JP2003052371A - Recombinant baculovirus and method for obtaining protein by using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a recombinant baculovirus capable of infecting both hosts of a Bm (bombycid insect) base and a Ac (noctuid insect) base and exhibiting a high infectivity titer, and to provide a method for producing a recombinant protein, capable of stably producing a large amount of the protein by using the same. SOLUTION: This recombinant baculovirus is obtained by subjecting a nuclear-polyhedrosis virus living in the bombycid insect as the host (BmNPV) and a nuclear-polyhedrosis virus living in the noctuid insect as the host (AcNPV) to homologous recombination and has features as follows: (1) the recombinant baculovirus infects an organism and cultured cells of both the bombycid insect and the noctuid insect as the host and multiplies therein; (2) the recombinant baculovirus has such a virus titer as 10 times the virus titer of the wild type BmNPV (nuclear-polyhedrosis virus living in the bombycid insect as the host), when the organism and cultured cells of the bombycid insect are infected with the recombinant baculovirus; and (3) the recombinant baculovirus retards expression of the cysteine by about 72 hr when compared with the wild type BmNPV (nuclear-polyhedrosis virus living in the bombycid insect as the host).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a recombinant baculovirus, and more particularly to a recombinant baculovirus having an expanded host region and a method for obtaining a protein using the same.

[0002]

2. Description of the Related Art In recent years, techniques for producing useful proteins using insects such as silkworms and Spodoptera litura and their cultured cells using gene recombination technology have been actively researched. In the gene recombination technique using these insects and insect cells, a method using a baculovirus expression system is generally used (Japanese Patent Laid-Open Nos. 61-9288 and 62-208).
No. 276).

In the method for producing a recombinant protein using such a baculovirus expression system, first, a recombinant baculovirus in which a gene of a protein to be produced is incorporated is prepared. Then, the produced recombinant baculovirus is introduced into a host, and this is propagated in the host to produce a recombinant protein.

Baculoviruses used for the production of recombinant proteins include, for example, Bombyx mori ( Bombyx mori ;
"Bm")-derived nuclear polyhedrosis virus (hereinafter,
"BmNPV") and the autographer of the family Noctuidae.
California ( Autographa califo
rnica ) -derived nuclear polyhedrosis virus (hereinafter referred to as "Ac
"NPV") is common. However, since baculovirus is a virus with high host specificity, BmNPV infects and propagates in the living organisms of Bombyx mori insects and their cultured cells as hosts, but in living organisms of the same Lepidopteran moths and their organisms. It cannot infect or propagate in cultured cells. Also for AcNPV, B
The same applies to mNPV.

That is, when BmNPV is used as the recombinant baculovirus, the host thereof is a living body of Bombyx mori insects capable of growing BmNPV and cultured cells thereof (Bm-cultured cells such as Bm-N and BoMo). is necessary. In addition, when AcNPV is used, the host is an insect of the family Noctuidae ( Autographa californica ( Autographa ca.
lifornica ; hereinafter referred to as “Ac”), Spodoptera frugiperda ( Spodoptera fr
Ugiperda ; hereinafter referred to as "Sf"), Trichoplusia ni (hereinafter referred to as "Tn") and the like, and their cultured cells (Sf-9, Sf-21, Tn-5, Tn-368, etc.) It is necessary that the cell line is an Ac-based cultured cell).

Therefore, when a BmNPV-derived recombinant baculovirus used for producing a recombinant protein is used, the recombinant protein can be produced only in the Bm-type cultured cells and the living organism of the host insect. In the same manner, when the AcNPV-derived one is used, the recombinant protein can be produced only in the Ac-based cultured cells and the living organism of the host insect. As described above, the conventional baculovirus host is limited, so that there is a problem that an optimal host that maximizes the performance of the recombinant baculovirus cannot be used. However, there was a problem that the recombinant baculovirus had to be produced separately when the above-mentioned production was performed.

By the way, in recent years, Maeda et al.
No. 0092) has developed a recombinant baculovirus capable of infecting both Bm-based and Ac-based hosts.
The infectious titer of this recombinant baculovirus in cultured insect cells was almost the same as the infectious titer of wild-type BmNPV (1 × 10 ⁸ PFU / ml) in the viral infective titer of Bm cultured cells. , The virus infecting titer to the Ac-type cultured cells was 5 × 10 ^{4 to} 5 × 10 ⁶ PFU / ml, which was 1/10 to 1/1000 more than the infectious titer of wild-type AcNPV (1 × 10 ⁷ PFU / ml). It was falling.

In addition, Mori et al. (MORI, H et.al. Journal o
f General Virology, vol.73, pp1877-1880, (1992))
Has also developed a recombinant baculovirus capable of infecting both hosts as well. The virus infectious titer of this recombinant baculovirus to the Ac-based cultured cells was
It was equivalent to cNPV. When the recombinant protein is produced using this recombinant baculovirus, the recombinant protein is expressed in an amount about 5 times as much as that in the case of using the Bm-type cultured cells when the Ac-type cultured cells are used as the production host. It was confirmed to do. When the silkworm larvae were infected, this recombinant baculovirus showed wild type B.
It was shown that the time to lethality was delayed and the expression time of the recombinant protein was delayed by about 2 days as compared with mNPV.

Thus, although recombinant baculoviruses capable of infecting both Bm-type and Ac-type hosts have been developed to enable expansion of the host region, they are compared with wild-type BmNPV or AcNPV. The infectious titer, recombinant protein expression ability, etc. were low or, at best, almost the same.

In the virus belonging to the baculovirus family, it is considered that some viral constituent proteins expressed in the late stage of infection are involved in the infection of cells and the growth in the infected cells (Lu, A. et.al.
J. Virol., Vol.69, pp975-982, (1995)). Among the viral constituent proteins expressed in the latter stage of infection, DNA helicase is replication of viral DNA in virus-infected cells, cysteine protease is lysis of host cells for virus release, and p35 is derived from the host in virus-infected cells. It has protein production control as a function, and these functions are also considered to be the cause of determining the specificity to the host.

[0011]

Therefore, the present invention is based on the Bm
Recombinant baculovirus that can infect both host system and Ac system and has significantly higher infectious titer than conventional baculovirus is prepared, and stable and large amount of the target recombinant protein can be produced by using this recombinant baculovirus. The task is to provide a method that enables production.

[0012]

[Means for Solving the Problems] The present inventors have prepared a large number of recombinant baculoviruses capable of infecting both Bm-type and Ac-type hosts and examined their properties. It was found that some of them can infect both hosts, their infectivity is extremely higher than that of wild-type baculovirus, and that the cysteine protease possessed by these viruses is also expressed slowly.

It has also been found that the above recombinant baculovirus can stably and in large quantity produce the target recombinant protein by incorporating a gene encoding the target protein into the recombinant baculovirus. Was completed.

That is, the present invention is obtained by homologous recombination of a nuclear polyhedrosis virus having a silkworm moth as a host with a nuclear polyhedrosis virus having a moth insect as a host, and having the following characteristics: Baculovirus (hereinafter,
"BANPV"). (1) It is possible to infect and proliferate both in the living body of Bombycidae and Bombycidae and their cultured cells. (2) The virus titer shown when infected with the living body and cultured cells of Bombycidae insects is 10 times higher than that of wild-type BmNPV. (3) The expression of cysteine protease of BANPV is delayed by about 72 hours as compared with wild-type BmNPV.

The present invention also provides the above-mentioned BANPV in which the structures of the viral constituent proteins gp64 and DNA helicase gene are mosaics of BmNPV-derived gene and AcNPV-derived gene.

Furthermore, in the present invention, a part of a viral constituent protein has a B sequence having a DNA sequence as shown in SEQ ID NO: 1.
It provides ANPV.

Furthermore, the present invention provides DN of SEQ ID NO: 1 above.
The present invention provides a BANPV genomic DNA having an A sequence.

Furthermore, the present invention obtains recombinant BANPV by introducing the genomic DNA of BANPV and a recombinant transfer vector incorporating a foreign gene into cultured cells of Bombyx mori or Insectidae, Then, a living body of Bombyx mori or Moth family insects or their cultured cells is infected with this recombinant BANPV to express the protein encoded by the foreign gene introduced into the recombinant BANPV and obtain the protein for collecting the protein. It provides a method.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The BANPV of the present invention comprises viral genomic DNA of baculovirus derived from nuclear polyhedrosis virus of lepidopteran silkworm moths such as Bombyx mori and Autographa californica ( Autographa californi
ca ), Spodoptera frugiperuda ( Spodop)
tera frugiperda ), Trichopurcia
It is obtained by homologous recombination with the viral genomic DNA of baculovirus derived from nuclear polyhedrosis virus of lepidopteran insects such as Trichoplusiani .

The homologous recombination for producing the BANPV of the present invention can be carried out by, for example, Maeda et al.
No.) can be performed according to the following procedure. (1) AcNPV DNA and BmNPV DNA digested with a restriction enzyme Bam HI were co-introduced into a living or monolayer Bm-N cell of Bombyx mori insects using a cationic lipid reagent such as lipofectin reagent.・
Transfect and incubate for a few days. (2) Next, using the culture supernatant of (1) above as a virus solution, Sf-9 cells are then infected. (3) Infection of Bm-N cells with virus solution and Sf-
Repeating the infection procedure to 9 cells, Bm also to Sf-9 cells
Obtain a viral fluid that can also infect N cells. (4) BANPV is isolated from the virus solution obtained in (3) above by the limiting dilution method. (5) The BANPV obtained in (4) above is infected with larvae of Bombyx mori family insects to obtain a final BANPV strain.

Next, the BANP obtained as described above
From the strains of V, those having a higher infectivity than the wild-type BmNPV are selected, and thus screening can be performed to obtain the BANPV strain of the present invention having a high infectious titer.

The screening described above is performed by King et al. (KIN
G, LA and POSSE, RD A Laboratry Guide Londo
n: Chapman and Hall, (1992)) 's limiting dilution method and Maeda et al.
(Maeda, S et, al. J. Gen. Vprol.vol.71, pp2631-263
9, (1990)) using the plaque assay method.
The infectious titer of the above, etc., and the infectious titer of 10 times or more of the infectious titer of the wild type BmNPV and the equivalent or more than the infectious titer of the wild type AcNPV may be the BANPV of the present invention.

The BANPV genome thus obtained can also be analyzed by using a known method. For example, an oligo primer is synthesized based on the gene information of the target BANPV and the target gene is amplified by the PCR method using the oligo primer. It is then cloned into a plasmid vector (eg pT7Blue T-Vecto
r; manufactured by Novagen)
NA sequence to a DNA sequencer (eg ABI Pr
ism 310 Genetic Analyzer;
PERKIN ELMER).

By such a method, the BANP of the present invention is
As a result of analysis of the V genome, the structures of the gp64 and DNA helicase genes, which are viral constituent proteins, were mosaic of BmNPV-derived genes and AcNPV-derived genes.

Expression of the recombinant protein using the BANPV of the present invention was performed by King et al. (KING, LA and POSSE, R.
D. A Laboratry Guide London: Chapman and Hall, (199
It can be performed according to the method of 2)). For example, a gene encoding a protein to be expressed is ligated to the 3'downstream portion of the polyhedron gene promoter of a transfer vector by ligation with a gene encoding a desired protein digested with an appropriate restriction enzyme as necessary. Prepare a transfer vector for recombination. Then, the transfer vector for recombination is used to co-transfect the cultured cells to transform the gene encoding the desired protein into BA.
Incorporation into NPV DNA creates recombinant BANPV. Then, the recombinant BANPV is infected and propagated in an insect living body or a cultured cell to express a desired protein.

Known methods can also be used for the method of infecting a host or cultured cells of the host with BANPV and the method of proliferation after infection. For example, the host is Bombix
In the case of larvae such as Bombyx mori ,
As an infection method, a method of inoculating BANPV and recombinant BANPV by subcutaneous injection can be used. Further, as a method for multiplying BANPV, a method of breeding the larva living body after infection for an appropriate period can be used.

The target protein expressed by the above-mentioned infection or proliferation method is isolated from the host or host cell,
As a method for collecting, a known method can be used. For example, when utilizing the larvae of Bombycidae as the host, the expressed protein is present in the body fluid of the larvae of Bombycidae, so the body fluid should be collected and purified by an appropriate means. The protein can be recovered. When the expressed target protein is present in Bm somatic cells, an appropriate buffer is added to the larvae of the silkworm, Bombycidae infected with recombinant BANPV, ground with a mixer, and centrifuged. It can be obtained by collecting Qing.

[0028] One of the features of the BANPV of the present invention is that the expression of the cysteine protease originally held by the baculovirus in the genome is delayed by about 72 hours from that of the wild-type BmNPV. However, this feature has great significance in obtaining the target protein. That is, when the larva of Bombycidae is used, the recombinant protein is produced in the body fluid and somatic cells of the larva as described above, but since cysteine protease is also expressed at about the same time, it is produced once. Recombinant proteins may be degraded. On the other hand, when the expression of cysteine protease is slow, the recombinant protein produced during that time is not decomposed and the target protein can be obtained in high yield.

Whether or not the BANPV of the present invention expresses the target protein can be confirmed by using, as the protein, a protein that emits light or fluorescence such as luciferase and GFP. When luciferase is used, it can be performed according to the method of Shimamura (JP-A-8-238086), and when GFP is used, it can be performed by a conventional method.

[0030]

The BANPV of the present invention is used for living organisms of Bombyx mori family insects and cultured Bm cells and living organisms of Bombyx mori family insects and A.
Both of the c-type cultured cells can be used,
Moreover, the ability of Bombyx mori insects to infect and proliferate living organisms or Bm culture cells is higher than that of conventional BmNPV. Also, the production time of cysteine protease is later than that of conventional BmNPV.

The property of BANPV is that the recombinant protein can be expressed in both Bm cells and Ac cells as a host, the expression amount is large, and the expressed recombinant protein is not easily decomposed. This means that BAPNV of the present invention can be extremely usefully utilized in a recombinant protein large-scale expression system utilizing baculovirus.

[0032]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 Acquisition of BANPV: 1. Baculovirus growth BmNPV-T3 strain (distributed by RIKEN) was Bm-.
The cells were grown using N cells (distributed by RIKEN).
The AcNPV-C2 strain (distributed by the Silkworm Insect Agriculture Research Institute) was grown using Sf-9 cells (manufactured by Invitrogen). Bm-N cells and Sf-9 cells are TC-100 insect cell culture medium containing 10% fetal bovine serum (manufactured by SIGMA; hereinafter referred to as "FBS") (hereinafter, referred to as "TC-
It was cultured at 25 ° C. in 100 medium ”.

2. Preparation of baculovirus DNA (a) Acquisition of BmNPV and AcNPV viral DNAs BmNPV-T3 strain viral DNAs can be obtained by Maeda method from virus-infected Bm-N cells (JP-A-4-370092). Was carried out according to. That is,
For preparation of BmNPV-T3 strain viral DNA, first, Bm
-Infecting N cells with BmNPV-T3 strain virus, 2
The cells were cultured at 5 ° C for 7 days. The obtained virus-infected cell culture supernatant was layered on a 40% sucrose solution and centrifuged for 1 hour at 5,000 xg and 15 ° C in a high-speed centrifuge to recover virus particles as a precipitate, and an appropriate amount of 10 mM was added. Tris
-HCl (pH 7.5) and 1 mM EDTA (hereinafter,
"TE buffer"). Then, the final concentration of the recovered virus particle solution was 1 mg / ml.
Protease K (manufactured by Wako Pure Chemical Industries) and 1% SDS (manufactured by Wako Pure Chemical Industries) are mixed so that
Protease treatment was carried out under the condition of 0 minutes. TE in this solution
An equal amount of phenol saturated with a buffer was added, the mixture was vigorously stirred, and then centrifuged at 15,000 rpm and 4 ° C. for 10 minutes to collect the supernatant. An equal amount of isopropanol was added to the obtained supernatant, and the mixture was allowed to stand at room temperature for 10 minutes, then 15,000 rp
The precipitate was recovered by centrifuging for 10 minutes under the conditions of m. To the obtained precipitate, 1 ml of 70% ethanol was added and lightly washed, followed by centrifugation at 15,000 rpm and 4 ° C. for 10 minutes to obtain a precipitate. The obtained precipitate was air-dried with a desiccator and then dissolved in TE buffer. This solution is BmNPV
-T3 strain viral DNA was stored at 4 ° C until use.

AcNPV-C2 strain viral DNA was obtained from virus-infected Sf-9 cells according to the above method.

(B) Restriction enzyme treatment of baculovirus DNA Using the restriction enzyme Bam HI, the method of Maeda et al. (MAEDA,
S et.al. Journal of General Virology, vol.71, pp185
1, (1990)) and subjected to restriction enzyme treatment. That is, B
Restriction enzyme Bam for 1 μg of mNPV virus DNA
3 at 37 ° C using 30 units of Hl (Takara Shuzo)
After digestion for a while, phenol treatment and ethanol precipitation were performed, and the obtained BmNPV virus DNA fragment was dissolved in 50 μl of TE buffer. This restriction enzyme did not cleave the gene DNA helicase involved in host specificity, but cleaved the whole site at 6 large sites.

(C) Co-transfection of baculovirus DNA into cells First, Bm-N was prepared using a 35 mm cell culture dish.
The cells were prepared by static culture to about 1 × 10 ⁶ monolayer confluent. Next, 0.1 μg of AcNPV DNA and all BmNPV DNA digested with the restriction enzyme Bam HI.
0.1 μg of the fragment was mixed with 100 μl of TC-100 medium without FBS in a 1.5 ml Eppendorf tube, and the mixture was allowed to stand at room temperature for 15 minutes. Further, 100 μl of TC-100 medium containing no FBS was mixed with 5 μl of lipofectin reagent (manufactured by GIBCO-BRL), and the mixture was allowed to stand at room temperature for 15 minutes.

Next, these two solutions were mixed and allowed to stand at room temperature for 15 minutes, and then 800 μm containing no further FBS.
1 of TC-100 medium was added to obtain a DNA solution. The DNA solution prepared above was added to Bm-N cells prepared as a monolayer confluent with a 35 mm cell culture dish, and cultured for 20 hours under a culture condition of 25 ° C., and then the DNA solution in the cell supernatant was removed. , T with 10% new FBS
2 ml of C-100 medium was added, and static culture was performed for 7 days under the culture condition of 25 ° C.

3. Isolation of BANPV Using the culture supernatant obtained above as a BANPV solution, Sf-9 cells were infected by the same method and cultured at 25 ° C. for 7 days, and the culture supernatant was collected. Recovered. Furthermore this BA
As an NPV solution, Bm-N cells were infected and cultured for 7 days under a culture condition of 25 ° C., and the culture supernatant was collected. This operation was repeated for 6 generations to obtain a BANPV solution capable of infecting both Sf-9 cells and Bm-N cells.

BANPV was isolated from the obtained BANPV solution by the method of King et al. (LA KING and RD POSSE).
A Laboratry Guide (1992)) was performed as follows. First, TC containing Bm-N cells containing 10% FBS was used.
Culturing in −100 medium, 1 × 10 ³ cells / well / 5
A 96-well plate with 0 μl was prepared. The BANPV solution thus obtained was diluted with TC-100 medium containing 10% FBS into 10 ^-6 , 10 ^-7 , 10 ^-8 and 10 ^-9 four-step dilutions, and these BANPV solutions were diluted to 1 of each plate. 5 per hole
The mixture was mixed with 0 μl to infect each other, and the plate was sealed to prevent drying and statically cultured under culture conditions of 25 ° C. Infection 7
On the day, virus strains that form polyhedra were selected under an optical microscope, and 6 strains of BANPV were obtained. Secondary selection is performed in the same manner using the virus infection solution obtained from these holes, and finally the result obtained by the secondary selection is infected into Sf-9 cells to form polyhedra. It was confirmed.

Furthermore, the 6 strains of BANPV obtained above
Was infected with Sf-9 cells at a multiplicity of infection (moi) of 5, and statically cultured under culture conditions of 25 ° C. for 7 days. Regarding this virus, it was confirmed on the 5th day after infection that a polyhedron was formed under an optical microscope.

Next, 6 strains of BANPV obtained above as the third selection were subcutaneously inoculated into a living body of Bombyx mori larvae (hereinafter referred to as “silkworm larvae”) immediately after molting, The animals were bred under the conditions of room temperature of 25 ° C. and humidity of 45%. To compare the number of polyhedra formed in the body of silkworm larvae on the 7th day after breeding, the larvae were dissected and fat body cells were collected. Then, a preparation is prepared and observed under an optical microscope, and a strain that forms a large number of polyhedra per cell is used as a final BANPV strain (BANPV strain).
PV9810N-6) was selected. The Patent Microbial Depositary Center of the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology does not accept the deposit of the virus, so this virus is not deposited. If there is a request for a sale that satisfies the conditions in Article 3 of the Article, the applicant, Katakura Industry Co., Ltd., guarantees that the sale will be in accordance with the conditions of that Article.

4. Recovery of virus-containing body fluid from silkworm larvae BANPV (BANPV98) obtained as described above.
10N-6) was subcutaneously injected to the silkworm larvae. Infected silkworm larvae at room temperature 25 ° C and humidity 45% for 7
Reared for days. Seven days after infection, the abdominal limbs were cut into scissors for dissection, body fluid was added dropwise, and collected in a 50 ml centrifuge tube cooled in ice. These tubes were immediately centrifuged at 3,000 rpm and 10 ° C for 10 minutes using an automatic low-speed cooling centrifuge, and the supernatant was collected and the BAN was collected.
It was PV liquid.

Test Example 1 Infectious titer measurement: BANPV was infected with both Bm-N cells and Sf-9 cells, wild-type BmNPV was infected with Bm-N cells, and wild-type AcNPV was infected with Sf-9 cells. The virus titer was measured over time by the dilution method. The results are shown in FIG. In addition, since wild-type AcNPV and wild-type BmNPV destroy cells 7 days after the final stage of infection, the infectious titer was measured up to 6 days.

As a result, the infectious titer of BANPV in Sf-9 cells was similar to that of wild-type AcNPV, but
-The infectious titer of BANPV in N cells is wild type BmNPV
About 10 times higher.

Test Example 2 Expression of Recombinant Protein Using BANPV: 1. Production of Recombinant BANPV Recombinant BANPV was produced by a conventional method. That is, B
ANPV DNA or BmNPV DNA and luciferase gene (distributed by Silkworm Insect Agricultural Technology Research Institute, Research Institute for Breeding and Cell Engineering) or GFP gene (Invitrog
transfer vector (pYN)
G: Katakura Industrial Co., Ltd. was used to co-transfect cultured cells to prepare BANPV and BmNPV incorporating these genes (hereinafter, BANPV and BmN incorporating luciferase gene).
PV is “BANPV / Luc” and “BmN”, respectively.
PV / Luc ”. In addition, BANPV and BmNPV in which the GFP gene has been incorporated are referred to as “BANPV /
GFP "and" BmNPV / GFP ").

2. Expression of Recombinant Protein in Silkworm Larvae Recombinant BANPV obtained above in silkworm larvae on the 5th day of 1st day
Was inoculated by subcutaneous injection, and then fed with artificial feed (Morus; manufactured by Katakura Kogyo Co., Ltd.) under conditions of 25 ° C. and 45% humidity, and 5 animals each were collected every 24 hours until the larvae of the silkworm die, − Stored at 80.

3. Preparation of Larval Ground Samples Larval ground samples were prepared by the following procedure. That is, 50 ml of frozen infected silkworm larvae was added with 50 ml of a grinding buffer having the following composition (hereinafter, referred to as "grinding buffer"), and ground for 5 minutes under ice-cooling conditions using a Waring blender (Inei Seieidou). . After filtering this grinding liquid with gauze, it was ground with a Teflon homogenizer (manufactured by Inai Seieido Co., Ltd.) under ice-cooling conditions for 3 minutes. This grinding fluid was put in a low-speed cooling centrifuge (Hitachi) at 4 ° C and 12,000 rpm for 2
After centrifugation for 0 minutes, the supernatant was collected and stored at -60 ° C.

<Grinding buffer composition><mM> EDTA (manufactured by Dojindo) 1 Sodium chloride (manufactured by Wako Pure Chemical Industries) 100 Benzamidine (manufactured by Wako Pure Chemical Industries) 10 PMSF (manufactured by Wako Pure Chemical Industries) 1 PIPES buffer solution (PH 6.6) (manufactured by Dojindo) 20 (including 1% Triton X-100 (manufactured by Wako Pure Chemical Industries) and 10% glycerol (manufactured by Wako Pure Chemical Industries))

4. Method for measuring luciferase activity The method for measuring luciferase activity is Shimamura (JP-A-8-
238086). That is, the larval ground sample prepared by the above method was diluted with an appropriate amount of grinding buffer. To 25 μl of the diluted larvae ground sample, 300 μl of 50 mM HEPES buffer (pH 7.8; manufactured by Dojindo) containing 10 mM magnesium sulfate, 200 μl of 3 mM ATP solution (manufactured by Oriental Yeast) and 25 μl of distilled water were added. Mix and further mix this solution with 0.125 mM D-luciferin (JBL
100 μl of Scientific INC.) Was added. Immediately after adding D-luciferin, a luminometer (LUMINESCENCE READER B
LR-201; manufactured by ALOKA) was used, and the number of photons for 30 seconds after standing for 30 seconds was cumulatively measured. The result is shown in FIG.

As a result, when the maximum expression level of luciferase, that is, the expression level of BANPV / Luc at 168 hours and the expression level of BmNPV / Luc at 120 hours were compared, BANPV / Luc was BmNP / Luc.
The expression level of luciferase was about 12 times higher than that of V / Luc.

5. Method for measuring the amount of GFP fluorescence The method for measuring the amount of GFP fluorescence was performed according to a conventional method. That is, the larval ground sample prepared by the above method was diluted with an appropriate amount of the ground buffer, and 50 μl of the diluted larval ground sample was further diluted 6-fold with distilled water. This diluted solution was rapidly transferred to a cell for fluorescence measurement, and the amount of fluorescence for 30 seconds after standing for 30 seconds under the conditions of excitation light wavelength 395 nm and fluorescence wavelength 509 nm using an Intelligen fluorescence detector (manufactured by JASCO Corporation) was measured. An integrated measurement was performed. The result is shown in FIG.

As a result, when the maximum expression level of GFP was compared, that is, the expression level of BANPV / GFP at 192 hours and the expression level of BmNPV / GFP at 144 hours were compared, the former expression level was higher than that of the latter. It was about 1.8 times higher than the expression level.

From the above results, when the recombinant protein was expressed using the BANPV of the present invention, the wild-type Bm was expressed.
It was found that the expression level of the recombinant protein was higher than that when the recombinant protein was expressed using NPV.

Test Example 3 Expression of Cysteine Protease in BANPV: 1. Preparation of virus-infected culture Bm-N cell extract solution In order to obtain 3 × 10 ⁵ Bm-N cells per well,
Two 24-well plates were prepared in which 500 μl of TC-100 medium containing 10% FBS was added. The BANPV solution and BmNPV solution were added dropwise to the media of these plates at a multiplicity of infection (moi) of 0.5 concentration, and the cells were cultured at 27 ° C. The virus-infected Bm-N cells were sampled by suspending the cells in a medium in a well, transferring to a microtube, centrifuging at 3,000 rpm for 10 minutes at 4 ° C., removing the supernatant, and precipitating virus. Infection Bm-N
This was done by freezing the cells. In addition, sampling was performed every 24 hours after virus inoculation up to 144 hours. The virus-infected Bm-N cells recovered and frozen had a concentration of 0.
5% Triton X-100 (manufactured by Wako Pure Chemical Industries),
0.1% NP40 (Nakarai Tesque) and 1 mM
Phosphate buffer solution containing PMSF (manufactured by Wako Pure Chemical Industries) (p
H6.8) was added in an amount of 50 μl, and the cells were disrupted by freeze-thawing treatment, and then centrifuged at 4 ° C. and 12,000 rpm for 10 minutes, and the supernatant was collected, and virus-infected culture Bm-N cell extract (hereinafter, "Cell extract").

2. Preparation of sample from virus-infected silkworm larvae body fluid BANPV solution and BmNP were applied to 5th-day-old silkworm larvae.
After inoculation of the V solution by subcutaneous injection, artificial feeding (manufactured by Katakura Kogyo Co., Ltd.) was given and raised under conditions of 25 ° C. and a humidity of 45%. Sampling was carried out by collecting body fluids from 5 animals every 24 hours until the silkworm larvae died. For collection of body fluid, cut the abdomen with scissors, collect the body fluid in a 50 ml centrifuge tube cooled in ice, and quickly using a low-speed cooling centrifuge (Hitachi) at 10 ° C for 3,000 rpm for 10 minutes. After centrifugation, the supernatant was collected and stored at -60 ° C. This supernatant was used as a body fluid sample.

3. Cysteine Protease Activity Measurement A microtube containing 3 mg / ml of Azocoll (S
250 μl of a 0.1 M succinic acid-sodium hydroxide buffer (pH 4.5) substrate solution containing IGMA) was mixed with 25 μl of the cell extract sample or 5 μl of the body fluid sample prepared above, and the mixture was mixed at 37 ° C. for 24 hours. Incubated for hours. Then, 500 μl of a 10% sodium dodecyl sulfate (SDS) solution was added to the reaction mixture to stop the enzymatic reaction. After stopping the reaction, 4 ℃, 10,000
After centrifuging for 10 minutes under the condition of rpm, the supernatant was put into a 50 μl cuvette, and the absorbance at a wavelength of 520 nm was measured by a U-3210 absorptiometer (manufactured by Hitachi). The results of the cell extract sample are shown in FIG. 4 and the results of the body fluid sample are shown in FIG.

As a result, as shown in FIG. 4, in the cell extract sample, although the time delay of the expression of cysteine protease of BANPV and BmNPV was not observed, the measured value of the activity of cysteine protease of BANPV was the measurement period ( Bm through 24-144 hours)
It was lower than 0.75 times the peak activity value of NPV.

Further, as shown in FIG. 5, in the body fluid sample, the expression time of cysteine protease of BANPV was delayed by about 72 hours from the expression time of BmNPV,
The activity values were almost the same.

Test Example 4 Structural analysis of BANPV gene: BANPV and BmNPV
-DNA helicase of T3 strain and AcNPV-C6 strain (MAEDA, S et. Al. J. Virol .., vol.67, pp6234-62)
38, (1993)), gp64 (OHKAWA, T et. Al. J. Viro.
l., vol.68, pp6619-6625, (1994)), cysteine protease (GOMI, S et. al., J. Virol., vol.68, pp132
3-1337, (1999)) and p35 gene (GOMI, S et. A.
l., J. Virol., vol.68, pp1323-1337, (1999)), and an oligo primer having the following sequence was synthesized.

<Sequence of primer> 1. DNA helicase 1) 5'-GTC CGA ACG AGA GGT
GCG CG-3 '2) 5'-ACA AAC GTT TAT CAT
TAA AC-3 '3) 5'-TCT CGA CCA AAA TGC
AAA TG-3 '4) 5'-ATA AAC AAT CGT TCG
GTG CG-3 '

2.gp64 1) 5'-AAA CGA ACT TGC GTC
TAC TG-3 '2) 5'-TGG TGT GTG CCG GCG
GAT TG-3 '3) 5'-GAG ACA CTG CCA CCA
AAG GC-3 '4) 5'-AAT TGG TAA TGA TGA
CCA CC-3 '

3. Cysteine Protease 1) 5'-CCG GCG TGC CGG GAA
TCG CG-3 '2) 5'-GTA ATA ATA AAA ATT
GTA TC-3 '

4.p35 1) 5'-ATT ACA CGC GCG TCG
TAA CG-3 '2) 5'-AAT TCT ACT CGT AAA
GCC AG-3 '

Using these primers, the TAKARA PCR Thermal Cycler was prepared under the following conditions.
The target gene was amplified by the PCR method using (Takara Shuzo).

[0066]   <PCR conditions>   Viral DNA 0.01 μg   2x 10 × Ex-Taq buffer (Takara Shuzo)   Primer (follow) 1ng   1 ng of primer   dNTP (Takara Shuzo) 1 μl   Ex-Taq (Takara Shuzo) 1 μl   Distilled water / 20μl

<PCR cycle> 1.94.0 ℃ 3 minutes 2.94.0 ℃ 30 seconds 3.60.0 ℃ 30 seconds 4.72.0 ℃ 30 seconds Repeat steps 5.2 and 3 25 times 6.72.0 ℃ 3 minutes

The amplified gene of interest was pT7Blue.
It was subcloned into T-Vector (Novagen). The amplified DNA sequence of the target gene is A
BI Prism 310 Genetic Analysis
er (manufactured by PERKIN ELMER).

As a result, the cysteine protease and p35 gene of BANPV had the same DNA sequence as AcNPV and were the cysteine protease and p35 gene derived from AcNPV. However, the DNA helicase gene and the gp64 gene are associated with AcNPV and BmNP.
It had the unique structure of V. A structural schematic diagram of gp64 and the DNA helicase gene is shown in FIG.

BANPV gp64 gene has a total length of 514
Of the amino acids, the first half of the 489 amino acids is AcN
It was highly homologous to PV and could be assumed to be derived from AcNPV.

The BANPV DNA helicase gene had a particularly unique structure. That is, BANPV
The homology of the nucleotide sequences of the DNA helicase genes of BmNPV and AcNPV was 98% and 96%, respectively (Figs. 7-a to 7-g). Also, BANPV and B
98% homology with the amino acid sequences deduced from the DNA helicase genes of mNPV and AcNPV, respectively.
And 95% (FIGS. 8-a to 8-c). In the BANPV DNA helicase, the first half of all 1222 amino acid residues, that is, the 1st to 1095th amino acid residues have high homology with BmNPV and BmNPV.
It could be guessed that it came from. However, the 71st amino acid residue among them was the same as AcNPV, and it could be inferred that it was derived from AcNPV. Further, the 1095th to 1222nd amino acid residues were the same as AcNPV, and it could be inferred that they were derived from AcNPV. However, the 1150th amino acid residue among them was the same as BmNPV and could be presumed to be derived from BmNPV. Furthermore, BANPV retained a unique mutation shown in Table 1 that was not found in AcNPV and BmNPV. That is, 73
The second amino acid residue is cysteine in both AcNPV and BmNPV, whereas it is tyrosine in BANPV, and the 79th amino acid residue is AcNPV and Bm.
In NPV, it is proline and alanine, respectively, whereas in BANPV, it is serine, and the 88th amino acid residue is threonine in both AcNPV and BmNPV, whereas in BANPV, it is serine and the 119th amino acid residue is AcNPVBmNPV. , Both are lysine, but in BANPV, asparagine, 4
The 10th amino acid residue is Alanine and Isoleucine in AcNPV and BmNPV, respectively, whereas BAN
It was threonine in PV, and the 643rd amino acid residue was valine in both AcNPV and BmNPV, whereas it was isoleucine in BANPV.

[0072]

[Table 1]

[0073]

The BANPV of the present invention comprises AcNPV and B
It was obtained by homologous recombination with mNPV, and is an expanded host range of nuclear polyhedrosis virus that infects cultured cells of Lepidoptera insects or living organisms of Lepidoptera insects. Not only that, but the ability of the Bombyx mori insects to infect and proliferate against larvae is
Compared to wild-type BmNPV, the time required for expression of cysteine protease originally held by baculovirus in the genome is significantly delayed when the larvae of Bombyx mori insects are used. It was

The properties of the BANPV of the present invention as described above are extremely advantageous for the production of recombinant proteins and peptides, mainly using Bm-type cultured cells or Bombyx mori insect living organisms. This is extremely economical when a large amount of useful peptides are produced in large quantities by an inexpensive insect larva such as silkworm.

[0075]

[Sequence list] SEQUENCE LISTING <110> KATAKURA INDUSTRIES CO., LTD. <120> Recombinant baculovirus and expression of the protein using       the same <130> 0110027 <140> <141> <160> 1 <170> PatentIn Ver. 2.1 <210> 1 <211> 3669 <212> DNA <213> BANPV9810-N6 <220> <223> gp64 gene <400> 1 atgattgaca acattttaca atttttttta aaagacgtgc ctgaagacaa aacgtacgag 60 attaacaact tgcaagatgc aaatcattta atcattagaa acacgcgaac aggaacacgc 120 agattgtttg agtacgttaa caattttcaa cagttcttaa acacaattag aaacaacttt 180 aacggtccgt gcgcaaaaca cgacatgggc gcatcttacg aagacaccga agaatctaca 240 gaaaaacagg cagttcaaca atcattagac ggccacgact gggtgctgga aagtaacgat 300 ttttgcattt ttgtaaaacc tttcattttg aagaagcact atgaagtcat acaaaactat 360 attaattttg aaaatttttt caagagcacc gaccctggat acattaacaa atgcgtgcaa 420 gcgggcgatt actattactg gcccaactgg cccaaagggc aagctttctc ttttaacgga 480 tggcagctgt ttttaaatat aaaatttgga attgtcattg aacccacgat accaatcata 540 cacaacaaaa aactaggccc tgtggatttg ttcgtgtttg accctaaata ttttctcaac 600 gtcgagctca gcctgcgcac gaatcacgat ccgccgcaaa cgctgtttgt aaatggaaaa 660 accaagtttg acgagtctca cgaagatttg tttatactca aaatggccga cggtactgtg 720 gttacgtgca aagttaatgg cgagctggtc aattcggata aaaatttttt caattacatt 780 agggacgaca ttaatttgga agaatgcatt acggtgccca aatacaagca tattgtcaac 840 gtcaacctca aaagcctcag agtgttcgaa gacaacaatt ttgacaaaaa cgacgtggac 900 ttgagcgaca cgcgttccag aaaaccgcgc attgtaccca ttatatcggc gagcagcgaa 960 aatgcagact acattcaaac gcaaatcaat ttggggctta tagcaattta cgagaacatg 1020 gttaaagttt tggcgaccca cgaacaagcc aacgatccaa accttctaca acaatacttt 1080 gaaaaaagca aatttaaaaa ttttgatttt ttaatttatg tactgtggaa gatattgacc 1140 aagaacgaaa atttctcgta cagagaaacc gacatcaaac tgtttttgga acttttgtgt 1200 gaatcattat ttgcctgcga caaagaaact ttaaatgaag ctttgaaacg atgtgagcct 1260 tacaaaaaac aagaaaaagt agtgtttaat aggacctgca atcattggtt tgactttgac 1320 gataccaaat tgtgcgtgtc gttgggctat tattatggca tacactacat gatatatctg 1380 actctgtcgg ctaaaaacga aactttagat gacgatgaat tgtgggcgta cacgtacgag 1440 aatgtaatgg cgttaaactt gccgcctgac attgtgtgta aaggattctt tagaaaatta 1500 gaaaacgtag tgaccggagt caatttggtt ttcaacggca aaaactatca aattgtaaag 1560 aaagaggatg acctattcaa attggttaaa agcaattgtt acaagttgag caacataaaa 1620 tttaacaatt ggaaatactt gtacttgaca acgtacggtg tgtacaattt gttcaccaac 1680 agctttcatt cgaattgtcc atttttgttg ggcaccacgt tgccgcagac actcaagaag 1740 cccaccgacg aagagtattt gcccgaggac gcgtttaatt acatgctatc tactagcacc 1800 gacgagctca gcatttatag aatttatcac atcgccaaaa tgtgccgaga tgtaaaaatg 1860 ctaaaaacta acatggccat agttaactac atgggcaatt gcaacacatg ccaagccgat 1920 atgcgaatcg cgttaaacaa cttgtttcgc gatttgtgga atttggacga tgaaaatttg 1980 attacgctcg ctttgtacgt aaacaagaac aaggtttccg acatgttgca caacttgaaa 2040 tgtaaaccgt gtcggtcaac ggtgtcggga tctcgaccaa aatgcaaatg ctacaaaaaa 2100 ataaaaatca atagaaaagc gctaaaagtc tgcttgatag cggacatgtt cggtaacgat 2160 gcagaattga gcaaattaat ttggatgcta atttttacca acaaaacgta cgtgtcgacc 2220 acgttgattc gcacgaacag cgaatttgtt aaccaacacg gagagttttt ttttaaagaa 2280 cacaacaaaa ttattcagta cttgtatcaa acaattcaca aaatcgaata cgttgacatg 2340 ttgatggaca agtttaatga taaacgtttg tttttaaccg agttgcgcga tgatgttgca 2400 cgcgaacccg atgtccagtt tgaagaatcg gataacattt gcaaatttta cacgcaccac 2460 accgacgcgt taataatctt gaaaaagtac aatgtgtggt gggacaaaat catattggcg 2520 cgcagcacgg acgatttgcc cacctggctg actcgattct acatgcgcat aatcatgtcc 2580 aaaatggact tgaaagaata ctcgtacaat tatttaaaaa agattgtcga aggctatttg 2640 tactttaaac gtttcaccaa ttttaatcac gcaaacgcga ttatgttgat gcatttcgcc 2700 gccagtctag ccattcccgt agattacgga aaaaaagcta tttacatgcc cggcgaaccg 2760 ggttcgggca agtcgtcgtt ctttgagctg ttggactatt tggtgttgat gcacaagttt 2820 gacgatgaca accacagcgg agagtccaac aaggaaacga gcgacaaaga agtgagcaaa 2880 ctcaattcgc agctgtacgt tatcaacgag ttgaaacaat gcagcgagtc gtactttaag 2940 aagcatgccg attcgagcaa gagcgattcc aagagcagaa aataccaagg gctgctaaag 3000 tatgaagcca actacaaaat gttaattgtt aacaacaagc cgctgtacgt ggacgattat 3060 gacgacggtg tacaagacag atttttgatt gtttacacga accataagtt tgtagacagc 3120 gtcaagtttg ccggctccgt gtacgaacac atcaagtcca aacagtttcc gatagaaagc 3180 atgtattacg agtcgcttgt gacgccggtg cgtttgtttt tgtcgcacgt actaatgtac 3240 agacgcgacc ccaaaacggg ctttgtagta tacaaaacgc tgctgagcaa cgaccctatg 3300 cataagcaca acctaatgtg cttgagcacc aacaatagtc cgctgtatgc gctcatttac 3360 attctaaaca ttaaaacggt gcgcagtgcc actattacaa taggagagga caaaatggag 3420 gagatgatag gtattgccgt gcagcatttg aaaaactttt tgcacccctc gtttgttcag 3480 tacaattata aaaagaatat aaacgcaagt agttcaaagt cttttgtatt taatgaacaa 3540 gttttattac agcaaattaa aaataaattt aaaaataatt acaataaaac tacaaatgtg 3600 ttttataaca tgacaatggc gttgaacaga aacgatttga acactagtgt accaaatttt 3660 gtatgttaa 3669

[Brief description of drawings]

FIG. 1 is a drawing showing infectious titers of BANPV, BmNPV and AcNPV.

FIG. 2 is a drawing showing luciferase luminescence in silkworm larvae by BANPV and BmNPV.

FIG. 3 is a drawing showing GFP expression levels in silkworm larvae by BANPV and BmNPV.

FIG. 4 is a drawing showing the expression levels of cysteine proteases of BANPV and BmNPV in culture extract samples.

FIG. 5: BANPV and Bm in body fluid samples
It is a figure which shows the expression level of the cysteine protease of NPV.

FIG. 6 is a diagram showing a structural schematic diagram of gp64 of BANPV and a DNA helicase gene.

FIG. 7-a: BANPV, BANPV and AcNP
It is drawing which shows the base sequence of the DNA helicase gene of V.

FIG. 7-b: BANPV, BANPV and AcNP
It is drawing which shows the base sequence of the DNA helicase gene of V.

FIG. 7-c: BANPV, BANPV and AcNP
It is drawing which shows the base sequence of the DNA helicase gene of V.

FIG. 7-d: BANPV, BANPV and AcNP
It is drawing which shows the base sequence of the DNA helicase gene of V.

FIG. 7-e: BANPV, BANPV and AcNP
It is drawing which shows the base sequence of the DNA helicase gene of V.

FIG. 7-f: BANPV, BANPV and AcNP
It is drawing which shows the base sequence of the DNA helicase gene of V.

FIG. 7-g: BANPV, BANPV and AcNP
It is drawing which shows the base sequence of the DNA helicase gene of V.

Figure 8-a: BANPV, BANPV and AcNP.
It is a figure which shows the amino acid sequence deduced from the DNA helicase gene of V.

FIG. 8-b BANPV, BANPV and AcNP
It is a figure which shows the amino acid sequence deduced from the DNA helicase gene of V.

FIG. 8-c: BANPV, BANPV and AcNP
It is a figure which shows the amino acid sequence deduced from the DNA helicase gene of V. that's all

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Teruyuki Koyama             1567 Shimookutomi, Sayama City, Saitama Prefecture F-term (reference) 4B024 AA20 BA80 CA01 DA02 EA02                       FA01 FA20 GA11 HA01                 4B064 AG01 CA10 CA19 CC24                 4B065 AA90X AA95Y BA01 CA24

Claims (5)

[Claims] 1. A nuclear polyhedrosis virus (hereinafter, referred to as “BmNPV”) having a silkworm moth as a host, and a nuclear polyhedrosis virus having a moth insect as a host (hereinafter, “AcNP”).
A recombinant baculovirus (hereinafter referred to as "BAN") obtained by homologous recombination of V.
PV ”). (1) It is possible to infect and proliferate both in the living body of Bombycidae and Bombycidae and their cultured cells. (2) The virus titer shown when infected with the living body and cultured cells of Bombycidae insects is 10 times higher than that of wild-type BmNPV. (3) Expression of cysteine protease is wild type BmNP
About 72 hours later than V. 2. The structure of the viral constituent protein gp64 and the DNA helicase gene is Bm.
The BANPV according to claim 1, which is a mosaic of NPV-derived genes and AcNPV-derived genes. 3. A BANPV in which a part of a viral constituent protein has a DNA sequence as shown in SEQ ID NO: 1. 4. A BAN having the DNA sequence of SEQ ID NO: 1.
Genomic DNA of PV. 5. A recombinant BANPV is obtained by introducing the genomic DNA of BANPV according to claim 1 and a transfer vector for recombination incorporating a foreign gene into cultured cells of Bombyx mori or Insectidae. Then, the recombinant BANPV is infected with a living body of Bombyx mori or moths or cultured cells thereof to express the protein encoded by the foreign gene introduced into the recombinant BANPV and collect the protein. A method for obtaining a protein characterized by: