U.S. patent application number 09/140719 was filed with the patent office on 2001-10-04 for megakaryocyte differentiation factor.
Invention is credited to ISHIDA, NOBUHIRO, IWASA, FUYUKI, KURIHARA, TATSUYA, MIURA, KENJU, NAKAZATO, HIROSHI, TSUJIMOTO, MASAFUMI, TSURUOKA, NOBUO, YAMAGUCHI, NOZOMI, YAMAICHI, KOZO.
Application Number | 20010026931 09/140719 |
Document ID | / |
Family ID | 26408534 |
Filed Date | 2001-10-04 |
United States Patent
Application |
20010026931 |
Kind Code |
A1 |
TSUJIMOTO, MASAFUMI ; et
al. |
October 4, 2001 |
MEGAKARYOCYTE DIFFERENTIATION FACTOR
Abstract
A novel megakaryocyte differentiation factor, for example,
consisting essentially of SEQ ID NO: 30; DNA coding for the
megakaryocyte differentiation factor, an expression vector
comprising the DNA, a host transformed with the expression vector,
and a process for production of the megakaryocyte differentiation
factor using the host. The megakaryocyte differentiation factor
accelerates differentiation of megakaryocytes in the presence of
IL-3, and acts as a thrombopoietin, and therefore an effective
medicament to various diseases involving a decrease in
platelets.
Inventors: |
TSUJIMOTO, MASAFUMI; (OSAKA,
JP) ; IWASA, FUYUKI; (OSAKA, JP) ; TSURUOKA,
NOBUO; (OSAKA, JP) ; NAKAZATO, HIROSHI;
(OSAKA, JP) ; MIURA, KENJU; (OSAKA, JP) ;
ISHIDA, NOBUHIRO; (NAGAOKAKYO-SHI, JP) ; KURIHARA,
TATSUYA; (OSAKA, JP) ; YAMAICHI, KOZO; (OSAKA,
JP) ; YAMAGUCHI, NOZOMI; (KYOTO-SHI, JP) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Family ID: |
26408534 |
Appl. No.: |
09/140719 |
Filed: |
August 26, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09140719 |
Aug 26, 1998 |
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08474661 |
Jun 7, 1995 |
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5874253 |
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08474661 |
Jun 7, 1995 |
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08091028 |
Jul 14, 1993 |
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Current U.S.
Class: |
435/69.1 ;
435/69.3 |
Current CPC
Class: |
A61P 7/00 20180101; A61K
38/00 20130101; C07K 14/475 20130101 |
Class at
Publication: |
435/69.1 ;
435/69.3 |
International
Class: |
C12P 021/06; C12N
015/09 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 17, 1992 |
JP |
4-212305 |
Mar 4, 1993 |
JP |
5-067339 |
Claims
1. A megakaryocyte differentiation factor having the following
properties; (1) stimulating differentiation of megakaryocytes; (2)
exhibiting a molecular weight of 55 to 57 kD as determined by gel
filtration and SDS-polyacrylamide gel electrophoresis (SDS-PAGE),
and having no intermolecular disulfide linkage; (3) exhibiting an
isoelectric point of 6.5.+-.0.5; and (4) having at least one of the
amino acid sequences shown in SEQ ID NO: 1 to 9 in the Sequence
Listing.
2. A megakaryocyte differentiation factor according to claim 1,
produced by human cells.
3. A megakaryocyte differentiation factor according to claim 2,
produced by human cancer cells.
4. A megakaryocyte differentiation factor according to claim 3,
produced by cells derived from human epidermoid carcinoma cell
A431.
5. A megakaryocyte differentiation factor according to claim 4,
produced by cells derived from human epidermoid carcinoma cell
A431, cultured in a protein-free medium.
6. A megakaryocyte differentiation factor having an amino acid
sequence the same as the native amino acid sequence of a
megakaryocyte differentiation factor according to claim 1, an amino
acid sequence wherein one or more than one amino acid residue in
said native amino acid sequence is deleted, an amino acid sequence
wherein one or more than one amino acid residue in said native
amino acid sequence is replaced with other amino acids, an amino
acid sequence wherein one or more than one amino acid is added to
said native amino acid sequence, or an amino acid sequence
including a combination of said amino acid modifications.
7. A megakaryocyte differentiation factor essentially consisting of
the amino acid sequence shown in SEQ ID NO: 30.
8. A megakaryocyte differentiation factor having an amino acid
sequence wherein one or more than one amino acid residue in the
amino acid sequence shown in SEQ ID NO: 30 is deleted, an amino
acid sequence wherein one or more than one amino acid residue in
the amino acid sequence shown in SEQ ID NO: 30 is replaced with
other amino acids, or an amino acid sequence wherein one or more
than one amino acid is added to the amino acid sequence shown in
SEQ ID NO: 30 or an amino acid sequence including a combination of
said amino acid modification.
9. A megakaryocyte differentiation factor according to claim 6,
which is glycosylated.
10. A megakaryocyte differentiation factor according to claim 7,
which is glycosylated.
11. A megakaryocyte differentiation factor according to claim 8,
which is glycosylated.
12. A megakaryocyte differentiation factor obtainable from a
culture of transformed cells constructed by a gene recombination
technique using a polynucleotide hybridizable with a polynucleotide
coding for at least one of the amino acid sequences shown in SEQ ID
NO: 1 to 9.
13. A megakaryocyte differentiation factor according to claim 6,
wherein the N-terminus of the factor is biochemically or chemically
modified, preferably the first methionine is deleted and the second
alanine is acethylated.
14. A megakaryocyte differentiation factor according to claim 8,
wherein the N-terminus of the factor is biochemically or chemically
modified, preferably the first methionine is deleted and the second
alanine is acethylated.
15. A megakaryocyte differentiation factor according to claim 12,
wherein the N-terminus of the factor is biochemically or chemically
modified, preferably the first methionine is deleted and the second
alanine is acethylated.
16. An isolated DNA coding for a megakaryocyte differentiation
factor according to claim 1.
17. An isolated DNA coding for a megakaryocyte differentiation
factor according to claim 6.
18. An expression vector comprising a DNA according to claim
16.
19. An expression vector comprising a DNA according to claim
17.
20. A host transformed with an expression vector according to claim
18.
21. A host transformed with an expression vector according to claim
19.
22. A pharmaceutical composition comprising a megakaryocyte
differentiation factor according to claim 1 and a pharmaceutically
acceptable carrier.
23. A pharmaceutical composition comprising a megakaryocyte
differentiation factor according to claim 6 and a pharmaceutically
acceptable carrier.
24. A pharmaceutical composition according to claim 22, for
treating thrombo cytopenia.
25. A pharmaceutical composition according to claim 23, for
treating thrombo cytopenia.
26. An antibody to megakaryocyte differentiation factor according
to claim 1.
27. An antibody to megakaryocyte differentiation factor according
to claim 6.
28. A process for production of a megakaryocyte differentiation
factor according to claim 1, comprising the steps of culturing a
host according to claim 20, and recovering the megakaryocyte
differentiation factor.
29. A process according to claim 28, wherein the host is silkworm
Bombyx mori.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a megakaryocyte
differentiation factor, gene coding for the factor and a process
for production thereof. The megakaryocyte differentiation factor is
useful as a hemopoietic stimulating factor for
megakaryocyte-platelet linease.
[0003] 2. Description of Related Art
[0004] It is well known that various hemopoietic factors inducing
the growth and differentiation of blood cells are involved in a
process from hemopoietic stem cells to mature blood cells.
[0005] Although the life time of platelet is as short as 9 to 10
days, concentration of platelets in the blood is maintained rather
constant during the stationary state. Moreover, when the number of
platelets is artificially reduced by one of various available
method in an experimental animals, the number of the platelets
recovers in the blood in a few days. From these facts, it is
supposed that factors which stimulate formation of platelets are
present, and so far a great effort has been made to identifycation
of the factors.
[0006] It is considered that at least two regulatory factors are
involved in the megakaryocyte-platelet hemopoietic lineage. The
first factor by itself stimulates formation of megakaryocyte
colonies and is called a megakaryocyte colony stimulating factor.
The second factor by itself does not have an activity to stimulate
formation of megakaryocyte colonies, but in combination with the
first factor, increases the number of megakaryocyte colonies and
stimulates the differentiation thereof, and is called a
megakaryocyte potentiator.
[0007] The former includes interleukin 3, and
granulocyte/macrophage colony stimulating factor, and the latter
includes erythropoietin, macrophage colony stimulating factor,
interleukin 6, 7 and 11, LIF, and the like. Some of these factors
actually exhibit in vivo effect of increasing the number of
platelets or shortening the time required to recover the number of
platelets (Hideaki Mizoguchi: Tanpakushitsu Kakusan Koso 36, 1195,
1991 in Japanese).
[0008] However, most of these factors exhibit a diversity of
biological activities other than participation in differentiation
of blood cells in various hemopoietic lineages including
differentiation in megakaryocyte-platelet lineages. For example,
although IL-6 and IL-11 actually exhibit in vivo thrombopoietic
action, they stimulate production of acute phase protein, and in
severe cases, cause cachexia. Moreover, IL-6 is accompanied with
various clinical problems; for example, it is possible for IL-6 to
stimulate the growth of mesangium cells in the kidney resulting in
renal failure (Tadashi Matsuda et al., Tanpakushitsu Kakusan Koso,
36, 1184, 1991 in Japanese). In addition, since IL-6 does not
exhibit a high blood level during a thrombcytopenic phase, it is
not considered as a physiological factor.
[0009] Platelets play an important role in a hemostatic mechanism.
Diseases involving decrease of platelets (Fanconi's syndrome,
megakaryocytic thrombocytopenia, aplastic anemia, and the like) are
clinically dangerous, and in particular hemorrhaging cannot be
controlled. Therefore, it is considered that isolation and
identification of a factor which stimulates production of platelets
is useful to prevent the above-mentioned danger.
[0010] Currently, bone marrow transplantation is becoming a
powerful therapeutic means for treating leukenia etc., and the
ratio of successful cases is increasing through the use of
cytokines such as erythropoietin (EPO), granulocyte colony
stimulating factor (G-CSF) etc. At present a problem in the bone
marrow transplantation is a decrease in the number of platelets,
and if a thrombopoietic factor is available, it is expected that
the ratio of successful cases will increase and a period of
hospitalization will be shortened. Not only hemopoietic diseases
but also thrombocytopenia in chemotherapy and radio isotopic
therapy of cancers may be controlled by thrombopoietin.
[0011] The present inventors, considering the various
above-mentioned difficulties with known factors, carried out
various research to find a factor which stimulates production of
platelets and is effective for treatment of patients having
thrombocytopenia or insufficient platelet function, and as a
result, the present inventors found a novel factor which stimulates
differentiation of megakaryocytes, cloned a gene coding for said
factor, constructed an expression vector, and succeeded in
expressing the gene to produce said factor.
SUMMARY OF THE INVENTION
[0012] Accordingly, the present invention provides a megakaryocyte
differentiation factor having the following properties;
[0013] (1) stimulating differentiation of megakaryocytes;
[0014] (2) exhibiting a molecular weight of 55 to 57 kD as
determined by gel filtration and SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), and having no intermolecular disulfide
linkage;
[0015] (3) exhibiting an isoelectric point of 6.5.+-.0.5; and
[0016] (4) having at least one of the amino acid sequences shown in
SEQ ID NO: 1 to 9 in the Sequence Listing.
[0017] The present invention also provides a gene coding for the
megakaryocyte differentiation factor.
[0018] The present invention further provides an expression vector
comprising the gene coding for the megakaryocyte differentiation
factor.
[0019] The present invention moreover provides a host transformed
with the expression vector.
[0020] The present invention still further provides a process for
production of the megakaryocyte differentiation factor using said
host.
BRIEF EXPLANATION OF DRAWINGS
[0021] FIG. 1 shows an elution profile of protein (A280
--.circle-solid.--) and a megakaryocyte differentiation factor
(acetylcholine esterase activity --.smallcircle.--) from a
Q-Sepharose column, wherein the elution was carried out by NaCl
concentration gradient (0 to 1.0M) to obtain fractions 1 to
120.
[0022] FIG. 2 shows an elution profile of protein (A280
--.circle-solid.--) and a megakaryocyte differentiation factor
(acetylcholine esterase activity --.smallcircle.--) from a
Phenyl-Sepharose column, wherein the elution was carried out by
ammonium sulfate concentration gradient (30 to 0%) and
ethyleneglycol concentration gradient (0 to 50%) to obtain
fractions Nos. 1 to 100, followed by 50% ethyleneglycol to obtain
fraction Nos. 101 to 120.
[0023] FIG. 3 shows an elution profile of protein (A280
--.circle-solid.--) and a megakaryocyte differentiation factor
(acetylcholine esterase activity --.smallcircle.--) from an
S-Sepharose column, wherein the elution was carried out by NaCl
concentration gradient (0 to 0.5M) to obtain fractions Nos. 1 to
100.
[0024] FIG. 4 shows an elution profile of protein (A280 --) and
megakaryocyte differentiation factor (acetylcholine esterase
activity --.smallcircle.--) from a Hiroad 26/60 Superdex 75 Pg
column, and a result of analysis of the resulting fractions by
SDS-PAGE (lower part of the Figure).
[0025] FIG. 5 is an electrophoretic pattern showing a result of
SDS-PAGE analysis of the purified megakaryocyte differentiation
factor.
[0026] FIG. 6 shows a result of an iselectroric focusing of the
purified megakaryocyte differentiation factor.
[0027] FIG. 7 is an electrophoretic pattern showing a result of
sugar chain analysis for the purified megakaryocyte differentiation
factor by SDS-PAGE, wherein the column 1 shows a result for a
non-treated megakaryocyte differentiation factor, and the column 2
shows a result for an endoglycosidase F-treated megakaryocyte
differentiation factor (note, the band near the 35 kD position is
derived from the enzyme preparation).
[0028] FIG. 8 is a graph comparing acetylcholine esterase activity
of megakaryocytes derived from mouse bone marrow cells cultured for
5 days in the presence or absence of purified megakaryocyte
differentiation factor (55 kDa protein) and with or without
addition of IL-6.
[0029] FIG. 9 shows a result of acetylcholine esterase staining of
mouse bone marrow cells cultured for 4 days after the addition of
IL-3, in the presence (B) or absence (A) of purified megakaryocyte
differentiation factor.
[0030] FIG. 10 shows a result of May-Gruenwald-Giemsa's staining of
mouse bone marrow cells cultured for 4 days after the addition of
IL-3, in the presence (B) or absence (A) of purified megacaryocyte
differentiation factor.
[0031] FIG. 11 shows detection of RNA with a DNA probe (PCR product
amplified with KY100 and N1065 described in Example 2.1) wherein
the RNA was extracted from cultured Bombyx moli cells infected with
recombinant virus (TPO55-Bm NPV) comprising a gene coding for
megakaryocyte differentiation factor (Lane B), cells infected with
wild type virus (B6E) (Lane A) or from non-transformed cells (Lane
C).
[0032] FIG. 12 is a graph showing an expression of megakaryocyte
differentiation factor (TP55) in hemolymph of Bombyx mori after
reparation by Matrex Blue A column chromatography.
DETAILED DESCRIPTION
[0033] As starting materials for isolating the present
megakaryocyte differentiation factor, human cells, such as human
cancer cells, preferably human epidermoid carcinoma cells A431,
particularly preferably human epidermoid carcinoma A431 grown in a
protein-free medium can be mentioned.
[0034] In addition to the above-defined megakaryocyte
differentiation factor, the present invention relates megakaryocyte
differentiation factors obtainable from transformants such as cells
or animals constructed by gene technology and having the same amino
acid sequence as the above-defined megakaryocyte differentiation
factor, an amino acid sequence wherein a portion of the
above-defined megakaryocyte differentiation factor is deleted, an
amino acid sequence wherein a portion of the above-defined
megakaryocyte differentiation factor is replaced with other amino
acid or amino acid sequence, or an amino acid sequence wherein one
or more than one amino acid sequence is added to the above-defined
megakaryocyte differentiation factor, or having an amino acid
sequence including a combination of said modifications.
[0035] Moreover, the present invention relates to megakaryocyte
differentiation factor having the amino acid sequence shown in SEQ
ID NO: 30, an amino acid sequence wherein a portion of the amino
acid sequence shown in SEQ ID NO: 30 is deleted, an amino acid
sequence wherein a portion of the amino acid sequence shown in SEQ
ID NO: 30 is replaced with an other amino acid or amino acid
sequence, or an amino acid sequence wherein one or more than one
amino acid sequence is added to the amino acid sequence shown in
SEQ ID NO: 30, or having an amino acid sequence including a
combination of said modifications.
[0036] The present invention also relates to genes coding for the
above-mentioned megakaryocyte differentiation factors. The present
invention further relates a process for production of the
megakaryocyte differentiation factors using the gene by means of
gene recombination technology. The gene recombination technology
follows conventional procedures by using a synthetic or natural
polynucleotide coding for the amino acid sequence of the native
megakaryocyte differentiation factor, an amino acid sequence
wherein a portion of the native amino acid sequence is deleted, an
amino acid sequence wherein a portion of the native amino acid
sequence is replaced with other amino acid or amino acid sequence,
or an amino acid sequence wherein one or more than one amino acid
is added to the native amino acid sequence, or coding for an amino
acid sequence including a combination of said modifications, but
not limited to the above.
[0037] The above-mentioned various modification can be carried out
by a conventional technique such as site-specific a
mutagenesis.
[0038] The number of amino acids involved in the modification such
as addition, deletion or replacement is not limited, but as for
addition the number of amino acid depends on the number of amino
acids, for example, that of the functional peptide used in a hybrid
protein with the megakaryocyte differentiation factor of the
present invention or that of a signal peptide added to the present
factor, namely depends on purpose of the modification, as for
deletion the number of amino acids may be designed or determined so
as to maintain megakaryocyte differentiation activity and it is,
for example, 1 to 30, preferably 1 to 20 or it can be that of
region other than the active region of the present factor, and as
for replacement the number of amino acids also may be designed or
determined so as to maintain megakaryocyte differentiation activity
and it is, for example, 1 to 10, preferably, 1 to 5.
[0039] An addition or improvement of an signal sequence, choice of
host-vector system, and improvement of expression regulatory region
may provide efficient expression. In addition, a host may be chosen
to provide a glycosylated product. Moreover, a polynucleotide
coding for at least one of the amino acid sequences shown in SEQ ID
NO: 1 to 9 may be used as a DNA probe for cloning a gene.
[0040] The present invention further provides a pharmaceutical
composition comprising a megakaryocyte differentiation factor as an
effective ingredient. The pharmaceutical composition is preferably
used as a medicament for thrombocytopenia.
[0041] In addition, the present megakaryocyte differentiation
factors can be used to obtain specific antibodies according to a
known procedure.
[0042] Now, the present invention is explained in more detail.
[0043] (1) Starting Material
[0044] As a starting material for obtaining a novel protein of the
present invention, a culture supernatant of cells derived from
human epidermoid carcinoma cell A431 (ATCC CRL 1555), rendered to
be capable of growing in a protein-free medium according to
Yamaguchi et al. method (Yamaguchi N. et al., Cancer Res. 50, 7008,
1991), is mentioned. This cell line was designated as Human
epidermoid carcinoma SBM 330, and deposited as FERM BP-3911 with
the Fermentation Research Institute Agency of Industrial Science
and Technology, 1-3, Higashi 1-chome, Tsukuba-shi Ibraki-ken (307)
Japan, on Jul. 1, 1992.
[0045] (2) Assay Method for Megakaryocyte Differentiation
Factor
[0046] To assay a megakaryocyte differentiation factor,
megakaryocyte-series cell lines (for example, CMK cells or cells
derived therefrom), or mouse bone marrow cells may be used. For
example, activity of acetylcholine esterase which is known to be
specifically detected in murine megakaryocytes is carried out using
mouse bone marrow cells according to the Ishibashi et al. method
(Ishibashi, T. et al., Proc. Natl. Acad. Sci. USA 86, 5953, 1989).
In addition, histochemical detection of megakaryocytes is carried
out by subjecting cultured bone marrow cells to acetylcholine
esterase staining and May-Gruenwald-Gremsa's staining and the
observing the shape of the stained cells.
[0047] (3) Purification of Megakaryocyte Differentiation Factor
[0048] The megakaryocyte differentiation factor can be purified,
for example, starting from a culture supernatant of A431 cells
cultured in a protein-free medium, concentration by
ultrafiltration, and column chromatography using, for example,
Matrex Blue A (Amicon), Q-Sepharose (Pharmacia), phenyl-Sepharose
(Pharmacia), S-Sepharose (Pharmacia) and Hiload 26/60 Superdex 75
(Pharmacia) alone or in combination. Protein is monitored by
measuring A280 nm.
[0049] (4) Determination of Partial Amino Acid Sequence of
Megakaryocyte Differentiation Factor
[0050] To determine an amino acid sequence, the megakaryocyte
differentiation factor purified in the section (3) is digested into
fragments with a protease such as Achromobacter Protease I (API) at
37.degree. C. for 2 hours. The resulting peptide fragments are
separated and recovered by reverse phase HPLC (acetonitrile
gradient in 0.1% trifluoroacetic acid) using YMC-Pack AM-303
column. The peptide fragments thus obtained are subjected to a
sequencer such as a gas-phase sequencer obtained from, for example,
Applied Biosystem. A definite purification process and detailed
properties of the megakaryocyte differentiation factor are
described in Example 1.
[0051] The present invention also provides gene coding for
megakaryocyte differentiation factor. The gene may be cDNA prepared
from mRNA, genomic DNA, and synthetic DNA. For example, cDNA can be
cloned by a polymerase chain reaction (PCR) using DNA (nucleotide)
primers designed on the basis of partial amino acid sequences as
shown in Example 1 of a megakaryocyte differentiation factor
purified from the above-mentioned human cells, such as human
epidermoid carcinoma cells, for example A431 cells. The cloning is
described in detail in Examples 1 and 2.
[0052] The gene of the present invention further includes DNAs
coding for protein or glycoprotein having megakaryocyte
differentiation activity and hybridizing with the nucleotide
sequence of SEQ ID NO: 30. Nucleotide sequence of DNA cloned in
Example 2 and an amino acid sequence predicted from the nucleotide
sequence are shown in SEQ ID NO: 30.
[0053] Thus once an amino acid sequence is determined, various
mutated megakaryocyte differentiation factors, such as a
polypeptide, wherein one or more than one amino acid is added to
the native amino acid sequence or the amino acid sequence shown in
SEQ ID NO: 30 and maintaining megakaryocyte differentiation factor
activity, a polypeptide wherein one or more than one amino acid is
deleted from the native amino acid sequence or the amino acid
sequence shown in SEQ ID NO: 30 and maintaining megakaryocyte
differentiation factor activity, a polypeptide wherein one ore more
than one amino acid is replaced with one or more than one other
amino acid, in the native amino acid sequecne or the amino acid
sequence shown in SEQ ID NO: 30, and maintaining megakaryocyte
differentiation factor activity, or a polypeptide including a
combination of the above-mentioned modifications such as addition,
deletion and/or replacement of amino acids maintaining
megakaryocyte differentiation factor activity can be designed and
produced.
[0054] According to the present invention, although the nucleotide
sequence shown in SEQ ID NO: 30 is disclosed, gene coding for the
present megakaryocyte differentiation factor is not limited
thereto. Once an amino acid sequence of the present native
megakaryocyte differentiation factor or an amino acid sequence of a
mutated megakaryocyte differentiation factor is determined,
according to the degeneracy of genetic code, various nucleotide
sequences coding for the same amino acid sequence can be designed
and prepared. In this case, codons used with a high frequency in a
chosen host are preferably used.
[0055] A gene coding for the present megakaryocyte differentiation
factor can be obtained as cDNA according to Example 2, but the gene
is not limited to cDNA. Namely, once a nucleotide sequence coding
for an amino acid sequence of native megakaryocyte differentiation
factor is determined, a gene coding for the native megakaryocyte
differentiation factor can be cloned as a cDNA according to a
strategy different from the strategy definitely described herein,
and moreover a desired gene can be cloned from the genome of a cell
producing the megakaryocyte differentiation factor.
[0056] Where a desired gene is cloned from the genome, various
primer nucleotides or probe nucleotides used in Example 2 can be
used as probes for screening genomic DNA fragments. Moreover, other
probes designed on the basis of the nucleotide sequence described
in SEQ ID NO: 30 can be used. General procedures for cloning a
desired DNA from a genome are well known in the art (see Current
Protocols In Molecular Biology, John Wiley & Sons, Chapters 5
and 6).
[0057] Gene coding for the native megakaryocyte differentiation
factor of the present invention can also be prepared by chemical
synthesis. It is easy in the art to chemically synthesize DNA using
an automated DNA synthesizer, for example, Applied Biosystems 396
DNA/RNA synthesizer. Accordingly a person with ordinary skill in
the art can easily synthesize DNA having the nucleotide sequence
shown in SEQ ID NO: 30.
[0058] A gene coding for the present native megakaryocyte
differentiation factor using codons different from native codons,
and a gene coding for a mutated megacaryocyte differentiation
factor can be prepared by chemical synthesis as described above.
Alternatively they can be obtained by site-specific mutagenesis
using as a templete a DNA or RNA having a nucleotide sequence shown
in SEQ ID NO: 30 together with mutagenic primers (for example, see,
Current Protocols In Molecular Biology, John Wiley & Sons,
Chapter 8).
[0059] Once a gene coding for the present megakaryocyte
differentiation factor is obtained, the gene can be used to produce
a recombinant megakaryocyte differentiation factor according to a
conventional gene recombination technology. Namely, a DNA coding
for the present megakaryocyte differentiation factor is inserted
into an appropriate expression vector, the vector is introduced to
appropriate host cells, the transformed host cells are cultured,
and the target megakaryocyte differentiation factor is recovered
from the culture (cells or medium). The present megakaryocyte
differentiation factor can be biochemically or chemically modified,
for example, N-terminal acylated.
[0060] In addition, on the basis of the nucleotide sequence shown
in SEQ ID NO: 30, a protein data base was searched with fasta
Program (GCG package). As a result, the megakaryocyte
differentiation factor belongs to the super family of serine
protease inhibitor. On the other hand, in human leucocyte elastase
inhibitor, chicken ovalbumin Y gene product, human plasminagen
activator inhibitor 2 and human squamous cell carcinoma antigen,
which are similar to the present megakaryocyte differentiation
factor in their expected steric structure, and distribution of
hydrophobic and hydrophilic amino acids, the N-terminal portion is
not cleaved and forms a signal peptide. Accordingly, there is a
possibility that in the present megakaryocyte differentiation
factor, the N-terminal portion may function as a signal peptide and
the megakaryocyte differentiation factor may be secreted without
cleavage of the signal peptide. Further, the present megakaryocyte
differentiation factor may be modified so that the first methionine
is deleted and the second alanine is acetylated.
[0061] As hosts, both the prokaryote and enkaryote can be used. As
prokaryote, bacteria such as Escherichia coli the genus Bacillus,
for example, B. subtilis and the like can be used. As eukaryote,
yeast such as the genus Saccharomyces, for example, S. serevisiae,
insect cells such as Spodoptera frugiperda cells, Cabbage looper
cells, Bombyx mori cells, animal cells such as human cells, monkey
cells, mouse cells and the like can be used. Moreover, insects per
se, such as Bombyx mori may be used.
[0062] As expression vectors, plasmid, phage, phagemid, virus such
as bacuro virus, vaccinia virus or the like can be used. A promoter
in an expression vector is selected depending on host used. For
example, lac promoter, trp promoter and the like can be used as
bacterial promoters, and adhl promoter, pqk promoter and the like
can be used as yeast promoters. On the other hand, baccuro virus
polyhedrin promoter can be used as insect promoter, and Simian
virus 40 early or late promoter can be used for animal cells.
[0063] Transformation of a host with an expression vector can be
carried out according to conventional procedures well known in the
art, and these procedures are described in, for example, Current
Protocols in Molecular Biology, John Wiley & Sons. Culturing of
a transformant also can be carried out according to a conventional
procedure.
[0064] Purification of a megakaryocyte differentiation factor from
a culture or an insect body can be carried out according to
conventional procedures used for isolation and purification of a
protein, for example, ultrafiltration, various types of column
chromatography such as Q-Sepharose column chromatography and the
like.
EXAMPLES
[0065] Next, the present invention is further explained by
Examples.
Example 1
[0066] Purification of megakaryocyte differentiation factor
[0067] (1) Culturing of A431 Cells
[0068] Frozen SBM 330 cells conditioned in a protein-free medium
from A431 cells were thawed, and cultured in a primary medium
(Ham's F12 medium containing 10% fetal bovine serum). Namely, the
cells were plated in 10 T flasks having a culture area of 150
cm.sup.2, and cultured to confluence at 37.degree. C. in the
presence of 5% CO.sub.2. Next, the cells were peeled off with a
0.25% trypsin solution (Chiba Kessei) and subcultured in 10 roller
bottles having a culture area of 850 cm.sup.2 at 37.degree. C. and
0.5 rpm for about 3 days to recover 1.8.times.10.sup.9 cells. The
cultured cells were attached to a ceramic core (S-451) of an
Opti-cell incubator (Charles River Inc. Wilmington, Mass.) and
perfusion culturing was started using 10L of a primary medium.
[0069] The perfusion culture was carried out at 37.degree. C. with
supplying oxygen at 150 mmHg. The primary medium was replaced with
a protein-free medium as follow. Namely, after culturing for about
7 days in a primary medium, a protein-free medium was supplied to
the culture at a rate of 20L/day, and simultaneously the culture
supernatant was recovered from the culture at the same rate. As a
result, the primary medium containing serum was substantially
completely replaced with the protein-free medium by supplying about
100 L of the protein-free medium. Thereafter, cell culture
supernatant was continuously recovered to obtain 1000 L of cell
culture supernatant. A part (about 300 L) of the cell culture
supernatant thus obtained was concentrated to 2 L using an
ultrafiltration membrane (Milipore, Bedford, Mass.; MW 10,000 cut)
and the concentrate was dialyzed against 20 mM Tris/HCl buffer (pH
7.4).
[0070] (2) Assay of Megakaryocyte Differentiation Factor Using
Mouse Bone Marrow Cells
[0071] Myeloid cells were pushed out of the femur of female
BDF.sub.1 mouse and suspended in an .alpha.-MEM medium (Flow
Laboratories, Inc. McLean, Va., USA). Percoll layers having
different densities (Pharmacia LKB Biotechnology, Tokyo) were
overlaied, and the bone marrow cell suspension was put thereon,
followed by centrifugation at 400.times. g for 20 minutes.
Mononuclear cells collected at the interface of a layer having a
density of 1.07 g/ml and a layer having a density of 1.08 g/ml were
recovered and washed once with .alpha.-MEM containing 10% FBS, and
resuspended in the same medium containing 0.5 mM
diisopropylfluorophospha- te. The suspension was then put into a
plastic cell culture dish (Corning, N.Y., USA) and cultured at
37.degree. C. in 5% carbon dioxide and 95% air for 2 hours. During
the culturing, at one hour from the start of culturing, the cell
culture dish was replaced with a new one. After the culturing,
cells were washed with 10% FBS/.alpha.-MEM three times.
[0072] The non-adherent bone marrow mononuclear cells thus obtained
were suspended in 10% FBS/1% BSA/0.1 mM
2-mercaptoethanol/.alpha.-MEM, and plated in a 96-well microplate
(Corning) in an amount of 5.times.10.sup.4 cell/well. If necessary,
to a test sample were added 25 U/ml mouse recombinant IL-3 (Genzyme
Corporation, Cambridge, Mass., USA) and 1 to 2 .mu.g/ml anti-IL-6
antibody (Boehringer Mannheim, Mannheim, FRG). Where anti-IL-6
antibody was added, the test sample and the antibody were
preincubated at 37.degree. C. for an hour prior to seeding the
cells.
[0073] The culturing was carried out at 37.degree. C. in 5%
CO.sub.2 - 5% O.sub.2 - 90% N.sub.2 for 4 to 5 days. After
culturing cells in each well of the microplate they were washed
twice with PBS, and lysed with 180 .mu.l of 0.2% (w/v) Triton
X-100, 1 mM EDTA, 0.12M NaCl, 50 mM HEPES (pH 7.5), and 20 .mu.l of
a substrate, 5.6 mM acetythiolcholine iodide, was added thereon.
After culturing with shaking at a room temperature for an hour, 20
.mu.l of the solution was transferred to a microplate for
fluorescent assay (Dynatech Micro FLUOR "B" Plate).
[0074] To the microplate were added 20 .mu.l of 0.4 mM
7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarine in
acetonitrile and 160 .mu.l of 0.2% (w/v) Triton X-100, 1 mM EDTA,
50 mM sodium acetate (pH 5.0), and fluorescent emission was
measured by a fluorometer (excitation 365 nm, emission 450 nm).
[0075] For acetylcholine esterase staining, the cells were
centrifuged by a Cytospin to adhere the cells onto a slide glass,
and fixed with 5% glutaraldehyde, 10 mM phosphate buffer (pH 6.7)
for 15 minutes, and as a substrate acetylthiocholine was used
according to Mizoguchi's method (Method for Culturing Hemopoietic
Stem Cells, Chugai Igaku, 1986, ed. Y. Miura, pp82-88). Namely,
after fixing the cells, the slide glass was washed with 0.1M
phosphate buffer (pH 6.0), and to each slide glass was layered a
mixture of 1.5 ml of 0.67 mg/ml acetylthiocholine iodide, 0.1M
phosphate buffer (pH 6.0), 0.2 ml of 30 mM CUSO.sub.4, 0.2 ml of 5
mM potassium ferricyanide and 0.1 ml of 0.1M sodium citrate. The
slide glass was incubated at a room temperature for 4 hours, and
washed with water. May-Gruenwald-Giemsa's staining is well known in
hematology and was carried out using reagents available from E.
Merck (Darmstadt, FRG) wherein May-Gruenwald's staining was carried
out for 4 minutes and Giemsa's staining was carried out for 10
minutes.
[0076] (3) Purification of Megakaryocyte Differentiation Factor
[0077] A culture supernatant concentrate of A431 cells was dialyzed
and centrifuged to obtain a supernatant which was then applied to a
Matrex Blue A Column equilibrated with 20 mM Tris/HCl buffer (pH
7.4), and after washing the column with the same buffer, a bound
fraction was eluted with the same buffer containing 2M NaCl.
Megakaryocyte differentiation activity detected by the
above-mentioned method was found in the bound fraction. Therefore,
the bound fraction was dialyzed against 20 mM Tris/HCl buffer (pH
7.4) and applied to a Q-Sepharose column equilibrated with the same
buffer. The column was thoroughly washed and a megakaryocyte
differentiation factor was eluted by NaCl gradient (see FIG. 1).
This factor was eluted near the position of 0.3 to 0.5M NaCl.
[0078] The active fraction obtained from Q-Sepharose was made
ammonium sulfate 30% saturation, and was applied to a phenyl
Sepharose column equilibrated with 20 mM Tris/HCl buffer (pH 7.4)
containing 30% saturation of ammonium sulfate. The megakaryocyte
differentiation factor was eluted by simultaneously forming
concentration gradients of ammonium sulfate (30% to 0%) and
ethyleneglycol (0 to 50%) (see FIG. 2). The megakaryocyte
differentiation factor measured in the presence of anti-IL-6
antibody was observed over several fractions in the beginning of
the concentration gradient formation.
[0079] The fractions thus obtained were combined, thoroughly
dialyzed against 50 mM MES/NaOH buffer (pH 6.0), and applied to an
S-Sepharose column equilibrated with the same buffer. A bound
fraction was eluted by a 0 to 0.5M NaCl concentration gradient (see
FIG. 3). Although activity was widely distributed, relatively high
activity was found in the beginning of the concentration gradient
formation. The fractions obtained from S-Sepharose were applied to
a Hilord 26/60 Super Dex 75 (Pharmacia) column for gel filtration.
The column used had be previously equilibrated with the same
buffer, and elution was carried out with the same buffer (see FIG.
4). Activity of megakaryocyte differentiation activity was eluted
near the position corresponding to a molecular weight of 55 to 57
kDa.
[0080] According to the above-mentioned steps, about 80 .mu.g of a
fraction showing two bands near 55 to 57 kDa as analyzed by
SDS-PAGE was obtained from 300L of a culture supernatant of A431
derived cells (see FIG. 5). The two bands were correlated with the
activity (FIG. 1). Accordingly, it was concluded that the two bands
observed in said fraction correspond to a desired megakaryocyte
differentiation factor.
[0081] (4) Properties of Megakaryocyte Differentiation Factor
[0082] The present megakaryocyte differentiation factor has the
following properties.
[0083] 1) Molecular weight: about 55 kDa (gel filtration and
SDS-PAGE) (FIGS. 4 and 5)
[0084] The present factor exhibits two bands in SDS-PAGE, and there
is no difference in mobility between reducing condition and a
non-reducing condition. Therefore the factor does not have
intermolecular disulfide linkage.
[0085] 2) Isoelectric point: 6.5.+-.0.5 (FIG. 6)
[0086] A several bands are observed in the above-mentioned
range.
[0087] 3) The above-mentioned heterogeneity of the present factor
can be explained as heterogeneity in a sugar chain structure of
glycoprotein. Namely, where the present factor is treated with
endoglycosidase F which is an asparagine linked sugar removing
enzyme, the molecular weight of the present factor decreased to a
molecular weight of about 40 kDa, and the heterogeneity also
decreased (FIG. 7), in SDS-PAGE. In addition, where a fraction
exhibiting a single band and a fraction exhibiting two bands were
digested with API and a peptide map was prepared by fractionation
by a reversed phase HPLC, the difference was not observed between
the two fractions.
[0088] 4) The present factor contains at least one of the amino
acid sequences shown in SEQ ID NO: 1 to 9.
[0089] 5) Biological activity
[0090] Where mouse bone marrow cells were cultured in the presence
of a purified megakaryocyte differentiation factor and IL-3, the
proliferation and differentiation of megakaryocytes are observed
(FIGS. 8, 9 and 10). FIG. 8 shows the result of measurement for
acetylcholine esterase activity of megakaryocytes; FIG. 9 shows the
result of acetylcholine esterase staining of the cultured cells
(.times.20); and FIG. 10 shows the result of May-Gruenwald-Giemsa's
staining of the cultured cells (.times.100). In both the FIGS. 9
and 10, it is seen that megakaryocytes increased in the presence of
a megakaryocyte differentiation factor (B) in comparison with in
the absence of the same (A).
[0091] (5) Structure of megakaryocyte differentiation factor
[0092] To characterize the structure of a purified megakaryocyte
differentiation factor, the factor was digested with API and the
structures of the resulting fragments were determined. After the
digestion of the factor with API, each fragment was recovered by
reversed phase HPLC, and the structures for appropriate fractions
were determined. As a result, the peptide fragments had amino acid
sequences shown in SEQ ID NO: 1 to 9.
Example 2
[0093] Determination of Structure of cDNA for Megakaryocyte
Differentiation Factor
[0094] 1. Analysis of cDNA nucleotide sequence of megakaryocyte
differentiation factor by PCR (1)
[0095] Oligonucleotides NI065 (SEQ ID NO: 10; corresponding to
449-486 of SEQ ID NO: 30) and NI067 (SEQ ID NO: 11; corresponding
to 1049-1080 of SEQ ID NO: 30) were synthesized by designing
nucleotide sequences on the basis of the amino acid sequences shown
in SEQ ID NO: 3 and 4 respectively.
[0096] Total RNA was purified from A431 cells using ISOGEN (Wako
Pure Chemical) according to the manufacturer's instructions. RNA
having poly A was purified from the total RNA, and a reaction was
carried out using a 3'-RACE Kit (Gibco BRL). Namely, the
above-mentioned oligomer N1065 and oligomer 3'-RACE adaptor primer
(SEQ ID NO: 12) attached to the 3'-RACE Kit (Gibco BRL) were used
to carry out polymerase chain reaction (PCR) according to
instructions included with the kit.
[0097] The reaction product was then subjected to second PCR using
the primer NI067 and the oligomer 3'-RACE adapter primer included
in the 3'-RACE Kit (Gibco BRL) to obtain a DNA fragment of about
900 base pairs. Next, using a direct nucleotide sequence
determination method for a PCR product, according to U. Gyllensten
et. al., Proc. Natl. Acad. Sci. USA 85: 7652 (1988), the DNA
fragment of about 900 base pairs was directly used as a reaction
substrate to determine a nucleotide sequence of a portion
representing protein and a portion downstream of the protein
portion using a Taqu Dye Deoxy Terminator Cycle Sequencing kit
available from Applied Biosystem and a fluorescent nucleotide
sequencer (Applied Biosystem, Type 370A) according to a
manufacturers instruction. As a result, a sequence from nucleotide
number 1081 to 1950 of SEQ ID NO: 30 was shown.
[0098] On the basis of this sequence, oligomer KY100 (SEQ ID NO:
13; corresponding to 1255-1236 of SEQ ID NO: 30) was synthesized.
The reaction product obtained by the PCR using NI065 and the
oligomer 3'-RACE adapter primer attached to the 3'-RACE Kit (Gibco
BRL) was used as a reaction substrate to carry out a further PCR
using NI065 and KY100. As a result, a DNA fragment of 807 base
paires was obtained.
[0099] This DNA fragment of 807 base paires was directly used as a
reaction substrate to determine its nucleotide sequence using Taq
Dye Deoxy Terminator Cycle Sequencing kit available from Applied
Biosystem and a fluorescent nucleotide sequencer according to a
manufactures instruction. As a result, a nucleotide sequence from
nucleotide number 487 to 1080 of SEQ ID NO: 30 was shown. On the
basis of this sequence, oligomers NI073 (SEQ ID NO: 14;
corresponding to 864-886 of SEQ ID NO: 30), NI074 (SEQ ID NO: 15;
corresponding to 1012 to 992 of SEQ ID NO: 30), and NI075 (SEQ ID
No: 16; corresponding to 302-782 of SEQ ID NO: 30) were
synthesized.
[0100] 2. Analysis of cDNA nucleotide sequence of megakaryocyte
differentiation factor by PCR (2)
[0101] A. Preparation of mRNA from megakaryocyte differentiation
factor expressing cell line (A431)
[0102] From 1.1 g of frozen cells of human epidermoid carcinoma
cell line (A431), 25 .mu.g of mRNA was extracted and purified using
an RNA extraction kit and an mRNA purification kit available from
Pharmacia-LKB.
[0103] B. Preparation of DNA phage library from megakaryocyte
differentiation factor expressing cell line (A431)
[0104] (1) Synthesis of cDNA
[0105] From 5 .mu.g of the mRNA derived from A431, cDNA was
synthesized using a cDNA synthesis kit Time Saver available from
Pharmacia-LKB. First 5 82 g of mRNA dissolved in 20 .mu.l of
distilled water treated with diethylpyrocarbonate (DEPC) was heated
at 65.degree. C. for 10 minutes and cooled on ice. 11 .mu.l of a
first strand reaction mixture, 1 .mu.l of DTT solution and 1 .mu.l
of 130 U/ml NotI/oligomer 18 primer solution (Pharmacia-LKB) were
added thereto, and the mixture was incubated at 37.degree. C. for
an hour.
[0106] The reaction mixture was added to a second strand reaction
mixture, and the mixture was incubated at 12.degree. C. for 30
minutes and 22.degree. C. for an hour, and heated at 65.degree. C.
for 10 minutes. 100 .mu.l of a mixture of
phenol/chloroform/isoanylalcahol (25:24:1; abbreviated as PC
hereinafter) was added thereto, and the mixture was vigorously
stired, centrifuged at 14,000.times.9 for one minute to obtain a
supernatant, which was then fractionated by a Sephacryl S-400 spin
column (Pharmacia LKB) to obtain 100 .mu.l of cDNA solution.
[0107] (2) Addition of EcoRI adaptor
[0108] To 100 .mu.l of the cDNA solution were added 5 .mu.l of 10
U/ml EcoRI adaptor (Pharmacia LKB), 30 .mu.l of polyethylene glycol
buffer, 1 .mu.l of 1/5 diluted ATP solution and 1 .mu.l of T4 DNA
ligase, and the mixture was incubated at 37.degree. C. for an hour.
After heating at 65.degree. C. for 10 minutes, 1.5 .mu.l of ATP
solution and 1 .mu.l of T4 polynucleotide kinase were added thereto
and the mixture was incubated at 37.degree. C. for 30 minutes.
After heating at 65.degree. C. for 10 minutes, 2 .mu.l of 20
U/.mu.l NotI was added to the mixture, which was then incubated at
37.degree. C. for an hour. 150 .mu.l of PC was added to the
mixture, which was then vigorously stirred and centrifuged at
14,000.times. g for a minute, and the supernatant was fractionated
on Sephadex S-400 spin column to obtain 150 .mu.l of cDNA
solution.
[0109] (3) Incorporation of cDNA into phage vector and in vitro
packaging
[0110] After digesting with EcoRI and NotI, to 15 .mu.l of the cDNA
solution were added 2 .mu.g of dephosphorylated .lambda.gt11D
(Pharmacia LKB). After ethanol precipitation, the precipitate was
dissolved in 8 .mu.l of ligase buffer solution, and 1 .mu.l of
1/75-diluted ATP solution and 1 .mu.l of T4 DNA ligase were added
to the solution, which was then incubated at 16.degree. C. for 30
minutes and stored on ice.
[0111] An in vitro packaging reaction was carried out using
Gigapack II Gold (Stratagene), and a library of 3.22.times.10.sup.6
pfu of recombinant phages was obtained from said 3 ligase reaction
products. Said library was amplified in an E. coli Y1090 r.sup.-
host to obtain 6.0.times.10.sup.10 pfu/ml of an A431 phage library
stock.
[0112] C. Identification and isolation of cDNA fragment for
megakaryocyte differentiation factor by PCR
[0113] (1) Amplification of cDNA insert fragment in A431 phage
library by PCR
[0114] 10 .mu.l of a stock solution of 6.0.times.10.sup.10 pfu/ml
A431 phage library (corresponding to 6.0.times.10.sup.8 pfu) was
used as a template DNA for PCR reaction, and 5 .mu.l of 10.times.
PCR buffer, 8 .mu.l of 1.25 mM 4 dNTPs, 2 .mu.l of 1 OD/ml
.lambda.gt11-forward primer (.lambda.gt 11F) (SEQ ID NO: 17), 2
.mu.l of 1 OD/ml .lambda.gt11-reverse primer (.lambda.gt 11R) (SEQ
ID NO: 18), and 1 .mu.l of 5 U/.mu.l Taq DNA polymerase (Perkin
Elmer Cetus) were added, and the total volume of the mixture was
made to be 50 .mu.l with DEPC-treated distilled water. 30 reaction
cycles of 93.degree. C. for a minute, 55.degree. C. for 2 minutes
and 72.degree. C. for 3 minutes were carried out, and the reaction
mixture was incubated at 72.degree. C. for 10 minutes. As a result
of an analysis by 1% Agarose gel electrophoresis, a smeary pattern
ranging 0.8 to 6 kb was shown.
[0115] (2) PCR analysis using cDNA insert DNA amplification
fragment mixture as a template and using TP7 (SEQ ID NO: 20;
corresponding to 683-703 of SEQ ID NO: 30), /TP10, TP7/TP6 (SEQ ID
NO: 19; corresponding to 1036-1001 of SEQ ID NO: 30), TP8 (SEQ ID
NO: 21; corresponding to 941-964 of SEQ ID NO: 30), TP10 (SEQ ID
NO: 22; corresponding to 1036-986 of SEQ ID NO: 30) and TP8/TP6 as
primers.
[0116] 1 .mu.l of a 1/5000-diluted solution of the above-mentioned
PCR reaction product was used as a template DNA for PCR reaction,
and 5 .mu.l of 10.times. PCR buffer, 8 .mu.l of 1.25 mM 4 dNTPs, 2
.mu.l each of 1 OD/ml primers in combination as described
hereinafter and 1 .mu.l of Perfect Match (Stratagene) were added,
and a total volume of the mixture was made to 49 .mu.l with
DEPC-treated distilled water.
[0117] The reaction mixture was heated at 95.degree. C. for 5
minutes and 60.degree. C. for 5 minutes, 1 .mu.l of 5 U/.mu.l Taq
DNA polymerase (Perkin Elmer Cetus) was added thereon, and 30
cycles of 94.degree. C. for a minute, 60.degree. C. for 2 minutes
and 72.degree. C. for 3 minutes were carried out, followed by an
incubation at 72.degree. C. for 10 minutes. As primers, TP7/TP10,
TP7/TP6, TP8/TP10, and TP8/TP6 were used.
[0118] As a result of an analysis of the PCR reaction product by 2%
agarose gel electrophoresis, bands of 354 bp, 354 bp, 96 bp and 96
bp were obtained corresponding to primers respectively.
[0119] (3) Analysis for primary sequence of PCR amplification
products (354 bp) obtained by using primers TP7/TP10 and
TP7/TP6
[0120] The bands of said PCR amplification products (354 bp)
obtained using primers TP7/TP10 and TP7/TP6 were excised from the
2% agarose gel after the electrophoresis, and to the excised
agarose gel pieces was added 50 .mu.l of DEPC-treated distilled
water, and the mixture was heated at 45.degree. C. for 30 minutes.
To 2 .mu.l of this solution as a template DNA, were added 5 .mu.l
of 10.times. PCR buffer, 8 .mu.l of 1.25 mM 4 dNTPs, 2 .mu.l each
of 1 OD/ml primers in combination and 1 .mu.l of Perfect Match
(Stratagene), and a total volume of the reaction mixture was made
to 49 .mu.l with DEPC-treated distilled water. The mixture was
heated at 95.degree. C. for 5 minutes and 60.degree. C. for 5
minutes, 1 .mu.l of 5 U/.mu.l Taq DNA polymerase (Perkin Elmer
Cetus) was added, and 30 reaction cycles of 94.degree. C. for a
minutes, 60.degree. C. for 2 minutes and 72.degree. C. 3 minutes
were carried out, followed by incubation at 72.degree. C. for 10
minutes. As primes for the above-mentioned PCR reaction, TP7/TP10
and TP7/TP6 were used. Bands of the PCR reaction products (each 354
bp) were excised from 2% agarose gel of the electrophoresis,
extracted and purified, and the product was inserted into pCR II
(Invitrogen), which was then used to transform E. coli INV.alpha.F'
(Invitrogen). Plasmid DNA was extracted and purified, and it was
confirmed by EcoRI digestion that DNA fragment of 354 bp had been
inserted.
[0121] Primary sequence of the DNA insert fragment was analyzed
using M13 forward primer (M13F) (SEQ ID NO: 23) and M13 reverse
primer (M13R) (SEQ ID NO: 24) (Aplied Biosystem's automated
sequencer, Model 370A). As a result, a 296 bp sequence
corresponding to the nucleotide number 704 to 999 of SEQ ID NO: 30
was found, and this sequence contained C-terminal 3 amino acids
(XRK; but ERK from DNA nucleotide sequence) of SEQ ID NO: 9
corresponding to the downstream portion of primer TP7, N-terminal 5
amino acids (ADLSG) of SEQ ID NO: 6 corresponding to the upstream
portion of primer TP6, and 8 amino acids (YLRALGLK) of SEQ ID NO: 5
corresponding to primer TP8, revealing that the PCR reaction
products (each 354 bp) is a part of cDNA coding for megakaryocyte
differentiation factor.
[0122] 3. Screening of cDNA Coding for Megakaryocyte
Differentiation Factor
[0123] A. Preparation of cDNA plasmid library from megakaryocyte
differentiation factor expressing cell line (A431)
[0124] (1) Synthesis of first strand cDNA
[0125] From 5 .mu.g of mRNA derived from A431 cell line, cDNA was
synthesized using a Super Script plasmid system available from
GIBCO. First 2 .mu.l of NotI primer adaptor was added to 5 .mu.g of
mRNA dissolved in 5 .mu.l of diethylpyrocarbonate (DEPC)-treated
distilled water, and the mixture was heated at 70.degree. C. for 10
minutes and cooled on ice. 4 .mu.l of 5.times. first strand buffer,
2 .mu.l of 0.1 M DTT solution, 1 .mu.l of 10 mM 4dNTPs and 1 .mu.l
of DEPC-treated distilled water were added thereto, and the mixture
was incubated at 37.degree. C. for 2 minutes. 5 .mu.l of a Super
Script reverse transcriptase was added to the reaction mixture,
which was then incubated at 37.degree. C. for one hour and then put
on ice to stop the reaction.
[0126] (2) Synthesis of second strand cDNA
[0127] To 18 .mu.l of 20 .mu.l reaction mixture for the first
strand cDNA synthesis were added 93 .mu.l of DEPC-treated distilled
water, 30 .mu.l of 5.times. second strand buffer, 3 .mu.l of 10 mM
4 NTPs, 1 .mu.l of 10 U/.mu.l E. coli DNA ligase, 4 .mu.l of 10
U/.mu.l E. coli DNA polymerase and 1 .mu.l of 2 U/.mu.l E. coli
RNase H, and the mixture was incubated at 16.degree. C. for 2
hours. 2 .mu.l (10 U) of T4 DNA polymerase was added to the
reaction mixture, which was then incubated at 16.degree. C. for 5
minutes.
[0128] The reaction mixture was put on ice, and after adding 10
.mu.l of 0.5M EDTA and 150 .mu.l of PC thereto, was vigorously
stirred and centrifuged at 14,000.times. g for 10 minutes, and 140
.mu.l of the supernatant was transferred to a fresh centrifuge
tube. 70 .mu.l of 7.5M ammonium acetate and 0.5 ml of ethanol were
added to the supernatant, which was then allowed to stand at
-80.degree. C. for 30 minutes. The mixture was centrifuged at
14,000.times. g for 10 minutes, and after removing the supernatant,
the precipitate was washed with 0.5 ml of 70% ethanol and dried
under a reduced pressure.
[0129] (3) Addition of BstXI adapter
[0130] The above-mentioned cDNA precipitate was dissolved in 25
.mu.l of DEPC-treated distilled water, 10 .mu.l of 5.times.T4 DNA
ligase buffer, 10 .mu.l of BstXI adapter (Invitrogen) and 5 .mu.l
of T4 DNA ligase were added to the solution, which were then
incubated at 16.degree. C. for 16 hours. 50 .mu.l of PC was added
to the mixture, which was the vigorously stirred, and centrifuged
at 14,000.times. g for 5 minutes. 45 .mu.l of the supernatant was
transferred to a fresh centrifuge tube. 25 .mu.l of 7.5M ammonium
acetate and 150 .mu.l of ethanol were added to the tube, which was
stirred and allowed to stand at -80.degree. C. for 30 minutes.
After centrifuging at 14,000.times. g for 10 minutes to remove
supernatant, the precipitate was washed with 0.5 ml of 70% ethanol
and dried under a reduced pressure.
[0131] (4) NotI digestion
[0132] The above-mentioned cDNA precipitate was dissolved in 41
.mu.l of DEPC-treated distilled water, and 5 .mu.l of REAct 7
buffer and 4 .mu.l of NotI were added to the solution, which was
incubated at 37.degree. C. for 2 hours. 50 .mu.l of PC was added to
the mixture, which was then vigorously stirred and centrifuged at
14,000.times. g for 10 minutes, and 45 .mu.l of the supernatant was
transferred into a centrifuge tube.
[0133] (5) Elimination of adapter and size fractionation of partial
cDNA
[0134] The above-mentioned cDNA solution was fractionated using a
Quick Spin Column Linker 5 (Boehringer Mannheim). 50 .mu.l of 40
.mu.g/.mu.l cDNA was obtained.
[0135] (6) Incorporation of cDNA into phagevector and
transformation of E. coli
[0136] To 37.5 .mu.l of the above-mentioned cDNA solution were
added 12.5 .mu.l of pCR/CMV (Invitrogen) vector (29 .mu.g/.mu.l)
digested with Not I and BstXI, and further added were 400 .mu.l of
Takara Ligation kit A solution and 50 .mu.l of B solution, and the
mixture was incubated at 16.degree. C. for 30 minutes, and 1 ml of
Max Efficiency DH5.alpha. competent cells (BRL) were transformed to
obtain 71,550 recombinant clones. All colonies were collected from
plate (2.86.times.10.sup.7 cells/ml) and stored at -80.degree. C.
in the presence of 20% glycerol.
[0137] B. Screening of megakaryocyte differentiation factor cDNA by
colony hybridization
[0138] Using the cDNA plasmid library derived from A431 cell line,
a total of 227,000 (3700/plate) colonies were formed on 60 plates
of 9 cm diameter, and the colonies were replicated to
nitrocellulose filters. A probe was prepared by carrying out PCR
(as described hereinbefore) using primer NI 067 and 3'-RACE adaptor
(GIBCO BRL) to obtain a 900 bp PCR product, digesting the PCR
product with Bam HI to obtain two DNA fragments (0.5 kb and 0.4 kb)
and nick-translating the DNA fragments with
[.alpha.-.sup.32P]dCTP.
[0139] For the colony hybridization, the filter was incubated in
5.times. SSC, 25 mM phosphate buffer (pH 7.4), 5.times. Denhaldt's
solution, 1% SDS, 100 .mu.g/ml heat denatured salmon sperm DNA and
50% formamide at 42.degree. C. for 18 hours, and washed with
5.times. SSC, 0.1% SDS at 40.degree. C. for 20 minutes and
45.degree. C. for 20 minutes. Detection was carried out by exposing
BAS 2000 (Fuji Film) for 18 hours.
[0140] First, second and third screening was carried out to obtain
4 clones, i.e., TP290, TP308, TP310 and TP317. The length of insert
cDNA was 1.2 kb, 1.1 kb, 1.2 kb and 1.2 kb respectively. The TP290,
TP310 and TP317 cover a region downstream from the nucleotide
number 685 of SEQ ID NO: 30.
[0141] 4. Analysis of cDNA Nucleotide Sequence Coding for
Megakaryocyte Differentiation Factor by PCR (3)
[0142] A. Preparation of mRNA from megakaryocyte differentiation
factor expressing cell line (HPC-Y11)
[0143] From 1.1 g of frozen cells of human pancreatic cancer cell
line (HPC-Y11), 50 .mu.g of mRNA was extracted and purified using
an RNA extraction kit and mRNA purification kit available from
Pharmacia-LKB.
[0144] B. Preparation of cDNA phage library from megakaryocyte
differentiation factor expressing cell line (HPC-Y11)
[0145] (1) Synthesis of cDNA
[0146] From 5 .mu.g of mRNA derived from HPC-Y11, cDNA was
synthesized using a Pharmacia-LKB's Time Saver cDNA synthesis kit.
First, 5 .mu.g of mRNA was dissolved 20 .mu.l of
diethylpyrocarbonate (DEPC)-treated distilled water, and the
solution was heated at 65.degree. C. for 10 minutes and cooled on
ice. 11 .mu.l of first strand reaction mixture, 1 .mu.l of DTT
solution and 1 .mu.l of NotI/oligomer 18 primer solution were added
to the mixture, which was then incubated at 37.degree. C. for an
hour.
[0147] 100 .mu.l of second strand reaction mixture was added to the
mixture, which was then incubated at 12.degree. C. for 30 minutes
and at 22.degree. C. for an hour, and heated at 65.degree. C. for
10 minutes. 100 .mu.l of phenol- chloroform-isoamyl alcohol
(25:24:1, abbreviated as PC) was added to the mixture, which was
then vigorously stirred and centrifuged at 14,000.times. g for a
minute, and the supernatant was fractionated using a Sephacryl
S-400 spin column (Pharmacia-LKB) to obtain 100 .mu.l of cDNA
solution.
[0148] (2) Addition of EcoRI adaptor
[0149] To 100 .mu.l of the cDNA solution, were added 5 .mu.l of
EcoRI adapter (Pharmacia-LKB), 30 .mu.l of polyethylene glycol, 1
.mu.l of ATP solution and 1 .mu.l of T4 DNA ligase, and the mixture
was incubated at 37.degree. C. for an hour. After heating at
65.degree. C. for 10 minutes, 1.5 .mu.l of ATP solution and 1 .mu.l
of T4 polynucleotide kinase were added to the mixture, which was
then incubated at 37.degree. C. for 30 minutes. After heating at
65.degree. C. for 10 minutes, 12 .mu.l of Not I was added to the
mixture, which was then incubated at 37.degree. C. for an hour. 150
.mu.l of PC was added to the mixture, which was then vigorously
stired and centrifuged at 14,000.times. g for a minute, and the
supernatant was fractionated using a Sephacryl S-400 spin column to
obtain 150 .mu.l of cDNA solution.
[0150] (3) Incorporation of cDNA into phage vector and in vitro
packaging
[0151] To 15 .mu.l of the cDNA solution, was added 2 .mu.g of
.lambda.gt 11D (Pharmacia-LKB) which had been digested with EcoRI
and Not I and dephosphorydated, and after ethanol precipitation,
the precipitate was dissolved in 8 .mu.l of ligase buffer. 1 .mu.l
of 1/75-diluted ATP solution and 1 .mu.l of T4 DNA ligase were
added to the mixture, which was then incubated at 16.degree. C. for
30 minutes and stored on ice. An in vitro packaging reaction was
carried out using a Giga Pack II Gold (Strotagene), and
5.34.times.10.sup.6 pfu of recombinant phages were obtained from
the above-mentioned 3 ligase reaction products. The library was
amplified in E. coli Y1090 r.sup.-1 host to obtain a stock of
1.7.times.10.sup.11 pfu/ml HPC-Y11 phage library.
[0152] C. Identification and isolation of 5'-portion of
megakaryocyte differentiation factor cDNA
[0153] (1) Amplification by PCR of HPC-Y11 phage library cDNA
insert DNA fragment primer NI074 upstream portion.
[0154] To 1 .mu.l (corresponding to 1.7.times.10.sup.9 pfu) of
6.0.times.10.sup.10 pfu/ml HPC-Y11 phage library stock solution as
a template DNA for PCR reaction, were added 5 .mu.l of 10.times.
PCR buffer, 8 .mu.l of 1.25 mM 4 dNTPs, 1 .mu.l of 10 OD/ml
.lambda.gt11-forward F1 primer (SEQ ID NO: 25), 1 .mu.l of 50 D/ml
NI074 primer and 1 .mu.l of Perfect Match (Stratagene), and a total
volume was made to be 49 .mu.l with DEPC-treated distilled
water.
[0155] After heating the reaction mixture at 95.degree. C. for 5
minutes and 60.degree. C. for 5 minutes, 1 .mu.l of 5 U/.mu.l Taq
DNA polymerase (Perkin Elmer Cetus) was added thereto, and 35
reaction cycles of 94.degree. C. for a minute, 60.degree. C. for a
minute and 72.degree. C. for 2 minutes were carried out, followed
by incubation at 72.degree. C. for 10 minutes. A result of analysis
by 2% agarose gel electrophoresis showed a smeary pattern ranging
from 0.3 to 6 kb.
[0156] (2) PCR analysis using a template which is a PCR
amplification fragment mixture prepared by using
.lambda.gt11F1/NI074 primer, and using as primers .lambda.gt11F2
(SEQ ID NO: 26)/NI075, .lambda.gt11F2/TP12 (SEQ ID NO: 28;
corresponding to 703-683 of SEQ ID NO: 30), .lambda.gt11F2/TP11(SEQ
ID NO: 27; corresponding to 619-599 of SEQ ID NO: 30),
.lambda.gt11F2/TP13 (SEQ ID NO: 29; corresponding to 595-575 of SEQ
ID NO: 30), TP7/NI074, TP7/NI075, and NI073/NI074
[0157] 1 .mu.l of a 1/100 diluted solution of the above-mentioned
PCR reaction product was used for a template DNA for PCR reaction,
and 5 .mu.l of 10.times. PCR buffer, 8 .mu.l of 1.25 mM 4 dNTPs,
0.5 .mu.l each of 10 OD/ml primers in combination as described
hereinafter, and 1 .mu.l of Perfect Match (Stratagene) were added
thereto, and a total of the reaction mixture was made to be 49
.mu.l with DEPC-treated distilled water. The reaction mixture was
heated at 95.degree. C. for 5 minutes and at 60.degree. C. for 5
minutes, 1 .mu.l of 5 U/.mu.l Taq DNA polymerase (Perkin Elmer
Cetus) was added thereto, and 35 reaction cycles of 94.degree. C.
for a minute, 60.degree. C. for 2 minutes and 72.degree. C. for 2
minutes were carried out, followed by an incubation at -72.degree.
C. for 10 minutes.
[0158] As primers, .lambda.gt11F2/NI075, .lambda.gt11F2/TP12,
.lambda.gt11F2/TP11, .lambda.gt11F2/TP13, TP7/NI074, TP7/NI075, and
NI073/NI074 were used. As a result of 2% agarose gel
electrophoresis, bands of 969 bp, 870 bp, 786 bp, 762 bp, 330 bp,
120 bp and 149 bp were obtained corresponding to the primers.
[0159] (3) PCR analysis and primary sequence analysis using
.lambda.gt11F/TP11 and .lambda.gt11F/TP13 primer, for PCR
amplification product (969 bp) prepared using F2/NI075 primers
[0160] 0.5 .mu.l of the above mentioned PCR reaction product (969
bp) prepared by using .lambda.gt11F2/NI075 as primers was used as a
template DNA for PCR reaction, and 5 .mu.l of 10.times. PCR buffer,
8 .mu.l of 1.25 mM 4 dNTPs, 1 .mu.l of 10 OD/ml .lambda.gt11F
primer, 1 .mu.l of 10 OD/ml TP11 primers or 1 .mu.l of 10 OD/ml
TP13 primers, and 1 .mu.l of Perfect Match (Stratagene) were added,
and the total volume was made to be 49 .mu.l with DEPC-treated
distilled water.
[0161] The reaction mixture was heated at 95.degree. C. for 5
minutes and 60.degree. C. for 5 minutes, 1 .mu.l of 5 U/.mu.l Taq
DNA polymerase (Perkin Elmer Setus) was added thereto, and 35
reaction cycles of 94.degree. C. for a minute, 60.degree. C. for 2
minutes and 72.degree. C. for 2 minutes were carried out, followed
by an incubation at 72.degree. C. for 10 minutes.
[0162] Bands of PCR reaction products (678 bp and 654 bp,
respectively) were excised from 2% agarose gel after
electrophoresis, extracted and purified, and inserted into PCRII
(Invitrogen), which was then used to transform E. coli IN
V.alpha.F' (Introgen). Plasmid DNA was extracted and purified from
the transformant, and digested with EcoRI to confirm that a 0.7 kb
DNA fragment was inserted. The primary sequence of the inserted DNA
fragment was analyzed by using M13 forward primer M13F and M13
reverse primer M13R (Applied Biosystems automated sequencer Model
370A).
[0163] As a result, a sequence of 619 bp corresponding to
nucleotide number 1 to 619 of SEQ ID NO: 30 was found, and the
nucleotide sequence of 133 nucleotides consisting of nucleotide
number 487 to 619 of SEQ ID NO: 30 conformed to the N-terminus of
the primary sequence found in Example 2.1. In this 619 bp sequence
there are 19 amino acids of SEQ ID NO: 3 (VERVDFTNHLEDTR RNINK from
DNA nucleotide sequence) and 5 amino acids (LYDAK) of SEQ ID NO: 7,
and it was clarified that this PCR reaction products (each 0.7 kb)
was a part of cDNA coding for megakaryocyte differentiation
factor.
[0164] It was considered that the translation starting methionine
corresponds to 74th nucleotide, and 5'-non translational region
consisted of 73 bp. Accordingly, it was clarified that these PCR
reaction products (each 0.7 kb) contain N-terminus of structural
gene for megakaryocyte differentiation factor.
[0165] 5. Analysis of cDNA Nucleotide Sequence Coding for
Megakaryocyte Differentiations Factor by PCR (4)
[0166] Among an N-terminal portion of structural gene for a
megakaryocyte differentiation factor derived from HPC-Y11 and a
sequence considered to be a 5'-non-translational region, obtained
in the section C(3), the sequence of nucleotide numbers 12 to 31 of
SEQ ID NO: 30 which is a sequence part considered to be a
5'-non-translational region was used as a basis to synthesize an
oligomer NI 083 (SEQ ID NO: 31).
[0167] The RNA having poly A prepared from A431 cells in the
section 1., a Preamplification System (Gibco BRL) and random
hexamers attached to the System were used to synthesize first
strand cDNA according to included instructions, and PCR was carried
out by using NI083 and NI074 as well as Ampli Taq (Takara). As a
result, a DNA fragment of 1001 bp which is a cDNA fragment for a
megakaryocyte differentiation factor was obtained.
[0168] This DNA fragment, a PCR product, of 1001 bp was directly
used as a substrate for sequencing on a Taq Dye Deoxy Terminator
Cycle Sequencing kit (Applied Biosystem) and a fluorescent
sequencer (Applied Biosystem Type 370A) according to the included
manufacturers instruction. As a result, a sequence of nucleotide
numbers 32-486 of SEQ ID NO: 30 was found. In addition, a result
obtained for nucleotide numbers 487 to 991 of SEQ ID NO: 30
conformed to the sequence obtained in the section 1.
[0169] By combining with the sequence obtained in the section 1.,
the nucleotide sequence 32-1950 of SEQ ID NO: 30 which is cDNA
nucleotide sequence coding for a megakaryocyte differentiation
factor of A431 cell was determined.
[0170] For this nucleotide sequence, all of possible 3 reading
frames were mechanically translated into amino acid sequences and
it was found that one of them has a region which can be translated
to a continuing amino acid sequence containing all the amino acid
sequences shown in SEQ ID NO: 1 to 9, and the reading frame of the
megakaryocyte differentiation factor was determined.
[0171] It was found that in this reading frame a codon for
methionine (nucleotide numbers 74 to 76) found at a translation
start position exists, and from this position a region which can be
translated to an amino acid sequence containing the amino acid
sequences shown in SEQ ID NO: 1 to 9 continues up to the nucleotide
position 1213 of SEQ ID NO: 30, and it was determined that the
nucleotide sequence from the position 74 to the position 1213 of
SEQ ID NO: 30 is the region translated to megakaryocyte
differentiation factor.
[0172] The nucleotide sequence GCAATGG (nucleotide numbers 71 to 77
of SEQ ID NO: 30) encompassing the methionine codon of nucleotide
numbers 74 to 76 of SEQ ID NO: 30 corresponds to a sequence
(G/A-N-N-A-T-G-G) encompassing a methionine codon frequently
present at a translation start site found by M. Kozak, Nucleic
Acids Research (1981) Vol. 9, p5233-5252.
[0173] Primary sequence of megakaryocyte differentiation factor was
thus clarified, the number of amino acids was 380 in the structural
gene, an expected molecular weight was 42904.43, and an expected
isoelectric point was 6.79. SEQ ID NO: 1 corresponds to amino acid
numbers 188 to 196 of SEQ ID NO: 30; SEQ ID NO: 2 corresponds to
amino acid numbers 181 to 187 of SEQ ID NO: 30; SEQ ID NO: 3
corresponds to the amino acid numbers 126 to 144 of SEQ ID NO: 30;
SEQ ID NO: 4 corresponds to the amino acid numbers 325 to 341 of
SEQ ID NO: 30; SEQ ID NO: 5 corresponds to the amino acid numbers
289 to 297 of SEQ ID NO: 30; SEQ ID NO: 6 corresponds to the amino
acid numbers 305 to 324 of SEQ ID NO: 30; SEQ ID NO: 7 corresponds
to the amino acid numbers 121 to 125 of SEQ ID NO: 30; and SEQ ID
NO: 8 corresponds to the amino acid numbers 284 to 288 of SEQ ID
NO: 30; and SEQ ID NO: 9 corresponds to the amino acid numbers 204
to 213 of SEQ ID NO: 30. In addition, A poly A addition signal
AATAAA sequence is present at nucleotide numbers 1933 to 1998 of
SEQ ID NO: 30.
Example 3
[0174] Isolation and Identification of cDNA Coding for
Megakaryocyte Differentiation Factor From A431 by PCR and
Construction of Expression Vector
[0175] Oligomers NI078 (SEQ ID NO: 32) and NI079 (SEQ ID NO: 33)
were synthesized on the basis of the sequence (SEQ ID NO: 30)
obtained in Example 2. Note in the NI078, the sequence of
nucleotide numbers 13 to 37 encompassing the translation start
methionine codon conforms to the sequence of the nucleotide numbers
74 to 98 of SEQ ID NO: 30 and an EcoRI recognizing site (nucleotide
numbers 4 to 9) and an Nru I recognizing site (nucleotide numbers 8
to 13) were artificially added; and in the NI079, the sequence of
the nucleotide numbers 17 to 49 conforms to the nucleotide sequence
of the nucleotide numbers 1237 to 1269 of SEQ ID NO: 30, and an
EcoRI recognizing site (nucleotide numbers 3 to 8) and an Not I
recognizing site (nucleotide numbers 9 to 16) were artificially
added.
[0176] The RNA having poly A prepared from A431 cells in Example 2
section 1., a Preamplification System (Gibco BRL), and Oligomers,
i.e., random hexamers included in the system were used according to
the included manufacturer's instructions to synthesize first strand
cDNA, and PCR was carried out using the synthesized DNA as a
template and NI078 and NI079 as primers and using Ampli Taq (Perkin
Elmer Cetus). As a result, a DNA fragment of 1224 base pairs which
is a cDNA fragment for a megakaryocyte differentiation factor and
has all information relating to megakaryocyte differentiation
factor, was obtained.
[0177] This DNA fragment was treated with EcoRI to generate EcoRI
cohesive sites at both the ends of the cDNA coding for a
megakaryocyte differentiation factor in virtue of EcoRI recognizing
sites artificially added to the oligomers NI078 (SEQ ID NO: 32) and
NI079 (SEQ ID NO: 33). This cDNA fragment coding for megakaryocyte
differentiation factor was introduced into a mammorian expression
vector pdKCR-DHFD at it's EcoRI recognizing site to obtain
pdKCR-DHFR-TPO55.
[0178] The animal cell expression vector pdKCR-dhfr (Oikawa, S. et.
al., Biochem. Biophys. Res. Commun. 164, 39, 1989) is a derivative
of PKCR (O'Hare et. al., Pro. Natl. Acad. Sci. USA, 78, 1527, 1981)
and has SU 40 early promoter and a rabbit .beta.-globin gene and
dhfr (dehydrofolate reductase) gene. Note, a host transformed with
the expression vector, was designated as Escherichia coli SBM 308,
and deposited with the Fermentation Research Institute, Agency of
Industrial Science and Technology, 1-3, Higashi 3-chome,
Tsukubashi, Ibaraki, Japan as FERM P-11506 on Jun. 7, 1990, and
transferred to an international deposition under the Budapest
treaty as FERM BP-4197 on Feb. 18, 1993.
[0179] The clone pdKCR-DHFR-TPO55 containing megakaryocyte
differentiation factor cDNA which was incorporated in to pdKCR-DHFR
was sequenced using a Taq Dye Deoxy Terminator Cycle Sequencing kit
(Applied Biosystem) and a fluorescent sequencer (Applied Biosystem
Type 370A) according to included instructions. As a result, the
determined nucleotide sequence conformed to the sequence of the
nucleotide numbers 99 to 1236 of SEQ ID NO: 30 and oligomers N1078
and N1079. In addition, it was confirmed by the sequencing that a
megakaryocyte differentiation factor cDNA inserted into the vector
is in correct orientation in relation to an expression vector
promoter.
[0180] As shown in the above, once the information of SEQ ID NO: 30
is provided, it is easy for a person skilled in the art that the
nucleotide sequence is determined by amplifying cDNA coding for
megakaryocyte differentiation factor in total or in a optional
portion on megakaryocyte differentiation factor expressing cell
line (for example, A431) and boned in a optional expression
vector.
Example 4
[0181] Expression of Megakaryocyte Differentiation Factor in Bombyx
mori
[0182] (1) Construction of Bombyx mori Expression Vector
[0183] A megakaryocyte differentiation factor cDNA clone
pdKCR-DHFR-TPO55 was digested with NotI to cleave the NotI
recognizing site artificially added to the NI079. The NotI cohesive
end thus generated was blunt-ended using a blunting kit available
from Takara Shuzo, and to the blunt end was added an XbaI linker
(Takara Shuzo) according to an attached instruction. The plasmid
thus obtained was digested simultaneously with NruI and XbaI to
cleave the NruI recognizing site artificially added to the NI078
and the XbaI recognizing site of the XbaI linker introduced to
prepare a megakaryocyte differentiation factor cDNA fragment having
an NurI cohesive end and an XbaI cohesive end. This DNA fragment
was inserted at the Nru I recognizing site into a baculovirus
transfer vector for Bombyx mori nuclear polyhedrosis virus, pBm4
(available from Department of Insect Genetics and Bleeding National
Institute of Sericultural and Entomological Science, Ohwashi,
Tukuba, Ibaraki 305, Japan) simultaneously digested with NruI and
XbaI to obtain pBm4-TPO55.
[0184] (2) Construction of TPO55 Recombinant Virus
[0185] A cell line derived from Bombyx mori embryonic, BoMo15AIIc
(available from Department of Insect Genetics and Bleeding National
Institute of Sericultural and Entomological Science, Ohwashi,
Tukuba, Ibaraki 305, Japan) was subcultured in a medium containing
10% fetal bovine serum (FBS: GIBCO BRL) and 500 .mu.g/ml gentamicin
in MGM 448 at 25.degree. C. TPO55 recombinant virus was constructed
by co-introducing Bombyx mori nuclear polyhedrosis virus gene DNA
and pBm4-TPO55 plasmid DNA into Bombyx mori cultured cells by, for
example, calcium phosphate co-precipitation method.
[0186] Namely, 2 .mu.g of genomic DNA of wild type virus B6E
(available from Department of Insect Genetics and Bleeding National
Institute of Sericultural and Entomological Science, Ohwashi,
Tukuba, Ibaraki 305, Japan) and 10 .mu.g of the transfer plasmid
pBm4-TPO55 were dissolved in 240 .mu.l of sterile purified water,
and to the solution was added the same volume of 0.5 M CaCl.sub.2
and 0.1M HEPES, and the mixture was mixed and allowed to stand at a
room temperature for 10 minutes. To the mixture was added 480 .mu.l
of 0.2M NaCl, 0.05M HEPES, 0.75 MM NaH.sub.2PO.sub.4 and 0.75 mM
Na.sub.2HPO.sub.4, and the mixture was stirred for a few second and
allowed to stand at a room temperature for 20 to 30 minutes to form
calcium phosphate gel containing the genomic DNA and the
plasmid.
[0187] Next, 960 .mu.l of calcium phosphate gel suspension
containing the viral genomic DNA and the transfer vector was added
to 4 ml of BoMo15AIIc cells in a 25 cm.sup.2 T flask (T25,
Corning), and the mixture was allowed to stand for 12 hours. The
medium was replaced with a fresh MGM448 (containing 10% FBS and the
antibiotics), and culturing was carried out for 25.degree. C. On
the sixth day the cultured medium was recovered as a viral
solution.
[0188] The cultured medium was centrifuged to obtain a clear
supernatant, which was then diluted, and added to BoMo15AIIc cells
cultured on the microtiter plate, and after 8 days a culturing
medium in which viral infection was microscopically observed but a
polyhedral body was not formed, was selected (by a limited dilution
method). The cultured medium was recovered. Contamination with a
wild virus in the viral solution factor was not observed.
[0189] A recombinant virus thus constructed, containing a DNA
coding for megakaryocyte differentiation factor was designated as
TPO55-BmNPV.
[0190] (3) Test for Expression of Recombinant Gene
[0191] About 1.times.10.sup.6 BoMo15A IIc cells were cultured in 4
ml of MGM448 medium containing 10% FBS on the bottom of 25 cm.sup.2
area of a flask for 2 days by plate culture. To the culture, 0.5
moi of wild type virus B6E or recombinant virus (TPO55-BmNPV)
containing a gene coding for megakaryocyte differentiation factor
were added and infected, BoMo15A IIc cells, and the cells were
cultured at 25.degree. C. for 3 days, and total RNA was extracted
using Isogen (Wako Pure Chemical). Similarly, total RNA was
extracted from non infected BoMo 15AIIc cells.
[0192] Next, 1 Mg of the RNA thus extracted was size-fractionated
by agarose gel electrophoresis, and the separated RNA was
transferred to a Zetaprobe membrane by the capillary action. The
membrane was soaked in a hybridization buffer containing
megakaryocyte differentiation factor cDNA (:PCR product amplified
with KY100 and NI065 described in Example 2.1) (TPO55 probe DNA)
labeled with digoxigenin (Boehringer Mannheim), and the mixture was
incubated at 42.degree. C. for 12 hours to allow the formation of
specific complex of recombinant megakaryocyte differentiation
factor mRNA and the TPO55 probe DNA thereof. The complex was then
reacted with an alkaline phosphatase-conjugated anti-digoxigenin
antibody (Boehringer Mannheim), and the complexed megakaryocyte
differentiation factor mRNA was ditected by chemoluminescence
generated by hydrolysis of Lumigen PPD (AMPPD) (Boehringer
Mannheim) according to manufacture's instructions with alkaline
phosphates.
[0193] As seen in FIG. 11, recombinant megakaryocyte
differentiation factor mRNA was detected in total RNA extracted
from TPO55-EmNPV-infected cells, and it was shown that the mRNA was
expressed in the TPO55-BmNPV-infected cells. On the other hand
expression of mRNA which hydridized with the probe DNA was not
observed in the B6E-infected cells and uninfected cells.
[0194] (4) Preparation of Solution of Recombinant Virus
[0195] About 1.times.10.sup.6 BoMo15AIIc cells were cultured in 4
ml of MGM448 containing 10% FBS on the bottom of a 25 cm.sup.2
flask for 2 days, and to this culture was added 10 .mu.l of the
culture medium of BoMo15AIIc cells containing the recombinant virus
cloned in the above section (2). After culturing at 25.degree. C.
for 14 days, the culture medium was centrifuged at 1000 rpm for 5
minutes to obtain a supernatant as a recombinant virus
solution.
[0196] (5) Preparation of Hemolymph of Bombyx mori
[0197] 50 .mu.l/head of a viral solution of the 10.sup.-1-diluted
recombinant virus solution obtained in the above section (3) or a
10.sup.-1-diluted wild type virus B6E solution was injected to
Bombyx mori larvae at 5th instar, and the silkworms were fed with
commercially available artificial feed (Morus; Katakura Kogyo) at
20.degree. C. for 4 to 5 days. The abdomens of 50 silkworms were
cut, and an extract containing the hemolymph and the content in the
central intestine was taken in a plastic tube cooled with ice, and
centrifuged to obtain a supernatant.
[0198] (6) Confirmation of Activity of Megakaryocyte
Differentiation Factor
[0199] 50 ml of the hemolymph of the silkworms obtained in the
above section (5) was thoroughly dialyzed against a 20 mM Tris/HCl
(pH 7.4) buffer, and applied to a Matrex Blue A column
(.phi.2.5.times.15 cm) equilibrated with the same buffer. The
column was thoroughly washed with the same buffer to eliminate a
unbound fraction, and a bound protein was eluted by a concentration
gradient of 0 to 1M NaCl. An elution profile for megakaryocyte
differentiation activity of the hemolymph obtained from silkworms
injected with the recombinant virus was compared with that for a
wild type virus.
[0200] As seen from FIG. 12, megakaryocyte differentiation activity
in the hemolymph from silkworms injected with the recombinant virus
was significantly higher than that for the wild type virus.
[0201] Although Bombyx mori vacurovirus transfer vector pBm4,
Bombyx mori nuclear polyhedrosis virus PbE and Bombyx mori cells
BoMo15AIIc were used in Example 4, the present invention is not
limited to the use of these materials. Namely, other baculovirus
transfer vector (such as pBK283, pBKblue, available from Funakoshi)
Bombyx mori nuclear polyhedrosis virus (such as purified DNA
available from Funakoshi), Bombyx mori cells (such as BmN4 cells,
available from Funakoshi) can be easily used by a person with
ordinary skill in the art to obtain a megakaryocyte differentiation
factor.
[0202] A megakaryocyte differentiation factor of the present
invention accelerates formation of megakaryocytes from myeloid
cells in the presence of IL-3. The present megakaryocyte
differentiation factor plays an important role in differentiation
of megakaryocytes and acts in vivo as a thrombopoietin.
Accordingly, the present megakaryocyte differentiation factor may
be medicaments effective to not only various diseases involving
decrease of platelets but also for control of the number of
platelets decreasing by radiation in the case of bone marrow
transradiation, or for control of the number of platelets in
chemotherapy of cancers.
Sequence CWU 1
1
* * * * *