JP2691323B2 - NF-IL6β gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to interleukin-6.
Nuclear factor (NF-) that induces the expression of the (IL-6) gene.
IL6β) gene, more specifically, it induces expression of IL-6, which is useful as a drug, by sequence-specifically binding to the nucleotide sequence of SEQ ID NO: 1, which is a palindromic structure existing in the transcriptional regulatory region of IL-6 gene. The present invention relates to a gene encoding a nuclear factor having the above-mentioned action, and the above nuclear factor can induce IL-6 production in vivo to perform hematopoiesis and the like.

[0002]

IL-6 is an important physiologically active factor which is involved not only in the immune system but also in the proliferation and differentiation of hematopoietic stem cells, the induction of acute phase proteins and the differentiation of nerve cells. The biological actions of the IL-6 can be roughly classified into (1) induction of cell differentiation or induction of certain gene expression, (2) induction of cell proliferation, and (3) suppression of cell proliferation. IL-6 has been shown to exert a completely different biological activity depending on the target cells (Hirano, Kishimoto, Experimental Medicine,
Vol.7, No.1, 1989, p11-12).

On the other hand, IL-6 is known to be abnormally produced in certain autoimmune diseases and cancer (Kishimoto T. and Hirano T., Molecular regulation.
of Blymphocyte response, Ann. Rev. Immunol. , 6, 48
5-512 (1988)). At the time of this disease associated with abnormal production of IL-6, as a result of analyzing the gene of cells that abnormally produce IL-6, no change such as gene mutation was found in the structural portion of the gene encoding IL-6 itself. It was speculated that the above-mentioned abnormal IL-6 production is caused as a result of abnormal nuclear factors that control gene transcription.

By the way, IL-6 is an interleukin-
It is known that its production is induced by stimulation with 1 (IL-1) (Kishimoto T. and Hirano T.,
Molecular regulation of B lymphocyte response, An
n. Rev. Immunol., 6 , 485-512 (1988)).

Therefore, the inventors of the present invention have investigated the tumor-forming glioblastoma cells of the brain glial cells (SK-MG4, K. Yasukawa e).
t al., EMBO Journal, vol.6, pp2939-2945 (1987)), which was convinced that there is a nuclear factor that directly acts on the gene of IL-6 by the action of IL-1, C-fos SRE (Tre
isman, R., Cell, 42 , 889-902 (1985)) exists and has a sequence-specific binding to the nucleotide sequence of SEQ ID NO: 1, which is a 14 bp palindromic structure in that region. It was confirmed that the nuclear factor was present, and the nuclear factor was first named NF-IL6 (1988).
Fiscal year, Japan Immunological Society, Akira et al., P. 281), and then changed the factor to C / EBP2 (Japanese Patent Application No. 2-27).
9650).

[0006]

The object of the present invention is to provide a novel nuclear factor belonging to the above C / EBP family, namely N.
The purpose is to isolate a gene encoding F-IL6β, determine its nucleotide sequence, and use the gene to produce the above nuclear factor by gene recombination technology.

[0007]

According to the present invention, IL-
A nuclear factor that binds in a sequence-specific manner to the nucleotide sequence of SEQ ID NO: 1, which is a palindromic structure existing in the transcriptional regulatory region of 6 genes, and encodes the amino acid sequence shown in SEQ ID NO: 2. IL-6 gene expression inducing nuclear factor gene (hereinafter referred to as "NF-IL6
"β gene") is provided.

The abbreviations for amino acids, peptides, base sequences, nucleic acids and the like in the following specification are IUPAC,
IUB regulations, "Guidelines for preparing specifications including nucleotide sequences or amino acid sequences" (edited by the Japan Patent Office), or the labeling methods commonly used in the art.

The gene of the present invention encodes an IL-6 gene expression-inducing nuclear factor (NF-IL6β) as described above, and enables production of a polypeptide having an amino acid sequence of the factor by a genetic engineering technique. The present invention also provides a technique for producing such recombinant NF-IL6β, which includes genetic information and can be used for producing the polypeptide. This recombinant has an action of inducing the expression of the IL-6 gene, and therefore, if this recombinant is applied to a living body, IL-6 is expressed in the living body.
Can be produced. Therefore, the above recombinants are useful as hematopoietic agents and the like by inducing IL-6 production.

The gene of the present invention will be described in detail below.

By the way, the base sequence shown in SEQ ID NO: 1 is
It has already been confirmed by the present inventors that it specifically binds to C / EBP2, and the base sequence can be synthesized by a generally known method as in Reference Example described later.

The determination of the gene sequence of the present invention and the production of the gene are specifically according to the examples described below, but generally as follows.

That is, first, the gene of the present invention is isolated by the method of Singh et al. (Singh, H. et al., Utilizing the binding property of the nuclear factor relating to the gene of the present invention to the specific base sequence.
Cell, Vol.52, 415-423, Febrary 12, 1988).

The mRNA necessary as a template for preparing the cDNA of the gene of the present invention can be extracted from appropriate animal-derived cells such as human placenta cells, human monocytic cells, human fibroblasts, etc. by a conventional method. it can. RNA above
Can be extracted using, for example, a guanidinium thiocyanate solution or a suitable surfactant such as SDS, NP-40, Triton X100, deoxycholic acid, or a physical method such as a method using a homogenizer or freeze-thawing. After complete or complete solubilization, chromosomal DN
A is for Polytron (POLYTRON, Kinematica Switzerlan
It can be carried out by shearing to some extent using a mixer or syringe such as d) and then separating the protein from the nucleic acid fraction. For this extraction operation, phenol
Chloroform extraction method and guanidinium / cesium chloride method [Guanidinium / Cesium Chloride method, T.
Maniatis, EF Fritsch and J. Sambrook, Molecular
Cloning, p194-196 (Cold Spring Harbor Laborator
y), 1982] etc. are generally used. In addition, in each of the above methods
To prevent RNA degradation by RNase, for example, RNase
Inhibitors, heparin, polyvinyl sulfate, diethylpyrocarbonate, vanadium complex, bentonite, macaloid and the like can be added and used.

MR from RNA obtained by the above procedure
NA is separated and purified by extracting the extract with, for example, oligo dT-cellulose [Collaborative Research Co.
Research Inc.)], poly U-sepharose [Pharmacia] or the like, or by a batch method.

The purified mRNA obtained as described above is usually unstable and is replaced by a stable complementary DNA (cDNA) type, and is ligated to a replicons derived from a microorganism to enable amplification of a target gene. The above mR in vitro
The conversion of NA into cDNA, that is, the synthesis of cDNA can be performed as follows.

That is, first, oligo dT is used as a primer (this primer may be a vector primer to which poly dT is added), and mRNA is used as a template for dNTP.
In the presence of (dATP, dGTP, dCTP or dTTP), reverse transcriptase is used to synthesize a single-stranded cDNA complementary thereto from mRNA. The next step differs depending on whether oligo dT or vector primer was used in the above, respectively.

In the former case, mRNA used as a template is decomposed and removed by alkali treatment or the like, and then double-stranded DNA is prepared by using reverse-transcriptase or DNA polymerase I with single-stranded DNA as a template. Double strand D obtained next
Both ends of NA are treated with exonuclease, a plurality of bases in a suitable linker DNA or an annealable combination is added to each, and this is incorporated into a suitable vector such as an EK-based plasmid vector or a λgt-based phage vector. In the latter case, an open circular plasmid in which a plurality of bases in the same annealable combination as described above are added while leaving the mRNA used as the template, and linker D
NA (often an autonomously replicating region and mRN in animal cells)
A DNA fragment containing the transcriptional promoter region of A is used) to form a closed circle, and then dNTP
RNase H and DNA polymerase I are allowed to coexist in the presence of DNA to replace mRNA with a DNA chain to obtain a complete plasmid DN.
Create A.

The DNA obtained as described above is used as a vector host, for example, Escherichia coli.
hia coli), Bacillus subtilis
s), Saccharomyces c.
erevisiae) and the like, and transformed into a suitable host. As a method for introducing this DNA into a host and a method for transforming the same, a generally used method, for example, collecting cells mainly in a logarithmic growth phase and treating with CaCl ₂ so that DNA is easily incorporated naturally, a plasmid is incorporated. It is possible to adopt a method of making it. In the above method, MgCl ₂ or RbCl can be further coexistent to further improve the efficiency of transformation, as is generally known. Alternatively, a method in which host cells are transformed into spheroplasts or protoplasts and then transformed can also be adopted. When a λ phage, which is generally used as a phage vector, is used as a vector, a cDNA library using λ phage can be prepared by in vitro packaging. As the cDNA library, commercially available cD
NA library, eg Clontech
It is also possible to use various cDNA libraries marketed by the company.

According to a preferred method of the invention, λgt
11 is used as a vector to prepare a cDNA library,
The lysogen was isolated by isopropyl β-
Immobilize on a nitrocellulose filter saturated with an inducer such as D-thiogalactopyranoside (IPTG).

The bacterium fixed on the nitrocellulose filter is cultivated and cultivated until the nuclear factor related to the gene of the present invention is sufficiently out of the bacterium. For example, when E. coli Y1090 used in the Examples described below is used as a host, it is generally cultivated at 42 ° C for 3 to 4 hours and then at 37 ° C overnight for lysis. Good. Then, the obtained nuclear factor can be immobilized on nitrocellulose and used for the following screening.

The nuclear factor relating to the gene of the present invention spotted on this filter is bound to the nucleotide sequence of SEQ ID NO: 1 which is preliminarily labeled with, for example, ³² P and specifically binds to the nuclear factor, Radioactive spots were identified by autoradiography, λ phage was eluted from the gel at that site, and the host, for example E. coli Y1090
And the same procedure is repeated to make a single clone.

From the isolated phage, a commonly known method (Molecular Cloning (A Laboratory Manual), T. Maniat
is, EFFritsch, J. Sambrook, ColdSpring Harbor Lab
oratory (1982)), etc., and DNA containing the gene of the present invention
Can be prepared.

A DNA restriction fragment can be prepared by digesting the gene obtained above with an appropriate restriction enzyme.

The fragment was transformed into a suitable plasmid vector such as pUC18 (C. Yanisch-Perron, J. Vieira and J. Mess.
ing, Gene, 33 , 103-119 (1985)) using a restriction enzyme and ligase to obtain a recombinant plasmid.

The obtained recombinant plasmid can be transduced into an appropriate host such as Escherichia coli, and the transformant thus obtained can be used in a generally known method such as Molecular Cloning (A Laboratory Manual). ),
T. Maniatis, EFFritsch, J. Sambrook, Cold Spring
According to the method described in Harbor Laboratory (1982) p104-106 and the like, a restriction enzyme map of a clone encoding the gene can be prepared.

The nucleotide sequence of the above clones can be determined by a known method, for example, the dideoxy method by Sanger et al. (Sanger F., Nicklen S. and Coulson AR, DNA se.
quencing with chain-terminating inhibitors, Proc.N
at.Acad.Sci., USA, 74 , 5463-5467 (1977)). Thus, it is possible to determine the total DNA sequence of NF-IL6β obtained above.

The above-mentioned gene of the present invention can be confirmed by a generally known method such as a footprinting method or a gel shift assay method (Aguilera, RJ et al., Cell, 51 , 909-917 (1).
987)) etc.

First, the confirmation method using the foot printing method can be performed as follows. That is, a clone containing the gene of the present invention isolated as described above is integrated into a suitable host, for example, E. coli Y1089,
In order to induce the expression of the gene of the present invention, it is cultured in a medium mixed with IPTG (Sigma) at 37 ° C. for about 1 hour, and after the culture is completed, freeze-thawing is repeated to obtain a cell extract. As a control, a crushed product that is not stimulated with IPTG is prepared. The amount of IPTG added is preferably about 10 mM.

C-containing a 14 bp palindrome to which the nuclear factor encoded by the gene of the present invention specifically binds
fos basal enhancer region (-17 of human IL-6 gene)
9-111 region, H. Isshiki et al., Mol. Cell.Bio
l., 10 , 2757-2764 (1990)) was labeled with a terminal end and partially methylated with dimethyl sulfate, and a mixture of this and the above-mentioned bacterial extract was electrophoresed in the same manner as the gel shift assay, The free band with no protein bound and the band with slow migration are collected, reacted with piperidine, and then run on a sequence gel. Thereby, the base to which the protein is bound can be specified.

Next, the confirmation by the gel shift assay method is performed as follows.

A part of the amino acid sequence of the DNA binding region of the NF-IL6β gene is chemically synthesized, and a polyclonal antibody using this as an antigen is prepared by a generally known method.
The portion that binds to DNA is C / EBP [Landschulz,
WH et al., Genes Dev., 2, 786-800 (1988); Lands
chulz, WH et al., Science, 240 , 1759-1764 (198
8)] can be presumed as a polypeptide portion corresponding to a gene portion having many basic amino acids having homology with the gene portion encoding the amino acid sequence binding to DNA, and NF-
As reported for IL6.

According to the reaction with the polyclonal antibody, NF-IL6β cannot bind to the DNA binding site on the gel shift assay, and the band with slow mobility is not formed.

The total DNA base sequence of the thus obtained NF-IL6β gene is shown in SEQ ID NO: 2.
The corresponding amino acid sequence is also shown in SEQ ID NO: 2.

The gene of the present invention can be directly isolated from a clone containing the gene of the present invention which has been already isolated by the present inventor, and can be easily obtained from the above-mentioned cDNA library using this as a probe. be able to.

Further, the gene of the present invention is based on the nucleotide sequence of SEQ ID NO: 2 and is, for example, phosphite triester
ture, 310, 105 (1984)) and the like can be used for chemical synthesis.

The gene of the present invention determined and produced as described above has the following characteristics. i) Encodes 269 amino acids. ii) The expression product has a leucine zipper structure on the C-terminal side. iii) The expression product has a portion rich in basic amino acids on the N-terminal side of the leucine zipper. iv) The expression product has a homology of 81.8% in the C / EBP-rich portion with the basic amino acids and 51.7% in the leucine zipper structure portion.

By using the thus obtained gene of the present invention, recombinant NF-IL6β related to the gene of the present invention can be easily produced and obtained in a large amount by a gene recombination technique.

This recombinant NF-IL6β of the present invention
The production method of can be basically according to various publicly known gene recombination techniques, using the above-mentioned gene (DNA) of the present invention as an essential [eg Science, 224 , 1431, (1984); B
iochem.Biophys.Res.Comm., 1 30, 692 (1985): Proc.N
atl. Acad. Sci., USA, 80, 5990 (1983): EP Publication No. 187991 Publication: Molecular Cloning, T. Maniatis
et al., Cold Spring Harbor Laboratory (1982), etc.].

More specifically, a recombinant DNA capable of expressing the gene of the present invention in a host cell may be prepared, introduced into a host cell for transformation, and the transformed strain may be cultured. Here, any of eukaryotes and prokaryotes can be used as host cells. The eukaryotic cells include cells such as vertebrates and yeasts. Examples of vertebrate cells include Jurkat cells and COS cells that are monkey cells [Y. Gluzman, Cell, 23 , 175-182
(1981)] and a dihydrofolate reductase-deficient strain of Chinese hamster ovary cells [G. Urlaub and LA Chas
in, Proc. Natl. Acad. Sci., USA, 77 , 4216-4220
(1980)] and the like are often used, but are not limited to these. As a vertebrate cell expression vector, a promoter located upstream of the gene to be normally expressed,
Those having an RNA splice site, a polyadenylation site, a transcription termination sequence and the like can be used, which may further have an origin of replication if necessary. As an example of the expression vector, CMV-NF-IL6 in which a CMV promoter, which is a cytomegalovirus-derived promoter, is ligated upstream of the gene of the present invention can be mentioned as a preferable example, but the present invention is not limited thereto. .

As the eukaryotic microorganism, yeast is commonly used, and among them, yeast of the genus Saccharomyces can be advantageously used. Examples of expression vectors for eukaryotic microorganisms such as yeast include pAM82 [A. Miyanohara et al, Proc.
Natl. Acad. Sci., USA, 80 , 1-5 (1983)] and the like can be used.

Escherichia coli and Bacillus subtilis are commonly used as prokaryotic hosts. In the present invention, a plasmid vector capable of replicating in the host bacterium is used, and a promoter and an SD (Shine-and-Synthesis) upstream of the gene are used so that the gene of the present invention can be expressed in the vector. It is preferable to use an expression plasmid having a Dalgarno) nucleotide sequence and a start codon (eg, ATG) necessary for initiation of protein synthesis. Examples of Escherichia coli as the host include Escherichia coli
) The K12 strain and the like are often used, and pBR322 is generally often used as the vector, but not limited to these and various known strains and vectors can also be used. The promoter, for instance the tryptophan (trp) promoter, lpp promoter, lac promoter, can be used P _L promoter, and the like, both capable of expressing the gene of the invention.

As a method for introducing the desired recombinant DNA thus obtained into a host cell and a method for transforming the same, various methods generally used can be adopted, and the expression plasmid into which the target gene has been inserted is the DEAE-dextran method. It can be incorporated into Jurkat cells by, for example, calcium phosphate-DNA co-precipitation method, and thus desired transduced cells can be easily obtained.

The desired transformant thus obtained can be cultured by a conventional method, and the nuclear factor related to the gene of the present invention is produced and accumulated by the culture. As the medium used for the culture, various types commonly used depending on the employed host cells can be appropriately selected. For example, culturing a transformant using Escherichia coli or the like as a host cell can be performed by using LB medium, E medium,
M9 medium, M63 medium, etc. can be used. In addition, various commonly known carbon sources, nitrogen sources, and
Inorganic salts, vitamins, natural product extracts, physiologically active substances and the like can also be added. Transduced cells using Jurkat cells as a host include, for example, RPMI-1640 medium and Dulbecco's modified Eagle's minimum essential medium (Dulbecc).
Culture can be performed using a medium such as o's modified Eagle's MEM) to which a serum component such as fetal calf serum (FCS) is added, if necessary. As the culture conditions of the transformant, conditions suitable for the growth of host cells can be adopted. In the case of Escherichia coli, for example, pH is about 5 to 8, preferably 7 or its vicinity, temperature is about 20 to 43 ° C., preferably 37. ℃ or the vicinity can be adopted. As described above, the target recombinant NF-IL6β is produced, accumulated or secreted into the transformant cell or outside the cell.

The NF-IL6β is subjected to various separation operations utilizing its physical and chemical properties (“Biochemical Data Book II”, pages 1175-1259, 1st edition, 1st edition, 1980, 6).
March 23rd, Tokyo Kagaku Doujinshi; Biochemistry, vol.
25, No.25, 8274-8277 (1986); Eur. J. Biochem., 163,
313-321 (1987), etc.) for separation and purification.
As the method, specifically, for example, a normal reconstruction process,
Treatment with protein precipitants (salting out method), centrifugation, osmotic shock method, ultrasonic disruption, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid Examples include various liquid chromatographies such as chromatography (HPLC), dialysis methods, and combinations thereof. As described above, the desired recombinant NF-IL6β can be easily produced in high yield and high purity on an industrial scale.

When the NF-IL6β obtained above is used as a medicine, it is preferable that the NF-IL6β is administered in such a dosage form that it is directly taken into the cell nucleus. Specifically, it is preferably used as a liposome preparation which is designed such that the surface thereof is appropriately modified and the drug directly encapsulated in the cell nucleus is incorporated.

[0047]

INDUSTRIAL APPLICABILITY According to the present invention, a gene for NF-IL6β is provided, which can be used to express and produce recombinant NF-IL6β by a genetic engineering technique, and the recombinant NF thus expressed is produced. −
IL6β is typically useful as a hematopoietic agent.

[0048]

EXAMPLES In order to explain the present invention in more detail, reference examples and examples will be given below.

[0049]

Example 1 Cloning of NF-IL6β gene (1) Plaque hybridization method [WD Benton, et al., Science, 1] using EMBL3 genomic library derived from human placenta (Clontech)
96 , 180 (1977)]. NF-IL6 cDNA as a probe [about 300 bp Spl I -Sac I fragment containing a DNA binding site: Akira, S., e
Tal., EMBO J., 9 , 1897-1906 (1990)] labeled with ³² P, hybridization was performed at 6 × S.
It was carried out in SC at 55 ° C. for 16 hours.

As a result, several positive clones were isolated, one of which was a clone (HPG13 clone).
Is a probe specific for NF-IL6 [of about 100 bp
Pst I / Eco RI fragment: Akira, S., et al., EMBO J., 9 ,
1897-1906 (1990)] and does not hybridize with NF-
It was considered to encode a novel IL-6 gene expression-inducing nuclear factor different from IL6.

Using the above HPG13 clone as a probe, NF- was prepared from the cDNA library as follows.
The IL6β cDNA was cloned. (2) NF-IL6β cDNA was obtained by screening using the antibody described in Reference Example below using a λgt11 expression library (Human Lung cDNA: Clontech).

That is, E. coli Y1090 and lambda gt11 phage were mixed for 15 minutes at 37 ° C. and then LB
It was plated on an LB plate together with agar and cultured at 42 ° C. for 3.5 hours. After that, a nitrocellulose filter saturated with 10 mM IPTG (isopropyl β-D-thiogalactopyranoside) was put on the plate and cultured overnight. Filter the filter with TBST (50 mM Tris-HCl buffer, pH 7.9,
Treated with 150 mM NaCl and 0.05% Tween-20) + 5% dry milk for 30 minutes at room temperature. After the TBST + dry milk was removed, the antibody described below was diluted 2000-fold into a buffer solution containing only TBST and treated for 1 hour. After washing with TBST, the cells were treated with TBST containing biotinylated anti-rabbit IgG for 1 hour. Finally, color was developed with avidin-conjugated alkaline phosphatase to obtain a positive clone. The agarose gel containing the periphery of the positive clone was dissolved, and the above operation was repeated to singlenize.

Thus, a positive clone designated as HLu5 was obtained.

By using the HLu5 cDNA insert as a probe, the same c
Several positive clones were obtained from the DNA library. The nucleotide sequences of these clones were determined by the method of Sanger et al. [Proc. Natl. Acad. Sci., USA, 74 , 5463-546.
7 (1977)].

By the analysis and ligation of the above clones, about 1.2 k coding for full-length NF-IL6β was obtained.
The cDNA of b was obtained.

The cDNA is a protein consisting of 269 amino acid residues whose molecular weight is calculated to be 28.4 kD, that is, NF-
It encodes IL6β. Due to the correspondence between the genomic sequence from the HPG13 clone and the above cDNA,
The NF-IL6β gene of the present invention includes NF-IL6 and C
As with / EBP, it was revealed that the intervening sequence was deleted. The nucleotide sequence of the NF-IL6β gene and the amino acid sequence encoded thereby are shown in SEQ ID NO: 2.

[0057]

Example 2 Construction of Expression Plasmid (1) The coding region of NF-IL6β was added to CMV-CAT.
After removing the CAT gene from the vector, it was inserted downstream of the cytomegalovirus promoter / enhancer to obtain the NF-IL6β expression plasmid CMV-N.
F-IL6β was obtained.

(2) Sac I -Pst I fragment from NF-IL6β cDNA clone was used as Sma of Bluscript SK ⁺ [purchased from Toyobo Co., Ltd., Stratagene Co.].
NF-IL6β was inserted into the I-Pst I site in frame with the β-galactosidase gene.
The expression plasmid BS-NF-IL6β was obtained.

(3) The above plasmid was transformed into E. coli
i JM105) and the fusion protein was synthesized by incubating the transformant strain to induce the synthesis of IPTG.

[0060]

[Example 3] Expression of NF-IL6β in Escherichia coli (1) Preparation of ptrpΔRBS Expression vector pTM1 having a tryptophan promoter / operator [Now the text man, metabolism 22:81,
1985] with the restriction enzymes Pst I and Bam HI,
A DNA fragment (I) of about 1.5 kb containing a tryptophan promoter operator was isolated.

On the other hand, plasmid pAT153 [Twigg,
AJand Sherratt, D., Nature, 283, 216 (1980)]
Was cleaved with restriction enzymes Pst I and Bam HI to isolate a DNA fragment (II) of about 2.6 kb. DNA thus obtained
Fragment (I) and DNA fragment (II) were ligated using T4 DNA ligase to obtain plasmid pNS1.

The plasmid pNS1 was digested with the restriction enzymes Hpa I and
Digested with Cla I and excluding 32 bp between Hpa I and Cla I D
The NA fragment (III) was isolated. This DNA fragment (III)
Oligonucleotide 5'-AACTAGTAC between Hpa I and Cla I of
GCAT-3 'and 5'-CGATGCGTACTAGTT-3' were ligated using T4 DNA ligase to obtain ptrpΔRBS.

The outline of the construction is shown in FIG. In the figure, Ap ^R indicates ampicillin resistance, Tc ^R indicates tetracycline resistance, and trp indicates tryptophan promoter. The same applies to the following figures.

From the second base of the transcription start point by the tryptophan promoter of pNS1
This vector lacks the Cla I recognition site.

(2) Preparation of various vectors having 5'untranslated region The outline of this method is as shown in FIG. 2, and was performed as follows.

That is, after digesting the plasmid ptrpΔRBS with the restriction enzyme Cla I, the alkaline phosphatase (Calf i
ntestine alkaline phosphatase) was used to obtain a DNA fragment (IV) having its 5'end dephosphorylated. Oligonucleotide linkers (shown in Table 1 below) constituting various 5'untranslated regions containing a ribosome binding site were ligated to this DNA fragment using T4 DNA ligase to obtain ptrp.
Ten plasmids of 690 to ptrp699 were obtained.

[0067]

[Table 1]

[0068]

[Table 2]

The series of plasmids thus obtained has a 5'untranslated region containing a ribosome binding site under the tryptophan promoter, and a restriction enzyme at its 3'end.
It has a ClaI site. Therefore, by connecting the foreign gene together with the initiation codon of the protein to the Cla I site or the Hind III site located immediately downstream thereof, a plasmid that most efficiently expresses the foreign gene product can be selected.

(3) Expression of NF-IL6β in Escherichia coli The plasmids obtained in (2) above and the expression plasmid prepared from the NF-IL6β gene were used for E. coli SG21058 [Maurizi, MR, Tr
isler, P. and Gottesman S., J. Bacteriol., 164 , 1124
(1985)].

Each of the resulting transformants was treated with 100 μg / m
LB medium containing 1 ampicillin [Maniatis, T., Frits
ch, EF and Sambrook, J., Molecular Cloning, A Labor
atory Manual, p440 (1982)]. 0.5 ml of this culture solution was added to 50 ml of a production medium [Bacto casamino acid (manufactured by Difco) 10 g / l, L-cysteine H).
Cl 20 mg / l and thiamine · HCl 10 mg / l in M9 medium (but containing glucose 4 g / l, see the above reference]], and shake culture at 37 ° C. in a 300 ml Erlenmeyer flask. 4.5 hours after the start of culture, 50 μl of 20 mg / ml 3β-indole acrylic acid (manufactured by Sigma) is added, and the culture is continued for another 4 hours.

After completion of the culture, NF-IL6β expressed and produced in Escherichia coli in each culture solution can be analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

[0073]

[Reference Example 1] Production of antibody 17 amino acids in the DNA binding domain of NF-IL6,
That is, Arg-Arg-Glu-Arg-Asn-Asn-Ile-Ala-Val-Arg-Lys-Se
A synthetic peptide corresponding to r-Arg-Asp-Lys-Ala-Lys was bound to ovalbumin, and this was immunized into a rabbit to obtain antiserum. Et al. Were purified by affinity chromatography using Sepharose 4B (Pharmacia) to which the above synthetic peptides were bound.

[Sequence list]

SEQ ID NO: 1 Sequence Length: 14 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: cDNA Sequence: ACATTGCACA ATCT 14

SEQ ID NO: 2 Sequence length: 1292 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA Sequence characteristics: Characteristic symbols: CDS Location: Characteristic determination Method: E Sequence: CACGTCAGCC GGGGCTAGAA AAGGCGGCGG GGCTGGGCCC AGCGAGGTGA CAGCCTCGCT 60 TGGACGCAGA GCCCGGCCCG ACGCCGCCCC ATG ACG GCC GCG CTC TTC AGC CTG 112 Met Thr Ala Ala Leu Phe Ser Leu 1 5 GAC GCC CGC GCT CGC GGC CGC GCT TTC 160 Asp Gly Pro Ala Gly Gly Ala Pro Trp Pro Ala Glu Pro Ala Pro Phe 10 15 20 TAC GAA CCG GGC CGG GCG GGC AAG CCG GGC CGC GGG GCC GAG CCA GGG 208 Tyr Glu Pro Gly Arg Ala Gly Lys Pro Gly Arg Gly Ala Glu Pro Gly 25 30 35 40 GCC CTA GGC GAG CCA GGC GCC GCC GCC CCC GCC ATG TAC GAC GAC GAG 256 Ala Leu Gly Glu Pro Gly Ala Ala Ala Pro Ala Met Tyr Asp Asp Glu 45 50 55 AGC GCC ATC GAC TTC AGC GCC TAC ATC GAC TCC ATG GCC GCC GTG CCC 304 Ser Ala Ile Asp Phe Ser Ala Tyr Ile Asp Ser Met Ala Al a Val Pro 60 65 70 ACC CTG GAG CTG TGC CAC GAC GAG CTC TTC GCC GAC CTC TTC AAC AGC 352 Thr Leu Glu Leu Cys His Asp Glu Leu Phe Ala Asp Leu Phe Asn Ser 75 80 85 AAT CAC AAG GCG GGC GGC GCG GGG CCC CTG GAG CTT CTT CCC GGC GGC 400 Asn His Lys Ala Gly Gly Ala Gly Pro Leu Glu Leu Leu Pro Gly Gly 90 95 100 CCC GCG CGC CCC TTG GGC CCG GGC CCT GCC GCT CCC CGC CTG CTC AAG 448 Pro Ala Arg Pro Leu Gly Pro Gly Pro Ala Ala Pro Arg Leu Leu Lys 105 110 115 120 CGC GAG CCC GAC TGG GGC GAC GGC GAC GCG CCC GGC TCG CTG TTG CCC 496 Arg Glu Pro Asp Trp Gly Asp Gly Asp Ala Pro Gly Ser Leu Leu Pro 125 130 135 GCG CAG GTG GGC CCG TGC GCA CAG ACC GTG GTG AGC TTG GCG GCC GCA 544 Ala Gln Val Gly Pro Cys Ala Gln Thr Val Val Ser Leu Ala Ala Ala 140 145 150 GGG CAG CCC ACC CCG CCC ACG TCG CGC GAG CCG CCG CGC AGC AGC CCC 592 Gly Gln Pro Thr Pro Pro Thr Ser Pro Glu Pro Pro Arg Ser Ser Pro 155 160 165 AGG CAG ACC CCC GCG CCC GGC CCC GCC CGG GAG AAG AGC GCC GGC AAG 640 Arg Gln Thr Pro Ala Pro Gly Pro Ala Arg Glu Lys Se r Ala Gly Lys 170 175 180 AGG GGC CCG GAC CGC GGC AGC CCC GAG TAC CGG CAG CGG CGC GAG CGC 688 Arg Gly Pro Asp Arg Gly Ser Pro Glu Tyr Arg Gln Arg Arg Glu Arg 185 190 195 200 AAC AAC ATC GCC GTG CGC AAG AGC CGC GAC AAG GCC AAG CGG CGC AAC 736 Asn Asn Ile Ala Val Arg Lys Ser Arg Asp Lys Ala Lys Arg Arg Asn 205 210 215 CAG GAG ATG CAG CAG AAG TTG GTG GAG CTG TCG GCT GAG AAC GAG AAG 784 Gln Glu Met Gln Gln Lys Leu Val Glu Leu Ser Ala Glu Asn Glu Lys 220 225 230 CTG CAC CAG CGC GTG GAG CAG CTC ACG CGG GAC CTG GCC GGC CTC CGG 832 Leu His Gln Arg Val Glu Gln Leu Thr Arg Asp Leu Ala Gly Leu Arg 235 240 245 CAG TTC TTC AAG CAG CTG CCC AGC CCG CCC TTC CTG CCG GCC GCC GGG 880 Gln Phe Phe Lys Gln Leu Pro Ser Pro Pro Phe Leu Pro Ala Ala Gly 250 255 260 ACA GCA GAC TGC CGG TAACGCGCGG CCGGGGCGGG AGAGACTCAG CAACGAlaCCA Asp Cys Arg 265 268 TACCTCAGAC CCGACGGCCC GGAGCGGACG CCCTGCTGCC GACGCCAGAG CCGCCGCGTG 995 CCCGCTGCAG TTTCTTGGAC ATAGACCAAA GAAGCTACAG CCTGGACTTA CCACCACTAA 1055 ACTGCGAGAG AA GCTAAACG TGTTTTTTTTT CCCTTAAATT ATTTTTGTAA TGGTAGCTTT 1015 TTCTACATCT TACTCCTGTT GATGCAGCTA AGGTACATTT GTAAAAAGAA AAAAAACCAG 1075 ACTTTTCAGA CAAACCCTTT GTATTGTAGA TAAGAGGAAA AGACTGAGCA TGCTCACTTT 1135 TTTATATACAATT

[Brief description of the drawings]

FIG. 1 shows a schematic diagram of the production of plasmid ptrpΔRBS carried out according to Example 1 (1).

FIG. 2 shows a schematic diagram of preparation of plasmids ptrp690 to ptrp699 performed according to Example 3 (2).

Claims (2)

(57) [Claims] 1. A gene for NF-IL6β, which encodes the amino acid sequence shown in SEQ ID NO: 2. 2. NF-IL containing the gene according to claim 1.
6β expression plasmid.