JP3412930B2 - Membrane protein polypeptide capable of supporting pre-B cell growth and gene thereof - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a gene and a novel membrane protein encoded by the gene, and more specifically, a gene encoding a novel membrane protein polypeptide capable of supporting pre-B cell growth, and the gene. The present invention relates to a vector containing the vector, a transformant using the vector, and a method for producing a novel membrane protein polypeptide using the gene. The present invention further relates to a monoclonal antibody that recognizes a novel membrane protein polypeptide capable of supporting pre-B cell growth.

The gene of the present invention is used for rheumatoid arthritis (R
A) Encodes a novel membrane protein that enhances the ability to support pre-B cell growth on patient synovial cells. By inserting the gene of the present invention into an appropriate vector and transforming a conventional host cell, it becomes possible to produce a large amount of a novel membrane protein polypeptide having a uniform pre-B cell growth supporting ability. From this, the present invention enables the identification of rheumatoid arthritis (RA) and the preparation of these clinical diagnostic reagents.

[0003]

2. Description of the Related Art Inflammatory cells in the synovium and synovial fluid of patients with rheumatoid arthritis (RA) are derived from peripheral blood, and the transfer of these cells to the synovium etc. has not been completely elucidated. It is thought that a complicated interaction between a chemical signal given to cells and a protein (adhesion molecule) on the cell membrane is involved.

Various studies have been conducted on the importance of these membrane proteins in arthritis. For example, cells that are ligands of the T cell surface molecule LFA-1 on the synovial lining and synovial blood vessels of patients with rheumatoid arthritis (RA) and are involved in both cell adhesion and emigration in the vessel wall. It is known that interadhesion molecule-1 (hereinafter referred to as ICAM-1) is expressed [Hale et al .; Arthritis Rheu
m. 32:22 (1989), and Haynes et al .;
Springer Semin. Immunopath
ol. 11: 163 (1989)].

Similarly, vascular cell adhesion molecule-1 (hereinafter VCAM-1), which is a ligand of integrin VLA-4 expressed on T lymphocytes (especially memory cells) and monocytes, is associated with rheumatoid arthritis (RA) and Expression on synovial membranes such as atypical arthritis and fibroblast-like synovial cells [Morales
-Ducret et al .; Immunol. , 149: 1
424 (1992)], and a membrane protein called VAP-1 is expressed in synovial hyperendothelial venules, suggesting that this protein may act as a specific recognition structure of leukocytes. [Salmi et al .; Science
e, 257, 1407 (1992)].

[0006] The present inventor has conducted research for the purpose of clarifying the pathological significance of the bone marrow microenvironment in diseases that cause B cell dysfunction, and has been studied in patients with rheumatoid arthritis (RA) and multiple myeloma (MM). ) In patients, compared to normal people,
From the finding that the pre-B cell proliferation supporting ability of bone marrow-derived stromal cells is enhanced and that direct contact between pre-B cells and stromal cells is essential for this supporting ability, Stroma cell lines of patients (RASV5-5, MMSV3-3) having factors that promote the proliferation of pre-B cells were established, and it was found that these stroma cell lines support growth of pre-B cells. , Known Stem cellfactor
(SCF), ICAM-1, CD44, VCAM-1,
LFA-1α, LFA-1β, NCAM, ELAM-1
It was found that an unknown adhesion factor other than that of the above is involved [J. Immunol. , 149: 4088 (199
2)].

[0007] Furthermore, a synovial cell line SynSV6-in which synovial cells of rheumatoid arthritis (RA) patients are made into cell lines
14 was also suggested to have the ability to support pre-B cell proliferation in the same manner as the bone marrow-derived stromal cell line RASV5-5 of patients with rheumatoid arthritis (RA), and therefore, it also reacted with these cell lines. , A novel monoclonal antibody that does not react with human bone marrow-derived stromal cell line NFSV1-1 that does not have the ability to support pre-B cell proliferation, and succeeded in cloning the gene encoding its antigenic membrane protein (Bst-1) ( Japanese Patent Application No. 5-141178).

[0008]

On the other hand, the inventor of the present invention recognizes various membrane proteins that are expressed in synovial cells derived from patients with rheumatoid arthritis (RA) but not in normal cells. A novel mouse that has a property of reacting with SynSV6-14 but not with normal bone marrow stromal cell line NFSV1-1 in the process of producing a mouse monoclonal antibody and recognizing a membrane protein different from Bst-1 described above. The monoclonal antibody RS38 was obtained. Next, using this RS38 antibody, a cDNA library prepared from a synovial cell line of a patient with rheumatoid arthritis (RA) is screened, and a clone encoding a novel membrane protein that reacts with the RS38 is isolated. Succeeded and completed the present invention.

That is, the present invention provides a novel membrane protein polypeptide having a pre-B cell growth supporting ability, a gene encoding the polypeptide, a vector containing the gene,
It is an object to provide a transformant using the vector and a method for producing a novel membrane protein using the gene.

Another object of the present invention is to provide a monoclonal antibody that recognizes a novel membrane protein having a pre-B cell growth supporting ability.

[0011]

The present invention for achieving the above object comprises the following (1) to ( 6 ). (1) The amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or its
Amino acid with partial substitution, deletion or addition of amino acid sequence
A polypeptide having an acid sequence and having a pre-B cell growth supporting ability .

(2) The amino acid sequence shown in SEQ ID NO: 1 of the sequence listing or the amino acid sequence thereof is partially replaced, deleted or added.
A DNA encoding a polypeptide having an added amino acid sequence and having a pre-B cell growth supporting ability .

[0013]

[0014] (3) a recombinant vector comprising the DNA according to (2).

( 4 ) A prokaryotic or eukaryotic host cell transformed with the recombinant vector according to ( 3 ) above.

( 5 ) Culturing the host cell according to ( 4 ) above
The isolated amino acid sequence shown in SEQ ID NO: 1 or its amino acid sequence.
Has a partially substituted, deleted or added amino acid sequence,
And a method for producing a polypeptide having a pre-B cell growth supporting ability .

( 6 ) Recognizing a polypeptide having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing or an amino acid sequence obtained by partially substituting, deleting or adding the amino acid sequence and having a pre-B cell proliferation supporting ability. Monoclonal antibody.

Next, the present invention will be described in detail.

The monoclonal antibody of the present invention can be basically prepared as follows. That is,
The antibody of the present invention uses synovial cells derived from a rheumatoid arthritis (RA) patient having pre-B cell proliferation supporting ability as a sensitizing antigen to immunize according to a usual immunization method, and according to a usual cell fusion method. It can be prepared by cell fusion and cloning according to a usual cloning method.

More specifically, the method for producing a monoclonal antibody of the present invention includes, for example, rheumatoid arthritis (RA) established as cultured cells as the sensitizing antigen.
Mammalian plasma cells (immune cells) immunized with the sensitizing antigen using the patient's synovial cell line SynSV6-14
Is fused with a myeloma cell of a mammal such as a mouse,
The resulting fused cells (hybridomas) are cloned,
A preferable method is, for example, a method in which a clone producing the antibody of the present invention that recognizes SynSV6-14 is selected from the clones, and the clone is cultured to recover the desired antibody.

In the above-mentioned method for producing a monoclonal antibody, the mammal immunized with the sensitizing antigen is not particularly limited, but is selected in consideration of compatibility with myeloma cells used for cell fusion. Is preferred,
Generally, mice, rats, hamsters and the like are used.

Immunization is carried out by a general method, for example, the synovial cell line Syn of the rheumatoid arthritis (RA) patient.
It is carried out by administering the cultured cells of SV6-14 to a mammal by intraperitoneal injection or the like. More specifically, PB
What was diluted and suspended in an appropriate amount with S, physiological saline or the like, and used in combination with a usual adjuvant as desired, was used for animals with 4 to 21
It is preferred to administer several times daily. In addition, a usual carrier (shreber) can be adopted for the administration. As the immune cells, splenocytes isolated after the final administration of the cell line are preferably used.

As the mammalian myeloma cell as the other parent cell to be fused with the above-mentioned immune cell, various known cell lines such as P3 (P3X63Ag8.6) are known.
53) [J. Immunol. , 123: 1548 (1
978)], p3-U1 [Current Topic
s in Micro-biology and Im
Munology, 81: 1-7 (1978)], NS
-1 [Eur. J. Immunol. , 6: 511-5
19 (1976)], MPC-11 [Cell, 8: 4].
05-415 (1976)], SP2 / 0 [Natur
e, 276: 269-270 (1978)], FO
[J. Immunol. Meth. , 35: 1-21
(1980)], S194 [J. Exp. Med. , 1
48: 313-323 (1978)], R210 [Na
True, 277: 131-133 (1979)] and the like are preferably used.

The cell fusion between the immune cells and myeloma cells is basically a known method, for example, the method of Milstein et al. [Met.
hods Enzymol. , 73: 3-46 (198
1)] and the like.

More specifically, the cell fusion is carried out in an ordinary nutrient medium in the presence of a fusion promoter, for example. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used, and if desired, an auxiliary agent such as dimethyl sulfoxide can be added and used to enhance the fusion efficiency.
The ratio of immune cells to myeloma cells used is preferably, for example, about 1 to 10 times more immune cells than myeloma cells. The medium used for the cell fusion,
For example, RPMI suitable for growth of the myeloma cell line
-1640 medium, MEM medium, and other ordinary mediums used for this type of cell culture can be used, and a serum supplement such as fetal calf serum (FCS) can also be used in combination.

For cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the medium and pre-heated to about 37 ° C. in a PEG solution, for example, an average molecular weight of 100
About 0-6,000 PEG is usually added to the medium at about 30-
It is performed by adding at a concentration of 60% (W / V) and mixing. Subsequently, the desired hybridoma is formed by repeating the operation of successively adding an appropriate medium and centrifuging to remove the supernatant.

The hybridoma is selected by culturing in a usual selection medium, for example, HAT medium (medium containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT medium is continued for a sufficient period of time to kill cells (unfused cells) other than the target hybridoma, usually for several days to several weeks. Then, according to a usual limiting dilution method, screening and monocloning of hybridomas producing the desired antibody are carried out.

The hybridoma producing the monoclonal antibody of the present invention thus produced can be subcultured in an ordinary medium and can be stored in liquid nitrogen for a long period of time.

To collect the monoclonal antibody of the present invention from the hybridoma, the hybridoma is cultured according to a conventional method to obtain a culture supernatant thereof, or the hybridoma is administered to a mammal compatible therewith and proliferated. Then, the method of obtaining the ascites is adopted. The former method is a method suitable for obtaining highly pure antibodies, while the latter method is a method suitable for mass production of antibodies.

Further, the antibody obtained by the above method is
It can be purified to a high degree of purity using ordinary purification means such as a salting-out method, a gel filtration method, and affinity chromatography.

The monoclonal antibody of the present invention thus produced is a radioimmunoassay method (RIA), an enzyme immunoassay method (EIA), or a fluorescent antibody method (Immunofluo).
It is possible to identify and identify synovial cells of patients with rheumatoid arthritis (RA) expressing a novel membrane protein of an antigen with high sensitivity and high accuracy by ordinary immunological means such as rescense analysis). To do.

The gene of the present invention is a rheumatoid arthritis (R) that expresses a membrane protein showing human pre-B cell growth supporting ability.
A) It is obtained by preparing mRNA from the synovial cells of a patient and then converting it into double-stranded cDNA by a known method. The cells serving as the source of this mRNA were SynSV6 used as an immunogen of hybridoma RS38.
-14 cell line and the like, but not limited to these cell lines, any cell may be used as long as it expresses a membrane protein having a pre-B cell growth supporting ability. In the present invention, SynSV6-8 was used.

The preparation of total RNA to obtain mRNA is
After guanidine thiocyanate treatment, cesium chloride density gradient centrifugation was performed [Chirgwin et al. , B
iochemistry, 18: 5294 (197).
9)], a method for obtaining total RNA, treatment with a surfactant in the presence of a ribonuclease inhibitor of a vanadium complex,
Phenol treatment [Berger & Birke
nmeier, Biochemistry, 18:51
43 (1979)] method, known means can be used.

Preparation of mRNA from total RNA is carried out by poly (A) + from total RNA by affinity column chromatography using an oligo (dT) -bound carrier such as sepharose or cellulose, or by a batch method.
This can be done by recovering RNA. Further, poly (A) + RNA can be further purified by a sucrose density gradient centrifugation method or the like. In addition, without preparing RNA once,
A method of directly obtaining poly (A) + RNA or a simple method using a commercially available kit can also be used.

From the mRNA obtained as described above, double-stranded c
To obtain DNA, for example, using mRNA as a template,
Oligo (dT) complementary to the poly A-chain at the 3'end is used as a primer to treat with reverse transcriptase to synthesize DNA (cDNA) complementary to mRNA.

After the mRNA is decomposed and removed by alkali treatment, the obtained single-stranded cDNA is used as a template for reverse transcriptase or DNA polymerase (for example, Klenow).
Fragments etc.), then treat with S1 nuclease,
Double-stranded cDNA can also be obtained by directly treating with RNase H and DNA polymerase [M
aniatis et al. , Molecular
Cloning, Cold Spring Harb
or Laboratory (1982), and Gub
ler & Hoffman, Gene, 25: 263.
(1983)]. Recently, simple kits are also commercially available, and double-stranded cDNA can be obtained using them.

The cDNA thus obtained is transformed into an appropriate vector, for example E of pBR322, pSC101 or the like.
After being incorporated into a K-type plasmid vector or a phage vector such as λgt10, E. coli (X1776, HB10
1, DH1, DH5, etc.) can be transformed to obtain a cDNA library (for example, “Mo described above”).
(See “Regular Cloning”).

On the other hand, by inserting the double-stranded cDNA obtained by the above method into an appropriate expression vector, other prokaryotic or eukaryotic host cells can be transformed.

In order to ligate the double-stranded cDNA with the vector, an appropriate chemically synthesized DNA adapter is added, and the vector DNA cleaved with a predetermined restriction enzyme is treated with T4 phage DNA ligase in the presence of ATP. It can be done by

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

As a promoter for gene expression in mammalian cells, viral promoters such as retrovirus, polyoma virus, adenovirus, simian virus (SV) 40 and human polypeptide chain elongation factor 1α (HEF- 1
A cell-derived promoter such as α) may be used. For example, when using the SV40 promoter, Mul
Ligan et al. [Nature, 277: 108].
(1979)], it can be easily implemented.

The origin of replication includes SV40 polyoma virus, adenovirus, bovine papilloma virus (BP).
V) or the like can be used, and the phosphotransferase APH (3 ′) I can be used as a selection marker.
I or I (neo) gene, thymidine kinase (T
K) gene, Escherichia coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (DHFR) gene and the like can be used.

To express a gene of interest using a prokaryotic cell as a host cell, the host cell is transformed with a replicon derived from a species compatible with the host, that is, a plasmid vector containing an origin of replication and regulatory sequences. Just do it. Further, the vector preferably has a marker gene capable of imparting phenotypic (phenotypic) selectivity to transformed cells. For example, in the case of Escherichia coli, it can be transformed with pBR322 which is a vector using it as a host [Boliver et al .: Gene,
2:95 (1975)]. The pBR322 contains genes for ampicillin and tetracycline resistance,
Transformed cells can be identified by utilizing the resistance of either.

As a promoter required for gene expression in a prokaryotic host cell, a β-lactamase gene promoter [Chang et al .: Nature 275: 615 (1
978)] or the lactose promoter [Goedde
L et al .: Nature, 281: 544 (1979)],
And tryptophan promoter [Goeddel et al .:
Nucleic Acid Res. , 8: 4057
(1980)], tac promoter and the like are preferred, but the present invention is not limited thereto.

Among the hosts used in the expression system of the present invention, prokaryotic host cells include, for example, Escherichia coli (Escherichia coli).
ia coli), Bacillus subtilis
tilis), Bacillus thermophilus (Bacil)
lus thermophilus) and the like are preferable, but the present invention is not limited thereto.

Examples of eukaryotic host cells include, for example, Saccharomyces.
es cerevisiae) and other eukaryotic microorganisms are preferable, and COS cells, Chinese hamster ovary (CHO) cells, C127 cells, 3T3 cells, Hela cells, BHK cells, Namalwa cells, human embryonic kidney cells (293). Cells derived from mammals such as cells) can also be preferably used, but the cells are not limited thereto. The culture of the transformant of the present invention may be carried out by appropriately selecting culture conditions suitable for the host cell.

To isolate a cDNA encoding a membrane protein having a pre-B cell growth supporting ability of the present invention, for example, pre-B cell growth supporting ability is used as an index, or direct expression cloning using an antibody is used. The method can be used. The pre-B cell growth supporting ability was measured by the mouse pre-B cell line DW34 [Eur. J. Immunol. , 18:
1767 (1988)]. That is, cells expressing a membrane protein capable of supporting pre-B cell growth in a 24-well plate were cultured until they became subconfluent (a density of about 50% is desirable), and after irradiating with an appropriate amount of radiation, per well 1-2 x 1
RPM with addition of 0 ³ DW34 and 10% FCS
4 at 37 ° C. under the condition of 5% CO ₂ in I-1640 medium.
Incubate for about 6 days. By examining the number of viable cells of DW34 in each well by trypan blue staining, the degree of enhancement of growth supporting ability can be known.

In the present invention, a membrane protein is expressed by flow cytometry by FACScan using a monoclonal antibody RS38 that recognizes a novel membrane protein on synovial cells of a rheumatoid arthritis (RA) patient. By selecting transformants, subdividing the plasmid DNA used for producing the transformants to produce transformants again, and repeating the screening of the transformants by flow cytometry, The gene could be cloned.

Specifically, after culturing the transduced transformant (293T cells), 0.02% of EDTA was added.
Peel off the plate with PBS containing 2% FCS, 0.
The cells are washed with a FACS buffer consisting of PBS containing 02% NaN ₃ and then reacted with RS38 as a primary antibody. Then, the cells were washed with FACS buffer to remove unreacted primary antibody, further reacted with a secondary antibody, FITC-labeled antibody (FITC-labeled goat anti-mouse Ig antibody), and then dead cells were stained with propidium iodide. Transformants that strongly reacted with RS38 were selected by identifying the phenotype and analyzing the viable cells by FACScan.

Further, Escherichia coli (DH5) containing the cDNA used for the preparation of the transformant reacted with the antibody was treated with alkali to select the plasmid group containing the target gene, and these plasmid groups were further added to several plasmid groups. After transducing into 293T cells again, the selection of transformants by FACScan analysis using the above-mentioned monoclonal antibody RS38 was repeated to obtain the novel preform shown in SEQ ID NO: 2 in the sequence listing. Full-length cDNA encoding a membrane protein polypeptide capable of supporting B cell growth
NA (pRS38-BOS) could be obtained.

This cDNA was inserted between the XbaI cleavage sites of pUC19 vector to obtain pRS38-pUC19.
The E. coli DH5α strain containing Escherichia coli was added to the Institute of Life Science and Technology, Institute of Industrial Science and Technology on October 5, 1993, by Escherichia coli.
DH5α (pRS38-pUC19), accession number FE
It has been deposited internationally under the Budapest Treaty as RM BP-4434.

In general, eukaryotic genes are considered to exhibit polymorphism, as is known for human interferon genes and the like [see, for example, Nishi et al., J. et al. Bioc
hem. , 97: 153 (1985)], one or more amino acids may be substituted by this polymorphism, or the amino acids may not be changed at all even if the base sequence is changed.

Further, a polypeptide lacking or adding one or more amino acids in the amino acid sequence of SEQ ID NO: 1 in the sequence listing or a polypeptide in which an amino acid is substituted with one or more amino acids It may have the same function (pre-B cell growth supporting ability) as the novel membrane protein of the invention. For example, human interleukin-2
It has already been known that a polypeptide obtained by converting a nucleotide sequence corresponding to cysteine of (IL-2) gene into a sequence corresponding to serine retains IL-2 activity [Wang et al., Science, 224: 1431 (1
984)].

Furthermore, the gene of the known protein and the gene of SEQ ID NO: 2 in the sequence listing can be ligated using an appropriate restriction enzyme or adapter to produce a polypeptide bound to the known protein. Examples of the gene of the known protein include immunoglobulin, and the gene shown in SEQ ID NO: 2 in the sequence listing may be used instead of the variable region portion thereof to bind to the Fc portion [(Z
ettlmeissl et al., DNA AND CELL
BIOLOGY, 9: 347-353 (1990)].

Further, when expressed in eukaryotic cells, most of them have a sugar chain added, and the sugar chain addition can be regulated by converting one or more amino acids. It may have the same function as the novel membrane protein polypeptide of the invention. Therefore, even if the gene encoding the membrane protein polypeptide of the present invention is artificially modified by various methods as described above, as long as the obtained polypeptide has the same function as the membrane protein polypeptide of the present invention, those It goes without saying that all the genes and polypeptides of the above are included in the present invention.

Furthermore, in the gene that hybridizes with the gene shown in SEQ ID NO: 2 in the sequence listing, the polypeptide expressed from that gene has the same function (pre-B cell growth supporting ability) as the membrane protein polypeptide of the present invention. It goes without saying that all such genes and polypeptides, as long as they have, are included in the present invention. In this case, the hybridization can be performed using ordinary hybridization conditions (for example, see the above-mentioned "Molecular Cloning").

In order to produce the desired membrane protein polypeptide capable of supporting the growth of pre-B cells, a transformant transformed with the gene encoding the polypeptide is cultured, and the produced polypeptide is appropriately transformed. A uniform soluble membrane protein polypeptide can be obtained by solubilizing with a solubilizing agent, followed by separation and purification. As the solubilizer, for example, Nonidet P-40 (NP
-40), Sodium Dodecyl Sulph
ate (SDS), Triton X-100, Twe
en 20 and the like are preferred.

The soluble membrane protein can also be produced by genetic engineering. That is, as shown in FIG. 3, since RS38 is expected to be a cell transmembrane protein having a cell transmembrane domain and an intracellular domain on the N-terminal side, the 49th A of SEQ ID NO: 1 in the sequence listing is shown.
Soluble RS38 having N-terminal at sn was subjected to PCR-mutagenesis [M. Kamman et al., Nucl. Acids Re
s. , 15: 5404 (1989)]. In that case, a known signal sequence may be used, for example, Bst-1 (Japanese Patent Application No.
-141178) and G-CSF (Japanese Patent Publication No. 2-539).
No. 5) signal sequence and the like.

As a means for separating and purifying the membrane protein polypeptide, a method used for ordinary proteins may be applied. For example, various chromatography such as affinity chromatography using the above-mentioned monoclonal antibody, and ultrafiltration The membrane protein polypeptide of the present invention can be separated and purified by appropriately selecting and combining filtration, salting out, dialysis and the like and separating and purifying according to a usual method.

[0060]

EXAMPLES The present invention will be described in detail below with reference to reference examples and examples, but the present invention is not limited to these examples.

Reference Example 1 Establishment of a Stromal Cell Line Derived from a Healthy Person A pAct-SVT plasmid [BBRC 186: 12] containing the large T antigen cDNA of SV40 and the chicken β-actin promoter in a stromal cell of a healthy person.
9-134 (1992)] by Gene Pulser
(Manufactured by BioLad) was used for electroporation. That is, 1 × stromal cells of a healthy person in PBS
A 0.8 ml aliquot of 10 ⁷ cells / ml and 10 μg of the plasmid were mixed and incubated in ice for 10 minutes, followed by electroporation under the conditions of 250 V and 250 μF capacitance, and further incubated in ice for 10 minutes. After doing, 10% FCS (Bioproducts
Were suspended in RPMI-1640 medium (manufactured by GIBCO) containing C.I. The culture medium was exchanged every 3 days, and after about 2 weeks, colonies of adherent cells with good growth were taken out with a piece of filter paper impregnated with trypsin, and the bone marrow-derived stromal cell line (NFSV1
-1) was obtained [J. Immunol. , 149: 408
8 (1992)].

Reference Example 2 Establishment of Synovial Cell Line Derived from Patients with Rheumatoid Arthritis (RA) The pAct-SVT plasmid containing the large T antigen cDNA of SV40 and the chicken β-actin promoter in synovial cells derived from rheumatoid arthritis (RA). [BBR
C, 186: 129-134 (1992)], Gen.
Electroporation was performed using ePulser (manufactured by BioLad). That is, a 0.8 ml aliquot of 1 × 10 ⁷ cells / ml of RA patient-derived synovial cells in PBS was mixed with 10 μg of the plasmid, incubated in ice for 10 minutes, and then subjected to 250 V, 250 μF capacitance. After electroporation and further incubation in ice for 10 minutes, 10% FCS (Bio
The cells were suspended in RPMI-1640 medium (manufactured by GIBCO) containing products) and cultured in a 10 cm culture dish. The culture medium was exchanged every 3 days, and after about 2 weeks, colonies of adherent cells with good growth were taken out with a piece of filter paper impregnated with trypsin, and the rheumatoid arthritis (RA) patient-derived synovial cell line (SynSV6- 8 and SynSV6
-14) was obtained.

Example 1 Preparation of Monoclonal Antibody 1) Sensitizing Antigen and Immunization Method As a sensitizing antigen, a rheumatoid arthritis (RA) patient-derived synovial cell line having the ability to support pre-B cell proliferation obtained in Reference Example 2 above. Antigen sensitization was performed using SynSV6-14. The cell line is 10% fetal calf serum (FCS, Bioprodu
(manufactured by cts) and RPMI-1640 containing 50 μM 2-mercaptoethanol (manufactured by GIBCO) were used as a medium, and subculture was performed under a temperature condition of 37 ° C. in a 5% CO ₂ incubator.

The cells were treated with 0.02% EDTA and PBS and recovered from the culture flask in the incubator by light pipetting. Approximately 1 x 10 ⁷ cells /
The cells were suspended in RPMI-1640 medium at a cell number of ml, suspended, and immunized into BALB / C mice (4 weeks old, ♀, Japan SLC, Inc.). Approximately 1 x 10 ^{7 for the} first immunization
Cells / ml of cells were injected intraperitoneally into the mice, and 2-3 weeks later, 1 × 10 ⁷ cells / ml of cells were boosted. Furthermore, 2 to
1 × 10 ⁷ cells / ml of cells were boosted 2-3 times at 3-week intervals, 3 days after the final immunization, the mice were sacrificed and their spleens were excised.

2) Cell fusion After slicing the spleen extracted from one mouse, the free spleen cells were spun down, and then RPMI-1640 medium (GIBCO).
(Manufactured by Mfg. Co., Ltd.), suspended and thoroughly washed. On the other hand, mouse myeloma cell line P3X63Ag8.65
3 [J. Immunol. , 123: 1548 (197)
9)], 10% fetal calf serum (FCS, FILTRON
Cells obtained by culturing in a DMEM (manufactured by GIBCO) medium containing the same) were similarly washed with the DMEM medium, and then 1 × 10 ⁷ cells and 1 × 10 ^{8 of the} splenocytes were centrifuged. And mix in polyethylene glycol 1500 (Bo
manufactured by Ehringer) by a conventional method [Clin. Ex
p. Immunol. 42: 458-462 (198).
0)] and the cells were fused.

The obtained fused cells were dispensed in DMEM medium containing 10% FCS into 96 well plates, and 5
Incubated at 37 ° C. in a% CO ₂ incubator. From the next day, HAT selection medium (1.0 × 10 ⁻⁴ M hypoxanthine, 4.0 × 10 ⁻⁷ M aminopterin, 1.6 × 10 ⁻⁵⁾
10% in complete RPMI-1640 medium containing M thymidine
% FCS and 50 μM 2-mercaptoethanol were added) and the culture was continued. After the start of the culture, half of the supernatant was added twice a week to fresh HA.
Instead of the T medium, the culture was continued and the growth was maintained.

The fused cells thus obtained were cloned using the limiting dilution method. That is, using the antibody in the culture supernatant of the fused cells, the binding to the sensitizing antigen was examined, and only clones having strong binding to the sensitizing antigen were cloned using the limiting dilution method by a conventional method. Formed.

The formation of clones was adjusted so that the above hybridoma and BALB / C mouse splenocytes were contained in a predetermined amount, and 9 to 10 hybridomas / well were prepared.
It was seeded on a 6-well plate and cultured at 37 ° C. in a 5% CO ₂ incubator. The procedure of cloning the growing hybridoma in the same manner was repeated according to the usual limiting dilution method until a theoretically single clone was obtained. Clones producing the desired antibody were screened using the sensitizing antigen.

3) Screening The screening of fused cells (hybridomas) was carried out by the indirect fluorescent antibody method using a flow cytometer (Flow Cytometer). Screening for clones producing the desired antibody was carried out using, as target cells, a) SynSV6-14 (sensitizing antigen), b) normal human bone marrow-derived stromal cell line NFSV1-1, and the like. That is, a synovial cell line (SynSV) of rheumatoid arthritis (RA) patient having pre-B cell proliferation supporting ability
6-14) by immunizing BALB / C mice with a normal human bone marrow-derived stromal cell line that reacts with SynSV6-14 but does not have the ability to support pre-B cell growth (N
The monoclonal antibody was screened by using the fact that it did not react with FSV1-1) as an index. The first screening is SynSV, which is a sensitizing antigen as a reaction cell.
6-14. First, in order to select a fused cell clone that reacts with SynSV6-14, the SynSV
The culture supernatant that reacts with 6-14 was selected and the primary screening was performed.

That is, the cells were treated with the reaction buffer [2% F
CS, PBS containing 0.02% NaN ₃ ] and suspended in 20 μl of hybridoma culture supernatant (about 5 × 1
0 ^5/20 [mu] l), was reacted for 20 minutes at 4 ° C.. After washing twice with the above buffer, FITC-labeled goat anti-mouse Ig antibody (manufactured by Cappel) was added and incubated for 20 minutes. After washing 3 times, flow cytometer (FAC)
Scan, Becton Dickinson).

Next, normal human bone marrow-derived stromal cell line NFSV1-1 was used as a reaction cell and analyzed by a flow cytometer as described above. As a result, a hybridoma producing an antibody that strongly reacts with SynSV6-14 was obtained.

In this manner, the normal human bone marrow-derived stromal cell line NFS, which reacts with SynSV6-14,
A hybridoma (RS38) that produces an antibody that does not react with V1-1 was isolated. The antibody produced by this hybridoma was of the IgM, κ type.

The hybridoma producing the above-mentioned monoclonal antibody RS38 was BALB / C mouse splenocytes and mouse myeloma P3X63Ag8.653.
A new fused cell produced by using as a parent cell, the Institute of Biotechnology, Institute of Industrial Science and Technology, which is a public microbial depository institution, dated October 5, 1993.
It has been internationally deposited according to the Budapest Treaty under the e-Mouse hybridoma RS38, accession number FERM BP-4433.

4) Purification of antibody The fused cells prepared in 2) above were cultured by a conventional method, and the antibody produced in the culture supernatant was separated and purified by a conventional method. That is, a hybridoma was collected from the well in which the antibody titer against the sensitizing antigen was the highest, and one of the wells in which cell proliferation was observed was taken,
The obtained cultured cells were spread in a 5% CO ₂ tissue culture flask and subcultured at 37 ° C. for growth. The obtained cells were intraperitoneally injected into BALB / C mice (6 weeks old, ♀, manufactured by Japan SLC, Inc.) that had been administered with pristane. After 10 to 14 days, the ascites produced was collected and
The product was salted out with ammonium sulfate, dialyzed with PBS, and purified with a QAE column. The antibody was further salted out and sufficiently dialyzed to obtain a purified product of about 6 mg / ml.

Example 2 Properties of monoclonal antibody 1) Distribution of cell lines R in various cell lines (various cell lines shown in Table 1)
The distribution of S38 antigen was analyzed using FACScan.
That is, various cell lines were used as primary antibodies, mouse monoclonal antibody RS38 obtained in Example 1, 10 μg.
The cells were suspended in a FACS buffer consisting of PBS containing 2% FCS and 0.02% NaN ₃ in the presence of 1 ml / ml, and incubated in ice for 20 minutes. After washing twice with a FACS buffer, FITC-labeled goat anti-mouse Ig antibody (manufactured by Cappel) was used as a secondary antibody, and further incubated on ice for 15 minutes. Propidium iodide
umIodide, PI) was added to a final concentration of 1 μg / ml and incubated for another 5 minutes on ice.
After washing three times with a FACS buffer, whether the cells were alive or dead was identified by the forward scattered light, and only the living cells were subjected to FACScan (Bect
on Dickinson). As a target, mouse monoclonal antibodies SG2 and RF3
(Japanese Patent Application No. 5-141178) was used. The results are shown in Table 1. As is clear from Table 1, the distribution of RS38 antigen was found to be different from SG2 and RF3.

[0076]

[Table 1]

Example 3 1. Preparation of cDNA library 1) Preparation of poly (A) + RNA Synovial cell line SynS derived from rheumatoid arthritis (RA) patient
Preparation of Poly (A) + RNA from V6-8 cells
Fast Track ^™ mRNA ISOLATIO
N KIT version 3.2 (Invitro
gen). That is, SynSV6-8 cells for 20 10 cm culture dishes were homogenized,
Total RNA was prepared according to the prescription attached to the kit. Furthermore,
Using oligo d (T) cellulose attached to the kit, poly (A) + RNA was purified according to the prescription attached to the kit.

2) Construction of cDNA library Using 5 μg of the above poly (A) + RNA as a material, cDNA was prepared.
A Synthesis Kit TimeSave ^™ cDNA Syn
Double-stranded cDNA was synthesized using the Thesis Kit (Pharmacia) according to the prescription attached to the kit,
BstXI adapter (manufactured by Invitrogen) D
NA ligation kit (manufactured by Takara Shuzo) was used for ligation according to the prescription attached to the kit. Free BstX
To remove the I adapter, use the Size Sep included in the kit.
Using a 400 Spin Column according to the prescription attached to the kit, about 1 double-stranded cDNA with an adapter added
00 μl was obtained.

Adapter addition 2 produced in this way
Ligatio of about 100 μl of the single-stranded cDNA
2 μl was used for n reaction, and the restriction enzyme BstXI
And pEF-BOS vector treated with alkaline phosphatase (Takara Shuzo) [Nuc. Acid Re
s. , 18: 5322 (1990)] and DNA lig.
ation kit (Takara Shuzo)
A DNA library was constructed. The constructed cDNA library is E. coli cell line DH5 (manufactured by Toyo Bo).
The total size was estimated to be about 2 × 10 ⁵ independent clones. 50 pools of transduced E. coli 2,000-4,000 clones
A pool was prepared and used in the following experiments.

2. Cloning by direct expression method 1) Transfection into 293T cells The pooled Escherichia coli was transformed into LB medium containing 50 μg / ml of ampicillin [Molecular Cloin.
g: A Laboratory Manual, Sam
Brook et al., Cold Spring Harbor
The cDNA was amplified by culturing with the Laboratory Press (1989)], and the alkaline method [Molecular Cloning: A Labo] was used.
ratory Manual, Sambrook et al., C
old Spring Harbor Laborat
ory Press (1989)] to recover the plasmid DNA from E. coli. Obtained plasmid DNA
Was purified by repeating ultracentrifugation with a cesium chloride / ethidium bromide density gradient twice, and 293T cells [293 cells (Trans
formed primary embryonal
kidney, human ATCC CRL157
3) a cell line into which SV40 Large T antigen cDNA was introduced].

That is, 2 μm of purified plasmid DNA
g 1 mM Tris-HCl, 0.1 mM EDTA
Dissolved in 100 μl of buffer solution containing 14 μl of 2M
After adding CaCl ₂ , 50 mM HEPES (pH
7.1) 280 mM NaCl, 1.5 mM Sod
It was gradually mixed with a buffer solution containing ium Phosphate, incubated at room temperature for 30 minutes, and added to 293T cells in a 24-well plate. 293T cells have 1
0% fetal bovine serum (PCS; manufactured by Bioproducts)
In DMEM (manufactured by GIBCO) medium containing
It was cultured for 2 days under the condition of% CO ₂ .

2) Analysis by FACScan Transduced 293T cells were detached from a 24-well plate with PBS containing 0.02% EDTA and 2% FC was removed.
FACS consisting of PBS containing S and 0.02% N _a N ₃
After washing the cells twice with buffer, 10
The cells were suspended in 20 µl of FACS buffer in the presence of µg / ml of the monoclonal antibody RS38, and incubated in ice for 20 minutes. After washing twice with FACS buffer, 2
FITC-labeled goat anti-mouse Ig antibody (Ca
(manufactured by Ppel) was used and further incubated for 15 minutes on ice. Propidium iodide
(Iodide, PI) was added to a final concentration of 1 μg / ml, and the mixture was further incubated on ice for 5 minutes, and then F
After washing three times with an ACS buffer solution, the living cells were identified by the forward scattered light, and then only the living cells were subjected to FACScan (Bec).
Ton Dickinson).

3) Cloning of cDNA library Plasmid DNA recovered by the alkaline method using 2,000 to 4,000 clones of E. coli as one pool.
Was transfected into 293T cells according to the method described above, and screened by the above FACS analysis. A peak strongly stained with mouse monoclonal antibody RS38 was observed in 293T cells expressing the 25th pool. E. coli DH5 (GIBCO BRL) was transduced again with this plasmid DNA pool, and
LB agar plates containing 0 μg / ml ampicillin were inoculated.

Approximately 2,000 clones that formed colonies were divided into meshes on the bottom surface of the plate so that the positions of the inoculated clones could be identified on an agar plate.
One of them was inoculated so as to give 0 clones / plate, and two same clones were prepared. Twenty pools were prepared from 100 clones as one pool, and Escherichia coli was cultured in an LB medium containing 50 μg / ml of ampicillin. After recovering the plasmid DNA by the alkaline method, 293T cells were transfected by the calcium phosphate method and FACScan analysis was performed in the same manner as described above. As a result of FACScan analysis, E. coli 10
0 clones were isolated one by one, each clone was cultured, and plasmid DNA was recovered by the alkaline method. 293T cells were transfected with each plasmid DNA by the calcium phosphate method, and FACS was performed as described above.
Can analysis was performed to obtain a single positive clone, and pRS
It was named 38-BOS.

About this clone, Auto Read
sequencing kit (Pharmacia
Made) and Auto Cycle Sequencing
A kit (manufactured by Pharmacia) was used to perform a sequence reaction according to the prescription attached to the kit. L. F.
^The nucleotide sequence was determined by using a ^TM DNA sequencer (Pharmacia). As a result, the longest Open
It is a gene with a total length of 996 bp (SEQ ID NO: 2 in the sequence listing) that is estimated to encode 180 amino acid residues from Reading Frame, and gene analysis software Gene
As a result of a homology search using the database SWISS PLO T and NBRL using tex, it was a novel gene.

4) Northern Blotting
Expression analysis by analysis FastTrack from RA patient-derived synovial cell line SynSV6-14, RA patient-derived bone marrow stromal cell line RASV5-5, and normal human-derived bone marrow stromal cell line NFSV1-1
^TM mRNA ISOLATION KIT vers
Pion using ion 3.2 (manufactured by Invitrogen)
ly (A) + RNA was prepared and Northern B was prepared.
Rotating analysis (Molecula
r Cloning; A Laboratory Man
ual, Sambrook et al., Cold Spring
g Harbor Laboratory Pres
s, 1989). That is, 10 cm culture dish 1
Each cell line cultured on 0 sheets was collected, homogenized, and then total RNA was prepared according to the prescription attached to the kit.
Furthermore, using oligo d (T) cellulose attached to the kit, according to the prescription attached to the kit, Poly (A) + RNA
Was purified.

The label of the probe is Multiprime.
DNA labeling system (Am
ershham). That is, pEF-
The insert considered to encode RS38 inserted into the BstXI site of BOS was restricted to the restriction enzyme Hind.
A 315 bp fragment was prepared by digestion with III, and a labeled probe was prepared according to the recipe attached to the kit. Sy
nSV6-14, RASV5-5, and NFSV1-1
After using 3 μg of Poly (A) + RNA prepared above per lane and performing agarose gel electrophoresis,
ybridization Transfer Mem
Gene Screen Plus ^™ as a lane
(Manufactured by DUPONT) was used for blotting by the capillary method. Hybridization is 0.5
M NaPO ₄ , 1 mM EDTA, 7% SDS, 1%
Gilbert & C composed of BSA and pH 7.0
It was carried out at 65 ° C. overnight using a hurch buffer. After hybridization, 2x membrane
Washed 4 times at room temperature with SSC, then 0.1 x SSC,
After washing twice with 0.1% SDS at 55 ° C., it was overlaid with an X-ray film and autoradiography was performed overnight. As a result, as shown in FIG. 1, a clear band of about 1.0 kb was detected in SynSV6-14.

3. Expression by BALB3T3 cells The novel molecule was introduced into BALB3T3 cells and examined for expression in animal cells. That is, 20 μg of pRS38-BO
PSV2n containing S and 2 μg of neomycin resistance gene
eo [P. J. Southem and P.S. Ber
g, J. Mol. Appl. Genet. , 1: 327
(1982)] was added to a 0.8 ml aliquot of 1 × 10 ⁷ cells / ml and incubated in ice for 10 minutes, and then a Gene Pulser (manufactured by BioLad) was used to adjust the capacitance to 250 V and 250 μF. Were transfected and co-transfected.

After further incubation in ice for 10 minutes, the cells were treated with 2 mg / ml G418 and 10% FCS.
(Manufactured by Bioproducts) containing DMEM medium (G
It was suspended in IBCO) and cultured in a 24-well plate.
The culture medium was exchanged every 3 days, and after about 2 weeks, the mouse monoclonal antibody RS38 was extracted from the hole forming a single colony of neomycin-resistant adherent cells that grew well.
A transformed cell line BALB3T3 38-1, which reacts with
38-3 and 38-9 were obtained. Also, as control cells,
A transformed cell line BALB3T3 38-4, which does not react with RS38 but is neomycin resistant, was obtained.

4. Biological Properties of Novel Molecule 1) Growth Support of Mouse Pre-B Cell Line The biological properties of the novel molecule are shown below using the mouse pre-B cell line DW34, which grows in a stromal cell-dependent manner, using the number of proliferating cells as an index. It was analyzed by the method. First, 2
Transfected cell line BALB3T3 38-
1, 38-3 and 38-9 and the control cell line BALB
3T3 and BALB3T3 38-4 were cultured to subconfluence, irradiated with 30 Gy of radiation, and then 1
Add 2 × 10 ³ DW34 per hole and add 10% FC
RPMI-16 containing S (manufactured by Bioproducts)
The cells were cultured in a 40 (GIBCO) medium at 37 ° C. and 5% CO ₂ for 4 days. The number of viable DW34 cells in each well was counted by trypan blue staining, and the growth supporting ability was analyzed.
As a result, as shown in FIG. 2, the transduced cell line BALB3
Proliferation of DW34 was promoted in T3 38-1, 38-3 and 38-9 compared to control cell lines BALB3T3 and BALB3T3 38-4.

5. Physico-chemical properties of the novel molecule 1) N-terminal analysis Using the DNA analysis software Gene Works, the hydrophobic region and hydrophilic region of the gene obtained as described above were analyzed. The results of the analysis are shown in FIG. As a result, the region consisting of 28 amino acid residues from the 21st amino acid Lys of the sequence listing to the 48th amino acid Ala of SEQ ID NO: 1 has the highest hydrophobicity, and the mature protein has a cell membrane penetrating N-terminal side. It was predicted to be a transmembrane protein having a domain and an intracellular domain.

[0092]

As described above in detail, according to the present invention, the pre-B
Gene encoding a novel membrane protein polypeptide capable of supporting cell growth, vector containing the gene, transformant using the vector, and pre-B using the gene
The present invention relates to a method for producing a novel membrane protein capable of supporting cell growth, wherein the gene of the present invention encodes a membrane protein on synovial cells of patients with rheumatoid arthritis (RA).

By inserting the gene of the present invention into an appropriate vector and transforming a conventional host cell, it is possible to produce a large amount of uniform novel membrane protein polypeptide. By using synovial cells derived from a rheumatoid (RA) patient as a sensitizing antigen, it is possible to produce a monoclonal antibody that recognizes the membrane protein polypeptide. Therefore, according to the present invention, rheumatoid arthritis (RA) can be obtained. And the production of these clinical diagnostic reagents.

[Sequence list]

SEQ ID NO: 1 Sequence length: 180 Sequence type: Amino acid Topology: linear Sequence type: Peptide Array Met Ala Ser Thr Ser Tyr Asp Tyr Cys Arg Val Pro Met Glu Asp Gly                   5 10 15 Asp Lys Arg Cys Lys Leu Leu Leu Gly Ile Gly Ile Leu Val Leu Leu              20 25 30 Ile Ile Val Ile Leu Gly Val Pro Leu Ile Ile Phe Thr Ile Lys Ala          35 40 45 Asn Ser Glu Ala Cys Arg Asp Gly Leu Arg Ala Val Met Glu Cys Arg      50 55 60 Asn Val Thr His Leu Leu Gln Gln Glu Leu Thr Glu Ala Gln Lys Gly  65 70 75 80 Phe Gln Asp Val Glu Ala Gln Ala Ala Thr Cys Asn His Thr Val Met                  85 90 95 Ala Leu Met Ala Ser Leu Asp Ala Glu Lys Ala Gln Gly Gln Lys Lys             100 105 110 Val Glu Glu Leu Glu Gly Glu Ile Thr Thr Leu Asn His Lys Leu Gln         115 120 125 Asp Ala Ser Ala Glu Val Glu Arg Leu Arg Arg Glu Asn Gln Val Leu     130 135 140 Ser Val Arg Ile Ala Asp Lys Lys Tyr Tyr Pro Ser Ser Gln Asp Ser 145 150 155 160 Ser Ser Ala Ala Ala Pro Gln Leu Leu Ile Val Leu Leu Gly Leu Ser                 165 170 175 Ala Leu Leu Gln SEQ ID NO: 2 Sequence length: 996 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence type: cDNA to mRNA How the characteristics were determined: E Array GTGGAATTC ATG GCA TCT ACT TCG TAT GAC TAT TGC AGA GTG CCC ATG GAA 51 GAC GGG GAT AAG CGC TGT AAG CTT CTG CTG GGG ATA GGA ATT CTG GTG 99 CTC CTG ATC ATC GTG ATT CTG GGG GTG CCC TTG ATT ATC TTC ACC ATC 147 AAG GCC AAC AGC GAG GCC TGC CGG GAC GGC CTT CGG GCA GTG ATG GAG 195 TGT CGC AAT GTC ACC CAT CTC CTG CAA CAA GAG CTG ACC GAG GCC CAG 243 AAG GGC TTT CAG GAT GTG GAG GCC CAG GCC GCC ACC TGC AAC CAC ACT 291 GTG ATG GCC CTA ATG GCT TCC CTG GAT GCA GAG AAG GCC CAA GGA CAA 339 AAG AAA GTG GAG GAG CTT GAG GGA GAG ATC ACT ACA TTA AAC CAT AAG 387 CTT CAG GAC GCG TCT GCA GAG GTG GAG CGA CTG AGA AGA GAA AAC CAG 435 GTC TTA AGC GTG AGA ATC GCG GAC AAG AAG TAC TAC CCC AGC TCC CAG 483 GAC TCC AGC TCC GCT GCG GCG CCC CAG CTG CTG ATT GTG CTG CTG GGC 531 CTC AGC GCT CTG CTG CAG TGAGATCCCA GGAAGCTGGC ACATCTTGGA AGGTCCGTCC 589 TGCTCGGCTT TTCGCTTGAA CATTCCCTTG ATCTCATCAG TTCTGAGCGG GTCATGGGGC 649 AACACGGTTA GCGGGGAGAG CACGGGGTAG CCGGAGAAGG GCCTCTGGAG CAGGTCTGGA 709 GGGGCCATGG GGCAGTCCTG GGTGTGGGGA CACAGTCGGG TTGACCCAGG GCTGTCTCCC 769 TCCAGAGCCT CCCTCCGGAC AATGAGTCCC CCCTCTTGTC TCCCACCCTG AGATTGGGCA 829 TGGGGTGCGG TGTGGGGGGC ATGTGCTGCC TGTTGTTATG GGTTTTTTTT GCGGGGGGGG 889 TTGCTTTTTT CTGGGGTCTT TGAGCTCCAA AAAATAAACA CTTCCTTTGA GGGAGAGCAA 949 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAGAATTC CACCACA 996

[Brief description of drawings]

FIG. 1 North of genes obtained in Examples of the present invention
The analysis result by ern Blotting analysis (agarose gel electrophoresis photograph) is shown.

FIG. 2 shows the ability of the membrane proteins obtained in the examples of the present invention to support the growth of mouse pre-B cell line DW34.

FIG. 3 shows the DNA hydrophobicity and hydrophilicity regions of the genes obtained in the examples of the present invention as DNA analysis software Gene Works.
The results of analysis using are shown.

Front page continuation (51) Int.Cl. ⁷ Identification code FI C12N 5/10 C12P 21/02 C 15/09 ZNA 21/08 C12P 21/02 21/02 21/08 C12R 1:91 // (C12P 21 / 02 C12N 5/00 A C12R 1:91) 15/00 ZNAA (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00-15/28 BIOSIS (DIALOG) SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / GeneSeq

Claims (6)

(57) [Claims] 1. An amino acid sequence represented by SEQ ID NO: 1 in the sequence listing or a partial substitution, deletion or addition of the amino acid sequence.
A polypeptide having a different amino acid sequence and having a pre-B cell growth supporting ability . 2. The amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing or the amino acid sequence thereof is partially replaced, deleted or added.
A DNA having a different amino acid sequence and encoding a polypeptide having a pre-B cell growth supporting ability . 3. A recombinant vector containing the DNA according to claim 2 . 4. A prokaryotic or eukaryotic host cell transformed with the recombinant vector according to claim 3 . 5. The host cell according to claim 4 is cultured and produced.
Amino acid sequence shown in SEQ ID NO: 1 or a part of the amino acid sequence characterized by separating the produced polypeptide
Has a modified, deleted or added amino acid sequence, and
A method for producing a polypeptide having the ability to support B cell proliferation . 6. The amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing or the amino acid sequence thereof is partially replaced, deleted or added.
A monoclonal antibody that recognizes a polypeptide having a different amino acid sequence and capable of supporting pre-B cell growth .