JPH02291272A - Production of peptides - Google Patents

Abstract

PURPOSE:To contrive the process for the production of useful peptide or polypeptide by the recombination of a specific polyhedrin gene of a nuclear polyhedrosis virus of common cutworm. CONSTITUTION:Polyhedrin gene of common cutworm nuclear polyhedrosis virus (SlNPV C-411) is produced by including a DNA having the restriction enzyme map of the Fig.1, having a size of 3 kBase and coding the amino acid sequence of the figure (70th to 249th are omitted, G: glycine, A: alanine, V: valine, L: leucine, I: isoleucine, S: serine, T: threonine, D: aspartic acid, E: glutamic acid, N: asparagine, Q: glutamine, K: lysine, R: arginine, C: cysteine, M: methionine, F: phenylalanine, Y: tyrosine, W: tryptophan, H: histidine, P: proline). S.Littoralis CLS-79-C7 strain cell, etc., is infected with a recombinant virus prepared by the use of the gene to produce a useful peptide such as urokinase and insulin.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing peptides, and relates to a method for producing peptides using genetic engineering techniques, particularly for producing substances useful for medicine, etc. A novel polyhedrin gene, which is used to produce various useful substances,
The present invention provides recombinant genes, useful vectors, methods for producing the same, recombinant viruses, methods for producing the same, virus-infected cells, methods for producing peptides, and fusion proteins.

[Conventional technology]

It is already known that useful substances can be produced using baculovirus through genetic recombination technology.

For example, in Japanese Patent Application Laid-Open No. 60-37988, Autograph 7 California
lifornica), Japanese Patent Application Laid-Open No. 61-9297, each nuclear polyhedrosis virus (hereinafter referred to as AcNPV,
It has been described that useful substances can be produced by recombining the polyhedrin gene of BmNPV (abbreviated as BmNPV).

[Problems to be solved by the invention] However, since the AcNPV and BmNPV vectors themselves are approximately 10 kilobases long, the transformation efficiency in Escherichia coli is low when cloning the gene of a useful substance into the vector, and the useful substance cannot be cloned into the vector. It is difficult to clone large genes.

The present inventors searched for a new polyhedrin gene different from the conventional polyhedrin gene from the Spodoptera nuclear polyhedrosis virus (S I NPV; obtained from the Ministry of Agriculture, Forestry and Fisheries, Agricultural Research Center), and assembled the polyhedrin gene. By changing the method, the conventional method was improved and the present invention was completed.

[Means to solve the problem] The present invention consists of the following configuration.

(1.) Spodoptera nuclear polyhedrosis virus (S P
．． Polyhedrin gene of NPV C-411).

Sn]゛W T R F M E D TRPNRCFRFL A Q H A L R C
D P + Mi (2) A polyhedrin gene of the Hasmonyo 1. Urchin nuclear polyhedrosis virus (S f IVC-411) containing DNA encoding the following amino acid sequence.

Y R I S L A K
K GGCPVMNLH △ KV IWYNF￥K I VYVGTDSA l《(In the formula, G is glycine, A is alanine, ■ is hanone, L
is leucine, I is isoleucine, S is serine, T is threonine, D is aspartic acid, E is glutamic acid, N is asparagine, Q is glutamine, K is lysine, R is arginine, C is cysteine, M is methionine, F is (3) Spodoptera nuclear polyhedrosis virus (5 4
2 NPV C-411) polyhedrin gene.

GATAAAAATTT TAAAATTTTA A
TCTATAGAT AGAAAAGATAAAATT
TTAATC TATAGATAΔΔ A G T
A A A G T A 'T' T T A G
A T A A A AG T T T C A T
G G8CATCGTCGAA CGCATCAGTC TGAACCTGCA GTGTACGTGG AGACAGCTTC CCGTCGTACG TCGCCAAGAA CGCCGAATAC GCACCGATTC CCCATCGTG8TCGGCACC AA AGGTGGCGGT ΔCCACTTCGT GGCCG88GAG ACGATCAAGA CAATGA8TAC TG'TCCCGTGA TTGAGAGTTT GAGGAGATCC TCCCATAATG CCCCATCTGG 8CAAAATT T ACACGATGCT TATAGTCGCT GCAAAACCTA AGGTCACGTG CTCGAACGCG 8CAGTGCCTA TGTATACGAT ATCAA888TG AGGCCGACG8CAATTATAGT AACAAGTATT CTAAGCGCAA GCGCGAGCTC TGGGTCCCGG TAAAAAATCAAA A
AGTTGACTC TGTTCAAGG8GATC
CGTAAC GTGAAACCCG ACACG
ATGAA GCTGATCGTCAAGTATAT
CG GCCACGTTGC TTTGTATAA
T TTGGCTGTTTT (4) Vector obtained by replacing at least part of the polyhedrin gene of Spodoptera nuclear polyhedrosis virus (S I!.NPVC-411) with a gene constituting a useful substance (5) Spodoptera nuclear polyhedrosis virus (S42NPνC- A vector (6) in which a gene constituting a useful substance is inherited after the fragment containing the promoter of the 5' upstream region of the polyhedrin gene of 411), and a DNA fragment of the 3' downstream region of the polyhedrin gene (6) (a) Spodoptera nucleus (b) 8K DN Insert the A fragment into a cloning vehicle to generate a recombinant cloning vehicle, (
C) A method for producing a vector, which comprises inserting at least one foreign DNA into the recombinant cloning vehicle so that the foreign DNA is under the transcriptional control of a polyhedrin promoter.

(7) Spodoptera nuclear polyhedrosis virus (INFVC)
A recombinant virus in which at least a portion of the polyhedrin gene of -411) is replaced with a gene constituting a useful substance.

(8) Spodoptera nuclear polyhedrosis virus (INFVC)
-411) useful substance {1! D in the 3' downstream region of the polyhedrin gene.
A recombinant virus that has a gene replaced with DNA inherited from an NA fragment.

(9) (a) Cutting the polyhedrin gene of Spodoptera nuclear polyhedrosis virus (S I NPV (:-411)) to produce a DNA fragment containing the polyhedrin promoter and sufficient adjacent DNA to enable gene recombination. (b) inserting the DNA fragment into a cloning vehicle to generate a recombinant cloning vehicle; (C)
inserting at least one foreign DNA into said recombinant cloning vehicle to produce a vector such that the foreign DNA is under the transcriptional control of a polyhedrin promoter; (d) inserting said vector in such a way as to enable genetic recombination; A method for producing a recombinant virus, which comprises contacting a virus (1!NPV C-411) with a virus.

00) Cells infected with the recombinant virus described in (7) above.

(11) A cell infected with the recombinant virus described in (8) above.

02) A method for producing peptides, which comprises culturing the cells described in 0ω or (11) above in a medium and collecting peptides from the culture.

03) (a) Foreign DNA is injected after the fragment containing the promoter of the 5' upstream region of the polyhedrin gene of Spodoptera nuclear polyhedrosis virus (iNPVC-411), and the D of the 3' downstream region of the polyhedrin gene is injected.
A method for producing peptides, which comprises producing a recombinant virus having a gene replaced with DNA inherited from an NA fragment, and (b) culturing cells infected with the recombinant virus.

En Spodoptera nuclear polyhedrosis virus (Sffi)
A recombinant gene in which at least a portion of the polyhedrin gene of NPVC-411) is replaced with foreign DNA.

05) Spodoptera nuclear polyhedrosis virus (SIN)
A recombinant gene obtained by inheriting a fragment containing a promoter in the 5' upstream region of the polyhedrin gene of FVC-411), followed by foreign DNA, and further inheriting a DNA fragment in the 3' downstream region of the polyhedrin gene.

06) Spodoptera nuclear polyhedrosis virus (!IINP
νG-411) has a gene in which foreign DNA is inherited after a fragment containing a promoter in the 5' upstream region of the polyhedrin gene, and the gene is replaced with DNA inheriting a DNA fragment in the 3' downstream region of the polyhedrin gene. A fusion protein produced by culturing cells infected with a recombinant virus.

θ'7) Spodoptera nuclear polyhedrosis virus (S ff
i A fusion protein comprising a polypeptide fused to at least a portion of a polyhedrin peptide of NPVC-411).

The present invention will be explained in detail below.

The inventors of the present invention
tura) nuclear polyhedrosis virus (S n NPV')
However, in the present invention, the synthesis of DNA fragments (chemical synthesis, cutting by restriction enzyme treatment), isolation and searching, analysis of DNA sequences, treatment of E. coli etc. (transformation, culture, (Acquisition of blasmid, etc.)
etc., the manipulation techniques normally practiced in genetic engineering are applied (T.MANIATrS et.al (198
2): Molecular cloning, Col.
d Spring Harbor Laboratory
) After purifying the polyhedra of S f NPV and dissolving it with alkali, the OV of S f NPV released was used as a cell CL derived from Subodobutella littoralis of Egyptian origin. S-
7. Infect and multiply 9 strains (manufactured by Kyushu University Faculty of Agriculture). NOV is a virus that is formed by budding from cells and passing through the cell membrane. Plaques were formed by a plaque formation method using Seaplaque agarose, and a single virus was obtained by repeating the same procedure three times from a single plaque, which was designated as SiNPV C-411. There is a method to obtain SffNPV C-411 DNA from NOV grown in cultured cells, but the following method is
You can get a lot of NA easily (old KOBAYAS,
M. (1984): 8 ppl. Ent. Zoo
．． 19(3), 280-287 and S. T. TZI
Eight et. al (1979): νirology,
99. 39! IQ-409). This Sj2NPV
After propagating C-411 with the CLS-79 strain, it is injected into the abdomen of 3rd instar Spodoptera larvae, which die about 7 days later, and the body fluid of the dissolved larvae, which contains virus polyhedra, is collected. By filtering and centrifuging this body fluid, the polyhedra are purified, dissolved with alkali, the virus is released, and Sea! Purify the virus by sugar density gradient centrifugation; rL ultrahigh-speed centrifugation. Is this virus separated from the protein with protease K? After that, the cloned virus Sρ of approximately 1308 o
Obtain NPV C-411 DNA.

After cutting this DNA with various restriction enzymes, 0.5
Electrophoresis is performed using ~1.5%, preferably 0.7% agarose, transferred to nitrocellulose, and Southern hybridization performed.

As a probe, the polyhedrin gene of nuclear polyhedrosis virus, which has been previously reported and is well conserved, such as AcMN.
PV. BmNPV, Orysia Pseudogata (Op.
Two DNAs are chemically synthesized based on the polyhedrin gene of SNPv, OpMNPV), and the ends are labeled with 32p for use. As a result, a positive fragment of approximately 3 kilobases was obtained, and most of its nucleotide sequence was determined by the guideeoxy method. The fragment contained synthetic D used as a probe.
Each has one base sequence similar to NA. Furthermore, the base sequence of 747 hese including these two parts can encode a protein of 249 residues. The size of the protein is 245 or 2
This protein is clearly different from known proteins because the N-terminal part is longer by 3 or 4 residues compared to the 46 residues. Furthermore, among the bebutide fragments obtained by cleaving purified polyhedrin with bromine cyanide, the amino acid sequences of two fragments were examined using the Edman degradation method, and the results showed that they completely matched the amino acid sequences that could be deduced from DNA. .

Taking these things into consideration, this DNA fragment is SIPV
It is recognized that it contains the polyhetrin gene of C-411. The known nuclear polyhedrosis viruses AcMNPV and BmNPV have completely identical 71 bases on the left side, that is, 5' upstream of their start codon (8TG), whereas the polyhedrin gene of the 12NPV C-411 of the present invention is completely identical to these. It is clearly different from the well-known OpSN
Both PV and opgNpv have different base sequences. Therefore, the polyhedrin gene of SffiNPV C-411 of the present invention is novel, unlike the polyhedrin gene of known nuclear polyhedrosis viruses.

Polyhedrin protein is not necessary for nuclear polyhedrosis virus to proliferate in cultured cells, so we created a recombinant virus by replacing the gene for this protein with the constituent genes of other useful substances, and transferred this virus to cells. It is possible to produce useful substances by infecting That is, Si! ．． NPVC
The region encoding the polyhedrin protein ~411 is located in the center of a positive fragment of about 3 kilohese, and about 1.1 kilobases 5' upstream from the start codon is a promoter-containing sequence, and 3' from the stop codon. Approximately 1.2 downstream
Kilohose is the part that contains the BoliA addition site. The DNA that encodes the polyhedrin protein in this central part
By inserting a gene encoding another useful substance instead, it is possible to produce the useful substance of the other gene under the control of the polyhedrin promoter.

This l! An embodiment of constructing a vector using the polyhedrin protein gene of NPV C-411 is illustrated. l!
The polyhedrin protein gene of NPV C-411 is inserted into a vector or vehicle such as E. coli vector pBR322 or pUc vector according to a conventional method. INFV
The polyhedrin gene of C-411 has a cleavage site with the restriction enzyme Acc I near the N-terminus about 20 amino acids,
There is a cleavage site by the restriction enzyme Sanl on the 3' side near the translation termination signal. A fusion protein can be created by removing the part between Acc I and Sai ■, inserting a linker there, and inheriting it with another gene.

In addition, by using the χbaI site upstream of the start codon, removing the region from the Xbar site to the AceT site, and inserting a polyrin group there, the gene of the useful substance can be read from its own start codon. It is also possible to create proteins containing only genes that are useful substances. In other words, l! Of the approximately 3 kilobase DNA fragment containing the polyhedrin-encoding DNA of NPV C-411, the old ndI[l-Accl fragment, which contains the promoter and approximately 1.1 kilobases, and the Sal fragment containing the polyA addition site.
I IIindIII fragment approx. 1. By inserting 2 kilohose into the commercially available plasmid pUc9 with the old Bam linker in between, a hector capable of producing a fusion protein can be created. The size of this hector is about 5 kilobases, and the gene of a useful substance is inserted in alignment with the reading frame at one site of the BamH I linker.

The vector of the present invention is a known AcNP of approximately 10 kilobases.
Since it is smaller than the V and BmNPV vectors, when cloning genes for useful substances, it has good transformation efficiency in E. coli, and larger genes for useful substances can be easily cloned.

The hector of the present invention can be directly implanted into chromosomes within cells. In addition, this vector DNA and wild virus DNA were cultured in insect cells, 3D and 1Ittoralis C.
Strain LS-79-C7 (Deposited with the Institute of Microbiology, Agency of Industrial Science and Technology: FAIKEN JO No. 2723) (Accession number FERFI-B)
P-2723), a recombinant virus can also be created by homologous recombination. The recombinant virus can be selected by plaque hybridization or by using appropriate markers. The promoter of the polyhedrin gene is strong and is regulated so that it functions during the late stage of virus infection, so wild virus l! The polyhedrin protein of NPV C-411 is produced in large amounts during the late stage of infection. For example, the selected recombinant virus can be used as S. Iittoralis
By infecting CLS-79-C7 cell lines and culturing them at 27°C, a large amount of protein transcribed and translated from the polyhedrin promoter can be obtained in the late stage of virus propagation. Also S. littoralis CL
S-79-C7 strain is the parent strain SLIittoralis
It is a suspension culture cloned from the CLS-79 strain, a cell line suitable for virus propagation, and is capable of culturing cells, propagating viruses, and producing large amounts of protein. The cells used in the present invention include the above-mentioned S. Iittora
lis CLS-79-C7 strain, Sodo tera
Examples include the fru i erda sf-9 strain.

Useful substances applied in the present invention, such as peptides or polypeptide producing genes, include enzymes such as urokinase and angiotensin converting enzyme; hormones such as insulin, somatomedin, human growth hormone, and various animal growth hormones; Interferon (α, β,
Cytokinins such as T), interleukin (1-7), granulocyte colony stimulating factor, and erythroboetin; AIDS virus gpl60 (gpl20, gp41)
, proteins of various viruses such as HBs of hepatitis B virus; receptor proteins of various cells such as CD4 of T cells; peptides such as various physiologically active substances, polybebutide, proteins, tJ! Examples include proteins, and by using the vector of the present invention, it is possible to express the genes of these useful substances. In addition, the outpatient D used in the present invention
Although NA corresponds to the gene constituting the useful substance described above, it is not particularly limited thereto, and other genes can also be used. In addition, the genes of many natural products that will be isolated in the future,
The Hector of the present invention is particularly useful for those that require protein modification. If the gene of the useful substance used in the present invention can be cloned in a vector, its size cannot be absolutely defined, but it is preferably 5 kilohese or less, more preferably in the range of 500 bases to 5 kilohese. belongs to.

〔Example〕

Next, examples of the present invention will be described.

Example l. Spodoptera nuclear polyhedrosis virus (1!NPVC-411) by Southern hybridization
Detection of the polyhedrin gene from DNA of approximately 0.0.
1 μg of DNA was digested with restriction enzyme 11indII [ (all restriction enzymes less than 2 manufactured by Takara Shuzo Co., Ltd. were used) and BamH I, 11indlll and EcoR I or old ndl [[ and Ilinc [I after cutting with 0.7 % agarose was used, and after transfer to nitrocellulose, hybridization was performed at 42°C. Probes 1 and 2 are nucleotide sequences of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) ( Based on the DNA (Fig. 1), chemical synthesis was performed using a nucleic acid synthesizer manufactured by Abride Hyosyste
Approximately 0.1 μg) was end-labeled with T''p-dATP and used.

As a result, as shown in Fig.
When cut and folded, a positive II piece of 4'13 kilobases was seen. As a result of cloning 1, this positive fragment was found to be a fragment cleaved only by H i n d■, so it was cloned into the old ndlI1 site of pUc9 (obtained from Kyushu University School of Medicine) to generate ps ffi +123. Obtained. This psg++23
DNA of the old ndIII fragment of (approximately 1.tt g)
was further fragmented with restriction enzymes and subjected to Southern hybridization in the same manner as above. As a result, one probe hybridized with the 0.7 kilobase old ncll fragment, and the other probe hybridized with the 0.7 kilobase old ncll fragment. 6km based old nc■
As a result, a restriction enzyme map (pS I H23) as shown in Figure 3 was obtained.
It was determined. This ps/}123 is E. coli JM83
strain (manually from Kyushu University School of Medicine), and this was transferred to the Institute of Microbial Technology, F4I Faculty of Industrial Technology.
A coli JM83: Deposited as psNH23. Its deposit number is FEIKEN JO deposit No. 2721 (accession number F
ERM BP-2721).

Example 2. Determination of the DNA base sequence Approximately 1 μg of the positive DNA fragment cloned into the old ndI site of ρUC9 was further cloned into smaller fragments using the restriction enzyme map in Figure 3, and the size of the base sequence was determined using the guideoxy method. decided on the part. As a result of computer analysis, as shown in Figure 4, a 747-hose DNA that can encode a protein consisting of 249 amino acids was found near the center of the approximately 3 kilobase fragment.
There was an A. The amino acid sequence deduced from the DNA by computer analysis showed more than 80% commonality with the conserved portions of previously reported polyhedrin genes. However, the polyhedrin of the nuclear polyhedrosis viruses of Autographa californica (Ac), Bombyx mori (Bm), and Orysia pseudotsugata (Op); AcMNPV (245 residues);
BmNPV (245 residues), OpMNPV (245
This one has a longer N-terminus by 3 or 4 residues than OpSNPV (246 residues). On the other hand, purified SλNP
VC-411's boly, trine protein (approximately 500μ
g) was fragmented by decomposition with bromine cyanide, purified by reverse-phase high-performance liquid chromatography, and the amino acid sequences of 10 amino acids from the N-terminus of two fragments were determined by Edman degradation method. As a result, the amino acid sequences of these fragments (from 2nd to 11th and 66th in Figure 4) were determined.
The amino acid sequence deduced from the DNA base sequence (amino acid sequence from position 75 to position 75) completely matched. These things indicate that this DNA fragment is Sffi NPVC-
This suggests that it contains the gene that coats the polyhedrin protein 411.

In addition, the polyhetrin protein was a novel substance that had not been described in any literature.

Regarding this DNA fragment, the part upstream from the start codon (ATG) has many A and T as in the case of polyhetrin of other nuclear polyhedrosis viruses, but AcMNPV and BmNPV
The 71 hese 5' upstream of the start codon is the same, whereas the sequence of S/2NPV C-411 is different from those of AcMNPV and BmNPV.
In addition, the SfNPV C-411 (7) sequence is OpMN
It is clearly different from PV and OpSNPV. Ko(7)S
The transcription start site of INPV C-411 is estimated to be the site indicated by the arrow in FIG. 4 as a result of S■masoping.

Example 3. Creation of Hector Hector consists of the 5' upstream part containing the start codon of the polyhedrin gene of INPV C-411 and the 3' part from the stop codon.
It was created by using the downstream part. Hector ■ contains the old ndIII-Accl fragment as the 5' upstream part, and the 3
'SalI-HindIIl as the downstream part, and Bamt as the cloning site between them.
l I linker was inserted. By inserting the gene for a useful substance into this region in the same frame (reading frame), it has been confirmed that a protein with the N-terminal 19 amino acids of the INPV C-411 polyhedrin protein can be produced (No. 6 Figure 1a).

Hector ■ has the old ndI [[ Xba
By inserting a polyline fragment in between, it is possible to express the gene of a useful substance with an initiation codon (ATG) as a single protein (Figure 6-b). ). Both of these vectors are approximately 5
There are kilometers.

Example 4. Cloning of the β-galactosidase gene l) Insertion of the β-galactosidase gene into vector I As shown in Figure 7, the BamH1 site of Hector I was inserted in order to align the reading frames of the β-galactosidase gene and the N-terminal portion of the polyhedrin gene. After cutting, it was filled in and a Bgffil linker was inserted.

1 The acZ gene was used by cutting out the relevant portion from commercially available plasmid pMc1871 (manufactured by Pharmacia). Six amino acids other than the polyhedrin and β-galactosidase genes were added using a linker and others (7th
psffiV2 1acZ25) in the figure. This psffν
2lacZ25 was introduced into Escherichia coli 11B 101 strain (manually obtained from Kyushu University School of Medicine), and this was transferred to the Institute of Microbial Technology, Agency of Industrial Science and Technology.
Deposited as IOI: psfV2 1acZ25. Its deposit number is FER No. 2722 (accession number FER
M BP-2722).

2) Insect cell S. littoralis CLS-7
Vector DNA and wild virus/I/S in strain 9-C7
Preparation of recombinant virus using gNpV C-411 DNA 2. 5 X I O5cells
/ml 1iLtoralis CLS-79-
Add 4.5 ml of C7 strain, leave it at room temperature for 1 hour, and then
0.1 μg of PV C-411 DNA, psfV 2
I! ．． 1 ml of H containing 10 μg of ac Z 25
E B S (0.137M tJacl, 5mM
KCI, 0. 7mM NazHPOa-2HzO, 5
．． 5mM glucose, 0.021M)ta
pes) pH7. 50μ to 0! 2.5MCaCj of
2z was added and the mixture was allowed to stand at room temperature for 20 minutes to aggregate the DNA, which was then dropped onto the medium.

After leaving this at 27°C for 4 hours, remove the medium and transfer to HEB.
Approximately 0.5 d of 15% glycerol in S was added and left to stand for 3 minutes. Thereafter, the glycerol solution was removed, and after washing twice with 5 ml of IPL4l medium (Goodwin, 1977) (Table 1) supplemented with 10% fetal calf serum (FCS), the same medium was mixed with 4. Added 5 ml. After culturing at 27°C for 4 days, the supernatant was diluted and cultured at 27°C for 6 days to form plaques. X-gan(5
-bromo4-k4-indolyl-β-D-galactocyto) and IPL41 (IOFCS) medium.
After overlaying 2 ml of 75% agarose on a Petri dish with plaques formed, culture at 37°C for 4 hours, remove blue plaques, repeat plaque formation 3 times, purify the virus, and incubate the recombinant virus SffiNPV. β-g
It was used in later experiments as affi2.

(Margin below this page) Table 1 NaHzPO4 112o NaHCO, KCI CaCI2 Mgso4 7HzO (NHa)6MOJza 4HZO COCI2 6HzO CuClz 2H20 MnClz 4H20 ZnCI2 FeSOs 7tlzO L-arginine hydrochloride L-aspartic acid L-asparagine β-alanine L-cystine L -Glutamic acid 1160.0 350.0 1200. O soo. o 1880.0 0,04 0.05 0.20 0,02 0.04 0.55 80Q. 0 1300.0 1300.0 300.0 100.0 1500.0 L-Glutamine L-Glycine L-Histidine L-Hydroxyproline-Isoleucine L-Leucine L-Lysine hydrochloride L-Methionine L-Proline L-Phenylalanine DL - Serine L - Tyrosine L - Tributophane L - Leonine L - Valin Sucrose Maltose Glucose Malate α - Ketoglutaric acid 1000.0 200.0 200.0 800.0 750.0 250.0 700.0 1000.0 500.0 1000.0 400.0 250.0 100.0 200.0 500.0 16500.0 1000.0 2500.0 53.6 29.6 Thiamine succinate fumarate hydrochloride Riboflavin pantothenate Calcium pyridoxy Hydrochloride para-aminobenzoic acid folic acid niacin isoinositol biotin cyanocobalamin choline chloride tryptose broth 4.8 o. oso O. 080 0.008 0.400 0.320 0,080 0.160 0.400 0.160 0.240 20.000 2600.0 3)! rl1 of insertion of β-galactosidase gene into INPVβ-gaj22 DNA! Add 5 pieces to a 1L capacity bottle.
raI is CLS-79-C7 strain at 5X10'c
cells/500mf (IPL-41 medium, 10% FC
S ), then the virus S f NP
Vβ-gal2 was infected with MOL1 (multiplicity of infection 1) and cultured at 27°C for 5 days. The virus was purified by precipitation with 10% polyethylene glycol i2 (PEG precipitated bt), sucrose density gradient centrifugation, ultra-high speed centrifugation, etc., followed by protease treatment and phenol treatment to obtain S j2 NPVβ-gaj22. DNA
Approximately 10 μg was obtained. Approximately 0.1 μg of this DNA was added to BamH
After cutting with I, EcoR I, Hind m, 0
．． Electrophoresis was performed using 7% acarose (Fig. 8 1a),
After transfer to nitrocellulose, Southern hybridization was performed using β-galactosidase gene DNA (about 0.1 μg) as a probe. The β-galactosidase gene used as a probe was labeled using α”p-dCTP and a multi-primer kit (manufactured by Amajam). For example, it has become clear that it is possible to introduce other genes into SlNPvC-411 in place of the polyhedrin DNA of SlNPvC-411 (Fig. 8-b), and that a recombinant virus can be created.

Example 5. Expression of β-galactosidase ■) Activity measurement S. littoral
isCLS-79-C7 cell line 5 XIO6cells
S ffi NPV C-411, SIJPV β-
gal2 was infected with Mol 25, the virus was adsorbed at 27°C for 2 hours, and the virus was incubated with IPL-41 medium (0% FCS) for 2 hours.
After washing twice, IPL-41 medium (10%FCS) 50m
Cultured in a spinner flask with a capacity of 100 mR containing 1 ml of ONPG (
β-galactosidase activity was examined using 0-nitrophenyl-β-D-galactopyranoside).

However, the activity is the total activity including the medium and cells, and the number of units was calculated using the following formula.

t　X　V　X　o. o. 6. .

L: Reaction time (minutes) V: M of the culture solution used for unit determination (d) 0.0
．． : Absorbance As a result, no β-galactosidase activity was observed in the cells used as a control alone or in the cells infected with Sj2NPV C-411, but in the cells used as a control and cells infected with Sj2NPVβ-gal2, 4 Activity peaked at 16,000 units on day 1 (
Figure 9). Furthermore, when we compared the intracellular and extracellular (in the medium) activities, we found that most of the activity was observed within the cells, and extracellular activity was observed from the third day onward when the cells began to break down, indicating that β-galactosidase It is thought that it is not released outside the cell but accumulates inside the cell.

2) Intracellular distribution of β-galactosidase Cells adhered to and cultured on a coverslip were infected with SffNPVC-411 and SINPνβ-gal2 at +101 50,
27°C, culture for 2 days, formaldehyde, Trito
After treatment with nX 100, blocking was performed with bovine serum albumin, and the primary antibody (β-galactosidase antibody)
and further treated with fluorescein isothiocyanate (F
After treatment with a secondary antibody labeled with ITC), the cells were observed using a fluorescence microscope (Figure 10-a). As a result, wild virus l!
While NPV C-411 was not stained at all, the recombinant virus S f NPVβ-gal2 was stained mainly in the nucleus, indicating the possibility of translocation to the nucleus (Fig. 10-b).

3) “Labeling of proteins with S-methionine355
- Proteins synthesized during virus proliferation were labeled with methionine, and the results were examined by electrophoresis using SOS-polyacrylamide, treatment with an exacerbating agent, and exposure to X-ray film. 1 xi placed in a 4-hole plate
o5cells 3yo1ittoralis CLS-
79-C7 strain was infected with the virus at an MOI of 50, and
Culture at 7°C for 2 hours, remove the virus solution after a certain period of time, and transfer to methionine-free IPL4L medium (10% FCS).
) lml once. Then 75 u Ci (
Methionine-free IPL-41 medium (10% FCS) containing 353-methionine (2775Bq)/ml
) was added, cultured for 12 or 24 hours, the medium was removed, 100 μl of sample buffer was added to lyse the cells, and the samples were frozen until used for analysis. (Sample Buffer - 62.5mM) Li-HCl (pH 6.8), 1% SOS, 0.005% Bromophenol Blue, 10% Glycerol, 10%2
-Mercaptoethanol) As shown in Figure 11, a polyhedrin band (approximately 32 kilodaltons) was observed in the wild virus-infected cells only after 24 hours between 12 and 36 o'clock, and in parallel, Sffi NPV β A β-galactosidase hand (approximately 116 kilodaltons) was observed in -gaQ2 virus-infected cells. From this,. : NoS
f NPVβ-gaj22 virus is Sj2NPV
It seems that β-galactosidase is clearly expressed instead of polyhedrin in C-411.

〔Effect of the invention〕

According to the present invention, a gene for a useful substance of 5 kilobases or less can be relatively easily cloned into a vector, and the gene can be relatively easily cloned into a recombinant gene that replaces the polyhedrin gene portion of S f NPVC-411. Viruses can be created. Furthermore, by propagating this recombinant virus in insect cells, it is possible to produce useful peptides, proteins, and glycoproteins.

[Brief explanation of drawings]

Figure 1 shows the nucleotide sequences of probes 1 and 2, which are part of the polyhedrin genes of AcMNPV, BmNPV, OpMNPV, and OpSNPV, and Figure 2 shows the base sequences of probes 1 and 2 of SfNPV C.
A photograph showing the restriction enzyme cleavage pattern and hybridization of the polyhedrin gene of -411, Figure 3 is from SI
Figure 4 shows the restriction enzyme map of the polyhedrin gene of NFV C-411. Figure 4 shows the base sequence and amino acid sequence of the polyhedrin gene of 51NPV C-411. Figure 5
The figure shows the 5' upstream nucleotide sequences of AcMNPν, BmNPV, OpMNPV and OpSNPV (7) polyhedrin genes.
Figure 7 shows the cloning of β-galactosidase, Figure 8 shows the construction of S i! ．． NPVβ-ga
A photograph showing the restriction enzyme cleavage pattern and hybridization of the ll2 gene, Figure 9 is S f NPVβ-
Diagram showing gaf2 β-galactosidase production, No. lθ
The figure shows SPNPV C-411 and S f NPVβ-g
Photographs showing af2(7)-infected cells and FIG. 11 are photographs showing expression of β-galactosidase by pulse labeling. Applicant Agrochemical Biotechnology Development Technology Research Association Agent Patent Attorney Yusuke Hiraki Patent Attorney Teiji Ishii Probe AclJ%PV ATG-----CJ Engf
fmouth 'A 'n'clATAαtechnical Tαhi Artificial AIY sword 0
Sχ7ACBrrNPV. ．． ．． −−−−−7− −
−． ．． ．． A. ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． AAC. ．． ．． ．．
．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． T. ．． ．． ．． ．． ．． ．．
．． ．． :． ．． ．． ．． T. ．． A. ．． T. OplJ Rabbit V.
．． ．． −−−−−1− −−. ．． A. ．． ．． ．． ．． C. ．． G
．． ．． ．． ．． ．． G. ．． G. ．． ．． ．． ．． T. ．． T. ．． C.
．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． A.
．． ．． ．． OpSNPV. ．． ．． -----1-T ATA
I go fl. ．． QaΩyu. , ℃． ．． σr'. Ac. G.
．． T. ．． A. ．． T. ．． T. ．． ．． ．． ．． ．． ．． ．． ．．
．． ．． ．． ．． ．． ．． ．． ．． T. ．． ．． ．． ．． ．． ．． ．． C. ．． G. ．． C.
GTC. C. ．． ．． ．． ．． A. ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． C.
．． G. ．． C-cho. ．． ．． ．． C. ．． ．． ．． ．． A. ．． ．． ．． ．． ．． ．．
．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． C. ．． T. ．． ．．
．． ．． T & Ne JPV. C. ．． ．． ．． ．． ．． ．． ．． Op Sho V. C. ．． ．． ．． ．． ．． ．． ．． 011+SNPV. ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．．
．． C. Person AT,. 、． ．． ．． ．． ．． ．． A. A.A. ．． G.
．． ．． ．． ．． ．． ．． ．． ．． C. TCTA. ．． ．． ．． ．． C. ．． AGA
G. ．． T. ．． ．． ．． ．． ．． ．． ．． ．． CAG ATr'. ．． G.
．． C. ．． A(χ:r....-CTC GAαC ecoliACAAcTACCr Aa■r-sing GATCCTT
T'Cf. 'l'GGGAccccG 200. Hmm℃
a Eikanto. ．． T. ″rT........,
,A. G,,T. ．． C. ．． A. ．． ．． ．． ．． C. ．． TT
．． A. ．． ．． ．． ．． G. ．． A. ．． OCA. ．． ．． C;. ．．
．． ．． ．． G. ．． ．． ．． ．． ．． C. ．． ．． ．． ．． A. C. ．． C
．． ．． C. ．． ．． ．． ．． C. ．． C. ．． ．． T. ．． ．． ．． C.
．． ．． ．． ．． ．． CAC. ．． G. ．． ．． ．． ．． ．． ．． ．． A. ．． G
．． ．． ．． ．． ．． A. ．． ．． ．． ．． ．． ．． ．． C. ．． C. ．． A
．． ．． ．． ．． ．． ．． ．． ．． 'rT. ．． ．． ．． ．． ．． ．． T. ．． A.C.
lvNPvσn冫SσK℃υAGAACTTCrA C
AAGCCC-Fu'C GrTTACATCG GrA
α Engineering BmNPV. ,,． ．． A. ．． ．． ．． 1. ．．
．． ．． ．． ．． ．． ．． ．． ．． ．． A. ．． ．． ．． ．． ．． ．． ．． ．． ．． ．．
．． ．． ．． ．． ．． ．． C. ．． A. ．． ．． ．． ．． ．． ．． ．． C. ．．
．． ．． ．． AOp Boar V. ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．．
．． ．． T. ．． T. ,,,． ．． ．． ．． ．． ．． ．． G. ．． ．． ．． ．．
T. ．． C. ．． G. ．． T. ．． GAGC. ．． G. ．． ．． O
pSNPV. ,T. ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．．
．． ．． ．． ．． ．． ．． A. ．． ．． ．． ．． ．． ．． ．． G. ．． ．． G.
．． ．． ．． C.. A. ．． T. ．． ．． ．． ．． C. ．． G. ．．
A probe 2 ErrNPV. ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．．
．． ．． ．． G. ．． ,,． 、． ,,,． ,,,,,,,,,,
A,,,qM■■. A. ．． ．． ．． ．． ．． ．． ．． G. ．． C. ．．
．． ．． ．． G. ．． C;r. ．． ．． ．． ．． ．． ．． C. ．． C. ．． ．． ．．
．． ．． ．． ．． ．． OpSNPV. ．． ．． ．． A. ．． ．． ．． ．． ．． ．． ．．
．． ．． 、． ．． ．． ．． ．． ．． Cr. ．． ．． A. T. A. ．． C. ．． ．．
．． ．． T. ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． C. ．． A.A. ．． ．． ．．
G. ．． ．． ．． ．． T. ．． C. ．． T. ．． ．． ．． ．． ．． A. ．．
．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． A. C. ．． G. ．． G. ．． G
．． ．． C. ．． ．． ．． ．． T. ．． G. ．． ．． ．． ．． ．． A. ．． ．． ．． ．． A
T. A. ．． ．． ．． ．． ．． ．． ．． ．． ．． ．． G. ．． T. ．． ．．
．． ．． ．． ．． ．． ．． A. T. Figure 1 χHindm 25:Sj! NPVC-41iDN
AHindm-BamHI3 6:SfNPvC-41
l DNA HindIIl-EcoRI4.7:SQ
NPVC-4N DNA Hindl[l-Hincl
Restriction enzyme map area of 7 genes 6-a Construction diagram of vector {1) 6-b IIll construction diagram of vector fill 7 β
- Cloning of galactosidase). Hind I [
I 5 SI NPVβ-901
2 days arnH f2, SfNPVC-411 days om
HI 6.5INPVβ-gof2 EcoRT
3, SR NPVC-41 lεcoRI7'sAN
Pβ-go12 Hindm4 SJ! NPVC-4
11 1-1ihdlU (number of culture days) Figure 9 β-galactosidase produced by SINPV p-9o12

Claims (1)

[Scope of Claims] 1. Spodoptera nuclear polyhedrosis virus (SlNPV) having the following restriction enzyme map and approximately 3 kilobases.
C-411) polyhedrin gene. [Gene sequence is available] 2. Spodoptera nuclear polyhedrosis virus (SlNPV C-41) containing DNA encoding the following amino acid sequence.
1) polyhedrin gene. [There is a gene sequence] (In the formula, G is glycine, A is alanine, V is valine, L
is leucine, I is isoleucine, S is serine, T is threonine, D is aspartic acid, E is glutamic acid, N is asparagine, Q is glutamine, K is lysine, R is arginine, C is cysteine, M is methionine, F is (phenylalanine, Y is tyrosine, W is tryptophan, H is histidine, P is proline) 3. Polyhedrin gene of Spodoptera nuclear polyhedrosis virus (Sl2NPV C-411) containing the following base sequence. [Gene sequence is available] 4. Spodoptera nuclear polyhedrosis virus (SlNPV)
A vector obtained by replacing at least a part of the polyhedrin gene of C-411) with a gene constituting a useful substance. 5. Spodoptera nuclear polyhedrosis virus (SlNPV)
C-411), a fragment containing a promoter in the 5' upstream region of the polyhedrin gene is injected with a gene constituting a useful substance, and further a DN in the 3' downstream region of the polyhedrin gene.
Vector inherited from A fragment. 6. (a) Spodoptera nuclear polyhedrosis virus (SIN)
PV C-411) by cleaving the polyhedrin gene to produce a DNA fragment containing the polyhedrin promoter and sufficient adjacent DNA to enable genetic recombination; (b) inserting the DNA fragment into a cloning vehicle and assembling it; A method for producing a vector, comprising: generating a recombinant cloning vehicle; and (c) inserting at least one foreign DNA into said recombinant cloning vehicle such that the foreign DNA is under the transcriptional control of a polyhedrin promoter. 7. Spodoptera nuclear polyhedrosis virus (SlNPV)
A recombinant virus in which at least a part of the polyhedrin gene of C-411) has been replaced with a gene constituting a useful substance. 8. Spodoptera nuclear polyhedrosis virus (SlNPV)
C-411), a fragment containing a promoter in the 5' upstream region of the polyhedrin gene is injected with a gene constituting a useful substance, and further a DN in the 3' downstream region of the polyhedrin gene.
A recombinant virus that has a gene replaced with DNA inherited from the A fragment. 9. (a) Spodoptera nuclear polyhedrosis virus (SIN)
PV C-411) by cleaving the polyhedrin gene to produce a DNA fragment containing the polyhedrin promoter and sufficient adjacent DNA to enable genetic recombination; (b) inserting the DNA fragment into a cloning vehicle and assembling it; (c) inserting at least one foreign DNA into said recombinant cloning vehicle such that the foreign DNA is under the transcriptional control of a polyhedrin promoter to produce a vector; (d) producing a gene. A method for producing a recombinant virus, which comprises bringing the vector into contact with a virus (SlNPV C-411) so as to make recombination effective. 10. A cell infected with the recombinant virus according to claim 7. 11. A cell infected with the recombinant virus according to claim 8. 12. A method for producing peptides, which comprises culturing the cell according to claim 10 or 11 in a medium and collecting peptides from the culture. 13. (a) Spodoptera nuclear polyhedrosis virus (Sl
Foreign DNA is injected after the fragment containing the promoter of the 5' upstream region of the polyhedrin gene of NPV C-411), and the DNA of the 3' downstream region of the polyhedrin gene is injected.
A method for producing peptides, which comprises producing a recombinant virus having a gene substituted with DNA inherited from the A fragment, and (b) culturing cells infected with the recombinant virus. 14. Spodoptera nuclear polyhedrosis virus (SlNPV)
A recombinant gene in which at least a portion of the polyhedrin gene of C-411) is replaced with foreign DNA. 15. Spodoptera nuclear polyhedrosis virus (SlNPV)
A recombinant gene obtained by inheriting a fragment containing a promoter in the 5' upstream region of the polyhedrin gene of C-411), followed by foreign DNA, and further inheriting a DNA fragment in the 3' downstream region of the polyhedrin gene. 16. Spodoptera nuclear polyhedrosis virus (SlNPV)
C-411) has a gene in which foreign DNA is inherited after a fragment containing a promoter in the 5' upstream region of the polyhedrin gene, and the gene is replaced with DNA inheriting a DNA fragment in the 3' downstream region of the polyhedrin gene. A fusion protein produced by culturing cells infected with a recombinant virus. 17. Spodoptera nuclear polyhedrosis virus (SlNPV)
A fusion protein in which at least a portion of the polyhedrin peptide of C-411) is replaced with another polypeptide.