U.S. patent application number 11/415575 was filed with the patent office on 2006-12-07 for antibody against a mast cell surface antigen.
Invention is credited to Fukiko Atsumi, Makoto Kawai, Motoki Kuhara, Tadashi Okada, Masao Shibata.
Application Number | 20060276631 11/415575 |
Document ID | / |
Family ID | 36570684 |
Filed Date | 2006-12-07 |
United States Patent
Application |
20060276631 |
Kind Code |
A1 |
Kawai; Makoto ; et
al. |
December 7, 2006 |
Antibody against a mast cell surface antigen
Abstract
A mast cell surface antigen, DNA thereof and an antibody against
the antigen are provided. The amino acid sequence of this mast cell
surface antigen is the translation of the coding region of its DNA.
The base sequence of this DNA has been clarified in the following
manner. Namely, mast cells obtained by incubating cord blood
monocular cells are co-incubated with primary culture of
fibroblasts to give connective tissue type mast cells (MC-TC). Then
mRNA is extracted from this MC-TC cell extraction and a cDNA
library is constructed therefrom. Immunological screening is
carried out with the use of anti-MC-TC antiserum and the base
sequence of the positive clone thus obtained is identified. Owing
to the clarification of the amino acid sequence of this mast cell
antigen, it becomes possible to reveal the role of mast cells in
the pathology of allergic diseases and thus an antibody against
mast cells can be easily obtained.
Inventors: |
Kawai; Makoto; (Nagoya-shi,
JP) ; Okada; Tadashi; (Nisshin-shi, JP) ;
Atsumi; Fukiko; (Nagoya-shi, JP) ; Shibata;
Masao; (Ina-shi, JP) ; Kuhara; Motoki;
(Ina-shi, JP) |
Correspondence
Address: |
DAVIS & BUJOLD, P.L.L.C.
112 PLEASANT STREET
CONCORD
NH
03301
US
|
Family ID: |
36570684 |
Appl. No.: |
11/415575 |
Filed: |
May 2, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10250644 |
Nov 4, 2003 |
7045597 |
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PCT/JP01/00005 |
Jan 4, 2001 |
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11415575 |
May 2, 2006 |
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09229932 |
Jan 13, 1999 |
6255107 |
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10250644 |
Nov 4, 2003 |
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Current U.S.
Class: |
530/350 ;
530/388.22; 536/23.5 |
Current CPC
Class: |
C07K 16/28 20130101;
C07K 14/705 20130101 |
Class at
Publication: |
530/350 ;
530/388.22; 536/023.5 |
International
Class: |
C07K 14/74 20060101
C07K014/74; C07K 16/28 20060101 C07K016/28; C07H 21/04 20060101
C07H021/04 |
Claims
1-3. (canceled)
4. An antibody that specifically reacts against an isolated mast
cell surface antigen comprising an amino acid sequence listed in
SEQ ID NO. 1.
Description
[0001] This application is a divisional application of Ser. No.
10/250,644 filed Jan. 4, 2001, now U.S. Pat. No. 7,045,597, which
is a national stage completion of PCT/JP01/00005 filed Jul. 3, 2001
which is a continuation-in-part of Ser. No. 09/229,932 filed Jan.
13, 1999, now U.S. Pat. No. 6,255,107.
TECHNICAL FIELD OF THE INVENTION
[0002] This invention relates to a mast cell surface antigen, DNA
thereof, and antibody against the antigen.
BACKGROUND OF THE INVENTION
[0003] Allergic diseases, such as bronchial asthma or allergic
rhinitis, are induced as follows: firstly, antigen-specific IgE
production is induced, and then, from mast cells, basophils and the
like, activated by the induced IgE, various chemical mediators such
as histamine, eosinophil chemotactic factor in allergy (ECF-A),
leukotrienes, platelet-activating factor (PAF) and thromboxane are
produced and released. Specifically in tissues, the mast cells
release these chemical mediators, and therefore play an important
role in development of allergic diseases.
[0004] Human mast cells are differentiated into tryptase positive
cell (MC-T) and both tryptase and chymase positive cells (MC-TC),
according to the granule content of proteolytic enzyme in the mast
cells. MC-T are mainly distributed in the lung tissues and the
gastrointestinal tract mucosa, whereas MC-TC are distributed in the
skin tissues. These mast cells, unlike cells of other leukocytes,
leave bone marrow for the peripheral environment as pluripotent
stem cells, and differentiate into MC-T or MC-TC, followed by
adhesion to either lung or skin fibroblasts. Since it is believed
that such mast cells play a major role in development of allergic
diseases, it is necessary to specifically detect and separate the
mast cells in order to clarify the physiological functions of the
mast cells.
[0005] However, heretofore, no cell surface antigen specific to the
mast cells has been known. Since the antibody against the cell
surface antigen specific to the mast cells will allow us to
specifically remove or eliminate the mast cells, identification of
the cell surface antigen specific to the mast cells has an
important meaning not only in clarification of the underlying cause
of allergic diseases but in treatment thereof.
[0006] An object of the present invention, which was made to solve
the above problem, is to provide a mast cell surface antigen, DNA
thereof, and an antibody against the antigen.
DISCLOSURE OF THE INVENTION
[0007] The mast cell surface antigen comprises an amino acid
sequence listed in SEQ ID NO. 1, or a substantially identical amino
acid sequence. This amino acid sequence is a translation of the
coding region of DNA of the mast cell surface antigen. The base
sequence of the DNA of the mast cell surface antigen has been
clarified in the following manner. Namely, as explained in detail
in an embodiment section, after mast cells are obtained from cord
blood monocular cells, mRNA is extracted from cell extraction of
these mast cells, and a cDNA library is constructed from the mRNA.
Immunological screening of the cDNA library is carried out using
the antiserum, and the base sequence of the positive clone thus
obtained is identified by means of a DNA sequencer. In the base
sequence listed in SEQ ID NO. 2, a sequence of 36-38, namely ATG,
is the initiation codon, and a sequence of 2394-2396, namely, TGA,
is the termination codon. In other words, a sequence of 36-2396 is
the coding region, and the base sequence in the range codes the
amino acid sequence listed in SEQ ID NO. 1. Identification of the
amino acid sequence of this mast cell antigen allows us to clarify
the role of the mast cells in development of allergic diseases, and
thus to obtain the antigen which specifically reacts to the mast
cells.
[0008] An antibody against the mast cell surface antigen can be
obtained in the following steps, for example. Firstly, the mast
cell antigen comprising the amino acid sequence listed in SEQ ID
NO. 1 is injected to a mammal (except human) for immunization, and
fused cells are prepared by fusing the antibody producing cells
obtained from the immunized mammal with myeloma cells. Then, from
the fused cells, a clone which produces an antibody that reacts
with the mast cell surface antigen is selected and cultured, and
the supernatant of the culture is purified. The antibody allows us
to specifically remove or eliminate the mast cells, and thus to
treat allergic diseases. In short, this antibody is expected to
work as antiallergic agent.
[0009] Cells that produce this antibody can be obtained in the
following steps, for example. Firstly, the mast cell antigen
comprising the amino acid sequence listed in SEQ ID NO. 1 is
injected to a mammal (except human) for immunization, and fused
cells are prepared by fusing the antibody producing cells obtained
from the immunized mammal with myeloma cells. Then, from the fused
cells, a clone which produces an antibody that reacts with the mast
cell surface antigen is selectively cultured.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a graph showing the ratio of chymase positive mast
cells and measured values of tryptase concentration for the mast
cells before and after coculture;
[0011] FIG. 2 is an explanatory view showing base Bcsc-1; and
[0012] FIG. 3 is an explanatory view showing a construction of a
cDNA expression vector BCMGSNeo.
BEST MODE FOR CARRYING OUT THE INVENTION
[0013] [1] Culture of MC-TC
[0014] Cord blood treated with heparin was layered over the Ficoll
Hypaque solution (specific gravity: 1.077, Sigma Inc.), centrifuged
at 300.times.g for 30 minutes at room temperature to separate the
mononuclear cells, which were then suspended in RPMI1640 medium
(Nissui Seiyaku) containing 10% of FBS (Gibco BRL), 50 M of
2-mercaptoethanol, 4 mM of L-glutamine, 100 U/ml of penicillin and
50 g/ml of streptomycin. The concentration of the mononuclear cells
in the suspension was adjusted to 5.times.10.sup.6/ml, and the
suspension was poured into a collagen coated culture dish (Iwaki
Glass) having a diameter of 10 cm, and, added with SCF (100 ng/ml,
PeproTech Inc.) and IL-6 (50 ng/ml, PeproTech Inc.), cultured for 2
weeks to obtain two-week cultured cells containing neutrophils,
lymphocytes, macrophages, basophils and precursor cells of mast
cell. SCF is a factor participating in differentiation and
proliferation of mast cells expressed on fibroblasts. It is an
abbreviation for stem cell factor.
[0015] The cord blood mononuclear cells obtained in the above were
cultured in the presence of 100 ng/ml of SCF and 50 ng/ml of IL-6
for 6 weeks, and when the human mast cells became predominant, that
is, when the number of human mast cells reached the order of 106,
the mast cells were further cocultured with a primary culture of
human fibroblasts. Specifically, the human mast cells were
transferred to a monolayer of human fibroblasts from either skin or
lung tissues, and cultured for 2 months in the presence of 50 ng/ml
SCF.
[0016] The ratio of chymase positive mast cells and the
concentration of tryptase were measured for the mast cells before
and after the coculture. The result is shown in FIG. 1. FIG. 1
shows that before the coculture of mast cells, i.e. for the human
mast cells cultured for 10-16 weeks in the presence of SCF and IL-6
(the left end of the graph in FIG. 1), the ratio of chymase
positive cells and the concentration of tryptase were both very
low, whereas those for the human mast cells after cocultured with
human fibroblasts for 6-8 weeks (the center of the graph in FIG. 1)
and those for the human mast cells cocultured with lung fibroblasts
(the right end of the graph in FIG. 1) showed remarkable increases,
especially for the human mast cells cocultured with skin
fibroblasts.
[0017] Staining was performed to the mast cells cultured for 15
weeks in the presence of SCF and IL-6 and the mast cells cocultured
with skin fibroblasts for 2 months after having been cultured for 6
weeks, using antibodies against tryptase and those against chymase.
As a result, in case of staining against tryptase, it was confirmed
that both types of mast cells were wholly stained, whereas in case
of staining against chymase, it was confirmed that most cells
cocultured with fibroblast were stained but cells which were not
cocultured were only partly stained. The results show that MC-T is
differentiated into MC-TC by the coculture.
[0018] As described above, connective tissue type-human mast cells,
or MC-TC, were obtained by culturing cord blood monocular cells in
the presence of SCF and IL-6 and subsequently coculturing them with
the primary culture of human skin fibroblasts.
[0019] [2] Preparation of mRNA
[0020] According to the manual of "Quick-Prep Micro mRNA
Purification Kit" of Pharmacia, poly(A)+mRNA was extracted from the
cell extraction of the above MC-TC in the following steps.
[0021] 2.times.10.sup.6 of MC-TC was centrifuged and washed with
PBS twice, added with 400 L of elution buffer accompanying the
aforementioned kit, stirred well by a vortex mixer (rotary beater),
further added with 800 L of elution buffer and stirred by the
vortex mixer. The solution obtained was transferred into an assist
tube (Assist) made of polypropyrene, and centrifuged at 14,000 rpm
for 5 minutes to separate the precipitate.
[0022] 1 mL of oligo dT cellulose was centrifuged at 14,000 rpm to
separate the supernatant.
[0023] The oligo dT cellulose in 2) was added to the cell
extraction in 1), and the solution was well mixed by inverting the
assist tube for 3 minutes, centrifuged at 14,000 rpm for 5 seconds
to separate the supernatant, which was then washed with 1 mL of
high salt concentration buffer 5 times and then in low salt
concentration buffer 2 times.
[0024] The precipitate was suspended in 300 L of low salt
concentration buffer, put in a microspin column accompanying the
aforementioned kit, which was centrifuged for 5 seconds, and washed
with 500 L of low salt concentration buffer 3 times.
[0025] 200 L of elution buffer warmed up to 65.degree. C. was added
to the column, and 10 L of glycogen solution and 400 L of potassium
acetate solution were further added. After 1 mL of 95% ethanol was
added, the column was centrifuged at 14,000 rpm for 1 hour at
4.degree. C., and dried under reduced pressure.
[0026] [3] Construction of cDNA Library
[0027] According to the manual of "Zap-cDNA synthesis kit" of
Stratagene, a cDNA library was constructed from mRNA obtained in
the above [2]. Size fractionation was carried out using CL-2B gel
accompanying this kit.
[0028] [4] Immunological Screening
[0029] A phage solution of the cDNA library constructed in the
above [3] was diluted with SM buffer (containing 50 mM of Tris-HCl
(pH7.5), 100 mM of NaCl, 10 mM of MgSO.sub.4.H.sub.2O, and 0.01%
gelatin), to give a phage concentration of 2.5-4.times.10.sup.5
pfu/mL. This solution was dispensed per 100 L to 10 centrifuging
tubes. Added with 500 L of overnight culture solution of
Escherichia coli (XL-1blue) (which was cultured overnight in 5 mL
of NZY medium so as to obtain O.D..sub.600=1.5), the solution was
stirred with a vortex mixer, and incubated at 37.degree. C. for 15
minutes. Further added with 7 mL of soft agarose (which was made to
give a concentration of 0.6%, by dissolving agarose gel for
electrophoretic migration in a culture medium), the solution was
stirred by the vortex mixer, and spread in a 14.times.10 cm square
dish. When the agarose was set, the dish was soaked in 20 mL of
IPTG, and with a dried nitrocellulose filter thereon, incubated at
37.degree. C. for 4 hours. For the NZY medium, 5 g of NaCl, 2 g of
MgSO.sub.4.7H.sub.2O, 5 g of yeast extract, and 5 g of NZamine
(casein hydrolysate) were dissolved in 1 liter of water, the pH was
adjusted to 7.5 with NaOH, the solution added with 15 g agar was
sterilized and dissolved in an autoclave.
[0030] After the incubation, the filter was removed, and washed
with shaking with 300 mL PBS for 15 minutes, which was repeated 2
times. Then, the PBS was discarded. Added with 300 mL of PBS
(PBS-SM) containing 0.5% skim milk, the filter was washed with
shaking for 1 hour at room temperature for blocking. Added with a
rat antiserum (which is an antiserum obtained from a rat which
provided its spleen cells for preparation of a later-described
hybridoma clone ahMC5C12 (Accession No. FERM BP-6070)) diluted
1:400 in PBS-SM, the filter was washed with shaking for 1 hour at
room temperature. The antiserum solution was discarded, and the
solution was washed with shaking with PBS plus 0.05% Tween (PBS-T)
for 5 minutes at room temperature, which was repeated 4 times.
Then, added with 200 mL of peroxydase labeled anti-rat IgG (Medical
& Biological Laboratories Co., Ltd.) diluted 1:1000 in PBS-SM,
the filter was washed with shaking for 1 hour at room temperature,
washed with shaking 4 times with 500 mL of PBS-T, and further twice
with 100 mL of PBS.
[0031] The filter was drained, soaked in a color substrate solution
(solution of 12 mg of diaminobenzidine/25 mL of PBS, added with 50
L of 2.5% cobalt chloride and 50 L of 2% nickel sulphate), and
further added with 80 L of 30% hydrogen peroxide solution for color
development. Based on the location of color development, associated
clones were selected.
[0032] [5] Determination of Base Sequence
[0033] According to the manual of the aforementioned "Zap-cDNA
synthesis kit" of Stratagene, in vivo excision of 2 positive clones
obtained by immunological screening from the Uni-ZAP XR vector was
carried out. The clones were subcloned in pBluescript phagemid and
grown with Escherichia coli. The base sequences of the respective
clones were determined using ABI PRISM 377 DNA sequencer of Perkin
Elmer.
[0034] In homology search by BLAST conducted against the database
for the obtained base sequences, the base sequence of one clone was
approximately coincided with that of tryptase III, and the base
sequence of the other clone was approximately coincided with that
of a gene named Breast cancer suppressor candidate-1 (Bcsc-1) (FIG.
2). In FIG. 2, SEQ ID NO. 8, a sequence of 1-2552 represents
Bcsc-1. BLAST is an abbreviation of Basic Local Alignment Search
Tool.
[0035] A clone having the approximately identical base sequence to
that of tryptase III was determined to have nothing to do with the
target cell surface antigen, since tryptase III is a well-known
enzyme which exists in mast cell granules. Then, it was determined
whether the clone having the approximately identical base sequence
to that of Bcsc-1 is the target cell surface protein.
[0036] [6] Transfection to BHK Cells
[0037] Among the cDNA obtained in the above, the part designated as
a region for coding protein of Bcsc-1 stored in the database was
inserted to an expression vector which was prepared according to
the method of Karasuyama et al. (see FIG. 3; Karasuyama, H. &
Melchers, F.: Eur. J. Immunol., 18, 97-104, 1988, Karasuyama, H.,
Tohyama, N. & Tada, T.: J. Exp. Med., 169, 13-35, 1989, Yagita,
H., Nakamura, T., Karasuyama, H. & Okumura, K.,: Proc. Natl.
Acad. Sci. USA, 86, 645-649, 1989, Karasuyama, H., Kudo, A. &
Melchers, F.: J. Exp. Med., 172, 969-972, 1990). Particularly, in
introduction of XhoI and NotI restriction enzyme fragments into
Bcsc-1, cagCTCGAGatggaggaggctctgggg (SEQ ID NO. 3) was used as 5'
XhoI primer, and tctggatGCGGCCGCtcaaaggcaaagat (SEQ ID NO. 4) was
used as 3' NotI primer. As PCR conditions, the Bcsc-1 was heated at
94.degree. C. for 4 minutes, reacted at 95.degree. C. for 1 minute
and 15 seconds, at 60.degree. C. for 1 minute and 10 seconds, and
72.degree. C. for 3 minutes, which was repeated 39 times, and then
reacted at 72.degree. C. for 5 minutes. The Bcsc-1 introduced with
XhoI and NotI site was treated with XhoI and NotI, and then
inserted to BCMGSNeo which was also treated with XhoI and NotI.
[0038] The BHK cells were introduced to Bcsc-1 by lipofection and
genetically transformed. Namely, the BHK cells were spread in 10%
FCS DMEM (Nissui Seiyaku) to give a density of 50-70%, and after
cultured overnight, cultured at 37.degree. C. for 2 hours in a
fresh medium. The BHK cells were washed 3 times with serum-free
DMEM. 100 L of serum-free DMEM containing 2.5 L (2.5 g) of Bcsc-1
introduced BCMGSNeo, and 200 L of equivalent mixture of 100 L of
serum-free DMEM containing 100 L of lipofectamin were added to 800
L of serum-free DMEM, and the BHK cells were cultured at 37.degree.
C. for 3 hours.
[0039] [7] Preparation of anti-Bcsc-1 Antibody
[0040] Bcsc-1 introduced pET-28a vector was expressed in
Escherichia coli as a fusion protein with His-tag. The fusion
protein was purified on a nickel chelate column, and a rabbit was
immunized with the purified protein in a usual manner, to prepare
an antiserum, namely, anti-Bcsc-1 polyclonal antibody. Introduction
of Bcsc-1 to pET-28a vector was carried out as follows. EcoRI and
XhoI restriction fragments were introduced to Bcsc-1, and after
treated with XhoI and EcoRI, the Bcsc-1 was inserted to pET-28a
vector which was also treated with XhoI and EcoRI. The introduction
of EcoRI and XhoI restriction fragments to Bcsc-1 were carried out
by PCR amplification using tcagGAATTCatggaggaggctct (SEQ ID No. 5)
as 5' EcoRI primer and ggtaCTCGAGaaaggcaaagatagc (SEQ ID No. 6) as
3' XhoI primer.
[0041] [8] Cell Staining and Western Blot
[0042] The BHK cells obtained in the above [6] (which were
expressed by insertion of Bcsc-1 gene) were cultured overnight in
10% FCS DMEM containing 500 g/mL of G418 to separate the culture.
The anti-Bcsc-1 polyclonal antibody (diluted 1:10.sup.3) obtained
in the above [7] was placed on the culture, which was then reacted
at 37.degree. C. for 1 hour, and rinsed with PBS. Further added
with FITC labeled anti-rabbit IgG (H+ L) (Medical & Biological
Laboratories Co., Ltd.) (diluted 1:100), the culture was reacted at
37.degree. C. for 1 hour to observe localization within the cells
by a fluorescence microscope.
[0043] Also, protein recognized by this anti-Bcsc-1 polyclonal
antibody was searched by Western blot, using mast cell extract,
Bcsc-1 introduced BHK cell extract, extract of eosinophil in which
differentiation was induced using IL-5, peripheral blood cell
extract, extract of HL-60, that is, a peripheral blood system cell
line, and extract of eosinophil differentiated from HL-60, as
antigens.
[0044] As a result, when the mast cell extract was stained, diffuse
fluorescence was recognized in the whole cytoplasma. Accordingly,
it seemed that the Bcsc-1 gene is not against the mast cell surface
antigen. In Western blot, when the Bcsc-1 introduced BHK cells were
antigens, a band was detected at around 45 kD, which was
substantially coincided with molecular weight expected from the
inserted Bcsc-1. On the other hand, when the mast cell was the
antigen, a band was recognized at around 90 kD. The difference in
the results between 2 cell extracts gives support to a fact that
the Bcsc-1 gene is not against the mast cell surface antigen.
[0045] [9] Determination of Base Sequence
[0046] The gene sequence found by the present inventors was
compared with that of Bcsc-1 (SEQ ID NO. 8) stored in the database
(FIG. 2). As a result, the base sequence obtained by the present
inventors had one extra base A (943 in FIG. 2 or 943 of SEQ ID NO.
2) around 140 bp upstream of the initiation codon (ATG of 1055-1057
in FIG. 2) of Bcsc-1. Accordingly, it became apparent that the
initiation codon of the base sequence obtained by the present
inventors (36-38 in FIG. 2 or 36-38 of SEQ ID NO. 2) is located
around 1000 bp upstream of the Bcsc-1 initiation codon.
Furthermore, the region containing a newly found initiation codon
was a typical transcription initiation sequence (ACCATGG). The
protein coded by the base sequence obtained by the present
inventors (SEQ ID NO. 2) was named MASA-1.
[0047] [10] Introduction of MASA-1 to BHK Cells
[0048] Transfection of the BHK cells by MASA-1 was carried out in
the same manner as in the above [6]. However,
tcttgcCTCGAGatggtgcacttctgtgg (SEQ ID NO. 7) was used as 5' XhoI
primer.
[0049] [11] Determination of MASA-1 Properties
[0050] Using the MASA-1 introduced BHK cells instead of the Bcsc-1
introduced BHK cells, cell staining and Western blot were performed
in the similar way to the above [8]. However, in cell staining, a
monoclonal antibody 5C12 produced by a below mentioned hybridoma
ahMC5C12 was used as a primary antibody. As a result, a band at
around 90 kD was observed by Western blot using the MASA-1
introduced BHK cells as an antigen, as in the case of using the
mast cells, and cell surface expression was observed as well by
cell staining. Expression in RBL-2H3 rat mast cells was also
observed. However, no expression was observed in cord blood,
eosinophil, peripheral blood lymphocyte (PBL), and HL-60 peripheral
blood cell line. From the above, it was confirmed that the protein
coded by MASA-1 is a mast cell surface specific protein.
[0051] [12] Hybridoma ahMC5C12 (Accession No. FERM BP-6070)
[0052] Here, as an example, a method for preparing the hybridoma
ahMC5C12 is explained. Firstly, the cells obtained from cord blood
cells cultured for 2 weeks in Embodiment 1 (10.sup.6 cells/0.10 ml)
was injected into the abdominal cavity of an infant rat 4 days
after birth to ablate the induction ability of antibody production
for all antigens in the cells. Then, 1.5 months later, the rat was
injected to its abdominal cavity with both the MC-TC (10.sup.6
cells/0.10 ml) obtained in the above [1] and complete adjuvant for
immunization. Further, the rat was injected to its abdominal cavity
with the cells alone 2 times every 2 weeks. The spleen was taken
out 4 days after the final immunization and the cell fusion was
carried out as follows.
[0053] Spleen cells taken out from the rat and myeloma cells of a
mouse were mixed in a ratio of 10:1, and added with 50%
polyethylene glycol 1500 as a fusion accelerator to carry out cell
fusion. After the cell fusion, the cells were suspended in HAT
medium containing 10% bovine serum in such a way that the cell
concentration for a spleen cell becomes
5.quadrature..about.10.sup.5 cells/ml, and distributed to a 96 well
microtiter plate (Nalge Nunc International) by 200 l for every
well. The hybridomas were cultured in a CO.sub.2 incubator (5%
CO.sub.2, 37.quadrature.{hacek over (Z)}), and grown in HAT medium.
Screening of the hybridomas of spleen cells and myeloma cells was
carried out. The cells were then adapted and cultured in IMDM
(Iscove's modified Dulbecco's medium) supplemented with 10% FCS
(Fetal Calf Serum). Among antibodies in the culture supernatant of
the hybridomas, by using MC-TC separated from skin lesion of
mastocytoma as antigens, clones which produce antibodies specific
to the antigens were separated by the fluorescent antibody method
and named ahMC5C12 (FERM BP-6070). The harvested clone cells were
suspended in 90% bovine serum containing 10% DMSO and kept in
liquid nitrogen. The monoclonal antibodies, specific to mast cell
surface antigens produced by the clones, were harvested by growing
the ahMC5C12 in the abdominal cavity of a nude mouse and purifying
the antibodies.
INDUSTRIAL AVAILABILITY
[0054] It is possible to make clear the role of the mast cells in
development of allergic diseases since the present invention
identifies the amino acid sequence of a mast cell surface antigen.
Furthermore, since use of an antibody to the mast cell surface
antigen of the present invention allows us to specifically remove
or eliminate the mast cells, treatment of allergic diseases becomes
possible.
DEPOSITARY INSTITUTION
[0055] Hybridoma ahMC5C12 has been deposited with the following
international depositary institution, under the following accession
number and deposit date.
[0056] Name: National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology, Ministry of
International Trade and Industry
[0057] Address: 1-3, Higashi 1-home, Tsukuba city, Ibaraki pref.,
Japan
[0058] (Zip Code 305-0046)
[0059] Accession Number and Deposit Date:
[0060] (1) hybridoma ahMC5C12
[0061] FERM BP-6070: Aug. 21, 1997
Sequence CWU 1
1
8 1 786 PRT human 1 Met Val His Phe Cys Gly Leu Leu Thr Leu His Arg
Glu Pro Val 1 5 10 15 Pro Leu Lys Ser Ile Ser Val Ser Val Asn Ile
Tyr Glu Phe Val 20 25 30 Ala Gly Val Ser Ala Thr Leu Asn Tyr Glu
Asn Glu Glu Lys Val 35 40 45 Pro Leu Glu Ala Phe Phe Val Phe Pro
Met Asp Glu Asp Ser Ala 50 55 60 Val Tyr Ser Phe Glu Ala Leu Val
Asp Gly Lys Lys Ile Val Ala 65 70 75 Glu Leu Gln Asp Lys Met Lys
Ala Arg Thr Asn Tyr Glu Lys Ala 80 85 90 Ile Ser Gln Gly His Gln
Ala Phe Leu Leu Glu Gly Asp Ser Ser 95 100 105 Ser Arg Asp Val Phe
Ser Cys Asn Val Gly Asn Leu Gln Pro Gly 110 115 120 Ser Lys Ala Ala
Val Thr Leu Lys Tyr Val Gln Glu Leu Pro Leu 125 130 135 Glu Ala Asp
Gly Ala Leu Arg Phe Val Leu Pro Ala Val Leu Asn 140 145 150 Pro Arg
Tyr Gln Phe Ser Gly Ser Ser Lys Asp Ser Cys Leu Asn 155 160 165 Val
Lys Thr Pro Ile Val Pro Val Glu Asp Leu Pro Tyr Thr Leu 170 175 180
Ser Met Val Ala Thr Ile Asp Ser Gln His Gly Ile Glu Lys Val 185 190
195 Gln Ser Asn Cys Pro Leu Ser Pro Thr Glu Tyr Leu Gly Glu Asp 200
205 210 Lys Thr Ser Ala Gln Val Ser Leu Ala Ala Gly His Lys Phe Asp
215 220 225 Arg Asp Val Glu Leu Leu Ile Tyr Tyr Asn Glu Val His Thr
Pro 230 235 240 Ser Val Val Leu Glu Met Gly Met Pro Asn Met Lys Pro
Gly His 245 250 255 Leu Met Gly Asp Pro Ser Ala Met Val Ser Phe Tyr
Pro Asn Ile 260 265 270 Pro Glu Asp Gln Pro Ser Asn Thr Cys Gly Glu
Phe Ile Phe Leu 275 280 285 Met Asp Arg Ser Gly Ser Met Gln Ser Pro
Met Ser Ser Gln Asp 290 295 300 Thr Ser Gln Leu Arg Ile Gln Ala Ala
Lys Glu Thr Leu Ile Leu 305 310 315 Leu Leu Lys Ser Leu Pro Ile Gly
Cys Tyr Phe Asn Ile Tyr Gly 320 325 330 Phe Gly Ser Ser Tyr Glu Ala
Cys Phe Pro Glu Ser Val Lys Tyr 335 340 345 Thr Gln Gln Thr Met Glu
Glu Ala Leu Gly Arg Val Lys Leu Met 350 355 360 Gln Ala Asp Leu Gly
Gly Thr Glu Ile Leu Ala Pro Leu Gln Asn 365 370 375 Ile Tyr Arg Gly
Pro Ser Ile Pro Gly His Pro Leu Gln Leu Phe 380 385 390 Val Phe Thr
Asp Gly Glu Val Thr Asp Thr Phe Ser Val Ile Lys 395 400 405 Glu Val
Arg Ile Asn Arg Gln Lys His Arg Cys Phe Ser Phe Gly 410 415 420 Ile
Gly Glu Gly Thr Ser Thr Ser Leu Ile Lys Gly Ile Ala Arg 425 430 435
Ala Ser Gly Gly Thr Ser Glu Phe Ile Thr Gly Lys Asp Arg Met 440 445
450 Gln Ser Lys Ala Leu Arg Thr Leu Lys Arg Ser Leu Gln Pro Val 455
460 465 Val Glu Asp Val Ser Leu Ser Trp His Leu Pro Pro Gly Leu Ser
470 475 480 Ala Lys Met Leu Ser Pro Glu Gln Thr Val Ile Phe Arg Gly
Gln 485 490 495 Arg Leu Ile Ser Tyr Ala Gln Leu Thr Gly Arg Met Pro
Ala Ala 500 505 510 Glu Thr Thr Gly Glu Val Cys Leu Lys Tyr Thr Leu
Gln Gly Lys 515 520 525 Thr Phe Glu Asp Lys Val Thr Phe Pro Leu Gln
Pro Lys Pro Asp 530 535 540 Val Asn Leu Thr Ile His Arg Leu Ala Ala
Lys Ser Leu Leu Gln 545 550 555 Thr Lys Asp Met Gly Leu Arg Glu Thr
Pro Ala Ser Asp Lys Lys 560 565 570 Asp Ala Leu Asn Leu Ser Leu Glu
Ser Gly Val Ile Ser Ser Phe 575 580 585 Thr Ala Phe Ile Ala Ile Asn
Lys Glu Leu Asn Lys Pro Val Gln 590 595 600 Gly Pro Leu Ala His Arg
Asp Val Pro Arg Pro Ile Leu Leu Gly 605 610 615 Ala Ser Ala Pro Leu
Lys Ile Lys Cys Gln Ser Gly Phe Arg Lys 620 625 630 Ala Leu His Ser
Asp Arg Pro Pro Ser Ala Ser Gln Pro Arg Gly 635 640 645 Glu Leu Met
Cys Tyr Lys Ala Lys Thr Phe Gln Met Asp Asp Tyr 650 655 660 Ser Leu
Cys Gly Leu Ile Ser His Lys Asp Gln His Ser Pro Gly 665 670 675 Phe
Gly Glu Asn His Leu Val Gln Leu Ile Tyr His Gln Asn Ala 680 685 690
Asn Gly Ser Trp Asp Leu Asn Glu Asp Leu Ala Lys Ile Leu Gly 695 700
705 Met Ser Leu Glu Glu Ile Met Ala Ala Gln Pro Ala Glu Leu Val 710
715 720 Asp Ser Ser Gly Trp Ala Thr Ile Leu Ala Val Ile Trp Leu His
725 730 735 Ser Asn Gly Lys Asp Leu Lys Cys Glu Trp Glu Leu Leu Glu
Arg 740 745 750 Lys Ala Val Ala Trp Met Arg Ala His Ala Gly Ser Thr
Met Pro 755 760 765 Ser Val Val Lys Ala Ala Ile Thr Phe Leu Lys Ser
Ser Val Asp 770 775 780 Pro Ala Ile Phe Ala Phe 785 2 2571 DNA
human unsure 2484 n=t or a 2 ccgggctgca ggagaggaga aaatcttgca
tcaccatggt gcacttctgt ggcctactca 60 ccctccaccg ggagccagtg
ccgctgaaga gtatctctgt gagcgtgaac atttacgagt 120 ttgtggctgg
tgtgtctgca actttgaact acgagaatga ggagaaagtt cctttggagg 180
ccttctttgt gttccccatg gatgaagact ctgctgttta cagctttgag gccttggtgg
240 atgggaagaa aattgtagca gaattacaag acaagatgaa ggcccgcacc
aactatgaga 300 aagccatctc ccagggccac caggccttct tattggaggg
ggacagcagc tccagggatg 360 tcttctcttg caatgtgggt aacctccaac
ctgggtcgaa ggcggcagtc accctgaagt 420 atgtgcagga gctgcctctg
gaagcagatg gggctctgcg ctttgtgctc ccagctgtcc 480 tgaatcctag
ataccagttc tctgggtcgt ctaaggacag ttgccttaat gtgaagactc 540
ctatagtccc tgtggaggac ctgccctaca cactcagcat ggtcgccacc atagattccc
600 agcatggcat tgagaaggtc caatccaact gccccttgag tcctaccgag
tacctaggag 660 aggacaagac ttctgctcag gtttccctgg ctgctggaca
caagtttgat cgggacgtgg 720 aactcctgat ttactacaat gaggtgcata
cccccagcgt ggttttggag atggggatgc 780 ctaacatgaa gccaggtcat
ttgatgggag atccatctgc aatggtgagt ttctatccaa 840 atatcccaga
agatcaacca tcaaatacct gtggagagtt tatctttctc atggaccgct 900
cgggaagtat gcagagcccc atgagtagcc aggatacatc tcagctgcga atacaggcag
960 ccaaggaaac actgattttg ctgctgaaga gtttacctat aggctgttat
ttcaacatct 1020 atggatttgg ctcttcctat gaggcatgct ttccggagag
tgtgaagtac actcagcaaa 1080 caatggagga ggctctgggg agagtgaagc
ttatgcaggc cgacctaggg ggcactgaaa 1140 tcttggcacc actccagaac
atttacaggg gaccctccat cccaggccac cccctacagc 1200 tttttgtctt
tacagatgga gaagttacag acacgtttag tgtaattaaa gaagttagga 1260
tcaacagaca gaaacacagg tgtttctcat ttggtattgg agaaggcacc tccaccagcc
1320 taataaaagg tattgcccgg gcatcagggg gcacctcaga atttatcaca
ggcaaagaca 1380 ggatgcagtc caaggctctc aggactctga aacgctctct
gcagcctgtg gtagaggatg 1440 tctctctgag ctggcatttg cctcctggtc
tgtctgctaa aatgctttcc ccagaacaga 1500 ctgtcatctt taggggtcag
agattaatca gctatgccca gctgaccggg aggatgccag 1560 cagcagagac
aacaggagaa gtatgcctca aatatacact ccagggcaag acttttgagg 1620
ataaggtgac atttcctcta caacccaagc ctgatgtcaa cctcaccatt caccgccttg
1680 ctgccaagtc cttgctccag accaaggaca tgggcctcag ggagactcca
gcaagtgata 1740 aaaaagatgc attgaacctt agccttgagt ctggtgtcat
aagctccttc acagctttca 1800 ttgctatcaa taaggagctc aacaagccgg
ttcaggggcc tctggctcat agggacgtcc 1860 caaggccaat tctgttgggt
gcttctgccc cattgaagat aaaatgccaa tcaggttttc 1920 gaaaggcctt
acactctgac cgtcctcctt ctgcatctca gcccagaggg gaacttatgt 1980
gttataaggc caagacattc cagatggacg attacagtct ctgtgggttg ataagtcaca
2040 aggaccagca cagtccaggc tttggagaga atcaccttgt gcagctgatt
taccaccaaa 2100 atgcaaatgg ttcctgggat ctgaatgaag atctagccaa
gatcctaggt atgagtttgg 2160 aagaaataat ggctgcacag cctgccgagc
ttgtggattc ctcaggctgg gccaccatcc 2220 tggccgtgat ctggctgcac
agcaatggta aggacttgaa gtgtgaatgg gagcttctgg 2280 aaaggaaggc
cgtggcctgg atgcgtgccc atgcaggctc caccatgcct tcggttgtga 2340
aagctgctat tactttcctg aagtcatctg tggatcctgc tatctttgcc ttttgaagat
2400 accatccaga aaaagaagtg cctttaattt gctactgtca tttcctctag
tatcactttt 2460 gctgtgatga tgtgttcttg tgtnttataa ctctttattt
tttgccataa aagtaaagga 2520 tgcttactcc acttcgaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa a 2571 3 27 DNA Artificial Sequence primer 3
cagctcgaga tggaggaggc tctgggg 27 4 29 DNA Artificial Sequence
primer 4 tctggatgcg gccgctcaaa ggcaaagat 29 5 24 DNA Artificial
Sequence primer 5 tcaggaattc atggaggagg ctct 24 6 25 DNA Artificial
Sequence primer 6 ggtactcgag aaaggcaaag atagc 25 7 29 DNA
Artificial Sequence primer 7 tcttgcctcg agatggtgca cttctgtgg 29 8
2551 DNA human 8 gcacaccatg gtgcacttct gtggcctact caccctccac
cgggagccag tgccgctgaa 60 gagtatctct gtgagcgtga acatttacga
gtttgtggct ggtgtgtctg caactttgaa 120 ctacgagaat gaggagaaag
ttcctttgga ggccttcttt gtgttcccca tggatgaaga 180 ctctgctgtt
tacagctttg aggccttggt ggatgggaag aaaattgtag cagaattaca 240
agacaagatg aaggcccgca ccaactatga gaaagccatc tcccagggcc accaggcctt
300 cttattggag ggggacagca gctccaggga tgtcttctct tgcaatgtgg
gtaacctcca 360 acctgggtcg aaggcggcag tcaccctgaa gtatgtgcag
gagctgcctc tggaagcaga 420 tggggctctg cgctttgtgc tcccagctgt
cctgaatcct agataccagt tctctgggtc 480 gtctaaggac agttgcctta
atgtgaagac tcctatagtc cctgtggagg acctgcccta 540 cacactcagc
atggtcgcca ccatagattc ccagcatggc attgagaagg tccaatccaa 600
ctgccccttg agtcctaccg agtacctagg agaggacaag acttctgctc aggtttccct
660 ggctgctgga cacaagtttg atcgggacgt ggaactcctg atttactaca
atgaggtgca 720 tacccccagc gtggttttgg agatggggat gcctaacatg
aagccaggtc atttgatggg 780 agatccatct gcaatggtga gtttctatcc
aaatatccca gaagatcaac catcaaatac 840 ctgtggagag tttatctttc
tcatggaccg ctcgggaagt atgcagagcc ccatgagtag 900 ccaggataca
tctcgctgcg aatacaggca gccaaggaaa cactgatttt gctgctgaag 960
agtttaccta taggctgtta tttcaacatc tatggatttg gctcttccta tgaggcatgc
1020 tttccggaga gtgtgaagta cactcagcaa acaatggagg aggctctggg
gagagtgaag 1080 cttatgcagg ccgacctagg gggcactgaa atcttggcac
cactccagaa catttacagg 1140 ggaccctcca tcccaggcca ccccctacag
ctttttgtct ttacagatgg agaagttaca 1200 gacacgttta gtgtaattaa
agaagttagg atcaacagac agaaacacag gtgtttctca 1260 tttggtattg
gagaaggcac ctccaccagc ctaataaaag gtattgcccg ggcatcaggg 1320
ggcacctcag aatttatcac aggcaaagac aggatgcagt ccaaggctct caggactctg
1380 aaacgctctc tgcagcctgt ggtagaggat gtctctctga gctggcattt
gcctcctggt 1440 ctgtctgcta aaatgctttc cccagaacag actgtcatct
ttaggggtca gagattaatc 1500 agctatgccc agctgaccgg gaggatgcca
gcagcagaga caacaggaga agtatgcctc 1560 aaatatacac tccagggcaa
gacttttgag gataaggtga catttcctct acaacccaag 1620 cctgatgtca
acctcaccat tcaccgcctt gctgccaagt ccttgctcca gaccaaggac 1680
atgggcctca gggagactcc agcaagtgat aaaaaagatg cattgaacct tagccttgag
1740 tctggtgtca taagctcctt cacagctttc attgctatca ataaggagct
caacaagccg 1800 gttcaggggc ctctggctca tagggacgtc ccaaggccaa
ttctgttggg tgcttctgcc 1860 ccattgaaga taaaatgcca atcaggtttt
cgaaaggcct tacactctga ccgtcctcct 1920 tctgcatctc agcccagagg
ggaacttatg tgttataagg ccaagacatt ccagatggac 1980 gattacagtc
tctgtgggtt gataagtcac aaggaccagc acagtccagg ctttggagag 2040
aatcaccttg tgcagctgat ttaccaccaa aatgcaaatg gttcctggga tctgaatgaa
2100 gatctagcca agatcctagg tatgagtttg gaagaaataa tggctgcaca
gcctgccgag 2160 cttgtggatt cctcaggctg ggccaccatc ctggccgtga
tctggctgca cagcaatggt 2220 aaggacttga agtgtgaatg ggagcttctg
gaaaggaagg ccgtggcctg gatgcgtgcc 2280 catgcaggct ccaccatgcc
ttcggttgtg aaagctgcta ttactttcct gaagtcatct 2340 gtggatcctg
ctatctttgc cttttgaaga taccatccag aaaaagaagt gcctttaatt 2400
tgctactgtc atttcctcta gtatcacttt tgctgtgatg atgtgttctt gtgtattata
2460 actctttatt ttttgccata aaagtaaagg atgcttactc cacttcgctt
ctctgctcca 2520 ggttcacttt ggatatgatc tttcttttcc c 2551
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