JPH11116600A - Guinea pig interferon gamma - Google Patents

Abstract

(57) Abstract: Guinea pig IFN-γ and its modified proteins, genes encoding these proteins, expression plasmids containing the genes, transformants into which the expression plasmids have been introduced, and transformants Methods for producing recombinant proteins to be used, and antibodies to these proteins and methods for producing the antibodies. [Effect] In guinea pigs which are supposed to elicit an immune response close to humans, I strongly reflects the activation status of macrophages, natural killer cells and T cells.
Means for analyzing the expression of FN-γ are provided. By this analysis, it is possible to evaluate and evaluate immunotherapeutic agents for diseases or cancer caused by abnormalities of the immune system.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to guinea pig interferon gamma (also referred to as guinea pig IFN-γ) and its gene. The present invention also provides an expression plasmid containing the gene, a transformant into which the expression plasmid has been introduced, a method for producing a recombinant protein using the transformant, an antibody against guinea pig IFN-γ, and a method for producing the antibody About.

[0002]

2. Description of the Related Art IFN-γ is a protein produced by activated lymphocytes and having an activity to induce an antiviral state in cells. At the same time, IFN-γ is known to play a central role in the cellular immune response during infection.

[0003] Macrophages recognize heterologous biological substances that have invaded the living body, and secrete factors that enhance vascular permeability, factors that induce neutrophils, and cytokines such as interleukin-12. Natural killer cells activated by interleukin 12 produce and activate IFN-γ. On the other hand, macrophages phagocytose the heterologous biological substance, induce the expression of major histocompatibility class II molecules, and present the peptide as a degradation product of the heterologous biological substance by the molecule. Major histocompatibility class II
T cells that recognize the complex of molecule and peptide are activated. At this time, certain CD4-positive T cells induced to differentiate by interleukin 12 and IFN-γ
-Activates macrophages and natural killer cells by secreting γ, and enhances the elimination function of these cells for removing heterologous biological materials.

[0004] Since a series of reactions elicited when such a heterologous biological substance invades have been studied mainly using mice, various cytokines including IFN-γ and their antibodies have been isolated from mice. ing. On the other hand, when analyzing the inflammatory response by skin reaction, guinea pigs (guinea pi
g) has been heavily used. Particularly in guinea pigs, a delayed hypersensitivity reaction is sharply observed. For example, Mycobacterium bovis BCG
Delayed hypersensitivity skin reaction can be observed in guinea pigs but not in mice. This suggests that, in one aspect, the immune response induced by invasion of heterologous biological material is more similar to humans in guinea pigs than in mice. As described above, an experimental system using a guinea pig is important in order to analyze a series of reactions caused when a different kind of biological material enters.

In analyzing the above reaction, IFN-γ
Is one of the essential cytokines. Already, the structures of human and mouse IFN-γ have been determined (R. Devos et al.
ucleic Acids Research vol.10, p2487 (1982) and P.
W. Gray et al., Proc. Natl. Acad. Sci. USA Vol. 8
0, p5842 (1983)), and their antibodies have also been isolated.
However, the structure of IFN-γ in guinea pigs has not been determined, which is a major obstacle in analyzing the above reaction using guinea pigs.

[0006]

The object of the present invention is to provide guinea pig IFN-γ and its modified proteins, genes encoding these proteins, expression plasmids containing the genes, transformants into which the expression plasmids have been introduced,
It is another object of the present invention to provide a method for producing recombinant proteins using the transformant, an antibody against these proteins, and a method for producing the antibody.

[0007]

Means for Solving the Problems The present inventors succeeded in cloning the guinea pig IFN-γ gene, and have completed further studies to complete the present invention.

The gist of the present invention is to provide (1) a DNA encoding the following protein (a) or (b): (a) a guinea pig IFN-γ protein having the amino acid sequence of SEQ ID NO: 2, ) Having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2, and
A protein having an activity, (2) a DNA having the nucleotide sequence of SEQ ID NO: 1,
Or a DNA that hybridizes with the DNA under stringent conditions and encodes a protein having IFN-γ activity in guinea pigs.
Or (4) a recombinant protein obtained by expressing the DNA of (2), (4) an expression plasmid that expresses the DNA of (1) or (2), (5) the (4)
(6) a method for producing a recombinant protein, comprising culturing the transformant according to (5) and collecting an expressed protein; (8) A method for producing an antibody against the protein according to (3), wherein the method uses the protein according to (3) or a fragment thereof as an antigen.

[0009]

DETAILED DESCRIPTION OF THE INVENTION A first aspect of the present invention relates to a guinea pig IFN having the amino acid sequence of SEQ ID NO: 2.
DNA encoding -γ or the guinea pig IFN
DNA encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of -γ, and having guinea pig IFN-γ activity. A second aspect of the present invention relates to a guinea pig IFN- having the nucleotide sequence of SEQ ID NO: 1.
DNA that hybridizes with γ DNA or the guinea pig IFN-γ DNA under stringent conditions and encodes a protein having guinea pig IFN-γ activity
A. Hereinafter, these DNAs will be sequentially described.

1) D encoding guinea pig IFN-γ
NA (guinea pig IFN-γ DNA) Among the above DNAs, “DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2”, more specifically, “DN having the base sequence of SEQ ID NO: 1”
"A" is DNA encoding guinea pig IFN-γ of the present invention. The DNA is, for example, Molecular Clonin
g 2nd Edt.Cold Spring Harbor Laboratory Press (198
According to 9) and the like, it can be obtained by the following method.
That is, first, total guinea pig RNA was prepared from guinea pig spleen cells stimulated with concanavalin A, and then total R
MRNA containing polyA is isolated from NA. It is known that spleen cells stimulated with concanavalin A produce high levels of IFN-γ. Total RNA is obtained by the AGPC method.
(acid guanidium thiocyanate-phenol-chloroform met
hod; Tuji, Nakamura: Experimental Medicine 9, p99 (1991)). For mRNA, an oligo dT cellulose column was used.
loning 2nd Edt.Cold Spring Harbor Laboratory Pres
s (1989).
Next, a cDNA library is prepared by reverse transcription of the obtained mRNA using a reverse transcriptase. cDN
The A library can be prepared, for example, by the Gubler & Hoffman method (Gene,
25, p263 (1983)). Next, this DNA is double-stranded with DNA polymerase I and inserted into an appropriate cloning vector. The resulting recombinant cloning vector is used to transform a suitable host. Plasmid DNA obtained from the transformed host contains radiolabeled mouse IFN.
-Hybridize with the γ cDNA probe. A pool of clones giving a positive signal to the probe is identified. By further dividing the clone pool and repeating the search by hybridization, a single transformed cell containing guinea pig IFN-γ cDNA can be finally obtained. Guinea pig I obtained
Plasmid DNA is prepared from the transformed cells containing the FN-γ cDNA, and the nucleotide sequence of the DNA is determined. For the determination of the nucleotide sequence, for example, the nucleotide sequence can be determined by a sequencer using an Auto Read Sequencing kit (manufactured by Pharmacia), or B using the dideoxy method.
The caBEST Sequencing kit (TAKARA) can be used to determine the nucleotide sequence using RI. Further, a corresponding amino acid sequence is determined from the base sequence.

[0011] The guinea pig IFN-
The nucleotide sequence of the γ gene is shown in SEQ ID NO: 1, and the amino acid sequence of guinea pig IFN-γ is shown in SEQ ID NO: 2. Guinea pig IFN-γ and human IFN-γ (GenBank A
ccession No. V00543), 72
%, And 58% homology in the amino acid sequence. Guinea pig IFN-γ and mouse IFN-γ (GenBa
(nk Accession No. K00083), a homology of 64% in the nucleotide sequence and 37% in the amino acid sequence was observed. By expressing the cloned DNA, guinea pig IFN-γ can be obtained. The details of the expression method will be described in the section on the protein of the third embodiment.

The expressed protein is guinea pig IFN-
Guinea pig IFN-γ activity is measured by a bioassay to check for γ. Guinea pig IFN
The -γ activity includes an antiviral activity or an anti-cell proliferation activity, and can be measured by the following method. 1. Antiviral activity Antiviral activity can be measured as the action of inhibiting cytopathy by virus. A guinea pig cultured cell line, for example, a GPC-16 cell line or a 104C1 cell line is inoculated at an appropriate concentration into a 96-well plate, and a test sample is added thereto at 37 ° C.
Cultivate in a humid atmosphere of 5% CO ₂ . After 24 hours, the medium is replaced with a fresh medium, and the virus (for example, EMCV (encephalomyocard
itis virus) or VSV (vesicular stomatitis viru
s)) Add the suspension and incubate for an additional 48 hours at 37 ° C, 5% CO _{2 in a} humid atmosphere. After the culture, the medium is removed, 50 μl of a 0.02% neutral red solution dissolved in the medium and insoluble matter removed by filtration is added, and the mixture is incubated at 37 ° C. in a 5% CO ₂ humid atmosphere for 45 minutes. Remove stain and add phosphate buffered saline
Wash twice. It is dissolved in 100 μl of a 0.05 M sodium phosphate solution containing 50% ethanol, and the absorbance at OD ₅₄₀ is measured with a microplate reader. The dilution ratio of the test sample that inhibits cell degeneration by 50% is determined, and the reciprocal thereof is used to calculate the antiviral activity unit. 2. Anti-cell Proliferating Activity Test cells suspended in culture medium are inoculated into plastic tissue culture plates at appropriate concentrations. The test cells may be any cells that can be grown in vitro on guinea pig-derived cells, and examples include GPC-16 cells and 104C1 cells. Next, a sample containing IFN appropriately diluted with a medium is added, and the cells are cultured under the conditions of 37 ° C and 5% CO _{2 in a} humid atmosphere. After culturing for 2 or 3 days, a cell suspension is prepared using 0.05% trypsin / 0.02% EDTA, and the cell concentration is measured with a coulter counter (Co
ulter Electronics Inc.) to calculate the proliferation rate.

2) DNA coding for modified protein of guinea pig IFN-γ Among the above-mentioned DNA, "an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2" Have and guinea pig IF
The term "DNA encoding a protein having N-γ activity" refers to artificially produced so-called modified proteins or allelic variants existing in vivo, such as guinea pig IFN.
-Means DNA encoding a protein having gamma activity. DNA encoding the protein can be obtained, for example, by site-directed mutagenesis (Methods in Enzymology, 100, p468).
(1983)) and PCR (Molecular Cloning 2nd Edt.
Chapters, Cold Spring Harbor Laboratory Press (1989)), etc., can be easily prepared by those skilled in the art. Here, the number of amino acid residues to be deleted, substituted or added refers to the number of amino acids that can be deleted, substituted or added by a well-known method such as site-directed mutagenesis.
Here, “having guinea pig IFN-γ activity” means
It means that it has properties unique to natural guinea pig IFN-γ, for example, it has antiviral activity or anti-cell proliferation activity in guinea pigs. Specifically, for example, guinea pig cells GPC-1 by EMCV or VSV
It means having the activity of inhibiting the cytopathic activity of 6 or 104C1 or the activity of inhibiting the growth of guinea pig cells GPC-16 or 104C1. By providing various modified proteins and the like to the aforementioned method for measuring guinea pig IFN-γ activity,
Modified proteins having guinea pig IFN-γ activity can be easily selected.

3) DNA that hybridizes with guinea pig IFN-γ DNA under stringent conditions Among the aforementioned DNAs, “a protein that hybridizes with guinea pig IFN-γ DNA under stringent conditions and has guinea pig IFN-γ activity The term "DNA encoding" means a DNA that hybridizes to guinea pig IFN-γ DNA having the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and at the same time, the protein encoded by the DNA has guinea pig IFN-γ activity. I do. Here, “DNA that hybridizes under stringent conditions” refers to, for example, formamide concentration:
Hybridization was carried out under the conditions of 50% (V / V), salt concentration: 5 × SSC, temperature: about 42 ° C., salt concentration: 2 × SSC,
Temperature: means DNA that hybridizes with guinea pig IFN-γ DNA when washed under conditions of about 42 ° C. Cloning of these DNAs can be performed by screening a cDNA library or a genomic library prepared from animal tissues using the DNA of SEQ ID NO: 1 as a probe. For example, Molecular Cloning 2nd Edt. Cold Spring Harbor La
boratory Press (1989) and the like. In particular,
For example, it can be carried out by the method described in TINS, 15, 319-323 (1992) and in the literature cited in this document, and more specifically, for example, by the following method.
That is, it can be carried out by screening a cDNA library or a genomic library prepared from various animal tissues, particularly guinea pig tissues, using a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 as a probe. As a screening method, for example, a method as described in Example 2 can be mentioned. As the cDNA library, it is preferable to use a cDNA library derived from guinea pig spleen. Hybridization conditions are the same as those described above, or TINS, 15, 319-3
23 (1992) and the conditions described in the literature cited in this literature. Further, “having guinea pig IFN-γ activity” is the same as the content described in 2) above.

The DNA of the present invention is useful for examining the state of immune cells in a guinea pig at the time of invasion of a different biological substance or at the time of cancer. For example, the expression of IFN-γ that strongly reflects the activation state of macrophages, natural killer cells, and T cells can be analyzed by Northern blot using the DNA of the present invention.

A third aspect of the present invention is the first aspect of the present invention.
Or a recombinant protein obtained by expressing the DNA according to the second aspect. A typical example of the protein included in the present embodiment is guinea pig IFN-γ having the amino acid sequence of SEQ ID NO: 2. The guinea pig IFN-γ has a signal sequence at the N-terminus, and the signal sequence is expected to correspond to positions 1 to 20 of the amino acid sequence shown in SEQ ID NO: 2. Since the signal sequence is processed and removed when translocating to the membrane, such a mature guinea pig IFN-γ from which the signal sequence has been removed is also included in this embodiment. Next, a method for preparing the protein of this embodiment will be described. For example, the cloned guinea pig IFN-γ cD
NA can be expressed and produced by ligating NA to a known expression vector such as pET or pCDM8, and then introducing it into an appropriate host. Either prokaryotic or eukaryotic cells may be used as the host. For example, Escherichia coli strains and animal cell strains are already widely available and can be obtained. For example, animal cell hosts include COS-1, COS
-7, CHO cells and the like. To transform an appropriate animal cell host using the expression plasmid, for example, DEAE
Dextran method (Current P compiled by FMAusubel et al.
rotocols in Molecular Biology, John Wiley & Sons
(1987)). The protein of the present embodiment is the guinea pig IFN of the eighth embodiment of the present invention described below.
-Can be used as an antigen when producing a γ-recognizing antibody. Further, the protein of this embodiment can be purified by affinity purification using a guinea pig IFN-γ-recognizing antibody or ordinary column chromatography.

A fourth aspect of the present invention is an expression plasmid for expressing the DNA of the first or second aspect of the present invention. A fifth aspect of the present invention is a transformant transformed with the expression plasmid of the fourth aspect. A sixth aspect of the present invention is a method for producing a recombinant protein, comprising culturing the transformant of the fifth aspect and collecting the expressed protein. The methods for producing these expression plasmids and transformants, or the methods for producing recombinant proteins, are all well known to those skilled in the art, as described in the third aspect of the present invention. A recombinant protein in the present invention is a polypeptide expressed in a transformed cell from a replicable expression plasmid obtained by recombinant DNA technology (regardless of whether or not it is glycosylated by the cell that produces it). Means
The polypeptide exhibits properties unique to natural guinea pig IFN-γ, for example, antiviral or antiproliferative activity in guinea pigs to some extent.

[0018] A seventh aspect of the present invention is an antibody against the protein of the third aspect of the present invention. The antibody is, for example,
Guinea pig IFN-γ or a fragment thereof can be obtained as an antigen, and can be obtained as an antiserum and an isolated anti-guinea pig IFN-γ antibody. As a method for producing an antiserum, for example, the new cell engineering experiment protocol p210 Shujunsha
(1993), immunizing an animal such as a rabbit or a mouse using the method described in (1993) and obtaining the serum thereof. In addition, as a method for isolating and producing an anti-guinea pig IFN-γ antibody, for example, the method described in Chapter 4 Protein Experiments for Molecular Biology Research, Yodosha (1994) can be easily used. Monoclonal antibodies can be produced. That is, a rabbit or mouse is immunized with guinea pig IFN-γ or a fragment thereof as an antigen, and its spleen cells are fused with myeloma cells to produce a hybridoma, and a hybridoma producing an antibody recognizing guinea pig IFN-γ from the hybridoma. Is selected and cultured, and the culture supernatant is obtained. In order to enhance antigenicity, a guinea pig IFN-γ may be bound to a suitable carrier protein as needed to serve as an immunogen. Various adjuvants can be used for immunization, but Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) are preferable. As animals immunizing with antigens,
Rabbits, mice, rats, cows, sheep, goats, chickens and the like can be used. Uses of the antibody include affinity chromatography, screening of a cDNA library, diagnostic reagents and experimental reagents. For example, it can be used to measure the concentration of IFN-γ in blood, body fluid, urine, etc.
It can be used as a research reagent for studying the relationship of -γ. In particular, in order to analyze a series of reactions elicited when foreign biological substances enter using a guinea pig, IFN-
It is useful for measuring the amount of γ.

An eighth aspect of the present invention is a method for producing an antibody against the protein according to the third aspect of the present invention, which comprises using the protein of the third aspect of the present invention or a fragment thereof as an antigen. The details are as described in the section on the antibody of the seventh aspect of the present invention.

[0020]

EXAMPLES The basic experimental method was described by Molecular Cloning 2nd Edt. (Cold Harbor La.) Compiled by Maniatis et al.
boratory Press, 1989), C compiled by Ausubel et al.
urrent Protocols in Molecular Biology (John Wiley
& Sons, 1987) and the Cell Engineering Laboratory Protocol (Shujunsha, 1991) compiled by the Cancer Research Division of the Institute of Medical Science, The University of Tokyo (Shujunsha, 1991). It should be noted that the present invention is not limited only to the following examples, and it is needless to say that ordinary changes in the technical field of the present invention can be made.

Example 1 (Preparation of cDNA library of guinea pig spleen cells)
Spleens were removed from Hartley-type guinea pig males, 23 weeks of age (purchased from Charles River), and cell suspensions were prepared in Hanks' solution using a slide glass. Cell clumps were removed by filtration. The cells were collected by centrifugation, suspended in 7 ml of 0.1 x phosphate buffered saline (PBS), and immediately added with 7 ml of 2 x PBS and 10 ml of Hank's solution to lyse the cells. After centrifugation, spleen cells were spiked at a concentration of 5 x 10 ⁶ cells / ml in RPMI medium (Osaka University Micro-Lab) containing 10% fetal bovine serum, 50 μM 2-mercaptoethanol, 50 U / ml penicillin, 50 μg / ml streptomycin. Suspended. Concanavalin A 5
μg / ml and 37% in a humid atmosphere of 5% CO _2.
Incubated at ℃. After 24 hours, cells were collected by centrifugation and stored frozen. TRIZOL reagent (GIBC
RNA is prepared from the frozen cells using an OBRL (manufactured by OBRL), and mRNA purification kit (mRNA puri
A polyA fraction was obtained using a fication kit (Pharmacia). For cDNA synthesis, use the cDNA synthesis kit (Supers
cript Choice System for cDNA Synthesis Kit) (GIBC
OBRL). Restriction enzyme EcoRI /
NotI adapter was ligated and ligated with phage vector Lambda Zap II (λZAPII) which had been digested with EcoRI,
Gigapack III Gold Packaging Extract
(Gigapack III Gold Packaging Extract) (Stratagene
(Made by the company).

Example 2 (Acquisition of guinea pig IFN-γ cDNA) The cDNA-encapsulated phage prepared in Example 1 was seeded on an agarose plate to which NZCYMA soft top agarose (GIBCO BRL) was added and cultured. The obtained plaques are filtered with a membrane filter colony / plaque screen (Colony / Pl
aqueScreen) (manufactured by NEN). Mouse IFN-γ
CDNA full length (PWGray et al., Proc. Natl. Acad.
Sci. USA Vol. 80, p5842 (1983)) with [α- ³² P] dCTP
And labeled as a probe. Hybridization buffer (6 x SSC (1 x SSC is 150 mM NaCl, 15 mM trisodium citrate), 30% formamide, 0.1% sodium dodecyl sulfate (SDS), 1 x Denhardt's solution, 20 mM Tris buffer (PH 7.0), 10% dextran sulfate), immersed in a hybridization buffer containing a ³² P-labeled probe and 50 μg / ml denatured salmon sperm DNA, and incubated at 37 ° C. for 3 hours (prehybridization). Hybridization was performed by incubating overnight at 37 ° C. The filter was washed twice with 6 × SSC solution at room temperature for 20 minutes and twice at 37 ° C. for 30 minutes, and autoradiographed at −80 ° C. Seven phages were selected from the phages that gave a strong positive signal, and these were infected with Escherichia coli XL1-BlueMRF (Stratagene) together with ExAssist helperphage (Stratagene). The culture supernatant is infected into Escherichia coli SOLR strain (manufactured by Stratagene), and the inserted portion of the phage that produces a positive signal is inserted into the plasmid vector “P Blue Script” (pBl
uescript) was obtained.

Example 3 (Analysis of the base sequence of guinea pig IFN-γ cDNA) Seven D-DNAs obtained in Example 2 and inserted into a plasmid
Use the ABI PRISM Dye Terminator Cycle Sequen
cing FS Ready Reaction Kit) (Applied Biosystems). The nucleotide sequence of the guinea pig IFN-γ cDNA was determined by comparing the determined DNA nucleotide sequence or the human IFN-γ cDNA. The determined DNA base sequence (1232 bases) is represented by SEQ ID NO:
1 and the deduced amino acid sequence (amino acid number: 166) are shown in SEQ ID NO: 2. Guinea pig IFN-γ and its gene thus determined were compared with human IFN-γ and its gene or mouse IFN-γ and its gene. As a result, guinea pig IFN-γ and human IFN-γ
-Γ (166 amino acids, GenBank Accession No. V00543)
Among them, a homology of 72% in the base sequence and 58% in the amino acid sequence was observed. Guinea pig IF
N-γ and mouse IFN-γ (155 amino acids, GenBank Ac
cession No. K00083), 64
%, And 37% homology in the amino acid sequence.

[0024]

Industrial Applicability According to the present invention, it is possible to clarify the state of immune cells in guinea pigs at the time of invasion of heterologous biological material or at the time of cancer-bearing. That is, the expression of IFN-γ, which strongly reflects the activation state of macrophages, natural killer cells, and T cells, can be analyzed by Northern blot using the DNA of the present invention and by ELISA using the antibody of the present invention. It has been speculated that upon invasion of a foreign biological substance, an immune response closer to humans is induced in guinea pigs. For example, a delayed hypersensitive skin reaction due to Mycobacterium bovis b.c. is observed in guinea pigs but not in mice. Here, it is meaningful to analyze the immune response using guinea pigs. By this analysis,
It is possible to evaluate and evaluate immunotherapeutic agents for diseases or cancer caused by abnormalities of the immune system.

[0025]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1232 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA Sequence characteristics Characteristic determination method: E sequence CTGAAGATCA GTCACTAGAA AAGGAAGATC AGTGAAGTTC TTTGGACCTG ATCGACTTAA 60 TACAAGAGCT ACTGATTTCA TCTTCTTTAG CCCAACTCTC TGAAACA ATG AAG TAT 116 Met Lys Tyr ACA AGT TCC ATC TTG GCT CTT CAG TTC TGT ATC ATT TTG AGT TTT TCT 164 Thr Ser Ser Ile Leu Ala Leu Gln Phe Cys Ile 5 Le Au Ser Phe Ser TAC TGC CAG TCA AGA TTT ACA AAT GAA ATA CGC ATT CTA AAG 212 Ser Tyr Tyr Cys Gln Ser Arg Phe Thr Asn Glu Ile Arg Ile Leu Lys 20 25 30 35 AAC TAC TTT AAT GCA GAT AAC TCA GAT GTA GGA GAC AAT GGG ACG CTC 260 Asn Tyr Phe Asn Ala Asp Asn Ser Asp Val Gly Asp Asn Gly Thr Leu 40 45 50 TTT GTA GGC ATT TTG AAG AAT TGC CAA GAG GAG AGT GAA AGA AAA ATA 308 Phe Val Gly Ile Leu Lys Asn Cys Gln Glu Glu Ser Glu Arg Lys Ile 55 60 65 TTT CAG AGC CAA ATC GTC TCC TTC TAC TTC AAA CTC TTT GAA AAA CAT 356 Phe Gl n Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Glu Lys His 70 75 80 TTT ACA GAC AAT CAG ACT GTC CAA AAT AGC ATG AAC ACC ATC AAG GAA 404 Phe Thr Asp Asn Gln Thr Val Gln Asn Ser Met Asn Thr Ile Lys Glu 85 90 95 CAA ATC ATT ACT AAG TTC TTC AAA GAC AAC AGC AGC AAC AAG GTG CAG 452 Gln Ile Ile Thr Lys Phe Phe Lys Asp Asn Ser Ser Asn Lys Val Gln 100 105 110 115 GCT TTC AAA AAC CTG ATT CAA ATT TCG GTC AAT GAC GAG CAT GTC CAG 500 Ala Phe Lys Asn Leu Ile Gln Ile Ser Val Asn Asp Glu His Val Gln 120 125 130 CGC CAA GCG ATC ATT GAA CTC AAA AAA GTG ATA GAT GAC CTG TCA CCG 548 Arg Gln Ala Ile Ile Glu Leu Lys Lys Val Ile Asp Asp Leu Ser Pro 135 140 145 AAC CAG AGG AAA CGA AGA AGG ACT CAG ATG CTG TTT CAG AGC CGG AGA 596 Asn Gln Arg Lys Arg Arg Arg Thr Gln Met Leu Phe Gln Ser Arg Arg 150 155 160 GCA TCC AAA TAAGAGTCAT TCTGCCTATA ATATTTGAAT TTTTAAATCA 645 Ala Ser Lys 165 AACCTATTTA TTAATATTTA AAATTATTTA TATGGAGAAT ATATTTTTAC TCATTAATCA 705 AAGAAGTATT TAATAGTAAG TTTAATGTAA TGAAAATGAA TATCTATTAT CTATTAATAT 765 ATGTATTATT TATAATTCCT GAATCCTGTC ATTGTTCTTC TTGTCCTTTA TCCTTTCTGA 825 TTAAGTAACA AGACCATGTG ATGATCCAGT TCTGTCTCAG GGGCCAGCTA AGCAGCTGAC 885 CTGAGCAAGA CCCTGAGGGT TGTGCATTTA TTTCACTTGA TGATACAAGA AATACTTATA 945 GATTAAATAA CACCATCTGG TTGCTGCCTA TTTGAGAACA TCTCTGCATT CTGAGCCAGT 1005 GCTTTAATGG CCTGGCAGAC AGCACTTGAA TATGTCAGGC GAAATGGCCT GGTACCCTGG 1065 TAAAAACACA GCATCTCAGG AAATGTCACA TCTGGTGCTT CTAAATATTG CTGACAACTG 1125 TGATTGAACT CAAATGGAAA TTACCTCACT TGTTTAGTTT ATTGATTTCT AATATATATA 1185 AATAAAATAT AATTTTACAA ACAAAAAAAA AAAAAAAAAA AAAAAAA 1232

SEQ ID NO: 2 Sequence length: 166 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Met Lys Tyr Thr Ser Ser Ile Leu Ala Leu Gln Phe Cys Ile Ile Leu 5 10 15 Ser Phe Ser Ser Tyr Tyr Cys Gln Ser Arg Phe Thr Asn Glu Ile Arg 20 25 30 Ile Leu Lys Asn Tyr Phe Asn Ala Asp Asn Ser Asp Val Gly Asp Asn 35 40 45 Gly Thr Leu Phe Val Gly Ile Leu Lys Asn Cys Gln Glu Glu Ser Glu 50 55 60 Arg Lys Ile Phe Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe 65 70 75 80 Glu Lys His Phe Thr Asp Asn Gln Thr Val Gln Asn Ser Met Asn Thr 85 90 95 Ile Lys Glu Gln Gle Ile Ile Thr Lys Phe Phe Lys Asp Asn Ser Ser Asn 100 105 110 Lys Val Gln Ala Phe Lys Asn Leu Ile Gln Ile Ser Val Asn Asp Glu 115 120 125 His Val Gln Arg Gln Ala Ile Ile Glu Leu Lys Lys Val Ile Asp Asp 130 135 140 Leu Ser Pro Asn Gln Arg Lys Arg Arg Arg Thr Gln Met Leu Phe Gln 145 150 155 160 Ser Arg Arg Ala Ser Lys 165

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 21/02 C12N 15/00 ZNAA // (C12N 1/21 C12R 1:19) (C12N 5/10 C12R 1:91) ( C12N 15/09 ZNA C12R 1:91) (C12P 21/02 C12R 1:91) (C12P 21/02 C12R 1:19)

Claims (8)

[Claims] 1. A DNA encoding the following protein (a) or (b): (A) A guinea pig interferon gamma protein having the amino acid sequence of SEQ ID NO: 2. (B) 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 of SEQ ID NO: 2, and having interferon gamma activity in guinea pigs; 2. A DNA having the nucleotide sequence of SEQ ID NO: 1, or a DNA that hybridizes with the DNA under stringent conditions and encodes a protein having interferon gamma activity in guinea pigs.
A. 3. A recombinant protein obtained by expressing the DNA according to claim 1 or 2. An expression plasmid which expresses the DNA according to claim 1 or 2. A transformant transformed by the expression plasmid according to claim 4. 6. A method for producing a recombinant protein, comprising culturing the transformant according to claim 5, and recovering the expressed protein. 7. An antibody against the protein according to claim 3. 8. A method for producing an antibody against the protein according to claim 3, wherein the protein according to claim 3 or a fragment thereof is used as an antigen.