JP2997770B2 - Monoclonal antibody against bovine c-Kit protein and cell separation method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a monoclonal antibody that specifically reacts with a bovine c-Kit protein, a hybridoma producing the monoclonal antibody, and a bovine c-Kit protein-positive cell using the monoclonal antibody. It relates to a separation method.

[0002]

2. Description of the Related Art The c-Kit protein is a c-kit gene [EMBO J.
6,3341 (1987), EMBO J. 7,1003 (1988)]. c-Kit protein is a c-Kit receptor [Genes & Dev
elopment 4, 390 (1990)], Kit receptor [EMBO Journa
l10, 2451 (1991)], SCF receptor [Japanese Patent Application No. 4-502628]
(1992), Advances in Immunology 55, 1 (1994)], or
CD117 [Leukocyte typing V, Oxford University Press
(1995)].

[0003] Such c-Kit protein is a stem cell factor which is one of cytokines.
r, hereinafter referred to as “SCF”) [Japanese Patent Application No. 4-502628 (1992), Cell
63, 167 (1990), Cell 63, 175 (1990), Cell 63, 195
(1990), Cell 63, 203 (1990)].
(1990), Cell 63, 225 (1990)].

[0004] Human [EMBO Journal 6, 3341 (1987)], mouse [EMBO Journal 7, 1003 (1988)], chicken [Gene 1
28, 257 (1993)], goats [Biochimica et Biophysica Act]
a 1352, 151 (1997)], and bovine [Gene 141, 305 (199
4)] The cDNA encoding the c-kit protein has been cloned and its nucleotide sequence has been determined. As a result, the c-Kit protein is a cell membrane protein consisting of a secretory signal sequence, an extracellular region, a transmembrane region, and an intracellular region, in order from the amino terminus. It has been clarified that there is a region responsible for protein tyrosine kinase activity (phosphorylase region).

[0005] In humans and mice, c-Kit protein is derived from hematopoietic progenitor cells of various blood cells.
expressed on the cell membrane surface of mature mast cells, germ cells, melanocytes, etc., and plays an essential role as a cell membrane surface receptor for SCF for the normal growth and differentiation of these c-Kit protein-positive cell types [Advances in Immunology]
55, 1 (1994)].

A monoclonal antibody against mouse-derived c-Kit protein [EMBO Journal 10, 2111 (1991)] and a monoclonal antibody against human-derived c-Kit protein [Japanese Patent Application No. 4-510017 (1992), B1ood 76, Supplement 295a (19)
90), Leukemia Research 12,923 (1988), Leukemia Res
earch 12, 929 (1988), Leukemia Research 11, 1329 (1
985), Blood 77, 1876 (1991), Blood 79, 338 (1992)]
These antibodies have been reported to be used for c-Kit such as blood progenitor cells, germ cells, melanocytes, etc.
It is used for identification and separation of protein positive cells.

However, the reactivity of these monoclonal antibodies to bovine c-Kit protein is unknown. From the knowledge on c-Kit protein in humans and the like, it is considered that c-Kit protein has a major role in the growth of blood progenitor cells, germ cells, melanocytes and the like in cattle. Therefore, it is considered that elucidation of the biological role of the bovine c-Kit protein is important in elucidating the formation process of the above cell types in cattle. In order to elucidate the biological role of c-Kit protein, a means for separating c-Kit protein-positive cell types such as blood progenitor cells and germ cells is required.

[0008] In order to examine the dynamics and function of c-Kit protein in cattle, and to isolate c-Kit protein-positive cells such as bovine blood progenitor cells and germ cells, specificity for bovine c-Kit protein is It is necessary to produce antibodies having high specificity, especially monoclonal antibodies which are excellent in specificity and reproducibility. However, production of monoclonal antibodies against bovine c-Kit protein has not been reported so far. Therefore, development of a monoclonal antibody against bovine c-Kit protein has been desired.

[0009]

SUMMARY OF THE INVENTION The present invention relates to a bovine c-
A monoclonal antibody specifically reacting with the Kit protein, a hybridoma producing the monoclonal antibody,
And a method for separating bovine c-Kit protein-positive cells using the monoclonal antibody.

[0010]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that bovine c-Kit
From a hybridoma obtained by fusing antibody-producing cells obtained from an animal immunized with the protein with myeloma cells, a monoclonal antibody specifically reacting with the bovine c-Kit protein was successfully obtained, and the monoclonal antibody was successfully obtained. Bovine-derived from bovine bone marrow cells using antibodies
The inventors succeeded in isolating c-Kit protein-positive cells, and completed the present invention. That is, the present invention is a monoclonal antibody that specifically reacts with a bovine c-Kit protein.

[0011] The present invention is also a hybridoma producing the above monoclonal antibody. Further, the present invention provides
Using the above monoclonal antibody, bovine c-Kit characterized by separating bovine c-Kit protein positive cells from a sample containing bovine c-Kit protein positive cells
This is a method for separating protein positive cells.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. 1. Monoclonal antibody of the present invention The monoclonal antibody of the present invention includes all monoclonal antibodies capable of specifically reacting with the bovine c-Kit protein. Among them, a monoclonal antibody that specifically reacts with the extracellular region of the bovine-derived c-Kit protein can be exemplified as a preferable one, and a monoclonal antibody produced by a hybridoma described later can be exemplified as the most preferable one.

2. Preparation of Monoclonal Antibody of the Present Invention The monoclonal antibody of the present invention that specifically reacts with the bovine c-Kit protein can be produced, for example, through the following steps. (1) Preparation of antigen (2) Collection of immune and antibody-producing cells (3) Cell fusion (4) Selection and cloning of hybridoma (5) Collection of monoclonal antibody Each step will be described below.

(1) Preparation of Antigen Preparation of the bovine c-Kit protein used as an antigen is not limited to a specific method, but cDNA encoding the bovine c-Kit protein is already known [Gene 141]. , 305-3
06 (1994)], bovine c-Ki as an antigen using the cDNA
Preferably, a t protein is prepared. As the cDNA encoding the bovine-derived c-Kit protein, for example, a cDNA having the nucleotide sequence of SEQ ID NO: 1 can be used. The above cD
Preparation of bovine c-Kit protein from NA, for example,
A recombinant vector containing a cDNA encoding a bovine c-Kit protein is prepared, a suitable host cell is transformed with the vector, and a culture obtained by culturing the transformant in a suitable medium is used. It can be performed by purification.

The cDNA used here may be the entire region of the cDNA encoding the bovine c-Kit protein shown in SEQ ID NO: 1, but may be the extracellular region of the bovine c-Kit protein (amino terminal side). Amino acid residues 25-521 from
Is preferred. When using the cDNA encoding the extracellular region of the bovine c-Kit protein, a part of the extracellular region of the bovine c-Kit protein (for example, amino acid residues 25 to 509 from the amino terminal side) is encoded. Alternatively, a cDNA may be used. The recombinant vector and the host cell are not particularly limited, and any known cells may be used. In particular, it is preferable to use a recombinant baculovirus and a cultured insect cell. Culture of the transformant and purification of the culture can be performed according to a conventional method.

(2) Immunization and collection of antibody-producing cells The bovine c-Kit protein obtained as described above is administered as an immunogen together with an adjuvant to mammals and birds. Here, examples of the adjuvant include commercially available Freund's complete adjuvant, Freund's incomplete adjuvant, BCG, Hunters, Titer Mac, keyhole limpet hemocyanin-containing oil, and the like, and these may be used alone. Or a mixture of two or more of these.

As mammals, horses, monkeys, dogs, pigs, goats, sheep, rabbits, guinea pigs, hamsters,
A mouse or the like can be used, and as a bird, a pigeon, a chicken, or the like can be used, and a mouse, a rat, or the like is particularly preferable. As a method of administration, any known method can be used, but intravenous administration,
Subcutaneous or intraperitoneal administration is preferred.

The immunizing dose of the antigen is usually 10 to 200 μg, preferably 25 to 100 μg per mouse at a time. The interval between immunizations is usually 1 to 4 weeks, preferably 2 weeks, and the number of immunizations is usually 2 to 5 times, preferably 3 to 4 times.

1-5 days after the last immunization, preferably 3 days
One day later, the antibody producing cells are collected. The antibody-producing cells to be collected include lymph node cells, spleen cells and the like, and are preferably foot lymph node cells.

(3) Cell fusion As the myeloma cells to be fused with the antibody-producing cells, cell lines derived from various animals such as mice, rats, and humans and generally available to those skilled in the art can be used. As a cell line to be used, a cell line having drug resistance, having a property that it cannot survive in a selection medium (for example, HAT medium) in an unfused state and can survive in a selection medium only in a state fused with an antibody-producing cell, preferable. Generally, 8-azaguanine resistant strains can be used. This cell line, hypoxanthine - deficient in guanine phosphoribosyl Bo transferase (HGPRT ^-), can not grow in HAT medium.

As such myeloma cells, P3 /
Mouse myeloma cell lines such as X63-Ag8-UI, 210.RCY.Ag1.
Rat myeloma cell lines such as 2.3 and human myeloma cell lines such as SKO-007 can be used. Cell fusion is
For example, mixing ratio of myeloma cells and antibody-producing cells 1: 5
It can be performed by bringing the cells into contact with each other for 2 to 5 minutes at room temperature in the presence of a fusion promoter in a medium such as RPMI1640 medium at a ratio of about 1:10. At this time, as a fusion promoter,
Polyethylene glycol, polyvinyl alcohol and the like having an average molecular weight of 1500 to 4000 can be used. Also, a fusion virus such as Sendai virus can be used.

(4) Selection and Cloning of Hybridoma After the cell fusion, the desired hybridoma is selected. The selection method may be in accordance with an ordinary method, and is not particularly limited. As a sorting method, for example, the following method can be used. That is, after the bovine c-Kit protein is adsorbed to each well of the microplate, it is blocked with Block Ace (Dainippon Pharmaceutical) or the like. The culture supernatant of the hybridoma was added to each well of the microplate and incubated at 37 ° C.
Leave for 1 hour, and then remove bovine c-Kit protein and bovine c-Ki.
React with a monoclonal antibody that specifically reacts with the t protein. After washing with physiological saline, an appropriately diluted alkaline phosphatase-conjugated goat anti-immunoglobulin is added. After washing with physiological saline, ALP
The activity of alkaline phosphatase is measured using Rose (Sinotest), and the well having the color of alkaline phosphatase is defined as a well containing cells producing an antibody specific to bovine c-Kit protein. The method for cloning the desired hybridoma from this well is as follows:
A normal method may be followed, and there is no particular limitation. The cloning of the hybridoma can be performed by, for example, a limiting dilution method, a soft agar method, a fibrin gel method, a fluorescence excitation cell sorter method, or the like.

(5) Collection of Monoclonal Antibody As a method of collecting a monoclonal antibody from the obtained hybridoma, a usual cell culture method, ascites formation method, or the like can be used. In the cell culture method, for example,
Hybridomas are cultured in animal cell media such as RPMI 1640 medium containing 10-20% calf serum, MEM medium, E-RDF medium or serum-free medium under normal culture conditions (e.g., 37 ° C., 5% CO ₂ concentration).
After culturing for 3 to 7 days, the desired monoclonal antibody can be obtained from the culture supernatant.

In the ascites formation method, for example, a mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) is administered intraperitoneally to an animal of the same species as a mammal derived from myeloma cells, and then the hybridoma is administered. 1 × 10 ⁶ 〜1 × 10
^7, preferably the administration of 5 × l0 ⁶ cells intraperitoneally. After breeding the administered mammal for 1 to 4 weeks, preferably 2 weeks, the target monoclonal antibody can be obtained by collecting ascites or serum.

In the above-mentioned method for collecting an antibody, when purification of the antibody is required, a sulfate analysis method, ion exchange chromatography using an anion exchanger such as DEAE-cellulose, protein A sepharose and the like are used. A known method such as affinity chromatography, molecular sieve chromatography that sifts according to molecular weight or structure, etc. is appropriately selected, and purification can be performed by using these alone or in combination.

3. Use of the monoclonal antibody of the present invention The monoclonal antibody of the present invention is, for example, bovine c-Ki
Purification of bovine c-Kit protein from a sample containing t protein, quantification of bovine c-Kit protein, or bovine c-Kit from a sample containing bovine c-Kit protein positive cells
-Can be used to separate Kit protein positive cells. Purification of bovine c-Kit protein from a sample containing bovine c-Kit protein can be performed, for example, by affinity chromatography using a support on which the monoclonal antibody of the present invention is immobilized. The sample containing the bovine c-Kit protein used at this time is not particularly limited as long as it contains the bovine c-Kit protein, and may be any sample. The quantification of the bovine c-Kit protein can be carried out, for example, by the sandwich ELISA method using the monoclonal antibody of the present invention.

The separation of bovine c-Kit protein-positive cells from a sample containing bovine c-Kit protein-positive cells can be performed, for example, as follows. That is, the monoclonal antibody of the present invention is labeled with biotin, and added to a sample containing bovine c-Kit protein-positive cells to react the monoclonal antibody of the present invention with bovine c-Kit protein-positive cells. . To this, streptavidin labeled with a fluorescent label (for example, a fluorescent dye phycoerythrin label) is added and reacted with the above-mentioned biotin-labeled monoclonal antibody. By doing so, bovine-derived c-Kit protein positive cells can be separated.
At this time, the sample containing bovine-derived c-Kit protein-positive cells to be used is not particularly limited as long as it contains bovine-derived c-Kit protein-positive cells, and may be any sample. In addition, the bovine-derived c-Kit protein-positive cell means a cell derived from bovine and expressing the c-Kit protein on the cell membrane surface.

[0028]

The present invention will now be described more specifically with reference to examples. However, the present invention is not limited to these examples. Example 1 Preparation of Monoclonal Antibody (1) Preparation of Antigen cDNA encoding bovine c-Kit protein (hereinafter referred to as “c-Kit”)
“Bovine c-Kit protein cDNA”) has already been cloned by the inventors and its nucleotide sequence has been determined [Gene 141, 305-306 (1994)]. In addition, cow origin
SEQ ID NO: 1 shows the nucleotide sequence of c-Kit protein cDNA.

Based on the cDNA sequence of the bovine c-kit protein cDNA, the bovine c-Kit protein consists of 977 amino acid residues.
It has been shown to have 90% and 83% homology. In addition, from the comparison of the amino acid sequences of the human c-Kit protein and mouse c-Kit protein, the bovine c-Kit protein has a secretory signal sequence (amino acid residues 1 to 24) and an extracellular region in order from the amino terminal side. (Amino acid residues 25 to 521), a transmembrane region (amino acids 522 to 545), and an intracellular region (amino acids 546 to 977)
And the region consisting of the amino acid residues 578 to 686 of the bovine-derived c-Kit protein, and furthermore, the region consisting of the amino acid residues 764 to 913, is a region responsible for tyrosine kinase activity (phosphonase region). It is speculated that there is (FIG. 1).

Thus, the extracellular region of the bovine c-Kit protein was used as an antigen for preparing a monoclonal antibody against the bovine c-Kit protein, in order to produce and purify the extracellular region in large quantities. Known methods [Virus Research 21, 123 (1991), Nucl.
es. 15, 10233 (1987)], a recombinant baculovirus incorporating a cDNA encoding most of the extracellular region of the bovine c-Kit protein (region from the 1st to the 509th amino acid residue). And a transformant transformed with the recombinant baculovirus was cultured.

In the region from the 1st to the 509th amino acid residue of the bovine c-Kit protein produced in the transformant by culturing, the secretory signal sequence portion (the 1st to the 24th amino acid residue) is removed. Region from the amino acid residue to the 509th amino acid residue (hereinafter referred to as “c-Kit protein extracellular region”) is considered to be secreted into the culture solution. The outer region was purified. The details are described below.

Preparation of Transfer Vector Containing cDNA Encoding Extracellular Region of c-Kit Protein Unless otherwise indicated below, standard methods or conditions refer to the methods described in Molecular Cloning 2nd Edition Or a condition. First, in order to prepare a cDNA encoding the extracellular region of the c-Kit protein, plasmid cl.75 [Gene 141, 305 (1994)] was used as a template, and the two synthetic oligonucleotides A and B shown in FIG. A polymerase chain reaction (hereinafter, referred to as “PCR”) was performed as a primer.

The plasmid cl.75 used as a template
This is a plasmid in which a bovine-derived c-Kit protein cDNA (about 3800 bases in length) was inserted into the restriction enzyme EcoRI cleavage site of plasmid pBluescriptSK (-) (Stratagene) (FIG. 3). In addition, E. coli JMbk incorporating plasmid cl.75
FERM P-1678 to the National Institute of Bioscience and Biotechnology
Deposited as 1 (Deposit date: April 23, 1998)
Day).

In the base sequence of primer A, 11 to
The 27th nucleotide sequence is 1 in the bovine c-Kit protein cDNA.
Although the same as the nucleotide sequence from 03 to 119, the nucleotide sequence from 1 to 10 is different from the nucleotide sequence of the bovine c-Kit protein cDNA, and the nucleotide sequence from 3 to 8 (CTCGAG) is particularly restricted. Sequence of the cleavage site by the enzyme XhoI. On the other hand, the base sequence of the primer B was 789 of the bovine c-Kit protein cDNA.
And 808th are complementary.

The PCR was performed under the following general reaction conditions. That is, PCR was performed using 1 μg / ml cl.75, 1 μg each.
M oligonucleotide primers, 1 × PCR buffer (PerkinElmer, ABI), and 0.2 mM each of deoxyadenosine-5′-3 phosphate, deoxyguanosine-5′-3phosphate, deoxycytidine-5′-3 Phosphoric acid and thymidine-5 '
Using 100 μl of a reaction solution composed of −3 phosphate and DNA Thermal Cycler 480 (Perkin Elmer, ABI) at 94 ° C.
This was repeated 20 times, with 1 minute, 2 minutes at 42 ° C. and 3 minutes at 72 ° C.

About 700 base pairs in length amplified by PCR
The DNA fragment was purified by agarose gel electrophoresis under standard conditions, and then digested with restriction enzymes XhoI and HindIII. The digest was ligated to plasmid cl.75 previously digested with restriction enzymes XhoI and HindIII using E. coli T4 ligase under standard conditions to generate plasmid pKitPCR.

Using the plasmid pKitPCR, E. coli JM109 [G
ene 33, 103 (1985)], the transformants were cultured by standard methods, and the plasmid pKitPCR was amplified. Next, pKitPCR was digested with restriction enzymes XhoI and DraI to prepare a cDNA fragment encoding amino acid residues 1 to 509 of bovine c-Kit protein, which was previously digested with XhoI and SmaI according to standard methods. Plasmid pBac
PAK8 (Clontech) [Nucleic Acids Res. 17, 23
66 (1989), J. Gen. Virol 68, 1233 (1987)] to create the transfer vector pBacPAK8CKIT.

A translation termination codon (TAA) exists 16 bases downstream from the SmaI cleavage site of pBacPAK8. Therefore, the transfer vector pBacPAK8CKIT contains bovine c-Ki
5 amino acid residues derived from pBacPAK8 (glycine-arginine-proline-leucine-
Asparagine) is considered to be encoded.

Preparation of Recombinant Baculovirus Producing Extracellular Region of c-Kit Protein Transfer vector pBacPAK8CKIT and genomic DNA of baculovirus BakPAK6 (Clontech) [Biotechn.
iques 14,810 (1993)] and cultured insect cells IPLB-SF-21AE
(Hereinafter referred to as “SF21AE cells”) [In vitro 13,213 (197
7)] to obtain cK by cotransfection.
A recombinant baculovirus BacCKIT strain producing the extracellular region of the it protein was produced [Virus Research 21,123 (1
991), Nucleic Acids Research 15, 10233 (1987)].

That is, the transfer vector pBacPA
BakP previously cut with K8CKIT (100 ng) and restriction enzyme Eco81I
AK6 virus genomic DNA (100 ng) and lipofectin
(Gibco BRL) (5 μg), and the mixture was added to a culture solution of SF21AE cells (1.5 × 10 ⁶ cells) cultured in a culture dish (35 mm) and cultured at 28 ° C. for 6 hours. Thereafter, fetal bovine serum was added to a final concentration of 10%, and the cells were further cultured for 3 days. Thereafter, the culture supernatant was collected and serially diluted with TC100 medium, and 0.2 ml of each diluted solution was adsorbed to 1.5 × 10 ⁶ SF21AE cells cultured in a 35 mm dish for 1 hour, and then the diluted solution was removed. . After adding 3 ml of TC100 medium containing 1% agarose to the SF21AE cells and culturing for 3 days, 20 μg / ml of 5-bromo-4-chloro-3-indoyl-3-D-galactoviranoside (Wako Pure Chemical Industries, Ltd.) ) Was added, and the cells were further cultured for 1 day. Of the plaques that appeared,
Collect several plaques that do not stain blue and
Each recombinant virus clone was suspended in 0.3 ml of TC100 medium to obtain a stock solution. Each suspension was infected to SF21AE cells cultured in a 35-mm dish, and cultured for 4 days to obtain a culture solution containing the recombinant virus.

A high molecular weight DNA was prepared from the virus-infected cells according to a standard method, and the two synthetic oligonucleotides C and D shown in FIG. 2 were used as primers under general conditions.
PCR was performed to examine the presence or absence of bovine c-Kit protein cDNA in the genomic DNA of each clone of the recombinant virus. The nucleotide sequence of primer C is identical to the nucleotide sequence at positions 1182 to 1202 of pBacPAK8, and the nucleotide sequence of primer D is derived from bovine
It is complementary to the sequence at positions 372 to 391 of the c-kit protein cDNA. As a result, the recombinant baculovirus clone Ba containing the bovine c-Kit protein cDNA as expected
cCKIT was obtained. By infecting the clone with a larger amount of cells, about 200 ml of the virus solution was obtained.

Production and Purification of Recombinant Bovine c-Kit Protein Using Recombinant Baculovirus Cultured in a 75 ml plastic bottle (Nunc)
After inoculating 1 × 10 ⁷ Tn-5 cells with the virus solution (1 × 10 ⁶ plaque-forming units) obtained in Example 1, 10 ml of the cells was added.
In TC100 medium at 28 ° C. for 4 days. After collecting the culture supernatant, it was centrifuged at 45,000 rpm for 1 hour to obtain a centrifuged supernatant. The supernatant was subjected to salt exchange by dialysis to obtain a 10 mM tris (hydroxyl) aminomethane hydrochloric acid solution (pH 8.0). This was adsorbed on an anion exchange column (MonoQ column, Pharmacia) at a flow rate of 1 ml / min.

When the fraction eluted without adsorption was analyzed by SDS-polyacrylamide gel electrophoresis (hereinafter referred to as "SDS-PAGE"), it was found that this fraction contained only the extracellular region of c-Kit protein. Since this was found, this was concentrated using a Macrosep 10K column (Filtran) to obtain a purified c-Kit protein extracellular region standard. The apparent molecular weight of the extracellular region of the c-Kit protein determined by SDS-PAGE was 65,000. From a total of 200 ml of culture supernatant, about 4.5 mg
The extracellular region of c-Kit protein was obtained.

(2) Collection of Immunization and Antibody-Producing Cells Immunization 25 μg of the extracellular region of c-Kit protein in complete Freund's adjuvant was placed in the footpad of a female Balb / c mouse (8-week-old, CLEA Japan). Three injections were given every two weeks. One week later, the same mouse was injected into the footpad with 30 μg of extracellular c-Kit protein in saline.
A total of four mice were immunized.

Cell fusion Three days after the final immunization, the mouse's foot lymph nodes were removed,
A single cell suspension was used. 1 × 10 ⁸ lymph node cells per mouse were replaced with 1 × 10 ⁷ 8-azaguanine-resistant myeloma cells P3 / X63-Ag8-U1 [Current Topics in Microbiology
and Immunology 81, 1 (1978)] using 50% polyethylene glycol (average molecular weight 1540, Boehringer Mannheim). Cells were plated in a 96-well plate at 100μ.
1 was dispensed. After 1 day, 2 days, and 3 days, HAT medium was added for 10 days each.
HAT resistant cells, ie, hybridomas, were grown by adding 0 μl each.

Selection and Monocloning of Hybridoma The extracellular region of the c-Kit protein was adsorbed, and 50 μl of the culture supernatant of the hybridoma was added to each well of the microplate which had been blocked with Block Ace (Dainippon Pharmaceutical).
The reaction was carried out at a temperature of 1 hour. After washing with physiological saline, appropriately diluted alkaline phosphatase-conjugated goat anti-mouse immunoglobulin (50 μl) was added and reacted at 37 ° C. for 1 hour. After washing with physiological saline, the activity of alkaline phosphatase was measured using ALP Rose (Sinotest). Alkaline phosphatase is colored with bovine c-Kit
Wells containing antibody-producing cells specific for the protein were used.

Monocloning of antibody-producing cells was carried out by subjecting selected hybridomas to limiting dilution three times using mouse intraperitoneal macrophages as feeder cells. The hybridoma clone established as described above was designated as bK-1 and deposited with the National Institute of Bioscience and Human Technology as FERM P-16780 (deposit date: April 23, 1998). The subclass of the antibody produced by bK-1 (hereinafter referred to as "bK-1 antibody") is Ig
G1.

Collection of Monoclonal Antibody The above hybridoma was injected into the abdominal cavity of Balb / c mice that had been injected intraperitoneally with pristane (2,6,10,14-tetramethylpentanedane) in advance. When the injected mice were bred for 2 weeks, ascites due to the hybridoma was formed in the mice and a high concentration of antibody was produced in the ascites, and the ascites of the mice was collected. Purification of the collected antibody was performed using a DEAE-Sephacel column (Pharmacia) after fractionation with 50% saturated ammonium sulfate.

Example 2 Expression of Recombinant Bovine c-Kit Protein in Cos-7 Cells and Investigation of Reactivity of Monoclonal Antibody Against It So far, human, mouse and chicken c-kit proteins have been encoded. The expression of these recombinant proteins in Cos cells transfected with the cDNA has been reported, and this expression system has been used as an experimental system to examine the biochemical properties of c-Kit proteins from these animal species. [Cell 63, 213 (1990), EMBO Journal 10, 2451 (19
91), Journal of Veterinary Medical Sciences 57, 23
1 (1995)].

In Cos cells into which an expression vector has been introduced, the c-Kit protein is known to be expressed on the cell membrane surface as a glycoprotein having a molecular weight of about 120,000 to 150,000, and to have activity as a receptor for SCF. The recombinant c-Kit protein has a binding activity with SCF and a protein tyrosine kinase activity, and the kinase activity is significantly increased by binding to SCF. Therefore, the recombinant bovine c-Kit protein was expressed in Cos-7 cells [Cell 23, 175 (1981)], and the reactivity of the monoclonal antibody against this was examined. The details are described below.

(1) Recombinant bovine c in Cos-7 cells
Expression of -Kit protein and measurement of binding activity to bovine-derived SCF Preparation of rabbit antiserum against bovine-derived c-Kit protein To detect expression of recombinant bovine-derived c-Kit protein in Cos-7 cells, the following is shown. Thus, a fusion protein of bovine c-Kit protein and bacterial glutathione-S-transferase (hereinafter, referred to as "GST-c-Kit") is expressed in Escherichia coli, and rabbit antiserum (hereinafter, "GST-c-Kit") is used as an antigen. Anti-GST-c-Kit antiserum ”).

The details will be described below. First, from cow
To produce a cDNA encoding the region from the 25th amino acid residue to the carboxyl terminus of the c-Kit protein,
Plasmid cl.75 having bovine c-Kit protein cDNA
[Gene 141, 305 (1994)] as a template, and two synthetic oligonucleotides B and E shown in FIG.
PCR was performed under general reaction conditions.

In the base sequence of primer E, 11 to
The 26th sequence is identical to the 173rd to 188th nucleotide sequence in the bovine c-Kit protein cDNA, but the 1st to 10th nucleotide sequence is different from the bovine c-Kit protein cDNA sequence, Especially the 3rd to 8th base sequence (CTCGAG) is a restriction enzyme
XhoI cleavage site sequence, 7th to 12th base sequence
(AGGCCT) is the sequence of the cleavage site by the restriction enzyme StuI.

About 650 base pairs in length amplified by PCR
The DNA fragment was purified by agarose gel electrophoresis under standard conditions, and then digested with restriction enzymes XhoI and HindIII. The digest was ligated with plasmid cl.75 previously digested with restriction enzymes XhoI and HindIII using E. coli T4 ligase under standard conditions to generate plasmid pBScKIT.

After transforming Escherichia coli JM109 using this plasmid, the transformant was cultured by a standard method to amplify the plasmid pBScKIT. Next, pBScKIT was digested with restriction enzymes StuI and EcoRI to prepare a cDNA fragment encoding the 25th amino acid residue to the carboxyl terminus of the bovine c-Kit protein. Prokaryotic gene expression plasmid pGEX-3X (Pharmacia Biotech) digested with [Gene 67,
31 (1998)] to prepare a plasmid pGEXbckit. This plasmid encodes a fusion protein (GST-c-Kit) in which the region from the 25th amino acid residue of the bovine c-Kit protein to the carboxyl terminus is linked to the carboxyl terminus of glutathione-S-transferase. it is conceivable that.

Escherichia coli strain JM109 was transformed using pGEXbckit to express GST-c-Kit. That is, E. coli transformed with pGEXbckit was transformed with 100 μg / ml ampicillin,
% Glucose containing 8 ml of 2 × TY medium (hereinafter referred to as “2 × TY-G
Medium "), added to 800 ml of 2xTY-G medium, and further cultured at 37 ° C. Cell density about 2 × 10 ⁸ ce
After growing Escherichia coli to lls / ml, 0.1 mM of isopropyl-β-D-thiogalactopyranoside was added to induce the expression of the fusion protein, and the cells were further cultured at 37 ° C. for 1 hour.
The culture was centrifuged at 4000 × g for 10 minutes to collect the E. coli cells, and the collected E. coli was dissolved in 50 ml of a lysis buffer containing 1 mg / ml lysozyme (10 mM Na ₂ HPO ₄ , 30 mM NaCl, 0.25% Tween 20, 10%).
mM 2-mercaptoethanol, 10 mM EDTA, 10 mM EGTA)
Suspended in water. The suspension was freeze-thawed four times, followed by sonication to dissolve the Escherichia coli body. This was centrifuged at 800 xg for 10 minutes, and the precipitate containing the fusion protein was recovered.
It was suspended in 6 ml of PAGE sample buffer. This sample is
Electrophoresis was performed in a preparative 7.5% SDS-polyacrylamide gel, and an eluted fraction containing only GST-c-Kit was collected. The eluted fraction obtained from a total of 4 liters of E. coli solution was
Concentration using a K column (Filtran) yielded about 9 mg of the purified fusion protein solution. Rabbits were immunized according to a standard method to prepare anti-GST-c-Kit antiserum.

Further, the obtained antiserum was subjected to affinity purification. That is, about 0.5 mg of the recombinant bovine-derived c-Kit protein produced using the recombinant baculovirus in Example 1 (1) was
An affinity column was prepared by binding to an ap NHS-activated column (Pharmacia). 1 ml of antiserum 10
mM tris (hydroxyl) aminomethane hydrochloride solution (pH
The mixture was diluted 10-fold with (7.4) and adsorbed to an affinity column at a flow rate of 1 ml / min. After washing the column with 10 mM tris (hydroxyl) aminomethane hydrochloric acid solution (pH 7.4),
The antibody bound to the column was combined with 4 ml of 100 mM glycine aqueous solution (p
H 2.5). This was dialyzed against PBS to obtain an affinity purified anti-bovine c-Kit antibody.

Preparation of Bovine c-Kit Protein Expression Vector and Gene Transfer into Cos-7 Cells An expression vector pSVLbkit for expressing bovine c-Kit protein in animal cells was prepared. First, plasmid cl.75 was replaced with restriction enzymes XhoI and Bho according to standard methods.
Bovine cK contained in cl.75 by digestion with amHI
A fragment containing the entire region of the it protein cDNA was prepared. This was connected to an eukaryotic expression plasmid pSVL (Pharmacia) previously cut with XhoI and BamHI, and the expression plasmid pSVL was
VLbKit was prepared.

Gene transfer into Cos-7 cells was performed using the liposome method [Biotechniqes 12, 643 (1987)]. That is, 4 μg of the expression plasmid pSVLbckit and 20 μl of a plus reagent (Gibco BRL) were mixed and allowed to stand at room temperature for 15 minutes. 30 μl of Lipofectamine (Gibco BRL)
Was further added and left at room temperature for 15 minutes. This mixture was added to Cos-7 cells which had been washed in advance with an Opti-MEM medium (Gibco Bethesda Research Laboratories) and left at 37 ° C. for 3 hours to carry out gene transfer. Thereafter, fetal bovine serum was added to a final concentration of 10%, and the mixture was further cultured at 37 ° C. for 2 days and used in the following experiments.

Detection of Expression of Recombinant Bovine c-Kit Protein in Cos-7 Cells Using Anti-GST-c-Kit Antiserum Recombinant bovine c-Kit in transfected Cos-7 cells
The expression of the Kit protein was determined by affinity-purified anti-bovine c-Kit antibody and anti-GST-cK as described in (1) of Example 2.
It was confirmed by indirect immunofluorescence staining and immunoprecipitation using it antiserum. Indirect immunofluorescence staining was performed as follows.

1 × 10 ⁵ Cos-7 cells into which the expression vector was transfected and 1 × 10 ⁵ Cos-7 cells without the transfection were suspended in 50 μl of an affinity-purified anti-bovine c-Kit antibody. Left for a minute. After washing with PBS containing 1% fetal bovine serum (hereinafter referred to as “FCS / PBS”), 50 μl
Was suspended in FCS / PBS, and 1 μl of anti-rabbit IgG goat antiserum (Coulter) labeled with the fluorescent dye fluorescein isothiocyanate was added thereto and left at 4 ° C. for 20 minutes. Next, after washing with FCS / PBS, the cells were suspended in 100 μl of FCS / PBS, and the fluorescence intensity of the cells was measured using a fluorescence activated cell sorter (EPICS Elite, Coulter).

As a result, it was shown that about 15% of the Cos-7 cells transfected with the expression plasmid were stained with the anti-GST-c-Kit antiserum. On the other hand, Cos-7 cells without gene transfer were not stained at all. On the other hand, immunoprecipitation was performed as follows.

That is, 2 × 10 ⁶ Cos-7 cells transfected with the expression vector pSVLbckit and 2 × 10 ⁶ Cos-7 cells not transfected were cultured in a 100 mm plastic dish. -Free cysteine-free DMEM supplemented with various dialysed fetal bovine serum
The medium was replaced with a medium (Gibco Bethesda Research Laboratory). To this was added 4.1 MBq of [ ³⁵ S] Express protein labeling mix (NEN), and the mixture was allowed to stand at 37 ° C. for 4 hours, thereby labeling cellular proteins with radioactive amino acids. After washing the cells with PBS, 1.5 ml of RIPA buffer [Molecular and Cellular Biology 8, 4896 (198
8)] to obtain a cell lysate. 50 μl of protein G Sepharose (Pharmacia Biotech) suspended in RIPA buffer at a volume ratio of 50% to 1.5 ml of the lysate
After the mixture was left at 4 ° C. overnight, the mixture was centrifuged at 150 × g for 30 minutes, and the supernatant was recovered. To 250 μl of this supernatant, 5 μl of anti-GST-c-Kit antiserum and 20 parts of protein G sepharose suspension were added.
μl was added and reacted at 4 ° C. overnight. After washing the immunoprecipitate obtained by centrifugation four times with RIPA buffer,
It was suspended in S-PAGE sample buffer and heated at 100 ° C. for 5 minutes. After electrophoresis in a 7.5% polyacrylamide gel, the gel was treated with a sensitizer (Enlightening, DuPont) for 15 minutes. The gel is then dried and X
The film was exposed to light.

As a result, in Cos-7 cells transfected with the expression plasmid, the recombinant bovine c-Kit protein was detected as two bands having a molecular weight of about 135,000 and 150,000 (FIG. 4). On the other hand, these bands were not observed in Cos-7 cells into which no gene had been introduced. In a study in which recombinant mouse-derived c-Kit protein was expressed in Cos cells, mouse-derived c-Kit protein was detected as two bands with molecular weights of 130,000 and 150,000 [EMBO Journal 10, 2451 (1991)]. . The molecular weight of the recombinant bovine c-Kit protein is in good agreement with this. From the above results obtained by the indirect immunofluorescence staining method and the immunoprecipitation method, it was confirmed that the recombinant bovine c-Kit protein was expressed on the cell membrane surface in Cos-7 cells into which pSVLbckit was introduced.

Measurement of Binding Activity of Recombinant Bovine c-Kit Protein to Bovine SCF Binding Activity of Recombinant Bovine c-Kit Protein Expressed on Cos-7 Cells Transfected with pSVLbcKit to Bovine SCF Was examined by binding experiments using radiolabeled SCF. First, as shown below, recombinant bovine SCF
Was prepared. The cDNA encoding bovine SCF (hereinafter referred to as "bovine SCF cDNA") has already been cloned by the inventors and its nucleotide sequence has been determined [Biochimica Biophysica Acta 1223, 148-150 (199
Four)]. The nucleotide sequence of cDNA encoding bovine SCF is shown in SEQ ID NO: 2.

The base sequence of bovine SCF cDNA [Bioc
himica Biophysica Acta 1223, 148-150 (1994)], the bovine SCF consists of 274 amino acid residues,
Compared to the human and mouse sequences,
%, 79.6%. Also, from comparison with the amino acid sequences of human SCF, mouse SCF and rat SCF, bovine SCF was found to have a secretory signal sequence (the amino acid residue from -25 to the first amino acid residue) and an extracellular region (+ 1 to 190 amino acid residues), a cell transmembrane region (191 to 213 amino acid residues), and an intracellular region (214 to 249 amino acid residues);
It was presumed that the region consisting of the amino acid residues from +1 to 165th or 166th is a region that functions as a soluble bovine SCF.

Therefore, for the purpose of producing and purifying a large amount of soluble bovine SCF, from the -25th to 1st bovine SCF,
Proteins containing up to 65 amino acid residues (hereinafter referred to as
CDN encoding "short-form bovine SCF")
A was prepared by a known method [Virus Research 21, 123 (19
91), Nucleic Acids Research 15, 10233 (1987)] to prepare a recombinant containing a cDNA encoding a truncated bovine SCF, and culturing the transformant transformed with the recombinant. Soluble bovine SCF in culture
Therefore, bovine SCF was purified from the culture solution by an ordinary method. The details are described below.

I) Preparation of Recombinant Baculovirus Producing Bovine SCF First, in order to prepare cDNA encoding truncated bovine SCF, plasmid pbSCF1 having bovine SCF cDNA [Biochimica Biophysica Acta 1223, 111-222]
(1994)] as a template, and a polymerase chain reaction (PCR) was performed using the two synthetic oligonucleotides F and G shown in FIG. 2 as primers. Plasmid pbSCF1 is obtained by combining bovine SCF cDNA with plasmid pBluescriptTM SK.
(-) (Stratagene) restriction enzyme BamHI cleavage site and X
A sequence of 16 bases between the BamHI cleavage site and the first base of the cDNA [(5 ′)-CAG
CGCTGCCTTTCCT- (3 ′)]. Escherichia coli harboring plasmid pbSCF1 was deposited as FERM P-16232 with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology (Deposit date: May 19, 1997).

In the nucleotide sequence of primer F, 9
The sequence from the 24th to the 24th is identical to the inserted sequence of 16 bases in pbSCF1. The 10th to 25th sequences from the 5 'side of the nucleotide sequence of the primer G are complementary to the nucleotide numbers 571 to 556 of the bovine SCF cDNA. However, the subsequent nucleotide sequence is different from the sequence of the cDNA.
The 9th to 9th sequences (TTA) are complementary to the protein synthesis stop codon TAA. The third to eighth sequences (AAGCTT) are cleavage site sequences by the restriction enzyme HindIII.

The PCR was performed under general reaction conditions. That is, PCR was performed using 2 μg / ml pbSCF, 1 μM each.
Oligonucleotide primers, 1 × PCR buffer (Perkin-Elmer ABI) and 0.2 mM deoxyadenosine-5′-3 phosphate, deoxyguanosine-5′-3phosphate, deoxycytidine-5′-3 100 μl of a reaction solution composed of phosphoric acid and thymidine-5′-3 phosphoric acid, and D
NA Thermal Cycler 480 (Perkin Elmer A
BI) for 15 cycles, with 1 cycle at 94 ° C., 1 minute at 52 ° C. and 2 minutes at 72 ° C.

A DNA fragment of about 600 base pairs in length amplified by PCR was purified by agarose gel electrophoresis under standard conditions, and then subjected to restriction enzymes BamHI and HindIII.
Digested. Plasmid pBluescript SK (-) (Stratagene) previously digested with the restriction enzymes BamHI and HindIII using E. coli T4 ligase under standard conditions
And the plasmid pBSbSCF was prepared. Escherichia coli JM109 [Gene 33,103-119 (198
5)] and transform plasmid pB using standard methods.
SbSCF was amplified. Next, pBSbSCF was replaced with the restriction enzyme HindII.
A cDNA fragment was prepared by digestion with I and SmaI, and both ends were blunted with E. coli DNA polymerase I according to standard methods, followed by plasmid pAcYM1 [Virus Research
21, 123 (1991)] to prepare a transfer vector pAcbSCF.

The genomic DNA of pAcbSCF and baculovirus BakPAK6 (Stratagene) is cotransfected into SF21AE cells (supra) [Virus Rese
arch 21, 123 (1991), Nucleic Acids Research 15, 1
[0233] Thus, a recombinant baculovirus AcSCF strain producing bovine SCF was prepared. That is, pAcbSCF 10ng and Ba previously cut with the restriction enzyme Eco81I.
100 ng of kPAK6 virus genomic DNA was mixed with 5 μg of ribofectin (Gibco-BRL) and the mixture was 3.5 cm
Was added to a culture solution of SF21AE cells (1 × 10 ⁶ cells) cultured in the culture dish described above, and cultured at 28 ° C. for 6 hours. Then add fetal bovine serum to a final concentration of 10%,
Culture was continued for another 3 days. After collecting the culture supernatant and serially diluting it with the TC100 culture solution, add 0.1 ml of the diluted solution to 3.
After adsorption for 1 hour to 5 10 ⁶ SF21AE cells cultured in cm dishes, except for the culture supernatant. 1.5 to this SF21AE cell
2 ml of TC100 medium containing 5% agarose was added, and the cells were cultured for 3 days. Thereafter, 10 μg / ml of 5-bromo-4-chloro-
1 ml of TC100 medium containing 3-indyl-β-D-galactopyranoside (Wako Pure Chemical Industries) was overlaid and cultured for another day. From the plaques that appeared, a plurality of plaques that did not stain blue were collected, and each was suspended in 0.3 ml of TC100 medium to prepare a stock solution of each recombinant virus clone. 3.5ml
Were infected with the respective suspensions, and cultured for 4 days to obtain a culture solution containing the recombinant virus. Remley method [Nature 227, 680
(1970)], a part of the culture solution (10 μl each) was subjected to SDS-polyacrylamide gel electrophoresis (hereinafter, referred to as “SDS-PA”).
GE "), and after the electrophoresis, the gel was stained with Coomassie Brilliant Blue to examine the presence or absence of bovine SCF having a molecular weight of about 20,000 in the culture solution. On the other hand, high molecular weight DNA was prepared according to standard methods from the virus-infected cells, it was digested with the DNA restriction enzyme BamHI, and agarose gel electrophoresis according to standard methods, was ³² P-labeled bovine
Analysis was performed by Southern blot hybridization using SCF cDNA as a probe, and the presence or absence of SCF cDNA in the genomic DNA of each clone of the recombinant virus was examined. As a result, a recombinant baculovirus clone AcbSCF strain containing SCF cDNA as expected and secreting bovine SCF in the culture supernatant was obtained. By infecting a larger amount of SF21AE cells with the culture supernatant of this clone, about 200 ml
Was obtained. The virus infectivity in this virus solution was 2 × 10 ⁷ plaque forming units (PFU) / ml.

Ii) Production and Purification of Recombinant Bovine SCF Using Recombinant Baculovirus 1 × 10 ⁷ Tn-5 cells cultured in a 75 ml culture plastic bottle (Nunc) were added to the above virus solution. (1 × 10 ⁶
PFU) and inoculate the cells in 10 ml of TC100 medium.
C. for 4 days. After recovering the culture supernatant, salt exchange was performed using a gel filtration column (PD-10 column, Pharmacia), and a 10 mM tris (hydroxyl) aminomethane hydrochloride (hereinafter, referred to as “Tris hydrochloride”) solution (pH 8.0) And
After adsorbing this to an anion exchange column (MonoQ column, Pharmacia) at a flow rate of 1 ml / min, the protein is eluted with a NaCl concentration gradient from 0 to 1 M in the presence of 10 mM Tris-HCl buffer (pH 8.0). I let it. A part of the eluted fraction of each 1 ml was analyzed by PAGE according to the Lemley method to find a fraction containing bovine SCF, collected, subjected to salt exchange with a PD-10 column, and subjected to a 10 mM sodium acetate solution (pH 4. 6).
After adsorbing this to a cation exchange column (MonoS column, Pharmacia) at a flow rate of 1 ml / min, the protein was eluted with a 0 to 1 M NaCl concentration gradient in the presence of 10 mM sodium acetate (pH 4.6). When a part of each eluted fraction was analyzed by PAGE, it was found that the fraction eluted at a NaCl concentration of about 0.5 M contained only bovine SCF, and these were collected and purified recombinant bovine SCF. It was a standard. This was analyzed by SDS-PAGE.
The molecular weight of F was shown to be between 19000 and 23000 (FIG. 8). The weight of purified bovine SCF was determined by quantitative amino acid analysis. As a result, about 2 mg of purified bovine SCF was obtained from about 400 ml of the culture supernatant.

The recombinant bovine SCF obtained by the above (i) and (ii) was subjected to the chloramine T method [Nature 194,495].
(1962)] by was labeled with ¹²⁵ I. Labeled bovine SC
F (hereinafter referred to as “ ¹²⁵ I-bSCF”) has a specific activity of 900 GBq / n
was mol. 20 fmol of Cos-7 cells transfected with pSVLbckit and non-transfected Cos-7 cells (2 × 10 ⁵ each)
And ¹²⁵ I-bSCF at 22 ° C. for 90 minutes. Thereafter, a ¹²⁵ I- BSCF which are free with ¹²⁵ I- BSCF bound to cells were separated using a phthalate oil and the radioactivity of ¹²⁵ I ^-bSCF bound to the cells was determined in a gamma counter. The results are shown in Table 1 below.

[0075]

[Table 1]

As shown in Table 1, ¹²⁵ I-bSCF bound to the Cos-7 cells transfected with the expression plasmid, but no significant binding to Cos-7 cells not transfected was observed. . In addition, when simultaneously reacting a 100-fold excess of unlabeled recombinant bovine SCF with 20 fmol of ¹²⁵ I-bSCF during the reaction, the transfected Cos-7 cells and ¹²⁵ I-bSCF
Since the binding to F was competitively inhibited, the recombinant bovine c-Kit protein expressed in Cos-7 cells and ¹²⁵ I-
It was confirmed that the binding to bSCF was specific. From the above results, it was shown that the recombinant bovine c-Kit protein expressed on the surface of Cos-7 cells into which pSVLbckit was introduced has an activity of specifically binding to bovine SCF.

(2) Examination of the reactivity of the monoclonal antibody to the recombinant bovine c-Kit protein The reactivity of the bK-1 antibody to the recombinant bovine c-Kit protein expressed on the Cos-7 cell surface was determined. The indirect immunofluorescence staining was examined according to the method of (1) of Example 2. At this time, 1 μg of the bK-1 antibody was used for 1 × 10 ⁵ cells.
The reaction was performed. As a result, it was observed that about 30% of the Cos-7 cells transfected with pSVLbckit were specifically stained. On the other hand, Co without gene transfer
The s-7 cells did not stain at all.

Next, the examination by the immunoprecipitation method was carried out according to the method of (1) of Example 2. At this time, 5 μg of the bK-1 antibody and protein G were added to 250 μl of the Cos-7 cell lysate.
After adding 20 μl of the Sepharose suspension and reacting at 4 ° C. overnight, the immunoprecipitate was detected. As a result, the molecular weight was about 135,000 in Cos-7 cells transfected with the expression plasmid.
And 150,000 two bands were detected (FIG. 5). The molecular weights of these bands were higher than those of Example 2 (1).
Recombinant bovine cK detected using GST-c-Kit antiserum
The agreement with the molecular weight of the it protein indicated that the bK-1 antibody specifically reacted with the recombinant bovine c-Kit protein. On the other hand, these bands were not detected in Cos-7 cells into which no gene was transfected.

Example 3 Detection of Bovine c-Kit Protein in Bovine Vascular Endothelial Cells Using Monoclonal Antibody It is known that human-derived c-Kit protein is expressed in vascular endothelial cells in humans. [Blood 83, 2145
(1994)]. Therefore, the reactivity of the monoclonal antibody to the bovine c-Kit protein expressed in bovine vascular endothelial cells was examined by indirect immunofluorescence staining and immunoprecipitation.

(1) Examination by Immunofluorescent Staining First, a biotin-labeled bK-1 antibody (hereinafter referred to as “biotin-labeled bK-1 antibody”) was prepared. That is, 2 mg
Was added with 0.2 mg of sulfo-NHS-LC-biotin (Pierce) and left at room temperature for 30 minutes. Thereafter, a fraction containing a biotin-labeled bK-1 antibody was collected through a gel filtration column (PD-10 column, Pharmacia Biotech).

EGG cells derived from bovine adrenal capillary endothelial cells [US Pat. No. 4,670,394] 1 × 10 ⁵ cells and 0.5 μg of biotin-labeled bK
The -1 antibody was reacted at 4 ° C for 20 minutes. After washing with FCS / PBS, this was reacted with streptavidin (Coulter) labeled with the fluorescent dye phycoerythrin at 4 ° C. for 20 minutes. This was washed with FCS / PBS, suspended in 100 μl of FCS / PBS, and the fluorescence intensity of the cells was measured using a fluorescence activated cell sorter (EPICS Elite, Coulter). At this time, a reaction obtained by reacting only streptavidin labeled with phycoerythrin without reacting the biotin-labeled bK-1 antibody was used as a negative control.

As a result, when the biotin-labeled bK-1 antibody was reacted, the fluorescence intensity of 64% of the EJG cells was significantly increased as compared with the negative control. The results showed that the biotin-labeled bK-1 antibody specifically reacted with the bovine c-Kit protein expressed in 64% of the EJG cells.

(2) Examination by immunoprecipitation The immunoprecipitation was performed according to the method described in (1) of Example 2. The medium of EJG cells (3 × 10 ⁶ ) previously cultured in a 100-mm dish was exchanged with a methionine-cysteine-free DMEM medium supplemented with 10% dialyzed fetal calf serum.
MBq [ ³⁵ S] Express protein labeling mix (New England Nuclear) was added and left at 37 ° C. for 4 hours. This was washed with PBS and solubilized with 1.5 ml of RIPA buffer to obtain a cell lysate. To 1.5 ml of the cell lysate was added 50 μl of a suspension of protein G Sepharose, and the mixture was allowed to stand at 4 ° C. overnight. In 500 μl of this supernatant, 5 μg of bK-1 antibody and 20 μl
Was added, and the mixture was reacted overnight at 4 ° C. to perform immunoprecipitation. Thereafter, the immunoprecipitate was detected by the method described in (1) of Example 2.

As a result, two of the molecular weights of 135,000 and 150,000
This band was specifically immunoprecipitated by the bK-1 antibody (FIG. 6). In contrast, when no reaction was carried out with the bK-1 antibody (negative control), these bands were not observed.
Since the molecular weights of these two bands correspond to the molecular weights of the recombinant bovine c-Kit protein detected using the bK-1 antibody in (2) of Example 2, the bK-1 antibody was EJG
It was shown to specifically react with bovine c-Kit protein in cells.

Example 4 Bovine Derivation from Bovine Bone Marrow Cells
Isolation of c-Kit protein-positive cells c-Kit protein-positive cells contained in human bone marrow have been separated using monoclonal antibodies, and blood progenitor cells capable of forming colonies have been identified in the c-Kit protein-positive cell fraction. Included in the frequency is a colony formation test (co
lony formationassay).
[Japanese Patent Application 4-510017, Blood 78, 30 (1991), Blood 78, 14
03 (1991)]. Therefore, bovine c-Kit protein-positive cells were separated from bovine bone marrow cells using the bK-1 antibody, and their colony forming ability was examined.

Bone marrow cells were collected from the femurs of normal newborn bulls, diluted with PBS supplemented with 0.15% EDTA, and then diluted with Ficoll Pak (Pharmacia Biotech).
The mononuclear cells were collected by density gradient centrifugation using a. After culturing this at 37 ° C. for 2 hours in RMPI1640 medium, only cells that did not adhere to the plastic dish were collected. This was suspended in Iscove's medium (Gibco BRL) containing 34% CP-1 (Farto Pharmaceutical) and 4% bovine serum albumin, and-
Stored at 80 ° C and used for experiments. Thawed bone marrow mononuclear cells 2
To 1 × 10 ⁷ cells, add 1 μg of biotin-labeled bK-1 antibody.
Left for a minute. After washing this with FCS / PBS, streptavidin labeled with the fluorescent dye phycoerythrin was added,
It was left at 4 ° C. for 20 minutes. After washing with FCS / PBS, the fluorescence-staining positive and negative cell fractions were collected using a fluorescence-activated cell sorter (EPICS Elite, Coulter).

As a result, 94% of all cells contained in the cell fraction positive for fluorescent staining were c-Kit protein positive cells (FIG. 7). From these results, it was found that c-Kit protein-positive cells and c-Kit protein-negative cells could be sorted as fluorescent staining positive and negative cell fractions, respectively. In order to examine the colony forming ability of the sorted cells, a previously reported method [Experimental Hematology
19, 226 (1991)].

That is, cells of each fraction (2 × 10 ⁴ cells) were
Resuspend in 1 ml of colony formation culture and
After culturing at 37 ° C. for 7 days in a plastic dish, the number of formed colonies was counted. 30% fetal bovine serum, 1.0% methylcellulose, 1% bovine serum albumin,
0.1 mM β-mercaptoethanol, 2 mM glutamine, 2.5
μg / ml amphotericin B, 50 units / ml penicillin,
Iscove's medium containing 50 µg / ml streptomycin and 100 ng / ml recombinant bovine SCF was used as a medium for the colony formation test.

As a result, an average of 30 colonies were formed per 2 × 10 ⁴ cells before the fractionation, an average of 224 cells were observed in the bovine c-Kit protein positive cell fraction, and an average of 3 colonies were formed in the negative cell fraction. Was observed. In addition, no colony formation was observed in any of the cell fractions under the conditions except for the bovine SCF from the culture solution for the colony formation test.
From the above results, it was shown that the c-Kit protein-positive cell fraction separated using the bK-1 antibody frequently contained blood progenitor cells having a colony forming ability dependent on bovine SCF.

[0090]

Industrial Applicability According to the present invention, a monoclonal antibody specifically reacting with a bovine c-Kit protein, a hybridoma producing the monoclonal antibody, and a method for separating bovine c-Kit protein positive cells using the monoclonal antibody Is provided. By using the monoclonal antibody of the present invention, purification of bovine c-Kit protein from a sample containing bovine c-Kit protein, quantification of bovine c-Kit protein, or bovine c-Kit protein positive cells Bovine c-Kit protein-positive cells can be efficiently and accurately separated from a sample containing the same.

[0091]

[Sequence list] SEQ ID NO: 1 Sequence length: 3069 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: mRNA to cDNA sequence CTTGGCGCTC GCGGCTCTGG GGGCTCGGCT TTGCCGCGCT CCCGGCACTC GGGCGAGAGC 60 CGGAACGTGG AACAGAGCTC CGGCCCCCC CG ATG AGA GGC GCT 114 Met Arg Gly Ala 1 CGC GGC GCC TGG GAT TTC CTC TTC GTT CTG CTG CTC CTG CTC CTC GTC 162 Arg Gly Ala Trp Asp Phe Leu Phe Val Leu Leu Leu Leu Leu Leu Val 5 10 15 20 CAG ACA GGC TCT TCT CAG CCA TCT GTG AGT CCA GGG GAA CTG TCT CTA 210 Gln Thr Gly Ser Ser Gln Pro Ser Val Ser Pro Gly Glu Leu Ser Leu 25 30 35 CCA TCT ATC CAC CCA GCA AAA TCA GAG TTA ATT GTC AGC GTT GGC GAC 258 Pro Ser Ile His Pro Ala Lys Ser Glu Leu Ile Val Ser Val Gly Asp 40 45 50 GAG ATT AGG CTG TTA TGC ACC GAT CCA GGA TTT GTC AAG TGG ACT TTT 306 Glu Ile Arg Leu Leu Cys Thr Asp Pro Gly Phe Val Lys Trp Thr Phe 55 60 65 GAG ATC CTG GGT CAA CTG AGT GAG AAA ACA AAC CCG GAA TGG ATC ACC 354 Glu Ile Leu Gly Gln Leu Ser Glu Lys Thr Asn Pro Glu Trp Ile Thr 70 75 80 GAG AAA GCA GAG GCC ACA AAT ACA GGC AAT TAC ACG TGC ACC AAT AAA 402 Glu Lys Ala Glu Ala Thr Asn Thr Gly Asn Tyr Thr Cys Thr Asn Lys 85 90 95 100 GGC GGC TTG AGC AGT TCC ATT TAT GTG TTT GTT AGA GAC CCC GAG AAG 450 Gly Gly Leu Ser Ser Ser Ile Tyr Val Phe Val Arg Asp Pro Glu Lys 105 110 115 CTT TTC CTG ATT GAC CTT CCC TTG TAC GGG AAA GAA GAA AAC GAC ACG 498 Leu Phe Leu Ile Asp Leu Pro Leu Tyr Gly Lys Glu Glu Asn Asp Thr 120 125 130 CTG GTT CGC TGT CCC CTG ACA GAC CCC GAG GTG ACC AAT TAC TCT CTT 546 Leu Val Arg Cys Pro Leu Thr Asp Pro Glu Val Thr Asn Tyr Ser Leu 135 140 145 ACG GGG TGT GAG GGG AAA CCT CTC CCC AAG GAT TTG ACA TTT GTG GCC 594 Thr Gly Cys Glu Gly Lys Pro Leu Pro Lys Asp Leu Thr Phe Val Ala 150 155 160 GAC CCC AAG GCA GGT ATC ACA ATC AGA AAT GTG AAG CGC GAG TAC CAT 642 Asp Pro Lys Ala Gly Ile Thr Ile Arg Asn Val Lys Arg Glu Tyr His 165 170 175 180 CGG CTC TGT CTG CAC TGC TCA GCG AAT CAG AGG GGC AAG TCC ATG CTG 690 Arg Leu Cys Leu His Cys Ser Ala A sn Gln Arg Gly Lys Ser Met Leu 185 190 195 TCG AAG AAA TTC ACT CTG AAA GTG CGG GCA GCC ATC AAA GCT GTG CCA 738 Ser Lys Lys Phe Thr Leu Lys Val Arg Ala Ala Ile Lys Ala Val Pro 200 205 210 GTT GTG TCT GTG TCC AAA ACC AGC TAT CTT CTC AGG GAA GGA GAG GAA 786 Val Val Ser Val Ser Lys Thr Ser Tyr Leu Leu Arg Glu Gly Glu Glu 215 220 225 TTT GCA GTG ACA TGC TTG ATT AAA GAC GTG TCT AGT TCC GTG GAC TCT 834 Phe Ala Val Thr Cys Leu Ile Lys Asp Val Ser Ser Ser Val Asp Ser 230 235 240 ATG TGG ATA AAG GAA AAC AGC CAG CAG ACT AAA GCA CAG ACG AAG AAG 882 Met Trp Ile Lys Glu Asn Ser Gln Gln Thr Lys Ala Gln Thr Lys Lys 245 250 255 260 AAT AGC TGG CAT CAG GGT GAC TTC AGT TAT CTC CGT CAG GAA AGG TTG 930 Asn Ser Trp His Gln Gly Asp Phe Ser Tyr Leu Arg Gln Glu Arg Leu 265 270 270 275 ACT ATC AGC TCA GCA AGA GTG AAT GAT TCC GGT GTG TTC ATG TGT TAC 978 Thr Ile Ser Ser Ala Arg Val Asn Asp Ser Gly Val Phe Met Cys Tyr 280 285 290 GCC AAT AAC ACT TTT GGA TCA GCA AAT GTC ACA ACA ACC TTA GAA GTA 1026 Ala Asn Asn Thr PheGly Ser Ala Asn Val Thr Thr Thr Leu Glu Val 295 300 305 GTA GAT AAA GGA TTC ATT AAT ATC TTC CCT ATG ATG AAC ACA ACA GTA 1074 Val Asp Lys Gly Phe Ile Asn Ile Phe Pro Met Met Asn Thr Thr Val 310 315 320 TTT GTA AAT GAT GGA GAG AAT GTG GAT CTG GTT GTT GAA TAT GAG GCA 1122 Phe Val Asn Asp Gly Glu Asn Val Asp Leu Val Val Glu Tyr Glu Ala 325 330 335 340 340 TAT CCC AAA CCT GTA CAC CGA CAA TGG ATA TAT ATG AAC AGA ACC TCC 1170 Tyr Pro Lys Pro Val His Arg Gln Trp Ile Tyr Met Asn Arg Thr Ser 345 350 355 ACT GAT AAG TGG GAC GAT TAT CCT AAG TCT GAA AAT GAA AGT AAT ATC 1218 Thr Asp Lys Trp Asp Asp Tyr Pro Lys Ser Glu Asn Glu Ser Asn Ile 360 365 370 AGA TAC GTA AAT GAA CTT CAT CTA ACC AGA TTA AAA GGG ACT GAA GGA 1266 Arg Tyr Val Asn Glu Leu His Leu Thr Arg Leu Lys Gly Thr Glu Gly 375 380 385 GGC ACT TAC ACA TTT CAC GTG TCC AAT TCT GAT GTC AAT TCT TCC GTG 1314 Gly Thr Tyr Thr Phe His Val Ser Asn Ser Asp Val Asn Ser Ser Val 390 395 400 ACA TTT AAT GTT TAC GTG AAC ACA AAA CCA GAA ATC CTG ACG CAT GAC 1362 T hr Phe Asn Val Tyr Val Asn Thr Lys Pro Glu Ile Leu Thr His Asp 405 410 415 420 AGG CTG GTG AAT GGC ATG CTA CAG TGC GTG GCC GCA GGG TTC CCG GAG 1410 Arg Leu Val Asn Gly Met Leu Gln Cys Val Ala Ala Gly Phe Pro Glu 425 430 435 CCA ACC ATC GAT TGG TAC TTT TGT CCA GGA ACT GAG CAG AGG TGT TCT 1458 Pro Thr Ile Asp Trp Tyr Phe Cys Pro Gly Thr Glu Gln Arg Cys Ser 440 445 450 GTT CCC GTT GGG CCA GTG GAT GTA CAG ATC CAA AAC TCA TCT GTC TCA 1506 Val Pro Val Gly Pro Val Asp Val Gln Ile Gln Asn Ser Ser Val Ser 455 460 465 CCA TTT GGA AAA CTA GTG GTT TAT AGC ACC ATT GAT GAC AGC ACA TTC 1554 Pro Phe Gly Lys Leu Val Val Tyr Ser Thr Ile Asp Asp Ser Thr Phe 470 475 480 AAA CAC AAT GGG ACG GTG GAG TGC AGG GCT TAT AAC GAT GTG GGC AAG 1602 Lys His Asn Gly Thr Val Glu Cys Arg Ala Tyr Asn Asp Val Gly Lys 485 490 495 500 AGT TCT GCC TCT TTT AAC TTT GCA TTT AAA GGT AAC AGC AAA GAA CAA 1650 Ser Ser Ala Ser Phe Asn Phe Ala Phe Lys Gly Asn Ser Lys Glu Gln 505 510 515 ATC CAT GCT CAC ACC CTG TTC ACG CCG TTG CTG ATT GGT TTT GTG ATC 1698 Ile His Ala His Thr Leu Phe Thr Pro Leu Leu Ile Gly Phe Val Ile 520 525 530 GCA GCT GGT TTA ATG TGT ATC TTC GTG ATG ATT CTT ACA TAC AAA TAT 1746 Ala Ala Gly Leu Met Cys Ile Phe Val Met Ile Leu Thr Tyr Lys Tyr 535 540 545 TTG CAG AAA CCC ATG TAT GAA GTA CAG TGG AAA GTT GTC GAG GAG ATA 1794 Leu Gln Lys Pro Met Tyr Glu Val Gln Trp Lys Val Val Glu Glu Ile 550 555 560 AAT GGA AAC AAT TAT GTT TAC ATA GAC CCA ACA CAA CTT CCT TAT GAT 1842 Asn Gly Asn Asn Tyr Val Tyr Ile Asp Pro Thr Gln Leu Pro Tyr Asp 565 570 575 580 580 CAC AAA TGG GAG TTT CCC AGG AAC AGG CTG AGT TTT GGG AAA ACC CTG 1890 His Lys Trp Glu Phe Pro Arg Asn Arg Leu Ser Phe Gly Lys Thr Leu 585 590 595 GGT GCT GGC GCC TTC GGG AAA GTT GTT GAG GCC ACC GCT TAT GGC TTA 1938 Gly Ala Gly Ala Phe Gly Lys Val Val Glu Ala Thr Ala Tyr Gly Leu 600 605 610 ATT AAA TCA GAT GCA GCC ATG ACT GTT GCT GTC AAG ATG CTC AAA CCA 1986 Ile Lys Ser Asp Ala Ala Met Thr Val Ala Val Lys Met Leu Lys Pro 615 620 625 AGC GCC CAT TTA ACC GAA CGA GAA GCC CTA ATG TCT GAA CTC AAA GTC 2034 Ser Ala His Leu Thr Glu Arg Glu Ala Leu Met Ser Glu Leu Lys Val 630 635 640 TTG AGT TAC CTC GGT AAT CAT ATG AAT ATT GTG AAT CTT CTG GGA GCG 2082 Leu Ser Tyr Leu Gly Asn His Met Asn Ile Val Asn Leu Leu Gly Ala 645 650 655 660 TGC ACC ATT GGA GGG CCC ACC CTG GTC ATT ACA GAA TAT TGT TGC TAT 2130 Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Thr Glu Tyr Cys Cys Tyr 665 670 675 GGT GAT CTT CTG AAT TTT TTG AGA AGA AAA CGT GAT TCA TTT ATT TGC 2178 Gly Asp Leu Leu Asn Phe Leu Arg Arg Lys Arg Asp Ser Phe Ile Cys 680 685 690 TCA AAG CAG GAA GAT CAC GCC GAA GTG GCG CTTAT AAG AAC CTT CTT 2226 Ser Lys Gln Glu Asp His Ala Glu Val Ala Leu Tyr Lys Asn Leu Leu 695 700 705 CAT TCA AAG GAG TCT TCC TGC AAT GAT AGT AGT ACT AAT GAG TAC ATG GAC 2274 His Ser Lys Glu Ser Ser Cys Asn Asp Ser Thr Asn Glu Tyr Met Asp 710 715 720 ATG AAA CCT GGA GTT TCT TAT GTT GTA CCA ACC AAG GCA GAC AAG AGG 2322 Met Lys Pro Gly Val Ser Tyr Val Val Pro Thr Lys Ala Asp Lys Arg 725 730 735 740 AGA TCT GCA AGA ATA GGT TCA TAC ATA GAA AGA GAC GTG ACT CCT GCT 2370 Arg Ser Ala Arg Ile Gly Ser Tyr Ile Glu Arg Asp Val Thr Pro Ala 745 750 755 ATC ATG GAA GAT GAT GAG CTG GCC CTG GAC CTG GAG GAC TTG CTG AGC 2418 Ile Met Glu Asp Asp Glu Leu Ala Leu Asp Leu Glu Asp Leu Leu Ser 760 765 770 TTT TCT TAC CAG GTG GCA AAA GGC ATG GCG TTC CTT GCC TCA AAG AAT 2466 Phe Ser Tyr Gln Val Ala Lys Gly Met Ala Phe Leu Ala Ser Lys Asn 775 780 785 TGT ATT CAT AGA GAC TTG GCA GCC AGA AAT ATC CTC CTT ACT CAT GGT 2514 Cys Ile His Arg Asp Leu Ala Ala Ala Arg Asn Ile Leu Leu Thr His Gly 790 795 800 CGA ATC ACA AAG ATT TGT GAT TTT GGT CTA GCC AGA GAC ATC AAG AAT 2562 Arg Ile Thr Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Lys Asn 805 810 815 820 GAT TCT AAT TAT GTG GTC AAA GGA AAC GCT CGA CTC CCT GTG AAG TGG 2610 Asp Ser Asn Tyr Val Val Lys Gly Asn Ala Arg Leu Pro Val Lys Trp 825 830 835 ATG GCA CCA GAG AGT ATT TTC AAC TGT GTA TAC ACA TTT GAA AGT GAT 2658 Met Ala Pro Glu Ser Ile Phe Asn Cys Val Tyr Thr Phe Glu Ser A sp 840 845 850 GTC TGG TCC TAT GGG ATT TTT CTG TGG GAG CTG TTC TCT TTA GGA AGC 2706 Val Trp Ser Tyr Gly Ile Phe Leu Trp Glu Leu Phe Ser Leu Gly Ser 855 860 865 865 AGC CCC TAC CCT GGA ATG CCA GTC GAT TCT AAG TTC TAC AAG ATG ATC 2754 Ser Pro Tyr Pro Gly Met Pro Val Asp Ser Lys Phe Tyr Lys Met Ile 870 875 880 AAG GAA GGT TTC CGA ATG CTC AGC CCC GAG CAT GCA CCT GCG GAA ATG 2802 Lys Glu Gly Phe Arg Met Leu Ser Pro Glu His Ala Pro Ala Glu Met 885 890 895 895 900 TAT GAC ATC ATG AAG ACC TGC TGG GAT GCT GAT CCC TTG AAA AGG CCA 2850 Tyr Asp Ile Met Lys Thr Cys Trp Asp Ala Asp Pro Leu Lys Arg Pro 905 910 915 ACA TTT AAG CAG ATT GTG CAG CTG ATT GAG AAG CAG ATC TCA GAG AGC 2898 Thr Phe Lys Gln Ile Val Gln Leu Ile Glu Lys Gln Ile Ser Glu Ser 920 925 930 ACC AAT CAT ATT TAT TCC AAC TTA GCA AAC TGC AGC CCC CAC CGG GAG 2946 Thr Asn His Ile Tyr Ser Asn Leu Ala Asn Cys Ser Pro His Arg Glu 935 940 945 AAC CCC GCC GTG GAC CAT TCT GTG CGC ATC AAC TCT GTG GGC AGC AGC 2994 Asn Pro Ala Val Asp His Ser Val Arg Ile A sn Ser Val Gly Ser Ser 950 955 960 GCC TCC TCC ACG CAG CCT CTG CTT GTC CAC GAA GAT GTC TGA 3036 Ala Ser Ser Thr Gln Pro Leu Leu Val His Glu Asp Val * 965 970 975 AGCAGTCTGC ATCTGGGGGT CTCCTGACAA CCC 3069

SEQ ID NO: 2 Sequence length: 911 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: mRNA to cDNA sequence T ATG AAG AAG ACA CAA ACT TGG ATT ATC ACT TGC ATT TAT CTT CAA 46 Met Lys Lys Thr Gln Thr Trp Ile Ile Thr Cys Ile Tyr Leu Gln 1 5 10 15 CTG CTC CTA TTT AAT CCT CTC GTC CAC ACT CAA GGG ATC TGC AGT AAC 94 Leu Leu Leu Phe Asn Pro Leu Val His Thr Gln Gly Ile Cys Ser Asn 20 25 30 CGT GTG ACT GAT GAT GTG AAA GAC GTT ACA AAA TTG GTG GCA AAT CTT 142 Arg Val Thr Asp Asp Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu 35 40 45 CCC AAA GAC TAT ATG ATA ACC CTC AAA TAT GTC CCC GGG ATG GAC GTT 190 Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val 50 55 60 TTG CCT AGT CAT TGT TGG ATA AGC GAG ATG GTG GAA CAA CTG TCA GTC 238 Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Glu Gln Leu Ser Val 65 70 75 AGC TTG ACT GAT CTT CTG GAC AAG TTT TCG AAT ATT TCT GAA GGC TTG 286 Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu 80 85 90 95 AGT AAT TAT TGT ATC ATA GAC AAA CTT GTG AAA ATA GTT GAT GAC CTT 334 Ser Asn Tyr Cys Ile Ile Asp Lys Leu Val Lys Ile Val Asp Asp Leu 100 105 110 GTG GAG TGC ATG GAA GAA CAC TCA TCT GAG AAT GTA AAA AAA TCA TCT 382 Val Glu Cys Met Glu Glu His Ser Ser Glu Asn Val Lys Lys Ser Ser 115 120 125 AAG AGC CCA GAA CCC AGG CAG TTT ACT CCT GAG AAA TTT TTT GGA ATT 430 Lys Ser Pro Glu Pro Arg Gln Phe Thr Pro Glu Lys Phe Phe Gly Ile 130 135 140 TTT AAT AAA TCC ATC GAT GCC TTC AAG GAC TTG GAG ATA GTG GCT TCT 478 Phe Asn Lys Ser Ile Asp Ala Phe Lys Asp Leu Glu Ile Val Ala Ser 145 150 155 AAA ATG AGT GAA TGT GTG ATT TCC TCA ACA TCA AGT CCT GAA AAA GAT 526 Lys Met Ser Glu Cys Val Ile Ser Ser Thr Ser Ser Pro Glu Lys Asp 160 165 170 175 TCC AGA GTC AGT GTC ACA AAA CCA TTT ATG TTA CCC CCT GTT GCA GCC 574 Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Ala 180 185 190 AGC TCC CTT AGG AAT GAC AGC AGT AGC AGT AAT AGG AAG GCC TCA AAT 622 Ser Ser Leu Arg Asn Asp Ser Ser Ser Ser Asn Arg Lys Ala Ser Asn 195 200 205 TCC ATT GAA GAT TCC AGC CTA CAA TGG GCA GCC GTA GCA TTG CCA GCA 670 Ser Ile Glu Asp Ser Ser Leu Gln Trp Ala Ala Ala Val Ala Leu Pro Ala 210 215 220 TTC TTT TCT CTT GTG ATC GGG TTT GCT TTT GGG GCC TTT TAC TGG AAG 718 Phe Phe Ser Leu Val Ile Gly Phe Ala Phe Gly Ala Phe Tyr Trp Lys 225 230 235 AAG AAA CAA CCA AAT CTT ACA AGG ACA GTT GAA AAC AGA CAG ATT AAT 766 Lys Lys Gln Pro Asn Leu Thr Arg Thr Val Glu Asn Arg Gln Ile Asn 240 245 250 255 GAA GAG GAT AAT GAA ATA AGT ATG TTG CAA GAG AAA GAG AGA GAG TTT 814 Glu Glu Asp Asn Glu Ile Ser Met Leu Gln Glu Lys Glu Arg Glu Phe 260 265 270 CAA GAA GTG TAA TT GTGGCTTCTA TCAACACTGT TACTTTCGTA CATTGGCGGG 868 Gln Glu Val * 275 TAACAGTTCA TGTTTGCTTC ATAAATGAAG CAGCTTTAAA CAA 911

[Brief description of the drawings]

FIG. 1 is a view showing each region of the amino acid sequence of a bovine c-Kit protein.

FIG. 2 is a diagram showing the base sequences and restriction enzyme sites of synthetic oligonucleotide primers A to G used for PCR.

FIG. 3 shows the structure of plasmid cl.75.

FIG. 4 is a photograph showing the results of electrophoresis of an immunoprecipitate obtained by immunoprecipitation using an anti-GST-c-Kit antiserum.

FIG. 5 is a photograph showing the result of electrophoresis of an immunoprecipitate obtained by an immunoprecipitation method using a bK-1 antibody.

FIG. 6 is a photograph showing the results of electrophoresis of an immunoprecipitate obtained by an immunoprecipitation method using a bK-1 antibody.

FIG. 7 is a view showing the results of separating bovine c-Kit protein-positive cells from bovine bone marrow cells.

FIG. 8 is a photograph showing the results of SDS-polyacrylamide electrophoresis of purified recombinant bovine SCF.

────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shigeki Inumaru 4-1-1, Namiki, Tsukuba, Ibaraki Pref. (56) References Gene, 141, p. 305-306, 1994 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 C07K 16/28 C12P 21/08

Claims (3)

(57) [Claims] 1. A hybrid having an accession number of FERM P-16780.
Monoclonal antibody produced by Doma . 2. A hybridoma that produces the monoclonal antibody according to claim 1. 3. A bovine c-Kit protein characterized by separating bovine c-Kit protein positive cells from a sample containing bovine c-Kit protein positive cells using the monoclonal antibody according to claim 1. A method for separating positive cells.