, 100 mM KCl at 37 ° C
I (manufactured by Toyobo Co., Ltd.) was treated for 2 hours and then subjected to agarose gel electrophoresis to recover a 1.9 Kbp DNA fragment. 10m of vector pCITE-2c (Novagen)
It was treated with a restriction enzyme XhoI (manufactured by Toyobo Co., Ltd.) for 2 hours at 37 ° C. in 20 mM Tris-HCl buffer (pH 8.5) containing M MgCl ₂ , 1 mM DTT, 100 mM KCl, and then alkaline phosphatase (Takara Shuzo Co., Ltd.) was added. DN obtained by dephosphorization by incubating at 65 ° C for 2 hours
A was purified by subjecting it to phenol treatment. After mixing this with the above 1.9 Kbp DNA fragment, 6.6 mM Mg
T4 DNA ligase was reacted overnight at 66 ° C. in 66 mM Tris-HCl buffer (pH 7.5) containing Cl ₂ , 5 mM DTT, 1 mM ATP, and introduced into Escherichia coli JM109 strain.
CITE / KPO27 was obtained (Fig. 5).

PCITE / KPO27 was added to 10 mM MgC
10 mM Tris-HCl (pH containing l _2, 1 mM DTT
7.5) Restriction enzyme NaeI (Toyobo Co., Ltd.) at 37 ° C
After 2 hours of treatment with DNA, the DNA was purified by phenol treatment. This template DNA contains 2 mM spermidi
ne, 6 mM MgCl ₂ , 10 mM NaCl, 1 unit / μ
l RNasin ribonuclease inhibitor (Promega),
40 including 0.5 mM ATP, GTP, CTP, UTP
T7 R in mM Tris-HCl buffer (pH 7.5) at 37 ℃
Transcription was performed by reacting with NA Polymerase. Purify this by phenol treatment,
RNA was collected as an ethanol precipitate. Continued Red
RNA was translated using Nova Lysate (Novagen) as a label containing ³⁵ S-methionine. When this reaction product was subjected to SDS polyacrylamide gel electrophoresis and autoradiography was performed, a band having a molecular weight of about 70,000 was recognized.

Example 20 . pMBPKPO27 vector
(For E. coli) pKPO27 obtained in Example 8 was added to 10 mM MgCl 2
Restriction enzyme Xho in 20 mM Tris-HCl buffer (pH 8.5) containing ₂ , 1 mM DTT, 100 mM KCl at 37 ° C
I (manufactured by Toyobo Co., Ltd.) for 2 hours to collect DNA as an ethanol precipitate, 5 mM MgCl ₂ , 10 mM DTT,
DN in 20 mM Tris-HCl buffer (pH 7.4) containing 1 mM dATP, dCTP, dGTP, dTTP at 10 ° C
The Klenow fragment of A polymerase was treated for 1 hour to blunt the ends. Then, agarose gel electrophoresis is performed and 1.
A 9 Kbp DNA fragment was recovered.

Vector pMAL-c (New England BioLa
bs) 10 mM MgCl ₂ , 1 mM DTT, 50 mM
20 mM Tris-HCl buffer containing NaCl (pH 7.5)
After treating with the restriction enzyme StuI for 2 hours at 37 ° C., alkaline phosphatase (Takara Shuzo) was added, and the mixture was kept at 65 ° C. for 2 hours for dephosphorylation. The DNA was deproteinized by treating with phenol and recovered as an ethanol precipitate.

After mixing this with the above 1.9 Kbp DNA fragment, 6.6 mM MgCl ₂ , 5 mM DTT, 1 mM ATP
T4 DNA ligase was reacted overnight at 66 ° C. in a 66 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing Escherichia coli and introduced into Escherichia coli JM109 strain to obtain pMBPKPO27 (FIG. 6). As shown in FIG. 6, this plasmid has an expression unit downstream of the tac promoter, the maltose binding protein (MBP) gene (malEΔ2-26), and Fac.
The recognition sequence of tor Xa and the cDNA after the 34th amino acid Ser of Meg-POT are connected so that their frames match.

Example 21 . Expression of Fusion Protein in Escherichia coli JM109 Strain Escherichia coli transformed with pMBPKPO27 in Example 20 was cultured in 5 ml of LB medium containing 50 ug / ml ampicillin and 0.2% glucose at 37 ° C. for 16 hours, and this culture solution was used. 4 ml was added to 400 ml LB medium containing 50 ug / ml ampicillin and 0.2% glucose.
After culturing at 37 ° C. for about 2 hours, isopropyl-β-D-thiogalactoside was added to 0.3 mM and culturing was continued for another 3 hours. The cultured cells were treated with S as in Example 10.
The above anti-Meg was subjected to DS polyacrylamide electrophoresis.
-Western blotting with POT peptide serum or anti-MBP serum showed MBP and Meg
-The expression of the fusion protein of POT was confirmed.

Expressed recombinant megakaryocyte amplification factor (Me
g-POT) was purified from the bacterial cells as follows.

The suspension disrupted by sonication (20 minutes) in 20 mM Tris-HCl buffer (pH 7.5) containing 10 mM EDTA was centrifuged (10,000 rpm x 30 min, 4 ° C, SA 60).
0 rotor, manufactured by Sorvall), and the collected precipitate was suspended in distilled water. Centrifuge the suspension again (10,000g x 90mi
n), and the collected precipitates were collected in 25 mM Tris-HCl buffer (p 2%) containing 1% 2-mercaptoethanol (2-ME) and 8M urea.
H8.0) and centrifuge again (35,000 rpm x 60 min) to remove insoluble matter, then 10 mM 2-ME, 10 mM EDTA, 200 m
It was diluted 2-fold with 10 mM Tris-HCl buffer (pH 8.0) containing M NaCl. This solution was added to an amylose column (75 ml, manufactured by BioLabs) equilibrated with the same buffer, and the column was washed with the same buffer. The protein bound to the column was eluted with the same buffer containing 10 mM maltose. This fraction is Fa
₂ mM CaCl ₂ and 150 mM NaCl, which are digestion buffers for ctor Xa
It was dialyzed against 20 mM Tris-HCl buffer (pH 7.4) containing Factor Xa was added to the dialyzed solution and enzymatically digested at 37 ° C for 16 hours. The reaction solution is a membrane filter PM-10.
After concentrating with (Amicon), it was passed through a PD-10 column equilibrated with the equilibration buffer of the above amylose column, then added again to the amylose column, and the column was washed with the same buffer and passed through. Fractions and wash fractions were collected. The above-mentioned fractions were concentrated with Centriprepo 10.

The recombinant megakaryocyte amplification factor thus obtained was sensitized to a rabbit with Freund's complete adjuvant five times every two weeks. On the 10th day after the final sensitization, whole blood was collected from the carotid artery to obtain antimegakaryocytic amplification factor antiserum.

Example 22. Construction of pEFDKPOf vector
Construction (for animal cells) pKPO27 was added to 10 mM MgCl ₂ , 1 mM DTT, 1
20 mM Tris-HCl buffer solution (pH 8.
5) After treatment with restriction enzymes EcoRI and BamHI (manufactured by Takara Shuzo Co., Ltd.) at 37 ° C., agarose gel electrophoresis was performed to recover a 1.8 kbp DNA fragment.

The vector DHFR-ΔE-RVh was transformed with EcoR
I and BamHI were similarly treated, and then treated with alkaline phosphatase (Takara Shuzo Co., Ltd.) at 60 ° C. for 2 hours to dephosphorylate and perform agarose gel electrophoresis to recover the recovered 7 kbp DNA of the above 1.8 kbp DNA. Ligation with the fragment gave pEFDKPO5 '.

This was mixed with 10 mM MgCl ₂ and 1 mM DT
T, 20 mM Tris-HCl buffer containing 100 mM KCl (p
H8.5) after treatment with BamHI restriction enzyme at 37 ° C., 6.7 mM MgCl ₂ , 16.6 mM (NH ₄ ) ₂ S
O ₄ , 10 mM 2-mercaptoethanol, 6.7 mM
The ends were blunted by treatment with T4 DNA polymerase in 67 mM Tris buffer containing EDTA and 330 mM dNTPs at 37 ° C. for 5 minutes. Furthermore, KpnI linker (Amers
ham company) to obtain pEFDKPO5′K. Newly added pKPO27 to 10 mM MgCl ₂ , 1 mM DT
T, 20 mM Tris-HCl buffer containing 100 mM KCl
After digestion with the restriction enzyme XhoI at 37 ° C. in (pH 8.5), 6.7 mM MgCl ₂ , 16.6 mM (NH ₄ ) ₂ S
O ₄ , 10 mM 2-mercaptoethanol, 6.7 mM
The ends were blunted by treatment with T4 DNA polymerase in 67 mM Tris buffer containing EDTA and 330 mM dNTPs at 37 ° C. for 5 minutes. This is further added with 10 mM MgCl ₂ , 1 m
Restriction enzyme SalI in 50 mM Tris-HCl buffer (pH 7.5) containing M DTT and 100 mM NaCl at 37 ° C
And agarose gel electrophoresis was performed to recover a 1.3 kbp DNA fragment. Vector pCDM8 (Invi
(made by Trogen) 10 mM MgCl ₂ , 1 mM DT
T, 20 mM Tris-HCl buffer containing 100 mM KCl
After treatment with the restriction enzyme XhoI in (pH 8.5) at 37 ° C., further 10 mM MgCl ₂ , 1 mM DTT, 100 m
20 mM Tris-HCl buffer containing M KCl (pH 8.5)
After digestion with the restriction enzyme HpaI at 37 ° C. in the medium, agarose gel electrophoresis was performed and the recovered 3.3 kbp DNA was ligated with the 1.3 kbp DNA fragment to obtain pCDMKPO3 ′.
Got The restriction enzyme Kpn was added to pEFDKPO5'K and pCDMKPO3 'in 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl ₂ , 1 mM DTT at 37 ° C.
After treatment with I and agarose gel electrophoresis, fragments of 10 kbp and 0.9 kbp were recovered and ligated to obtain pEFDKPOf (FIG. 7). As shown in the figure, this plasmid contains the dihydrofolate reductase gene (dhfr) and the EF1α promoter and SV40p.
Human Meg-POT cDNA during olya signal
Are connected.

The vector DHFR-ΔE-RV
h is DH described in WO92 / 19759
FR-ΔE-PMh-gγ1 and RVh-PM1f-4
Was prepared as follows.

DHFR-ΔE-PMh-gγl and RV
h-PM1f-4 was added to 10 mM MgCl ₂ , 1 mM DT
T, 100 mM KCl, 20 mM containing 0.01% BSA
It was treated with restriction enzymes PvuI and BamHI (manufactured by Takara Shuzo Co., Ltd.) at 37 ° C. in a Tris-hydrochloric acid buffer solution (pH 8.5), subjected to agarose gel electrophoresis to recover 4 kbp and 3 kbp fragments, and ligating them to DHFR. -ΔE-R
Vh was prepared.

Example 23. Factors for amplification of megakaryocytes in CHO cells
Offspring (Meg-POT) gene expression CHO cells DXB-11 were phosphate-buffered sali at 1 × 10 ⁷ cells / ml.
After suspending in ne (PBS), 10 μg of pEFDKPOf was added to 0.8 ml of this cell suspension. 1900V, 25
Electroporation using GenePulsar (manufactured by Bio Rad) under the condition of μF.
on) to introduce the plasmid into CHO cells. After a recovery period of 10 minutes at room temperature, the electroporated cells were added to 25 ml of α-MEM medium (GIBCO) containing 10% fetal bovine serum. After culturing at 37 ° C for 24 hours, the cells recovered by trypsin treatment were diluted 100-fold, and 10% fetal bovine serum-free ribonucleoside- and deoxyribonucleoside-free α-MEM medium (G
The cells were cultured for 3 weeks in IBCO. At this time, the medium was exchanged once every 3 to 4 days. Since colonies had grown to the extent that they could be visually confirmed, clones were picked up, and culture was continued for each clone. After that, it is added to the previous medium with methotrex.
ate (MTX) (manufactured by Sigma) was added, and selective culture was continued. The MTX concentration at the time of primary selection was 10 nM,
Secondary selection was performed on the obtained resistant clones in a medium having an MTX concentration of 50 nM. 5 clones finally obtained
IMDM medium containing 0 nM MTX and 2% FCS (G
A large amount was cultured in IBCO), and the culture supernatant was used for purification.

The culture supernatant was analyzed by SDS / PAGE,
Meg-POT by Western blotting method using an antibody prepared by using a synthetic peptide near the N-terminus as an antigen
Was detected. As the molecular weight marker, Example 1 was used.
The pre-stained molecular weight marker manufactured by Bio-Rad shown in 5 was used. As shown in FIG. 8, the molecular weight is about 33 kd,
Three bands were detected at 30 kd and 27.5 kd.

Example 24. From culture supernatant of CHO cells
Recombinant megakaryocyte amplification factor (Purification of Meg-POT I) Tw was added to 10 l of the culture supernatant of the CHO cells prepared in Example 23.
Add een20 to a final concentration of 0.01%, and add 5
The insoluble matter was removed through a μm membrane filter (manufactured by Fuji Film Co., Ltd.). A recombinant megakaryocyte amplification factor was purified from this culture supernatant by the following procedure.

(I) Add 0.01% Tween to the above culture supernatant.
Blue-Sepha equilibrated with 50 mM Tris-HCl buffer (pH 8) containing 20 and 0.2 M NaCl.
Rose fast flow column (5.0 x 20c
m, manufactured by Pharmacia). After washing the column with the same buffer, 0.01% Tween 20 and 2M
The protein adsorbed on the column was eluted with 50 mM Tris-HCl buffer (pH 9) containing KCl. Collect this fraction,
Concentrate with Minitan (manufactured by millipore),
20 mM acetate buffer containing 01% Tween 20 (pH
The procedure of adding 5) and diluting is repeated to desalt, and the resulting insoluble matter is centrifuged (10,000 rpm × 30 mi).
n) removed. The supernatant was subjected to subsequent cation exchange chromatography purification.

(Ii) 2 containing 0.01% Tween 20
S-Sep equilibrated with 0 mM acetate buffer (pH 5)
harose fast flow column (5.0 x 1
2 cm), the above fraction was added, and the column was washed with the same buffer solution, and the NaCl concentration in the same buffer solution was adjusted to 0.1, 0.
The protein adsorbed on the column was eluted by increasing the concentration to 2, 0.3, 0.5M.

(Iii) The 0.1 M NaCl fraction containing the recombinant Meg-POT was prepared in advance with 0.1% TFA.
Vydac equilibrated with 24% acetonitrile containing
After adding to a C4 column (10 × 250 mm) and washing the column with the same eluent, the concentration of acetonitrile in 0.1% TFA was linearly increased to 24-48% in 48 minutes to remove the protein adsorbed on the column. It was eluted. Flow rate is 1 ml
/ Min, protein detection is 220 nm and 280
nm at two wavelengths. Acetonitrile concentration is 40-4
Fractions containing 5% recombinant Meg-POT were pooled and the next Gel Permeation Chromatogr was collected.
I put it on aphy.

(Iv) A fraction containing recombinant Meg-POT was equilibrated with 40% acetonitrile containing 0.01% TFA, and a TSKgel G3000SW column (21.
5 × 60 cm). The flow rate was 3 ml / min, and the protein was detected at 280 nm. The main peaks eluted at 37 to 44 minutes were collected, diluted with 0.1% TFA, and then subjected to reverse phase HPLC under the same conditions as (iii) to collect the main peaks.

The recombinant megakaryocyte amplification factor (Meg-POT) thus obtained was analyzed by SDS / PAGE.
Gel concentration 12%, Laemmli method Nature
e, 227, 680 (1970), 2D
-The protein was stained using the silver stain reagent "Daiichi" (manufactured by Daiichi Pure Chemicals). The molecular weight marker is a low molecular weight marker [Phosphorylase B (92.5
kd), bovine serum albumin (66.2 kd), ovalbumin (45.0 kd), carbonic anhydrase (31.0)
kd), soybean trypsin inhibitor (21.5k
d), lysozyme (14.4 kd)] was used.

As a result, purified recombinant Meg-P
OT gave a single band with a molecular weight of approximately 33,000.

Example 25. Recombinant megakaryocyte amplification factor (M
N-terminal and C-terminal amino acid sequence analysis of ( eg-POT) (i) Recombinant Meg-POT obtained in Example 24
The N-terminal amino acid sequence analysis was performed using a gas phase protein sequencer type 476A (manufactured by Applied Biosystems). As a result, the following three types of N-terminal amino acid sequences (a) to (c) were recognized.

(A) Ser-Arg-Thr-Leu-Ala-Gly-Glu-Thr-
Gly-Gln-Glu-Ala-Ala -... (b) Leu-Ala-Gly-Glu-Thr-Gly-Gln-Glu-Ala-Ala-Pro-
Leu-Asp -... (c) Gly-Glu-Thr-Gly-Gln-Glu-Ala-Ala-Pro-Leu-Asp-
Gly-Val -... These sequences are the sequences (a) to in Example 13.
Corresponds to (c).

(Ii) The C-terminal amino acid sequence of recombinant Meg-POT was analyzed.

10 mg / ml in recombinant Meg-POT
100 μl of 70% formic acid solution containing cyanogen bromide of
After decomposing cyanogen bromide at room temperature for 24 hours, an excess reagent was removed by a centrifugal concentrator. The residue was dissolved in 0.1% TFA and equilibrated in 0.1% TFA Vydac C4
Add to the column (4.6 x 250 mm), 0.1% TF
The concentration of acetonitrile in A was linearly increased to 80% in 40 minutes to elute the cyanogen bromide fragment adsorbed on the column. Of the two obtained peaks, the C-terminal peptide fragment was further digested with Endo Asp-N. The C-terminal peptide was dissolved in 50 mM phosphate buffer (pH 8.0), Endo Asp-N was added, and enzyme digestion was performed at room temperature for 16 hours. After adding 10% TFA to the reaction solution to adjust the pH to 3, Vydac C1 equilibrated with 0.1% TFA was used.
Add to 8 columns and adjust the acetonitrile concentration to 0 for 48 minutes.
The peptide adsorbed on the column was eluted by linearly increasing to ˜48%. The obtained fragment was analyzed by a gas phase protein sequencer type 476A, and the amino acid sequence of the C-terminal fragment was as shown below.

Asp-Pro-Ser-Trp-Arg-Gln-Pro-Glu-Arg Example 26. Megakaryocyte amplification factor in culture supernatant of CHO cells
Measurement of (Meg-POT) activity The culture supernatant of the CHO cells obtained in Example 23 and the megakaryocyte amplification factor activity of the recombinant megakaryocyte amplification factor purified in Example 24 were measured according to the method described above.

As a result, as shown in Table 6, the culture supernatant of the CHO cells into which the vector containing the cDNA for megakaryocyte amplification factor was introduced clearly showed megakaryocyte amplification factor activity, and the purified recombinant megakaryocytes were purified from it. The sphere amplification factor also showed megakaryocyte amplification factor activity.

The protein content of the purified product was calculated by amino acid analysis with Ala = 28.

[0165]

[Table 6] Megakaryocyte amplification factor activity in culture supernatant of CHO cells and of purified recombinant Meg-POT Final concentration of test body megakaryocyte colony number CHO cell culture supernatant 0.08% 4 ^a) 0.31% 11 ^a) 1.25% 10.5 ^a) 5% 12 ^a) Purified product 0.01 ng / ml 0 ^b) 0.1 ng / ml 6 ^b) 1 ng / ml 8 ^b) 10 ng / ml 11 ^b) a) A value obtained by subtracting the number of colonies formed by IL-3 alone (25 colonies) from the number of colonies formed.

B) From the number of colonies formed, IL-3
The value obtained by subtracting the number of colonies formed independently (20.5).

Example 27. From culture supernatant of CHO cells
Purification of Recombinant Megakaryocyte Amplification Factor (Meg-POT) II II 10 l of CHO cell culture supernatant prepared in Example 23 (1
0 liter) with Tween 20 at a final concentration of 0.01%
Insoluble matter was removed through a 5 μm membrane filter (manufactured by Fuji Film Co.). A recombinant megakaryocyte amplification factor was purified from this culture supernatant by the following procedure.

(I) The above culture supernatant was concentrated 20 times using a spiral cartridge (manufactured by Amicon). Ammonium sulfate was added to the concentrate at a final 50% saturation concentration while stirring the concentrated solution at 4 ° C, and the precipitated protein was centrifuged (10,000gx
Collected as a precipitate by 30 min) and 10 mM Tris-HCl buffer
It was dissolved in (pH 7.4). Ammonium sulfate was added to the fraction so that the final concentration was 1 M, and the fraction was subjected to the following hydrophobic chromatography.

(Ii) It was added to a Phenyl-sepharose 6FF column (5.0 x 15 cm, manufactured by Pharmacia) equilibrated with 10 mM Tris-HCl buffer (pH 7.4) containing 1 M ammonium sulfate. After washing the column with the same buffer, the ammonium sulfate concentration was lowered to 0.1 M and the column was washed again, and then the recombinant Meg-POT was eluted with 10 mM Tris-HCl buffer (pH 8.5) containing 0.1% Tween 20. . This fraction was concentrated with a membrane filter PM-10 (manufactured by Amicon), diluted 10-fold with 10 mM Tris-HCl buffer (pH 8.5) containing 0.01% Tween 20, and subjected to anion exchange chromatography.

(Iii) DEAE-sepharose fast flow column (5 x 13c) equilibrated with the buffer used for dilution of the above fractions
m), the column was washed with the same buffer, and the NaCl concentration in the same buffer was increased to 0.1 M to elute the protein adsorbed on the column. This fraction containing recombinant Meg-POT was treated with 10% TF
The pH was adjusted to 3 by adding A, and then subjected to reverse phase HPLC under the conditions shown below.

(Iv) The above fraction was added to a Vydac C4 column (10 x 250 mm) equilibrated with 32% acetonitrile containing 0.1% TFA, the column was washed with the same eluent, and the acetonitrile concentration in 0.1% TFA was added. Was linearly increased from 32 to 48% in 64 minutes to elute the protein adsorbed on the column. The flow rate is 1.0 ml / min,
The protein was detected at two wavelengths of 220 nm and 280 nm. 40-45%
Collect the fractions eluted at acetonitrile concentration of 0.1% T
Diluted 2-fold with FA. Reverse phase HPLC of this solution under the same conditions
The main peaks eluting at 40-45% acetonitrile concentration were collected and subjected to the next Gel Permeation Chromatography.

(V) The above fractions were added to a TSK G3000SW column (21.5 x 60 cm) equilibrated with 40% acetonitrile containing 0.1% TFA. Protein detection was performed at 280 nm at a flow rate of 3 ml / min. Collect the major peaks eluting between 42-47 minutes and remove 0.1% TF
Diluted with A.

(Vi) The above fraction was subjected to reverse phase HP under the same conditions as (iv).
LC was performed and the main fraction was collected.

If necessary, this fraction was subjected to cation exchange chromatography to remove the organic solvent and TFA.
20 mM acetate buffer containing 0.01% Tween 20 (pH
S-sepharos diluted 10 times with 5.0) and equilibrated with the same buffer
Add to e fast flow column, wash the column with the same buffer, and then use recombinant Meg-P with the same buffer containing 0.3M NaCl.
The OT was eluted.

The recombinant megakaryocyte amplification factor (Meg-POT) thus obtained was analyzed by SDS / PAGE.
Gel concentration 12%, Laemmli's method Nature, 227, 680
Electrophoresis according to (1970), 2D-silver stain reagent "Daiichi"
(Manufactured by Daiichi Pure Chemicals) was used to stain the protein. As the molecular weight marker, the low molecular weight marker manufactured by Bio-Rad and used in Example 15 was used.

As a result, purified recombinant Meg-P
OT gave a single band with a molecular weight of about 30,000.

The megakaryocyte amplification factor activity of this product was measured according to the method described above, and the molecular weight of about 33, obtained in Example 24,
000 compared to that. As a result, as shown in Table 7, those having a molecular weight of about 30,000 showed no activity at any of the measured concentrations.

[0178]

Table 7 Megakaryocyte amplification factor activity of purified megakaryocyte amplification factor Molecular weight Megakaryocyte amplification factor activity ^a) Final concentration (ng / ml) 40 10 2.5 0.625 About 33,000 11 8.5 5 7 About 30,000 1 0.5 0 1.5 a) The value obtained by subtracting the number of colonies formed by IL-3 alone (25) from the number of formed colonies.

Example 28 . Recombinant megakaryocyte amplification factor (M
N-terminal and C-terminal amino acid sequence analysis of ( eg-POT) II (i) Regarding the recombinant Meg-POT having a molecular weight of about 30,000 obtained in Example 27, a gas phase protein sequencer type 476A (manufactured by Applied Biosystems) ) With N
Terminal amino acid sequence analysis was performed. As a result, the following three types of N-terminal amino acid sequences (a) to (c) were recognized.

(A) Ser-Arg-Thr-Leu-Ala-Gly-Glu-Thr-
Gly-Gln-Glu-Ala-Ala -... (b) Leu-Ala-Gly-Glu-Thr-Gly-Gln-Glu-Ala-Ala-Pro-
Leu-Asp -... (c) Gly-Glu-Thr-Gly-Gln-Glu-Ala-Ala-Pro-Leu-Asp-
Gly-Val -... These sequences are the sequences (a) to
Corresponds to (c).

(Ii) The C-terminal amino acid sequence of recombinant Meg-POT was analyzed.

Recombinant Meg- having a molecular weight of about 30,000
100 μl of a 70% formic acid solution containing 10 mg / ml of cyanogen bromide was added to POT, and after decomposing cyanogen bromide at room temperature for 24 hours, excess reagents were removed by a centrifugal concentrator. The residue was dissolved in 0.1% TFA and equilibrated with 0.1% TFA Vy
Addition to dac C4 column (4.6 x 250mm), 0.1% TF
The concentration of acetonitrile in A was linearly increased to 80% in 40 minutes to elute the cyanogen bromide fragment adsorbed on the column. Flow rate is 1ml / min, peptide detection is 220nm and 280nm
I went there. The obtained two peaks were analyzed by a gas phase protein sequencer type 476A, and the amino acid sequence of the C-terminal fragment was as shown below.

Asp-Ala-Leu-Arg-Gly-Leu-Leu-Pro-Val-Le
u-Gly-Gln-Pro-Ile-Ile-Arg From Example 25 to Example 28, the molecular weight is about 33,000.
And the N-terminal of the amino acid sequence having a molecular weight of about 30,000 are equivalent, but differ only in the C-terminal. On the other hand, regarding the megakaryocyte amplification factor activity, those with a molecular weight of about 33,000 show activity, but those with a molecular weight of about 30,000 do not show activity. Therefore, the molecular weight is about 33,000 and about 30,000.
It is considered that there is an important site involved in megakaryocyte amplification factor activity in the C-terminal amino acid sequence between 0.

Example 29 . Recombinant megakaryocyte amplification factor
Purification of (Meg-POT) III (i) The Meg-POT fraction eluted from the Blue sepharose fast flow column of Example 24 with 2M KCl was desalted with minitan (millipore) and ammonium sulfate was added at 4 ° C. The mixture was added to 50% saturation and stirred for 30 minutes. The resulting precipitate was collected by centrifugation (10,000 g x 30 minutes) and dissolved in distilled water. Add 0.01% Tween 20 to this solution overnight at 4 ° C.
Q-sepharose fast flow column (5) dialyzed against 0 mM Tris-HCl buffer (pH 8.0) and equilibrated with the same buffer
x 10 cm, Pharmacia). After washing the column with the same buffer, adjust the NaCl concentration in the same buffer to 0.1, 0.2, 0.3, 0.5,
The protein adsorbed on the column was eluted by gradually increasing it to 1.0M. After each fraction was subjected to SDS / PAGE, Meg-POT was detected by Western blotting using the antibody against Meg-POT prepared in Example 23. The molecular weight marker at this time was a prestained molecular weight marker [phosphorylase B (10
6kd), bovine serum albumin (80.0kd), ovalbumin (49.5kd), carbonic anhydrase (32.5kd)
kd), soybean trypsin inhibitor (27.5k)
d), lysozyme (18.5 kd)] was used. As a result, a band was stained even at a molecular weight of about 70,000 (Fig. 9). In order to confirm whether or not Meg-POT activity was observed in this molecular species, the following purification was performed to separate it from the molecular species having a molecular weight of about 33,000.

(Ii) The pH was adjusted to 5 by adding acetic acid to a 0.2 M NaCl fraction containing a molecular species having a molecular weight of about 70,000.
Vydac C4 equilibrated with 32% acetonitrile containing% TFA
Added to the column. After washing the column with the same eluent, 0.1%
The concentration of acetonitrile in TFA was linearly increased to 32-48% for 40 minutes to elute the protein adsorbed on the column. Fractions with an acetonitrile concentration of 39-41% were collected and subjected to subsequent gel filtration.

(Iii) The above fraction was added to PB containing 0.01% Tween 20.
It was diluted with S, concentrated with Centriprep-10, and added to a TSK G3000SW column equilibrated with the same buffer. The flow rate was 3 ml / min, and the protein was detected at two wavelengths of 220 nm and 280 nm. The Meg-POT activity was measured by a colony formation method, and Meg-POT was detected by Western blotting after SDS / PAGE.
As a result, the molecular weight of about 70,000 is 42-4.
It was eluted at 6 minutes, and no molecular species having a molecular weight of about 33,000 was detected in the fraction (Fig. 10). As the molecular weight marker, the aforementioned molecular weight marker manufactured by Bio-Rad was used.

The Meg-POT activity of the 42-46 minute fraction was measured in the same manner as in Example 26. As shown in Table 8, the Meg-POT activity was also observed in the 42-46 minute fraction. Therefore, it was estimated that the molecular species having a molecular weight of about 70,000 also has Meg-POT activity.

[0188]

[Table 8] Megakaryocyte amplification factor activity of each purified fraction of CHO cell culture supernatant TSK G3000SW Fraction (min) 42-43 43-44 44-45 45-46 Megakaryocyte amplification factor activity ^a) 12 9 10.5 5.5 a): A value obtained by subtracting the number of colonies formed by IL-3 alone (19.5) from the number of formed colonies.

Example 30 . Structure of modified expression vector
Construction and expression in COS cells (1) Construction of pEFNKPOS vector PCR was used to obtain the modified Meg-POT gene. PCR
381A based on the nucleotide sequence of the gene shown in SEQ ID NO: 10 as the primer used for
It was synthesized using a DNA Synthesizer (manufactured by Applied Biosystems).

Primer-3S: 5'-CTGGCTCACCGGCTCTCTGA-3 ' C-terminal of Meg-POT purified from cultured CHO cells
Furin, a processing enzyme in the animal cell that exists near the edge,
Recognition sequence Arg-x-Arg-Arg²⁹⁵Meg-POT remains to code
Make a gene and use this to construct its expression vector
It was

Using the pKPO27 as a template, the DNA Thermal Cycler (P
PCR was performed using erkin Elmer Cetus). pKPO2
7 1 μg to 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 6 mM (NH ₄ ) ₂ SO
₄ , 2mM MgCl ₂ , 0.1% Triton X-100,0.1mg / ml BSA, 100mM d
eoxynucleotide triphosphate (dNTP), primer-3S and
Amplification was carried out in 100 μl of PCR reaction solution containing 100 pmol of G982A each. First, after denaturing at 95 ° C for 6 minutes, it was cooled to 85 ° C and 2.5 units of Pfu DNA Polymerase (manufactured by Stratagene) was added. Then, denaturation 94 ° C for 1 minute, annealing 60 ° C for 30 seconds, extension 72
PCR was performed by repeating the cycle of 2 minutes at 30 ° C. 30 times (FIG. 11). The sample was subjected to agarose gel electrophoresis, and the amplified 590 bp DNA fragment was extracted. Polynucleotide kinase
Was used to phosphorylate the 5'end of the DNA and ligated with the vector pSP73 which had been previously treated with the restriction enzymes SmaI and alkaline phosphatase to obtain pSP982A. This inserted nucleotide sequence is Dideoxy s
determined by the equencing method, the 982nd G is replaced by T
It was confirmed that there is a BamHI site on the 3'side and no other mutations.

PSP982A and pRVHKPO27 were digested with restriction enzymes SalI and BamH
After treatment with I, agarose gel electrophoresis was carried out to recover 190 bp and 9.5 kbp DNA fragments from each, and these were ligated to obtain pEFNKPOS (FIG. 12).

(2) Expression in COS cells COS cells were suspended in PBS at 1 × 10 ⁷ cells / ml, and 20 μg of pEFNKPOS was added to 0.8 ml of this cell suspension. 1500V, 25
The plasmid was introduced into COS cells by electroporation using GenePulsar (manufactured by BioRad) under μFD conditions. The electroporated cells were added to a DMEM medium (GIBCO) containing 1% fetal bovine serum, and after culturing for 72 hours, the culture supernatant was collected. Each culture supernatant was concentrated about 10 times with Centriprep-10 and subjected to SDS polyacrylamide gel electrophoresis. Western blotting was performed using the anti-megakaryocytic amplification factor antiserum obtained by immunizing a rabbit with the Meg-POT protein expressed in Escherichia coli and purified in Example 21 as an antigen, and then added to the culture supernatant. Meg-POT
Accumulation of modified form was confirmed.

The megakaryocyte amplification factor activity of this culture supernatant was measured according to the method described above. The results are shown in Table 9.

[0195]

[Table 9] Megakaryocyte amplification factor activity in culture supernatant of COS cells COS cells Megakaryocyte amplification factor activity ^a) Culture supernatant concentration (%) 10 5 2.5 1.25 0.625 Control COS 1 0 0 1 1 pRVHKPO27f Introduced COS 9.5 9.5 6.5 12 0 pEFNKPOS Introduced COS 9.5 0.5 1 0 0 a) A value obtained by subtracting the number of colonies formed by IL-3 alone (40.5) from the number of colonies formed.

(3) Construction of amino acid-substituted Meg-POT expression vector Based on the nucleotide sequence of the gene shown in SEQ ID NO: 10, the following oligonucleotides were used as primers for PCR.
It was synthesized using 381A DNA Synthesizer (manufactured by Applied Biosystems).

Primer-T3-2: 5'-CATGATTACGCCAAGCTCGAA-3 'Primer-754GA: 5'-GCTGCCTCCTCCTGGTCCTGGTCCAGG
GGTCC-3 'Primer-754GS: 5'-CCAGGACCAGGAGGAGGCAGCCAGGGC
GGC-3 'Primer 850GA: 5'-AGCACGGGCACCAGGCCCCGCAGAGCG
TCC-3 'Primer 850GS: 5'-CTGCGGGGCCTGGTGCCCGTGCTGGGC
A Meg-POT modified expression vector-pEFNKPOL252V in which Leu at the 252nd position of CAGCCC-3 'pEFNKPOS was replaced with Val was constructed. Under the same conditions as above, pKPO27 was used in combination with primer-T3-2 and 850GA.
PCR was performed with each combination of 0GS and G982A. The sample was subjected to gel electrophoresis, 0.9kbp and 150bpD, respectively.
NA was extracted. After mixing these DNA fragments, primer T3-
PCR was performed again using 2 and G982A (FIG. 11). Agarose gel electrophoresis was performed to extract the amplified 1 kbp DNA. The 5'end of DNA was phosphorylated using Polynucleotide kinase and ligated with vector pSP73 which had been previously treated with restriction enzymes SmaI and alkaline phosphatase to obtain pSP850G. The inserted nucleotide sequence was determined by the Dideoxy sequencing method, and 850
The 3rd C is replaced by G, and the 982nd G is replaced by T.
It was confirmed that there are HI sites and no other mutations.
After treating pSP850G and pRVHKPO27 with restriction enzymes SalI and BamHI, agarose gel electrophoresis was performed to obtain 190b
DNA fragments of p and 9.5 kbp were recovered and ligated to pEFNKP
OL252V was obtained (Fig. 12).

Further, a Meg-POT modified expression vector-pEFNKPOQ220E in which the 220th Gln of pEFNKPOS was replaced with Glu was constructed. Under the same conditions as above, pKPO27 was primed with primers T3-2 and 754.
With the combination of GA and the combination of 754GS and G982A respectively
PCR was performed. The sample was subjected to gel electrophoresis to extract 0.9 kbp and 150 bp DNA, respectively. After mixing these DNA fragments, PCR was performed again using the primers T3-2 and G982A. Amplified 1kbp DN after agarose gel electrophoresis
A was extracted. 5'of DNA using Polynucleotide kinase
The ends were phosphorylated and ligated with the vector pSP73 which had been previously treated with the restriction enzymes SmaI and alkaline phosphatase to obtain pSP754G. The inserted nucleotide sequence was determined by the Dideoxy sequencing method. The C at the 754th position was replaced with a G, and the G at the 982nd position was replaced with a T.
It was confirmed that there was a BamHI site on the side and there were no other mutations. After treating pSP754G and pRVHKPO27 with restriction enzymes EcoRI and BamHI, agarose gel electrophoresis was performed to recover 1 kbp and 8.7 kbp DNA fragments, respectively, and ligate them together.
EFNKPOQ220E was obtained (Fig. 12).

(4) Expression of amino acid-substituted Meg-POT in COS cells In the same manner as above, pEFNKPOL252V and pEFNKPOQ220E were introduced into COS cells by electroporation.
Centriprep-10 each about 72 hours culture supernatant
It was concentrated 10-fold and subjected to SDS polyacrylamide gel electrophoresis. Similarly, Western blotting was performed using an anti-megakaryocytic amplification factor antiserum obtained by immunizing a rabbit with the Meg-POT protein expressed and purified in Escherichia coli obtained in Example 21 as an antigen, and The accumulation of modified Meg-POT was confirmed.

[0200]

[Sequence list]

SEQ ID NO: 1 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Leu Ala Gly Glu Xaa Gly Gln Glu Ala Ala Pro Leu Asp Gly Val Leu 1 5 10 15 SEQ ID NO: 2 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu Asp Gly Val Leu Ala 1 5 10 15 SEQ ID NO: 3 Sequence length : 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu Asp Gly Val Leu Ala Asn 1 5 10 15 SEQ ID NO: 4 Sequence length: 14 Sequence Type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu Asp Gly Val Leu 1 5 10 SEQ ID NO: 5 Sequence length: 35 Sequence type: Amino acid Topology: Linear Sequence Type: Peptide Sequence: Leu Ala Va l Ala Leu Ala Gln Lys Asn Val Lys Leu Ser Thr Glu 1 5 10 15 Gln Leu Arg Xaa Leu Ala His Arg Leu Ser Glu Pro Pro Glu Asp 20 25 30 Leu Asp Ala Leu Pro 35 SEQ ID NO: 6 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: DNA Sequence: CTCAACAGAG CAGCTGCGCT GTCTGGCTCA CCGGCTCTCT 40 SEQ ID NO: 7 Sequence length: 69 Sequence type: Nucleic acid Number of strands: Two Chain topology: Linear Sequence type: cDNA Sequence: CGC CAA CTC CTT GGC TTC CCG TGT GCG GAG GTG TCC GGC CTG AGC 45 Arg Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser 1 5 10 15 ACG GAG CGT GTC CGG GAG CTG GCT 69 Thr Glu Arg Val Arg Glu Leu Ala 20 SEQ ID NO: 8 Sequence length: 75 Sequence type: Nucleic acid Strand number: Double strand Topology: Linear Sequence type: cDNA Sequence: CTC TCT GAG CCC CCC GAG GAC CTG GAC GCC CTC CCA TTG GAC CTG 45 Leu Ser Glu Pro Pro Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu 1 5 10 15 CTG CTA TTC CTC AAC CCA GAT GCG TTC TCG 75 Leu Leu Phe Leu Asn Pro Asp Ala Phe Ser 20 25 SEQ ID NO: 9 Sequence length: 197 Sequence type: Nucleic acid Strand number: Double strand Topology: Linear Type of sequence: cDNA Origin organism name: human Characteristics: cDNA derived from cultured cells obtained from lymph nodes of human pancreatic cancer patients and encoding a part of a polypeptide having human megakaryocyte amplification factor activity.

Sequences: AA CTC CTT GGC TTC CCG TGT GCG GAG GTG TCC GGC CTG AGC ACG 44 Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr 1 5 10 GAG CGT GTC CGG GAG CTG GCT GTG GCC TTG GCA CAG AAG AAT GTC 89 Glu Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val 15 20 25 AAG CTC TCA ACA GAG CAG CTG CGC TGT CTG GCT CAC CGG CTC TCT 134 Lys Leu Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser 30 35 40 GAG CCC CCC GAG GAC CTG GAC GCC CTC CCA TTG GAC CTG CTG CTA 179 Glu Pro Pro Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu 45 50 55 TTC CTC AAC CCA GAT GCG 197 Phe Leu Asn Pro Asp Ala 60 65 SEQ ID NO: 10 Sequence length: 2129 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA Origin organism name: Human Direct origin: Plasmid pKPO27 Features: A polypeptide having human megakaryocyte amplification factor activity, which is derived from cultured cells obtained from lymph nodes of a human pancreatic cancer patient, The encoding cDNA.

Sequence: GAATTCGGCA CGAGGCCACT CCCGTCTGCT GTGACGCGCG GACAGAGAGC 50 TACCGGTGGA CCCACGGTGC CTCCCTCCCT GGGATCTACA CAGACC ATG GCC 102 Met Ala 1 TTG CCA ACG GCT CGA CCC CTG TTG GGG TCC TGT GGG LeuGu ACC Le Prou Thr Pro Ala 5 10 15 CTC GGC AGC CTC CTG TTC CTG CTC TTC AGC CTC GGA TGG GTG CAG 192 Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln 20 25 30 CCC TCG AGG ACC CTG GCT GGA GAG ACA GGG CAG GAG GCT GCA CCC 237 Pro Ser Arg Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro 35 40 45 CTG GAC GGA GTC CTG GCC AAC CCA CCT AAC ATT TCC AGC CTC TCC 282 Leu Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser 50 55 60 CCT CGC CAA CTC CTT GGC TTC CCG TGT GCG GAG GTG TCC GGC CTG 327 Pro Arg Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu 65 70 75 AGC ACG GAG CGT GTC CGG GAG CTG GCT GTG GCC TTG GCA CAG AAG 372 Ser Thr Glu Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys 80 85 90 AAT GTC AAG CTC TCA ACA GAG CAG CTG CGC TG T CTG GCT CAC CGG 417 Asn Val Lys Leu Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg 95 100 105 CTC TCT GAG CCC CCC GAG GAC CTG GAC GCC CTC CCA TTG GAC CTG 462 Leu Ser Glu Pro Pro Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu 110 115 120 CTG CTA TTC CTC AAC CCA GAT GCG TTC TCG GGG CCC CAG GCC TGC 507 Leu Leu Phe Leu Asn Pro Asp Ala Phe Ser Gly Pro Gln Ala Cys 125 130 135 ACC CGT TTC TTC TCC CGC ATC ACG AAG GCC AAT GTG GAC CTG CTC 552 Thr Arg Phe Phe Ser Arg Ile Thr Lys Ala Asn Val Asp Leu Leu 140 145 150 CCG AGG GGG GCT CCC GAG CGA CAG CGG CTG CTG CCT GCG GCT CTG 597 Pro Arg Gly Ala Pro Glu Arg Gln Arg Leu Leu Pro Ala Ala Leu 155 160 165 GCC TGC TGG GGT GTG CGG GGG TCT CTG CTG AGC GAG GCT GAT GTG 642 Ala Cys Trp Gly Val Arg Gly Ser Leu Leu Ser Glu Ala Asp Val 170 175 180 CGG GCT CTG GGA GGC CTG GCT TGC GAC CTG CCT GGG CGC TTT GTG 687 Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu Pro Gly Arg Phe Val 185 190 195 GCC GAG TCG GCC GAA GTG CTG CTA CCC CGG CTG GTG AGC TGC CCG 732 Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu Val Ser Cys Pro 200 205 210 GGA CCC CTG GAC CAG GAC CAG CAG GAG GCA GCC AGG GCG GCT CTG 777 Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg Ala Ala Leu 215 220 225 CAG GGC GGG GGA CCC CCC TAC GGC CCC CCG TCG ACA TGG TCT GTC 822 Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp Ser Val 230 235 240 TCC ACG ATG GAC GCT CTG CGG GGC CTG CTG CCC GTG CTG GGC CAG 867 Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly Gln 245 250 255 CCC ATC ATC CGC AGC ATC CCG CAG GGC ATC GTG GCC GCG TGG CGG 912 Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg 260 265 270 CAA CGC TCC TCT CGG GAC CCA TCC TGG CGG CAG CCT GAA CGG ACC 957 Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr 275 280 285 ATC CTC CGG CCG CGG TTC CGG CGG GAA GTG GAG AAG ACA GCC TGT 1002 Ile Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys 290 295 300 CCT TCA GGC AAG AAG GCC CGC GAG ATA GAC GAG AGC CTC ATC TTC 1047 Pro Ser Gly Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe 305 310 315 T AC AAG AAG TGG GAG CTG GAA GCC TGC GTG GAT GCG GCC CTG CTG 1092 Tyr Lys Lys Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu 320 325 330 GCC ACC CAG ATG GAC CGC GTG AAC GCC ATC CCC TTC ACC TAC GAG 1137 Ala Thr Gln Met Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu 335 340 345 CAG CTG GAC GTC CTA AAG CAT AAA CTG GAT GAG CTC TAC CCA CAA 1182 Gln Leu Asp Val Leu Lys His Lys Leu Asp Glu Leu Tyr Pro Gln 350 355 360 GGT TAC CCC GAG TCT GTG ATC CAG CAC CTG GGC TAC CTC TTC CTC 1227 Gly Tyr Pro Glu Ser Val Ile Gln His Leu Gly Tyr Leu Phe Leu 365 370 375 AAG ATG AGC CCT GAG GAC ATT CGC AAG TGG AAT GTG ACG TCC CTG 1272 Lys Met Ser Pro Glu Asp Ile Arg Lys Trp Asn Val Thr Ser Leu 380 385 390 GAG ACC CTG AAG GCT TTG CTT GAA GTC AAC AAA GGG CAC GAA ATG 1317 Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys Gly His Glu Met 395 400 405 AGT CCT CAG GTG GCC ACC CTG ATC GAC CGC TTT GTG AAG GGA AGG 1362 Ser Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg 410 415 420 GGC CAG CTA GAC AAA GAC ACC CTA GAC ACC C TG ACC GCC TTC TAC 1407 Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr 425 430 435 CCT GGG TAC CTG TGC TCC CTC AGC CCC GAG GAG CTG AGC TCC GTG 1452 Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val 440 445 450 CCC CCC AGC AGC ATC TGG GCG GTC AGG CCC CAG GAC CTG GAC ACG 1497 Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr 455 460 465 TGT GAC CCA AGG CAG CTG GAC GTC CTC TAT CCC AAG GCC CGC CTT 1542 Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu 470 475 480 GCT TTC CAG AAC ATG AAC GGG TCC GAA TAC TTC GTG AAG ATC CAG 1587 Ala Phe Gln Asn Met Asn Gly Ser Glu Tyr Phe Val Lys Ile Gln 485 490 495 TCC TTC CTG GGT GGG GCC CCC ACG GAG GAT TTG AAG GCG CTC AGT 1632 Ser Phe Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser 500 505 510 CAG CAG AAT GTG AGC ATG GAC TTG GCC ACG TTC ATG AAG CTG CGG 1677 Gln Gln Asn Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg 515 520 525 ACG GAT GCG GTG CTG CCG TTG ACT GTG GCT GAG GTG CAG AAA CTT 1722 Thr Asp Ala Val L eu Pro Leu Thr Val Ala Glu Val Gln Lys Leu 530 535 540 CTG GGA CCC CAC GTG GAG GGC CTG AAG GCG GAG GAG CGG CAC CGC 1767 Leu Gly Pro His Val Glu Gly Leu Lys Ala Glu Glu Arg His Arg 545 550 555 CCG GTG CGG GAC TGG ATC CTA CGG CAG CGG CAG GAC GAC CTG GAC 1812 Pro Val Arg Asp Trp Ile Leu Arg Gln Arg Gln Asp Asp Leu Asp 560 565 570 ACG CTG GGG CTG GGG CTA CAG GGC GGC ATC CCC AAC GGC TAC CTG 1857 Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile Pro Asn Gly Tyr Leu 575 580 585 GTC CTA GAC CTC AGC GTG CAA GAG GCC CTC TCG GGG ACG CCC TGC 1902 Val Leu Asp Leu Ser Val Gln Glu Ala Leu Ser Gly Thr Pro Cys 590 595 600 CTC CTA GGA CCT GGA CCT GTT CTC ACC GTC CTG GCA CTG CTC CTA 1947 Leu Leu Gly Pro Gly Pro Val Leu Thr Val Leu Ala Leu Leu Leu 605 610 615 GCC TCC ACC CTG GCC TGA 1965 Ala Ser Thr Leu Ala 620 GGGCCCCACT CCCTCCCTCTCT GCTGGGGATC CCCGCCTGGC 2015 CAGGAGCAGG CACGGGTGAT CCCCGTTCCA CCCCAAGAGA ACTCGCGCTC 2065 AGTAAACGGG AACATGCCCC CTGCAGACAC GTAAAAAAAA AAAAAAAAAA 2115 AAAAAAAACT CGAG 2129 No .: 11 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Ser Arg Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu 1 5 10 15 Asp Gly SEQ ID NO: 12 Sequence length: 584 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Glu Thr Gly Gln Glu Ala Ala Pro 1 5 Leu Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser 10 15 20 Pro Arg Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu 25 30 35 Ser Thr Glu Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys 40 45 50 Asn Val Lys Leu Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg 55 60 65 Leu Ser Glu Pro Pro Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu 70 75 80 Leu Leu Phe Leu Asn Pro Asp Ala Phe Ser Gly Pro Gln Ala Cys 85 90 95 Thr Arg Phe Phe Ser Arg Ile Thr Lys Ala Asn Val Asp Leu Leu 100 105 110 Pro Arg Gly Ala Pro Glu Arg Gln Arg Leu Leu Pro Ala Ala Leu 115 120 125 Ala Cys Trp Gly Val Arg Gly Ser Leu Leu Ser Glu Ala Asp Val 130 135 140 Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu Pro Gly Arg Phe Val 145 150 155 Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu Val Ser Cys Pro 160 165 170 Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg Ala Ala Leu 175 180 185 Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp Ser Val 190 195 200 Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly Gln 205 210 215 Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg 220 225 230 Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr 235 240 245 Ile Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys 250 255 260 Pro Ser Gly Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe 265 270 275 Tyr Lys Lys Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu 280 285 290 Ala Thr Gln Met Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu 295 300 305 Gln Leu Asp Val Leu Lys His Lys Leu Asp Glu Leu Tyr Pro Gln 310 315 320 Gly Tyr Pro Glu Ser Val Ile Gln His Leu Gly Tyr Leu Phe Leu 325 330 335 Lys Met Ser Pro Glu Asp Ile Arg Lys Trp Asn Val Thr Ser Leu 340 345 350 Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys Gly His Glu Met 355 360 365 Ser Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg 370 375 380 Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr 385 390 395 Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val 400 405 410 Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr 415 420 425 Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu 430 435 440 Ala Phe Gln Asn Met Asn Gly Ser Glu Tyr Phe Val Lys Ile Gln 445 450 455 Ser Phe Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser 460 465 470 Gln Gln Asn Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg 475 480 485 Thr Asp Ala Val Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu 490 495 500 Leu Gly Pro His Val Glu Gly Leu Lys Ala Glu Glu Arg His Arg 505 510 515 Pro Val Arg Asp Trp Ile Leu Arg Gln Arg Gln Asp Asp Leu Asp 520 525 530 Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile Pro Asn Gly Tyr Leu 535 540 545 Val Leu Asp Leu Ser Val Gln Glu Ala Leu Ser Gly Thr Pro Cys 550 555 560 Leu Leu Gly Pro Gly Pro Val Leu Thr Val Leu Ala Leu Leu Leu 565 570 575 Ala Ser Thr Leu Ala 580 584 SEQ ID NO: 13 Sequence length: 248 Sequence type : Amino acid Topology: Linear Sequence type: Peptide sequence Gly Glu Thr Gly Gln Glu Ala Ala Pro 1 5 Leu Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser 10 15 20 Pro Arg Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu 25 30 35 Ser Thr Glu Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys 40 45 50 Asn Val Lys Leu Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg 55 60 65 Leu Ser Glu Pro Pro Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu 70 75 80 Leu Leu Phe Leu Asn Pro Asp Ala Phe Ser Gly Pro Gln Ala Cys 85 90 95 Thr Arg Phe Phe Ser Arg Ile Thr Lys Ala Asn Val Asp Leu Leu 100 105 110 Pro Arg Gly Ala Pro Glu Arg Gln Arg Leu Leu Pro Ala Ala Leu 115 120 125 Ala Cys Trp Gly Val Arg Gly Ser Leu Leu Ser Glu Ala Asp Val 130 135 140 Arg Ala Leu Gly Gly Leu Ala Cys Asp L eu Pro Gly Arg Phe Val 145 150 155 Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu Val Ser Cys Pro 160 165 170 Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg Ala Ala Leu 175 180 185 Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp Ser Val 190 195 200 Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly Gln 205 210 215 Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg 220 225 230 Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg 235 240 245 248

[Brief description of drawings]

FIG. 1 is a reversed phase H of Step-7 in a third embodiment.
The result of PLC (III) is shown.

FIG. 2 shows the structure of plasmid pKPO27.

FIG. 3 shows the structure of plasmid pRVHKPO27.

FIG. 4 shows the structure of plasmid pRVHKPO27f.

FIG. 5 shows the structure of plasmid pCITE.KPO27.

FIG. 6 shows the structure of plasmid pMBPKPO27.

FIG. 7 shows the structure of plasmid pEFDKPOf.

FIG. 8 is a diagram of Western blot using an antibody prepared by using SDS / PAGE to analyze the culture supernatant expressed in CHO cells and using a synthetic peptide near the N-terminus as an antigen (electrophoresis). Figure) is shown. Lane 1: molecular weight marker, lane 2: sample.

FIG. 9 shows SDS / PA of purified fractions (see Example 29 (i)) of the culture supernatant expressed using CHO cells.
Analyzed by GE, Meg-PO prepared in Example 24
The figure (electrophoretic diagram) of the western blot performed using the antibody with respect to T is shown. Lane 1: molecular weight marker, Lane 2: flow through and wash fractions, Lane 3: 0.1M.
NaCl fraction, lane 4: 0.2M NaCl fraction, lane 5: 0.3M NaCl fraction, lane 6: 0.5M
NaCl fraction, lane 7: 1.0 M NaCl fraction.

FIG. 10 shows SDS of purified fractions (see Example 29 (iii)) of the culture supernatant expressed using CHO cells.
The figure (electrophoresis figure) of the western blot performed after applying to / PAGE is shown. Lane 1: Elution time 42-43
Fraction, Lane 2: Fraction 43-44 min, Lane 3: Fraction 44-45, Lane 4: Fraction 45-46.

[Fig. 11] Fig. 11 shows the positions of the primers of the PCR performed for constructing the modified Meg-POT expression vector.

FIG. 12 shows modified Meg-POT expression vectors pEFNKPOS, pEFNKPOL252V, pE.
3 shows the structure of FNKPOQ220E.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12P 21/02 C 8214-4B // A61K 37/02 ABY 8314-4C C07K 13/00 8318-4H C12Q 1/68 A 7823-4B (C12N 1/21 C12R 1:19) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1:91) (72 ) Inventor Arihiro Hattori 1-135 Komamon, Gotemba City, Shizuoka Prefecture Chugai Pharmaceutical Co., Ltd.

Claims (10)

[Claims] 1. A DNA containing a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 12, a nucleotide sequence partially substituted, deleted or added, or a nucleotide sequence hybridizing to these. 2. A recombinant vector containing the DNA according to claim 1. 3. A prokaryotic or eukaryotic host cell transformed with the recombinant vector according to claim 2. 4. A method for producing a recombinant protein, which comprises culturing the host cell according to claim 3 and separating the produced protein. 5. A method for producing a recombinant protein, wherein the recombinant protein according to claim 4 is a protein having megakaryocyte amplification factor activity. 6. A DNA containing a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 13, a nucleotide sequence partially substituted, deleted or added, or a nucleotide sequence hybridizing to these. 7. A recombinant vector containing the DNA according to claim 6. 8. A prokaryotic or eukaryotic host cell transformed with the recombinant vector according to claim 7. 9. A method for producing a recombinant protein, which comprises culturing the host cell according to claim 8 and separating the produced protein. 10. A method for producing a recombinant protein, wherein the recombinant protein according to claim 9 is a protein having megakaryocyte amplification factor activity.