JP2676341B2 - Method for producing B cell differentiation factor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial applications]   This invention is a B cell differentiation factor having activity in humans.
(Hereinafter referred to as BCDF) gene
Production of human BCDF using prokaryotes such as Escherichia coli bacteria
Regarding construction method. Human BCDF according to the present invention is associated with immunodeficiency.
As a general therapeutic drug for diseases such as cancer, autoimmune diseases, etc.
It is a useful substance that can be used. [Conventional technology]   Soluble with biological and pharmacological activity produced by lymphocytes
Of Lymphokine, a Proteinaceous Factor, in Humans
It has the effect of regulating the immune response mechanism of the body. Re
Nphokines are more common than their immunologically active nature.
Antitumor agent, antiviral agent, antibacterial agent, immunodeficiency therapeutic agent, self
Expected to be useful as a therapeutic agent for autoimmune diseases (Ad
v. in Immunopharm. 507, 1980). The BCDF according to the present invention is
It is a type of nhokine, and from such a viewpoint
Expected to be used.   By the way, mature B cells that have been activated by the antigen stimulation are
It divides and proliferates with the help of cells, but B cells are antibodies.
To finally differentiate into a production cell, one or more
The above T cell-derived differentiation-inducing substances are essential
It has been known. The existence of this substance is described by R.W.Dutton et al., Tran.
splant.Rev.twenty three, 66 (1975), A. Schimpl and E. Wecker et al., Na
ture New Biology237, 15 (1972), revealed
Was. They are in the culture supernatant after culturing a mixture of mouse lymphocytes
Alternatively, mice stimulated with antigen or mitogens
Lymphoid depleted of mouse T cells
Sheep red blood of lymphocytes derived from lymphocytes and nude mice
Seen to amplify primary immune response against spheres (SRBC)
And the T lymphocytes are added to the active substance having such an action.
The substitute factor, or TRF, was given. Since then TRF
Is a non-antigen-specific major histocompatibility complex (hereinafter,
Abbreviated as MHC. B cells in a manner that does not require
B-cell antibodies that act on B cells and do not induce B-cell division amplification
Is defined as a humoral factor that induces differentiation into producer cells
ing.   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.   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.   In addition, human T-cell leukocyte virus (hereinafter referred to as "HTLV")
Method for producing BCDF by human T cells transformed with
It has also been reported (Japanese Patent Application No. 59-235199, 60-25301).   However, at this time, Escherichia coli bacteria
BCDF production method by prokaryotes such as
DNA in a prokaryotic vector
Produces BCDFs in prokaryotes after replication, transcription, and translation in
And the BCDF produced by this method
Not reported. [Problems to be solved by the invention]   Therefore, the object of the present invention is to obtain a DNA encoding human BCDF.
Humans with prokaryotic organisms such as Escherichia coli
A method for producing a polypeptide having BCDF activity is provided.
Things. [Means for solving the problem]   The inventors of the present invention conducted extensive research in order to solve the above problems.
As a result of envy, it encodes a polypeptide having human BCDF activity
Gene and a vector capable of replicating in a prokaryotic cell
Prokaryotes transformed with recombinant DNA consisting of DNA
Culture the cells in culture medium and collect the produced human BCDF.
As a result, the present invention has been completely accomplished. The following
Ming will be described in detail.   By the way, the present inventors have found that HTLV (human T cell leukocyte virus)
Which is one of the human T cells transformed by VT-1 cells.
Note that the cell (IFO 50096) produces large amounts of human BCDF
By looking at the gene encoding human BCDF,
Have been identified (Japanese Patent Application No. 61-184858).   In addition, as a reminder, the coding of human BCDF is described in Examples 1 to 7.
Details of the gene cloning are given. Well, human BC
Regarding the regulation of prokaryote that produces DF, first of all, human BCDF
Gene that encodes a gene capable of replicating in prokaryotic cells.
Incorporate into DNA.   At this time, the DNA encoding human BCDF
Insert it downstream of the promoter sequence or
The DNA fragment containing the vector sequence before insertion of the cDNA in the expression vector.
Alternatively, later, and upstream of the DNA encoding human BCDF
You can insert it in. In this case, the cD encoding human BCDF
NA is a group consisting of 212 amino acids as shown in Fig. 5.
It encodes a polypeptide, but contains 28
The N-terminal region corresponding to the mino acid is extremely hydrophobic,
It is called a signal sequence and is cleaved during the secretory process.
You. Therefore, from the 184 amino acids starting from the N-terminal PRO
CDNA fragment encoding mature human BCDF polypeptide
It is desirable to express it.   The method of integration is to cut the vector with an appropriate restriction enzyme.
And, if necessary, a suitable linker or anneal is possible
Polymerize multiple chlorines in combination. Processed like this
Mixed double-stranded DNA and vector DNA and ligase
Connect it.   The recombinant DNA thus obtained is used as a prokaryotic host.
Human BCDF production from the transformed microorganisms obtained by introducing
Just select the stock.   In the present invention, as a prokaryote, S.
Li, Bacillus subtilis, etc. can be used,
Preferably, Escherichia coli is used.   In addition, Escherichia coli which can be used in the present invention
EK type plasmid vector (ST
Linsen type: pSC101, pRK353, pRK646, pRK248, pDF41 etc.), E
K type plasmid vector (relaxed type: ColE
1, pVH51, pAC105, RSK2124, pCR1, pMB9, pBR313, pBR322, pBR
324, pBR325, pBR327, pBR328, pKY2289, pKY2700, pKN80, pKC
7, pKB158, pMK2004, pACYC1, pACYC184, dul etc.). λgt Thailand
Phage vector: λgt.λc.λgt.λB, λWES, λC, λ
WES, λB, λZJ vir., ΛB ′, λALO, λB, λWES.Ts622, λD
am etc. are included.   As a promoter, trp, lac, tac, tufB, β-lac
Tamase, lpp promoter and other Escherichia
· All promoters that work in E. coli are available
is there.   Transformation of host cells with recombinant DNA usually involves
The following methods are commonly used. Of Escherichia coli
If a prokaryote such as
The competent cells that can be generated are those that are transformed in the logarithmic growth phase.
After collection, well-known CaCl_TwoCan be transformed by the method
You. MgCl in the transformation reaction solution_TwoOr if RbCl coexists
Transformation efficiency is improved. Also the host cell protoplus
It is also possible to transform after preparation.   Incorporation of the human BCDF gene thus obtained
Recombinant prokaryotic cells are routinely cultured.
I just need.   The BCDF thus obtained was subjected to salting out, vacuum dialysis, and ultrafiltration.
Filtration, gel filtration chromatography, ion exchange chromatography
Chromatography, affinity chromatography,
Romatofocusing, reverse phase chromatography, focus
Culture by various methods such as electrophoresis and gel electrophoresis
It can be concentrated and purified from the culture supernatant.   Human BCDF accumulated in the transformed microbial cells.
If so, BCDF will be
It is recovered and purified by a possible method. The short description is as follows
It is. After culturing, collect the cells by centrifugation and include lysozyme
Suspend cells in a solution or other detergent-containing solution and react
After that, freeze and thaw are repeated and the cell extract is collected.
Using the above-mentioned purification method and / or an anti-BCDF antibody-immobilized column
It is easily purified by affinity chromatography.   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, 100 μg of streptomycin,
Gentamicin 10g μg and NaHCO_Three(Including 16 mM)
It is. This mixture was placed in a 96-well microplate for 3 days,
5% CO_TwoIncubate at 37 ℃ in the presence of
It is measured by an immunoassay method. Maximum in this condition
BCDF activity showing 50% of IgM production (highest CL4 response)
Was set as lU / 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 to be measured and 1 x 10^FourEach CL4 contains 200μ of 10% FCS.
Put in RPMI 1640 medium (additives are the same as above). This
5% CO in 96 well microplate for 3 days_Two
Incubate at 37 ° C in the presence. Cultured cells, complement, anti-human IgM
Antibody protein A-bound sheep's stored blood was dissolved in HANKS solution
Mix with agarose, spread on a petri dish and harden, and keep at 37 ℃ for 5
% CO_TwoThe number of hemolytic plaques after overnight culture in the presence of BCDF
The number of cells differentiated into human IgM-producing cells by the action is measured.   By the way, the gene for eukaryote is human interferon.
It is known to show polymorphism as is known for genes
(Taniguchi et al., Gene.Ten, 11〜15 (1980), Ohno, Tani
Mouth, Proc.Natl.Acad.Sci.USA,77, 5305 ~ 5309 (1981), Gr
ay et al, Nature,295, 501-508 (1981). This polymorphic phenomenon
Replaces some of the amino acids in the protein product
In some cases, even if there is a change in the base sequence, it is completely different
It may not be. Therefore, the human BCDF activity is also used in the present invention.
As long as it has sex, it starts from Pro at the 29th position in FIG.
One or more amino acids in the amino acid sequence is 1
Polypeptides substituted with one or more amino acids
And DNA encoding this polypeptide are also included in the present invention.
Is done. Furthermore, as shown in the examples, human BCDF activity
A polypeptide lacking one or more amino acids
Rui is a po to which one or more amino acids have been added.
Lipeptide (however, Ala is located upstream of the Pro at position 29 in Fig. 5)
(Except when adding
Polypeptides (including amino acid substitutions) and these
DNA with a nucleotide sequence encoding a polypeptide of
Embraced. Polypeptide function as human BCDF
Even if there are modified regions with additional amino acid sequences that block
Book if the newly added area can be easily removed
It can be used as the polypeptide and gene of the invention. Toes
And a polypeptide having human BCDF activity according to the invention.
It is human BCDF. (Effect)   The clinical application of BCDF manufactured in this way 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
It can be used for analysis of immunological pathology and
For repairing B cell dysfunction that is often seen in immune diseases
Can 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 antibody production
Given with other lymphokines and immunotherapeutic agents
It is considered that the antibody-producing function is restored to normal.   Focusing on the differentiation activity of BCDF, the malignant tumor cell line was
Anti-inflammatory by differentiating with DF and causing growth inhibition
It can also be used as a sex tumor agent.   Furthermore, the following are possible applications of BCDF. B thin
Cell growth factor (BCDF) (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
Cultured cells in combination with BCDF and other lymphokines
Nourishing and acidifying useful monoclonal antibodies.
come. These antibodies can be used for infectious diseases, cancer and diagnosis
You.   Thus BCDF is a very broadly effective substance.   The present invention will be specifically described below with reference to examples. 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 2 ME, 100 units
/ ml penicillin, 100 μg / ml streptomycin, 20 μg /
ml gentamicin and 16 mM NaHCO3), 2 x 10^Five
Contact VT-1 so that the number of cells per ml becomes 8 rpm.
The cells were incubated at 37 ° C for 3 days while rotating at 37 ° C. After culture, culture
The cells were collected by centrifugation and the cells were collected twice in RPMI1640 medium.
After washing the cells, 1 RPMI1640 medium in a 2 volume roller
1 × 10⁶The cells were suspended at a cell concentration of / ml. The roller
Incubate at 37 ° C for 2 days with rotation 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 ± 0.5 x 10^Four
Being included in the fraction corresponding to Dalton
all right. The gel filtration column has the following Pharmacia
Molecular weight mer manufactured by Fine Chemicals (Sweden)
Tested with a car. That is, Blue Dextran 2000 2 × 10
⁶, Ferritin 4.5 × 10^Five, Aldolase 1.58 × 10^Five,of
Albumin 4.5 x 10^Four, Chymotrypsinogen 2.5 × 10^FourPassing
Bit Cytochrome C1.17 × 10^Four. In addition, the fluxi containing BCDF
Collected and installed an ultrafiltration membrane (Amicon YM-10)
25 mM piperazine-hydrochloric acid buffer solution (pH
It was replaced with 6.3).   BCDF fractionated by AcA-34 column chromatography
The fraction was preliminarily 25 mM piperazine-hydrochloric acid buffer solution (pH 6.
3) Equilibrated with MonoP column (Pharmacia Fine)
Chemicals, Sweden). 25 this column
After washing with mM piperazine-HCl buffer, adjust to pH 4.5 with hydrochloric acid.
Prepared 40 ml of 1/10 diluted polybuffer 74 (Pharma
A Fine Chemicals, Sweden).
Column operation is fast protein liquid black
Matography, FPLC (Pharmacia Fine Chemica)
Rus, Sweden) with a flow rate of 0.5 ml / min.
Was. Collect 1 ml of the eluate and measure the BCDF activity and pH.
Was. BCDF activity was eluted at pH 4.9-5.1.   The BCDF active fraction obtained from the MonoP 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, Syn
chromatographic high performance liquid chromatography,
Eluent 0.1% TFA with acetonitrile concentration 0 to 60%
BCDF was eluted by increasing linearly up to. Acetonitrile
O.D.eluting at 50-55%.₂₈₀The peak of other O.D.₂₈₀of
It is completely separated from the peak and corresponds to this peak.
BCDF activity was detected. This peak is freeze-dried and BCDF
I got a 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 x 4.1 mm, Synchrom)
HPLC, and after elution, concentrated acetonitrile in 0.1% TFA.
Elute BCDF by increasing the linearity 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 of
BCDF activity was detected corresponding to the peak of. This peak
Was 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
(Applied Biosystem Co., Calf, Model 470A)
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).
The amino acid sequence is determined by J. Biol. Chem.,193, 265 ~ 275 (1
951).   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. Removal of protecting groups from conventional methods
And then C₁₈Reversed-phase column HPLC
The target oligonucleotide is purified using
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, 1
00 units / ml penicillin, 100 μg / ml streptomycin,
20 μg / ml gentamicin, 16 mM NaHCO_Three2 x 1)
0^FiveInoculate VT-1 to the number of cells / ml and rotate at 8 rpm for 3
Culture was carried out at 37 ° C for a day. After culturing, centrifuge the culture
After collecting the cells and washing the cells twice with PBS, the cells (1.8 x 10⁹Fine
Cells) in 800 ml of PBS solution and centrifuge the cells twice.
After washing, the nuclease inhibitor Ribonucleosi
RSB solution (10mM containing des-Vanadvl complex (10mM)
Tris-HCl, ph7.5, 10 mM NaCl, 1.5 mM MgCl_Two) Suspended in 800 ml
did. Next, add NP-40 to 0.05% and then
After stirring gently, centrifuge at 3000 rpm for 5 minutes to deucleate the cells.
Remove ris and add SDS (0.5% final concentration) and BDT to the supernatant.
Equal amount of phenol immediately after adding A (final concentration 5 mM)
In addition, cytoplasmic RNA was extracted. 3 times phenol extraction
After repeating the procedure, precipitate the RNA with 2 volumes of ethanol and
This precipitate was collected by heart and dissolved in 10 mM Tris-HCl, pH 7.5.
Was. The amount of RNA thus obtained from VT-1 cells was 30 m
It was g.   Next, oligo (dT) was used to collect mRAN from this RNA.
-Using cellulose (P.L.Biochemicals, Type7),
Chromatography was performed. Adsorption is 20 mM Tris-H
Dissolve RNA in Cl, pH 7.5, 0.5M Nacl, 1mM EDTA, 0.5% SDS solution
Buffer solution (20 mM Tris-HCl, pH 7.5, 0.5 M Na
Cl, 1mM BDTA) and then elution was performed with water and 10mM Tris-HCl (p
H7.5) by alternately eluting mRNA. This
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
Nick and Gap formed 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. This small overhang at the 3'end is the T4 DNA
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 cNA 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 one of the two
The cDNA-encoded sequence in COS cells
You can express Petit Takidomari. Also in E.Coli
However, it can be replicated and selected as tetracycline resistant
be able to.   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-cDNA
Dissolve 20 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,
Incubated at 37 ° C for 60 minutes and then at room temperature for 60 minutes.
And this annealed DNA is a competent E.Col
Introduced in iMC1061. Next, the MC1061 competent cells
The method of making and the method of introduction are shown below.   E. Coli MC1061 with 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 the cells by centrifugation, and remove the TfbI (30 mM acetate
Lithium, 100 mM RbCl, 10 mM CaCl_Two, 50mM MnCl_Two, 15% green
Suspend in 40 ml of serine, pH 5.8) and leave at 0 ° C for 5 minutes
Was.   The cells were collected again by centrifugation and the Tfb II (10 mM MOPS
of 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
Was stored at 0 ° C for 15 minutes, 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, 0.1% glucose) on agar plate, 37 ℃
Colonies appeared after overnight incubation at. (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 Enzymology, 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 the 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 analysis using probe-5
And hybridized with any of the probes.
Was. It did not hybridize with other probes.   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) For large-scale preparation of pBSF2-38 plasmid DNA
MC1061 containing this pBSF2-38 was treated with 20 μg / ml tetracyte.
100m of Ψ medium containing lint 25μg / ml streptomycin
1 and inoculated with shaking at 37 ° C. for 5 to 7 hours. Then final
Contains chloramphenicol to a concentration of 170 μg / ml.
100 ml of new Ψ medium was added, and the mixture was further shake-cultured overnight.   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 keep at 0 ℃ for 10 minutes.
Let stand for a while. After that, TritonX-100 (final concentration 0.1%)
Is added and the mixture is allowed to stand at 0 ° C. for 60 minutes. Then 30,000 rpm, 30
Centrifuge for minutes and add an equal volume of water-saturated phenol to the supernatant.
To process. Treat the aqueous layer with an equal volume of chloroform.
Then extract the aqueous layer to a final concentration of 20 μg / ml.
RNase and incubate for 60 minutes at 37 ° C.
Was.   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,
Collect the DNA precipitate by centrifugation.   Next, dissolve the precipitate in 3.8 ml of water, add 4 g of CsCl,
After dissolution, add 200μ of 10mg / ml EtBr and add 40,000rpm, 1
Perform ultracentrifugation fractionation at 20 ° C for 6 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,
After drying well, the precipitate was washed with 50 mM Tris-Hcl, pH 7.5.
Lysed in μ and used for monkey cell transfection
Pulled. (B) Method for infecting monkey COS-7 cells with plasmid COS-
1 cell for 7 cells^FiveRPMI with 10% fetal calf serum
3 ml of this was suspended in 6 cm char and 5% carbon dioxide gas was added.
The cells were cultured overnight at 37 ° C in an incubator. Remove culture supernatant
Then, add 3 ml of RPMI containing 10% fetal bovine serum, and add 37 ° C.
The cells were cultured in a 5% carbon dioxide gas incubator for 2 hours. Culture
After culturing, the supernatant was removed and TBS (25 mM Tris-HCl, pH 7.5, 13
0mM Nacl, 5mM KCl, 0.6mM Na_TwoHPO_Four) Wash once with 2.5 ml
Was cleaned. The COS-7 cells were mixed with the plasmid mixture (TBS
(+) (0.7 mM CaCl in TBS_Two, 0.5 mM MgCl_TwoAlso added
1.0 ml, 2 μg of plasmid DNA and 10 mg / ml DEAE-d
extran50μ) and add 5% CO2 incubate at 37 ℃.
Incubate in a water bath for 4 hours, remove the supernatant, and then TB
Washed off with S2.5ml, 10% fetal bovine containing 150μM chloroquine
2.5 ml of RPMI containing fetal serum was added. 37 ℃, 5% carbon dioxide
After incubating for 5 hours in an incubator, remove the supernatant
And washed twice with 2.5 ml of TBS. RPMI containing 10% fetal bovine serum
Add 3 ml and mix at 37 ℃ in a 5 carbon dioxide incubator.
Cultured at night. After removing the supernatant, add 3 ml of the same RPMI, 37 ° C 5%
The cells were cultured in a carbon dioxide gas incubator for 2 days. And this
After centrifuging the culture supernatant of, the supernatant is used as a sample for measuring BCDF activity.
And   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-7 cells showed a clear BCDF activity compared with the control.
did.   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 is used for N-glycosylation in cDNA production.
It contains sugar chains from the position of the position,
The physicochemical properties of the VT-1 culture supernatant were determined by the method of Example 1.
It was identical to that of the purified BCDF purified by. 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.
Of human tonsil cells and BCDF activity stimulated by I1788 and TPA
CL4, Jurkat, CEM and unstimulated human tonsils not found
Prepared from glandular cells by the same method as in Example 4 (a)
It is.   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)³²Bam of P-labeled pBSF2-38
Hybridize at 42 ° C overnight using the H I cDNA insert as a probe.
It was soybean.   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, CESS (BCDF producing strain), RPMI 1788
The pBSF2-38 cDNA probe was hybridized to the derived mRNA.
I did it. CL-4, Jurkat, C that seems not to produce BCDF
Hybridizes to mRNA from CESS that does not produce EM and BCDF
Did not. It also hybridizes with the pBSF2-38 cDNA probe.
The size of soybean mRNA was estimated to be approximately 15-16S. 1
The parts are 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 (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.74By the method of 5,5463 ('77)
Decided. The determined nucleotide sequence and amino acid sequence
As shown in the figure. The restriction map 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. Example 8 (1) The vector used to express the human BCDF gene
The vector DNA was constructed as follows. (Fig. 7)   First, a DNA fragment (a) having the DNA sequence shown in FIG.
(L) were each synthesized by the solid-state phosphate triester method.
Was. Except for (a) and (g), T4 Polynucleotide
The 5'end was phosphorylated with Nase and ATP.   Next, mix (a) to (l), anneal, and then
Double-stranded synthetic DNA (A) was formed using gauze. one
Digest pT79-11 with restriction enzymes Hpa I and Xba I,
Large DNA fragments were separated by rose gel electrophoresis.
The resulting pT9-11 fragment and synthetic DNA (A) (see Figure 8)
) Were mixed and ligated using T4 DNA ligase. Profit
Introduced recombinant DNA into Escherichia coli HB101 strain
Then, a strain having ampicillin resistance was selected. Separate
From the isolated strain via the plasmid DNA,
Synthetic HIL
Select a bacterium that holds pT135 (Nco) with -2 cDNA
Was. (2) Use plasmids pT13S (Nco) and pBSF2-38
Recombinant DNA expressing human BCDF was constructed as follows.
did. (Figs. 9 and 10) i) The plasmid pT13S (Nco) was digested with the restriction enzymes Nco I and Xba.
 Cleavage with I and large DNA by agarose gel electrophoresis
The fragment was simply purified. On the other hand, it controls the plasmid pBSF2-38.
Cleave with limited BamHI and perform agarose gel electrophoresis.
Recover a small DNA fragment containing the BCDF cDNA insert
Was. And restrict the BamH I human BCDF cDNA insert
Completely cleaved with the enzyme Vba I and further partially cleaved with Mva I.   Then tryptophan promoter / operator
PT13S (Nco) fragment containing (trp p / o), and BCDF cDNA
Mva I-Xba I fragment-containing DNA mixture and synthetic DNA (B)
[⁵′ CATGCCAGTACCACC^ThreePhosphorylate the 5'and 5'ends
Was⁵′ TGGTGGTACTGG^Three’] Are mixed and TsDNA ligase is used.
I combined them. The recombinant DNA obtained was transformed into Escherichia
・ Introduced into Escherichia coli HB101 strain and has ampicillin resistance
Selected strains. Colony hybridase from the obtained strain
Hybridization with synthetic DNA (B)
A strain with NA was selected. Plasmid DN from the isolated strain
A to obtain a restriction enzyme digestion test and
Retains pTBCDF-01 by setting the base sequence
Bacteria to be selected were selected (pTBCD-01 / HB101). ii) The plasmid pBSF2-38 was cleaved with the restriction enzyme Ban I and DN
Treated with A polymerase I (Klenow) and cleaved with Xba I.
Separation of about 150 base pair DNA fragments by gelose gel electrophoresis
did. iii) Digest pTBCDF-01 obtained in i) with restriction enzyme BamHI
Then, after treatment with DNA polymerase I (Klenow), cut with Xba I
And then run the larger DNA fragment on agarose gel electrophoresis.
Received. iv) The two types of DNA fragments obtained in ii) and iii) were converted into T4 DNA fragments.
It was bound using gauze. The recombinant DNA obtained is
Escherichia coli HB101 strain introduced and ampicillin resistant
A strain having Plasmid DNA from the obtained strain
PTBC
A bacterium that retains DF-02 was selected (pTBCDF-02 / HB101, (F
ERM p-9061)). v) Holds plasmid pTBCDF-01 or pTBCDF-02
25 μg / ml of Escherichia coli HB101 strain
L medium containing syn and 25 μg / ml ampicillin (1%
Totryptone, 0.5% yeast extract, 0.5% NaCl, 0.1% glue
Grows overnight at 37 ° C. in 10 ml of course, pH 7.5. Incidentally
5 ml of the culture suspension was added to M9-casamino acid medium (0.6% Na_TwoHPO_Four・ 1
2H_TwoO, 0.3% KH_TwoPO_Four, 0.05% NaCl, 0.1% NH_FourCl, 0.05% MgSO_Four
・ 7H_TwoO, 0.00147% CaCl_Two, 0.2% glucose, 0.2% thistle
Acid, 0.02% L-leucine, 0.02% L-proline, 0.0002
% Thiamine hydrochloride, 100 μg / ml ampicillin, 25 μg / ml
Inoculate treptomycin, pH7.4) and grow at 28 ℃ for 3 hours
Nourished. After that, it will be 25 μg / ml 3-Indole Acry
Acid (IAA) was added and induction culture was performed at 23 ° C. for 21 hours.
The cultured cells were centrifuged, and 20 mM Tris-HCl (pH 7.5, 30 mM
 It was washed with NaCl) and suspended in 8 ml of the same buffer. Or
1% of protein produced in cells in the presence of 50 mM EDTA
Sodium dodecyl sulfate (SDS) or 1 mg / ml lysochy
Sonic treatment (50W, 30 seconds) following digestion
Extracted.   As shown in Table 1, part of the 3'end of human BCDF cDNA
It was deleted, and a part of human IL-2 cDNA bound to the deleted part.
I have. pTBCDF-01 / HB101 and complete human BCDF cDNA
Both pTBCDF-02 / HB101 culture extracts containing BCDF
Showed activity.   The recombinant BCDF obtained here is the same as in Example 1.
Purified and concentrated by the method of
Rough Column Synchropak RP-P (C₁₈) HPLC
It was eluted at an acetonitrile concentration of 50-55%. That a
The mino acid sequence was also determined as in Example 1, confirming the N-terminal structure.
did.   That is, the polypeptide produced by pTBCDF-02 / HB101 is
It has the following amino acid sequence: PRO VAL PRO PRO GLY GLU ASP SER LYS ASP VAL ALA ALA PRO HIS ARG GLN PRO LEU THR SER SER GLU ARG ILE ASP LYS GLN ILE ARG TYR ILE LEU ASP GLY ILE SER ALA LEU ARG LYS GLU THR CYS ASN LYS SER ASN MET CYS GLU SER SER LYS GLU ALA LEU ALA GLU ASN ASN LEU ASN LEU PRO LYS MET ALA GLU LYS ASP GLY CYS PHE GLN SER GLY PHE ASN GLU GLU THR CYS LEU VAL LYS ILE ILE THR GLY LEU LEU GLU PHE GLU VAL TYR LEU GLU TYR LEU GLN ASN ARG PHE GLU SER SER GLU GLU GLN ALA ARG ALA VAL GLN MET SER THR LYS VAL LEU ILE GLN PHE LEU GLN LYS LYS ALA LYS ASN LEU ASP ALA ILE THR THR PRO ASP PRO THR THRASN ALA
 SER LEU LEU THR LYS LEU GLN ALA GLN ASN GLN TRP LEU GLN ASP MET THR THR HIS LEU ILE LEU ARG SER PHE LYS GLU PHE LEU GLN SER SER LEU ARG ALA LEU ARG GLN MET Example 9 (1) Plasmid pT13S (Nco) (described in Example 8) and
And human interleukin-2 (HIL-
Produces human BCDF fusion protein (△ HIL-2-BCDF) with 2)
Recombinant DNA to be constructed was constructed as follows. (11th, 12th
(Fig.) i) The plasmid pT13S (Nco) was digested with the restriction enzymes Bgl II and Xb.
Cleavage with a I and agarose gel electrophoresis for large DNA
The fragment was isolated and purified. On the other hand, it controls the plasmid pBSF2-38.
Cleavage with the restriction enzyme BamHI and agarose gel electrophoresis
Recover a small DNA fragment containing the human BCDF cDNA insert
Was. Then, control the BamHI and human BCDF cDNA insert.
It was completely digested with the restriction enzyme Xba I and further partially digested with Mva I.   Next, pT13S (Nco) fragment containing the above promoter, human
DNA mixture containing Mva I-Xba I fragment containing BCDF cDNA and
Synthetic DNA (C) [⁵′ GATCTCTTCAGAGCCCCAGTACCCCC^Three'When
Phosphorylated at the 5'end⁵′ TGGGGGTACTGGGGCTCTGAAG
A^Three'] Were mixed and ligated using T4 DNA gauze. Profit
Introduced recombinant DNA into Escherichia coli HB101 strain
Then, a strain having ampicillin resistance was selected. Got
Synthesized DN by colony hybridization method by strain
Select a strain that has a DNA that hybridizes with A (C)
Was. Obtain plasmid DNA from the isolated strain and
Cleavage test and determination of the nucleotide sequence near the binding site.
The strains carrying pTBCDF-11 were selected (pT
BCDF-11 / HB101). ii) The plasmid pBSF2-38 was cleaved with the restriction enzyme Ban I and DN
Treated with A polymerase I (Klenow) and cleaved with Xba I.
Separation of about 150 base pair DNA fragments by gelose gel electrophoresis
did. iii) Digest pTBCDF-11 obtained in i) with restriction enzyme BamHI
Then, after treatment with DNA polymerase I (Klenow), cut with Xba I
And then run the larger DNA fragment on agarose gel electrophoresis.
Received. iv) The two types of DNA fragments obtained in ii) and iii) were converted into T4 DNA fragments.
It was bound using gauze. The recombinant DNA obtained is
Escherichia coli HB101 strain introduced and ampicillin resistant
A strain having Plasmid DNA from the obtained strain
PTBC
Bacteria retaining DF-12 were selected (pTBCDF-12 / HB101, (F
ERM P-9062). v) A part of human IL-2 cDNA is attached to the 5'end of BCDF cDNA.
And a part of the 3'end is deleted,
Plasmid pTBCDF-11 in which a part of IL-2 cDNA is bound
At the 5'end of HB101 and the complete BCDF cDNA containing
Plasmid pTBCDF-1 in which part of the IL-2 cDNA is bound
Both HB101 carrying 2 were cultivated and extracted according to Example 8.
A liquid was obtained. As shown in Table 2, both culture extracts showed BCDF activity.
Indicated. Example 10   PTBCDF-12 / HB101 according to Example 8 (described in Example 9)
Cultivate the cells and extract the granules formed in the cells by the following procedure.
Was.   Collect the cells by centrifugation and concentrate 10 times,
Add 20 mM Tris-Hcl buffer (pH 7.5) containing 30 mM NaCl
After suspension, add 1 mg / ml lysozyme and 0.05 M EDTA to the suspension.
After stirring and stirring, the mixture was left in ice for 1 hour. Then super
The cells were disrupted by sonication and centrifuged at 10,000 rpm for 5 minutes.
The granules were collected at.   The granules were solubilized with 6M guanidine hydrochloride and
-BCDF concentration of 100 μg / ml and 2M guanidine hydrochloride solution
Concentration is adjusted so that
Thion 1 mM and reduced glutathione 10 mM were added, pH 8.0,
It was left at room temperature for 10-16 hours. Next by Sephadex G-25
At the same time as removing guanidine hydrochloride by gel filtration,
Equivalent to △ HIL-2-BCDF, which became a buffer solution for rain reaction
Fractions were obtained. This substance was charged with SDS-polyacrylamide gel.
By electrophoresis, the molecular weight is the value calculated from the amino acid composition.
It is almost the same as the above, and it is N-terminal at the protein sequencer.
As a result of testing the amino acid sequence on the end side, it was found to be the HIL-2 sequence.
It was confirmed that there was.   That is, the polypeptide having this BCDF activity is
It has the amino acid sequence of ALA PRO THR SER SER SER THR LYS LYS THR GLN LEU GLN LEU GLU HIS LEU LEU LEU ASP LEU PHE ARG ALA PRO VAL PRO PRO GLY GLU ASP SER LYS ASP VAL ALA ALA PRO HIS ARG GLN PRO LEU THR SER SER GLU ARG ILE ASP LYS GLN ILE ARG TYR ILE LEU ASP GLY ILE SER ALA LEU ARG LYS GLU THR CYS ASN LYS SER ASN MET CYS GLU SER SER LYS GLU ALA LEU ALA GLU ASN ASN LEU ASN LEU PRO LYS MET ALA GLU LYS ASP GLY CYS PHE GLN SER GLY PHE ASN GLU GLU THR CYS LEU VAL LYS ILE ILE THR GLY LEU LEU GLU PHE GLU VAL TYR LEU GLU TYR LEU GLN ASN ARG PHE GLU SER SER GLU GLU GLN ALA ARG ALA VAL GLN MET SER THR LYS VAL LEU ILE GLN PHE LEU GLN LYS LYS ALA LYS ASN LEU ASP ALA ILE THR THR PRO ASP PRO THR THR ASN ALA SER LEU LEU THR LYS LEU GLN ALA GLN ASN GLN TRP LEU GLN ASP MET THR THR HIS LEU ILE LEU ARG SER PHE LYS GLU PHE LEU GLN SER SER LEU ARG ALA LEU ARG GLN MET (2) Cutting with kallikrein (reference example)   In 50 mM Tris-HCl buffer, pH 7.8, containing 113 mM NaCl
△ HIL-2-BCDF 80μg obtained in
Claine was reacted at 37 ° C for 15 hours and then analyzed by reverse phase HPLC with Ala-BCDF.
Equivalent fractions were collected. This is a protein sequencer
Results of analysis of amino acid sequence near the N-terminal with △ HIL
-2-BCDF was quantitatively converted to Ala-BCDF protein.
Was confirmed. “Ala-BCDF” is the N-end of BCDF.
One Ala is added to the end, specifically, the following
It has an amino acid sequence. ALA PRO VAL PRO PRO GLY GLU ASP SER LYS ASP VAL ALA ALA PRO HIS ARG GLN PRO LEU THR SER SER GLU ARG ILE ASP LYS GLN ILE ARG TYR ILE LEU ASP GLY ILE SER ALA LEU ARG LYS GLU THR CYS ASN LYS SER ASN MET CYS GLU SER SER LYS GLU ALA LEU ALA GLU ASN ASN LEU ASN LEU PRO LYS MET ALA GLU LYS ASP GLY CYS PHE GLN SER GLY PHE ASN GLU GLU THR CYS LEU VAL LYS ILE ILE THR GLY LEU LEU GLU PHE GLU VAL TYR LEU GLU TYR LEU GLN ASN ARG PHE GLU SER SER GLU GLU GLN ALA ARG ALA VAL GLN MET SER THR LYS VAL LEU ILE GLN PHE LEU GLN LYS LYS ANA LYS ASN LEU ASP ALA ILE THR THR PRO ASP PRO THR THR ASN ALA SER LEU LEU THR LYS LEU GLN ALA GLN ASN GLN TRP LEU GLN ASP MET THR THR HIS LEU ILE LEU ARG SER PHE LYS GLU PHE LEU GLN SER SER LEU ARG ALA LEU ARG GLN MET (3) Removal of N-terminal Ala by aminopeptidase P   Aminopeptidase P is available from Methods Enzsymol.19, 521
Purification was performed by the method described in (1970).   The Ala-BCDF solution obtained in (2) was added to 0.4 mM MnCl 2._TwoIncluding
Sephade equilibrated with 50 mM Tris-HCl buffer (pH 8.0)
Ala-BCDF equivalent fraction was obtained by gel filtration with xG-25. This
50 μg of Ala-BCDF obtained as described above was added to aminopeptidase
, Pase was added and reacted at 37 ℃ for 16 hours, then BCDF was applied by reverse phase HPLC.
Equivalent fractions were collected. In addition, a protein sequencer
As a result of analyzing the amino acid sequence on the N-terminal side with Ala-B
It was confirmed that CDF was quantitatively converted to BCDF. What
Table 3 shows the activities of Ala-BCDF and BCDF. BCDF activity is shown. Example 11   Plasmids pTBCDF-01 and pT9-11 were used to
The recombinant DNA expressing the missing human BCDF was
It was constructed. (Fig. 13) i) The plasmid pTBCDF-01 was cleaved with the restriction enzyme Xba I to give D
Treated with NA polymerase I (Klenow) and digested with PvvI, then
By gelose gel electrophoresis, trp p / o and 3'deficient
A small DNA fragment containing the isolated BCDF cDNA was isolated. On the other hand,
Cleavage of rasmid pT9-11 with restriction enzymes BamHI and pvvI
Then, agarose gel electrophoresis is used to determine the trpA terminator.
A large DNA fragment containing was recovered.   Both DNA fragments and synthetic DNA prepared in this way (C) [⁵′ CCTAGCCCGGGTGAATGAAT^Three'When⁵′ GATCATT
CATTCACCCGGGCTAGG^Three’] Are mixed and T4 DNA ligase is used.
I combined them. The recombinant DNA obtained was transformed into Escherichia
・ Introduced into Escherichia coli HB101 strain and has ampicillin resistance
Selected strains. Control by obtaining plasmid DNA from the isolated strain
Cleavage test with a restriction enzyme and the nucleotide sequence near the binding site
By carrying out the determination, a bacterium holding pTBCDF-03 was selected.
(PTBCDF-03 / HB101).   In addition, pTBCDF-03 lacks a part of the 3'end of BCDF cDNA.
It is a plasmid having a missing gene.   The polypeptide produced by pTBCDF-03 / HB101 is
It is considered to have the following amino acid sequence. PRO VAL PRO PRO GLY GLU ASP SER LVS ASP VAL ALA ALA PRO HIS ARG GLN PRO LEU THR SER SER GLU ARG ILE ASP LYS GLN ILE ARG TYR ILE LEU ASP GLY ILE SER ALA LEU ARG LYS GLU THR CYS ASN LYS SER ASN MET CYS GLU SER SER LYS GLU ALA LEU ALA GLU ASN ASN LEU ASN LEU PRO LYS MET ALA GLU LYS ASP GLY CYS PHE GLN SER GLY PHE ASN GLU GLU THR CYS LEU VAL LYS ILE ILE THR GLY LEU LEU GLU PHE GLU VAL TYR LEU GLU TYR LEU GLN ASN ARG PHE GLU SER SER GLU GLU GLN ALA ARG ALA VAL GLN MET SER THR LYS VAL LUE ILE GLN PHE LEU GLN LYS LYS ALA LYS ASN LEU ALA ii) Culture extract from pTBCDF-03 / HB101 in the same manner as in Example 8.
Was prepared. As shown in Table 4, culture of pTBCDF-03 / HB101
The extract showed BCDF activity.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a method for constructing recombinant DNA for expression in monkey cells. Second
The figure shows BCDF activity of the culture supernatant of monkey cell COS-7 into which the pBSF 2-38 cDNA was introduced, which was measured by Elisa method. Figure 3 shows pBSF 2-38 c measured by the reverse plaque method.
BCDF of culture supernatant of COS-7, a transgenic monkey cell
Activity. FIG. 4 is a Northern blotting analysis of pBSF 2-38 cDNA insert. Figure 5 shows the BCDF chlorine and amino acid sequences. Figure 6 is a restriction map. FIG. 7 is a construction diagram of plasmid pT13S (Nco). FIG. 8 shows the nucleotide sequence and restriction enzyme map of synthetic human interleukin 2 (HIL-2). FIG. 9 is a construction diagram of plasmid pTBCDF-01. Fig. 10 is a construction diagram of plasmid pTBCDF-02. Figure 11 shows plasmid pT
Construction drawing of BCDF-11. FIG. 12 is a construction diagram of plasmid pTBCDF-12. FIG. 13 is a construction diagram of plasmid pTBCDF-03.

   ────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Toshio Hirano               19 Mihogaoka, Ibaraki City

Claims (1)

(57) [Claims] Prokaryotic cells transformed with a recombinant DNA consisting of a gene encoding a polypeptide represented by the following amino acid sequence (I) and a vector DNA replicable in prokaryotic cells are produced by culturing in a medium. A method for producing a purified polypeptide having human B cell differentiation factor activity, having no natural signal peptide at its N-terminus and having no sugar chain, characterized by collecting human BCDF.