JP2001054395A - PROTEIN INDUCING FORMATION OF INTERFERON-gamma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new protein having respectively specific molecular weight, isoelectric point and partial amino acid sequence, inducing the formation of interferon-γ in an immunopathologically mediated tissue and useful e.g. as a treating and preventing agent for viral diseases, malignant tumor, immunologic diseases, etc. SOLUTION: The objective protein is a new protein having a molecular weight of 19,000±5,000 Dalton measured by SDS polyacrylamide gel electrophoresis or gel-filtration method and an isoelectric point of 4.8±1.0 measured by chromatofocusing method and exhibiting a biological action to induce the formation of interferon-γ in an immunopathologically mediated tissue or a protein immunologically equivalent to the above protein. The protein is useful e.g. as a treating agent or preventing agent for viral diseases, malignant tumor and immunologic diseases in general. The protein can be produced by preparing a cDNA library using an mRNA collected from mouse liver cell, searching the library with a probe, inserting the obtained gene into a vector and expressing the product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel protein that induces the production of interferon-γ (hereinafter abbreviated as “IFN-γ”) in immunocompetent cells.

[0002]

2. Description of the Related Art IFN-γ is known as a protein having an antiviral action, an antitumor action, and an immunoregulatory action, and is said to be produced by immunocompetent cells stimulated by an antigen or mitogen. Because of these biological effects, IFN-
Since its discovery, γ has been put to practical use as an antitumor agent, and is currently being vigorously studied in clinical trials as a therapeutic agent for general malignant tumors such as brain tumors. Currently available IFN-γ is natural IFN produced by immunocompetent cells.
-Γ, and recombinant IFN-γ produced by a transformant obtained by introducing DNA encoding IFN-γ collected from an immunocompetent cell into Escherichia coli. Are administered as “foreign IFN-γ”.

[0003] Among them, natural IFN-γ is usually produced by culturing immunocompetent cells established in a culture in a culture medium containing an IFN-γ inducer and purifying the culture. In this method, the type of IFN-γ inducer is IFN-γ
-It is said that it has a great effect on the production amount of γ, the ease of purification, and the safety of the product. Usually, concanavalin A, lentil lectin, American pokeweed lectin, endotoxin, lipopolysaccharide, etc. Mitogens are frequently used. However, all of these substances have a problem in that the molecules are diverse, the quality is easily varied depending on the source and the purification method, and it is difficult to obtain a desired amount of an IFN-γ inducer having a constant inducibility. In addition, many of the above substances show remarkable side effects when administered to a living body or even show toxicity depending on the substance, and it is extremely difficult to induce IFN-γ production by directly administering to a living body. there were.

[0004]

In view of such circumstances, an object of the present invention is to provide a novel protein that induces the production of IFN-γ in immunocompetent cells.

[0005] Another object of the present invention is to provide a DNA encoding such a protein.

[0006] Still another object of the present invention is to provide such a DN.
A to provide a replicable recombinant DNA comprising A and an autonomously replicable vector.

[0007] Still another object of the present invention is to provide a transformant obtained by appropriately introducing such a recombinant DNA into a host.

[0008] Still another object of the present invention is to provide a recombinant DN
An object of the present invention is to provide a method for producing the protein by applying the technology A.

[0009]

According to the present invention, the above first object is solved by a protein having the following physicochemical properties. (1) Molecular weight It shows a molecular weight of 19,000 ± 5,000 daltons when measured by SDS-polyacrylamide gel electrophoresis or gel filtration. (2) Isoelectric point When measured by the chromatofocusing method, 4.8 ± 1.
0 indicates the isoelectric point. (3) Partial amino acid sequence It has a partial amino acid sequence shown in SEQ ID NOS: 1 and 2 in the sequence listing. (4) Biological action Induces IFN-γ production in immunocompetent cells.

[0010] The present invention solves the above second problem by a DNA encoding such a protein.

The present invention solves the above-mentioned third problem by using the D
The problem is solved by a replicable recombinant DNA comprising NA and an autonomously replicable vector.

The present invention solves the above-mentioned fourth problem by using the D
The problem is solved by a transformant obtained by appropriately introducing a replicable recombinant DNA containing NA and an autonomously replicable vector into a host.

The present invention solves the fifth problem by a method for producing a protein, which comprises culturing a transformant capable of producing the protein in a nutrient medium and collecting the produced protein from the culture. is there.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, the protein of the present invention has unique physicochemical properties not found in conventionally known proteins.
Induces production of FN-γ.

The DNA of the present invention is inserted into an appropriate autonomously replicable vector to obtain a recombinant DNA.
The production of the protein is expressed by introducing A into a host that does not naturally produce the protein but can be easily grown to obtain a transformant.

The replicable recombinant DNA of the present invention expresses the production of the protein when introduced into a host which does not naturally produce the protein but can be easily propagated to obtain a transformant. .

The transformant of the present invention produces the protein when cultured.

When such a transformant is cultured according to the production method of the present invention, a desired amount of protein can be obtained relatively easily.

Hereinafter, the present invention will be described with reference to Examples, Experimental Examples, and the like.
This is based on the discovery of a novel protein that induces N-γ production. The present inventors have studied cytokines produced by mammalian cells, and have found that there is a completely unknown substance that induces IFN-γ production in mouse liver. When this substance was isolated by combining various purification methods, mainly column chromatography, and its properties and properties were examined, it was found that the substance was a protein and had the following physicochemical properties. found. (1) Molecular weight It shows a molecular weight of 19,000 ± 5,000 daltons when measured by SDS-polyacrylamide gel electrophoresis or gel filtration. (2) Isoelectric point When measured by the chromatofocusing method, 4.8 ± 1.
0 indicates the isoelectric point. (3) Partial amino acid sequence It has a partial amino acid sequence shown in SEQ ID NOS: 1 and 2 in the sequence listing. (4) Biological action Induces IFN-γ production in immunocompetent cells.

Next, a series of experiments for elucidating these physicochemical properties will be described.

[0021]

[Experimental example 1] <Preparation of purified protein> 8-week-old female CD-1
1 mg / animal of dead cells prepared by heating Corynebacterium parvum (ATCC11827) at 60 ° C. for 1 hour was injected into the abdominal cavity of 600 mice. Escherichia coli-derived purified lipopolysaccharide was injected at 1 μg / animal. One to two hours later, the mice were sacrificed by dislocating the cervical vertebrae, and after collecting blood from the heart, the liver was excised.
It was crushed and extracted with a homogenizer in a double volume of 50 mM phosphate buffer (pH 7.3). About 8,000 extracts
After centrifugation at 20 rpm for 20 minutes, about 9 l of the obtained supernatant was added to a 50 mM phosphate buffer (pH
7.3) was added so that ammonium sulfate would be 45% saturated, and allowed to stand at 4 ° C. for 18 hours.
After centrifugation for 0 minutes, about 19 l of a supernatant containing the protein was obtained.

About 4.6 of "Phenyl Sepharose" manufactured by Pharmacia, which was previously equilibrated with a 50 mM phosphate buffer (pH 7.3) containing 1 M ammonium sulfate.
After washing the column with fresh identical buffer, 50 mM phosphate buffer (pH 7.3) was added to the SV under a gradient of ammonium sulfate from 1 M to 0.2 M.
The solution was passed at 0.57. 0.8M ammonium sulfate concentration
Fraction containing the protein eluted at around 4.8 l
Was collected, membrane-concentrated, and 20 mM phosphate buffer (pH 6.
After dialyzing against 5) at 4 ° C. for 18 hours, the mixture was loaded on a column of about 250 ml of “DEAE-Sepharose” manufactured by Pharmacia which had been equilibrated with 20 mM phosphate buffer (pH 6.5) in advance. After washing the column with fresh identical buffer,
Under a concentration gradient of sodium chloride rising from
20 mM phosphate buffer (pH 6.5) was added to the column with SV1.
When the solution was passed through step 2, the protein was eluted at a sodium chloride concentration of about 0.13 M.

Approximately 260 ml of the eluate containing the protein is collected, concentrated, and added to a 25 mM bis-Tris buffer (pH 7.0).
After dialyzing against 1) at 4 ° C. for 18 hours, Pharmacia “Mono” previously equilibrated with the same fresh buffer solution
-P "is loaded on a column of about 24 ml and the pH is changed from pH7 to pH4.
When 10% (v / v) polybuffer 74 (pH 4.0) was passed through the column under a pH gradient falling to
Was about 4.8, the protein was eluted. About 23 ml of the eluate containing the protein is collected, concentrated, and
A mixture of disodium hydrogen phosphate, 3 mM sodium dihydrogen phosphate and 139 mM sodium chloride (pH 7.
The column was loaded on a column of "Superdex 75" manufactured by Pharmacia, which had been equilibrated in 2), and the same mixture was passed through a fresh mixture and subjected to gel filtration chromatography. As a result, the protein was eluted at around 19,000 daltons in molecular weight. . The fraction containing the protein was collected, concentrated, and subjected to the following Experimental Example 2
Was served. The yield was about 0.6 μg per mouse.

[0024]

[Experimental example 2] <Physicochemical properties of protein>

[0025]

[Experimental example 2-1] <Molecular weight> The purified protein prepared in Experimental example 1 was prepared by U.K.
Volume, pages 680 to 685 (1970), followed by SDS-polyacrylamide gel electrophoresis in the absence of a reducing agent.
A major band capable of inducing IFN-γ was observed at a position corresponding to 5,000 daltons. The molecular weight markers at this time were bovine serum albumin (67,000 daltons), ovalbumin (45,000 daltons), soybean trypsin inhibitor (20,100 daltons) and α-lactalbumin (14,400 daltons). Was.

[0026]

[Experimental example 2-2] <Isoelectric point> The purified protein was subjected to chromatofocusing according to a conventional method.
The isoelectric point is shown at 0.

[0027]

[Experimental example 2-3] <Partial amino acid sequence> An aliquot of the aqueous solution containing the purified protein prepared in Experimental example 1 was taken to about 50 μl.
Concentrated. 3% (w / v) SDS in concentrate, 60%
(V / v) glycerol and dithiothreitol 60 m
After adding 25 μl of a mixed solution consisting of g / ml and incubating at 50 ° C. for 30 minutes, the mixture was transferred onto a 15% (w / v) polyacrylamide gel and electrophoresed according to a conventional method. Thereafter, the gel was immersed in a mixture of 50% (v / v) aqueous methanol containing 0.1% (w / v) Coomassie Brilliant Blue R250 and a 10% (v / v) acetic acid aqueous solution to be stained, and then stained. (V / v) aqueous methanol and 7% (v / v)
Decolorize by repeatedly rinsing with a mixed solution of acetic acid aqueous solution.
After washing by immersion for 8 hours, a part containing the protein stained with Coomassie brilliant blue was cut out from the gel and freeze-dried.

Next, the dried gel was washed with 100 mM sodium bicarbonate containing 0.5 μg / ml of “TPCK trypsin” manufactured by Sigma, 0.5 mM calcium chloride and 0.02% (v / v).
v) The protein was immersed in 0.6 ml of a mixture of Tween 20 aqueous solution and incubated at 37 ° C. for 18 hours to digest the protein with trypsin. Then, the digest was centrifuged to collect the supernatant, while the sediment was removed by 0.001% (v / v) T
It was immersed in 1 ml of 1% (v / v) aqueous trifluoroacetic acid containing ween 20, shaken at room temperature for 4 hours, and centrifuged to collect the supernatant. 0.001% of newly formed precipitate
70% (v / v) aqueous trifluoroacetic acid containing (v / v) Tween 20, 0.001% (v / v) Tween
n20 containing 50% (v / v) aqueous trifluoroacetic acid and 50% (v / v) aqueous acetonitrile were treated in the same manner as described above, and the obtained supernatant and the supernatant obtained above were pooled. After concentrating to 250 μl, the mixture was centrifugally filtered.

The thus obtained aqueous solution containing the peptide fragment was previously equilibrated with 0.1% (v / v) aqueous trifluoroacetic acid, a column for high performance liquid chromatography manufactured by Tosoh “HPLC ODS-120T”. ]
After the column was washed with 0.1% (v / v) aqueous trifluoroacetic acid, the peptide concentration in the eluate was measured using an absorptiometer.
0% while monitoring under wavelengths of 4 nm and 280 nm
Under a concentration gradient of aqueous acetonitrile increasing from (v / v) to 70% (v / v), 0.1% (v / v) trifluoroacetic acid was passed through the column at a flow rate of 0.5 ml / min. Then, fractions eluted about 75 minutes or about 55 minutes after the start of the passage (hereinafter, referred to as “peptide fragment A” or “peptide fragment B”, respectively) were separately collected. The elution pattern at this time is shown in FIG.

The amino acid sequences of these peptide fragments A and B were examined using a protein sequencer “473A” manufactured by Perkin-Elmer according to a conventional method.
Each had the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing.

[0031]

[Experimental example 2-4] <Biological action>

[0032]

[Experimental example 2-4] <(a) IFN in immunocompetent cells
Induction of -γ Production Spleens were excised from female BDF1 mice> 8 weeks of age, and serum-free RPMI1640 medium (pH 7.
4), washed with the same fresh medium, and immersed in a gay buffer (pH 8.0) to cause hemolysis. RPMI1 obtained by supplementing the obtained splenocytes with 10% (v / v) fetal bovine serum
Cell density of 1 × 10 ⁷ cells / m in 640 medium (pH 7.4)
After the suspension, the suspension was loaded on a nylon wool column for cell separation manufactured by Wako Pure Chemical Industries, Ltd., and incubated at 37 ° C. for 1 hour in a 5% CO ₂ incubator. afterwards,
RPM with column supplemented with 10% (v / v) fetal calf serum
T cells were collected by passing through an I1640 medium (pH 7.4), washed with fresh identical medium, and subjected to the IFN-γ induction test described below.

Mouse T cells suspended in RPMI 1640 medium (pH 7.4) at a cell density of 1 × 10 ⁷ cells / ml were placed on a 96-well microplate at 0.15 m
The purified protein was appropriately diluted with an RPMI1640 medium (pH 7.4) supplemented with 10% (v / v) fetal bovine serum, and 0.05 ml of the diluted protein was added. Then, in the presence of 0.5 μg / ml concanavalin A or The cells were cultured in the absence of a 5% CO ₂ incubator at 37 ° C. for 24 hours. Thereafter, 0.1 ml of the culture supernatant was collected from each well, and the produced I
FN-γ was measured by a conventional enzyme immunoassay. At the same time, the same system was provided except that the purified protein was omitted, and this was treated in the same manner as above to serve as a control. Note that IFN-γ
As a standard, a standard mouse IFN-γ (Gg02-901-533) obtained from the United States National Institutes of Public Health was used and converted to international units (IU) for display.

As a result, significant IFN-
No production of γ was observed, whereas in the system to which the purified protein was added, significant production of IFN-γ was observed.
At a dose of 02 to 10 μg / ml, in the absence of Concanavalin A, about 2 to 2 per 1 × 10 ⁶ mouse T cells
00IU, about 2 to 2.0 in the presence of Concanavalin A
00IU of IFN-γ was produced. This supports that the protein has an effect of inducing the production of IFN-γ in immunocompetent cells.

Throughout the present invention, one unit of the protein is defined as the amount of a protein that induces 160 IU of IFN-γ when tested in the presence of concanavalin A as described above.

[0036]

Experimental Example 2-4 <(b) Enhancement of killer cell cytotoxicity> 100 μg / ml kanamycin, 5 × 10 ⁻⁵ M 2
-Mouse splenocytes prepared in the same manner as in Experimental Example 2-4 (a) in RPMI1640 medium (pH 7.2) containing mercaptoethanol and 10% (v / v) fetal calf serum, at a cell density of 1 × 10 ⁷ cells / ml. After adding 0, 1, 5 or 10 u / ml of recombinant human interleukin 2, the mixture was placed in a 25 ml culture flask. Purified protein was added to cell suspension in culture flask at 0, 0.8, 4, 2
Add 0 or 100 units / ml and incubate at 37 ° C. for 72 hours in a 5% CO ₂ incubator to obtain fresh RPMI164
After washing with 0 medium (pH 7.2), spleen cells were collected from YAC-1 cells (ATCC) previously labeled with radioactive sodium chromate.
TIB160) with effector / target cell ratio 20:
The cells were suspended in a fresh RPMI 1640 medium (pH 7.2) at a ratio of 1 or 40: 1. The cell suspension was placed in a 96-well microplate, cultured at 37 ° C. for 4 hours in a 5% CO ₂ incubator, and the radioactivity due to ⁵¹ Cr in the culture supernatant was measured by a gamma counter. Table 1 shows the results
Shown in

The results in Table 1 show that the protein of the present invention has the property of significantly enhancing the cytotoxicity of killer cells, and that the property is significantly enhanced by interleukin 2.

[0038]

[Table 1]

Proteins having the above physicochemical properties have not been known yet, and are considered to be novel substances.
Therefore, the present inventors isolated mRNA from mouse hepatocytes, and performed RT-PCR using the mRNA as a template in the presence of a primer chemically synthesized based on the partial amino acid sequence identified in Experimental Example 2-3. A DNA fragment partially encoding a protein was collected, and this was used as a probe to prepare the above mRN.
As a result of earnestly searching a cDNA library separately prepared from A, a DNA fragment consisting of 471 base pairs and having a base sequence from the 5 'end shown in SEQ ID NO: 4 in the sequence listing was obtained. Decoding this nucleotide sequence revealed that the protein was:
It was found to have an amino acid sequence consisting of 157 amino acids from the N-terminal shown in SEQ ID NO: 3 in the sequence listing. In addition, in SEQ ID NO: 3 in the sequence listing,
Amino acids denoted by the symbol “Xaa” shall mean methionine or threonine.

A series of operations leading to elucidation of the amino acid sequence and base sequence shown in SEQ ID NOs: 3 and 4 in the sequence listing are summarized as follows. (1) The protein was isolated from mouse hepatocytes by a combination of various purification methods centering on chromatography, and highly purified. (2) The purified protein was digested with trypsin, two types of peptide fragments were isolated from the digest, and their amino acid sequences were determined. (3) mRNA is collected from mouse hepatocytes, and is used as a template to perform a RT-PCR reaction in the presence of an oligonucleotide primer chemically synthesized based on the above partial amino acid sequence to prepare a DNA fragment. The DNA fragments were searched using oligonucleotides chemically synthesized based on the above as a probe, and DNA fragments partially encoding the protein were collected. (4) Separately, a cDNA library was prepared using the mRNA as a template and hybridized with the DNA fragment prepared above as a probe, and a transformant showing a remarkable association was collected. (5) cDNA is collected from the transformant, its base sequence is determined, decoded, and the decoded amino acid sequence is compared with the partial amino acid sequence to confirm that the base sequence encodes the protein. confirmed.

In the following Experimental Example 3, the steps (3) to (5) will be specifically described. However, the method itself used in this example is well known in the art. It is also described in detail in "Molecular Cloning A Laboratory Manual", 1989, published by Cold Spring Harbor and Masami Muramatsu, "Lab Manual Genetic Engineering", 1988, published by Maruzen Shuppan.

[0042]

[Experimental example 3] <Base sequence of DNA and amino acid sequence of protein>

[0043]

[Experimental example 3-1] <Preparation of total RNA> 3 g of mouse hepatocytes prepared in the same manner as in Experimental example 1 were taken by wet weight and weighed 6 g.
It was immersed in 20 ml of a mixed solution (pH 7.0) composed of M guanidine isothiocyanate, 10 mM sodium citrate and 0.5% (w / v) SDS, and crushed with a homogenizer. Next, 0.1 M EDTA containing 5.7 M cesium chloride (pH
7.5) was injected in an amount of 25 ml.
0 ml, and in this state, 20 ° C., 25,000 rpm
After ultracentrifugation at for 20 hours, the RNA fraction was collected. This RNA fraction is placed in a 15 ml centrifuge tube, an equal volume of a chloroform / isobutanol mixture (4: 1) is added, the mixture is shaken for 5 minutes, and centrifuged at 10,000 rpm at 4 ° C. for 10 minutes. The sample was collected, 2.5 volumes of ethanol was added, and the mixture was allowed to stand at −20 ° C. for 2 hours to precipitate total RNA.
The precipitate was collected, washed with 75% (v / v) aqueous ethanol, dissolved in 0.5 ml of sterilized distilled water, and subjected to the following experiment. The yield of total RNA was about 4 mg.

[0044]

[Experimental Example 3-2] <Preparation of DNA fragment partially encoding protein> 25 μm was added to 1 μg of the total RNA obtained in Experimental Example 3-1.
4 μl of M magnesium chloride, 10 × PCR buffer (1
00 mM Tris-HCl buffer (pH 8.3), 500 m
M potassium chloride), 8 μl of 1 mM dNTP mix, 1 unit / μl of RNase inhibitor
μl, 2.5 units / μl reverse transcriptase, 1 μl and 2.
1 μl of 5 μM random hexamer was added and made up to 20 μl with sterile distilled water. Take the mixture into a 0.5 ml reaction tube,
Incubation was performed at 25 ° C. for 10 minutes, at 42 ° C. for 30 minutes, at 99 ° C. for 5 minutes, and at 5 ° C. for 5 minutes according to a conventional method, and a reverse transcriptase reaction was performed to obtain an aqueous solution containing first-strand cDNA.

The first strand cDNA aqueous solution 20 μm
4 μl of 25 mM magnesium chloride per l, 10 × PCR
8 μl of buffer, 2.5 units / μl Amplitac DN
A polymerase was added in an amount of 0.5 μl, and primers 1 and 2 were added at 1 pmol each as a sense primer or an antisense primer, and then adjusted to 100 μl with sterile distilled water. According to a conventional method, a cycle of incubating the mixture at 94 ° C. for 1 minute, 45 ° C. for 2 minutes, and 72 ° C. for 3 minutes was repeated 40 times, and the reaction was carried out. The encoding DNA fragment was amplified. In addition, the primer 1 and the primer 2 are Pro-Glu-Asn-Ile-Asp-Asp in SEQ ID NOS: 1 and 2 in the sequence listing.
-Ile or Phe-Glu-Asp-Met-Thr
Oligonucleotides chemically synthesized based on the amino acid sequence represented by -Asp-Ile,
-ATRTCRTCDATRTTYTCNGG-3 'or 5'-TTYGARGAYATGACNGAYAT-
It had the base sequence represented by 3 '.

A portion of the PCR product thus obtained was fractionated by electrophoresis on a 2% (w / v) agarose gel according to a conventional method, transferred to a nylon membrane and taken up in 0.4N water. Fixed with sodium oxide, washed with 2 × SSC, air-dried, 5 × SSPE, 5 × Denhardt's solution, 0.5% (w /
v) dipped in a prehybridization mixture containing SDS and 100 μg / ml denatured salmon sperm DNA,
For 3 hours. Separately, as probe 1, Phe-Glu-Gl in SEQ ID NO: 1 in the sequence listing
Based on the amino acid sequence represented by u-Met-Asp-Pro, 5'-TTYGARGARATGGAYCC-
An oligonucleotide having a base sequence represented by 3 ′ was chemically synthesized, and was isotopically labeled with [γ- ³² P] ATP and T4 polynucleotide kinase. This probe 1
mol, 5x SSPE, 5x Denhardt's solution,
A nylon membrane is immersed in a mixture containing 0.5% (w / v) SDS and 100 μg / ml denatured salmon sperm DNA, and
And hybridized for 24 hours.
The nylon membrane was washed with 6 × SSC and autoradiographed by a conventional method.
It was included in the CR product.

Next, a plasmid vector “pT7 blue T” manufactured by Takara Shuzo was added to the remaining PCR product at 50 ng and an appropriate amount of T.
4 Add DNA ligase and add 100 mM AT
After adding P to a final concentration of 1 mM, the mixture was incubated at 16 ° C. for 18 hours to insert a DNA fragment into a plasmid vector, and the obtained recombinant DNA was introduced into E. coli “NoVa Blue” manufactured by Pharmacia by a competent cell method. To obtain a transformant. 10 g of the obtained transformant
/ L bactotryptone, 2.5 g / l sodium chloride,
15 g / l Bacto agar, 100 mg / l ampicillin, 40 mg / l X-Gal and 23.8 mg / l isopropyl-β-D-thiogalactopyranoside (hereinafter, referred to as
Abbreviated as "IPTG". ) Was inoculated into a plate medium containing the cells, and cultured at 37 ° C. for 24 hours to form colonies.
According to a conventional method, a nylon membrane was placed on a plate medium, and allowed to stand for about 30 seconds to transfer colonies. Thereafter, the nylon membrane was peeled off, and 0.5N sodium hydroxide and 1.5M
The cells were immersed in a mixed solution containing sodium chloride for 7 minutes to lyse the cells.
Then, the nylon membrane is immersed in 0.5 M Tris-HCl buffer (pH 7.2) containing 1.5 M sodium chloride for 3 minutes, washed with 2 × SSC, and immersed in 0.4 N sodium hydroxide for 20 minutes. After fixation, further washing with 5 × SSC, air drying, 5 × SSPE, 5 × Denhardt's solution, 0.5
% (W / v) SDS and 100 μg / ml denatured salmon sperm DNA were immersed in a prehybridization mixture and incubated at 65 ° C. for 3 hours. Thereafter, the probe 1 was hybridized to a nylon membrane according to a conventional method, washed with 6 × SSC, and subjected to autoradiography in the same manner as described above, and a transformant showing significant association with the probe 1 was collected from the plate medium. .

This transformant was treated with 100 μg of ampicillin.
/ Ml of L-broth medium (pH 7.2),
After culturing at 37 ° C. for 18 hours, cells were collected from the culture, and recombinant DNA was collected by the usual alkaline-SDS method. Examination by the dideoxy method revealed that this recombinant DN
A is No. 85 in the nucleotide sequence shown in SEQ ID NO: 4 in the sequence list.
And a DNA fragment having a nucleotide sequence corresponding to the 281st to 281st nucleotides.

[0049]

Experimental Example 3-3 <Preparation of mRNA> Take 0.05 ml of the aqueous solution containing the total RNA obtained in Experimental Example 3-1 and add
mM disodium-EDTA and 0.1% (w / v) SD
0.5 ml of 10 mM Tris-HCl buffer (pH 7.5) containing S was added to make the total volume 1 ml. To the mixture was added 1 ml of Nippon Roche Oligo (dT) ₃₀ latex “Oligotex-dT30 Super”, denatured by heating at 65 ° C. for 5 minutes, and immediately cooled in an ice bath for 3 minutes.
0.2 ml of 5M sodium chloride was added, and 10
After centrifuging at 10,000 rpm for 10 minutes at 25 ° C., the supernatant obtained by removing the supernatant was suspended in 0.5 ml of sterilized distilled water by adding 0.5 ml of sterilized distilled water.
Incubate for 5 min.
NA was eluted. The recovered mRNA was about 5 μg.

[0050]

[Experimental example 3-4] <Preparation of cDNA library> Using the cDNA cloning kit “cDNA synthesis system plus” manufactured by Amersham, m prepared in Experimental example 3-3
A cDNA library was prepared from the RNA. That is, 4 μl of the first strand cDNA synthesis solution, 1 μl of sodium pyrophosphate solution, 1 μl of human placental ribonuclease inhibitor solution, 2 μl of deoxynucleotide triphosphate mixed solution and 1 μl of oligo dT primer solution were added in this order to a 1.5 ml reaction tube, Experimental example 3
After adding 2 μg of the mRNA obtained in -3, 1 ml of sterilized distilled water was added.
The volume was 9 μl. 1 μl of a solution containing 20 units of reverse transcriptase was added to the mixture, and the mixture was incubated at 42 ° C. for 40 minutes to obtain a reaction product containing first-strand cDNA.

To the reaction product, 37.5 μl of the second strand cDNA synthesis solution, 0.8 unit of ribonuclease H derived from Escherichia coli and 23 units of DNA polymerase I were added in this order, and the mixture was made up to 100 μl with sterile distilled water. 6 at ℃
Incubate for 0 min at 22 ° C. for 60 min.
Two units of NA polymerase were added and incubated at 37 ° C. for another 10 minutes to obtain a reaction containing the second strand cDNA. The reaction was treated with 0.25M EDTA (pH 8.
After stopping the reaction by adding 4 μl of 0), phenol / chloroform extraction was carried out by a conventional method, and ethanol was precipitated to collect cDNA.

The cDNA thus obtained was added with 2 μl of L / K buffer, 250 pmoles of Eco RI adapter and 2.5 units of T4 DNA ligase in this order, and adjusted to 20 μl with sterile distilled water. And Eco RI adapters were ligated to both ends of the cDNA. Add 2μ 0.25M EDTA to the reaction
to remove the unreacted Eco RI adapter by molecular sieve chromatography, add 40 μl of L / K buffer and 80 units of T4 polynucleotide kinase, and make up to 400 μl with sterile distilled water. Incubate at 37 ° C. for 30 minutes, Eco RI
After methylation of the cleavage site, the reaction was phenol / chloroform extracted and ethanol precipitated to collect DNA. 1.5 μl of L / K buffer containing an appropriate amount of λgt10 arm and 2.5 units of T4 DNA ligase were added to the DNA, and the total volume was adjusted to 15 μl with sterile distilled water. The mixture was incubated at 15 ° C. for 16 hours and ligated. Phage containing recombinant λ DNA was obtained by applying in vitro packaging.

[0053]

[Experimental Example 3-5] <Cloning of Recombinant DNA> The phage prepared in Experimental Example 3-4 was infected with Amersham Escherichia coli NM514 by a conventional method, and then 10 g / l bactotryptone and 5 g / l bactoyeast. Extract,
The cells were inoculated on an agar medium (pH 7.0) containing 10 g / l sodium chloride and 15 g / l bactoagagar, and cultured at 37 ° C. for 6 hours to form plaques. The nylon membrane was placed on the agar medium, left standing for about 30 seconds to transfer the plaque onto the nylon membrane, and then the nylon membrane was peeled off.
2 minutes in an aqueous solution containing sodium hydroxide and 1.5 M sodium chloride, then 0.1 ml containing 1.5 M sodium chloride.
It was immersed in 5M Tris-HCl buffer (pH 7.0) for 5 minutes. Rinse the nylon membrane with 5 × SSC, air dry and 5 × SS
PE, 5x Denhardt's solution, 0.5% (w / v) SDS
And salmon sperm DNA were immersed in a mixed solution containing 100 μg / ml, and incubated at 65 ° C. for 3 hours. Thereafter, a nylon membrane was labeled with 32P using Amersham's DNA labeling kit “Ready Prime DNA Labeling System” and a suitable amount of the DNA fragment as the probe 2 obtained in Experimental Example 3-2, 5 × SSPE, 5 × Denhardt solution, 0.5% (w /
v) Hybridization by incubating at 60 ° C. for 20 hours in a mixture containing SDS and salmon sperm DNA at 100 μg / ml, followed by autoradiography in the same manner as described above, and phage DN showing significant association with probe 2
The A clone was collected.

This clone was amplified in Escherichia coli according to a conventional method, and a recombinant DNA was extracted from the cells. Recombinant D
While NA was digested with the restriction enzyme EcoRI, the plasmid vector pUC19 (ATCC37254) was digested with the same restriction enzymes, and the obtained DNA fragment and plasmid fragment were ligated with DNA ligase by a conventional method to obtain recombinant DNA. Then, this recombinant DNA was subjected to E. coli JM109 strain (ATCC 5332) by a usual competent cell method.
3) to obtain a transformant.

[0055]

[Experimental example 3-6] <Determination of base sequence and amino acid sequence> The transformant prepared in Experimental example 3-5 was subjected to L-broth medium (p
H7.2), and cultured with shaking at 37 ° C. for 18 hours.
The transformant was collected from the culture and subjected to normal alkaline-SD
Recombinant DN containing DNA of the present invention after treatment by Method S
A was obtained. When analyzed by an automatic sequencer using a fluorimeter, this recombinant DNA contained the base sequence from the 5 ′ end shown in SEQ ID NO: 5 in the sequence listing,
Decoding the nucleotide sequence suggested that it encodes the amino acid sequence from the N-terminus shown in SEQ ID NO: 5. In this amino acid sequence, the partial amino acid sequence shown in SEQ ID NOs: 1 and 2 in the sequence listing is contained at positions 79 to 103 or positions 26 to 43, which means that the protein of the present invention is It may have the amino acid sequence from the N-terminal shown in SEQ ID NO: 3, indicating that the protein is encoded by DNA having the nucleotide sequence from the 5'-terminal shown in SEQ ID NO: 4 in mouse liver in mouse liver. ing.

As described above, the protein that induces the production of IFN-γ in immunocompetent cells has been found as a result of a long-term study of the present inventors. It has unique physicochemical properties that cannot be seen. The present invention seeks to create this protein by applying recombinant DNA technology. Hereinafter, the protein of the present invention and a method for producing the same will be specifically described with reference to examples and the like.

The term "protein" as used herein means a naturally occurring protein or a recombinant DNA having specific physicochemical properties.
Means all proteins created by technology. The protein of the present invention usually has an amino acid sequence partially or wholly elucidated. For example, for example, the amino acid sequence from the N-terminal shown in SEQ ID NO: 3 in the sequence listing or an amino acid homologous thereto Sequences. A variant having an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 3 can be obtained by replacing one or more of the amino acids in the amino acid sequence of SEQ ID NO: 3 with another amino acid without substantially altering the intended biological action. Can be obtained by substituting Even if the same DNA is used, depending on the host into which the DNA is introduced, the components and composition of the nutrient medium used for culturing the transformant containing the DNA, the culture temperature and pH, etc. Although the desired biological action is retained by modification after expression, etc., one or two or more amino acids near the N-terminal in the amino acid sequence of SEQ ID NO: 3 are deleted, or one or two amino acids are deleted at the N-terminal. Mutants in which more than one amino acid is newly added may be produced. Such variants are, of course, also included in the proteins of the present invention, as long as they induce IFN-γ production in immunocompetent cells.

The protein of the present invention has a D
It can be produced by culturing a transformant containing NA in a nutrient medium and collecting the produced protein from the culture. In the transformant used in the present invention, for example, a DNA having a base sequence from the 5 'end shown in SEQ ID NO: 4 in the sequence listing, a base sequence homologous thereto, or a base sequence complementary thereto is appropriately introduced into a host. Can be obtained. In the above base sequence, one or more bases may be replaced with another base without changing the amino acid sequence to be encoded by utilizing the degeneracy of the gene.
Further, in order for the DNA to actually express the production of the protein in the host, one or more bases in the base sequence encoding the protein or a homologous mutant thereof can be appropriately substituted with another base. Needless to say.

The DNA used in the present invention does not matter whether it is derived from nature or artificially synthesized, as long as it has the above-mentioned sequence. Natural sources include, for example, mouse liver, the cells of which provide genes containing the DNA of the present invention. That is, for example, the liver is excised from a mouse that has been stimulated with a reticuloendothelial stimulating substance such as Corynebacterium parvum, BCG, mitogen, or lipopolysaccharide, and crushed, and then the total RNA is isolated. This total RNA was converted to oligo (d
T) After treatment with cellulose, oligo (dT) latex or the like to obtain poly (A) ⁺ RNA, mRNA is isolated by fractionation using a sucrose concentration gradient or the like. Using this mRNA as a template, a reverse transcriptase and a polymerase are allowed to act to form a double-stranded cDN.
A, and insert this into an appropriate autonomously replicable vector,
The resulting recombinant DNA is introduced into an appropriate host such as Escherichia coli to obtain a transformant. The transformant is cultured in a nutrient medium, and a transformant containing a DNA encoding the protein of the present invention is collected by applying a colony hybridization method to the culture. When the thus obtained transformant is treated by a general method, the DNA of the present invention can be obtained. On the other hand, in order to artificially synthesize the DNA of the present invention,
For example, chemically synthesized based on the base sequence shown in SEQ ID NO: 4 in the sequence listing, or inserted into an appropriate vector capable of autonomously replicating a DNA encoding the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing to obtain a recombinant DNA, A transformant obtained by introducing this into an appropriate host is cultured, the cells are separated from the culture, and the plasmid containing the DNA may be collected from the cells.

Such a DNA is usually introduced into a host in the form of a recombinant DNA. Recombinant DNA is usually DNA
If DNA is available, it can be prepared relatively easily by ordinary recombinant DNA technology. Examples of such vectors include, for example, pKK223-2, pGEX-2T, pR
L-λ, pBTrp2 DNA, pUB110, YEp
13, Ti plasmid, Ri plasmid, pBI121
And plasmid vectors such as pKK223-2, pGEX-2T, pGEX-2T and pKK223-2 for expressing the DNA of the present invention in prokaryotes such as Escherichia coli, Bacillus subtilis, and yeast.
RL-λ, pBTrp2 DNA, pUB110, YE
p12 is also required for expression in cells derived from animals and plants.
i plasmid, Ri plasmid and pBI121 are preferred.

For inserting the DNA of the present invention into such a vector, a method generally used in the art is generally employed.
Specifically, first, the gene containing the DNA of the present invention and an autonomously replicable vector are cut with a restriction enzyme and / or ultrasonic waves, and then the generated DNA fragment and the vector fragment are ligated. Restriction enzymes that specifically act on nucleotides in the cleavage of genes and vectors, especially type II restriction enzymes, specifically Sau 3AI, Eco RI, H
ind III, Bam HI, Sal I, Xba
If I, Sac I, Pst I, etc. are used, DNA
It is easier to ligate the fragment and the vector fragment. DNA
To ligate the fragment and the vector fragment, if necessary, they may be annealed and then subjected to DNA ligase in vivo or in vitro. Recombinant D thus obtained
NA can be replicated indefinitely by introducing it into a host as appropriate to obtain a transformant and culturing the transformant.

The recombinant DNA according to the present invention is E. coli,
It can be introduced into an appropriate host such as Bacillus subtilis, actinomycetes and yeast. When the host is Escherichia coli, the host may be cultured in the presence of recombinant DNA and calcium ions. On the other hand, when the host is Bacillus subtilis, the competent cell method or the protoplast method may be applied. To clone the transformant, a colony hybridization method may be applied, or the transformant may be cultured in a nutrient medium, and those that produce a protein that induces the production of IFN-γ in the immunocompetent cells may be selected.

When the transformant thus obtained is cultured in a nutrient medium, the protein is produced inside or outside the cells or cells. A nutrient medium usually contains a carbon source, a nitrogen source, minerals,
Furthermore, if necessary, a general liquid medium supplemented with micronutrients such as amino acids and vitamins is used, and individual carbon sources include starch, starch hydrolyzate, glucose, fructose, and sucrose. However, as a nitrogen source,
For example, nitrogen-containing inorganic or organic substances such as ammonia or ammonium salt, urea, nitrate, peptone, yeast extract, defatted soybean, corn steep liquor, meat extract and the like can be mentioned. When the transformant is inoculated into the nutrient medium and cultured for about 1 to 10 days under aerobic conditions such as aeration and agitation while maintaining the nutrient medium at a temperature of 25 to 65 ° C. and a pH of 2 to 8, the protein is obtained. Is obtained. This culture can be used as it is as an IFN-γ inducer, but usually, prior to use, if necessary, disrupt cells by ultrasonication or cell wall lysing enzyme, and then filter, centrifuge, or the like to remove the protein. Is separated from the cells or the disrupted cells and purified. For purification, the cells or the cultures from which the cells were disrupted were removed, for example, concentration, salting-out, dialysis, fractional precipitation, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, chromatofocusing, Gel electrophoresis,
A general method in the art for purifying a physiologically active substance such as isoelectric focusing can be adopted, and if necessary,
These methods may be appropriately combined. Then, depending on the final use form, the purified protein may be concentrated and lyophilized to be in a liquid or solid state.

As described above, the protein of the present invention has a property of inducing the production of IFN-γ in immunocompetent cells. Due to this property, the protein of the present invention can be used as an inducer in the production of IFN-γ by a cell culture method, and further has a sensitivity to IFN-γ, for example, viral diseases such as AIDS and condyloma acuminatum, Kidney cancer, granuloma,
It is useful as a therapeutic or preventive agent for mycosis fungoides, malignant tumors such as brain tumors, and immune diseases such as rheumatoid arthritis and allergy.

The protein of the present invention is usually co-present in a culture medium for producing IFN-γ by culturing immunocompetent cells, or directly into the body of mammals for treatment and prevention of IFN-γ-sensitive diseases. Is administered. That is, in the former use, leukocytes separated from mammalian peripheral blood,
For example, immunocompetent cells established as a culture such as HBL-38 cells, MO cells, Jurkat cells, EL-4 cells, L12-R4 cells, etc. are suspended in an appropriate culture medium containing the protein of the present invention. If necessary, a T cell stimulator such as mitogen, interleukin 2, or anti-CD3 antibody is added to the culture medium, and the culture medium is heated at a temperature of about 30 to 40 ° C.
While maintaining the H at about 5 to 8, the culture medium is cultivated for about 1 to 100 hours by an ordinary method while appropriately replacing the culture medium with fresh one. The culture thus obtained is generally used to purify a physiologically active substance, namely, concentration, salting out, dialysis, fractional precipitation, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography. , Chromatofocusing, gel electrophoresis, isoelectric focusing, etc.
IFN-γ can be collected.

On the other hand, for the treatment and prevention of IFN-γ-sensitive diseases, the IFN-γ according to the present invention is introduced into the body of mammals.
The inducer may be administered directly. Specifically, the IFN-γ inducer of the present invention is prepared into an appropriate dosage form suitable for administration and then orally administered to mammals, for example, intradermal, subcutaneous, intramuscular,
Inject intravenously or intraperitoneally. The mammal to which the protein of the present invention can be administered is not limited to humans. For example, mice, rats, hamsters, rabbits, dogs, cats, cattle,
Mammals such as horses, goats, sheep, pigs and monkeys may be used. Since the protein of the present invention has a strong IFN-γ-inducing ability, it can generally induce a desired IFN-γ production in a small amount, and since it has extremely low toxicity, serious side effects can be caused even when a large amount is administered. No cause. Therefore, the protein of the present invention has an advantage that the desired IFN-γ production can be rapidly induced without strictly controlling the dose when used.

In addition, since the protein of the present invention has a remarkable property of enhancing cytotoxicity caused by killer cells, it can be appropriately used in combination with interleukin 2 or tumor necrosis factor to obtain lung cancer, kidney cancer, It has a remarkable effect on the improvement of therapeutic effects and side effects in the treatment of malignant tumors including solid cancers such as breast cancer.

Hereinafter, the production of the protein according to the present invention using the transformant will be specifically described based on Examples.

[0069]

[Example 1] <Replicable recombinant DNA and transformant>
Takara Shuzo PCR kit "GeneAmp RNA PC
Using R Kit, a first-strand cDNA was prepared from the total RNA obtained by the method of Experimental Example 3-1. That is, 4 μl of 25 mM magnesium chloride, 2 μl of 10 × PCR buffer, and 1 mM dN were placed in a 0.5 ml reaction tube.
8 μl of TP mix, 1 μl of 1 unit / μl RNase inhibitor and 1 unit of 2.5 units / μl reverse transcriptase
1 μl of 2.5 μM random hexamer and 1 μg of total RNA obtained by the method of Experimental Example 3-1 were taken up to 20 μl with sterile distilled water. The mixture is then incubated in this order for 10 minutes at 25 ° C., 30 minutes at 42 ° C., 5 minutes at 99 ° C., 5 minutes at 5 ° C.
A reaction containing DNA was obtained.

Take 20 μl of this reaction product and add
4 μl of M magnesium chloride, 8 × 10 × PCR buffer
μl, 2.5 units / μl 0.5 μl of amplitac DNA polymerase was chemically synthesized based on the amino acid sequence near the N-terminus or C-terminus in SEQ ID NO: 3 in the sequence listing.
'-CGAGGGATCGAACTTTGGCCGAC
TTC-3 'or 5'-CGAGGAATTCCTAA
Appropriate amounts of a sense primer and an antisense primer having the nucleotide sequence represented by CTTTGATGTAAG-3 ′ were added, and the mixture was made up to 100 μl with sterile distilled water. Next, according to a conventional method, the mixture was heated at 94 ° C. for 1 minute, at 55 ° C. for 2 minutes,
A cycle of incubation at 72 ° C. for 3 minutes in this order was repeated 40 times, and the obtained PCR product was digested with the restriction enzyme Bam.
Bam HI-Ec digested with HI and Eco RI
o An RI DNA fragment was obtained.

This DNA fragment was dissolved in an appropriate amount of
10 ng of the plasmid vector “pGEX-2T” manufactured by Pharmacia, which had been cut with the restriction enzymes Bam HI and Eco RI in advance, and an appropriate amount of T4.
DNA ligase and 10 mM ATP at a final concentration of 1 m
M, and then incubated at 16 ° C. for 18 hours. The obtained recombinant DNA was introduced into Escherichia coli DH5 (ATCC53868) strain by a competent cell method to obtain a transformant, which was then used as a 50 μg / ml ampicillin.
And inoculated into an L-broth medium (pH 7.2) containing
After 18 hours, the recombinant DNA was extracted by the usual alkaline-SDS method.

This recombinant DNA was named "pMGTG-1" and its structure was examined by the dideoxy method. As shown in FIG. 2, this pMGTG-1 was
In the above, MGTG cDNA having the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing was linked downstream of the Tac promoter and the glutathione S transferase gene.

[0073]

[Example 2] <Production of protein using transformant> The transformant obtained by the method of Example 1 was used to prepare ampicillin 50 µg / m2.
of L-broth medium (pH 7.2) containing the same, and seed-cultured at 37 ° C. for 18 hours with shaking. 1% seed culture
(V / v) inoculate fresh 18 l of the same medium,
The cells were cultured at 37 ° C. with aeration and stirring. Then, when the absorbance of the culture at a wavelength of 650 nm reached about 0.6, IPT
G was added to a final concentration of 1 mM, and the cells were further cultured for 5 hours.
Thereafter, cells were collected from the culture by centrifugation, and 15
A mixed solution containing 0 mM sodium chloride, 16 mM disodium hydrogen phosphate and 4 mM sodium dihydrogen phosphate (pH 7.
After suspending the suspension in 3) and sonicating by a conventional method, the disrupted bacterial cells were centrifuged, and the supernatant was collected.

The supernatant was loaded on a Pharmacia "Glutathione Sepharose 4B" column which had been equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing 150 mM sodium chloride in advance, and was loaded with a fresh buffer. After washing, the column contains 5 mM reduced glutathione in 50 m
The protein was eluted by passing through an M Tris-HCl buffer (pH 8.0). Then, calcium chloride was added to the protein-containing fraction to a concentration of 2.5 mM, 1,000 units of thrombin was added, the mixture was incubated at 25 ° C. for 18 hours, and the reaction product was previously treated with 50 mM sodium chloride containing 150 mM sodium chloride. Tris-HCl buffer (pH 7.
The liquid was passed through a glutathione-Sepharose 4B column equilibrated in 5) to collect a non-adsorbed fraction. afterwards,
This fraction was concentrated and lyophilized to give a specific activity of about 5 ×.
A solid containing 10 ⁵ units / mg protein was obtained in a yield of about 3 mg / l of culture.

When the physicochemical properties of this purified protein were examined in the same manner as in Experimental Example 2, the purified protein was found to be SDS
A molecular weight of 19,000 ± 5,000 daltons as determined by polyacrylamide gel electrophoresis or gel filtration, and an isoelectric point of 4.8 ± 1.0 as determined by chromatofocusing. Furthermore, when tested by the method of Experimental Example 2-4, the purified protein was found to induce IFN-γ production in immunocompetent cells well in the absence and presence of concanavalin A, and also showed that killer cells had cytotoxicity. Markedly enhanced. This is a recombinant DNA
The technique also supports that the protein can be produced.

[0076]

According to the present invention, IF
This is based on the discovery of a novel protein that induces N-γ production. The protein of the present invention is usually a substance whose amino acid sequence has been partially or entirely elucidated, and exhibits a stable IFN-γ-inducing ability in immunocompetent cells. As a result, the protein of the present invention can be converted to IFN-γ by a cell culture method.
As an IFN-γ inducer for producing
It will have a wide variety of uses as a therapeutic or prophylactic agent for viral diseases, malignant tumors, and general immune diseases that are sensitive to IFN-γ.

Since the protein of the present invention has a strong IFN-γ-inducing ability, it can generally induce a desired IFN-γ production in a small amount, and has extremely low toxicity. It does not cause any side effects. Therefore, the protein of the present invention has an advantage that the desired IFN-γ production can be rapidly induced without strictly controlling the dose when used. In addition, since the protein of the present invention has a remarkable property of enhancing cytotoxicity caused by killer cells, by appropriately using it with interleukin 2 or tumor necrosis factor, it is possible to adopt lung cancer, kidney cancer, breast cancer, etc. by adoptive immunotherapy. It has a remarkable effect on the improvement of therapeutic effects and side effects in the treatment of malignant tumors including solid cancers.

The protein of the present invention which is also useful is obtained by utilizing the DNA of the present invention encoding the protein.
The desired amount can be easily manufactured.

The present invention exerts such remarkable functions and effects, and it can be said that the present invention is very significant in that it contributes to the art.

[0080]

[Sequence List] SEQUENCE LISTING <110> Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo <120> Protein which induces interferon-γ production <130> 10047102 <150> JP 184,162 / 94 <151> 1994-07-14 <160> 5 <210 > 1 <211> 25 <212> PRT <213> Mouse <400> 1 Ile Ile Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile 1 5 10 15 Gln Ser Asp Leu Ile Phe Phe Gln Lys 20 25 < 210> 2 <211> 18 <212> PRT <213> Mouse <400> 2 Gln Pro Val Phe Glu Asp Met Thr Asp Ile Asp Gln Ser Ala Ser Glu 1 5 10 15 Pro Gln <210> 3 <211> 157 < 212> PRT <213> Mouse <220> <221> Unsure <222> (70) <223> Xaa is Met or Thr <400> 3 Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn 1 5 10 15 Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met 20 25 30 Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile 35 40 45 Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser 50 55 60 Val Lys Asp Ser Lys Xaa Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile 65 70 75 80 Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser 85 90 95 Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asn Lys Met Glu 100 105 110 Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu 115 120 125 Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp 130 135 140 Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser 145 150 155 <210> 4 <211> 471 <212> DNA <213> Mouse <220> <221> CDS <222> (70) <223> Xaa is Met or Thr <400> 4 aactttggcc gacttcactg tacaaccgca gtaatacgga atataaatga ccaagttctc 60 ttcgttgaca aaagacagcc tgtgttcgag gatatgactg atattgatca aagtgccagt 120 gaaccccaga ccagactgat aatatacatg tacaaagaca gtgaagtaag aggactggct 180 gtgaccctct ctgtgaagga tagtaaaayg tctaccctct cctgtaagaa caagatcatt 240 tcctttgagg aaatggatcc acctgaaaat attgatgata tacaaagtga tctcatattc 300 tttcagaaac gtgttccagg acacaacaag atggagtttg aatcttcact gtatgaagga 360 cactttcttg cttgccaaaa ggaagatgat gctttcaaac tcattctgaa aaaaaaggat 420 gaaaatgggg ataaatctgt aatgttcact ctcactaact tacatcaaag t 471 <210> 5 <211> 471 <212> DNA <213> Mouse <220> <221> CDS <222> (1) ... (471) <220> <210> mat peptide <222> (1) ... (471) <220> <221> Unsure <222> (208) ... (210) <223> Xaa is Met or Thr <400> 5 aac ttt ggc cga ctt cac tgt aca acc gca gta ata cgg aat ata aat 48 Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn 1 5 10 15 gac caa gtt ctc ttc gtt gac aaa aga cag cct gtg ttc gag gat atg 96 Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met 20 25 30 act gat att gat caa agt gcc agt gaa ccc cag acc aga ctg ata ata 144 Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile 35 40 45 tac atg tac aaa gac agt gaa gta aga gga ctg gct gtg acc ctc tct 192 Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser 50 55 60 gtg aag gat agt aaa ayg tct acc ctc tcc tgt aag aac aag atc att 240 Val Lys Asp Ser Lys Xaa Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile 65 70 75 80 tcc ttt gag gaa atg gat cca cct gaa aat att gat gat ata caa agt 288 Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser 85 90 95 gat ctc ata ttc ttt cag aaa cgt gtt cca gga cac aac aag atg gag 336 Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asn Lys Met Glu 100 105 110 ttt gaa tct tca ctg tat gaa gga cac ttt ctt gct tgc caa aag gaa 384 Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu 115 120 125 gat gat gct ttc aaa ctc att ctg aaa aaa aag gat gaa aat ggg gat 432 Asp Asp Au Phe Lys Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp 130 135 140 aaa tct gta atg ttc act ctc act aac tta cat caa agt 471 Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser 145 150 155

[Brief description of the drawings]

FIG. 1 is a view showing an elution pattern of a peptide fragment obtained by digesting the protein of the present invention with trypsin in high performance liquid chromatography.

FIG. 2 pMGTG which is a recombinant DNA according to the present invention
FIG. 2 is a diagram showing a structure of -1.

[Explanation of symbols]

MGTG cDNA: cDNA encoding the protein of the present invention Ptac: tac promoter GST: glutathione S-transferase gene AmpR: ampicillin resistance gene pBR322ori: origin of replication in E. coli

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12N 15/09 ZNA C12R 1:91) (C12N 1/21 C12R 1:19)

Claims (14)

[Claims] 1. A protein having the following physicochemical properties or an immunological equivalent thereof. (1) Molecular weight It shows a molecular weight of 19,000 ± 5,000 daltons when measured by SDS-polyacrylamide gel electrophoresis or gel filtration. (2) Isoelectric point When measured by the chromatofocusing method, 4.8 ±
The isoelectric point is shown at 1.0. (3) Partial amino acid sequence It has a partial amino acid sequence shown in SEQ ID NOS: 1 and 2 in the sequence listing. (4) Biological action Induces the production of interferon-γ in immunocompetent cells. 2. Sequence number 3 in the sequence listing (provided that
“Xaa” shall mean methionine or threonine. ) Or a protein having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence described above, and which induces the production of interferon-γ in immunocompetent cells, or an immunological protein thereof. Equivalent. 3. SEQ ID NO: 3 in the sequence listing (provided that
“Xaa” shall mean methionine or threonine. A) a peptide fragment consisting of a part of the amino acid sequence shown in) 4. D encoding a protein, immunological equivalent or peptide fragment according to any one of claims 1 to 3
NA. 5. The base sequence shown in SEQ ID NO: 4 in the sequence listing, or a base sequence in which one or more bases are deleted, substituted or added in the base sequence, or
Claims containing a nucleotide sequence complementary to them or one or more of those nucleotide sequences substituted with other nucleotides based on the degeneracy of the gene without changing the amino acid sequence encoded thereby. DN according to item 4
A. 6. The method according to claim 4, which is derived from a mouse liver.
DNA according to 1. 7. The DNA according to claim 4, which is inserted as a replicable recombinant DNA into an autonomously replicable vector. 8. The method according to claim 4, which is appropriately introduced into a host.
Or the DNA of 7 above. 9. The D according to claim 8, wherein the host is Escherichia coli.
NA. 10. A transformant obtained by introducing the DNA according to claim 4 into a suitable host, and culturing the transformant in a nutrient medium.
A method for producing a protein, comprising collecting the produced protein from a culture. 11. A DNA having a base sequence shown in SEQ ID NO: 4 in the sequence listing, a base sequence in which one or more bases are deleted, substituted or added in the base sequence, or a base sequence complementary thereto Or a base sequence obtained by substituting one or two or more of these base sequences with other bases without changing the amino acid sequence encoded by the same based on the degeneracy of the gene, and a vector capable of autonomous replication. The method for producing a protein according to claim 10, which is in the form of a recombinant DNA. 12. The method for producing a protein according to claim 10, wherein the DNA is derived from mouse liver. 13. The method according to claim 10, wherein the host is Escherichia coli.
Or the method for producing a protein according to 12. 14. The produced protein is concentrated, salted out, dialyzed,
One or two selected from fractional precipitation, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, chromatography, gel electrophoresis and isoelectric focusing.
11. The method according to claim 10, wherein the sample is collected by more than one kind of purification method.
Or a method for producing a protein according to item 13.