JP2676336B2 - Human B cell differentiation factor gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial applications]   The present invention provides a B cell differentiation factor having activity in humans.
Gene for polypeptide (hereinafter referred to as "BCDF")
It is about. Human BCDF is a substance according to the present invention.
The existence of all
It is a useful substance that can be used as a therapeutic drug. [Conventional technology]   Mature B cells activated by antigen stimulation are
It divides and proliferates with help, but B cells are more
To ultimately differentiate into cells, one or more
It is known that T-cell-derived differentiation-inducing substances are essential.
Have been. The existence of this substance is described by R.W.Dutton et al., Transplan.
t.Rev.twenty three 66 (1975), A. Schimpl and E. Wecker et al., Nature.
N. Biol.237,15 (1972). they
In the culture supernatant after culturing the mouse lymphocyte mixture or
Lymphocyte culture of mice stimulated by hara and mitogen
The supernatant is a lymphoid cell population from which mouse T cells have been removed
Sheep erythrocytes (SRBs) of lymphocytes derived from group and nude mice
Found to amplify a primary immune response against C),
The active substance having such an action is a substitute factor for T lymphocytes.
The child, or TRF, was given. Since then TRF has
Non-antigen non-specific major histocompatibility complex (hereinafter MHC
Abbreviated. ) To B cells in a manner that does not require
Used to induce B cell antibody production without inducing B cell division proliferation
It is defined as a humoral factor that induces differentiation into cells
You.   Thereafter, a function indicating the presence of such a B cell differentiation factor
The above evidence is accumulated, and it is similar to the mouse in humans.
The presence of differentiation factors has been suggested. Currently as mentioned above
The factors that differentiate the B cells defined in 1. into antibody-producing cells
It came to be collectively called BCDF.   In this way, BCDF plays a role in B cell antibody production in the human body.
It plays an important role. The clinical application of BCDF is roughly classified.
There are three possibilities. The first is to make BCDF antibody by BCDF
Using BCDF immunoassay system with DF and anti-BCDF antibody
Immunological analysis of pathological conditions can be used and
For repair of B cell dysfunction that is often found in autoimmune diseases
Can also be used. The second application is for the treatment of various diseases.
You. For example, B cells associated with decreased helper function of T cells
BCDF alone or in patients with immunodeficiency due to decreased cytoplasmic antibody production
For administration with other lymphokines and immunotherapeutic agents
It is considered that the antibody production function is restored to normal.   Furthermore, the following are possible applications of BCDF. B thin
Cell growth factor (BCGF) (K. Yoshizaki et al., J. of Immunol.13
0, 1241 (1983), other T cells containing lymphokines
Normal B cells can be cultured for a long time by adding the factor to the medium.
Have been reported (B. Sredni et al., J. Exp. Med.,1
54, 1500 (1981)). These are normal B cells in culture
When appropriate for B cells transformed with EB virus
Antibody is produced in vitro by the action of BCDF during
I can make it. Specific antibodies, eg pathogenic bacteria, disease
Specific virus on the surface of protovirus, pathogen, cancer cell, etc.
Monoclonal B cells producing antibodies that recognize the original
And transformed with cloned normal B cells or EB virus
Cultivated cells in combination with BCDF and other lymphokines
To produce useful monoclonal antibodies.
come. These antibodies are used for treatment and diagnosis of infectious diseases and cancer
it can.   Conventionally, in order to obtain BCDF, normal normal cells separated from human peripheral blood etc.
BCDF is produced by stimulating human T cells with mitogen.
The method of making has been adopted. With this method, enough T cells
It is difficult to obtain BC due to the use of mitogen.
DF contains harmful mitogens, and it is difficult to remove them.
Difficulties, and for T cell culture, serum such as fetal bovine serum
It is necessary to add ingredients to the medium,
The quality and BCDF cannot be sufficiently separated, and BCDF is used for medical purposes.
To obtain purified BCDF is an obstacle
There are many problems such as points, and BCDF cannot be industrially produced.
Did not. In addition, human T cells are fused with human cancer cells by human fusion.
A method for obtaining fused cells and thereby producing BCDF is also reported.
(Okada et al., J.Exp.Med.,157, 583 (198
3)). However, human fused cells were produced by lymphokines during passage.
Practically BCDF producer fusion
There are no cells yet. [Problems to be solved by the invention]   Therefore, the object of the present invention is to target the polypeptide of human BCDF.
It is to provide the gene that does. Encodes human BCDF
DNA is derived from human BCDF by eukaryotic or prokaryotic cells.
It is indispensable and useful for mass production. [Means for solving the problem]   The present inventors have found that human T cell leukemia virus (hereinafter referred to as “HTL
Human T cells transformed with (V) are highly efficient.
Have already been found to produce BCDF in
Child activity is 5 × 10⁶A protein preparation having a unit / ml or more was obtained.   The human BCDF-producing human T cell line is prepared as follows.
You can Fill from human peripheral blood, tonsils, cord blood, etc.
Lymphocytes by density gradient centrifugation etc. using call pack etc.
, N. Yamamoto, science217Method of 737 (1982)
Transformation of human T cells using HTLV according to
Information). For example, use the following method
Can be. X-ray irradiation (12
1 x 10 cells inactivated at 000-14,000 rads)⁷/ ml and above
1 × 10 human lymphocytes obtained as⁷/ ml 20% FCS, 100
μg / ml kanamycin, 2 μg / ml NaHCO_Three, 25mM N-2-
hydroxyethyl piperazine-N'-2-ethanesulfonic
acid (HEPES) in RPMI1640 medium
Inoculate Kushale (Falcon # 3003) with 5% CO_TwoExistence
Incubate at 37 ° C below. Twice a week, add half the medium to fresh
Limiting after culturing for 2-3 months while changing the medium
Established a stock by the dilution method. Culture of established cells
The BCDF activity of the nutrient supernatant was measured to obtain a strain having BCDF activity.
You.   As cells established by this method, for example, VT-1
Labeled human T cell lines can be used. VT-1
There are no special conditions for growth and it is commonly used.
The culture conditions may be appropriately adopted. Also, BCDF
Can be produced by a general method, but is preferably
It should be done with a medium that does not contain proteins.   For example, as an optimal medium for increasing the number of VT-1 cells,
For example, use a medium supplemented with 1-30%, preferably 20% FCS
I have. FCS is the optimal medium for BCDF production.
The above-mentioned medium without addition may be used. Complete synthesis without FCS addition
Viability of VT-1 remains above 70% even after 48 hours of culture in the medium.
Is held.   In the case of producing BCDF from T cells, conventionally, FCS was added to the medium.
Added protein like or added mitogen
Was essential. (T. Teranishi et al., J. of Im
munol.128, 1093 (1982), A. Muraguchi et al., J. of Immuno.
l.127, 412 (1981)). VT-1 is used for this
When BCDF is produced in this way, serum such as FCS or blood is added to the medium.
Add protein components and other protein components in liquid
It is not necessary and commonly used T cells or B cells
It is worth noting that it is not necessary to add mitogen to the cell
Deserve. Therefore, BCDF can be inexpensively used without using expensive FCS.
Not only can it be produced, but it is harmful to the human body.
Easily create safe BCDFs that are protein and mitogen free
You can get it.   The above method of producing BCDF using VT-1 has various environments.
It is performed under specific conditions. However, preferably a VT-1 culture
Is about 5-10% carbon dioxide in the temperature range of about 35-38 ℃
It should be kept in humidity-controlled air containing. Also ideal
The pH of the medium is about 7.0-7.4, which is slightly alkaline.
Should hold down. VT-1 is a flat bottom microplate
Various types of incubator such as 100μ unit
Inoculated in quantity. Falcon Loveware Divisi
Beckton Dickinson End Corp.
(Falcon Labware, Div.Becton, Dickinson and C
o.) commercially available flask No. 3013 or 3024
Such tissue culture flasks can also be used. Alternatively above
Bottle No. 3 sold by Falcon Loveware
A rotary bottle such as 027 can also be used as a culture container.   Optimal conditions for culturing VT-1 to increase cell number
The initial density of cells is 1 x 10 per ml of medium.^FourCells or
5 × 10^FiveCells, preferably 2 x 10^FiveCells. Article above
When VT-1 is cultivated in a given condition, it usually takes 2 to 7 days per 1 ml of medium
5 × 10^Five2 x 10 from cells⁶Cell density increases up to the level of cells
Therefore, add new medium again and add 1 x 10 per 1 ml of medium.^Four~
5 × 10^FiveReduce cell density to cells and continue culturing again
You. In this way, VT-1
After continuing the culturing, the cells are separated by centrifugation or the like. cell
Wash with a protein-free synthetic medium
Inoculate complete synthetic medium. The initial density of cells at this time was
About 1 x 10 per 1 ml of ground^FourCells or 1 × 10⁷Being a cell
Is preferred, and ideally 1 x 10 per 1 ml of medium⁶In cells
is there.   The amount of BCDF produced by culturing VT-1 is
It changes with time. For example, 1 x 10 per 1 ml⁶At initial cell density
VT-1 was added to RPMI 1640 medium (100 units of penicillin per ml, suspension
100 μg of treptomycin, 10 μg of gentamicin and
And NaHCO_ThreeWhen cultured in 16 mM, the BCDF activity is 48 hours.
The peak level is reached later. Plus, present in the next 24 hours
BCDF activity is slightly reduced. In this way RPMI 1640 culture
Optimal culture time for producing BCDF with VT-1 in the ground is about 24 to 78
Time.   BCDF is used for salting out, vacuum dialysis, ultrafiltration and gel filtration chromatography.
Chromatography, ion exchange chromatography, affini
Tea chromatography, chromatofocusing,
Reversed-phase chromatography, focus electrophoresis and gel electrophoresis
Concentration from the above culture supernatant by various methods such as electrophoresis
Can be purified.   Next, the BCDF activity measurement method is as follows.
You.   Human B cell line CL4 (T.Hi which produces IgM in response to human BCDF
rano et al., Proc.Natl.Acad.Sci.,82, 5490.1985)
BCDF activity is measured. 6 × 10 with test solution to measure BCDF activity
^ThreeRPMI 1640 medium containing 200 μl of 10% FCS (1 ml
100 units of penicillin, streptomycin 100 μg,
Gentamicin 10 μg and NaHCO_Three(Including 16 mM)
You. This mixture was placed in a 96-well microplate for 3 days for 5 days.
% CO_TwoIncubate at 37 ° C in the presence of enzyme to remove the IgM amount of the culture supernatant.
Measure according to the epidemiological measurement method. Maximum Ig in this condition
BCDF activity showing 50% of M production (highest CL4 response)
It was set to 1 U / ml.   In addition, human B cell line CL4 that produces IgM in response to human BCDF
(O.SAIKI et al., Eur.J.Immunol13, 31 (1983))
(BCDF activity can also be measured by the reverse PFC method. BCDF activity
Test solution and 1 x 10^FourCL4 of 200μ 10% FCS
Put in RPMI 1640 medium containing the above (additives are the same as above). This
5% CO in 96 well microplate for 3 days_Two
Cultured at 37 ℃ in the presence of cultured cells, complement, anti-human IgM
Body protein A-bound sheep stored blood was dissolved in HANKS solution
Mix with galose, spread on a petri dish and harden, 37 ℃, 5%
CO_TwoThe number of hemolytic spots after overnight culture in the presence of BCDF
The number of cells differentiated into human IgM-producing cells is measured.   To identify and collect the gene encoding human BCDF
Are VT-1 cells cultured under the optimal conditions for BCDF production described above.
RNA was extracted and collected, and a cDNA library was created.
Clone cDNA encoding BCDF from library
I do. For this reason, the present inventors have shown that VT-
Human BCDF produced by 1 cell was completely purified, and its N-terminal
Amino acid partial sequence and the same BCDF as lysyl endopeptida
The resulting fragment peptide
Determine the amino acid sequence of peptide and correspond to each peptide
To synthesize this oligonucleotide.
Using the otide probe, the cDNA library
Clones that hybridize to several synthetic probes
The BCDF cDNA was identified by collecting the protein. here
The nucleotide sequence of the obtained cDNA is determined by a conventional method and the BCDF is
The substance of the gene to be
The more human BCDF is a polypeptide composed of 184 amino acids.
It was found to be Petit. At the same time got here
Link cDNA to expression vector for expression in eukaryotic cells
After that, the gene is introduced into the same cells and the cells are cultured.
And produce BCDF in the supernatant and purify human BC by purification.
Reconstitutes the DF identified and collected and corresponds to the BCDF gene structure.
Producing Vinant Human BCDF, and this BCDF
The same physicochemical and biological properties as human BCDF obtained from other human cells
It was clarified that it has physical properties. Than this
The identified gene certainly encodes the human BCDF protein.
It was finally proven.   On the other hand, human BCDF can also be produced in prokaryotes. sand
In other words, it is necessary to express the gene encoding human BCDF.
The recombinant DNA obtained by introducing the
Introduce it into the host and culture the resulting transformed microorganism.
No.   The gene encoding BCDF has an amino acid sequence (I), (I
I) or at least the base sequence corresponding to the partial structure
Have Prokaryote into which recombinant DNA is introduced
The host cells are Escherichia coli and Bacillus subtilis.
There are squirrels and other microorganisms, and current genetic engineering production
That can be easily done by those skilled in the field of
It is.   Culture medium for culturing transformed microorganisms (prokaryotes)
The culture method may be an ordinary medium or method.   Accumulation of human BCDF in transformed microbial cells
If so, BCDF will make it easier for the vendor in the field.
It is collected and purified by the method described below. The following is a brief description.
You. After culturing, centrifuge the cells to collect the lysozyme-containing solution.
Suspend the cells in a liquid containing other detergents, and after reaction, freeze.
Repeat cell binding and thawing to collect cell extract,
Affinity using purification method and / or anti-BCDF antibody immobilized column
Conveniently purified by infinity chromatography. Example 1   2 volume plastic roller incubator (Falcon # 30
27) (hereinafter referred to as roller) 1 containing 20% FCS R
PMI1640 medium (2 mM glutamine, 5 x 10^-FiveM 2ME, 100 single
Position / ml penicillin, 100 μg / ml streptomycin, 20 μ
g / ml gentamicin and 16 mM NaHCO_Three2 x)
Ten^Fiveinoculate VT-1 to 8 cells / ml and use 8r roller
The cells were incubated at 37 ° C for 3 days while rotating at pm. After the culture,
Collect the cells by centrifuging the nutrients and use the RPMI1640 medium twice to collect the cells.
After washing the cells, culture the cells in 1 volume of RPMI1640 in a 2-volume roller.
1 × 10 on the ground⁶The cells were suspended at a cell concentration of / ml. roller
Are cultivated at 37 ° C. for 2 days while rotating at 8 rpm. After culture
The culture was centrifuged to obtain a culture supernatant.   In culture containing BCDF obtained by culturing VT-1 as described above
BCDF was purified by Seiko from the following method. Cell-free supernatant 10
Ultrafiltration membrane (Amicon YM-10, Amicon Corp.
, Massachusetts, USA)
Equipment (Amicon mass processing cell 2000 type, Amicon Co
Nitrogen using Poration, Massachusetts, USA)
4 kg / cm depending on gas^TwoWas applied and filtered. Above the filtration membrane
The remaining 100 ml of concentrated solution was added to the ultrafiltration membrane (Amicon Y
Ultrafiltration device equipped with M-10) (Amicon, Stander
Type cell 52 type) and 4 kg / cm with nitrogen gas^TwoPressure of
And filtered. 5 ml of concentrated liquid remaining on the top of the filtration membrane
It was collected.   The concentrated supernatant described above was transferred to an AcA-34 gel filtration column (LKB P
roduker.Sweden, 2.6 × 90 cm). The gel
The filtration column is pre-assembled with PBS (phosphate buffer).
0.01M phosphate containing 0.15M sodium chloride
Equilibrated with buffer, pH 7.0). Dissolve the concentrated supernatant with PBS
Dispense and collect 5 ml of the eluate, and measure the BCDF activity of the collected liquid.
Specified. Fractions with BCDF activity have a molecular weight of 3.5 ± 10^FourDalt
Found to be contained in the fraction corresponding to
Was. The gel filtration column is shown below in Pharmacia Phi
Molecular weight marker manufactured by Chemicals (Sweden)
Tested. That is, Blue Dextran 2000 2 × 10⁶,
Ferritin 4.5 × 10^Five, Aldolase 1.58 × 10^Five, Of a
Lubumin 4.5 × 10^Four, Chymotrypsinogen 2.5 × 10^Fouras well as
Cytochrome C1.17 × 10^Four. In addition, the fraction including BCDF
Of the ultrafiltration membrane (Amicon YM-10)
Using an external filtration device, 25 mM piperazine-hydrochloric acid buffer solution (pH 6.
Replaced with 3).   BCDF fractionated by AcA-34 column chromatography
The fraction was preliminarily 25 mM piperazine-hydrochloric acid buffer solution (pH 6.
3) Equilibrated with Mono P column (Pharmacia phi)
Chemicals, Sweden). This column
After washing with 25 mM piperazine-hydrochloric acid buffer, pH 4.5 with hydrochloric acid
40 ml of 1/10 diluted polybuffer 74 (Pharma
(Shea Fine Chemicals, Sweden)
Was. Column operation is fast protein liquid
Chromatography, FPLC (Pharmacia Fineke
Flow rate is 0.5 ml / min.
Done. Collect 1 ml of the eluate and measure the BCDF activity and pH.
Specified. BCDF activity was eluted at pH 4.9-5.1.   The BCDF active fraction obtained from the Mono P column was added to 0.1% TFA
Reversed-phase chromatograph buffered with aqueous fluoroacetic acid)
Column for Synchropak RPP (C₁₈) (250 x 4, 1 mm, S
ynchrom) or high performance liquid chromatography?
Eluent 0.1% TFA acetonitrile concentration 0 to 60%
BCDF was eluted by increasing linearly up to. Acetonitrile
Elute at 50-55%. O.D.₂₈₀The peak of other O.D.₂₈₀
Is completely separated from the peak of
BCDF activity was detected. Lyophilize this peak and BC
I got a DF standard.   This preparation was subjected to SDS-polyacrylamide gel under reducing conditions.
(12% gel) electrophoresis was performed. Phase of 21,000
Cut out the gel section to hit, and in the Eppendorf tube
0.05% SDS, 10mM NH_FourHCO_ThreeExtract by shaking at 37 ° C overnight
Was.   This eluate is directly again applied to the column Sy for reverse phase chromatography.
nchropak RP-P (C₁₈) (250 × 4, 1mm, Synchrom)
Subjected to HPLC used and, after elution, acetonitrile in 0.1% TFA
Elute BCDF by increasing the concentration linearly from 0 to 60%
Was.   O.D.eluted with 50-55% acetonitrile.₂₈₀The pea
Ku is another O.D.₂₈₀It is completely separated from the peak,
BCDF activity was detected corresponding to the peak. This peak
Lyophilized to obtain purified BCDF.   As described above to determine the amino acid sequence of the BCDF protein.
6 μg of purified BCDF obtained in
Enser (Applied Biosystem Co., Calf, Model470A)
Introduced. The method for determining the amino acid sequence is described in J. Biol. Chem.,19
Three, 265-275 (1951)
Was.   The amino acid sequence from the N-terminus was as follows. Pro Val Pro Pro Gly Glu Asp Ser Lys Asp
Val Ala Ala Example 2   Purified BCDF preparation 20 prepared by the method described in Example 1
μg was dissolved in 5 mM Tris-HCl, pH 9.5 buffer and reconstituted.
Lysyl Endopeptidase (Japanese)
Light) (Lysyl endopeptidase: BCDF standard = 1: 200, mol
Ratio) and react at 37 ° C for 6 hours to add BCDF to fragmen
Was converted to The reaction mixture is a column for reverse-phase chromatography μ
Elute by applying HPLC using Bondo Pack (0.21 x 30 cm)
Solution, 0.06% TFA in acetonitrile concentration from 0 to 60%.
The fragments were separated and eluted in a linear increase with. This
As a result, HPLC elution peaks 1 to 9 were obtained. Of each peak part
Lyophilize the eluate and use a protein sequencer (Ap
plied Biosystem Co., Calf, Model 4704). A
The determination of the mino acid sequence is performed by J. Biol. Chem.,193, 265 ~ 275 (195
It was performed by the method described in 1).   Fragments with confirmed amino acid sequences and amino acid sequences
List the columns. Fragment 3 Lys-Glu-Ala-Leu-Ala-Glu Fragment 8 Lys-Leu-X-Ala-Gln-Asn-Gln-Trp-Leu-Gln-
Y-Met Fragment 2 Pro-Val-Pro-Pro-Gly-Glu-XY-Lys Fragment 6 Asp-Val-Ala-Ala-Pro-X   In addition, X and Y are amino acids that could not be identified   Fragments 2 and 6 correspond to the N-terminal sequence of Example 1.
Was. Example 3   Encodes the amino acid sequence of BCDF obtained in Examples 1 and 2.
The oligonucleotides were synthesized as follows.   Oligonucleotide synthesis is based on an applied biosystem.
Silica using a DNA synthesizer model 380A manufactured by
Using the gel as the solid phase carrier and the phosphite triester method
Nucleotide binding reaction was performed. The protecting group is removed by the usual method.
C after leaving₁₈Reversed-phase column HPLC
Use the gradient to purify the desired oligonucleotide
Was. Example 4 (A) 2-volume plastic roller incubator (Falco
# 3027) (1) 20% FC in (hereinafter referred to as roller)
RPMI 1640 medium containing S (2 mM glutamine, 5 x 10^-FiveM 2ME,
100 units / ml penicillin, 100 μg / ml streptomyces
20 μg / ml gentamicin, 16 mM NaHCO_ThreeContaining)
2 × 10^FiveInoculate VT-1 to the number of cells / ml and rotate at 8 rpm.
The cells were cultured for 3 days at 37 ° C. After culturing, centrifuge the culture
Cells were collected and washed twice with PBS, then cells (1.8 x 10
⁹Cells) in 800 ml of PBS solution and centrifuge the cells
Ribonucl, a nuclease inhibitor, after washing twice
RSB solution containing eosides-Vanadyl complex (10 mM) (1
0 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1.5 mM MgCl_Two) 800 ml
Suspended in water. Next, NP-40 was added to 0.05%.
After gently stirring, centrifuge at 3000 rpm for 5 minutes to contain the nucleus
Remove cell debris and add SDS (final concentration 0.5
%) And EDTA (final concentration 5 mM), and immediately after
And the cytoplasmic RNA was extracted. Total 3 times
Repeat the extraction with Nol and then RNA with 2 volumes of ethanol.
Precipitate, collect this precipitate by centrifugation, and use 10 mM Tris-HCl, pH 7.5.
And dissolved. RN thus obtained from VT-1 cells
The amount of A was 30 mg.   Next, oligo (dT) is used to obtain mRNA from this RNA.
-Using cellulose (P.L.Biochemicals, Type7),
Chromatography was performed. Adsorption is 20 mM Tris-H
RNA in Cl, pH 7.5, 0.5 M NaCl, 1 mM EDTA, 0.5% SDS solution
Dissolve the solution in a buffer (20 mM Tris-HCl, pH 7.5, 0.5
M NaCl, 1 mM EDTA), and then eluate water and 10 mM Tris-H
Performed by alternating elution of mRNA with Cl (pH 7.5)
Was. As a result, the amount of mRNA eluted was 576 μg. (B) Double-stranded cDN using 5 μg of mRNA prepared in (a)
A was produced. GUBLER, U and HOFFMAN, B, J., (Genetwenty five, 263,1
983), cDNA synthesis kit (Amersham)
Double-stranded cDNA was prepared using the Amersham protocol.
Made. That is, single transcripts from mRNA rather than reverse transcriptase
Synthesize strand cDNA to create a hybrid of mRNA and cDNA
RNA using E. coli ribonuclease H as a substrate
Formed Nick and Gabb on the chain. In addition, E. coli DNA poly
Nick translation type by Melase I
By replacing the mRNA with DNA to prepare double-stranded DNA
Was. The small overhang at this 3'end is T4DNA
It was removed using a polymerase to make double-stranded cDNA.
The double-stranded cDNA finally obtained was 1.08 μg. (C) 1.08 μg of the obtained double-stranded cDNA was added to the sucrose gradient.
Heart method (in a solution containing 50 mM Tris-HCl, 1 mM EDTA, pH 7.5
Sucrose density gradient 5-25%, 40,000 rpm, 4 ° C for 13 hours)
Further fractionation, and a part of it was subjected to agarose gel electrophoresis.
Analysis of the double-stranded cDNA
Collects fractions of 500 bp or more by ethanol precipitation method
did. The recovered double-stranded cDNA was about 0.6 μg. (D) 0.1M potassium cacodylate (pH adjusted to 7.
2) 10mM DTT, 2mM CoCl_Two, 0.5mM³²P-dCTP
(Specific activity 1 × 10⁶cpm / n mole), 0.6 μg double-stranded cDNA and
And 50 units of deoxynucleotidyl terminal trans
Mix ferase (BRL) and incubate at 24 ℃ for 20 minutes.
Sephadex G after treatment with phenol
Collect the cDNA fractions through a -50 column and
As a result, 0.24 μg of dC-tailed cDNA was obtained. This cDNA
Had about 13 dCMP residues added at both 3'ends. (E) On the other hand, as shown in Fig. 1, monkey cells (COS cells
CDNA expression vector pQ in pCEIL-2 (Nature,302,
305, (1983)). pQ directs cDNA to SV40 in both directions
Which has a cDNA in the early promoter and which has a cDNA
Pep encoded by cDNA in COS cells even if inserted in the opposite direction
Tide protein can be expressed. Also in E.coli
Can also be replicated and selected for tetracycline resistance.
Can be.   This pQ was cut with Pst I and the 3'end of the ds-cDNA
Just like putting dC tail on both ends, dG tail 13
Around one piece was given.   Next, this dG-tailed pQ100ng and dC-tailed ds-cDNA2
Dissolve 0 ng of 50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 1 mM EDTA.
Mix in the liquid, first at 65 ° C for 2 minutes, then at 45 ° C for 60 minutes,
Incubate for 60 minutes at 37 ° C and 60 minutes at room temperature
Was. And make this annealed DNA competent
It was introduced into E. coli MC1061. Next, MC1061 competent
The method of making cells and the method of introduction are shown below.   E. coli MC1061 was added to 100 ml of Ψ medium (2% tryptone, 0.
5% yeast extract, 0.5% MgSO_Four・ 7H_TwoO, pH 7.6),
37 ℃ until the absorbance of the culture solution is around 0.3-0.5 at 550nm
The culture was performed with shaking. After culturing, the culture solution is kept at 0 ℃ for 5 minutes.
Retain, collect cells by centrifugation, and remove Tfb I (30 mM acetic acid
Potassium, 100 mM RbCl, 10 mM CaCl_Two, 50mM MnCl_Two, 15%
Suspend in 40 ml of lyserine, pH 5.8) and let stand at 0 ℃ for 5 minutes
Was.   The cells were collected again by centrifugation and the Tfb II (10 mM MOPS
or PIPES, 75mM CaCl_Two, 10 mM RbCl, 10% glycerin, pH 6.
It was suspended in 40 ml of 5) and allowed to stand at 0 ° C for 15 minutes. This suspension
The liquid was dispensed and stored at -70 ° C.   Next, 100 μ of the competent cells prepared in this way
For 15 minutes at 0 ° C, in which dG-tailed pQ
10 μm standard prepared by annealing vector and dC-tailed cDNA
And 50 mM MgCl_Two, 10mM CaCl_Two90 μ of the solution of
Let stand for 20 minutes at 0 ° C. Then, after heat-treating at 37 ℃ for 60 seconds,
Hold at 0 ° C for 1-2 minutes, add 1 ml of Ψ medium,
The cells were cultured at 7 ° C for 60 minutes with shaking. 15 μg / ml of this culture
Contains tetracycline and 25 μg / ml streptomycin
Mu L medium (1% tryptone, 0.5% yeast extract, 0.5% Na
Cl) on agar plate and incubate at 37 ℃ overnight.
A colony appeared. (F) Paired with the obtained transformants of approximately 150,000 clones
Then, using probes 8-1 and 8-2, Grunstein, M
Et al. In Methods in Engymology, 68,379 (1979).
When knee hybridization was performed, it was 8-1.
Ten clones were found to hybridize. further
Probes 3-1 to 3-6 against these 10 clones
Colony hybridization in a similar manner using
Which hybridizes with probe 3-2 when
One clone was obtained. Plasmid DN carried by this clone
A is roughly extracted and purified, cleaved with restriction enzyme Pst I, and
The product was separated from the pQ vector by agarose gel electrophoresis.   Probe 8-1, probe 3-2, probe N-2 or
Southern hybridization using probe N-5
As a result of analysis, it hybridized with both probes.
I did Does not hybridize with other probes
Was.   This plasmid DNA was named pBSF2-38. Ie
The cDNA insert of this pBSF2-38 is a protein with BCDF activity.
Gene corresponding to the revealed partial amino acid sequence in white
It is clear that the cDNA has the sequence
Was identified as Example 5 (B) To prepare a large amount of pBSF2-38 plasmid DNA
MC1061 containing pBSF2-38 was added to 20 μg / ml tetrasaline.
Ψ medium containing Irin and 25 μg / ml sprepomycin 10
0 ml was inoculated and cultured with shaking at 37 ° C for 5 to 7 hours. Next
Chloramphenicol was added to a final concentration of 170 μg / ml.
Add 100 ml of new Ψ medium, and cultivate with shaking overnight.
Was.   The plasmid DNA amplified in this way was
It was purified as   Collect only the bacterial cells by centrifuging the culture solution, and add 50 mM Tris-H
Suspend in 5 ml of Cl, pH 7.5, freeze at -80 ° C, thaw and then
Add lysozyme (final concentration, 2mg / ml) for 10 minutes at 0 ℃
Allow to stand, add EDTA (final concentration 0.1M), and add 10 ℃ at 0 ℃.
Let stand for a minute. Then Triton X-100 (final concentration 0.1
%) And then left to stand at 0 ° C. for 60 minutes. Then 30,000 rp
Centrifuge for 30 minutes at room temperature and add an equal volume of water saturated
Treat with knoll. The water layer is added to an equal volume of chloroform.
And then extract the aqueous layer with a final concentration of 20 μg /
Add RNase so that the amount becomes ml, and incubate at 37 ° C for 60 minutes.
I did it.   Then 0.2 volume of 5M NaCl and 1/3 volume of polyethylene glycol
And left at 0 ° C for 60 minutes, 10,000 rpm for 20 minutes
During that time, the DNA precipitate is collected by centrifugation.   Then dissolve the precipitate in 3.8 ml of water and add 4 g of CsCl.
After dissolution, add 200 μl of 10 mg / ml EtBr to 40,000 rp
Perform ultracentrifugation fractionation at 20 ° C for 16 hours.   After centrifugation, extract the plasma DNA fraction and saturate with water.
Remove EtBr by extracting 4 times with 1-2 volumes of butanol.
Good. Then H_TwoAfter dialysis in O to remove CsCl, 3M acetic acid
Add 1/10 volume of soda pH 5.6 and add 2 volumes of cold ethanol
And then leave it overnight at -20 ° C. This ethanol precipitation
After collecting the cells by centrifugation and washing with 80% ethanol aqueous solution,
Dry well and precipitate this precipitate with 50 mM 10 mM Tris-HCl, pH 7.5.
Lysed in μ and used for monkey cell transfection.
Sampled. (B) Method for infecting monkey COS-7 cells with plasmid   1 x 10 COS-7 cells^Five10% fetal bovine serum so that it becomes / ml
Suspend in RPMI containing 3 ml of this 5% charcoal in a 6 cm petri dish.
The cells were cultured overnight at 37 ° C in an acid gas incubator. On culture
Clean and remove, and add 3 ml of new RPMI containing 10% fetal bovine serum.
Eat, cultivate for 2 hours at 37 ℃, 5% carbon dioxide incubator
Nourished. After culturing, the supernatant was removed and TBS (25 mM Tris-HC
l, pH 7.5, 130 mM NaCl, 5 mM KCl, 0.6 mM Na_TwoHPO_Four) 2.5m
It was washed once with l.   Plasmid mixture (TBS (+) (0.7 mM CaCl in TBS_Two,
0.5 mM MgCl_Two1.0 ml, plasmid DNA 2 μg
And add 10mg / ml DEAE-dextran 50μ, 37 ℃, 5
% Incubation for 4 hours in carbon dioxide incubator
After removing the supernatant and supernatant, wash and remove with TBS 2.5 ml, and remove 150 μM
2.5 ml of RPMI containing 10% fetal calf serum containing roroquine was added. Three
Ink for 5 hours in a 5% carbon dioxide incubator at 7 ℃
After the incubation, the supernatant was removed and washed twice with 2.5 ml of TBS. Ten
3% RPMI containing 5% fetal bovine serum was added at 37 ° C, 5% carbon dioxide
Cultured overnight in an incubator. Same RP after removing the supernatant
Add 3 ml of MI and in a 37 ° C 5% carbon dioxide incubator
Cultured for 2 days. And after centrifuging this culture supernatant,
The sample was used for BCDF activity measurement.   The results of measuring BCDF activity of COS culture supernatant using CL4
It is shown in FIGS. 2 and 3. Introduces pBSF2-38 plasmid DNA
The injected COS showed a clear BCDF activity as compared with the control.   Thus, the reconstituted cells produced by the eukaryotic cell COS-7
Binant human BCDF is a culture solution that is loaded with anti-BCDF antibody-immobilized column.
Reverse phase HPLC (Synchron C₁₈)
Refined. This BCDF has N-glycosylase in the cDNA structure.
It contains sugar chains due to the presence of
The physicochemical properties of the VT-1 culture supernatant were determined by the method of Example 1.
In agreement with that of the more purified purified BCDF. Ie Molecular weight: 3.5 ± 0.5 × 10^FourDalton (gel filtration method) 2.2 ± 0.2 x 10^FourDalton (SDS-Polyacrylamide Electric
Electrophoresis method) Isoelectric point: pH 4.9 to 5.1 Met. Example 6   Restriction fermentation from pBSF2-38 prepared according to Example 5 (a)
The BCDF cDNA insert cut out with elementary BamH I was used as a probe.
BCDF mRNA size, molecular species, BCDF mRNA producing strains
Done. The mRNA used was VT-1 cells that produce this BCDF.
First of all, CESS and RPM which are thought to produce BCDF.
CL-4, Jurkat, CEM cells with no I1788 and BCDF activity
Prepared in the same manner as in Example 4 (a).
You.   MRNA 10μg / 3.6μ, 5 × MOPS buffer (0.1M MOPS)
(PH7.0) 75mM NaOAc, 5mM EDTA) 6.0μ, formal
Dehydrate 5.4μ and formamide 15.0μ at 60 ℃ for 15 minutes
Incubate for 10 min.
80% containing Nol Blue and 0.05% xylene cyanol
Glycerol solution) 3 μm was used as the adjustment sample
did. This sample is 1 x MOPS buffer, 1.8% formaldehyde
Electrophoresis on a 1.6% agarose gel containing dehydration
After that, blot the nitrocellulose filter by a conventional method.
I did it. Then bake this filter at 80 ℃ for 3 hours.
Was. The filter prepared in this way is used in 3 x SS containing 0.1% SDS.
After soaking in C, 1 x Denhardt's solution, 50% formamide,
5 × SSC, 250 mM sodium phosphorus containing 250 μg / ml herring DNA
Prehybridize overnight at 42 ° C in acid buffer (pH 6.5)
did. And 1 x Denhardt's solution, 50% formami
5 × SSC, 250 μg / ml herring DNA-containing 50 mM sodium
In phosphate buffer (pH 6.5)³²P labeling_PBSF2-38 B
Hive at 42 ℃ overnight using amH I cDNA insert as a probe.
It was lysed.   Hybridized filter contains 0.2% SDS at room temperature
5 times 4 times with 2 x SSC, 0.1 x SSC containing 0.2% SDS at 50 ° C
At room temperature, wash twice for 30 minutes, air dry, and then autoradiogram
Created.   As a result, VT-1, CESSBCDF producing strain), RPMI1788 origin
CDNA of pBSF2-38 hybridizes to the mRNA of
did. CL-4, Jurkat, CEM, which seems not to produce BCDF,
And does not hybridize to mRNA from BCESS non-producing CESS
won. It also hybridizes with the cDNA probe of pBSF2-38.
It was estimated that the size of the mRNA to be transferred was about 15-16S. 1 copy
As shown in FIG. Example 7   From MC1061 holding pBSF2-38 to (a) of Example 5.
Obtain plasmid DNA as indicated and cut with the restriction enzyme BamHI.
The BCDF cDNA was prepared by exporting. And the restriction enzyme
The map and base sequence were determined. Base sequence is Maxam-Gilbert
Chemistry of (Meth.Enzym.65, 499 ('80) and M13 fur
J (Messing et al., Gene, 19,269 ('82)) was used
Dideoxynucleotide chain assembly method (F. Sanger et al., Pr
oc.Natl.Acad.Sci.U.S.A.74,By the method of 5463 ('77)
Decided. Show the determined nucleotide sequence and restriction map.
It is shown in FIG.   The human BCDF nucleotide sequence determined here was opened in Examples 1 and 2.
All of the amino acid subsequence structures shown are included exactly.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a method for constructing recombinant DNA for expression in monkey cells. Second
The figure shows the BCDF activity of the culture supernatant of monkey cell COS-7 into which pBSF2-38 DNA was introduced, which was measured by Elisa method. FIG.
BCDF activity of the culture supernatant of monkey cell COS-7 into which the pBSF2-38 cDNA was introduced, which was measured by the reverse plaque method. FIG. 4 is a Northern blotting analysis using the pBSF2-38 cDNA insert as a probe. Figure 5 shows the nucleotide sequence of BCDF. FIG. 6 shows a restriction map of BCDF.

Continuation of front page    (72) Inventor Yoshiyuki Takahara               1-1, Suzuki-cho, Kawasaki-ku, Kawasaki Ajinomoto Co., Inc.               Company Central Research Institute

Claims (1)

(57) [Claims] A human B cell differentiation factor gene having a base sequence corresponding to the following amino acid sequence (II). Amino acid sequence (II): PRO VAL PRO PRO GLY GLU ASP SER LYS ASP VAL ALA AL
A PRO HIS ARG GLN PRO LEU THR SER SER GLU ARG ILE
ASP LYS GLN ILE ARG TYR ILE LEU ASP GLY ILE SER AL
A LEU ARG LYS GLU THR CYS ASN LYS SER ASN MET CYS
GLU SER SER LYS GLU ALA LEU ALA GLU ASN ASN LEU AS
N LEU PRO LYS MET ALA GLU LYS ASP GLY CYS PHE GLN
SER GLY PHE ASN GLU GLU THR CYS LEU VAL LYS ILE IL
E THR GLY LEU LEU GLU PHE GLU VAL TYR LEU GLU TYR
LEU GLN ASN ARG PHE GLU SER SER GLU GLU GLN ALA AR
G ALA VAL GLN MET SER THR LYS VAL LEU ILE GLN PHE
LEU GLN LYS LYS ALA LYS ASN LEU ASP ALA ILE THR TH
R PRO ASP PRO THR THR ASN ALA SER LEU LEU THR LYS
LEU GLN ALA GLN ASN GLN TRP LEU GLN ASP MET THR TH
R HIS LEU ILE LEU ARG SER PHE LYS GLU PHE LEU GLN
SER SER LEU ARG ALA LEU ARG GLN MET 2. The gene according to claim 1, which is represented by the following nucleotide sequence. CCA GTA CCC CCA GGA GAA GAT TCC AAA GAT GTA GCC GC
C CCA CAC AGA CAG CCA CTC ACC TCT TCA GAA CGA ATT
GAC AAA CAA ATT CGG TAC ATC CTC GAC GGC ATC TCA GC
C CTG AGA AAG GAG ACA TGT AAC AAG AGT AAC ATG TGT
GAA AGC AGC AAA GAG GCA CTG GCA GAA AAC AAC CTG AA
C CTT CCA AAG ATG GCT GAA AAA GAT GGA TGC TTC CAA
TCT GGA TTC AAT GAG GAG ACT TGC CTG GTG AAA ATC AT
C ACT GGT CTT TTG GAG TTT GAG GTA TAC CTA GAG TAC
CTC CAG AAC AGA TTT GAG AGT AGT GAG GAA CAA GCC AG
A GCT GTG CAG ATG AGT ACA AAA GTC CTG ATC CAG TTC,
CTG CAG AAA AAG GCA AAG AAT CTA GAT GCA ATA ACC AC
C CCT GAC CCA ACC ACA AAT GCC AGC CTG CTG ACG AAG
CTG CAG GCA CAG AAC CAG TGG CTG CAG GAC ATG ACA AC
T CAT CTC ATT CTG CGC AGC TTT AAG GAG TTC CTG CAG
TCC AGC CTG AGG GCT CTT CGG CAA ATG