U.S. patent application number 10/730034 was filed with the patent office on 2004-12-16 for genomic dna encoding a polypeptide capable of inducing the production of interferon-gamma.
This patent application is currently assigned to KABUSHIKI KAISHA HAYASHIBARA SEIBUTSU KAGAKU KENKYUJO. Invention is credited to Kurimoto, Masashi, Okura, Takanori, Torigoe, Kakuji.
Application Number | 20040253303 10/730034 |
Document ID | / |
Family ID | 32929517 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040253303 |
Kind Code |
A1 |
Okura, Takanori ; et
al. |
December 16, 2004 |
Genomic DNA encoding a polypeptide capable of inducing the
production of interferon-gamma
Abstract
Disclosed is a genomic DNA encoding a polypeptide capable of
inducing the production of interferon-.gamma. by immunocompetent
cells. The genomic DNA efficiently expresses the polypeptide with
high biological activities of such as inducing the production of
interferon-.gamma. immunocompetent cells, enhancing killer cells'
cytotoxicity and inducing killer cells' formation, when introduced
into mammalian host cells. The high biological activities of the
polypeptide facilitate its uses to treat and/or prevent malignant
tumors, viral diseases, bacterial infectious diseases and immune
diseases without serious side effects when administered to
humans.
Inventors: |
Okura, Takanori; (Okayama,
JP) ; Torigoe, Kakuji; (Okayama, JP) ;
Kurimoto, Masashi; (Okayama, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
PATENT AND TRADEMARK CAUSES
SUITE 300
624 NINTH STREET, N.W.
WASHINGTON
DC
20001-5303
US
|
Assignee: |
KABUSHIKI KAISHA HAYASHIBARA
SEIBUTSU KAGAKU KENKYUJO
Okayama-shi
JP
|
Family ID: |
32929517 |
Appl. No.: |
10/730034 |
Filed: |
December 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10730034 |
Dec 9, 2003 |
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09479862 |
Jan 10, 2000 |
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6790442 |
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09479862 |
Jan 10, 2000 |
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08884324 |
Jun 27, 1997 |
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6060283 |
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Current U.S.
Class: |
424/450 ;
424/93.2; 514/44R |
Current CPC
Class: |
C07K 14/54 20130101 |
Class at
Publication: |
424/450 ;
514/044; 424/093.2 |
International
Class: |
A61K 048/00; A61K
009/127 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 27, 1996 |
JP |
185305/1996 |
Claims
What is claimed is:
1. A composition, comprising: (i) isolated DNA molecule comprising
a nucleotide sequence encoding the amino acid sequence of SEQ ID
NO:1, where Xaa is isoleucine or threonine; and (ii) a carrier
capable of introducing the isolated DNA molecule into a mammalian
cell, wherein said nucleotide sequence is of a fragment of human
genomic DNA.
2. The composition of claim 1, wherein the nucleotide sequence
comprises an exon having the nucleotide sequence shown in SEQ ID
NO:3, 4, 5, 6, or 7.
3. The composition of claim 1, wherein the nucleotide sequence
comprises an intron having the nucleotide sequence of SEQ ID NO:8,
9, 10, 11, or 12.
4. The composition of claim 1, wherein the nucleotide sequence is
of SEQ ID NO:13, 14 or 15.
5. The composition of claim 1, wherein the carrier is a virus or
liposome.
6. The composition of claim 1, wherein the isolated DNA molecule is
linked to a heterologous nucleotide sequence.
7. The composition of claim 6, wherein the heterologous sequence is
of a virus vector.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a division of application Ser.
No. 09/479,862, filed Jan. 10, 2000, which is a division of
application Ser. No. 08/884,324, filed Jun. 27, 1997, the entire
contents of both applications being hereby incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a genomic DNA, more
particularly, a genomic DNA encoding a polypeptide capable of
inducing the production of interferon-.gamma. (hereinafter
abbreviated as "IFN-.gamma.") by immunocompetent cells.
[0004] 2. Description of the Prior Art
[0005] The present inventors successfully isolated a polypeptide
capable of inducing the production of IFN-.gamma. by
immunocompetent cells and cloned a cDNA encoding the polypeptide,
which is disclosed in Japanese Patent Kokai No.27,189/96 and
193,098/96. Because the present polypeptide possesses the
properties of enhancing killer cells' cytotoxicity and inducing
killer cells' formation as well as inducing IFN-.gamma., a useful
biologically active protein, it is expected to be widely used as an
agent for viral diseases, microbial diseases, tumors and/or
immunopathies, etc.
[0006] It is said that a polypeptide generated by a gene expression
may be partially cleaved and/or glycosylated by processing with
intracellular enzymes in human cells. A polypeptide to be used in
therapeutic agents should be preferably processed similarly as in
human cells, whereas human cell lines generally have a disadvantage
of less producing the present polypeptide, as described in Japanese
Patent Application No.269,105/96. Therefore, recombinant DNA
techniques should be applied to obtain the present polypeptide in a
desired amount. To produce the polypeptide processed similarly as
in human cells using recombinant DNA techniques, mammalian cells
should be used as the hosts.
SUMMARY OF THE INVENTION
[0007] In view of foregoing, the first object of the present
invention is to provide a DNA which efficiently expresses the
polypeptide production when introduced into a mammalian host
cell.
[0008] The second object of the present invention is to provide a
transformant into which the DNA is introduced.
[0009] The third object of the present invention is to provide a
process for preparing a polypeptide, using the transformant.
[0010] [Means to Attain the Object]
[0011] The present inventors' energetic studies to attain the above
objects succeeded in the finding that a genomic DNA encoding the
present polypeptide efficiently expresses the polypeptide
production when introduced into mammalian host cells. They found
that the polypeptide thus obtained possessed significantly higher
biological activities than that obtained by expressing a cDNA
encoding the polypeptide in Escherichia coli.
[0012] The first object of the present invention is attained by a
genomic DNA encoding a polypeptide with the amino acid sequence of
SEQ ID NO:1 (where the symbol "Xaa" means "isoleucine" or
"threonine") or its homologous one, which induces
interferon-.gamma. production by immunocompetent cells.
[0013] The second object of the present invention is attained by a
transformant formed by introducing the genomic DNA into a mammalian
host cell.
[0014] The third object of the present invention is attained by a
process for preparing a polypeptide, which comprises (a) culturing
the transformant in a nutrient medium, and (b) collecting the
polypeptide from the resultant culture.
BRIEF EXPLANATION OF THE ACCOMPANYING DRAWINGS
[0015] FIG. 1 is a restriction map of a recombinant DNA containing
a genomic DNA according to the present invention.
[0016] Explanation of the symbols are as follows: The symbol "Hin
dIII" indicates a cleavage site by a restriction enzyme Hin dIII,
and the symbol "HuIGIF" indicates a genomic DNA according to the
present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The followings are the preferred embodiments according to
the present invention. This invention is made based on the
identification of a genomic DNA encoding the polypeptide with the
amino acid sequence of SEQ ID NO:1 or its homologous one, and the
finding that the genomic DNA efficiently expresses the polypeptide
with high biological activities when introduced into mammalian host
cells. The genomic DNA of the present invention usually contains
two or more exons, at least one of which possesses a part of or the
whole of the nucleotide sequence of SEQ ID NO:2. The wording "a
part" includes a nucleotide and a sequential nucleotides consisting
of two or more nucleotides in SEQ ID NO:2. Examples of the exons
are SEQ ID NOs:3 and 4. Human genomic DNA may contain additional
exons with SEQ ID NOs:5 to 7. Since the present genomic DNA is
derived from a mammalian genomic DNA, it contains introns, as a
distinctive feature in mammalian genomic DNAs. The present genomic
DNA usually has two or more introns such as SEQ ID NOs:8 to 12.
[0018] More particular examples of the present genomic DNA include
DNAs with SEQ ID NOs:13 and 14 or complementary sequences
thereunto. The DNAs with SEQ ID NOs:13 and 14 are substantially the
same. The DNA with SEQ ID NO:14 contains coding regions for a
leader peptide, consisting of the nucleotides 15,607th-15,685th,
17,057th-17,068th and 20,452nd-20,468th, coding regions for the
present polypeptide, consisting of the nucleotides
20,469th-20,586th, 21,921st-22,054th and 26,828th-27,046th, and
regions as introns, consisting of the nucleotides
15,686th-17,056th, 17,069-20,451st, 20,587th-21,920th and
22,055th-26,827th. The genomic DNA with SEQ ID NO:13 is suitable
for expressing the polypeptide in mammalian host cells.
[0019] Generally in this field, when artificially expressing a DNA
encoding a polypeptide in a host, one or more nucleotides in a DNA
may be replaced by different ones, and appropriate promoter(s)
and/or enhancer(s) may be linked to the DNA to improve the
expressing efficiency or the properties of the expressed
polypeptide. The present genomic DNA can be altered similarly as
above. Therefore, as far as not substantially changing in the
biological activities of the expressed polypeptides, the present
genomic DNA should include DNAs encoding functional equivalents of
the polypeptide, formed as follows: One or more nucleotides in SEQ
ID NOs:3 to 14 are replaced by different ones, the untranslated
regions and/or the coding region for a leader peptide in the 5'-
and/or 3'-termini of SEQ ID NOs:3, 4, 5, 6, 7, 13 and 14 are
deleted, and appropriate oligonucleotides are linked to either or
both ends of SEQ ID NO:13.
[0020] The present genomic DNA includes general DNAs which are
derived from a genome containing the nucleotide sequences as above,
and it is not restricted to its sources or origins as far as it is
once isolated from its original organisms. For example, the present
genomic DNA can be obtained by chemically synthesizing based on SEQ
ID NOs:2 to 14, or by isolating from a human genomic DNA. The
isolation of the present genomic DNA from such a human genomic DNA
comprises (a) isolating a genomic DNA from human cells by
conventional methods, (b) screening the genomic DNA with probes or
primers, which are chemically synthesized oligonucleotides with a
part of or the whole of the nucleotide sequence of SEQ ID NO:2, and
(c) collecting a DNA to which the probes or primers specifically
hybridize. Once the present genomic DNA is obtained, it can be
unlimitedly replicated by constructing a recombinant DNA with an
autonomously replicable vector by conventional method and then
introducing the recombinant DNA into an appropriate host such as a
microorganism or an animal cell before culturing the transformant
or by applying a PCR method.
[0021] The present genomic DNA is very useful in producing the
polypeptide by recombinant DNA techniques since it efficiently
expresses the polypeptide with high biological activities when
introduced into mammalian host cells. The present invention further
provides a process for preparing a polypeptide using a specific
genomic DNA, comprising the steps of (a) culturing a transformant
formed by introducing the present genomic DNA into mammalian host
cells, and (b) collecting the polypeptide which induces IFN-.gamma.
production by immunocompetent cells from the resultant culture.
[0022] The following explains the process for preparing the
polypeptide according to the present invention. The present genomic
DNA is usually introduced into host cells in the form of a
recombinant DNA. The recombinant DNA, comprising the present
genomic DNA and an autonomously replicable vector, can be
relatively easily prepared by conventional recombinant DNA
techniques when the genomic DNA is available. The vectors, into
which the present genomic DNA can be inserted, include plasmid
vectors such as pcD, pcDL-SR.alpha., pKY4, pCDM8, pCEV4 and pME18S.
The autonomously replicable vectors usually further contain
appropriate nucleotide sequences for the expression of the present
recombinant DNA in each host cell, which include sequences for
promoters, enhancers, replication origins, transcription
termination sites, splicing sequences and/or selective markers.
Heat shock protein promoters or IFN-.alpha. promoters, as disclosed
in Japanese Patent Kokai No.163,368/95 by the same applicant of
this invention, enables to artificially regulate the present
genomic DNA expression by external stimuli.
[0023] To insert the present genomic DNA into vectors, conventional
methods used in this field can be arbitrarily used: Genes
containing the present genomic DNA and autonomously replicable
vectors are cleaved with restriction enzymes and/or ultrasonic, and
the resultant DNA fragments and the resultant vector fragments are
ligated. To cleave genes and vectors by restriction enzymes, which
specifically act on nucleotides, more particularly, AccI, BamHI,
BglII, BstXI, EcoRI, HindIII, NotI, PstI, SacI, SalI, SmaI, SpeI,
XbaI, XhoI, etc., facilitate the ligation of the DNA fragments and
the vector fragments. To ligate the DNA fragments and the vector
fragments, they are, if necessary, first annealed, then treated
with a DNA ligase in vivo or in vitro. The recombinant DNAs thus
obtained can be unlimitedly replicated in hosts derived from
microorganisms or animals.
[0024] Any cells conventionally used as hosts in this field can be
used as the host cells: Examples of such are epithelial,
interstitial and hemopoietic cells, derived from human, monkey,
mouse and hamster, more particularly, 3T3 cells, C127 cells, CHO
cells, CV-1 cells, COS cells, HeLa cells, MOP cells and their
mutants. Cells which inherently produce the present polypeptide
also can be used as the host cells: Example of such are human
hemopoietic cells such as lymphoblasts, lymphocytes, monoblasts,
monocytes, myeloblasts, myelocytes, granulocytes and macrophages,
and human epithelial and interstitial cells derived from solid
tumors such as pulmonary carcinoma, large bowel cancer and colon
cancer. More particular examples of the latter hemopoietic cells
are leukemia cell lines such as HBL-38 cells, HL-60 cells ATCC
CCL240, K-562 cells ATCC CCL243, KG-1 cells ATCC CCL246, Mo cells
ATCC CRL8066, THP-1 cells ATCC TIB202, U-937 cells ATCC CRL1593.2,
described by J. Minowada et al. in "Cancer Research", Vol.10,
pp.1-18 (1988), derived from leukemias or lymphoma including
myelogenous leukemias, promyelocytic leukemias, monocytic
leukemias, adult T-cell leukemias and hairy cell leukemias, and
their mutants. The present polypeptide-processibility of these
leukemia cell lines and their mutants is so distinguished that they
can easily yield the polypeptide with higher biological activities
when used as hosts.
[0025] To introduce the present DNA into the hosts, conventional
methods such as DEAE-dextran method, calcium phosphate transfection
method, electroporation method, lipofection method, microinjection
method, and viral infection method as using retrovirus, adenovirus,
herpesvirus and vaccinia virus, can be used. The
polypeptide-producing clones in the transformants can be selected
by applying the colony hybridization method or by observing the
polypeptide production after culturing the transformants in culture
media. For example, the recombinant DNA techniques using mammalian
cells as hosts are detailed in "Jikken-Igaku-Bessatsu Saibo-Kogaku
Handbook (The handbook for the cell engineering)" (1992), edited by
Toshio KUROKI, Masaru TANIGUCHI and Mitsuo OSHIMURA, published by
YODOSHA. CO., LTD., Tokyo, Japan, and "Jikken-Igaku-Bessatsu
Biomanual Series 3 Idenshi Cloning Jikken-Ho (The experimental
methods for the gene cloning)" (1993), edited by Takahi YOKOTA and
Ken-ichi ARAI, published by YODOSHA CO., LTD., Tokyo, Japan.
[0026] The transformants thus obtained secrete the present
polypeptide intracellularly and/or extracellularly when cultured in
culture media. As the culture media, conventional ones used for
mammalian cells can be used. The culture media generally comprise
(a) buffers as a base, (b) inorganic ions such as sodium ion,
potassium ion, calcium ion, phosphoric ion and chloric ion, (c)
micronutrients, carbon sources, nitrogen sources, amino acids and
vitamins, which are added depending on the metabolic ability of the
cells, and (d) sera, hormones, cell growth factors and cell
adhesion factors, which are added if necessary. Examples of
individual media include 199 medium, DMEM medium, Ham's F12 medium,
IMDM medium, MCDB 104 medium, MCDB 153 medium, MEM medium, RD
medium, RITC 80-7 medium, RPMI-1630 medium, RPMI-1640 medium and
WAJC 404 medium. The cultures containing the present polypeptide
are obtainable by inoculating the transformants into the culture
media to give a cell density of 1.times.10.sup.4-1.times.10.sup.7
cells/ml, more preferably, 1.times.10.sup.5-1.times.10.sup.6
cells/ml, and then subjecting to suspension- or monolayer-cultures
at about 37.degree. C. for 1-7 days, more preferably, 2-4 days,
while appropriately replacing the culture media with a fresh
preparation of the culture media. The cultures thus obtained
usually contain the present polypeptide in a concentration of about
1-100 .mu.g/ml, which may vary depending on the types of the
transformants or the culture conditions used.
[0027] While the cultures thus obtained can be used intact as an
IFN-.gamma. inducer, they are usually subjected to a step for
separating the present polypeptide from the cells or the cell
debris using filtration, centrifugation, etc. before use, which may
follow a step for disrupting the cells with supersonication,
cell-lytic enzymes and/or detergents if desired, and to a step for
purifying the polypeptide. The cultures from which the cells or
cell debris are removed are usually subjected to conventional
methods used in this field for purifying biologically active
polypeptides, such as salting-out, dialysis, filtration,
concentration, separatory sedimentation, ion-exchange
chromatography, gel filtration chromatography, adsorption
chromatography, chromatofocusing, hydrophobic chromatography,
reversed phase chromatography, affinity chromatography, gel
electrophoresis and/or isoelectric focusing. The resultant purified
polypeptide can be concentrated and/or lyophilized into liquids or
solids depending on final uses. The monoclonal antibodies disclosed
in Japanese Patent Kokai No.231,598/96 by the same applicant of
this invention are extremely useful to purify the present
polypeptide. Immunoaffinity chromatography using monoclonal
antibodies yields the present polypeptide in a relatively high
purity at the lowest costs and labors.
[0028] The polypeptide obtainable by the process according to the
present invention exerts strong effects in the treatment and/or the
prevention for IFN-.gamma.- and/or killer cell-susceptive diseases
since it possesses the properties of enhancing killer cells'
cytotoxicity and inducing killer cells' formation as well as
inducing IFN-.gamma., a useful biologically active protein, as
described above. The polypeptide according to the present invention
has a high activity of inducing IFN-.gamma., and this enables a
desired amount of IFN-.gamma. production with only a small amount.
The polypeptide is so low toxic that it scarcely causes serious
side effects even when administered in a relatively-high dose.
Therefore, the polypeptide has an advantage that it can readily
induce IFN-.gamma. in a desired amount without strictly controlling
the dosage. The uses as agents for susceptive diseases are detailed
in Japanese Patent Application No.28,722/96 by the same applicant
of this invention.
[0029] The present genomic DNA is also useful for so-called "gene
therapy". According to conventional gene therapy, the present DNA
can be introduced into patients with IFN-.gamma.- and/or killer
cell-susceptive diseases by directly injecting after the DNA is
inserted into vectors derived from viruses such as retrovirus,
adenovirus and adeno-associated virus or is incorporated into
cationic- or membrane fusible-liposomes, or by self-transplanting
lymphocytes which are collected from patients before the DNA is
introduced. In adoptive immunotherapy with gene therapy, the
present DNA is introduced into effector cells similarly as in
conventional gene therapy. This can enhance the cytotoxicity of the
effector cells to tumor cells, resulting in improvement of the
adoptive immunotherapy. In tumor vaccine therapy with gene therapy,
tumor cells from patients, into which the present genomic DNA is
introduced similarly as in conventional gene therapy, are
self-transplanted after proliferated ex vivo up to give a desired
cell number. The transplanted tumor cells act as vaccines in the
patients to exert a strong antitumor immunity specifically to
antigens. Thus, the present genomic DNA exhibits considerable
effects in gene therapy for diseases including viral diseases,
microbial diseases, malignant tumors and immunopathies. The general
procedures for gene therapy are detailed in "Jikken-Igaku-Bessatsu
Biomanual UP Series Idenshichiryo-no-Kisogijutsu (Basic techniques
for the gene therapy)" (1996), edited by Takashi ODAJIMA, Izumi
SAITO and Keiya OZAWA, published by YODOSHA CO., LTD., Tokyo,
Japan.
[0030] The following examples explain the present invention, and
the techniques used therein are conventional ones used in this
field: For example, the techniques are described in
"Jikken-Igaku-Bessatsu Saibo-Kogaku Handbook (The handbook for the
cell engineering)", (1992), edited by Toshio KUROKI, Masaru
TANIGUCHI and Mitsuo OSHIMURA, published by YODOSHA CO., LTD.,
Tokyo, Japan, and "Jikken-Igaku-Bessatsu Biomanual Series 3 Idenshi
Clonong Jikken-Ho (The experimental methods for the gene cloning)"
(1993), edited by Takahi YOKOTA and Ken-ichi ARAI, published by
YODOSHA CO., LTD., Tokyo, Japan.
EXAMPLE 1
Cloning Genomic DNA and Determination of Nucleotide Sequence
EXAMPLE 1-1
Determination of Partial Nucleotide Sequence
[0031] Five ng of "PromoterFinder.TM. DNA PvuII LIBRARY", a human
placental genomic DNA library commercialized by CLONTECH
Laboratories, Inc., California, USA, 5 .mu.l of 10.times.Tth PCR
reaction solution, 2.2 .mu.l of 25 mM magnesium acetate, 4 .mu.l of
2.5 mM dNTP-mixed solution, one .mu.l of the mixed solution of 2
unit/.mu.l rTth DNA polymerase XL and 2.2 .mu.g/.mu.l Tth Start
Antibody in a ratio of 4:1 by volume, 10 pmol of an oligonucleotide
with the nucleotide sequence of 5'-CCATCCTAA TACGACTCACTATAGGGC-3'
(SEQ ID NO:16) as an adaptor primer, and 10 pmol of an
oligonucleotide with the nucleotide sequence of
5'-TTCCTCTTCCCGAAGCTGTGTAGACTGC-3' (SEQ ID NO:17) as an anti-sense
primer, which was chemically synthesized based on the sequence of
the nucleotides 88th-115th in SEQ ID NO:2, were mixed and volumed
up to 50 .mu.l with sterilized distilled water. After incubating at
94.degree. C. for one min, the mixture was subjected to 7 cycles of
incubations at 94.degree. C. for 25 sec and at 72.degree. C. for 4
min, followed by 32 cycles of incubations at 94.degree. C. for 25
sec at 67.degree. C. for 4 min to perform PCR.
[0032] The reaction mixture was diluted by 100 folds with
sterilized distilled water. One .mu.l of the dilution, 5 .mu.l of
10.times.Tth PCR reaction solution, 2.2 .mu.l of 25 mM magnesium
acetate, 4 .mu.l of 2.5 mM dNTP-mixed solution, one .mu.l of the
mixed solution of 2 unit/.mu.l rTth DNA polymerase XL and 2.2
.mu.g/.mu.l Tth Start Antibody in a ratio of 4:1 by volume, 10 pmol
of an oligonucleotide with the nucleotide sequence of 5'-CTA
TAGGGCACGCGTGGT-3' (SEQ ID NO:18) as a nested primer, and 10 pmol
of an oligonucleotide with the nucleotide sequence of
5'-TTCCTCTTCCCGAAGCTGTGTAGACTGC-3' (SEQ ID NO:19) as an anti-sense
primer, which was chemically synthesized similarly as above, were
mixed and volumed up to 50 .mu.l with sterilized distilled water.
After incubating at 94.degree. C. for one min, the mixture was
subjected to 5 cycles of incubations at 94.degree. C. for 25 sec
and at 72.degree. C. for 4 min, followed by 22 cycles of
incubations at 94.degree. C. for 25 sec and at 67.degree. C. for 4
min to perform PCR for amplifying a DNA fragment of the present
genomic DNA. The genomic DNA library and reagents for PCR used
above were mainly from "PromoterFinder.TM. DNA WALKING KITS",
commercialized by CLONTECH Laboratories, Inc., California, USA
[0033] An adequate amount of the PCR product thus obtained was
mixed with 50 ng of "pT7 Blue(R)", a plasmid vector commercialized
by Novagen, Inc., WI, USA, and an adequate amount of T4 DNA ligase,
and 100 mM ATP was added to give a final concentration of one mM,
followed by incubating at 16.degree. C. for 18 hr to insert the DNA
fragment into the plasmid vector. The obtained recombinant DNA was
introduced into an Escherichia coli JM109 strain by the competent
cell method to form a transformant, which was then inoculated into
L-broth medium (pH 7.2) containing 50 .mu.g/ml ampicillin and
cultured at 37.degree. C. for 18 hr. The cells were isolated from
the resulting culture, and then subjected to the conventional
alkali-SDS method to collect a recombinant DNA. The dideoxy method
analysis confirmed that the recombinant DNA contained the DNA
fragment with a sequence of the nucleotides 5,150th-6,709th in SEQ
ID NO:14.
EXAMPLE 1-2
Determination of Partial Nucleotide Sequence
[0034] PCR was performed in the same conditions as the first PCR in
Example 1-1, but an oligonucleotide with the nucleotide sequence of
5'-GTAAGTTTTCACCTTCCAACTGTAGAGTCC-3' (SEQ ID NO:20), which was
chemically synthesized based on the nucleotide sequence of the DNA
fragment in Example 1-1, was used as an anti-sense primer.
[0035] The reaction mixture was diluted by 100 folds with
sterilized distilled water. One .mu.l of the dilution was placed
into a reaction tube, and PCR was performed in the same conditions
as used in the second PCR in Example 1-1 to amplify another DNA
fragment of the present genomic DNA, but an oligonucleotide with
the nucleotide sequence of 5'-GGGATCAAGT AGTGATCAGAAGCAGCACAC-3'
(SEQ ID NO:21), which was chemically synthesized based on the
nucleotide sequence of the DNA fragment in Example 1-1, was used as
an anti-sense primer.
[0036] The DNA fragment was inserted into the plasmid vector
similarly as in Example 1-1 to obtain a recombinant DNA. The
recombinant DNA was replicated in Escherichia coli before being
collected. The analysis of the collected recombinant DNA confirmed
that it contained the DNA fragment with a sequence of the
nucleotides 1st-5, 228th in SEQ ID NO:14.
EXAMPLE 1-3
Determination of Partial Nucleotide Sequence
[0037] 0.5 .mu.g of a human placental genomic DNA, commercialized
by CLONTECH Laboratories, Inc., California, USA, 5 .mu.l of
10.times.PCR reaction solution, 8 .mu.l of 2.5 mM dNTP-mixed
solution, one .mu.l of the mixed solution of 5 unit/.mu.l "TAKARA
LA Taq POLYMERASE" and 1.1 .mu.g/.mu.l "TaqStart ANTIBODY" in a
ratio of 1:1 by volume, both of them are commercialized by Takara
Syuzo Co., Tokyo, Japan, 10 pmol of an oligonucleotide with the
nucleotide sequence of 5'-CCTGGCTG CCAACTCTGGCTGCTAAAGCGG-3' (SEQ
ID NO:22) as a sense primer, chemically synthesized based on a
sequence of the nucleotides 46th-75th in SEQ ID NO:2, and 10 pmol
of an oligonucleotide with the nucleotide sequence of
5'-GTATTGTCAATAAATTTCATTGC CACAAAGTTG-3' (SEQ ID NO:23) as an
anti-sense primer, chemically synthesized based on a sequence of
the nucleotides 210th-242nd in SEQ ID NO:2, were mixed and volumed
up to 50 .mu.l with sterilized distilled water. After incubating at
94.degree. C. for one min, the mixture was subjected to 5 cycles of
incubations at 98.degree. C. for 20 sec and at 68.degree. C. for 10
min, followed by 25 cycles of incubations at 98.degree. C. for 20
sec and 68.degree. C. for 10 min, with adding 5 sec in times to
every cycle, and finally incubated at 72.degree. C. for 10 min to
amplify further DNA fragment of the present genomic DNA. The
reagents for PCR used above were mainly from "TAKARA LA PCR KIT
VERSION 2", commercialized by Takara Syuzo Co., Tokyo, Japan.
[0038] The DNA fragment was inserted into the plasmid vector
similarly as in Example 1-1 to obtain a recombinant DNA. The
recombinant DNA was replicated in Escherichia coli before being
collected. The analysis of the collected recombinant DNA confirmed
that it contained the DNA fragment with a sequence of the
nucleotides 6,640th-15,671st in SEQ ID NO:14.
EXPERIMENT 1-4
Determination of Partial Nucleotide Sequence
[0039] PCR was performed in the same conditions as the PCR in
Example 1-3 to amplify further another DNA fragment of the present
genomic DNA; but an oligonucleotide with the nucleotide sequence of
5'-AAGATGGCTGCTGAACCAGTAGAAGACAATTGC-3' (SEQ ID NO:24), chemically
synthesized based on a sequence of the nucleotide 175th-207th in
SEQ ID NO:2, was used as a sense primer, an oligonucleotide with
the nucleotide sequence of 5'-TCCTTGGTCAATGAAGAGAACTTGGTC-3' (SEQ
ID NO:25), chemically synthesized based on a sequence of
nucleotides 334th-360th in the SEQ ID NO:2, was used as an
anti-sense primer, and after incubating at 98.degree. C. for 20
sec, the reaction mixture was subjected to 30 cycles of incubations
at 98.degree. C. for 20 sec and at 68.degree. C. for 3 min,
followed by incubating at 72.degree. C. for 10 min.
[0040] The DNA fragment was inserted into the plasmid vector
similarly as in Example 1-1 to obtain a recombinant DNA. The
recombinant DNA was replicated in Escherichia coli before being
collected. The analysis of the collected recombinant DNA confirmed
that it contained the DNA fragment with a sequence of the
nucleotides 15,604th-20,543rd in SEQ ID NO:14.
EXAMPLE 1-5
Determination of Partial Nucleotide Sequence
[0041] PCR was performed in the same conditions as the PCR in
Example 1-4 to amplify further another DNA fragment of the present
genomic DNA, but an oligonucleotide with the nucleotide sequence of
5'-CCTGGAATCAGATTACTTTGGCAAGCTTGAATC-3' (SEQ ID NO:26), chemically
synthesized based on the sequence of the nucleotide 273rd-305th in
SEQ ID NO:2, was used as a sense primer, and an oligonucleotide
with the nucleotide sequence of
5'-GGAAATAATTTTGTTCTCACAGGAGAGAGTTG-3' (SEQ ID NO:27), chemically
synthesized based on the sequence of nucleotides 500th-531st in the
SEQ ID NO:2, was used as an anti-sense primer.
[0042] The DNA fragment was inserted into the plasmid vector
similarly as in Example 1-1 to obtain a recombinant DNA. The
recombinant DNA was replicated in Escherichia coli before being
collected. The analysis of the collected recombinant DNA confirmed
that it contained the DNA fragment with a sequence of the
nucleotides 20,456th-22,048th in SEQ ID NO:14.
EXAMPLE 1-6
Determination of Partial Nucleotide Sequence
[0043] PCR was performed in the same conditions as the PCR in
Example 1-4 to amplify further another DNA fragment of the present
genomic DNA, but an oligonucleotide with the nucleotide sequence of
5'-GCCAGCCTAGAGGTATGGCTGTAACTATCTC-3' (SEQ ID NO:28), chemically
synthesized based on the sequence of the nucleotide 449th-479th in
SEQ ID NO:2, was used as a sense primer, and an oligonucleotide
with the nucleotide sequence of
5'-GGCATGAAATTTTAATAGCTAGTCTTCGTTTTG-3' (SEQ ID NO:29), chemically
synthesized based on the sequence of nucleotides 745th-777th in the
SEQ ID NO:2, was used as an anti-sense primer.
[0044] The DNA fragment was inserted into the plasmid vector
similarly as in Example 1-1 to obtain a recombinant DNA. The
recombinant DNA was replicated in Escherichia coli before being
collected. The analysis of the collected recombinant DNA confirmed
that it contained the DNA fragment with a sequence of the
nucleotides 21,996th-27,067th in SEQ ID NO:14.
EXAMPLE 1-7
Determination of Partial Nucleotide Sequence
[0045] PCR was performed in the same conditions as the first PCR in
Example 1-2 to amplify further another DNA fragment in the present
genomic DNA, but an oligonucleotide with the nucleotide sequence of
5'-GTGACATCATATTCTTTCAGA GAAGTGTCC-3' (SEQ ID NO:30), chemically
synthesized based on the sequence of the nucleotide 575th-604th in
SEQ ID NO:2, was used as a sense primer.
[0046] The reaction mixture was diluted by 100 folds with
sterilized distilled water. One .mu.l of the dilution was placed
into a reaction tube, and PCR was performed in the same conditions
as the second PCR in Example 1-2 to amplify further another DNA
fragment of the present genomic DNA, but an oligonucleotide with
the sequence of 5'-GCAATTTGAATCTTCATC ATACGAAGGATAC-3' (SEQ ID
NO:31), chemically synthesized based on a sequence of the
nucleotides 624th-654th in SEQ ID NO:2, was used as a sense
primer.
[0047] The DNA fragment was inserted into the plasmid vector
similarly as in Example 1-1 to obtain a recombinant DNA. The
recombinant DNA was replicated in Escherichia coli before being
collected. The analysis of the collected recombinant DNA confirmed
that it contained the DNA fragment with a sequence of the
nucleotides 26,914th-28,994th in SEQ ID NO:14.
EXAMPLE 1-8
Determination of Complete Nucleotide Sequence
[0048] Comparing the nucleotide sequence of SEQ ID NO:2, which was
proved to encode the present polypeptide, as disclosed in Japanese
Patent Kokai No.193,098/96 by the same applicant of this invention,
with the partial nucleotide sequences identified in Examples 1-1 to
1-7, it was proved that the present genomic DNA contained the
nucleotide sequence of SEQ ID NO:14. SEQ ID NO:14, consisting of
28,994 base pairs (bp), was extremely longer than the SEQ ID NO:2,
consisting of only 471 bp. This suggested that SEQ ID NO:14
contained introns, a characteristic of eukaryotic cells.
[0049] It was examined where partial nucleotide sequences of SEQ ID
NO:2, i.e., exons, and the donor and acceptor sites in introns,
respectively consisting of the nucleotides of GT and AG, located in
SEQ ID NO:14. Consequently, it was proved that SEQ ID NO:14
contained at least 5 introns, which located in the order of SEQ ID
NOs:10, 11, 12, 8 and 9 in the direction from the 5'- to the
3'-termini. Therefore, the sequences between the neighboring
introns must be exons, which were thought to be located in the
order of SEQ ID NOs:5, 6, 3, 4 and 7 in the direction from the 5'-
to the 3'-termini. It was also proved that SEQ ID NO:7 contained
the 3'-untranslated region other than the exons. The features of
the sequence elucidated as this are arranged in SEQ ID NO:14.
[0050] As disclosed in Japanese patent application by the same
applicant of this invention, the present polypeptide is produced as
a polypeptide with N-terminal amino acid of tyrosine other than
methionine in human cells, which is observed in SEQ ID NO:1. This
suggests that the present genomic DNA contains a leader peptide
region in the upstream of the 5'-terminus of the present
polypeptide-encoding region. A sequence consisting of 36 amino
acids encoded by the upstream of the nucleotides 20,469th-20,471st.
which is the nucleotides of TAC, are described as a leader peptide
in SEQ ID NO:14.
EXAMPLE 2
Preparation of Recombinant DNA pBGHuGF for Expression
[0051] 0.06 ng of the DNA fragment in Example 1-4 in a
concentration of 3 ng/50 .mu.l, 0.02 ng of the DNA fragment,
obtained by the methods in Example 1-5, 5 .mu.l of 10.times.LA PCR
reaction solution, 8 .mu.l of 2.5 mM dNTP-mixed solution, one .mu.l
of the mixed solution of 5 unit/.mu.l TAKARA LA Taq polymerase and
1.1 .mu.g/.mu.l TaqStart Antibody in a ratio of 1:1 by volume, 10
pmol of an oligonucleotide with the sequence of 5'-TCC
GAAGCTTAAGATGGCTGCTGAACCAGTA-3' (SEQ ID NO:32) as a sense primer,
chemically synthesized based on the nucleotide sequence of the DNA
fragment in Example 1-4, and 10 pmol of an oligonucleotide with the
nucleotide sequence of 5'-GGAAATAA TTTTGTTCTCACAGGAGAGAGTTG-3' (SEQ
ID NO:33) as an anti-sense primer, chemically synthesized based on
the nucleotide sequence of the DNA fragment in Example 1-5, were
mixed and volumed up to 50 .mu.l with sterilized distilled water.
After incubating at 94.degree. C. for one min, the mixture was
subjected to 5 cycles of incubations at 98.degree. C. for 20 sec
and at 72.degree. C. for 7 min, followed by 25 cycles of
incubations at 98.degree. C. for 20 sec and 68.degree. C. for 7 min
to perform PCR. The reaction mixture was cleaved by restriction
enzymes HindIII and SphI to obtain a DNA fragment of about 5,900
bp, with cleavage sites by HindIII and SphI in its both
termini.
[0052] PCR was performed in the same condition as above, but 0.02
ng of the DNA fragment in Example 1-5, 0.06 ng of the DNA fragment
obtained in Example 1-6, an oligonucleotide with the nucleotide
sequence of 5'-ATGTAGCGGCCGCGGCATGAAATTTTAA TAGCTAGTC-3' (SEQ ID
NO:34) as an anti-sense primer, chemically synthesized based on the
nucleotide sequence of the DNA fragment in Example 1-6, and an
oligonucleotide with the sequence of
5'-CCTGGAATCAGATTACTTTGGCAAGCTTGAATC-3' (SEQ ID NO:35) as a sense
primer, chemically synthesized based on the DNA fragment in Example
1-6, were used. The reaction mixture was cleaved by restriction
enzymes NotI and SphI to obtain a DNA fragment of about 5,600 bp,
with cleavage sites by NotI and SphI in its both termini.
[0053] A plasmid vector "pRc/CMV", containing a cytomegalovirus
promoter, commercialized by Invitrogen Corporation, San Diego, USA,
was cleaved by restriction enzymes HindIII and NotI to obtain a
vector fragment of about 5,500 bp. The vector fragment was mixed
with the above two DNA fragments of about 5,900 bp and 5,600 bp,
and reacted with T4 DNA ligase to insert the two DNA fragments into
the plasmid vector. An Escherichia coli JM109 strain was
transformed with the obtained recombinant DNA, and the transformant
with the plasmid vector was selected by the colony hybridization
method. The selected recombinant DNA was named as "pBGHuGF". As
shown in FIG. 1, the present genomic DNA, with the nucleotide
sequence of SEQ ID NO:13, was ligated in the downstream of the
cleavage site by the restriction enzyme HindIII in the recombinant
DNA.
EXAMPLE 3
Preparation of Transformant Using CHO Cell as Host
[0054] CHO-K1 cells ATCC CCL61 were inoculated into Ham's F12
medium (pH 7.2) containing 10 v/v % bovine fetal serum and
proliferated by conventional manner. The proliferated cells were
collected and washed with phosphate-buffered saline (hereinafter
abbreviated as "PBS") followed by suspending in PBS to give a cell
density of 1.times.10.sup.7 cells/ml.
[0055] 10 .mu.g of the recombinant DNA pBGHuGF in Example 2 and 0.8
ml of the above cell suspension were placed in a cuvette and
ice-chilled for 10 min. The cuvette was installed in "GENE PULSER",
an electroporation device commercialized by Bio-Rad Laboratories
Inc., Brussels, Belgium, and then pulsed once with an electric
discharge. After pulsing, the cuvette was immediately took out and
ice-chilled for 10 min. The cell suspension from the cuvette was
inoculated into Ham's F12 medium (pH 7.2) containing 10 v/v %
bovine fetal serum and cultured under an ambient condition of 5 v/v
% CO.sub.2 at 37.degree. C. for 3 days. To the culture medium was
added G-418 to give a final concentration of 400 .mu.g/ml, and the
culturing was continued further 3 weeks under the same conditions.
From about 100 colonies formed, 48 colonies were selected, and a
portion of each was inoculated into a well of culturing plates with
Ham's F12 medium (pH7.2) containing 400 .mu.g/ml G-418 and 10 v/v %
bovine fetal serum and cultured similarly as above. Thereafter, to
each well of the culturing plates was added 10 mM Tris-HCl buffer
(pH 8.5) containing 5.1 mM magnesium chloride, 0.5 w/v % sodium
deoxycholate, 1 w/v % NONIDET P-40, 10 .mu.g/ml aprotinin and 0.1
w/v % SDS to lyse the cells.
[0056] 50 .mu.l aliquot of the cell lysates was mixed with one ml
of glycerol and incubated at 37.degree. C. for one hour, before the
polypeptides in the cell lysates were separated by the
SDS-polyacrylamide gel electrophoresis. The separated polypeptides
were transferred to a nitrocellulose membrane in usual manner, and
the membrane was soaked in the culture supernatant of the hybridoma
H-1, disclosed in Japanese Patent Kokai No.231,598/96 by the same
applicant of this invention, followed by washing with 50 mM
Tris-HCl buffer containing 0.05 v/v % TWEEN 20 to remove an
excessive mount of the monoclonal antibody. Thereafter, the
nitrocellulose membrane was soaked in PBS containing rabbit-derived
anti-mouse immunoglobulin antibody for one hr, which was labeled
with horseradish peroxidase, followed by washing 50 mM Tris-HCl
buffer (pH 7.5) containing 0.05 v/v % TWEEN 20 and soaking in 50 mM
Tris-HCl buffer (pH 7.5) containing 0.005 v/v % hydrogen peroxide
and 0.3 mg/ml diaminobenzidine to develop colorations. The clone,
which highly produced the polypeptide, was selected based on the
color development and named "BGHuGF".
EXAMPLE 4
Production of Polypeptide by Transformant and its Physicochemical
Properties
[0057] The transformant BGHuGF in Experiment 3 was inoculated into
Ham's F12 medium (pH 7.2) containing 400 .mu.g/ml G-418 and 10 v/v
% bovine fetal serum, and cultured under an ambient condition of 5
v/v % CO.sub.2 at 37.degree. C. for one week. The proliferated
cells were collected, washed with PBS, and then washing with
10-fold volumes of ice-chilled 20 mM Hepes buffer (pH 7.4),
containing 10 mM potassium chloride and 0.1 mM
ethylendiaminetetraacetate bisodium salt, according to the method
described in "Proceedings of The National Academy of The Sciences
of The USA", vol.86, pp.5,227-5,231 (1989), by M. J. Kostura et al.
The cells thus obtained were allowed to stand in 3-fold volumes of
a fresh preparation of the same buffer under an ice-chilling
condition for 20 min and freezed at -80.degree. C., succeeded by
thawing to disrupt the cells. The resulting cells were centrifuged
to collect the supernatant.
[0058] In parallel, THP-1 cells ATCC TIB 202, derived from a human
acute monocytic leukemia, was similarly cultured and disrupted.
Supernatant, obtained by centrifuging the resulting cells, was
mixed with the supernatant obtained from the transformant BGHuGF
and incubated at 37.degree. C. for 3 hr to react. The reaction
mixture was applied to a column with "DEAE-SEPHAROSE", a gel for
ion-exchange chromatography, commercialized by Pharmacia LKB
Biotechnology AB, Upsalla, Sweden, equilibrated with 10 mM
phosphate buffer (pH 6.6) before use. After washing the column with
10 mM phosphate buffer (pH 6.6), 10 mM phosphate buffer (pH 6.6)
with a stepwise gradient of NaCl increasing from 0 M to 0.5 M was
fed to the column, and fractions eluted by about 0.2 M NaCl were
collected. The fractions were dialyzed against 10 mM phosphate
buffer (pH 6.8) before applied to a column with "DEAE 5PW", a gel
for ion-exchange chromatography, commercialized by TOSOH
Corporation, Tokyo, Japan. To the column was fed 10 mM phosphate
buffer (pH 6.8) with a linear gradient of NaCl increasing from 0 M
to 0.5 M, and fractions eluted by about 0.2-0.3 M NaCl were
collected.
[0059] While the obtained fractions were pooled and dialyzed
against PBS, a gel for immunoaffinity chromatography with the
monoclonal antibody were prepared according to the method disclosed
in Japanese Patent Kokai No.231,598/96 by the same applicant of
this invention. After the gel were charged into a plastic column
and washed with PBS, the above dialyzed solution was applied to the
column. To the column was fed 100 mM glycine-HCl buffer (pH 2.5),
and the eluted fractions, which contained a polypeptide capable of
inducing the production of IFN-.gamma. by immunocompetent cells,
were collected. After the collected fractions were dialyzed against
sterilized distilled water and concentrated with a membrane
filtration, the resultant was lyophilized to obtain a purified
solid polypeptide in a yield of about 15 mg/l-culture.
EXAMPLE FOR REFERENCE
Expression in Escherichia coli
[0060] As disclosed in Japanese Patent Kokai No.193,098/96, a
transformant pKHuGF which was obtained by introducing a cDNA with
the nucleotide sequence of SEQ ID NO:2 into Escherichia coli as a
host, was inoculated into L-broth medium containing 50 .mu.g/ml
ampicillin and cultured at 37.degree. C. for 18 hr under shaking
conditions. The cells were collected by centrifuging the resulting
culture, and then suspended in a mixture solution (pH 7.2) of 139
mM NaCl, 7 mM NaH.sub.2PO.sub.4 and 3 mM Na.sub.2HPO.sub.4,
followed by supersonicating to disrupt the cells. After the cell
disruptants were centrifuged, the supernatant was subjected to
purifying steps similarly as in Example 4-1 to obtain a purified
solid polypeptide in a yield of about 5 mg/l-culture.
[0061] Comparing the yields of the polypeptides in Example for
Reference and in Example 4-1 shows that the use of a transformant,
which is formed by introducing a genomic DNA encoding the present
polypeptide into a mammalian cell as a host, strongly elevates the
yield of the polypeptide per culture.
EXAMPLE 4-2
Physicochemical Property of Polypeptide
EXAMPLE 4-2(a)
Biological Activity
[0062] Blood were collected from a healthy donor by using a syringe
containing heparin, and then diluted with 2-fold volume of
serum-free RPMI-1640 medium (pH 7.4). The blood was overlaid on
ficoll, commercialized by Pharmacia LKB Biotechnology AB, Upsalla,
Sweden, and centrifuged to obtain lymphocytes, which were then
washed with RPMI-1640 medium containing 10 v/v % bovine fetal serum
before being suspended in a fresh preparation of the same medium to
give a cell density of 5.times.10.sup.6 cells/ml. 0.15 ml aliquots
of the cell suspension was distributed into wells of micro plates
with 96 wells.
[0063] To the wells with the cells were distributed 0.05 ml
aliquots of solutions of the polypeptide in Example 4-1, diluted
with RPMI-1640 medium (pH 7.4) containing 10 v/v % bovine fetal
serum to give desired concentrations. 0.05 ml aliquots of fresh
preparations of the same medium with 2.5 .mu.g/ml concanavalin A
were further added to the wells, before culturing in a 5 v/v %
CO.sub.2 incubator at 37.degree. C. for 24 hr. After the
cultivation, 0.1 ml of the culture supernatant was collected from
each well and examined on IFN-.gamma. by usual enzyme immunoassay.
In parallel, a systems as a control using the polypeptide in
Reference for that in Example 4-1 or using no polypeptide was
treated similarly as above. The results were in Table 1.
IFN-.gamma. in Table 1 were expressed with international units
(IU), calculated based on the IFN-.gamma. standard, Gg23-901-530,
obtained from the International Institute of Health, USA
1 TABLE 1 Sample of polypeptide IFN-.gamma. production (IU/ml)
Example 4-2 (a) 3.4 .times. 10.sup.5 Example for Reference 1.7
.times. 10.sup.5
[0064] Table 1 indicates that the lymphocytes as immunocompetent
cells produce IFN-.gamma. by the action of the present
polypeptide.
[0065] It is more remarkable that the polypeptide in Example 4-1
could induce IFN-.gamma. production more than that in Example for
Reference. Considering this and the difference in the yields of the
polypeptides, described in Example for Reference, it can be
presumed: Even if DNAs could be substantially equivalent in
encoding the same amino acid sequence, not only the expressing
efficiencies of the DNAs may differ, but the products expressed by
them may significantly differ in their biological activities as a
result of post-translational modifications by intracellular
enzymes, depending on types of the DNAs and their hosts; (a) one
type is used a transformant formed by introducing a DNA, which is a
cDNA, into a microorganisms as a host, and (b) other type is used a
transformant formed by introducing the present genomic DNA into a
mammalian cell as a host.
EXAMPLE 4-2(b)
Molecular Weight
[0066] SDS-polyacrylamide gel electrophoresis of the polypeptide in
Example 4-1 in the presence of 2 w/v % dithiothreitol as a reducing
agent, according to the method reported by U. K. Laemli et al., in
"Nature", Vol.227, pp.680-685 (1970), exhibited a main band of a
protein capable of inducing IFN-.gamma. in a position corresponding
to a molecular weight of about 18,000-19,500 daltons. The molecular
weight makers used in the analysis were bovine serum albumin
(67,000 daltons), ovalbumin (45,000 daltons), carbonic anhydrase
(30,000 daltons), soy bean trypsin inhibitor (20,100 daltons) and
.alpha.-lactoalbumin (14,000 daltons).
EXAMPLE 4-2(c)
N-Terminal Amino Acid Sequence
[0067] Conventional analysis using "MODEL 473A", a protein
sequencer commercialized by Perkin-Elmer Corp., Norwalk, USA,
revealed that the polypeptide in Example 4-1 had the amino acid
sequence of SEQ ID NO:15 in the N-terminal region.
[0068] Judging collectively from this result as well as the
information that SDS-polyacrylamide gel electrophresis exhibited a
main band in a position corresponding to a molecular weight of
about 18,000-19,500 daltons, and that the molecular weight
calculated from the amino acid sequence of SEQ ID NO:1 was 18,199
daltons, it can be concluded that the polypeptide in Example 4-1
has the amino acid sequence of SEQ ID NO:6.
[0069] As is described above, the present invention is made based
on the identification of a genomic DNA encoding the polypeptide
which induces the production of IFN-.gamma. by immunocompetent
cells. The present genomic DNA efficiently express the present
polypeptide when introduced into mammalian host cells. The
polypeptide features higher biological activities than that
obtained by the cDNA expression in Escherichia coli. Therefore, the
present genomic DNA is useful for the recombinant DNA techniques to
prepare the polypeptide capable of inducing IFN-.gamma. production
by immunocompetent cells. The present genomic DNA is useful to gene
therapy for diseases including viral diseases, bacterial-infectious
diseases, malignant tumors and immunopathies.
[0070] Thus, the present invention is a significant invention which
has a remarkable effect and gives a great contribution to this
field.
[0071] While there has been described what is at present considered
to be the preferred embodiments of the present invention, it will
be understood the various modifications may be made therein, and it
is intended to cover in the appended claims all such modifications
as fall within the true spirits and scope of the invention.
Sequence CWU 1
1
* * * * *