JP3016756B2 - Method for producing human B cell differentiation factor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] TECHNICAL FIELD The present invention relates to a human B cell differentiation activity
The present invention relates to a method for producing a sex factor. [0002] 2. Description of the Related Art B cell differentiati
on Factor: BCD F: T cell Replacing especially in mice
 Factor (TRF), and recently, IL-5
Are also produced from the T cell lineage,
Act directly on the cells and differentiate them into antibody-producing cells.
It is a factor consisting of a repeptide. [0003] Antibodies are used to detect bacteria and viruses that invade living organisms.
Reacts with xenobiotics such as cancer cells and inactivates them
It has the function of becoming or eliminating. B cell differentiation
The factor is a B cell clone specific to a specific antigen (xenobiotic).
That is, antibody production from B cell clones sensitized to a specific antigen
Inducing cells to produce antibodies against the antigen
Is BCDF an important aspect of the treatment of various infectious diseases and cancers?
It is a useful substance. That is, BCDF is the production of this factor.
Self considered to be caused by a shortage in the body
Diagnosis and treatment of immune diseases and immunodeficiencies as well as various
It is expected that it can be used to treat infectious diseases and cancer. [0004] It is said that the activity of BCDF has species specificity.
Have been done. Therefore, this factor is
For diagnosis or treatment of diseases, infectious diseases, cancer, etc.
It is desired to use human-derived BCDF.
Until now, some BCDF or TRF
Research is being done. First, mouse BCDF (or
Is TRF), see R.W.Dutton et al. (Prog. Immunol.,
1, 355 (1971)) and A. Schimpl and E. WecKer
(Nature,237, 15 (1972)). That
Later, even in humans, mouse BCDF (or TR
F), the presence of a substance corresponding to Geha, R.S. et al. (J. Exp. Me
d.,138, 1230 (1973)), Fauci, A.S. et al. (J. Immunol.,
117, 2100 (1976)) and Hirano, T. et al. (J. Immunol.,
119, 1235 (1977)), but
There are many unclear points about the structure as described in
It is chaotic because the genes involved are unknown
(Kishimoto, T., Ann.Rev.Immunol.,3, 133 (198
Five)) . Conventionally, to obtain human BCDF, human peripheral
Mitogen stimulation of normal human T-cells isolated from blood
The method of obtaining from the culture supernatant by culturing
(Hirano, T. et al., J. Immunol.,126, 517 (198
1) and Ralph, P. et al., J. Immunol.132, 1858 (198
Four)). The above Ralph, P. et al. Showed that mitogen-stimulated normal
Ammonium sulfate precipitation, DEAE from 3 l culture supernatant of human T cells
Cellulose column chromatography (DE-52),
Ultrogel column chromatography (AcA4
4), by blue agarose and red agarose
Affinity chromatography, reversed-phase high-performance liquid chromatography
Purified to about 11,000 times by chromatography
The molecular weight by gel filtration is about 20,000.
Was measured, but until the pure BCDF was obtained
And the amount obtained is very small (from 3 l of culture supernatant)
4.2 μg). Also, Okada, M. et al. (J. Exp. Med.,157,
583 (1983)) describes normal human T cells and human T cell line CEM.
-AG^RCulture of human T cell fusion strain obtained by fusing
Has reported a method for obtaining human BCDF from
Fusion cell line has reduced ability to produce substances during subculture
Tend to be unsuitable for practical material production.
You. There are two types of human BCDF, BCDFI and BCDFII.
And their properties are reported as follows (Te
ranishi, T. et al. J. Immunol.,128, 1903 (1982) and
Hirano, T. et al., J. Immunol.132, 229 (1984)). Immediately
Chi [0007] BCDFI: Molecular weight: 20,000 (gel filtration method) PI: 6.5 to 8.0 Action: Staphylococcus aureus cowan I (St
aphylococcus aureusCowan I (SAC)) stimulated B cells
(Blasted B cells) is used for immunoglobulin (Ig) producing cells.
Differentiates and induces cells. BCDF II: Molecular weight: 22,000, 36,000 (gel filtration method) PI: 5-6 Action 1) B-protein transformed with EB virus
Induces IgG production in lymphoblastoid cells (B-LCL)
You. 2) I of BACs stimulated with SAC in the presence of BCDFI
enhance differentiation into g-producing cells (BCDFII alone does not
Has no effect). Recently, human T-cell leukemia virus (HTL)
V) one strain of human T cells transformed by V)
A method for producing human BCDF using T-1 has been reported.
(Tadazo Kishimoto, Toshio Hirano, JP-A-61-115024
And JP-A-61-115025). The filing of these applications
The authors reported that from 10 l of serum-free culture supernatant of VT-1 cells,
Ultrafiltration, AcA-34 gel filtration column chromatograph
Chromatography, reverse phase chromatography
The product was purified to 1042 times (1.8% yield) using
To BCDF has a molecular weight of 3.5 ± 0.5 × 10^Four Dalton
(Gel filtration method) or 2.2 ± 0.2 × 10^Four Dalton
(SDS polyacrylamide electrophoresis), isoelectric point
4.9 to 5.1, and the amino acid sequence of the N-terminal part is Pr
o-Val-Pro-Pro-Gly-Glu-Asp-Ser-Lys-Asp-Val-Ala-Ala-
Is reported. This purified BCDF exhibits its activity in an EB
Russ-transformed cells, CESS I
The point of purification using gG production as an index and the molecular weight and
It is considered to be BCDFII from the PI value and the like. On the other hand,
The molecular weight of human BCDF reported by Ralph, p.
It is 20,000 daltons by law. This is BCDFI
But its structure and other physicochemical properties
Not disclosed. In addition, the above VT-1 cells were used.
The method for producing BCDF has been described in detail above.
Improvement compared to the method using vesicles or human T-fused cells
However, the amount of BCDF obtained from a large amount of culture supernatant is extremely small.
And using HTLV-infected cells as starting material
Therefore, it is not suitable for industrial production for pharmaceutical purposes.
Thus, for human B cell differentiation factor (BCDF)
Is not only revealed its structure, but also
There are many unclear points about the chemical properties and functions. Also,
Producing it in large quantities and putting it into practical use is not possible with conventional technology
It was impossible. [0010] SUMMARY OF THE INVENTION The present invention relates to a gene
To solve these problems by using replacement technology
It is. That is, the present invention relates to a human BCDF-high producing cell.
Isolate BCDF mRNA and govern BCDF production
BCDF gene (ie DNA sequence) or alternatively staining
In addition to clarifying the body-derived BCDF gene,
By clarifying the structure (amino acid sequence) of F molecule
Mass production of BCDF and medical
It is intended to provide the possibility of application to medicines, etc.
You. The human B cell differentiation factor according to the present invention is BCDF.
I, but by the same method as in the present invention.
Other BCDF structures could also be elucidated. [0011] [Means for Solving the Problems]Preparation of human BCDF gene and determination of nucleotide sequence The human BCDF cDNA of the present invention has already been reported to the present inventors.
Thus, the mouse B cell differentiation factor gene (c
DNA) cloned plasmid pSP6K-
mTRF23 (Japanese Patent Application No. 61-157227)
BamHI-Accl fragment (of mouse B cell differentiation factor)
Probe containing the entire coding region of the gene)
As described below. still,
The plasmid pSP6K-mTRF23 has been introduced.
E. coli (HB101 / pSP6K-mTRF23)
Named SBM285 and FERM P-88
28 deposit numbers have been deposited. First, mouse B chromosome was added to human chromosomal DNA.
There is a sequence that hybridizes with the vesicle differentiation factor gene
For example, from normal human placental cells
Digest the extracted DNA with PvuII or PstI,
BamH of the above plasmid pSP6K-mTRF23
The I-AccI fragment was obtained by the nick translation method.
³²After labeling with P, use this as a probe
Hybridization (Southern blot hybridizat)
ion). Digestion of DNA from human placenta with PvuII
In this case, a 3.0 kb DNA fragment was erased by PstI.
In this case, a 4.1 kb DNA fragment was
It has been observed that it hybridizes
DNA having a sequence similar to that of B cell differentiation factor gene
It is confirmed that there is. The result is the above probe
Can be used to clone human BCDF cDNA.
It indicates that [0013] Next, based on the above findings, the human BCDF cD
Prepare NA library. First, Maeda, M. et al. (J.
Exp.Med.,162, 2169 (1985))
From the cultured cells of the human T cell line ATL-2, a conventional method (Nikaido,
 T. et al., Nature,311, 631 (1984)).
(A)⁺Prepare RNA and use Okay with pCD vector
ama-Berg method (Okayama, H. and Berg, P., Mol. cell. Bio
l.,3, 280 (1983)).
Prepare. Next, several cDNA libraries were prepared.
, And plasmid DNA was separated therefrom, and S
After digestion with all or BamHI,
( ³²BamHI-AccI DNA labeled with P
Fragment) and Southern blot hybridization (So
uthern, E.M., J. Mol. Biol.,98, 503 (1975))
Hybridization with mouse B cell differentiation factor gene
The group having the DNA fragment to be selected is selected. Then, positive
Probe for each clone in the
Similarly using Southern blot hybridization
To hybridize with the above probe
Select a clone. The positive clones include human BC
Contains plasmid with DF cDNA gene
it is conceivable that. The positive positives obtained in this way
The plausibility of one of the loans (ph-IL-5-30)
Was named pCDVTRF and the plasmid was
A transformant (HB101) introduced into E. coli HB101 strain
/ PCDVTRF) is named SBM286,
FERM BP-1171 accession number and deposited
ing. Next, the plasmid of the positive clone
Restriction enzyme analysis and hybridization with the probe
As a result, the mouse B cell differentiation factor gene
A human BCDF cDNA similar to
The base of the DNA as described below.
The sequence is determined and the DNA is
A gene that produces a protein (polypeptide)
Find out if there is. First, the plasmid of the clone is prepared according to a conventional method.
(Eg, ph-IL-5-30)
After examining the cD, cD inserted into ph-IL-5
NA (including human BCDF cDNA gene)
Click on the BamHI site of an appropriate plasmid such as UC18.
Loaning was performed again using the dideoxy method (Sanger, F. et al., Pr.
oc.Natl.Acad.Sci. USA,74, 5463 (1977))
The nucleotide sequence of the above cDNA is determined. In our experiments
According to the above method, human BCDF cDNA was
The resulting cDNA base sequence of FIG. 1 was obtained. [0017]Determination of the region encoding human B cell differentiation factor
Set A human B cell differentiation factor is added to the cDNA base sequence obtained above.
Is determined to be included as follows.
Specified. That is, the open reading frame of this nucleotide sequence
Frame, and have already been determined by the present inventors.
Of the mouse B cell differentiation factor gene
The human B cell differentiation factor (human BCDF)
Determine the polypeptide-coding region and amino acid sequence
(FIG. 2). And the amino acids in the coding region
Sequence and human BCDF is a protein secreted extracellularly
Thus, the precursor polypeptide of human BCDF is
Signal peptide consisting of 19 amino acids on the N-terminal side
(Leader sequence) consisting of 134 amino acids
It was found to be a polypeptide (see FIG. 3). That is,
The mature human is the 20th to 134th amino acids in the amino acid sequence of FIG.
BCDF polypeptide. In the amino acid sequence of the precursor, two positions N-
Glycosylation site (Asn at positions 47 and 90)
And at least one O-glycosyl as described below
Site (Thr of No. 22), where the sugar chain is
In view of the addition, the mature human BCDF
The calculated molecular weight (13,149) of the repeptide has already been reported.
Of human BCDFI (to which sugar chains are added)
There is no contradiction with the molecular weight (about 20,000). Note that FIG.
Met (methionine) at the N-terminus of the polypeptide
Post translational modification pr
ocess) and may be formylated or acetylated.
In some cases, Met may be removed. Ma
In addition, the N-terminal a
Mino acids may be acetylated in a similar process. On the other hand, the plasmid ph-IL-5-
The cDNA inserted in No. 30, ie, identified as described above.
DNA having a human BCDF activity (polypep
A gene that produces
The host cell transformed by the inserted expression vector
Knowing by measuring the activity of the protein produced
But can also be checked as follows. That is, first, the plasmid ph-IL-5-3
B containing the entire human BCDF cDNA inserted at 0
The amHI DNA fragment was recloned into the pSP64 vector.
On. pSP64 has an inserted foreign gene of SP6.
Modified to be expressed only under the control of the promoter
Vector, which is used for in vitro SP6 RNA
MRNA of a foreign gene inserted in the presence of limerase
It can be synthesized (Krieg, P.A. and Melton, D.
A., Nucl.Acid.Res.,12, 7057 (1984), and Konars
ka, M.M., Cell,38, 731 (1984)). Next, from the above cDNA,
The mRNA solution prepared in vitro is
Xenopus oocytes (oocy)
te), and culture is performed.
Measures BCDF activity of translation products (proteins) from RNA
I do. Human BCDF (particularly human BCDFI) activity has already been
As described in Staphylococcus aureus Cowan I
(SAC) stimulated human B cells to induce IgM production
It is measured by checking in comparison with the control.
In this way, the above-mentioned cDNA enhances human BCDF activity.
Genes that produce proteins (polypeptides)
It was confirmed that the DNA of the present invention
A human BCDF polypeptide as set forth in the claims.
Shows the potential for mass production. [0022]Expression of human BCDF According to the present invention, a gene prepared as described above
By using the sequence, human BCDF can be produced.
And it is possible. For example, a human BCDF encoding
When using cDNA as a gene,
A suitable promoter (eg, SV40) is located 5 ′ upstream.
Expression vector with an inserted promoter
And suitable cells (eg, yeast cells, E. coli cells, or
Represents animal cells such as COS-I cells and CHO cells) and human B
It can also make proteins that differentiate cells. This place
Use the DNA region encoding the precursor protein
Is preferred, but encodes the mature form of the protein (polypeptide)
Alternatively, a cDNA region to be used may be used. In addition, the protein
Appropriate restriction enzymes are located immediately 5 'upstream of the encoding DNA.
(E.g., HindIII).
Site-directed mutagenesis
nesis) method to introduce foreign promoters.
Easy to enter. According to the present invention, instead of using cDNA,
The human BCDF gene containing introns from the human chromosome
A human B cell was prepared and used in the same manner as described above.
Proteins that differentiate vesicles can also be produced. That is,
DNA fragment containing the BCDF gene
As a lobe, use a suitable human gene library (eg,
For example, the human gene library of Maniatis
Library) and then, as with cDNA,
Suitable 5 'upstream of the DNA region encoding human BCDF
Construct an expression vector with a unique promoter
Transform or transfect with expression vector
By culturing the modified cells, human BCDF
Can be manufactured efficiently. The host cell in this case is preferably
A cell capable of chair is used. Animal cells are particularly preferred
New The BCDF gene derived from human chromosome is integrated.
Expression vector (pdKCR-hIL-5gen)
e) into which E. coli (HB101 / pdKCR) has been introduced.
-HIL-5gene) was named SBM 293.
International Deposit Number FERM BP-1477
Obtained and deposited. [0025]Purification of recombinant human BCDF Recombination produced by animal cells constructed as described above
Human BCDF can be prepared by any suitable method, for example,
Chromatography and gel filtration
Purified by reversed-phase high-performance liquid chromatography
can do. We purified in this way
-Amino acid sequence analysis of purified recombinant human BCDF
And electrophoresis analysis, and the results of this amino acid sequence analysis
And the nucleotide sequence of the gene encoding human BCDF
Based on the estimates (see eg FIG. 2), mature human BCD
The structure of the polypeptide portion of F is as shown in FIG.
There is an array from Ile at No. 20 to Ser at No. 134.
And estimating the two-dimensional array.
(Figure 16). Hereinafter, the present invention will be described by way of examples.
explain. [0026] 【Example】 (1)Confirmation of human BCDF gene Previously, the present inventors generated mouse BCDF (TRF).
, A DNA sequence (Japanese Patent Application No. Sho 61)
-157227). And by this gene
The mouse TRF produced has TRF activity [1).
B-cell leukemia cells (BCL₁ ) Was separated into IgM antibody-producing cells.
2) antigen (DNP-KLH) sensitization
B cells in mouse spleen are treated with antigen (DNP-ovalbumin)
Stimulated and separated into cells producing specific antibodies (anti-DNP-IgG).
Activity to activate 3) B cells activated in vivo
IgM synthesis-inducing activity of blast] and BCGFII activity
[1] BCL₁ Cell division promotion 2) Dextran sulfate
Mitogenesis of stimulated rest B cells]
I made it. CDN complementary to mouse TRF mRNA
A containing plasmid pSP6K-mTRF23 in Bam
After digestion with HI and AccI, a 654 bp Ba
The mHI-AccI fragment was separated. Mouse BC
This includes the entire coding region of the DF (TRF).
Fragment by nick translation ³²
Human chromosomal DNA using P as a probe
Southern blot hybridization of
hern, E.M., J.Mol.Biol.,98, 503 (1975))
went. Human chromosomal DNA was obtained from normal human placenta
ta, Y. and Honjo, T. (Biomed.Res.,1, 164 (198
0)), 2 μg of each was extracted with PvuII or
Is digested with PstI and subjected to Southern blot hybridization.
Run on a 0.6% agarose gel for
Was. After electrophoresis, the DNA in the gel is
(Schleicher & Schuel (Dassel))
Hybridized with robe. The washing conditions were 2 × SS containing 0.1% SDS.
C (SSC: 0.15 M NaCl-0.015 M que
PvuII) at 50 ° C. for 45 minutes.
When digested with, a DNA fragment of 3.0 kb,
When digested with PstI, a 4.1 kb DNA fragment was digested.
The fragment hybridizes with the mouse BCDF probe
It turned out to be. This probe is used for mouse chromosome D
The fragments of NA are more stringent (st
(hybridizing)
Mouse BCDF (TRF) c in human chromosomal DNA
There is a sequence that is not completely identical to DNA but has high homology.
Using this probe, the cD of human BCDF was determined.
Judging that screening of NA clones is possible,
Was. (2)Of the human BCDF cDNA clone
Separation The human BCDF cDNA library is prepared as follows
did. M.Maeda et al. (J.Exp.Med.,162, 2169 (1985))
Human T cells established from the blood of a patient with adult T cell leukemia
Strain ATL-2 was supplemented with RPMI 1640 + 10% fetal bovine serum
Medium, 37 ° C, 5% CO_Two Collect cells by culturing in
Was. From these cells, a conventional method (Nikaido, T. et al., Nature,311
, 631 (1984)).⁺Prepare RNA
Was. This poly (A)⁺CDNA using 3 μg of RNA
A library was created, but using a pCD vector
Okayama-Berg method (Okayama, H. and Berg, P., Mo
l.Cell.Biol.,3, 280 (1983)) and E. coli (E.
E. coli HB101 strain)
^Five Independent cDNA clones were obtained. This 2 × 10^Five CDNA clones
Whether the cDNA corresponding to the human BCDF gene exists
To determine if the plasmid was
DNA was prepared, and 2 μg of each DNA was added to SalI or
Is digested with BamHI, and digested with the above method.
Hybridization was performed. As a result, pCD
4.1 kb for SalI digestion, which inserts one cut into the vector
DNA fragments hybridize, whereas
Cut at almost both ends of the inserted cDNA.
In the BamHI digestion, a DNA fragment of 1.0 kb was generated.
It turned out to hybridize. Based on these results, the cDNA library
With a DNA sequence corresponding to the human BCDF gene
Was suggested to be included. So the mau
2 × 10^Five CDNA cDNA
Loan to conventional colony hybridization (Hana)
han, D. and Meserlson M., Gene,Ten, 63 (1980))
Screening was performed according to the procedure. Screening results 29
Clone is mouse BCDF (TRF) cDNA probe
And the plasmid D of the 29 clones
Endonuclease cleavage map of NA and endonuclei
Probe for DNA fragment digested with Raase
As a result of Southern hybridization using
All nine clones were determined to be identical. (3)Nucleotide sequence of human BCDF cDNA
Determination and Analysis of Polypeptides of Human BCDF cDNA
Amino acid sequence One of the 29 clones, ph-IL-5-30
Further investigation was made. Restriction endonuclease cleavage map
Was examined according to a conventional method, and inserted into ph-IL-5-30.
The cDNA thus obtained was once transformed into BamHI of pUC18 plasmid.
Subcloning into the site
According to a conventional method, the dideoxy method (Sanger, F. et al., Proc. Natl. Ac
ad.Sci. USA,74, 5463 (1977)). So
As a result, the inserted cDNA was 81% excluding the poly-A tail.
It was found to consist of 6 base pairs. This result was compared with that of mouse BCDF (TRF).
By comparing DNA sequences, human BCDF
Coding region was determined and the precursor of human BCDF was 134
Amino acids. Human BCDF is T
This 134 amino acid sequence to be secreted extracellularly
It is expected that the N-terminal sequence contains a signal sequence,
Mature human BCDF has the amino acid sequence of the above 134 amino acids.
From the 20th amino acid to the 134th amino acid
It is considered to be a molecule containing the polypeptide In this amino acid sequence, two N-G
A site capable of lycosylation (A in positions 47 and 90 in FIG. 3)
sn) and at least one O-glycosylation possibility
There is a site (the 22nd Thr in FIG. 3), and this additional
Potential sugar and polypeptide molecular weights (20
Add 13,149) for polypeptides # 134
Then, it has been reported as the molecular weight of BCDFI 20,
000. Comparison with mouse BCDF (TRF)
Then, in the precursor, the number of amino acids of human BCDF is one more.
No. Possible site of N-glycosylation at two positions of human BCDF
The first and third N-glycosides of mouse BCDF
Matches the site that can be customized. Mouse BCDF (TRF)
Of the three cysteine residues in the
Stain residues are also conserved in human BCDF
You. Of the coding region of the mouse and human BCDF
The nucleotide and amino acid sequences are 78% and 7%, respectively.
Has 0% homology. The PstI fragment of ph-IL-5-30
(515 bp) by nick translation
³²Using the P-labeled probe as a probe
DNA digested with PvuII or PstI of chromosomal DNA
Southern blot high as described above for fragments
As a result of hybridization, mouse BCDF pro
4.1 kb and 3.0 kb, respectively, as when using
Hybridized with the kb fragment. Human plow
When using a cleaning solution, the washing condition is 0.1 × SSC−0.1%
Stringent conditions of SDS, 65 ° C, 45 minutes
Even so, enough hybridization was done. The ATL was prepared using this human probe.
-2 poly (A)⁺RNA was prepared according to standard methods (Thomas, D.D.,
Proc.Natl.Acad.Sci. USA,77, 5201 (1980)), Noosa
The result of blot hybridization was 1.0.
Only one band of kb was observed. This means that the inventor
Identified the human BCDF gene as a human chromosomal DNA sequence.
In column, specific homology to mouse BCDF (TRF) gene
Gene, and from this human BCDF gene
That only one type of mRNA is translated
Is shown. (4)Measurement of human BCDF activity The human BCDF gene identified as described above is
The polypeptide that governs production or its polypeptide
That human BCDFI activity is present in a substance consisting of
Confirmed by the method. Plasmid ph-IL-5-30
Vectors for all and small human BCDF cDNA inserts
BamHI DNA fragment containing DNA
Recloned into the P64 vector. This plasmid is called S
After digestion with all, using SP6 RNA polymerase
MRNA was synthesized in vitro (Krieg,
P.A. and Melton, D.A., Nucleic Acid Res.,12, 7
057 (1984); and Konarska, M.M. et al., Cell,38, 731
 (1984)). The thus prepared mRNA solution is
Xenopus oocyte (oo)
cyte) and after culturing at 20 ° C. for 48 hours,
The product secreted in is collected and centrifuged.
Concentrate 4 times with a concentrator (Centricon 10; Amicon)
Was used as human recombinant BCDF,
Used for experiments. This human recombinant BCDF is B
Human B cells stimulated with SAC to determine whether they have CDFI activity
The vesicles were examined for their ability to induce IgM production. Hula rich in human B cells
Of normal human blood from Saiki, O. and Ralph,
P., Eur.J.Immunol.,13, 31 (1984))
And 1 × 10^Five Cells / 100 μl of cells were applied to SAC (0.
(001-0.0025%). Then the above human
・ Add recombinant BCDF to 15% concentration
And 37 ° C, 5% CO _Two And cultured for 6 days. The amount of IgM in the culture supernatant was determined by enzyme immunoassay.
As measured with the assay kit, 110 ng / well
Value was observed and the control was approximately 50 ng / well.
In contrast, IgM production was significantly induced. This induction
Is 135 ng / well by adding IL-2 (50 u / ml).
Up to IgM production was observed in the control
What was obtained was a B cell fraction completely free of T cells.
This is probably because it is difficult to obtain (5)Isolation of human BCDF chromosome gene
Identification Already in Example (1), the human chromosome
Although the existence of the BCDF gene was recognized,
The human BCDF chromosomal gene was isolated from a source. That is,
The source is AluI-H of human fetal liver DNA.
aeIII Sharon 4A phage line with partially digested fragment
Brally (Maniatis, T. et al., Cell,15, 687-702 (197
8)), having a partially digested EcoRI fragment of human placental DNA
Sharon 4A phage library (Yaoita, Y. and
Honjo, T., Biomed.Res.1, 164 (1980))
And D of human IgE-producing myeloma cell line 266B1
Sharon 4A file with a partially digested EcoRI fragment of NA
Library (Nishida, Y. et al., Proc. Natl. Acad. S.
ci.USA,79, 3833-3837 (1982)). Next, using these three libraries,
PstI-PstI cleavage of the human BCDF cDNA described above
Screening of phage using a piece (515 bp) as a probe
Becton and Davis (Becton, W.D. and
Davis, R.W., Science,196, 180-182 (1977))
I went according to. Fetal liver, placenta and myeloma
5 × 10 each from library^Five Phage plaques
3 that screen and hybridize with the probe
Clone λ12 (from fetal liver library), λ2
2 (from the placenta library) and λ38 (myeloma)
From the library). Subsequently, the restriction ends of these three clones
A nuclease cleavage map is prepared according to a conventional method, and human BCD is prepared.
Using the PstI-PstI fragment of F cDNA as a probe
Southern blot hybridization (So
Uthern blot hybridizatio
n) Analyzed. Analytical strategies and human BCDF
The gene structure is shown in FIG. 4, where E, H and B are
Cutting with EcoRI, HindIII and BamHI
The cleavage site is shown, and the black box indicates the exon region. This
According to the results of this analysis, the inserts of these three clones were
The coats overlap each other, and each coat shown in FIG.
Only the 3.2 kb BamHI fragment common to loans is
Hybridized with the probe. Therefore, this 3.2 kb
The BamHI fragment contains all of the DNA encoding human BCDF.
This 3.2 kb BamHI
The fragments were separated and duplicated after digestion with HindIII.
Each of the 6 kb fragments was subcloned into the pUC18 vector.
I did it. Next, Yanisch-Perron, C. et al. (Gene,Three
Three, 103-119 (1985)).
b.Extract each of the cloned plasmids from both ends.
Digest with Sonuclease III and VII
Shonarudeirion (unidirectionaliona
l deletion) Series of mutant clones
Was produced. Insertion of this mutant plasmid
Nucleotide sequence can be obtained by standard methods (Sanger, F. et al., Proc. N
atl.Acad.Sci.USA,74, 5463-5469 (1977)
Oxychain termination (dioxy c)
h-termination) method.
Was. Restriction endonuclei of human BCDF chromosome gene
Crease map and 4 exons and 3 introns
The organization of the nucleotides
FIG. 4 shows the sequence analysis strategy. Also, actually
BamHI 3.2 kb fragment containing the human BCDF gene
Are shown in FIGS. 5 to 9. Clear here
Of the exon part on the human BCDF chromosome gene
The nucleotide sequence is the human BCDF identified above.
It completely matched the nucleotide sequence of the cDNA. From the above results, in the chromosomal gene,
The first exon is the human BCDF precursor polypeptide chain
N-terminal first amino acid (Met) to 48th amino acid
Until (Glu), the second exon is the 49th amino acid (Th
r) to the 59th amino acid (Asn) in the third exon
Is amino acid No. 60 (His) to amino acid No. 102 (L
ys), and the fourth exon is the 103rd amino acid
From (Lys) to the 134th amino acid (Ser)
It was found that it was code. That is, human B fine
The vesicle differentiation factor is Ba of the human chromosome gene shown in FIGS.
In the mHI 3.2 kb fragment, A at position 535 in FIG.
, And is coded during the 2213th T in FIG.
It has been found. (6)Construction of human BCDF expression vector Human BCDF cDNA obtained in Example (2) and
Using the human BCDF chromosome gene obtained in Example (5)
Then, the following four types of animal cell expression vectors were prepared.   pdKCR-hIL-5 cDNA   pdKCR-hIL-5 cDNA-dhfr   pdKCR-hIL-5gene   pdKCR-hIL-5gene-dhfr A)pdKCR-hIL-5 cDNA and
Of pdKCR-hIL-5 cDNA-dhfr
(FIG. 10) In Example (2), the Okayama-Berg method (Okayama, H.
And Berg, P., Mol. Cell Biol.,3, 280 (1983))
First, the cDNA was cloned into a pCD vector.
E. coli HB101 transformed clone ph-IL-5
-30 was obtained. Isolate the plasmid from this clone,
A BamHI digestion and a partial digestion with PstI were performed.
CDNA containing the entire coding region of BCDF
A BamHI-PstI fragment was obtained. Phage
Between BamHI and PstI sites of M13mp19 DNA
And cloned into M13mp19-hIL-5cDN.
A was obtained. Next, the human BCDF coding region
The purpose of introducing a HindIII site immediately upstream of 5 '
The DNA sequence of the human BCDF cDNA shown in FIG.
The double underlined part in the opposite direction to the sequence shown in the figure
30mer that hybridizes with DNA strands
Oligonucleotides: 5'-GCAGAACGTTTCAAGCTTATGAGGATGCTT-3 ' (The underlined AAGCTT indicates the HindIII cleavage sequence.
(Messing, J. in Methods in Enzymo)
logy, vol. 101 Part C, pp62-65 (Academic Press, E
d. Wu, R.)), site-specific mutagenesis
And clone M13mp19-hIL-5 cDNA
(HindIII) was obtained. This phage DNA is digested with HindIII.
And a cD corresponding to the entire coding region of human BCDF
The HindIII fragment containing NA is transferred to the vector pdKCR (Ni
kaido, T. et al., Nature,311, 631-635 (1984): pdK
The CR vector is the pBR322 part of the pKCR vector.
HindII) (the vector was replaced with pBR327).
Introduction into the I site in the correct direction, the expression vector pdKC
R-hIL-5-cDNA was prepared. Next, the expression vector pdKCR-hI
Dihydrofolate reductase is added to the SalI site of L-5 cDNA.
Dase (dihydrofolate reducta)
se: dhfr) Purpose of introducing gene expression unit
The pdKCR-hIL-5 cDNA plasmid was
After digestion with II, the cleavage site was digested with T4 DNA polymerase.
It was a terminating end. The dhfr gene expression unit is pSV
_Two B on dhfr (BRL Inc.) PvuII site
Plasmid pSV with amHI linker_Twodhf
After preparing r-BL and digesting it with BamHI, T4D
Dhf
The r gene expression unit fragment was isolated. This fragment first
The obtained pdKCR-hIL-5 cDNA containing one cut
To produce pdKCR-hIL-5-dhfr
did. B)pdKCR-hIL-5gene and
Production of pdKCR-hIL-5-gene-dhfr
(FIG. 12) Fur having the human BCDF gene obtained in Example (5)
Complete gene encoding human BCDF from Ziclone λ12
The 3.2 kb BamHI fragment containing the gene region was isolated and pU
Subcloned into C18 vector and transformant pU
C18-hIL-5gene was obtained. Of this transformant
The plasmid was digested with BamHI and PstI and
BamHI- containing the entire gene region encoding BCDF
A PstI fragment (2.2 kb) was obtained. Phage
Between BamHI and PstI sites of M13mp19 DNA
And cloned into M13mp19-hIL-5gen
e was obtained. Next, in the same manner as in A), site-specific
Data Genesis and human BCDF coding
HindIII site was introduced 5 'upstream of the area
Clone M13mp19-hIL-5gene (Hin
dIII) was obtained. This phage DNA was converted to BamHI and P
After digestion with stI, the human BCDF gene was added to pUC19.
Plasmid pUC19-hIL obtained by cloning
-5gene (HindIII) is further divided by HindIII.
The whole gene region encoding human BCDF,
Correct the region to the HindIII site of the vector pdKCR
The expression vector pdKCR-hIL-5
gene was prepared. Next, the NruI site of this expression vector
To introduce a dhfr gene expression unit into pdK
CR-hIL-5gene plasmid digested with NruI
Then, the dhfr residue obtained in the above A) was added to the formed sticky end.
The gene expression unit fragments were ligated and pdKCR-hI
L-5gene-dhfr was prepared. Made above
The four expression vectors are SV40 (Simian vi)
rus40) human BCDF by early promoter
And dhfr expression, and SV40
Contains a replication origin. In addition, increased expression
The human BCDF cDNA and the human BCDF
Unnecessary parts of the 5 'and 3' untranslated regions are removed for both genes.
Has been left. (7)Generation of human BCDF by animal cells
Present The human BCDF expression vector prepared in Example (6)
Using human BCD in COS-I and CHO cells
Expression of F was performed. Animal cell line COS-I (monkey kidney)
Cells) are Dulbecco's modified with 10% fetal calf serum
Passage in an essential medium, 50-70% confluence
Using cells in ruent state, Graham, FL and
van der Eb, A. J. (Virology,52, 456-467 (1973))
Transfection by calcium phosphate method according to
Was done. Each cell in a culture dish with a diameter of 10 cm
10 μg of the four expression vectors obtained in Example (6)
Was used. 3 days, 37 ° C., 5% CO_Two Culture under
In addition to examining the BCDF activity of the supernatant,
man et al. (Proc. Natl. Acad. Sci. USA,80, 4094 (1983))
RNA was extracted according to the following procedure. Using the obtained RAN,
Nambu, J.R. et al. (Cell,35, 47-56 (1983))
Blot analysis (20 μg RNA / lane) and
The expression level of BCDF mRNA was examined. Probe used
Is M13m containing the entire coding region of human BCDF
Hi of p19-hIL-5 cDNA (HindIII)
ndIII-PstI fragment. CHO (Chinese
Expression in hamster ovary) cells is CHOdhfr^-Shares
It was performed using. Cells were MEM containing 10% fetal calf serum
α⁺(GIBCO) and transform as described above.
The transfection was performed. The expression vector used was pdKC
R-hIL-5 cDNA-dhfr and pdKCR-
hIL-5gene-dhfr. Culture was performed for 48 hours after transfection.
I, weissman, C. (Nucleic Acids Res.11, 687 (198
According to 3)), MEMα containing 10% dialyzed fetal bovine serum
^-(GIBCO), followed by methotrexe
0.1 μM, and after culturing for several days,
A colony resistant to rexate was isolated, and after passage, the supernatant
Was examined for BCDF activity. In order to obtain high expression cell lines,
Simonsen, C.C. and Levinson, A.D. (Proc. Natl. Aca
d.Sci.USA,80, 2495-2499 (1983)).
Solution with higher concentration of methotrexate in steps up to 1 mM
In addition, a human BCDF producing cell line was established. The assay method for human BCDF is described in T cell r
Kinashi, T., et al.
(Nature, 324, 70-73 (1986)). Assay
The cells used for BCL₁ Mouse chronic B leukemia cells (in
 vivo passage or BCL₁Clone 5B₁ b (ATC
C TIB197)) and dilute the cells
Mix with series, 5 × 10^-396 holes flat per 200μl
5% CO for 48 hours in bottom plate_Two Cultured at 37 ° C
Was. The medium was RPMI-containing 10% fetal calf serum.
1640, 5 × 10^-Five2-mercaptoethano of M
, 50 U / ml penicillin G, 50 μg / ml
Contains Leptomycin. After 48 hours, centrifuge the cells
After washing with Hanks' solution, suspend in 200 µl / well
Then, using half of the cells (100 μl),
The plaque assay was performed according to Gronowicz, E. et al. (Eur. J. Immunol.,
6588-590 (1976)).
Was measured. BCL₁ Since the cells are mouse-derived,
In this assay, the sensitivity of human BCDF was determined by mouse BC
Inferior to DF (about 1 /
100 were measurable. The COS-I cells were transformed into pdKCR-hIL-5
Expression vector for cDNA and pdKCR-hIL-5gene
And transfected in the cells three days later.
BCDF mRNA was analyzed by Northern blot analysis.
When analyzed, both hybridized at the position corresponding to 1 kb.
A zation band was observed. This means that COS-I cells
Transcript of the human BCDF gene is spliced correctly in
Has been shown. Also, generation of human BCDF mRNA
Surprisingly, the current amount was COS-I / pdKCR-h
IL-5gene has COS-I / pdKCR-hI
It was about 20 times higher than L-5 cDNA. Further, protein A.
In the plaque assay (PFC No.),
A trend similar to the blot analysis was observed. That is, both shots
Transfected with the current vector and cultured for 3 days
BC cultured with 20% of the culture supernatant of COS-I cells
L₁ Table 1 shows the number of plaques formed in the cells.
As compared with the control (host cell COS-I),
By comparison, COS-I / pdKCR-hIL-5gen
e is COS-I / pdKCR-hIL-5cDN
It gave a plaque formation number about 17 times higher than A. [0062] [Table 1] This means that pdKCR-hIL-5ge
ne is better than pdKCR-hIL-5 cDNA
Possibility due to high transfection efficiency
Can not be denied, but rather the inversion of the human BCDF gene
Unknown enhancement function acting on SV40 promoter in the tron section
Implies that there is. (8)Purification and properties of human BCDF COS-I cells were treated with pdKCR-hIL-5gene.
After transfection, 640 ml of culture solution after 3 days
(For 64 culture dishes with a diameter of 10 cm)
DF was purified. After centrifuging the culture, the supernatant was
Mouse BCDF (IL-5) monoclonal antibody as carrier
Affine bonded to Cellulofine (Chisso Corporation)
Apply to a human column (3 mL bed capacity)
Was adsorbed to the column (BCL by human BCDF).₁ Fine
Conversion of vesicles to IgM-producing cells can be achieved by using an anti-mouse
Was completely suppressed by this affinity column.
Was used for purification). Next, the column was washed in the following order.
did.   50 ml of 1M NaCl,   0.5% NP-40, 50 ml,   PBS (ph 7.2), 50 ml,   H_Two O, 50 ml. Next, it was adsorbed using 4 ml of 1M acetic acid.
Elute human BCDF and elute the solution
The solution was concentrated to 100 μl with a centrifuge. This dark
Connect the condensed liquid to two Superose 12 columns (Pharmacia)
HPLC and purification by gel filtration. Kara
Pre-equilibrate with PBS (pH 7.2)
It was eluted with the buffer (flow rate 0.3 ml / min). OD eluate
₂₁₀ And bioassay to monitor the active fraction for molecular weight
Collected around 40K. The elution pattern is shown in FIG.
You. In the figure, hatched portions indicate fractions having BCDF activity.
The solid line shows the protein, the dotted line shows the BCDF activity,
Arrows indicate elution positions of standard molecular weight proteins (molecular weight markers)
Is shown. This active fraction was subjected to Senshu Pack VP-3.
04-1251 (Sensyu Science, Protein C_Four Phase
). Adsorbed human BC
DF was dissolved in the presence of 0.1% trifluoroacetic acid (TFA).
The acetonitrile concentration of the effluent is linearly reduced from 0% to 80%.
Eluted by raising (flow rate 0.5 ml / min).
The elution pattern is shown in FIG. In the figure, the shaded area is
The fraction having BCDF activity is shown, and the dotted line is acetonitrile.
Shows the concentration of Purified human BCDF is acetonitrile
It was recovered at an elution position near 55% of ril. The yield is about 3μ
g. Thus, the purified human BCDF is
0.15 μg / 50 μl / lane at SDS-Polymer
The molecular weight was measured by acrylamide gel electrophoresis.
The migration pattern is shown in FIG. In the figure, 4) is the molecular weight
Marker lanes, lanes 1) to 3) have the following
The treated purified human BCDF was electrophoresed. 1) Sample immediately after purification, 2-mercaptoethanol and
Heat and heat treatment, 2) Sample immediately after purification, 2-mercaptoethanol treatment
Process, no heat treatment, 3) A sample stored at 4 ° C. for 2 weeks in the eluate after purification,
Not treated with 2-mercaptoethanol and heat
I As shown in FIG. 15, 2-mercaptoeta
With human BCDF sample without knoll treatment and heat treatment
Shows a single band of about 40K in agreement with the results of gel filtration.
However, this sample was treated with 2-mercaptoethanol.
(Without heat treatment), the molecular weight on the gel is about 20
It became a single band of K. The eluate (0.1% TF
A, about 55% acetonitrile) at 4 ° C for 2 weeks
Even after oxidizing conditions, the molecular weight on the gel is about
It became a single band of 20K. This is the active human
BCDF is a dimer (molecular weight of about 20K)
(About 40K). 2 μg of purified human BCDF (monomers and
N-terminal amino acid sequence using about 100 pmole)
Hewick, R. M. et al. (J. Biol. Chem.,256, 799
0-7997 (1981)), gas-phase protein
This was performed using a sequencer. Obtained in each cycle
Phenylthiohydantoin (PTH) amino acids are reversed-phase
Analyzed by HPLC, the N-terminal 27th amino acid
The sequence was determined as shown in Table 2. [0071] [Table 2]This result is similar to that of human B shown in Example (3).
13 predicted from the nucleotide sequence of CDF cDNA
A human BCDF precursor consisting of four amino acids is
19th amino acid from N-terminus when secreted from vesicle
The leader sequence was cleaved at the C-terminus of alanine (FIG. 3),
COS-I cells or perhaps their natural saw
Isoleucine from the N-terminal amino acid
And the monomer is a dimer consisting of 115 amino acids
It indicates that it is secreted as a glycoprotein. Note that the seventh, fourteenth, and twentieth
Despite threonine (Thr) being detected, salt
The 3rd amino acid judged as threonine from the base sequence is
What was not detected is that this third amino acid residue
This strongly indicates that a sugar chain has been added. This sugar chain
Is considered to be added via an O-glycoside bond. Less than
Mature recombination expressed by COS-I cells from above
(Recombinant) The characteristics of human BCDF
The two-dimensional estimated amino acid sequence of the monomer
The sequence is shown in FIG. The N-terminal amino acid sequence is different from isoleucine
The molecular weight in the natural state is about 4
0K, 2-mercaptoethanol treatment or 0.1
% TFA-55% after prolonged treatment with acetonitrile
About 20K on SDS-polyacrylamide electrophoresis gel
Indicates that the peptide portion of human BCDF is
Consists of immers.   The peptide portion of mature human BCDF is isoleuc
Consists of 115 amino acids starting with a
The molecular weight of the peptide moiety is 13,149. Mature human BCDF is a dimeric sugar
It is a protein. This means that the amino acids of mature human BCDF
28th and 71st asparagi from the N-terminus in the acid sequence
Is a sequence where N-glycosidation is likely to occur
And that the third threonine is
O-glycosidation due to undetectable
That the molecular weight of the monomer is
About 20K on acrylamide gel electrophoresis gel
The peptide portion has a molecular weight of 13,149
Inferred from Thus, the mature human BCDF monomer
Sugar chains with a total molecular weight of less than 7,000 are bound to the body
It is thought that there is. In the amino acid sequence of mature human BCDF,
The status of the cysteines at positions 44 and 86 from the N-terminus is
Unknown, 0.1% TFA-55% acetonitrile
SDS-poly
Equivalent to monomer on acrylamide gel electrophoresis gel
Because of the molecular weight, the formation of the dimer is due to the intermolecular SS
It is not due to crosslinking. However, 2-me
SDS polyacrylic quickly by lecaptoethanol treatment
Dissociation into monomers on an amide gel electrophoresis gel
And an SS crosslink exists in the molecule of the monomer to form a dimer
Is responsible for maintaining the conformation necessary for
available. From these observations, cysteine at position 44 and 8
The sixth cysteine may form an intramolecular crosslink
It is presumed that there is not. [0077] Analysis by analysis of human BCDF gene
From the exon-intron structure, the mature human BCDF
From 1 amino acid (Ile) to 29th amino acid (G1u)
Is the first exon, 30th amino acid (Thr) to 40th
Amino acids (Asn) up to exon 2, amino acid 41
(His) to 83rd amino acid (Lys)
Song, 84th amino acid (Lys) to 115th amino acid
(Ser) is derived from exon 4.   Purification of human BCDF is achieved by transfection of COS-I cells.
Ent expression (transient e
xpression) starting from 640 ml of culture
As a result, about 3 μg of pure human BCDF was obtained. COS-I fine
Cell transformation efficiency is about 10^-3Is
Considering that, CHO / pdKCR-hIL-5ge
The expression level of the ne-dhfr cell line is determined by the medical treatment of human BCDF, etc.
Could be a production strain intended for wider use in
Conceivable. [0078] According to the present invention, human BCDFI activity is demonstrated.
Sequence encoding polypeptide and polypeptide thereof
The structure of the part is clarified, using DNA recombination technology
The potential for mass production of human BCDF has been provided. Departure
The recombinant human BCDF obtained by the method described in
As a useful drug for the treatment of epidemics,
Is administered as a pharmaceutical composition with a pharmaceutically acceptable carrier.
It can be administered to a person.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the cloned ph-IL-5-3.
FIG. 2 is a nucleotide sequence diagram of a cDNA containing a human BCDF cDNA gene inserted in 0. FIG. 2 is a view showing a region encoding a human BCDF polypeptide in the nucleotide sequence of FIG. 1 together with the amino acid sequence encoded by the region. FIG. 3 is a diagram for explaining a mature human BCDF polypeptide portion and a leader sequence portion in the amino acid sequence shown in the lower part of FIG. 2 (amino acid sequence of human BCDF precursor). FIG. 4 is a diagram showing a strategy for analyzing the nucleotide sequence of a human BCDF gene and the structure of the gene. FIG. 5 shows the human BCDF chromosome gene (BamH
1 is a diagram showing a part of the entire base sequence of I 3.2 kb fragment). FIG. 6 shows the human BCDF chromosome gene (BamH
1 is a diagram showing a part of the entire base sequence of I 3.2 kb fragment). FIG. 7 shows the human BCDF chromosome gene (BamH
1 is a diagram showing a part of the entire base sequence of I 3.2 kb fragment). FIG. 8 shows the human BCDF chromosome gene (BamH
1 is a diagram showing a part of the entire base sequence of I 3.2 kb fragment). FIG. 9 shows the human BCDF chromosome gene (BamH
1 is a diagram showing a part of the entire base sequence of I 3.2 kb fragment). FIG. 10 shows a cDNA expression vector pdKCR.
-HIL-5 cDNA and pdKCR-hIL-5c
It is a figure which shows the process of DNA-dhfr construction. FIG. 11 shows human BCDF inserted into BamHI and PstI sites of phage M13mp19 DNA.
It is a figure which shows the base sequence of cDNA. FIG. 12 shows a human BCDF expression vector pdK.
CR-hIL-5gene and pdKCR-hIL-
It is a figure which shows the process of 5gene-dhfr construction. FIG. 13 shows the recombinant human BCDF Super.
It is a graph which shows the elution pattern by the gel filtration in anose12HPLC column. FIG. 14 shows reverse phase HPLC of a recombinant human BCDF on a Sensepak VP-304-1251 column.
4 is a graph showing an elution pattern according to Example 1. FIG. 15 shows purified mature recombinant human BCD.
It is a figure which shows the SDS polyacrylamide gel electrophoresis staining pattern of F. FIG. 16 is a diagram showing a deduced two-dimensional structure of the amino acid sequence of the polypeptide portion of the mature recombinant human BCDF monomer.

────────────────────────────────────────────────── ⁷ Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI (C12P 21/02 C12R 1:91) (56) References JP-A-63-146798 (JP, A) Nucleic Acids Res. , Vol. 14, No. 22 (1986) p. 9149-9158 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 21/02 C12N 15/24 CA (STN) REGISTRY (STN) BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. The following amino acid sequence (I): Embedded imageFor producing human B cell differentiation factor or its precursor having
Comprising a DNA encoding the above amino acid sequence.
Culturing host cells transformed with a vector consisting of
And the cultureNourishmentSaid human B-cell differentiation factor or its precursor
A method comprising collecting a body. 2. The DNA has the following base sequence (II): Embedded image The method of claim 1, comprising: 3. The said vector is ph-IL-5-30
Item 3. The method according to Item 1 or 2. 4. The following amino acid sequence (I ') Embedded imageA method for producing a human B cell differentiation factor having
Encoding the amino acid sequence (I '), and
Comprising DNA from a human chromosome containing
Culturing animal cells transformed with the vector,
Of human B cell differentiation factor from culture
To beClaim 1Method. 5. Human B cell differentiation activating factor, the molecular weight of the monomer
5. The method of claim 4, wherein the protein is about 20,000 glycoproteins. 6. Expression vector is pdKCR-hIL-5gene
Or expressed as pdKCR-hIL-5gene-dhfr
The method according to claim 4, wherein the method is performed. 7. Gene for human B cell differentiation activating factor including intron
Has the following formula (III): Embedded imageEmbedded imageEmbedded image (In the formula, the region together with the amino acid sequence indicates an exon.
However, the region in which only the base sequence is shown indicates an intron. )
Characterized by a base sequence represented byRuRequestTerm
4ToThe described method. 8. If the animal cell line is COS-I, CHO or CHO
dhfr ^-A cell represented by the formula:
Or the method of claim 1.