JPH1198997A - Production of interferon-gamma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain the subject compound useful for an antitumor agent, an antiviral agent, etc., by treating a recombinant Baculovirus containing interferon-γ DNA in the extract of the body fluid of the larva of a silkworm infected with the recombinant Baculovirus under specific conditions. SOLUTION: A recombinant Baculovirus contained in the culture supernatant liquid of a cultured cell of an insect pest subjected to gene recombination with a DNA end an interferon-γ protein or the body fluid of the larva of a silkworm infected with the recombinant Baculovirus is treated under acidic or alkali conditions and inactivated to inexpensively and efficiently obtain a large mount of the objective interferon-γ subjected to gene recombination useful as a medicine such as an antitumor agent, an antiviral agent, etc. A nuclear polyhedrosis virus of a recombinant silkworm by gene recombination with a DNA encoding interferon-γ is preferably used as the recombinant Baculovirus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a useful protein, which is produced as a pharmaceutical by producing interferon-γ, which is a candidate for an antitumor agent, an antiviral agent and an antiallergic agent, using a genetic recombination technique. In the process, it is possible to efficiently produce the recombinant interferon-γ by confining the recombinant and recovering the activity of the inactivated interferon-γ, thereby enabling the interferon-γ to be produced in large quantities and at low cost. The present invention relates to a method for producing interferon-γ for production as a pharmaceutical.

[0002]

2. Description of the Related Art Many techniques are known for producing useful proteins using a genetic recombination technique. For example, a method for producing a large amount of useful proteins using a genetically modified baculovirus is known as feline interferon. (Reference 1). When a useful protein produced using such a genetic recombination technique is produced as a pharmaceutical, it is necessary to contain (inactivate) the genetically modified product from the viewpoint of safety. For example, when a useful protein is produced using a genetically modified baculovirus, inactivation of the recombinant virus is essential because a large amount of the recombinant virus is present in a cell culture solution or an insect body fluid. Regarding the baculovirus inactivation method, various inactivation methods by a chemical or physical method are reported as in Reference 2, and a genetically modified product by a hydrochloric acid treatment (genetically modified silkworm nuclear polyhedrosis virus) An example of inactivation of is also reported in the production of feline interferon (JP-A-4-207198). In the case of producing useful proteins, it is necessary to maintain the activity at the same time as inactivating the recombinant virus.
Most of the conventional methods deactivate useful proteins. Since interferon-γ is extremely weak to chemical and physical treatments and has extremely low pH stability, it has been difficult to produce it as a pharmaceutical using a highly productive baculovirus expression system.

[0003]

SUMMARY OF THE INVENTION Interferon-γ
Is an extremely unstable protein that is inactivated by all physicochemical treatments such as heat treatment and acid / alkali treatment. Therefore, when interferon-γ is produced as a pharmaceutical using a genetic recombination technique, it is very often inactivated during the production process, and the production method is considerably limited.

[0004] Therefore, if the activity of interferon-γ once inactivated can be restored, the production of recombinant interferon-γ becomes easy, and interferon-γ can be efficiently used as a drug by a wide variety of production methods. It is expected that it will be possible to manufacture.

[0005]

Means for Solving the Problems In view of such circumstances, the present inventor aimed at efficient production of recombinant interferon-γ as a drug, devised an ingenuity, and developed interferon-γ recombinant baculovirus. Is inactivated by treating it under acidic or alkaline conditions, and surprisingly complete recovery of the activity by storing interferon-γ, which has completely lost its biological activity, under neutral and low temperatures. Thus, a method for efficiently producing large amounts of interferon-γ was established, thus completing the present invention.

That is, the present invention relates to interferon-γ
Provided are a method for inactivating a genetically modified baculovirus by an acid or alkali treatment, a method for restoring the activity of interferon-γ having lost biological activity by an acid or alkali treatment, and interferon-γ thus obtained. Things.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

[0008] The interferon-γ of the present invention includes all animal species. The method of the present invention is preferably used particularly when producing canine interferon-γ. Canine interferon-γ has a sugar chain attached thereto,
All those having the activity of canine interferon-γ, such as those having no sugar chain and those lacking a part of the C-terminal structure, are included.

The DNA encoding the canine interferon-γ protein can be produced, for example, as follows. That is, after extracting poly (A) RNA from dog cells, it is converted into cDNA, and canine interferon-γ
A gene encoding canine interferon-γ can be obtained by performing a polymerase chain reaction (hereinafter abbreviated as PCR) using a primer based on the gene sequence encoding the canine interferon-γ. As a method for obtaining RNA from canine cells, for example, canine lymphocytes stimulated with mitogen, etc., usual methods such as polysomal separation, sucrose density gradient centrifugation and electrophoresis are used. can give. As a method for extracting RNA from the above-mentioned dog cells, guanidine-thiocyanate-treated CsCl
Guanidine-thiocyanate-cesium chloride method of performing density gradient centrifugation (Reference 2) A method of treating with a surfactant using a vanadium complex in the presence of ribonuclease inhibitor and then extracting phenol (Reference 3), A guanidine thiocyanate-hot phenol method, a guanidine thiocyanate-guanidine hydrochloride method, a guanidine thiocyanate-phenol-chloroform method, a treatment with guanidine thiocyanate, and a treatment with lithium chloride. An appropriate method can be selected from methods for precipitating RNA by using the method.

[0010] The mR is determined by a conventional method such as canine lymphocyte, for example, lithium chloride / urea method, guanidine isothiocyanate method, oligo dT cellulose column method and the like.
NA is isolated, and a normal method is used from the obtained mRNA, for example, the method of Gubler et al. Okaya
cDNA is synthesized by the method of Ma et al.
In order to synthesize cDNA from the obtained mRNA, a method using a reverse transcriptase such as avian osteoblast virus (AMV) or the like and a method using a DNA polymerase or the like using a partial primer are basically used. However, it is convenient to use a commercially available synthesis or cloning kit. By performing PCR using this cDNA as a template and a primer based on the nucleotide sequence of canine interferon-γ, DNA encoding canine interferon-γ protein can be obtained.

[0011] Canine interferon-γ can also be produced using a silkworm expression system by preparing a recombinant silkworm nuclear polyhedrosis virus that infects silkworms. The recombinant silkworm nucleopolyhedrovirus is a recombinant plasmid prepared by ligating a DNA encoding the protein of canine interferon-γ to a silkworm cloning vector (Reference 6) and a silkworm nucleopolyhedrovirus virus DNA. Can be prepared by co-transfection of silkworm established cells. Therefore, the recombinant virus has i
It can be produced by an n vivo method. That is, D encoding the canine interferon-γ protein
A recombinant plasmid can be prepared according to a general genetic operation in which the NA portion is ligated downstream of the expression control portion of a silkworm cloning vector such as pBM030 (Reference 6). After co-transfecting the recombinant plasmid and the silkworm nuclear polyhedrosis virus DNA (Reference 6) into a silkworm-established cell, for example, the BM-N strain (Reference 6), according to the method described in the literature, culture is continued. A recombinant virus can be cloned from a non-recombinant (wild-type) and a recombinant virus that has appeared in a culture solution by a general method such as a limiting dilution method or a plaque method. Since the recombinant virus has no polyhedron-forming ability, it can be easily distinguished from the wild-type virus. The production of canine interferon-γ is carried out by growing the above-mentioned recombinant silkworm nuclear polyhedrosis virus in a silkworm established cell or a silkworm living body.

In the case of using silkworm established cells, BM-N cells are infected with a culture solution containing the above-mentioned recombinant virus, and cultured by flat culture or suspension culture. BM-
As a medium for culturing N cells, for example, a TC-10 medium (Reference 7) or a TC-100 medium (manufactured by Nippon Agricultural Industry Co., Ltd.) supplemented with bovine serum can be used. The culture temperature is suitably from 25 to 28 ° C. After the culture, the culture solution is centrifuged, and dog interferon-γ is recovered from the supernatant.

In the case of using a silkworm living body, a culture solution containing the above-mentioned recombinant virus is injected into a silkworm larva and fed with an artificial feed for breeding. After breeding, a body fluid is collected, and dog interferon-γ is recovered from the supernatant.

The acid or alkali used for inactivating the canine interferon-γ recombinant baculovirus includes, but is not limited to, hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, formic acid, and sodium hydroxide.

The pH of the acid or alkali solution used may be a value sufficient for inactivating the canine interferon-γ recombinant baculovirus, and usually 3 or less or 9 or more is appropriate. The method is also carried out at a temperature above the freezing point, preferably at 4 to 40 ° C. The processing time is at least one minute, but longer processing is also possible, and good results can be obtained by processing for 1 to 12 hours.

The activity can be improved by treating canine interferon-γ, which has lost its biological activity by treatment, at a neutral and low temperature. Here, neutral means pH 6
It is preferable to carry out the treatment at ~ 8. It is preferable to neutralize the solution that has been treated with acid or alkali in the baculovirus inactivation step, centrifuge, and incubate the supernatant at 0 to 15 ° C. Incubation time should be at least 1
The time is preferably 2 hours or more, more preferably 1 to 7 days.

[0017]

The present invention will now be described more specifically with reference to examples.

Reference Example 1 <Preparation of Canine Interferon-γ Gene Recombinant Silkworm Nucleopolyhedrovirus (1)> Preparation of Canine cDNA Lymphocytes were isolated from canine peripheral blood, and phytohemagglutinin (PHA) was isolated at 50 μg / PHA. Stimulation was performed for 48 hours at a final concentration of ml. After stimulation, ISOGEN (Nippon Gene Co., Ltd.)
Was used to prepare total RNA. The obtained RNA is
10 mM Tris-HCl buffer containing 10 mM EDTA (p
H7.5) (hereinafter abbreviated as TE), treated at 70 ° C. for 5 minutes, and added the same amount of TE containing 1 M LiCl. The RNA solution was applied to an oligo dT cellulose column equilibrated with TE containing 0.5 M LiCl, and washed with the same buffer. After further washing with TE containing 0.3 M LiCl, 2 mM E containing 0.01% SDS was used.
The adsorbed poly (A) RNA was eluted with DTA (pH 7.0). Using the poly (A) RNA thus obtained, a single-stranded cDNA was synthesized. That is, sterilized 0.5m
In a microcentrifuge tube, 5 μg of poly (A) RNA and 0.5 μg of oligo dT primer (12-18 me
r) was added, and sterilized water treated with diethylpyrocarbonate was added to make 12 μl, and the mixture was incubated at 70 ° C. for 10 minutes and then placed on ice for 1 minute. 200 μM Tris-HCl (pH 8.4) and 500 mM KCl solution
1, 25 mM MgCl2 2 μl, 10 mM dN
After adding 1 μl of TP and 2 μl of 0.1 M DTT, respectively, incubating at 42 ° C. for 5 minutes,
Unit reverse transcriptase (manufactured by GibcoBRL, Sup
erScript II) and add another 5 at 42 ° C.
After incubating for 0 minutes, a cDNA synthesis reaction was performed.
The reaction was further stopped by incubating at 70 ° C. for 15 minutes and placed on ice for 5 minutes. 1 μl of E. coli was added to the reaction solution. c
Add oligo RNaseH (2 units / ml)
Incubation was performed at 37 ° C for 20 minutes.

(2) Preparation of canine interferon-γ gene Based on the nucleotide sequence of canine interferon-γ (Reference 2), 5'GCGAATTCATGAATTATACAAA
GCTATATCTTAGCT3 '(SEQ ID NO: 1) and 5
'GCGAATTCTTTATTCGATGCTCTG
A primer having an EcoRI cleavage site added to the two ends of CGGCCTCGAAA3 ′ (SEQ ID NO: 2) was named DN.
Synthesized using an A synthesizer. CD obtained in (1)
Take 2 μl of NA into a 0.5 ml microcentrifuge tube, and add each primer to 20 pmol, 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgCl 2, 25 mM
mM KCl, 100 μg / ml gelatin, 50 μM
Each reagent was added to make each dNTP, 4 units ExTaq DNA polymerase (manufactured by Takara Shuzo Co., Ltd.).
Make it 00 μl. DNA denaturation conditions were 94 ° C for 1 minute, primer annealing conditions were 55 ° C for 2 minutes, and primer extension conditions were 72 ° C for 3 minutes.
Elmer Cetus DNA Thermal Cycler
The reaction was carried out for 30 cycles. 1% agaro
After electrophoresis on a sgel, a DNA fragment of about 560 bp was prepared according to a conventional method (Reference 8). This DNA fragment is
Takara Shuzo Co., Ltd. was added to T-Vector of Vitrogen.
DNA Ligation Kit Ver. The reaction was carried out at 16 ° C. for 2 hours using No. 1 and ligated. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant according to a conventional method. Next, insertion of the PCR fragment into this plasmid was confirmed by PCR under the same conditions as described above, and a fluorescent DNA sequencer (DNA sequencer 373 manufactured by PerkinElmer) was used.
S), and according to the attached protocol, it was confirmed that the obtained DNA fragment was a nucleotide sequence of canine interferon-γ DNA using a dye terminator cycle sequencing kit manufactured by PerkinElmer.

(3) Preparation of Plasmid for Silkworm Expression After digestion of 1 μg of vector pBM030 (Reference 7) with 30 units of restriction enzyme EcoRI at 37 ° C. for 16 hours,
The terminal was dephosphorylated with 1 unit of alkaline phosphatase derived from bacteria (Takara Shuzo Co., Ltd.). This one
% Agarose gel, and the D of about 11.3 Kb
The NA fragment was prepared according to a conventional method.

DNA Ligation Kit Ve
r. The ligation reaction was carried out at 16 ° C. for 16 hours using No. 1 and pBM030 prepared as described above and (2)
The DNA fragment of canine interferon-γ prepared in the above was ligated. Using this, Escherichia coli HB101 was transformed according to a conventional method. L containing 100 μg / ml ampicillin
From the colonies growing on the B plate, 2 codons from the initiation codon of the DNA encoding canine interferon-γ
Primers containing up to 7 bp, ie, 5'ATGA
ATTATACAAGCTATATCTTAGCT3 '
(SEQ ID NO: 3) and cloning site E of pBM030
26 bp primer downstream from coRI, ie
5'ATCAACAACGCACAGAATCTAAC
Using two kinds of primers of GCT3 ′ (SEQ ID NO: 7), the DNA denaturation condition was 94 ° C. for 1 minute, the primer annealing condition was 55 ° C. for 2 minutes, the primer extension condition was 72 ° C. Perkin-Elmer for 3 minutes
Using a DNA thermal cycler from Cetus, 30
PCR was performed in each cycle, and a DNA fragment of about 650 bp was obtained.
A plasmid was obtained in which A was integrated in the forward direction into pBM030. This recombinant plasmid was designated as pBMγ.
E. coli containing this plasmid was transformed into E. coli. coli (pB
Mγ).

Further, based on the nucleotide sequence of canine IFN-γ, 5′GCAGATCTATGAATTATATACAA
GCTATATCTTAGCT3 '(SEQ ID NO: 8) and 5'GCGAATTCTTATTTCGATGCTCT
Two types of primers, GCGGCCTCCGAAAA3 ′ (SEQ ID NO: 9), were synthesized by requesting Japan Bioservices Co., Ltd. 2 μl of the cDNA obtained in (1) was placed in a 0.5 ml microcentrifuge tube, and each primer was placed in a 20 ml microcentrifuge tube.
pmol, 20 mM Tris-HCl buffer (pH 8.0),
1.5 mM MgCl ₂ , 25 mM KCl, 100
μg / ml gelatin, 50 μM each dNTP, 4 units
Each reagent is added to make ExTaq DNA polymerase (manufactured by Takara Shuzo Co., Ltd.) to make the total volume 100 μl. DNA
Denaturation conditions at 94 ° C for 1 minute, primer annealing conditions at 55 ° C for 2 minutes, primer extension conditions at 72 ° C,
Perkin-Elmer Cetus for 3 minutes
The reaction was carried out for 30 cycles using a DNA thermal cycler manufactured by the company. This was electrophoresed on a 1% agarose gel,
A 517 bp DNA fragment (SEQ ID NO: 10) was prepared according to a conventional method. This DNA fragment was used for Invitrogen
Was connected to Takara Shuzo Co., Ltd. according to a conventional method. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant according to a conventional method. Next, using a fluorescent DNA sequencer (DNA sequencer 373S manufactured by PerkinElmer) and a DNA terminator encoding canine IFN-γ using a dye terminator cycle sequencing kit manufactured by PerkinElmer according to the attached protocol.
Was confirmed.

Next, using this DNA fragment as a template, PCR was carried out under the same conditions as above using three combinations of primers (SEQ ID NOS: 11 to 16), and three types of PCR amplified fragments (SEQ ID NOs: 17 to 19) I got These were collected according to a conventional method, and the fragment shown in SEQ ID NO: 17 was replaced with the restriction enzyme BamH.
I and EcoRV, the fragment shown in SEQ ID NO: 18 was digested with restriction enzymes HincII and SnabI, and the fragment shown in SEQ ID NO: 19 was digested with restriction enzymes EcoRV and EcoRV.
SEQ ID NO: digested with RI and treated with restriction enzymes
No. 17 and the fragment shown in SEQ ID NO: 19 treated with restriction enzymes were mixed, and EcoRI and BamHI of pUC19 were mixed.
This was inserted into the site according to a conventional method to obtain a recombinant vector. Further, after digesting this vector with the restriction enzyme EcoRV, the fragment shown in SEQ ID NO: 18 was inserted according to a conventional method to obtain a recombinant vector, and the base sequence of the inserted DNA (SEQ ID NO: 2)
0) was confirmed in the same manner as described above. Then, restriction enzyme B
The DNA inserted with amHI and EcoRI was recovered and inserted into pBM030 cut with restriction enzymes BglII and EcoRI in a conventional manner to prepare a recombinant vector pBMγS2 (−) for silkworm expression. Also, p
PCR was carried out using BMγS2 (−) as a template and primers shown in SEQ ID NO: 21 and SEQ ID NO: 22 to obtain a DNA fragment shown in SEQ ID NO: 23, which was similarly treated with a restriction enzyme, and then treated with BglII of pBM030. And Eco
It was inserted into the RI site to create pBMγS2 (−) / − 20.

(4) Preparation of Recombinant Silkworm Nuclear Polyhedrosis Virus Recombined with DNA Encoding Canine Interferon-γ Recombinant virus was prepared by the method of Reference 2. That is,
50 mM HEPES buffer (pH 7.1);
28 M NaCl, 0.7 mM Na ₂ HPO ₄ , 0.7
To a solution of 2.5ml consisting mM NaH ₂ PO _{4, 2.}
5 ml of a DNA mixture (0.25 M CaCl ₂ , 10 μg of silkworm nuclear polyhedrosis virus BmNPV T3 strain (Reference 2) DNA, DNA of recombinant plasmid pBMγ
65 μg), and 0.5 ml of the resulting suspension was cultured in a 25 cm ² flask in a TC-10 medium supplemented with 5 ml of 10% FBS (Reference 2) in a 25 cm ² flask.
In addition to the culture medium of 0 ^five BM-N cells, D silkworm cells
NA was introduced. After 20 hours, the medium was replaced with a fresh medium, and after culturing for further 7 days, the culture solution was collected. The culture solution was centrifuged, and the clarified supernatant was diluted to culture BM-
After adding to the culture medium of N cells and culturing for 8 days, a culture medium in which virus infection was observed by microscopy and polyhedra were not formed was selected (limit dilution method).

The limiting dilution method was repeated seven times, and the recombinant virus was cloned. The recombinant virus containing the DNA encoding the canine interferon-γ and the canine interferon-γ mutant prepared here was rBNVγ,
rBNVγS2 (−) and rBNVγS2 (−) / − 20.

(5) Preparation of Recombinant Virus Solution In the bottom of a 75 cm ² flask, 15 ml of 10% FBS
A 50 μl culture medium of the BM-N cells containing the recombinant virus cloned in the above (4) was added to about 3 × 10 ⁶ BM-N cells cultured in a plane in a TC-10 medium containing
After adding to M-N cells and culturing at 27 ° C. for 5 days, the culture was centrifuged at 3,000 rpm for 5 minutes to obtain a centrifuged supernatant as a recombinant virus liquid. The virus solution was diluted ^10-7 times, 1 ml of the diluted solution was added to the culture medium of BM-N cells, and the culture was continued at 27 ° C. for 7 days. Microscopic observation revealed that the BM-N cells in the culture medium were infected with the virus.

Reference Example 2 <Method for measuring antiviral activity> The virus was Vesicula.
r Stomatis Virus, susceptible cells were measured for antiviral activity using canine MDCK (ATCC CCL-34) according to the CPE method of Reference 11.

Reference Example 3 <Production of Canine Interferon-γ in Silkworm Established Cells> TC virus containing 10% of FBS was added to each of the recombinant virus solutions obtained in Example 1 in a 0.5 cm ² flask in a 25 cm ² flask.
Approximately 3 × 10 ⁶ BMs cultured in plane in 100 medium
Added to N cells. 30 minutes later, fresh 5 ml of 10% FB
The medium was replaced with a TC-10 medium containing S and cultured at 27 ° C. for 3 days. As a result of taking the centrifuged supernatant of the culture solution and examining the activity, 1
0 ⁴ Antiviral activity of the dilution units / ml was obtained.

Reference Example 4 <Production of canine interferon-γ in silkworm living body>
The virus solution of the recombinant virus obtained in Example 1 was injected into the silkworm larvae on the second day of the 5th day at 50 μl / head, and the virus solution was injected at 25 ° C. for 4 hours.
After feeding a commercially available artificial feed (Kanebo Silk Elegance) for 10 days, the abdomen of 10 silkworms was cut off, the body fluid was collected in an ice-cooled Eppendorf tube, and the supernatant after centrifugation was obtained. After sterilization by filtration through a 22 μm filter, the activity was measured. As a result, an antiviral activity of 10 ⁷ dilution units / ml was obtained.

Reference Example 5 <Quantification of Virus Concentration Due to Cytopathic Effect> According to the method of Reference 12, the concentration of the canine interferon-γ recombinant virus was quantified. That is, the culture supernatant of the silkworm BM-N cells infected with the recombinant virus or the silkworm larval body fluid extract was diluted and added to 5 × 10 ⁵ BM-N cell culture solutions. After culturing at 27 ° C for 10 days, the cytopathic effect on BM-N cells was confirmed by microscopic observation, and the amount of infectious virus was calculated. Infectious virus load was determined by determining TCID ₅₀ according to Ref.

Example 1 <Inactivation of Canine Interferon-γ Recombinant Silkworm Nucleopolyhedrovirus> The silkworm body fluid obtained in Example 4 was immersed in 100 ml of 0.1N hydrochloric acid and incubated at 4 ° C. for 6 hours. did. The obtained extract was adjusted to pH 7 with 2N sodium hydroxide to obtain a supernatant after centrifugation at 10,000 g for 30 minutes. As a result of examining the amount of the recombinant silkworm nucleopolyhedrovirus using the supernatant, the recombinant silkworm nucleopolyhedrovirus was completely inactivated. As a result of measuring the antiviral activity of the supernatant, no antiviral activity was observed. From this result, it was found that the canine interferon-γ recombinant silkworm nuclear polyhedrosis virus was inactivated by the hydrochloric acid treatment, but the biological activity of canine interferon-γ was lost.

Example 2 <Recovery of activity of canine interferon-γ that lost biological activity> The supernatant obtained in Example 6 was further subjected to pH 7 at 4
Incubated for 1 week at ° C. After the incubation, the antiviral activity was measured. As a result, an antiviral activity of 10 ⁷ dilution units / ml was obtained. This was almost equivalent to the activity before the treatment with hydrochloric acid, indicating that the biological activity of canine interferon-γ was completely restored.

[0033]

According to the present invention, it is possible to efficiently produce recombinant interferon-γ, and thus it is possible to produce interferon-γ as a medicine in large quantities and at low cost.

References 1. Sakurai et al .: J. Vet. Med. Sci. , 54, 5
63 (1992). 2. Chirgwin et al .: Biochemistry,
18, 5294 (1979). 3. Berger et al .: Biochemistry, 1
8, 5143 (1979). 4. Gubler et al .: Gene. 25,236-269
(1983). 5. Okayama et al .: Mol. Cell. Bio
l. , 2,161, (1982) & 3,280,
(1983). 6. T. Horiuchi et al .: Agic. Biol. C
hem. , 51, 1573-1580, (1987). 7. Molecular Cloning. Cold
Spring Harbor Laboratory.
New York. 1982.

[0035]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 32 Sequence type: nucleic acid Number of strands: single-stranded Topology-: linear Sequence type: other nucleic acid Synthetic DNA sequence GAATTCATGAATTATACAAGCTATATCTTAGC

SEQ ID NO: 2 Sequence length: 35 Sequence type: Number of nucleic acid chains: Single-stranded Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCGAATTCTTATTTCGATGCTCTGCGGCCTCGAAA

SEQ ID NO: 3 Sequence length: 27 Sequence type: Number of nucleic acid chains: Single-stranded Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATGAATTATACAAGCTATATCTTAGCT

SEQ ID NO: 4 Sequence length: 30 Sequence type: Number of nucleic acid chains: Single-stranded Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TTTTCACTGCATTCTAGTTGTGGTTTGTCC

SEQ ID NO: 5 Sequence length: 42 Sequence type: Number of nucleic acid chains: Single-stranded Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCGACCATGGCTCAGGCCATGTTTTTTAAAGAAATAGAAAAC

SEQ ID NO: 6 Sequence length: 36 Sequence type: Number of nucleic acid chains: Single-stranded Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGATCCTTATTTCGATGCTCTGCGGCCTCGAAACAG

SEQ ID NO: 7 Sequence length: 26 Sequence type: Number of nucleic acid chains: Single-stranded Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATCAACAACGCACAGAATCTAACGCT

SEQ ID NO: 8 Sequence length: 35 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCAGATCTATCAATTATACAAGCTATATCTTAGCT

SEQ ID NO: 9 Sequence length: 35 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCGAATTCTTATTTCGATGCTCTGCGGCCTCGAAA

SEQ ID NO: 10 Sequence length: 517 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCAGATCTAT GAATTATACA AGCTATATCT TAGCTTTTCA GCTTTGCGTG ATTTTGTGTT 60 CTTCTGGCTG TAACTGTCAG GCCATGTTTT TTAAAGAAAT AGAAAACCTA AAGGAATATT 120 TTCAGGCAAG TAATCCAGAT GTATCGGACG GTGGGTCTCT TTTCGTAGAT ATTTTGAAGA 180 AATGGAGAGA GGAGAGTGAC AAAACAATCA TTCAGAGCCA AATTGTCTCT TTCTACTTGA 240 AACTGTTTGA CAACTTTAAA GATAACCAGA TCATTCAAAG GAGCATGGAT ACCATCAAGG 300 AAGACATGCT TGGCAAGTTC TTACAGTCAT CCACCAGTAA GAGGGAGGAC TTCCTTAAGC 360 TGATTCAAAT TCCTGTGAAC GATCTGCAGG TCCAGCGCAA GGCGATAAAT GAACTCATCA 420 AAGTGATGAA TGATCTCTCA CCAAGATCCA ACCTAAGGAA GCGGAAAAGG AGTCAGAATC 480 TGTTTCGAGG CCGCAGAGCA TCGAAATAAG AATTCGC

SEQ ID NO: 11 Sequence length: 30 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATAGGATCCATGAATTATACAAGCTATATC

SEQ ID NO: 12 Sequence length: 33 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CTGGATATCTGGATTACTTGCCTGAAAATATTC

SEQ ID NO: 13 Sequence length: 27 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCATACGTATCGGACGGTGGGTCTCTT

SEQ ID NO: 14 Sequence length: 36 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGTGGTCGACTGTAAGAACTTGCCAAGCATGTCTTC

SEQ ID NO: 15 Sequence length: 36 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCGATATCCACCAGTAAGAGGGAGGACTTCCTTAAG

SEQ ID NO: 16 Sequence length: 33 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CTCGAATTCTTATTTCGATGCTCTGCGGCCTCG

SEQ ID NO: 17 Sequence length: 147 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATAGGATCCA TGAATTATAC AAGCTATATC TTAGCTTTTC AGCTTTGCGT GATTTTGTGT TCTTCTGGCT GTAACTGTCA GGCCATGTTT TTTTTAAAAA TAGAAAACCT AAAGGAATAT TTTCAGGCAA GTAATCCAGA TATCCAG

SEQ ID NO: 18 Sequence length: 201 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA sequence CCATACGTAT CGGACGGTGG GTCTCTTTTC GTAGATATTT TGAAGAAATG GAGAGAGGAG AGTGACAAAA CAATCATTCA GAGCCAAATT GTCTCTCT ACTTGAAACT GTTTGACAAC TTTAAAGATA ACCAGATCAT TCAAAGGAGC ATGGATACCA TCAAGGAAGA CATGCTTGGC AAGTTCTTAC AGTCGACCAC C

SEQ ID NO: 19 Sequence length: 195 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCGATATCCA CCAGTAAGAG GGAGGACTTC CTTAAGCTGA TTCAAATTCC TGTGAACGAT CTGCAGGTCC AGCGCAAGGC GATAAATGAA CTCATCAAG TGATGAATGA TCTCTCACCA AGATCCAACC TAAGGAAGCG GAAAAGGAGT CAGAATCTGT TTCGAGGCCG CAGAGCATCG AAATAAGAAT TCGAG

SEQ ID NO: 20 Sequence length: 517 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCAGATCTAT GAATTATACA AGCTATATCT TAGCTTTTCA GCTTTGCGTG ATTTTGTGTT 60 CTTCTGGCTG TAACTGTCAG GCCATGTTTT TTAAAGAAAT AGAAAACCTA AAGGAATATT 120 TTAATGCAAG TAATCCAGAT GTATCGGACG GTGGGTCTCT TTTCGTAGAT ATTTTGAAGA 180 AATGGAGAGA GGAGAGTGAC AAAACAATCA TTCAGAGCCA AATTGTCTCT TTCTACTTGA 240 AACTGTTTGA CAACTTTAAA GATAACCAGA TCATTCAAAG GAGCATGGAT ACCATCAAGG 300 AAGACATGCT TGGCAAGTTC TTAAATAGCA GCACCAGTAA GAGGGAGGAC TTCCTTAAGC 360 TGATTCAAAT TCCTGTGAAC GATCTGCAGG TCCAGCGCAA GGCGATAAAT GAACTCATCA 420 AAGTGATGAA TGATCTCTCA CCAAGATCCA ACCTAAGGAA GCGGAAAAGG AGTCAGAATC 480 TGTTTCGAGG CCGCAGAGCA TCGAAATAAG AATTCGC

SEQ ID NO: 21 Sequence length: 42 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCGGAATTCTTATCTTGGTGAGAGATCATTCATCACTTTGAT

SEQ ID NO: 22 Sequence length: 42 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCGGGATCCTTATCTTGGTGAGAGATCATTCATCACTTTGAT

SEQ ID NO: 23 Sequence length: 457 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCAGATCTAT GAATTATACA AGCTATATCT TAGCTTTTCA GCTTTGCGTG ATTTTGTGTT 60 CTTCTGGCTG TAACTGTCAG GCCATGTTTT TTAAAGAAAT AGAAAACCTA AAGGAATATT 120 TTCAGGCAAG TAATCCAGAT GTATCGGACG GTGGGTCTCT TTTCGTAGAT ATTTTGAAGA 180 AATGGAGAGA GGAGAGTGAC AAAACAATCA TTCAGAGCCA AATTGTCTCT TTCTACTTGA 240 AACTGTTTGA CAACTTTAAA GATAACCAGA TCATTCAAAG GAGCATGGAT ACCATCAAGG 300 AAGACATGCT TGGCAAGTTC TTACAGTCAT CCACCAGTAA GAGGGAGGAC TTCCTTAAGC 360 TGATTCAAAT TCCTGTGAAC GATCTGCAGG TCCAGCGCAA GGCGATAAAT GAACTCATCA 420 AAGTGATGAA TGATCTCTCA CCAAGATAAG AATTCGC

SEQ ID NO: 24 Sequence length: 501 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism: canine Sequence features Sequence features Symbol indicating: sig peptide Location: 1..72 Characteristic determination method: S Symbol indicating characteristic: mat peptide Location: 73..498 Characterization determination method: S sequence ATG AAT TAT ACA AGC TAT ATC TTA GCT TTT CAG CTT TGC GTG ATT TTG 48 Met Asn Tyr Thr Ser Tyr Ile Leu Ala Phe Gln Leu Cys Val Ile Leu TGT TCT TCT GGC TGT AAC TGT CAG GCC ATG TTT TTT AAA GAA ATA GAA 96 Cys Ser Ser Gly Cys Asn Cys Gln Ala Met Phe Phe Lys Glu Ile Glu AAC CTA AAG GAA TAT TTT CAG GCA AGT AAT CCA GAT GTA TCG GAC GGT 144 Asn Leu Lys Glu Tyr Phe Gln Ala Ser Asn Pro Asp Val Ser Asp Gly GGG TCT CTT TTC GTA GAT ATT TTG AAG AAA TGG AGA GAG GAG AGT GAC 192 Gly Ser Leu Phe Val Asp Ile Leu Lys Lys Trp Arg Glu Glu Ser Asp AAA ACA ATC ATT CAG AGC CAA ATT GTC TCT TTC TAC TTG AAA CT G TTT 240 Lys Thr Ile Ile Gln Ser Gln Ile Val Ser Phe Tyr Leu Lys Leu Phe GAC AAC TTT AAA GAT AAC CAG ATC ATT CAA AGG AGC ATG GAT ACC ATC 288 Asp Asn Phe Lys Asp Asn Gln Ile Ile Gln Arg Ser Met Asp Thr Ile AAG GAA GAC ATG CTT GGC AAG TTC TTA CAG TCA TCC ACC AGT AAG AGG 336 Lys Glu Asp Met Leu Gly Lys Phe Leu Gln Ser Ser Thr Ser Lys Arg GAG GAC TTC CTT AAG CTG ATT CAA ATT CCT GTG AAC GAT CTG CAG GTC 384 Glu Asp Phe Leu Lys Leu Ile Gln Ile Pro Val Asn Asp Leu Gln Val CAG CGC AAG GCG ATA AAT GAA CTC ATC AAA GTG ATG AAT GAT CTC TCA 432 Gln Arg Lys Ala Ile Asn Glu Leu Ile Lys Val Met Asn Asp Leu Ser CCA AGA TCC AAC CTA AGG AAG CGG AAA AGG AGT CAG AAT CTG TTT CGA 480 Pro Arg Ser Asn Leu Arg Lys Arg Lys Arg Ser Gln Asn Leu Phe Arg GGC CGC AGA GCA TCG AAA TAA 501 Gly Arg Arg Ala Ser Lys ** *

SEQ ID NO: 25 Sequence length: 441 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism: Canine Sequence characteristics Characteristic symbols: Location of sig peptide: 1..72 Characteristic determination method: S Symbol indicating characteristic: mat peptide Location: 73..438 Characterization determination method: S sequence ATG AAT TAT ACA AGC TAT ATC TTA GCT TTT CAG CTT TGC GTG ATT TTG 48 Met Asn Tyr Thr Ser Tyr Ile Leu Ala Phe Gln Leu Cys Val Ile Leu TGT TCT TCT GGC TGT AAC TGT CAG GCC ATG TTT TTT AAA GAA ATA GAA 96 Cys Ser Ser Gly Cys Asn Cys Gln Ala Met Phe Phe Lys Glu Ile Glu AAC CTA AAG GAA TAT TTT CAG GCA AGT AAT CCA GAT GTA TCG GAC GGT 144 Asn Leu Lys Glu Tyr Phe Gln Ala Ser Asn Pro Asp Val Ser Asp Gly GGG TCT CTT TTC GTA GAT ATT TTG AAG AAA TGG AGA GAG GAG AGT GAC 192 Gly Ser Leu Phe Val Asp Ile Leu Lys Lys Trp Arg Glu Glu Ser Asp AAA ACA ATC ATT CAG AGC CAA ATT GTC TCT TTC TAC TTG AAA CTG TTT 240 Lys Th r Ile Ile Gln Ser Gln Ile Val Ser Phe Tyr Leu Lys Leu Phe GAC AAC TTT AAA GAT AAC CAG ATC ATT CAA AGG AGC ATG GAT ACC ATC 288 Asp Asn Phe Lys Asp Asn Gln Ile Ile Gln Arg Ser Met Asp Thr Ile AAG GAA GAC ATG CTT GGC AAG TTC TTA CAG TCA TCC ACC AGT AAG AGG 336 Lys Glu Asp Met Leu Gly Lys Phe Leu Gln Ser Ser Thr Ser Lys Arg GAG GAC TTC CTT AAG CTG ATT CAA ATT CCT GTG AAC GAT CTG CAG GTC 384 Glu Asp Phe Leu Lys Leu Ile Gln Ile Pro Val Asn Asp Leu Gln Val CAG CGC AAG GCG ATA AAT GAA CTC ATC AAA GTG ATG AAT GAT CTC TCA 432 Gln Arg Lys Ala Ile Asn Glu Leu Ile Lys Val Met Asn Asp Leu Ser CCA AGA TAA 441 Pro Arg ***

SEQ ID NO: 26 Sequence length: 453 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism name: Canine Sequence characteristics Characteristic symbols: Location of sig peptide: 1..72 Characteristic determination method: S Symbol indicating feature: mat peptide Location: 73..450 Characterization determination method: S sequence ATG AAT TAT ACA AGC TAT ATC TTA GCT TTT CAG CTT TGC GTG ATT TTG 48 Met Asn Tyr Thr Ser Tyr Ile Leu Ala Phe Gln Leu Cys Val Ile Leu TGT TCT TCT GGC TGT AAC TGT CAG GCC ATG TTT TTT AAA GAA ATA GAA 96 Cys Ser Ser Gly Cys Asn Cys Gln Ala Met Phe Phe Lys Glu Ile Glu AAC CTA AAG GAA TAT TTT CAG GCA AGT AAT CCA GAT GTA TCG GAC GGT 144 Asn Leu Lys Glu Tyr Phe Gln Ala Ser Asn Pro Asp Val Ser Asp Gly GGG TCT CTT TTC GTA GAT ATT TTG AAG AAA TGG AGA GAG GAG AGT GAC 192 Gly Ser Leu Phe Val Asp Ile Leu Lys Lys Trp Arg Glu Glu Ser Asp AAA ACA ATC ATT CAG AGC CAA ATT GTC TCT TTC TAC TTG AAA CTG TTT 240 Lys Th r Ile Ile Gln Ser Gln Ile Val Ser Phe Tyr Leu Lys Leu Phe GAC AAC TTT AAA GAT AAC CAG ATC ATT CAA AGG AGC ATG GAT ACC ATC 288 Asp Asn Phe Lys Asp Asn Gln Ile Ile Gln Arg Ser Met Asp Thr Ile AAG GAA GAC ATG CTT GGC AAG TTC TTA CAG TCA TCC ACC AGT AAG AGG 336 Lys Glu Asp Met Leu Gly Lys Phe Leu Gln Ser Ser Thr Ser Lys Arg GAG GAC TTC CTT AAG CTG ATT CAA ATT CCT GTG AAC GAT CTG CAG GTC 384 Glu Asp Phe Leu Lys Leu Ile Gln Ile Pro Val Asn Asp Leu Gln Val CAG CGC AAG GCG ATA AAT GAA CTC ATC AAA GTG ATG AAT GAT CTC TCA 432 Gln Arg Lys Ala Ile Asn Glu Leu Ile Lys Val Met Asn Asp Leu Ser CCA AGA TCC AAC CTA AGG TAA 453 Pro Arg Ser Asn Leu Arg ***

SEQ ID NO: 27 Sequence length: 501 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism: Canine Sequence features Sequence features Sequence features Symbol indicating: sig peptide Location: 1..72 Characteristic determination method: S Symbol indicating characteristic: mat peptide Location: 73..498 Characterization determination method: S sequence ATG AAT TAT ACA AGC TAT ATC TTA GCT TTT CAG CTT TGC GTG ATT TTG 48 Met Asn Tyr Thr Ser Tyr Ile Leu Ala Phe Gln Leu Cys Val Ile Leu TGT TCT TCT GGC TGT AAC TGT CAG GCC ATG TTT TTT AAA GAA ATA GAA 96 Cys Ser Ser Gly Cys Asn Cys Gln Ala Met Phe Phe Lys Glu Ile Glu AAC CTA AAG GAA TAT TTT AAT GCA AGT AAT CCA GAT GTA TCG GAC GGT 144 Asn Leu Lys Glu Tyr Phe Asn Ala Ser Asn Pro Asp Val Ser Asp Gly GGG TCT CTT TTC GTA GAT ATT TTG AAG AAA TGG AGA GAG GAG AGT GAC 192 Gly Ser Leu Phe Val Asp Ile Leu Lys Lys Trp Arg Glu Glu Ser Asp AAA ACA ATC ATT CAG AGC CAA ATT GTC TCT TTC TAC TTG AAA CT G TTT 240 Lys Thr Ile Ile Gln Ser Gln Ile Val Ser Phe Tyr Leu Lys Leu Phe GAC AAC TTT AAA GAT AAC CAG ATC ATT CAA AGG AGC ATG GAT ACC ATC 288 Asp Asn Phe Lys Asp Asn Gln Ile Ile Gln Arg Ser Met Asp Thr Ile AAG GAA GAC ATG CTT GGC AAG TTC TTA AAT AGC AGC ACC AGT AAG AGG 336 Lys Glu Asp Met Leu Gly Lys Phe Leu Asn Ser Ser Thr Ser Lys Arg GAG GAC TTC CTT AAG CTG ATT CAA ATT CCT GTG AAC GAT CTG CAG GTC 384 Glu Asp Phe Leu Lys Leu Ile Gln Ile Pro Val Asn Asp Leu Gln Val CAG CGC AAG GCG ATA AAT GAA CTC ATC AAA GTG ATG AAT GAT CTC TCA 432 Gln Arg Lys Ala Ile Asn Glu Leu Ile Lys Val Met Asn Asp Leu Ser CCA AGA TCC AAC CTA AGG AAG CGG AAA AGG AGT CAG AAT CTG TTT CGA 480 Pro Arg Ser Asn Leu Arg Lys Arg Lys Arg Ser Gln Asn Leu Phe Arg GGC CGC AGA GCA TCG AAA TAA 501 Gly Arg Arg Ala Ser Lys ** *

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification symbol FI (C12P 21/02 C12R 1:91) (C12N 5/10 C12R 1:91)

Claims (8)

[Claims] 1. Included in a culture supernatant of cultured insect cells infected with a baculovirus transfected with a DNA encoding a protein of interferon-γ or a body fluid extract of a silkworm larva infected with the baculovirus. A method for producing interferon-γ, comprising a step of inactivating a recombinant baculovirus by treating it under acidic or alkaline conditions. 2. An acid used for treatment under acidic conditions is at least one selected from hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid and formic acid.
The method for producing interferon-γ according to claim 1, which is a seed. 3. The method for producing interferon-γ according to claim 1, wherein the pH is adjusted to 3 or less. 4. A method for producing interferon-γ, wherein interferon-γ having lost biological activity is treated under neutral and low temperature conditions. 5. The method for producing interferon-γ according to claim 4, wherein the treatment is carried out at pH 6 to 8. 6. The method for producing interferon-γ according to claim 4, wherein the treatment is carried out at 15 ° C. or lower. 7. A culture supernatant of cultured insect cells infected with a baculovirus transfected with a DNA encoding a protein of interferon-γ or contained in a body fluid extract of a silkworm larva infected with the baculovirus. The method for producing interferon-γ according to any one of claims 4 to 6, wherein the recombinant baculovirus is an interferon-γ that has lost its activity by treating it under acidic or alkaline conditions. 8. The DNA according to any one of SEQ ID NO: 24 to SEQ ID NO: 27, wherein the genetically modified baculovirus is
The method for producing interferon-γ according to any one of claims 1 to 7, wherein the method is a recombinant silkworm nuclear polyhedrosis virus genetically modified by a sequence.