JPH06336497A - B cell differentiating factor and its production - Google Patents

Abstract

PURPOSE:To produce a polypeptide having human BCDF activity from a procaryote such as bacterial cell of Escherichia coli having a DNA encoding human BCDF and to produce a useful polypeptide. CONSTITUTION:A polypeptide which is a purified polypeptide having human B cell differentiating factor (hereinafter abbreviated as BCDF), no natural signal peptide at its N end and no saccharide chain, contains AlaPro as an N end amino acid and Met as a C end amino acid and is produced from cells of procaryote.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a human B-cell differentiation factor (hereinafter referred to as BCDF) and human BC.
The present invention relates to a method for producing human BCDF using a prokaryote such as Escherichia coli bacterium in which a DF gene is incorporated.
Human BCDF according to the present invention is a disease caused by immunodeficiency,
It is a useful substance that can be generally used as a therapeutic drug for cancer and autoimmune diseases.

[0002]

2. Description of the Related Art Lymphokine, which is a soluble protein factor having biological activity and pharmacological activity produced by lymphocytes, has an action of regulating a small amount of immune response mechanism in a living body. Lymphokine is expected to be useful as an antitumor agent, an antiviral agent, an antibacterial agent, a therapeutic agent for immunodeficiency, and a therapeutic agent for autoimmune disease in general because of its nature as an immunoactive substance (Adv. In Immunopharm. 507, 1980). BCDF according to the present invention is a kind of lymphokine, and from such a viewpoint, application to medicine is expected. Now, mature B cells activated by antigen stimulation undergo mitotic proliferation with the help of T cells, but in order for B cells to finally differentiate into antibody-producing cells, one or more T cells It is known that the origin-derived differentiation-inducing substance is essential.
The existence of this substance is described in RWDutton et al., Transplant. Rev. 2
3 , 66 (1975), A. Schimpl and E. Wecker et al., Na.
ture New Biology 237 , 15 (1972). They found that in the culture supernatant of mouse lymphocyte mixture culture or in the culture supernatant of mouse lymphocytes stimulated by antigens and mitogens, lymphoid cell populations from which mouse T cells were removed and lymphs derived from nude mice. It was found to amplify the primary immune response of spheres to sheep red blood cells (SRBC), and the active entity with such an action was given the designation T lymphocyte replacement factor, or TRF. Since then, TRF acts on B cells in a non-antigen-specific manner that does not require concordance of major histocompatibility complex (MHC), does not induce mitotic proliferation of B cells, and It is defined as a humoral factor that induces the differentiation of cells into antibody-producing cells.

Since then, functional evidence showing the existence of such a B cell differentiation factor has been accumulated, and the existence of a differentiation factor similar to that of the mouse has been suggested in humans. At present, the factors for differentiating B cells defined as described above into antibody-producing cells have been generically called BCDF. As described above, BCDF plays an important role in the antibody producing function of B cells in the human body. Conventionally, in order to obtain BCDF, a method of producing BCDF by stimulating normal human T cells separated from human peripheral blood or the like with mitogen has been adopted. In this method, it is difficult to obtain T cells sufficiently, and since mitogen is used, harmful mitogen is mixed into BCDF and it is difficult to remove it, and fetal bovine serum is used for T cell culture. It is necessary to add serum components such as, etc. to the medium, and these added proteins and BCDF cannot be sufficiently separated, and the fact that purified BCDF cannot be obtained is an obstacle to using BCDF in medicine. There were many problems, and BCDF could not be industrially produced. In addition, a method has been reported in which human T cells are fused with human cancer cells to obtain human T fused cells, and thereby BCDF is produced (Okada et al., J. Exp. Med., 157 , 583).
(1983)). However, human fused cells often lose their lymphokine-producing ability during passage, and thus practical BCD
There are no F-producing human fusion cells. Also, a method for producing BCDF by human T cells transformed with human T cell leukocyte virus (hereinafter referred to as "HTLV") has been reported (Japanese Patent Application No. 59-235199, 60-25301). However, at the present time, a method for producing BCDF by a prokaryote such as Escherichia coli bacterium, that is, a DNA corresponding to BCDF is incorporated into a prokaryotic vector to be replicated, transcribed, translated in a prokaryotic cell, and BC
Method for producing DF and BC produced by this method
No DF has been reported.

[0004]

Therefore, an object of the present invention is to provide a method for producing a polypeptide having human BCDF activity by a prokaryote such as Escherichia coli bacterium having a DNA encoding human BCDF, and a useful polypeptide. It is provided as a substance.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a gene encoding a polypeptide having human BCDF activity and replication in prokaryotic cells are possible. The present invention was completed by culturing prokaryotic cells transformed with a recombinant DNA consisting of vector DNA in a medium and collecting the produced human BCDF. The present invention will be described in detail below. Now, the present inventors have noted that VT-1 cells (IFO 50096), which is one of human T cells transformed by HTLV (human T cell leukemia virus), produce large amounts of human BCDF. Have already identified the gene encoding human BCDF (Japanese Patent Application No. 61-18).
4858). In addition, as a precaution, Examples 1 and 2 and Reference Examples 1 to 1
Details of gene cloning encoding human BCDF are described in 6. Now, regarding the regulation of prokaryotes that produce human BCDF, first, a vector DNA capable of replicating the gene encoding human BCDF in the cells of prokaryotes.
Built in.

At this time, DNA encoding human BCDF
Is inserted downstream of the promoter sequence of the expression vector, or
Alternatively, a piece of DNA having a promoter sequence may be added before or after the insertion of the cDNA into the expression vector, and the human BC
It may be inserted upstream of the DNA encoding DF. In this case, the cDNA encoding human BCDF encodes a polypeptide consisting of 212 amino acids as shown in FIG. 5, but the N-terminal region corresponding to 27 or 28 amino acids of this polypeptide is extremely It is hydrophobic and is called a so-called signal sequence, which is cleaved during the secretory process,
Mature human BCDF polypeptide consisting of 185 amino acids starting from N-terminal AlaPRO and mature human BCDF consisting of 184 amino acids starting from N-terminal PRO
A polypeptide is obtained. Of these, the present invention is directed to the mature human BCDF polypeptide consisting of 185 amino acids starting from the N-terminal Ala PRO and the cDNA encoding this polypeptide. How to incorporate is
The vector is cleaved with a suitable restriction enzyme, and if necessary, a plurality of suitable linkers or bases in an annealable combination are polymerized. The double-stranded DNA processed in this way and the vector DNA are mixed and ligated with each other. The recombinant DNA thus obtained is introduced into a prokaryotic host, and human BC is selected from among the transformed microorganisms obtained.
A DF producing strain may be selected. In the present invention, Escherichia coli, Bacillus subtilis and the like can be used as the prokaryote, but preferably Escherichia coli
It is better to use stiffness.

As the Escherichia coli vector which can be used in the present invention, EK type plasmid vector (stringent type: pSC101, pRK353, pRK646,
pRK248, pDF41 etc.), EK type plasmid vector (relaxed type: ColE1, pVH51, pAC105, RSF21
24, pCR1, pMB9, pBR313, pBR322, pBR324, pBR325, pB
R327, pBR328, pKY2289, pKY2700, pKN80, pKC7, pKB15
8, pMK2004, pACYC1, pACYC184, dul, etc.). λgt type phage vector: λgt. λc. λgt. λB, λWES, λ
C, λWES, λB, λZJvir., ΛB ', λALO, λB, λWES.
Includes Ts622, λDam, etc. As the promoter, all promoters that function in Escherichia coli including trp, lac, tac, tufB, β-lactamase and lpp promoter can be used. Recombinant D
For the transformation of host cells using NA, the following methods are commonly used. When a prokaryote such as Escherichia coli is the host, competent cells capable of incorporating this DNA can be transformed by the well-known CaCl ₂ method after recovering the cells in the logarithmic growth phase. The coexistence of MgCl ₂ or RbCl in the transformation reaction solution improves the transformation efficiency. It is also possible to transform the host cells after preparing the protoplasts.

The recombinant prokaryotic cells in which the human BCDF gene has been obtained as described above may be cultured by a conventional method. BCD thus obtained
F is on the culture by various methods such as salting out, vacuum dialysis, ultrafiltration, gel filtration chromatography, ion exchange chromatography, affinity chromatography, chromatofocusing, reverse phase chromatography, focus electrophoresis and gel electrophoresis. It can be purified by concentrating from Sei. In addition, human BCDF was transformed into microbial cells.
In case of accumulation of BCDF, BCDF is recovered and purified by a method easily performed by a person skilled in the art. The following is a brief description. After culturing, the cells are collected by centrifugation, the cells are suspended in a solution containing lysozyme or a solution containing other detergents, and after the reaction, freezing and thawing are repeated, and a cell extract is collected. Alternatively, it is simply purified by affinity chromatography using an anti-BCDF antibody-immobilized column. Next, there is the following method for measuring BCDF activity. Human B cell line CL4 that produces IgM in response to human BCDF (T. Hirano et al., Proc. Natl. Ac
BCD using ad. Sci., 82 , 5490, 1985).
F activity is measured. 6 × with test solution for measuring BCDF activity
R containing 10 ³ CL4 containing 200 μl of 10% FCS
PMI1640 medium (100 units of penicillin per 100 ml, streptomycin 100 μg, gentamicin 1
Containing 0 μg and 16 mM NaHCO ₃ ). This mixture was placed in a 96-well microplate for 3 days with 5% CO ₂
The cells are cultured in the presence at 37 ° C., and the IgM amount of the culture supernatant is measured by enzyme immunoassay. Maximum I under these conditions
BC showing 50% of gM production (highest CL4 response)
The activity of DF was set to 1 U / ml.

Human B cell line CL4 (O. SAIKI et al., Eur. J. Immunol 1) which produces IgM in response to human BCDF
The BCDF activity can also be measured by the reverse PFC method using 3 , 31 (1983)). Test solution for measuring BCDF activity and 200 μl of 10% FCS with 1 × 10 ⁴ CL4
In RPMI1640 medium (additives are the same as above). This mixture is incubated in a 96-well microplate for 3 days at 37 ° C. in the presence of 5% CO ₂ . Cultured cells, complement, anti-human IgM antibody protein A-bound sheep preserved blood for H
The number of cells differentiated into human IgM-producing cells by the action of BCDF based on the number of hemolytic plaques after mixing with agarose dissolved in ANKS solution, spreading and solidifying on a petri dish, and culturing overnight at 37 ° C in the presence of 5% CO _2. To measure. By the way, eukaryotic genes are known to exhibit polymorphism as is known for the human interferon gene (Taniguchi et al., Gen.
e. 10 , 11-15 (1980), Ohno, Taniguchi, Proc. N
atl. Acad. Sci., USA, 77 , 5305-5309 (1
981), Gray et al., Nature, 295 , 501-50.
8 (1981). Due to this polymorphism, some of the amino acids in the protein product may be substituted, or there may be changes in the nucleotide sequence but no change at all. Therefore, also in the present invention, as long as it has human BCDF activity, a polypeptide in which one or more amino acids in the amino acid sequence of FIG. 5 are substituted with one or more amino acids (provided that Ala at position 28 is used). (Excluding the case of substituting for.) And DNA encoding this polypeptide are also included in the present invention. Furthermore, a polypeptide lacking one or more amino acids having human BCDF activity (provided that
(Except when Ala at position 28 is deleted), or a polypeptide added with one or more amino acids, a polypeptide having a combination sequence thereof (including substitution of amino acids), and a code for these polypeptides The present invention also includes a DNA having a nucleotide sequence that Human BC
Even if there is a modified region having an additional amino acid sequence that inhibits the polypeptide function as DF, the newly added region can be easily removed and used as the polypeptide and gene of the present invention. That is, the polypeptide having human BCDF activity is human BCDF according to the present invention.

[0010]

EFFECTS OF THE INVENTION The clinical application of BCDF thus produced can be roughly classified into three. Firstly, BCDF antibody is produced by BCDF, and it can be used for analysis of immunological pathology by using BCDF immunoassay system by BCDF and anti-BCDF antibody, and repair of B cell dysfunction that is scattered in autoimmune diseases. Can also be used. The second application is to treat various diseases. For example, T
It is considered that BCDF alone or other lymphokines or immunotherapeutic agents may be administered to patients with immunodeficiency due to a decrease in B cell antibody production due to decreased helper function of cells to restore the antibody production function to normal.

Focusing on the differentiation activity of BCDF, it can also be used as an anti-malignant tumor agent by differentiating a malignant tumor cell line with BCDF to induce growth inhibition. Furthermore, the following are possible applications of BCDF. B cell growth factor (BCGF) (K. Yoshizaki et al., J.
of Immunol. 130 , 1241 (1983)), and it has been reported that normal B cells can be cultured for a long period by adding other T cell factors containing lymphokines to the medium.
(B. Sredni et al., J. Exp. Med., 154 , 1500 (1
981)). These cultured normal B cells or EBs
For B cells transformed with virus, B
Antibodies can be produced in vitro by the action of CDF. Monocloning B cells that produce specific antibodies, eg, antibodies that recognize specific antigens on the surface of pathogenic bacteria, pathogenic viruses, pathogens, cancer cells, etc.,
Cloned normal B cells or cells transformed with EB virus can be cultured in combination with BCDF and other lymphokines to acidify useful monoclonal antibodies. These antibodies can be used for infectious diseases, cancer and diagnosis. Thus BCDF is a very broadly effective substance. The present invention will be specifically described with reference to the following reference examples and examples.

[0012]

【Example】

Reference Example 1 1 liter of 20% FCS-containing RPMI1640 medium (2 mM glutamine, 5 × 10 ⁻⁵ M 2ME, 100 units / ml penicillin) in a 2 liter plastic roller incubator (Falcon # 3027) (hereinafter referred to as roller). 100 μg / ml streptomycin, 20 μ
containing g / ml gentamicin and 16 mM NaHCO ₃ )
VT-1 was inoculated to obtain 2 × 10 ⁵ cells / ml, and the roller was rotated at 8 rpm for 3 days at 37 ° C.
It was cultured in. After culturing, the culture was centrifuged to collect the cells, and the cells were washed twice with RPMI1640 medium.
The cells were suspended in 1 liter of RPMI1640 medium in a liter roller at a cell concentration of 1 × 10 ⁶ / ml. Incubate at 37 ° C. for 2 days with the roller rotating at 8 rpm. After the culture, the culture was centrifuged to obtain a culture supernatant. From the culture supernatant containing BCDF obtained by culturing VT-1 as described above, BCDF was purified by the following method. 10 liters of cell-free supernatant were placed on an ultrafiltration membrane (Amicon YM-10, Amicon Corporation, Massachusetts, US
Using an ultrafiltration device equipped with A) (Amicon mass treatment cell type 2000, Amicon Corporation, Massachusetts, USA), nitrogen gas was applied at a pressure of 4 kg / cm ² for filtration. The 100 ml of concentrated liquid remaining on the upper part of the filtration membrane was further equipped with an ultrafiltration membrane (Amicon YM-10) to perform ultrafiltration (Amicon, Standard Cell 52).
Type) was used and a pressure of 4 kg / cm ² was applied with nitrogen gas for filtration. 5 ml of the concentrated liquid remaining on the upper part of the filtration membrane was collected.

The concentrated supernatant described above was processed on an AcA-34 gel filtration column (LKB Produker, Sweden, 2.6 × 90 cm). The gel filtration column was preliminarily equilibrated with PBS (phosphate buffered saline, 0.01M phosphate buffer containing 0.15M sodium chloride, pH 7.0). The concentrated supernatant is eluted with PBS and the eluate is 5m
Each aliquot was taken and the BCDF activity of the aliquot was measured. BC
It was found that the fraction having DF activity was contained in the fraction corresponding to the molecular weight of 3.5 ± 0.5 × 10 ⁴ daltons. The gel filtration column was tested with the following molecular weight markers manufactured by Pharmacia Fine Chemicals (Sweden). That is, Blue Dextran 2000 2x
10 ⁶ , ferritin 4.5 × 10 ⁵ , aldolase 1.58
× 10 ⁵ , ovalbumin 4.5 × 10 ⁴ , chymotrypsinogen 2.5 × 10 ⁴ and cytochrome C 1.17 × 10
⁴ . Fractions containing BCDF were collected and replaced with 25 mM piperazine-hydrochloric acid buffer (pH 6.3) using an ultrafiltration device equipped with an ultrafiltration membrane (Amicon YM-10).

The BCDF fraction fractionated by the AcA-34 column chromatography was equilibrated with 25 mM piperazine-hydrochloric acid buffer (pH 6.3) in advance on a MonoP column (Pharmacia Fine Chemicals, Sweden).
Passed through. The column was washed with 25 mM piperazine-hydrochloric acid buffer and then adjusted to pH 4.5 with hydrochloric acid.
Elution with / 10 dilution polybuffer 74 (Pharmacia Fine Chemicals, Sweden). Column operation is fast protein liquid chromatography, FPLC (Pharmacia Fine Chemicals,
(Sweden) and the flow rate was 0.5 ml / min. The eluate was collected in 1 ml portions, and BCDF activity and pH were measured. BCDF activity was eluted at pH 4.9-5.1. The BCDF active fraction obtained from the MonoP column was 0.1
% Reverse phase chromatography column Synchropak RP-P (C ₁₈ ) buffered with TFA (aqueous trifluoroacetic acid solution)
(250 × 4.1mm, Synchrom) was subjected to high performance liquid chromatography, and the acetonitrile concentration in the eluent 0.1% TFA was linearly increased from 0 to 60% BC
The DF was eluted. O.C. eluted with 50-55% acetonitrile. The peak of D. ₂₈₀ is different from that of other O. It was completely separated from the D. ₂₈₀ peak, and BCDF activity was detected corresponding to this peak. This peak was freeze-dried to obtain a BCDF standard.

This sample was subjected to SDS-polyacrylamide gel (12% gel) electrophoresis under reducing conditions. A gel section corresponding to a molecular weight of 21,000 was cut out, and 0.05% SDS, 10 mM NH ₄ HCO _{3 in} an Eppendorf tube was cut out.
The mixture was shaken at 37 ° C. overnight and extracted. This eluate is directly again used for the reversed phase chromatographic column Synchropak RP-P.
HPL using (C ₁₈ ) (250 × 4.1 mm, Synchrom)
After being subjected to C, BCDF was eluted by linearly increasing the acetonitrile concentration in 0.1% TFA from 0 to 60% after elution. O.C. eluted with 50-55% acetonitrile.
The peak of D. ₂₈₀ is different from that of other O. D. The _{2 80} peaks are completely separated, BCDF activity was detected in response to this peak. This peak was freeze-dried to obtain purified BCDF.
To determine the amino acid sequence of the BCDF protein, 6 μg of purified BCDF obtained as described above was added to the protein.
Sequencer (Applied Biosystem Co., Calf, Model
470A). The method for determining the amino acid sequence is described in J. Biol.
Chem., 193 , 265-275 (1951). The amino acid sequence from the N-terminus was as follows. Pro Val Pro Pro Gly Glu Asp Ser Lys Asp Val Ala Ala

Reference Example 2 20 μg of the purified BCDF preparation prepared by the method described in Reference Example 1 was dissolved in a buffer containing 5 mM Tris-HCl and pH 9.5 to prepare a lysyl endopeptidase (LysylEndopepti).
dase) (Wako) (Lysyl endopeptidase: BCDF standard)
(1: 200, molar ratio) was added and reacted at 37 ° C. for 6 hours to fragment BCDF. The reaction mixture was applied to a reversed-phase chromatography column μBondo Pack (0.21 x 30c
m), HPLC, eluate, 0.06% TFA
Fragments were separated and eluted with a linear increase in acetonitrile concentration from 0 to 60%. This results in HPL
C elution peaks 1-9 were obtained. The eluate of each peak is freeze-dried and then applied to the protein sequencer (Applied Biscel).
Osystem Co., Calf, Model 4704). The amino acid sequence is determined by J. Biol. Chem. 193 , 265-275.
It was performed by the method described in (1951). The fragment for which the amino acid sequence was confirmed and the amino acid sequence are described.

[0017]

Table 1 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-X-Y-Lys fragment 6 Asp-Val-Ala-Ala-Pro-X Note that X and Y could not be identified. Nos. 2 and 6 matched the N-terminal sequence of Reference Example 1.

Reference Example 3 Oligonucleotides encoding the amino acid sequence of BCDF obtained in Reference Examples 1 and 2 were synthesized as follows. Oligonucleotides were synthesized using a DNA synthesizer model 380A manufactured by Applied Biosystems, using silica gel as a solid phase carrier, and nucleotide binding reaction was carried out using the phosphite triester method. After removing the protecting group by a conventional method, the desired oligonucleotide was purified by C ₁₈ reverse-phase column HPLC using an acetonitrile gradient.

[0019]

[Chemical 1]

[0020]

[Chemical 2]

[0021]

[Chemical 3]

Reference Example 4 (a) 1 in 2 liter plastic roller incubator (Falcon # 3027) (hereinafter referred to as roller)
1 liter of RPMI1640 medium containing 20% FCS (2
mM glutamine, 5 × 10 ^-5 M 2ME, 100 units /
ml penicillin, 100 μg / ml streptomycin, 20 μg / ml gentamicin, 16 mM NaHCO ₃
Cells) were inoculated with 2 × 10 ⁵ cells / ml of VT-1 and cultured at 37 ° C. for 3 days while rotating at 8 rpm.
After culturing, the culture was centrifuged to collect the cells, and the cells were washed twice with PBS. The cells (1.8 × 10 ⁹ cells) were suspended in 800 ml of PBS solution and the cells were washed twice by centrifugation. Ribonucleosides-Van, a nuclease inhibitor
RSB solution containing adyl complex (10 mM) (10 m
M Tris-HCl, pH 7.5, 10 mM NaCl, 1.5 mM
gCl ₂ ) was suspended in 800 ml. Next, set NP-40 to 0.0
After adding 5%, gently stir 3000
Centrifuge at rpm for 5 minutes to remove cell debris containing nuclei, add SDS (final concentration 0.5%) and EDTA (final concentration 5 mM) to the supernatant, and immediately add an equal amount of phenol to the cytoplasm. RNA was extracted. After repeating phenol extraction three times in total, RNA was precipitated with 2 volumes of ethanol,
The precipitate was collected by centrifugation, 10 mM Tris-HCl, pH 7.5
It was dissolved in. The amount of RNA thus obtained from VT-1 cells was 30 mg.

Next, in order to obtain mRNA from this RNA, oligo (dT) -cellulose (PL Biochemical
s, Type 7) was used for column chromatography. Adsorption is 20 mM Tris-HCl, pH 7.5, 0.5M N
RNA was dissolved in aCl, 1 mM EDTA, 0.5% SDS solution, and the buffer solution (20 mM Tris-HCl, pH 7.
After washing with 5, 0.5 M NaCl, 1 mM EDTA), elution was performed with water and 10 mM Tris-HCl (pH 7.5) alternately with mRN.
This was done by eluting A. As a result, the amount of mRNA eluted was 576 μg. (B) Double-stranded cDNA was prepared using 5 μg of the mRNA prepared in (a). GUBLER, U and HOFFMAN, B, J.,
According to the method of (Gene 25 , 263, 1983), a double-stranded cDNA was prepared by the Amersham protocol using a cDNA synthesis kit (Amersham). That is, mRNA
Further, single-stranded cDNA was synthesized by reverse transcriptase, and nick and gap were formed in the RNA chain by using E. coli ribonuclease H with the hybrid of mRNA and cDNA as a substrate. Furthermore, double-stranded DNA was prepared by replacing the mRNA with DNA by a nick translation type reaction with E. coli DNA polymerase I. The small overhang at the 3'end is T
4 Double stranded cDNA was removed using DNA polymerase
A was produced. The final double-stranded cDNA was 1.08μ
It was g.

(C) The obtained double-stranded cDNA was 1.08 μm
sucrose density gradient centrifugation (50 mM Tris-HCl, 1 mM
Sucrose density gradient 5-2 in a solution containing EDTA and pH 7.5
5%, 40,000 rpm, 4 ° C for 13 hours), and a part of the fraction was analyzed by autoradiogram by agarose gel electrophoresis. Fractions with double-stranded cDNA size of 500 bp or more were collected. It was recovered by the ethanol precipitation method. The recovered double-stranded cDNA was about 0.6 μg. (D) 0.1M potassium cacodylate (Tris Base pH
7.2) 10 mM DTT, 2 mM CoCl ₂ ,
0.5 mM ³² P-dCTP (specific activity 1 × 10 ⁶ cpm / n mol
e), 0.6 μg double stranded cDNA and 50 units of deoxynucleotidyl terminal transferase (BR
L) was mixed and incubated at 24 ° C. for 20 minutes, then treated with phenol, and the cDNA fraction was collected through a Sephadex G-50 column to obtain 0.24 μg of dC-tailed cDNA as an ethanol precipitate. This cDNA had about 13 dCMP residues added to both 3'ends.

(E) On the other hand, as shown in FIG. 1, the cDNA expression vector pQ in monkey cells (COS cells) was replaced with pC.
EIL-2 (Nature, 302 , 305, (1983))
Built from. pQ can pinch the cDNA into the SV40 early promoter in both directions, and can express the peptide protein encoded by the cDNA in COS cells regardless of the direction of insertion of the cDNA. Also,
E. It is replicable in Coli and can be selected for resistance to tetracycline. This pQ was cleaved with PstI, and about 13 dG tails were added in exactly the same manner as the dC tail was attached to both ends of the 3'end of ds-cDNA. Next, this dG-tailed p Q 100ng and dC-tailed dS
-20 ng of cDNA was added to 50 mM Tris-HCl, pH 7.5, 0.1M N
Mix in a solution of aCl 1 mM EDTA, first at 65 ° C. for 2 minutes, then 45 ° C. for 60 minutes, 37 ° C. for 60 minutes,
Then, it was incubated at room temperature for 60 minutes. Then, the annealed DNA was treated with competent E. coli. Coli M
It was introduced into C1061. Next, the method for preparing competent cells of MC1061 and the method of introduction will be described below.

E. Coli MC1061 with 100 ml of Ψ
Medium (2% tryptone, 0.5 yeast extract, 0.5% MgSO 4
₄・ 7H ₂ O, pH 7.6) and the absorbance of the culture solution was 55
The cells were cultivated with shaking at 37 ° C. until they reached about 0.3 to 0.5 at 0 nm. After completion of the culture, the culture solution was kept at 0 ° C. for 5 minutes, the bacterial cells were collected by centrifugation, and TfbI (30 mM potassium acetate, 100 mM RbCl, 10 mM CaCl ₂ , 50 mM MnCl was added.
_2, 15% glycerol, was suspended in 40ml of pH 5.8),
It was left at 0 ° C. for 5 minutes. The cells were collected again by centrifugation, and Tfb II (10 mM MOPS or PIPES, 75 mM CaC
l ₂ , 10 mM RbCl, 10% glycerin, pH 6.5) 4
It was suspended in 0 ml and allowed to stand at 0 ° C for 15 minutes. This suspension was dispensed and stored at -70 ° C. Next, 100 μl of the competent cells thus prepared was kept at 0 ° C. for 15 minutes, in which the dG-tailed pQ vector and dC-tailed cells were prepared.
10 μl of preparation annealed with cDNA and 50 mM Mg
Mix 90 μl of Cl ₂ and 10 mM CaCl ₂ solution at 0 ° C.
Let stand for 20 minutes. Then, after heat treatment at 37 ° C. for 60 seconds, the mixture was kept at 0 ° C. for 1 to 2 minutes, 1 ml of Ψ medium was added thereto, and shaking culture was performed at 37 ° C. for 60 minutes. This culture medium was mixed with L medium containing 1 μg / ml tetracycline and 25 μg / ml streptomycin (1% tryptone, 0.
5% yeast extract, 0.5% NaCl, 0.1% glucose) was spread on an agar plate and incubated overnight at 37 ° C., whereby colonies appeared.

(F) About 150,000 transformed strains obtained
0 clones, using probes 8-1 and 8-2, Grunstein, M et al. Methods in Enzymology, 68, 3
When colony hybridization was performed by the method of 79 (1979), 10 clones that hybridized with 8-1 were recognized. Further, colony hybridization was carried out on these 10 clones using the probes 3-1 to 3-6 in the same manner to obtain one clone of the strain which hybridizes with the probe 3-2. The plasmid DNA carried by this clone was roughly extracted and purified, and after digestion with the restriction enzyme PstI, the cDNA insert was separated from the pQ vector by agarose gel electrophoresis. When Southern hybridization analysis was performed using probe 8-1, probe 3-2, probe N-2, or probe N-5, it hybridized with any probe. It did not hybridize with other probes. This plasmid DNA was named pBSF2-38. That is, it was revealed that the cDNA insert of the present pBSF2-38 had a gene sequence corresponding to the revealed partial amino acid sequence of the protein having BCDF activity, and the present cDNA was identified as the BCDF gene.

Reference Example 5 (a) MC1061 carrying the pBSF2-38 for preparing a large amount of pBSF2-38 plasmid DNA
20 μg / ml tetracycline and 25 μg / ml
100 μl of Ψ medium containing streptomycin was cultured and shake-cultured at 37 ° C. for 5 to 7 hours. Then final concentration 1
100 ml of a new Ψ medium containing chloramphenicol was added so that the concentration became 70 μg / ml, and the cells were further shake-cultured overnight. The plasmid DNA thus amplified was purified as follows. The culture solution was collected only by centrifugation and suspended in 5 ml of 50 mM Tris-HCl, pH 7.5-
After freezing at 80 ℃, thaw and then lysozyme (final concentration,
2 mg / ml), and left still at 0 ° C for 10 minutes, and then ED
Add TA (final concentration 0.1 M) and let stand at 0 ° C. for 10 minutes. Then add TritonX-100 (final concentration 0.1%)
Let stand for 60 minutes at 0 ° C. Then 30000 rpm, 30
Centrifuge for minutes and treat the supernatant with an equal volume of water saturated phenol. The aqueous layer was further treated with an equal volume of chloroform, and the aqueous layer was extracted, and the final concentration was 20 μg / m 2.
RNase was added to give 1 and incubated at 37 ° C. for 60 minutes. After that, 0.2 volume of 5M NaCl and 1/3 volume of polyethylene glycol are added, and the mixture is allowed to stand at 0 ° C. for 60 minutes and then centrifuged at 10,000 rpm for 20 minutes to recover the DNA precipitate.

Next, this precipitate was dissolved in 3.8 ml of water, and
After dissolution by adding g CsCl, 200 mg of 10 mg / ml EtBr
Ultracentrifugation fractionation is carried out at 40,000 rpm for 16 hours at 20 ° C. After the centrifugation is completed, the plasma DNA fraction is extracted, and EtBr is removed by performing extraction operation four times with 1 to 2 volumes of water-saturated n-butanol. After dialysis in H ₂ O to remove CsCl, add 1/10 volume of 3M sodium acetate pH 5.6,
Add another 2 volumes of cold ethanol and let stand overnight at -20 ° C. 80% of this ethanol precipitate was collected by centrifugation.
After washing with an aqueous ethanol solution and drying well, the precipitate was dissolved in 50 μl of 10 mM Tris-HCl, pH 7.5 to obtain a sample for monkey cell transfection.

(B) Method of Infecting Monkey COS-7 Cells with Plasmid COS-7 cells were suspended in RPMI containing 10% fetal calf serum at 1 × 10 ⁵ / ml, and 3 ml of this was suspended in a 6 cm Petri dish. And cultured overnight at 37 ° C. in a 5% carbon dioxide gas incubator. The culture supernatant was removed, fresh 3% RPMI containing 10% fetal bovine serum was added, and the cells were cultured at 37 ° C. in a 5% carbon dioxide gas incubator for 2 hours. After culturing, the supernatant was removed, and TBS (25 mM Tris-HCl, pH 7.5, 130
mM NaCl, 5 mM KCl, 0.6 mM Na ₂ HPO ₄ ) 2.5 m
It was washed once with l. The COS-7 cells were mixed with the plasmid mixture (TBS (+) (TBS containing 0.7 mM CaCl ₂ , 0.5.
(to which mM MgCl ₂ was added) 1.0 ml, plasmid DN
A2 μg and 10 mg / ml DEAE-dextran (50 μl) were added, and the mixture was incubated at 37 ° C. in a 5% carbon dioxide gas incubator for 4 hours. 2.5 ml of RPMI was added. After incubating at 37 ° C. in a 5% carbon dioxide gas incubator for 5 hours, the supernatant was removed and the cells were washed twice with 2.5 ml of TBS. 10 ml of RPMI containing fetal bovine serum was added, and the cells were cultured overnight at 37 ° C. in a 5% carbon dioxide gas incubator. The same RP after removing the supernatant
MI3 ml was added and the cells were cultured for 2 days in a 37 ° C. 5% carbon dioxide gas incubator. After this culture supernatant was spun down, the supernatant was used as a sample for measuring BCDF activity. The results of measuring BCDF activity of COS culture supernatant using CL4 are shown in FIGS. 2 and 3. COS-7 cells introduced with pBSF-38 plasmid DNA showed clear BCDF activity as compared with the control.

As described above, the recombinant human BCDF produced in the eukaryotic cell COS-7 has a culture solution containing anti-BCD.
F antibody immobilized column, then reverse phase HPLC as described above
(Synchron _C18 ). This BCDF
The cDNA structure contains a sugar chain because of its N-glycosylation position, and its physicochemical properties were in agreement with those of the purified BCDF purified by the VT-1 culture supernatant by the method of Reference Example 1. . That is, molecular weight: 3.5 ± 0.5 × 10 ⁴ Dalton (gel filtration method) 2.2 ± 0.2 × 10 ⁴ Dalton (SDS-polyacrylamide gel electrophoresis method) Isoelectric point: pH 4.9 to 5.1 Met.

Reference Example 6 BCDF cDNA insert cleaved with the restriction enzyme BamHI from pBSF2-38 prepared in Reference Example 5 (a).
As a probe, BCDF mRNA size, molecular species, B
The CDF mRNA producing strain was evaluated. MR used
NA is CESS, RP which is considered to produce BCDF in addition to VT-1 cells which produce this BCDF.
It was prepared from human tonsil cells stimulated with MI1788 and TPA, CL4, Jurkat, CEM having no BCDF activity, and unstimulated human tonsil cells by the same method as in (a) of Reference Example 4. Each mRNA 10 μg / 3.
6 μl, 5 × MOPS buffer (0.1 M MOPS (pH 7.0) 75
mM NaOAc, 5 mM EDTA) 6.0 μl, formaldehyde 5.4 μl and formamide 15.0 μl at 60 ° C.
The mixture was incubated for 15 minutes, and 3 μl of a dye solution (80% glycerol solution containing 0.05% bromophenol blue and 0.05% xylene cyanol) was added to this to prepare a control sample. This sample is 1 x MOPS buffer, 1.8
Electrophoresis was performed on a 1.6% agarose gel containing% formaldehyde, and then blotting was performed on a nitrocellulose filter by a conventional method. And this filter is 80 ℃
Bake for 3 hours. The filter prepared in this way is 0.1
After immersion in 3 × SSC containing 1% SDS, 1 × Denhardt's solution, 50% formamide, 5 × SSC, 250 μg / m
Prehybridization was carried out overnight at 42 ° C. in 50 mM sodium phosphate buffer (pH 6.5) containing 1 herring DNA. And 1 × Denhardt's solution, 50% formamide, 5 × SSC, 250 μg / ml herring DNA-containing 5
Hybridization was carried out overnight at 42 ° C. in 0 mM sodium phosphate buffer (pH 6.5) using the BamHI cDNA insert of ³² P-labeled pBSF2-38 as a probe.

Hybridized filter at room temperature at 0.
0.4 times at 50 ° C for 5 minutes 4 times in 2 × SSC containing 2% SDS.
Wash twice with 0.1% SSC containing 2% SDS for 30 minutes,
After air drying, an autoradiogram was prepared. As a result, V
T-1, CESS (BCDF producing strain), RPMI178
The cDNA probe of pBSF2-38 hybridized to the mRNA derived from 8. It did not hybridize to mRNAs derived from CL-4, Jurkat, CEM and BCESS non-producing BCDF which do not seem to produce BCDF. The size of mRNA that hybridizes with the pBSF2-38 cDNA probe was estimated to be approximately 15 to 16S. Part 1 is shown in FIG.

Example 1 Reference example 5 from MC1061 holding pBSF2-38
A plasmid DNA was obtained as described in (a) above and excised with a restriction enzyme BamHI to prepare BCDF cDNA. And the restriction enzyme map and the base sequence were determined. Nucleotide sequence is Maxam-Gilbert's chemical method (Meth. Enzym. 65 , 4,
99 ('80) and M13 phage (J. Messing el al.,
Gene, 19 , 269 ('82)) dideoxynucleotide chain assembly method (F. Sanger et. Al., Proc. Nat
l. Acad. Sci., USA 74, 5463 ('77). The determined nucleotide sequence and amino acid sequence are shown in FIG. A restriction enzyme map is shown in FIG. The human BCDF nucleotide sequence determined here correctly includes all the amino acid partial sequence structures disclosed in Reference Examples 1 and 2.

Reference Example 7 (1) The vector DNA used for expressing the human BCDF gene was constructed as follows. (FIG. 7) First, a DNA fragment (a) having the DNA sequence shown in FIG.
Each of (l) was synthesized by the solid phase phosphate triester method. Except for (a) and (g), the 5'end was phosphorylated using T4 polynucleotide kinase and ATP. Next, (a) to (l) are mixed, and after annealing, T4D
Double-stranded synthetic DNA (A) was formed using NA ligase. On the other hand, pT9-11 was modified with restriction enzymes HpaI and XbaI.
Large DNA by agarose gel electrophoresis
The fragments were separated. Next, the obtained pT9-11 fragment and synthetic DNA (A) (see FIG. 8) were mixed and ligated using T4 DNA ligase. The resulting recombinant DNA was introduced into the Escherichia coli HB101 strain, and a strain having ampicillin resistance was selected. From the isolated strains, a plasmid DNA was obtained, a digestion test with a restriction enzyme and a nucleotide sequence were determined to select a bacterium having pT13S (Nco) having synthetic HIL-2 cDNA.

(2) The plasmid pT13S (Nco) and
Recombinant DNA expressing human BCDF was constructed using pBSF2-38 as follows (FIGS. 9 and 10) i) Plasmid pT13S (Nco) was digested with restriction enzyme NcoI.
And digested with XbaI and the large DNA fragment was isolated and purified by agarose gel electrophoresis. On the other hand, plasmid p
BSF2-38 was cleaved with the restriction enzyme BamHI, and a small DNA fragment containing the human BCDF cDNA insert was recovered by agarose gel electrophoresis. And the BamH
The I human BCDF cDNA insert was completely digested with the restriction enzyme XbaI and further partially digested with MvaI. Next, a pT13S (Nco) fragment containing a tryptophan promoter / operator (trp p / o), an MvaI-XbaI fragment-containing DNA mixture containing human BCDF cDNA, and synthetic DNA.
(B) mixing the ^[5 'CATGCCAGTACCACC ^3' and 5 'ends were phosphorylated ^5' TGGTGGTACTGG ³ '], it was coupled with a T4DNA ligase. The resulting recombinant DNA was introduced into the Escherichia coli HB101 strain, and a strain having ampicillin resistance was selected. A strain having a DNA that hybridizes with the synthetic DNA (B) was selected from the obtained strains by the colony hybridization method. Bacteria that retain pTBCDF-01 were selected by obtaining a plasmid DNA from the isolated strain and performing a cleavage test with a restriction enzyme and determination of the nucleotide sequence near the binding site (p.
TBCDF / HB101).

Ii) The plasmid pBSF2-38 was digested with the restriction enzyme BanI, treated with DNA polymerase I (Klenow), digested with XbaI, and then separated by agarose gel electrophoresis to separate a DNA fragment of about 150 base pairs. iii) Use pTBCDF-01 obtained in i) with the restriction enzyme BamH
Cleaved with I, treated with DNA polymerase I (Klenow), and then X
Cleaved with baI and larger D for agarose gel electrophoresis
The NA fragment was recovered. iv) The two types of DNA fragments obtained in ii) and iii) were treated with T4
Ligation was done using DNA ligase. Obtained recombinant D
NA was introduced into the Escherichia coli HB101 strain, and a strain having ampicillin resistance was selected. Bacteria retaining pTBCDF-02 were selected by obtaining a plasmid DNA from the obtained strain and performing a cleavage test with a restriction enzyme (pTBCDF-02 / HB101 (FERM p-
9061)).

V) Escherichia coli HB carrying the plasmid pTBCDF-01 or pTBCDF-02
101 strains with 25 μg / ml streptomycin and 2
The cells were grown overnight at 37 ° C. in 10 ml of L medium (1% bactotryptone, 0.5% yeast extract, 0.5% NaCl, 0.1% glucose, pH 7.5) containing 5 μg / ml ampicillin. Then, 5 ml of the culture suspension was added to M9-casamino acid medium (0.6% Na ₂ HPO ₄ · 12H ₂ O, 0.3% KH ₂ PO ₄ , 0.05% N).
_{aCl, 0.1% NH 4 Cl,} 0.05% MgSO 4 · 7H 2 O, 0.001
47% CaCl ₂ , 0.2% glucose, 0.2% casamino acid,
0.02% L-leucine, 0.02% L-proline, 0.00
02% thiamine hydrochloride, 100 μg / ml ampicillin, 25 μg / ml streptomycin, pH 7.4) was inoculated and cultured at 28 ° C. for 3 hours. 25 μg /
3-indole acrylic acid (IAA) was added so that the amount became ml, and induction culture was performed at 23 ° C. for 21 hours. The cultured cells were centrifuged, and 20 mM Tris-hydrochloric acid (pH 7.5, 30 mM
It was washed with NaCl) and suspended in 8 ml of the same buffer. Thus, the protein produced in the bacterial cells is treated with 50 mM ED
Digestion with 1% sodium dodecyl sulfate (SDS) or 1 mg / ml lysozyme in the presence of TA followed by sonication (5
(0 W, 30 seconds). As shown in Table 1, a part of human BCDF cDNA on the 3'-end side is deleted, and a part of human IL-2 cDNA is bound to the deleted part. pTBCDF-01 / HB101 and pTBCDF-02 with the complete human BCDF cDNA
Both culture extracts of / HB101 showed BCDF activity.

[0039]

[Table 2] Table 1 BCDF activity (by reverse plaque method) of the culture extract of recombinant DNA-bearing bacteria ─────────────────────────── ────── Recombinant DNA-carrying bacteria BCDF activity (U / ml) ───────────────────────────────── pTBCDF -01 / HB101 800 pTBCDF-02 / HB101 28,000 ───────────────────────────────── Recombinant BCDF was purified and concentrated in the same manner as in Reference Example 1, and a single protein preparation was prepared using HPL using a reverse phase chromatographic column Synchropak RP-P (C ₁₈ ).
At C, it was eluted with an acetonitrile concentration of 50-55%. The amino acid sequence was also determined in the same manner as in Reference Example 1, and the N-terminal structure was confirmed. That is, pTBCDF-02 / HB1
The polypeptide produced by 01 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 LHE GLU 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 ALA PRO TLA 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

Reference Example 8 (1) Plasmid pT13S (Nco) (described in Reference Example 8)
And a human BCDF fusion protein (ΔHI with human interleukin-2 (HIL-2) using pBSF2-38.
Recombinant DNA producing L-2-BCDF) was constructed as follows. (FIGS. 11 and 12) i) The plasmid pT13S (Nco) was digested with the restriction enzyme BglII.
And XbaI, and a large DNA fragment was isolated and purified by agarose gel electrophoresis. On the other hand, plasmid pB
SF2-38 was cleaved with a restriction enzyme BamHI, and a small DNA fragment containing a human BCDF cDNA insert was recovered by agarose gel electrophoresis. And the BamHI
The human BCDF cDNA insert was completely digested with the restriction enzyme XbaI and partially digested with MvaI.

Next, pT13S containing the above promoter
(Nco) fragment, MvaI-X containing human BCDF cDNA
DNA mixture containing baI fragment and synthetic DNA (C) [ ⁵ '
JiATCTCTTCAGAGCCCCAGTACCCCC ³ 'and 5' ends were mixed and phosphorylated ⁵ 'TGGGGGTACTGGGGCTCTGAAGA ^3'] a, T
Ligation was performed using 4 DNA ligase. The resulting recombinant DNA was introduced into the Escherichia coli HB101 strain,
A strain having ampicillin resistance was selected. Synthetic DN from the obtained strain by colony hybridization method
A strain having a DNA that hybridizes with A (C) was selected. A bacterium carrying pTBCDF-11 was selected by obtaining a plasmid DNA from the isolated strain and carrying out a cleavage test with a restriction enzyme and determination of the base sequence in the vicinity of the binding site (pTBCDF-11 / HB101). ii) The plasmid pBSF2-38 was digested with the restriction enzyme BanI, treated with DNA polymerase I (Klenow), digested with XbaI, and then separated by agarose gel electrophoresis to separate a DNA fragment of about 150 base pairs. iii) The pTBCDF-11 obtained in i) was digested with the restriction enzyme BamH
Cleaved with I, treated with DNA polymerase I (Klenow), and then X
Cleaved with baI and larger D for agarose gel electrophoresis
The NA fragment was recovered.

Iv) Two types of DN obtained in ii) and iii)
The A fragment was ligated using T4 DNA ligase. The resulting recombinant DNA was introduced into the Escherichia coli HB101 strain, and a strain having ampicillin resistance was selected. Bacteria retaining pTBCDF-12 were selected by obtaining a plasmid DNA from the obtained strain and performing a cleavage test with a restriction enzyme (pTBCDF-12 / HB101,
(FERM P-9062). v) Human IL-2 cDN at the 5'end of BCDF cDNA
A part of A and a part of the 3'-terminal side are deleted, and a part of human IL-2 cDNA is linked to the deleted part of HB101 harboring plasmid pTBCDF-11 and complete BCDF cDNA. Human IL-2c on the 5'end side
Plasmid pTBCDF- in which a part of DNA is bound
Both HB101 holding 12 were cultured according to Reference Example 8 to obtain an extract. As shown in Table 2, both culture extracts contained B
It showed CDF activity.

[0044]

[Table 3] Table 2 BCDF activity of culture extract of recombinant DNA-bearing bacteria (by reverse plaque method) ─────────────────────────── ────── Recombinant DNA-carrying bacteria BCDF activity (U / ml) ───────────────────────────────── pTBCDF -11 / HB101 500 pTBCDF-12 / HB101> 25,600 ─────────────────────────────────

Example 2 pTBCDF-12 / HB101 (described in Reference Example 8) was cultured according to Reference Example 7, and the granules formed in the cells were extracted by the following procedure. The bacterial cells were collected by centrifugation, and concentrated to 10 times, 20 mM Tris- containing 30 mM NaCl.
After adding an HCl buffer solution (pH 7.5) and suspending it, 1 mg / ml of lysozyme and 0.05 M of EDTA were added thereto, and the mixture was stirred and allowed to stand on ice for 1 hour. Then, the cells were disrupted by ultrasonic disruption, and the granules were recovered by centrifugation at 10,000 rpm for 5 minutes. The granules were solubilized with 6M guanidine hydrochloride, and the ΔHIL-2-BCDF concentration was 100 μg / ml,
Then, the concentration was adjusted so as to obtain a 2M guanidine hydrochloride solution, and 1 mM of oxidized glutathione and 10 mM of reduced glutathione were added thereto, and the pH was adjusted to 8.0 at room temperature and the concentration was adjusted to 10-1.
It was left for 6 hours. Then, guanidine hydrochloride was removed by gel filtration with Sephadex G-25, and at the same time, a ΔHIL-2-BCDF-equivalent fraction which became a kallikrein reaction buffer solution was obtained. The molecular weight of this substance was almost the same as the value calculated from the amino acid composition by SDS-polyacrylamide gel electrophoresis.
As a result of assaying the amino acid sequence on the N-terminal side, it was confirmed to be the sequence of HIL-2. That is, this BCDF
The active polypeptide has the following amino acid sequence.

ALA PRO PHR 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 CYS ALA G 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 THLNLY MET 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 PHE ARG SERIES LEU GLN SER SER LEU ARG ALA LEU ARG GLN MET

(2) Cleavage with kallikrein 50 mM Tris-HCl buffer containing 113 mM NaCl,
80 μg of ΔHIL-2-BCDF obtained in pH 7.8
After reacting human plasma kallikrein with 37 ° C. for 15 hours, a fraction corresponding to Ala-BCDF was collected by reverse phase HPLC. As a result of analyzing the amino acid sequence near the N terminus with a protein sequencer, ΔHIL-2-BCDF
Was quantitatively converted to Ala-BCDF protein. In addition, "Ala-BCDF" is one in which one Ala is added to the N-terminus of BCDF, and specifically has the following 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 TVAL GLU LEU LEU 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 TLA THR A PRO APR 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) N-terminal A by aminopeptidase P
Removal of la Aminopeptidase P is described in Methods Enzymol. 19 , 52.
1 (1970). The Ala-BCDF solution obtained in (2) was added to 0.4 mM.
50 mM Tris-HCl buffer containing MnCl ₂ (pH 8.0)
Gel filtration through Sephadex G-25 equilibrated with
-The fraction equivalent to BCDF was obtained. A thus obtained
Aminopeptidase P was added to 50 μg of la-BCDF, reacted at 37 ° C. for 16 hours, and then BCDF was measured by reverse phase HPLC.
Equivalent fractions were collected. Furthermore, as a result of analyzing the amino acid sequence on the N-terminal side with a protein sequencer, Ala-
It was confirmed that BCDF was quantitatively converted to BCDF. Table 3 shows the activities of Ala-BCDF and BCDF.

[0050]

[Table 4] Table 3 BCDF activity of pTBCDF-12 / HB101 product (by reverse plaque method) ──────────────────────────── Produced protein BCDF activity (U / μg protein) ─────────────────────────── Ala-BCDF 20000 BCDF 20000 ─── ──────────────────────── BCDF activity is shown.

Reference Example 9 Recombinant D expressing human BCDF lacking C-terminal using plasmids pTBCDF-01 and pT9-11
NA was constructed as follows (Fig. 13) i) The plasmid pTBCDF-01 was digested with the restriction enzyme XbaI.
Cleaved with DNA, treated with DNA polymerase I (Klenow), Pvu
Small DNA containing trop / o and 3'deficient human BCDF cDNA by agarose gel electrophoresis after digestion with I
The fragments were separated. On the other hand, plasmid pT9-11 was digested with restriction enzymes BamHI and PvuI, and a large DNA containing trp A terminator was obtained by agarose gel electrophoresis.
The fragments were collected. Synthetic DNA both DNA fragments were adjusted in this way (C) ^[5 'CCTAGCCCGGGTGAATGAAT ^3'
When mixed with ⁵ 'GATCATTCATTCACCCGGGCTAGG ^3'], T4
Ligation was done using DNA ligase. Obtained recombinant D
NA was introduced into the Escherichia coli HB101 strain, and a strain having ampicillin resistance was selected. By obtaining plasmid DNA from the isolated strain and carrying out a restriction enzyme digestion test and determination of the nucleotide sequence near the binding site,
A bacterium carrying pTBCDF-03 was selected (pTBC
DF-03 / HB101). In addition, pTBCDF-03 is a plasmid having a gene in which part of the 3'end side of BCDF cDNA is deleted. In addition, pTBCDF-
The polypeptide produced by 03 / HB101 is considered to have 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 LHE GLU 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 ALA Same as Reference PHCBD-03 Then, a culture extract was prepared. As shown in Table 4, pTBC
The culture extract of DF-03 / HB101 showed BCDF activity.

[0053]

[Table 5] Table 4 BCDF activity (by reverse plaque method) of culture extract of recombinant DNA-bearing bacteria ─────────────────────────── ────Recombinant DNA-carrying bacteria BCDF activity (U / ml) ────────────────────────────── pTBCDF-03 / HB101 400 ──────────────────────────────

[0054]

[Brief description of drawings]

FIG. 1 shows a method for constructing a recombinant DNA for expression in monkey cells.

FIG. 2 pBSF 2-38 cD measured by Elisa method
8 shows BCDF activity of the culture supernatant of COS-7, a monkey cell into which NA was introduced.

FIG. 3 pBS measured by the reverse plaque method
Monkey cell COS into which F 2-38 cDNA has been introduced
The BCDF activity of the culture supernatant of -7 is shown.

FIG. 4 shows the northern blotting analysis results of pBSF 2-38 cDNA insert.

FIG. 5 shows the chlorine and amino acid sequences of BCDF.

FIG. 6 shows a restriction enzyme map.

FIG. 7 shows a construction diagram of plasmid pT13S (Nco).

FIG. 8: Synthetic human interleukin 2 (HIL-
The base sequence and restriction enzyme map of 2) are shown.

FIG. 9 shows a construction diagram of plasmid pTBCDF-01.

FIG. 10 shows a construction diagram of plasmid pTBCDF-02.

FIG. 11 shows a construction diagram of plasmid pTBCDF-11.

FIG. 12 shows a construction diagram of plasmid pTBCDF-12.

FIG. 13 shows a construction diagram of plasmid pTBCDF-03.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location // A61K 37/02 ABD 8314-4C ADU (C12N 1/21 C12R 1:19) (C12P 21 / 02 C12R 1:19) (72) Inventor Tadamitsu Kishimoto 3-5-31 Nakano, Tomitabayashi-shi, Osaka (72) Inventor Toshio Hirano 19 Mihogaoka, Ibaraki-shi, Osaka

Claims (6)

[Claims] 1. A purified polypeptide having human B cell differentiation factor (hereinafter referred to as BCDF) activity, having no natural signal peptide at its N-terminus and having no sugar chain,
The above polypeptide having Ala Pro as the N-terminal amino acid and Met as the C-terminal amino acid, and produced from a prokaryotic cell. 2. A purified polypeptide produced from a prokaryotic cell and having human B cell differentiation factor activity, comprising the following amino acid sequence (I). Amino acid sequence (I): 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 TRU CYS LEU VAL LEU GLU GILE 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 THR ASP R 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. A DNA encoding a polypeptide having the following amino acid sequence (I). Amino acid sequence (I): 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 TRU CYS LEU VAL LEU GLU GILE 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 THR ASP R 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 4. A recombinant DNA containing a gene encoding a polypeptide having the following amino acid sequence (I) and a vector DNA replicable in prokaryotic cells. Amino acid sequence (I): 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 TRU CYS LEU VAL LEU GLU GILE 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 THR ASP R 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 5. A prokaryotic cell transformed with a recombinant DNA containing a gene encoding a polypeptide having the following amino acid sequence (I) and a vector DNA replicable in a prokaryotic cell. Amino acid sequence (I): 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 TRU CYS LEU VAL LEU GLU GILE 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 THR ASP R 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 6. A prokaryotic cell transformed with a recombinant DNA containing a gene encoding a polypeptide having the following amino acid sequence (I) and a vector DNA replicable in a prokaryotic cell is cultured 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 in that the produced human BCDF is collected. . Amino acid sequence (I): 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 TRU CYS LEU VAL LEU GLU GILE 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 THR ASP R 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