JP5313529B2 - Baculovirus transfer vector, recombinant baculovirus, recombinant baculovirus expression system, and method for producing recombinant protein using them - Google Patents

Description

  The present invention relates to a baculovirus transfer vector, a recombinant baculovirus obtained using the baculovirus transfer vector, a recombinant baculovirus expression system using the same, and a method for producing a recombinant protein.

  Production of useful proteins using gene recombination techniques is a widely used technique today, and a baculovirus expression system is one of the techniques.

  In this baculovirus expression system, a baculovirus having a coding sequence in which a foreign gene is inserted instead of polh after the promoter of polyhedrin (AcMNPV orf8; BmNPV orf1: hereinafter referred to as “polh”), which is a non-essential gene for baculovirus. A foreign gene expression virus is produced by homologous recombination of a recombinant vector with baculovirus (Patent Document 1). Similar to this polh, a promoter derived from the p10 (AcMNPV orf137; BmNPV orf114) gene is often used.

  The baculovirus genes are expressed one after another while being adjusted over time during the cycles after the first phase among the four different viral replication cycles (Non-patent Document 1). Therefore, during the course of expression, different baculovirus genes are expressed in the immediate early (immediate-early phase), late early (delayed-early phase), late phase (late phase), and late phase (very late phase), depending on the viral replication cycle. ).

  The polh gene and the p10 gene are genes classified in the last stage. Thus, transcription from terminal promoters such as polh, p10 would require a large number of previously unknown viral products as well as cell products. For this reason, the expression of a foreign gene using the last-stage promoter, which is a conventional technique, is performed after the virus infection has sufficiently progressed. In addition, these terminal promoters are characterized by rapid protein expression in a short period of time.

  However, it is known that a rapid and rapid expression of a protein causes the formation of aggregates. For this reason, even if the amount of protein expression is large, the amount of active target protein is often very low. The formation of such aggregates is thought to be caused by the fact that the translated polypeptide chain is entangled with other polypeptide chains by intermolecular interactions at the intermediate stage before folding into the correct conformation. Finally, it is thought that a huge insoluble complex is formed. That is, it is speculated that rapid protein expression in a short time increases the chance of contact between polypeptides, promotes entanglement, and leads to a state in which aggregate formation is likely to occur.

  In order to solve this problem, various methods have been developed in other expression systems (Takara Bio Cold Shock Promoter; Patent Document 2, etc.), but the same contrivance has not been successful in the baculovirus expression system. In addition, when a foreign gene is expressed by a terminal expression promoter such as a polh promoter, it will be expressed after the virus infection and propagation have sufficiently progressed, and the protein quality control function and expression ability inherent in the cell are There is also a problem that it cannot be expected sufficiently (Non-Patent Document 2).

  The applicants have traditionally provided consigned protein production services using a baculovirus expression system. In this project, the soluble foreign protein contained in the protein was purified from a sample that exhibited significant aggregation of the foreign protein. In this case, the company has experienced many cases where it is unstable and agglomerates. On the other hand, when purified from samples with little aggregation, we have also experienced relatively good stability of the purified product, and there is some relationship between the amount of aggregation and the quality of the protein produced in the cell. I understood that.

  In order to solve these problems, the use of a promoter of the baculovirus pre-early gene ee-1 (AcMNPV orf147; BmNPV orf123) or a baculovirus post-early gene 39k (AcMNPV orf36; BmNPV orf27) has been studied. However, when used in a baculovirus expression system, the expression level was not sufficient.

  In addition, the expression level is increased by placing a baculovirus homologous DNA region (hereinafter abbreviated as “HRs”) known as an enhancer for the early and late early promoters upstream of the 39k promoter. Although it is known (Non-patent Document 3), it is mainly used for transformation in cells. These HRs have an enhancer function, a palindrome centered on an EcoRI recognition sequence is conserved (Non-patent Document 4), and it is considered that the ie-1 gene product binds to this region. In addition, it is known that the expression level increases only with the preserved palindrome, and is more prominent with HR5 (6 repetitions) (Non-patent Document 5).

  Furthermore, there is an example (pAcJP1, currently not commercially available) using a promoter of the baculovirus late gene vp39 (AcMNPV orf89; BmNPV orf72). Although the expression level is sufficient, it is inferior to the polh promoter. No longer used.

  Furthermore, a hybrid promoter with a polh promoter has been developed to improve this (Non-patent Document 6), but the problem of intracellular quality control has not been solved. Although gp64 (AcMNPV orf128; BmNPV orf105) which is a late gene but slightly expressed in the early stage as well as vp39 is sometimes used, it is known that the expression level is small and the expression level is increased by HRs. (Non-Patent Document 7), but not enough.

  From the above technical background, a baculovirus expression vector that expresses a sufficient amount of protein while suppressing aggregation has been demanded.

U.S. Pat. No. 4,745,051 JP 2006-340729 A JP 2003-52371 A P.D.Friesen and L.K.Miller, Curr.Top.Microbiol.Immunol., 131: 31 49 (1986) X.Du, S.M.Thiem (1997) J. Virol. 71: 7866-7872 L.A.Guarino, M.D.Summers, J.Virol., 60: 215-223 (1986b) L.A.Guarino, M.A.Gonzalez, M.D.Summers, J.Virol., 60: 224-229 (1986) V. Olson, J. Wetter, P. Friesen, J. Virol., 77: 5668-5677 (2003) Thiem et al., Gene 91 (1): p87 (1990) ZHOU et al., Acta Biochimica et biophysica sinica 2003, 35 (1): p18

  Accordingly, an object of the present invention is to provide a technique for expressing a protein in an appropriate amount while suppressing the occurrence of aggregation in a baculovirus expression system so that the target protein can be obtained in an active form.

  The present inventors first expressed baculovirus expression using 350 bp upstream of the start codon including the promoter regions of ie-1, vp39 and gp64 genes, which are representative of genes having early, late, and late early and late expression. The expression of green florescent protein (hereinafter referred to as “GFP”) in the system was examined over time using BmN cells. As a result, in the vp39 promoter, the fluorescence almost equivalent to that of the polh promoter was confirmed, but it was rapidly expressed in the later stage, and the ee-1 and gp64 showed slightly less fluorescence than the polh promoter as was known so far. I could only confirm.

  Therefore, in order to increase the expression level, among HRs known as enhancers, the combination of HR3, each promoter region, their distances, and polh 5′UTR known as a translation promoting sequence was examined, and GFP As a result of investigation on the expression, only a slight increase was observed in ie-1 and gp64. On the other hand, the combination of HR3, which is an HRs-conserved balindrome sequence, and vp39 does not change the expression level so much, but it has been found that protein expression is accelerated and aggregation is suppressed, and the present invention has been completed. .

That is, the present invention is a baculovirus transfer vector comprising the following constitutions b) and c) from left to right.
b) Enhancer DNA region including at least one baculovirus HRs conserved palindromic sequence described in SEQ ID NO: 1 c) DNA region including a promoter region derived from the baculovirus vp39 gene

  The present invention also relates to a recombinant baculovirus in which a foreign gene is introduced by the baculovirus transfer vector.

  Furthermore, the present invention is a recombinant baculovirus expression system comprising lepidopteran insect cells infected with the recombinant baculovirus.

  Furthermore, the present invention is a method for producing a recombinant protein and / or polypeptide comprising the above recombinant baculovirus expression system.

  According to the present invention, the protein or peptide can be expressed in an appropriate amount by advancing the expression time of the protein or peptide, so that foreign protein or polypeptide can be produced with relatively little aggregation. it can.

In order to construct the recombinant baculovirus of the present invention, it is first necessary to construct a baculovirus transfer vector. This baculovirus transfer vector consists of the following b) and c) from left to right:
b) Enhancer DNA region containing at least one baculovirus HRs conserved palindromic sequence (TTTACNNGTAGAATTCTACNNGTAAA: N represents A, C, G or T) described in SEQ ID NO: 1 c) derived from baculovirus vp39 gene It contains two DNA regions including a promoter region and can be obtained by the following method. In the present specification, the direction from left to right is the direction in which transcription proceeds from the promoter region derived from the baculovirus vp39 gene. Moreover, the baculovirus HRs conserved palindromic sequence included in the enhancer DNA region may be reversed.

  The baculovirus transfer vector is obtained by cloning an enhancer DNA region containing at least one HRs conserved palindromic sequence described in SEQ ID NO: 1 by means of PCR or the like using baculovirus genomic DNA as a template according to a conventional method, and then the same Can be constructed by cloning the region containing the vp39 promoter. As an enhancer DNA region including at least one of the above conserved palindromic sequences, for example, a DNA sequence having a homology of 80% or more, preferably 90% or more with a baculovirus HRs consensus sequence (CTAAACTCGCTTTACGAGTAGAATTCTACTTGTAAAACACAATCAAG: SEQ ID NO: 2) , DNA regions including baculovirus HRs consensus sequences, DNA regions including baculovirus HR3 (SEQ ID NO: 29), and the like. The DNA region including the DNA sequence having the above homology with the specific baculovirus HRs common sequence and the DNA region including the baculovirus HRs common sequence include the DNA sequence and the HRs common sequence itself or these sequences and both ends thereof at 100 bp. Or a sequence obtained by repeating these sequences a plurality of times. Examples of the DNA region encompassing baculovirus HR3 include baculovirus HR3 itself or HR3Long fragment (SEQ ID NO: 30) consisting of baculovirus HR3 and about 700 bp downstream thereof, and among these, HR3Long fragment is particularly preferred.

  On the other hand, as a DNA region including a promoter region derived from the baculovirus vp39 gene, the baculovirus vp39 gene start codon upstream 56-60 bp and the baculovirus late promoter motif (DTAAG: where D is A, Including T) or G). As a specific DNA region including a promoter region derived from a baculovirus vp39 gene, vp39pro (SEQ ID NO: 31) comprising a baculovirus vp39 gene start codon upstream of −1 to −110 bp, preferably −1 to −350 bp is preferable.

  In the recombinant baculovirus of the present invention, a DNA region including a cloning restriction enzyme cleavage site into which a DNA sequence encoding a desired protein or polypeptide can be inserted downstream of the sequence c). desirable.

  Examples of the DNA region including a cloning restriction enzyme cleavage site into which a DNA sequence encoding the desired protein or polypeptide can be inserted include a multi-cloning cassette capable of inserting a desired foreign gene in an appropriate direction. The sequence of this multi-cloning cassette can be obtained from several companies (Takara Bio, Toyobo, etc.), or can be synthesized using a DNA synthesizer. Both methods are used on a daily basis, and the use of these multiple cloning cassette units is also known in the art.

  Further, it is desirable that the downstream side of the multicloning cassette is followed by a 3 'terminal region containing a polyA signal, preferably a polyA signal derived from a polyh gene. This polyA signal can also be obtained from commercially available vectors, and can also be synthesized.

As a preferred embodiment of the baculovirus transfer vector, the following can be mentioned. That is, on the upstream side of the enhancer side, the following DNA sequence a)
a) A viral DNA sequence of baculovirus encoding the 5 ′ terminal flanking sequence of the baculovirus non-essential gene site is bound, and the following sequence e) is further provided downstream of the promoter side:
e) A viral DNA sequence of baculovirus encoding the 3 ′ terminal flanking sequence of the baculovirus non-essential gene site may be ligated.

  As a suitable example of the baculovirus non-essential gene site of the sequences a) and e), polh can be mentioned. That is, the baculovirus DNA that can replace the polh gene region by homologous recombination is easily available. Examples of such baculovirus DNA include BmNPV DNA derived from BmNPV that infects silkworm organisms and cultured cells established from silkworms (BmN, BoMo, etc.), 1 Cut (Nippon Agricultural Industries), etc., Spodoptera frugiperda Derived from AcMNPV that infects living organisms such as Spodoptera frugiperda and Trichoplusia ni and insect culture cells established from them (Sf-9, Sf-21, Tn-5, Tn-368, etc.) Examples include BacVector-1000 Triple Cut DNA (Novagen), BacVector-2000 Triple Cut DNA (Novagen), and other ABv baculoviruses (Katakura Kogyo: Patent Document 3) that infect both BmNPV and AcMNPV hosts.

  The 5'-end flanking sequence and the 3'-end flanking sequence of the sequences a) and e) can be interchanged. That is, in the transfer vector, a 5′-end flanking sequence and a 3′-end flanking sequence may be present so that homologous recombination can be successfully performed, and sequences b) and c) sandwiched between them. The constructed sequence may be reversed. Specific examples of the 5′-end flanking sequence and the 3′-end flanking sequence of a) and e) include 500 bp to 5 kbp further upstream, preferably 2 bp including the termination codon of the essential gene ref-2 upstream of polh. A sequence containing ˜3 kbp and a sequence containing 500 bp to 5 kbp, preferably 2 to 3 kbp, including the stop codon of the essential gene orf1629 downstream of polh and further downstream (the direction of CDS of orf1629 is opposite to ref-2) A combination etc. are mentioned. In addition, it is preferable that the sequence further includes a region containing a polyA signal of each gene (30 to 70 bp after the termination codon).

  Another preferred example of the baculovirus non-essential gene site of the sequences a) and e) is a baculovirus transfer vector in which Tn7R and Tn7L are inserted in place of the flanking region. Such a vector can be used in a Bac-to-Bac system (Invitrogen) to obtain a recombinant baculovirus. In this baculovirus transfer vector, Tn7R and Tn7L may be interchanged.

  In the baculovirus transfer vector, a DNA region of a foreign gene / foreign DNA encoding a desired protein or polypeptide (hereinafter collectively referred to as “protein”) can be incorporated as a sequence d) downstream of the sequence c). it can. The exogenous genes / DNAs that express this protein include prokaryotic genes / DNA including viruses-derived genes / DNA and eukaryotic genes / DNA. Preferred examples of the gene / DNA include antibody genes. When a cloning restriction enzyme cleavage site is incorporated in the vector, the DNA region of the foreign gene / foreign DNA to be encoded can be incorporated. By homologous recombination of the viral transfer vector thus made with viral DNA and infecting the host cell with the obtained recombinant baculovirus, it becomes possible to express a desired protein in the cell. In addition, when expressing an antibody as a protein, not only the whole antibody gene is used to infect a host cell by the above-described method and the antibody is expressed in the cell, but also the light chain gene and heavy chain gene of the antibody. Can be incorporated into separate baculoviruses, they can be mixed infecting host cells and antibodies can be expressed in the cells.

An optimal embodiment of the baculovirus transfer vector is pMVPLR (SEQ ID NO: 32), which has the following constitution from left to right.
1) DNA sequence derived from baculovirus BmNPV (T3 strain), bro-e, orf
133, region containing orf134, ref-2 gene (5582-7692)
2) A DNA sequence derived from baculovirus BmNPV (T3 strain) that does not encode a protein and upstream of the polyhedrin gene (7625 to 7692)
3) A DNA sequence derived from baculovirus BmNPV (T3 strain), which is HR3 (7698-
8733th) DNA region (HR3 Long fragment: 7698-9394)
Th)
4) DNA region including promoter region of vp39 gene of baculovirus BmNPV (T3 strain) (vp39pro: 9442 to 9791)
5) A DNA sequence derived from baculovirus BmNPV (T3 strain), which contains the orf1629, PK1, and orf4 genes from the downstream of the polyhedrin gene stop codon (positions 98 to 2885).

  In homologous recombination between a viral transfer vector and viral DNA, it is desirable to use a linearized baculovirus DNA in order to increase the recombination efficiency. As a restriction enzyme for digesting or cleaving baculovirus DNA used for this purpose, Eco81I can be mentioned, and digestion and cleavage sites in one baculovirus DNA are preferably 1 to 3 sites.

  Furthermore, as a host for producing a protein by the recombinant baculovirus of the present invention, Spodoptera frugiperda, Bombyx mori, Bombyx mandarina, Heliosis virulence (Heliothis virescens), Heliothis zea, Mamestra brassicas, Estigmene acrea or Trichoplusia ni, including lepidopterous insect cells, culture It may be a cell or a living body such as a larva or a moth.

  The culture time of the host cell infected with the recombinant baculovirus differs depending on the type of protein to be produced, etc., but generally 2 to 5 days for cultured cells, and 5 for larvae or pupae. It is about 7 days from.

  As an optimal embodiment of the present invention, a recombinant ABv baculovirus constructed based on ABv baculovirus and incorporating a desired foreign gene is used to infect silkworm cultured cells or larvae and pupae, and the desired foreign The method of expressing a gene can be mentioned.

  In the above-described optimal embodiment, particularly when an antibody gene is expressed as a foreign gene, superior effects such as an increase in the expression level and suppression of the shape of the aggregate are achieved as compared with the case where expression is performed using a conventional promoter. Is obtained.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. It will be readily understood by those skilled in the art that various modifications and changes in the configuration and implementation of the present invention can be made based on the contents described in the present specification, and these are within the spirit and scope of the present invention. . The starting materials and methods used in the following examples are as follows.

(Acquisition of promoter)
Using the genomic DNA of the BmNPV-T3 strain (Accession No. NC — 001962) as a template DNA, the HR3 and vp39 promoters and the ie-1 and gp64 promoters used for comparative studies were obtained by PCR.

(Acquisition of linearized baculovirus DNA)
The linearized baculovirus DNA for producing the recombinant virus was ABv baculovirus (Katakura Kogyo; JP 2003-52371). More specifically, silkworm larvae on the first day of age 5 were subjected to low temperature anesthesia with ice water or the like for about 10 minutes, and ABx baculovirus solution having a concentration of 1 × 10 ⁶ cells / 100 μl was attached to an injection needle such as G-26. Inject subcutaneously using a syringe. After that, baculovirus particles were grown by breeding for 7 days at 25 ° C and 45% humidity using an artificial feed for silkworms 3 (Morus: Katakura Kogyo). Viral particles were obtained.

  The obtained baculovirus particles were subjected to proteinase K digestion and phenol extraction to purify genomic DNA. Furthermore, a restriction enzyme was allowed to act on this to digest and cleave to obtain linearized baculovirus DNA. For example, ABv baculovirus genomic DNA is digested and cleaved by reacting 200 μg of baculovirus DNA with 30 units of restriction enzyme Eco81I at 37 ° C. for 20 hours.

(General method)
Among the operations described in this specification, basic operations such as plasmid preparation and restriction enzyme digestion are described in “Molecular Cloning: A Laboratory Manual 3rd ed.” (2001, issued by Cold Spring Harbor Laboratory). According to the method. Restriction enzymes and other enzymes used for the construction of the plasmid were obtained from either Takara Bio or Toyobo.

  In addition, unless otherwise specified, plasmids were constructed by ligating 5 fmol of vector and 15 fmol of insert, adding the same amount (5 to 10 μL) of solution I solution of ligation kit (Takara Bio), and at 16 ° C. for 1 hour. Reacted. All reaction solutions were used to transform E. coli DH5α and selected on LB medium containing ampicillin. After selecting the clone, the plasmid was purified. Using this BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems), this purified product was subjected to a sequencing reaction under the following conditions and analyzed with DNA Sequencer 3100-avant (Applied Biosystems).

<Sequence reaction>
1.96.0 ° C. 1 minute 2.96.0 ° C. 10 seconds 3.50.0 ° C. 5 seconds 4.60.0 ° C. 4 minutes Repeat 5.2 to 4 25 times

  Unless otherwise specified, PCR was performed using KOD-Plus (Toyobo) under the following conditions with DNA-Engine (Bio-Rad), and then purified with QIAquick PCR Purification kit (Qiagen).

<PCR reaction>
1.94.0 ° C. 2 minutes 2.94.0 ° C. 15 seconds 3.50.0 ° C. 30 seconds 4.68.0 ° C. 1 minute per 1 kbp 5.2-4 repeated 30 times

Recombinant baculovirus was prepared by co-transfection of linearized ABv baculovirus DNA and a foreign gene-transferred baculovirus transfer vector. That is, first, BmN cells were prepared by static culture in about 1 × 10 ⁶ monolayers using a 35 mm cell culture dish. 0.2 μg of linearized ABv baculovirus DNA (Katakura Kogyo) and 0.5 μg of a foreign gene-transferred baculovirus transfer vector were mixed with 100 μl of TC-100 (serum-free) in a 1.5 ml tube, and then at room temperature for 15 minutes. Left to stand. 4 μl of a cationic lipid reagent (Lipofectin; Invitrogen) was mixed with 100 μl of TC-100 (serum-free) and allowed to stand at room temperature for 15 minutes. The two solutions were then mixed and further allowed to stand at room temperature for 15 minutes before adding 800 μl of TC-100 (serum free). This mixed solution is added to BmN cells prepared as a monolayer in a 35 mm cell culture dish. After culturing at 25 ° C. for 16 hours, the mixed solution is removed, and 2 ml of new TC-100 (containing 10% FBS) is added. And static culture at 25 ° C. for 7 days. The culture supernatant was used as a recombinant virus stock solution.

Isolation of the exogenous recombinant ABv baculovirus from the recombinant virus stock solution was performed by the method of “A Laboratory Guide” (LA KING and RDPOSSE (1992)). That is, a 96-well plate was prepared in which BmN cells were cultured at 1.5 × 10 ³ cells / well / 50 μl TC-100 (10% FBS). A virus diluted solution obtained by diluting the recombinant virus stock solution into 10 ⁻³ , 10 ⁻⁴ , 10 ⁻⁵ , 10 ⁻⁶ , 10 ⁻⁷ , and 10 ⁻⁸ using TC-100 (10% FBS), 50 μl per well of each plate was mixed and infected. These plates were sealed and dried at 25 ° C. to prevent drying. The selection of the recombinant virus was performed on the seventh day of infection by confirming the absence of polyhedron formation under a light microscope.

Example 1
Investigation of expression level over time by GFP (1) Construction of plasmid for preparation of baculovirus transfer vector for comparison (pMProXX):
As a starting material for constructing the baculovirus transfer vector of the present invention, pM000001 (Katakura Industry, see FIG. 1, hereinafter abbreviated as “pM01”) was used. This vector was also used as a conventional baculovirus expression system, that is, as a positive control group. Since this includes a polh promoter, this was removed by the following method, and a plurality of restriction enzyme recognition sites useful for construction were further introduced.

  That is, PCR was performed using pM01 as a template, primer 1 (5′-aacaatgtaccgcgcggcgg-3 ′) and primer 2 (5′-agatcttaagctagcaagcatatggccatggtatttccatttgtatcgttaataaaaaac-3 ′) to obtain a DNA fragment. This DNA fragment was digested with restriction enzymes BstXI and BglII. On the other hand, pM01 was digested with BstXI and BglII, and the cut surface was dephosphorylated with alkaline phosphatase. PM01 linearized with the DNA fragment was ligated to transform E. coli. This transformant clone was plasmid purified and pM. The base sequence was confirmed using a rev (5'-caacgcacagaatctaacgc-3 ') primer, and pMProXX was selected. The outline of plasmid construction from (2) to (5) following is shown in FIG.

(2) Acquisition of HR3 and integration into vector Using virus genomic DNA of BmNPV-T3 strain (Accession No. NC — 001962) as a template, primer 3 (5′-ccatggaatattagacaacaaagatttattttattcatg-3 ′) and primer 4 (5′-aatattacgtccctgccagaa-3 ') Was used to obtain a PCR fragment (hereinafter referred to as "HR3Long fragment"). The HR3Long fragment was ligated with pT7-Blue T-Vector (Takara Bio) with Ex-Taq and dATP protruding one adenine at the end, and E. coli was transformed.

  This transformant clone was plasmid purified and M13. Selection was performed by confirming the base sequence using a rev (5'-tcacacaggaaacagctatg-3 ') primer. At this time, HR3Long acquired two types, 5 ′ → 3 ′ direction (HR3LR / pT7) and 3 ′ → 5 ′ direction (HR3LL / pT7).

  The above HR3LR / pT7 was digested with restriction enzymes NcoI and NheI to excise the HR3Long fragment. On the other hand, pMProXX prepared in (1) above was digested with restriction enzymes NcoI and NheI, and the cut surface was dephosphorylated with alkaline phosphatase. PMProXX linearized with the HR3Long fragment was ligated to transform E. coli. A clone of this transformant (pMProLR) was plasmid purified and pM. Selection was performed by confirming the base sequence using a rev (5'-caacgcacagaatctaacgc-3 ') primer.

  Similarly, the above HR3LL / pT7 was digested with restriction enzymes NcoI and BamHI. This was blunt-ended with a DNA branding kit (Takara Bio) to cut out the HR3 Long fragment. On the other hand, pMProXX prepared in (1) was digested with restriction enzymes NcoI and NdeI. This was similarly blunt ended, and the cut surface was dephosphorylated. PMProXX linearized with the HR3Long fragment was ligated to transform E. coli. This transformant clone (pMProLL) was plasmid purified and pM. Selection was performed by confirming the base sequence using a rev (5'-caacgcacagaatctaacgc-3 ') primer.

  The above HR3LR / pT7 was digested with restriction enzymes NcoI and NdeI, and a DNA fragment was excised to obtain baculovirus HR3 (hereinafter referred to as “HR3 Short fragment”). On the other hand, pMProXX prepared in (1) was digested with restriction enzymes NcoI and NdeI, and the cut surface was dephosphorylated. PMProXX linearized with the HR3 Short fragment was ligated to transform E. coli. A clone of this transformant (pMProSR) was plasmid purified and pM. Selection was performed by confirming the base sequence using a rev (5'-caacgcacagaatctaacgc-3 ') primer.

  Similarly, HR3LL / pT7 was digested with the restriction enzyme NcoI, the cut surface was blunted with a DNA branding kit (Takara Bio), and further digested with the restriction enzyme NdeI. On the other hand, pMProXX prepared in (1) is digested with restriction enzyme NheI, the cut surface is blunted with a DNA branding kit, further digested with restriction enzyme NdeI, and the cut surface is dephosphorylated with alkaline phosphatase. did. PMProXX linearized with the HR3 Short fragment was ligated to transform E. coli. A clone of this transformant (pMProSL) was plasmid purified and pM. Selection was performed by confirming the base sequence using a rev (5'-caacgcacagaatctaacgc-3 ') primer.

(3) Acquisition of baculovirus-derived promoters with different expression times and integration into vectors Using the viral genomic DNA of BmNPV-T3 strain (Accession No. NC_001962) as a template, primer 5 (5'-gctagcatcccaacggcgcagtg-3 ') and primer 6 PCR was performed using (5′-agatcttaagagtcgtttggttgttcacgat-3 ′) to obtain an ie-1 promoter fragment (350 bp upstream from the ee-1 start codon). Similarly, PCR was performed using primer 7 (5′-gctagcttggtaaacgtacactttaattattttac-3 ′) and primer 8 (5′-agatcttaagattgttgccgttataaatatggac-3 ′) to obtain a vp39 promoter fragment. PCR was performed using primer 9 (5′-gctagctttaaataaaccaaacacatgaatataatttt-3 ′) and primer 10 (5′-agatcttaagtgaggcatcttatatacccg-3 ′) to obtain a gp64 promoter fragment. These three types of promoter fragments were made to project one adenine at the end by Ex-Taq and dATP, and ligated with pT7-Blue T-Vector (Takara Bio), respectively, to transform Escherichia coli.

  Plasmid clones of these transformants (IE / pT7, VP / pT7, GP / pT7) were purified, and M13. Selection was performed by confirming the base sequence using a rev (5'-tcacacaggaaacagctatg-3 ') primer. These plasmids were digested with restriction enzymes NheI and BglII, and the respective promoter fragments (IE, VP, GP) were excised.

  The polh promoter fragment (PH) synthesized only the core sequence. That is, 1 μM synthetic DNA 1 (5′-CTAGCaaataataaccatctcgcaaataataCTTAA-3 ′) and 1 μM synthetic DNA 2 (5′-GATCTTAAGtatttatttgcgagatggttattatttG-3 ′) are mixed in 50 mM NaCl, heat-denatured, and then slowly cooled to form double-stranded DNA. I let you. Furthermore, the 5 'end was phosphorylated by T4 polynucleotide kinase.

  On the other hand, the four types of plasmids (pMProLR, pMProLL, pMProSR, and pMProLL) prepared in (1) above and (2) were digested with the restriction enzymes NheI and BglII, and the cut surface was removed with alkaline phosphatase. Phosphorylated. The above four types of promoter fragments (IE, VP, GP, PH) and a total of five types of linearized plasmids were ligated in a total of 20 types to transform E. coli.

  This transformant clone was plasmid purified and pM. Selection was performed by confirming the base sequence using a rev (5'-caacgcacagaatctaacgc-3 ') primer.

(4) Acquisition of Polh gene 5′UTR and integration into vector Using pM01 (Katakura Kogyo) as a template, primer 11 (5′-cttaagaagtattttactgttttcgtaacagtt-3 ′) and primer 12 (5′-atagtccagccatcggtttg-3 ′) PCR was performed to obtain a polh gene 5′UTR fragment. This 5′UTR fragment was made to project one adenine at the end by Ex-Taq and dATP, and ligated with pT7-Blue T-Vector to transform Escherichia coli. This transformant clone (5UTR / pT7) was plasmid purified, and M13. Selection was performed by confirming the base sequence using a rev (5'-tcacacaggaaacagctatg-3 ') primer.

  The 5UTR / pT7 was digested with restriction enzymes AflII and SnaBI, and the polh gene 5'UTR fragment was excised. On the other hand, the 20 types of plasmids prepared in (3) were digested with restriction enzymes AflII and SnaBI, and the cut surfaces were dephosphorylated with alkaline phosphatase. The 5 'UTR fragment and 20 types of linearized plasmids were ligated to transform E. coli. This transformant clone was plasmid purified and pM. Selection was performed by confirming the base sequence using a rev (5'-caacgcacagaatctaacgc-3 ') primer. Table 1 shows the names of baculovirus transfer vectors used in the construction of these plasmids, promoters and enhancers used, insertion directions, and the like.

(5) Construction of baculovirus transfer vector inserted with Aequorea jellyfish green fluorescent protein gene The Aequorea jellyfish green fluorescent protein (wild type, hereinafter referred to as “GFP”) gene (Accession No. M62653) was transferred to restriction enzymes BglII and XbaI. The GFP gene fragment was excised. On the other hand, 40 types of plasmids prepared in (3) and (4) and pM01 (Katakura Kogyo) as a positive control were digested with restriction enzymes BglII and XbaI, and the cut surface was dephosphorylated with alkaline phosphatase. The GFP gene fragment and a total of 41 types of linearized plasmids were ligated to transform E. coli. This transformant clone was plasmid purified and pM. Selection was performed by confirming the base sequence using a rev (5'-caacgcacagaatctaacgc-3 ') primer.

(6) Production of Promoter and HRs Recombinant Baculovirus The 41 types of GFP-introduced baculovirus transfer vectors obtained in (5) above and pM01 (Katakura Kogyo Co., Ltd.) without GFP introduced as a negative control group were referred to as ABv baculovirus. The recombinant virus stock solution was obtained by performing co-transfection.

GFP recombinant ABv baculovirus was isolated from each virus stock solution obtained above by the method of “A Laboratory Guide” (LA KING and RD POSSE A Laboratry Guide (1992)). That is, a 96-well plate was prepared in which BmN cells were cultured at 1.5 × 10 ³ cells / well / 50 μl TC-100 (10% FBS). Virus prepared by diluting the recombinant virus stock solution into 10 ⁻³ , 10 ⁻⁴ , 10 ⁻⁵ , 10 ⁻⁶ , 10 ⁻⁷ , and 10 ⁻⁸ using TC-100 (10% FBS) The diluted solution was mixed and infected by 50 μl per well of each plate. These plates were sealed and dried at 25 ° C. to prevent drying. The selection of the recombinant virus was performed on the seventh day of infection by confirming the absence of polyhedron formation under a light microscope. Except for the negative control group, recombinant viruses showing fluorescence due to GFP protein were further selected and cloned. At this time, recombinant viruses could not be obtained for the two types (pVPXX5, pGPLL5).

(7) Each promoter and HR3 and polh5′UTR by GFP fluorescence measurement
Analysis of the effect of 96-well plates were prepared by culturing BmN cells at 1.5 × 10 ³ cells / well of 50 μl TC-100 (10% FBS) per well. The 40 types of GFP gene expression viruses obtained in the above (6) are m. o. i. Infected with = 5. Every 2 hours after infection, GFP fluorescence was measured over time under conditions of an excitation wavelength of 398 nm, a cutoff of 455 nm, and a measurement wavelength of 509 nm.

  The RFU of the negative control group was subtracted from the obtained fluorescence intensity (RFU) data, and the GFP expression curve of each recombinant virus was “Clin. Chem.”, 23, 112. According to the method of (Rodbard, D. and McClean, S.W. (1977)), it returned to the following 4 coefficient logistic curves using the logit transformation.

  The 4-coefficient logistic curve is used to represent a sigmoid curve appearing in an enzyme immunoassay (ELISA) calibration curve, an organism growth curve, a protein expression curve over time, and the like. The data obtained above regressed as follows: At this time, when A = 0, D represents the maximum expression level (RFU), C represents the time (h) for hitting the S-shaped inflection point, and B represents the S-shaped curvature.

The regression equation used is as follows.

  As a result, with only the promoters and polh core sequences of both the ie-1 and gp64 genes, the maximum expression level, that is, the coefficient D, is less than 10% of the conventional technique, that is, pM01, regardless of the presence or absence of addition of HR3 etc. I understood. Therefore, when these promoters were used alone or in combination with HR3 or the like, it was judged that they were inefficient as a means for protein production. On the other hand, when the vp39 promoter was used, since a relatively high expression level was observed, further investigation was conducted.

The formula of the above four coefficient logistic regression curve was modified as follows, and 10% and 90% expression times were calculated. Table 2 below shows the composition of the vectors in the order of 10% expression time.

  When the HR3 enhancer was inserted, the 10% expression time was faster in all cases compared to the vp39 promoter alone (pMVPXXX above). Moreover, it was found that polh 5'UTR hardly affects the expression time. Therefore, it was suggested that the effect of HR3 is to accelerate the onset time. In particular, it was shown that the expression time was early in pMVPLR. For easy understanding, the measured data and their regression curves were graphed together with the vp39 promoter alone (above pMVPXXX) and the conventional polh promoter (above pM01) in FIG.

  In the following examples, expression comparison was performed using these three types of baculovirus transfer vectors as a Gateway destination vector. That is, the control group as a conventional technique (polh promoter) is based on pM01, the control group for verifying the effect of baculovirus HR3 is based on pMVPXX, and the vector of the present invention is based on pMVPLR.

Example 2
(1) C-terminal 6xHis-tagged destination vectorization of pM01, pMVPXXX, pMVPLR pK0CHT15 (Katakura Industry: pM01 C-terminal 6xHis-tagged fusion protein expression vector) The digested surface was digested with EcoRI, and the cut surface was blunted with a DNA branding kit (Takara Bio). This was digested with the restriction enzyme EcoRV, and then the cut surface was dephosphorylated with alkaline phosphatase.

  25 ng of gateway reading frame cassette B (Invitrogen, hereinafter abbreviated as “RfB”) and 50 ng of the linearized plasmid were ligated, and the reaction solution was diluted 5 times. 1 μL was used to transform E. coli DB3.1 (Invitrogen). This chloramphenicol resistant transformant clone was plasmid purified and pM. The nucleotide sequence was confirmed by using the rev (5′-caacgcacagaatctaacgc-3 ′) primer and selected to obtain pM01-DestCH.

  pM01-DestCH was digested with restriction enzymes KpnI and SnaBI to excise the RfB fragment and the subsequent 6xHis-tagged sequence. On the other hand, the above pMVPXXX and pMVPLR were digested with restriction enzymes KpnI and SnaBI, and the cut surface was dephosphorylated with alkaline phosphatase. The 6xHis-tagged RfB fragment and the linearized plasmid were ligated to transform E. coli DB3.1 (Invitrogen). This chloramphenicol resistant transformant clone was plasmid purified and pM. The rev (5'-caacgcacagaatctaacgc-3 ') primer was used for selection by confirming the base sequence, which were designated as pMVPXXX-DestCH and pMVPLR-DestCH, respectively. Differences around these promoters are shown in FIG.

(2) Construction of each baculovirus transfer vector inserted with PDE4C (phosphodiesterase 4C) gene An entry clone vector containing PDE4C gene (Get Gateway entry clone from JBIC Outcome Consort I: No. FLJ38065AAAF), pM01-DestCH, pMVPXXX- An LR clonase reaction was performed with each destination vector of DestCH and pMVPLR-DestCH and Gateway LR clonase (Invitrogen) according to the manufacturer's manual. All reaction solutions were used to transform 80 μL of E. coli DH5α. This transformant clone was plasmid purified and pM. Selection was performed by confirming the base sequence using a rev (5'-caacgcacagaatctaacgc-3 ') primer. In addition, the name of the plasmid was changed as follows so that it could be easily compared. This replacement is the same in the following embodiments.

(3) Preparation of PDE4C genetically modified baculovirus The above three types of vectors were recombined with ABv baculovirus by co-transfection. Next, the virus-infected cells were collected, and a recombinant virus in which the expression of the His fusion protein was observed and the PDE4C gene was introduced into the virus genome was selected by Western blotting analysis using an anti-His antibody. And cloned.

(4) Recovery and analysis over time in pupae and pupae Using the cloned virus solution obtained above, 9 types of virus were inoculated into silkworm larvae on the first day of 5th day and pupae one day after hatching. Two animals were collected over time every 64, 72, 88, 96, 112, 120, 136, and 144 hours after virus infection and frozen in a freezer at -80 ° C.

  Tris buffered saline (TBS) is added to the collected silkworms and cocoons to a total volume of 30 mL, and a homogenizer (Hitachi) and an ultrasonic crusher (Vibracell) are added in this order together with one protease inhibitor cocktail Complete (Roche). Used and ground. This grinding liquid was centrifuged for 10 minutes at 3,000 rpm and 4 ° C. with a low speed centrifuge (Hitachi), and the supernatant was filtered with gauze. This filtrate was diluted 2-fold with TBS, and 1 mL was centrifuged for 1 hour at 45,000 rpm at 4 ° C. with an ultracentrifuge, optima MAX (Beckman), rotor TLA-100.3, and separated into a supernatant and a precipitate. . 1 mL of TBS was added to the precipitate and completely resuspended with an ultrasonic crusher (BRASON).

  The ground sample obtained as described above was separated by SDS-PAGE (Ato), and then the protein was transferred from the gel to a PVDF membrane (Pall) by a blotting apparatus (Ato). After the transfer, the protein-adsorbed PVDF membrane was blocked with T-TBS containing 1% skim milk (Wako Pure Chemical Industries) (TBS containing 0.1% Tween 20) and diluted 2,000 times as a primary antibody. Western blot analysis using anti-His rabbit antibody G-18 (Santa Cruz) and anti-rabbit IgG goat antibody ALP label (Nacalai Tesque) diluted 20,000 times as the secondary antibody was performed. Signal detection was performed by color development with BCIP-NBT reagent (Nacalai Tesque).

  The ultracentrifugation supernatant and the precipitation band at each recovery time were subjected to densitometry using ImageJ (NIH) to obtain the optical density. The expression in the pupae is shown in FIG. 5, and the expression in the larvae is shown in FIG. 6 in relation to the expression time and the densitometry result.

  As a result, it was shown that the optimal recovery time for each baculovirus transfer vector was 138 to 144 hours after infection. 5 and 6, it was shown that when the polh promoter (pM01), which is the prior art, was used (pM01), there were significantly more aggregates. In the case of expression in larvae (FIG. 6), when the vp39 promoter was used alone (pMVPXXX), the expression was slight. In order to confirm whether this is a phenomenon peculiar to larvae or due to a foreign protein, a similar investigation was conducted on other foreign genes.

Example 3
SPHK1 (sphingosine kinase 1) and ASB6 (ankyrin repeat and SOCS
Analysis of time-dependent expression results in box-containing 6) larvae For SPHK1 and ASB6 (JBIC results Conso I clones No.FLJ12340AAAF and No.FLJ12942AAAF), as in Example 2, recombinant virus production, expression, sample recovery, Soluble insoluble separation and analysis were performed.

  As a result, when SPHK1 was expressed (FIG. 7), the pMVPLR of the present invention had a significantly higher amount of soluble expression than the conventional one, and a better result was obtained than in Example 2, but the same result was obtained for pMVPXXX. It became.

  On the other hand, when ASB6 was expressed (FIG. 8), pMVPXXX showed the best results at 138 hours after virus infection. Therefore, it was considered that the decrease in the expression of pMVPXX in the larvae in Example 2 (FIG. 6) and FIG. 7 depends on the type of foreign protein.

Example 4
Percentage of soluble baculovirus transfer vectors (1) Expression level in BmN cells UGDH (UDP-glucose dehydrogenase, JBIC results Conso I clone No. FLJ40611AAAF) and BCKDHA (branched chain keto acid dehydrogenase E1, alpha polypeptide, JBIC results) Conso I clone No. FLJ45695AAAF) was prepared in the same manner as in Example 2. In combination with the recombinant viruses (SPHK1, ASB6) prepared so far, expression in BmN cells was performed using a total of 12 types of recombinant viruses. That is, a 6-well plate in which BmN cells were cultured at 1.0 × 10 ⁶ cells / 2 ml TC-100 (10% FBS) per well was prepared. Each recombinant virus solution is then m.p. o. i. Infected with = 5.

  120 hours after infection, the cells were peeled off by pipetting, and each culture supernatant was added so as to be 0.25% CHAPS, and allowed to stand on ice for 10 minutes to perforate the cell membrane. Soluble and insoluble separation was performed from each 1 mL by ultracentrifugation in the same manner as in Example 2, and analysis was performed by densitometry from the results of Western blotting.

(2) Analysis of soluble ratio in expression in pupae, larvae, and BmN cells In addition to the analysis results in BmN above, each of the results of temporal expression tests in pupae and larvae of Example 2 and Example 3 The solubility ratio was also calculated for the best expression time in baculovirus transfer vectors and genes. The results are shown below.

表中、「−」は、発現量が低すぎ、デンシトメトリーによる解析が不能であった例を示す。
また、ゴシック体表記は本発明の例を示す。

In the table, “-” indicates an example in which the expression level is too low to be analyzed by densitometry.
The Gothic notation indicates an example of the present invention.

  As a result, compared with the case where each gene is expressed by pM01 having the polh promoter which is the prior art, the pMVPLR which is an example of the present invention has an improved solubility ratio and a reduced aggregation ratio. It was shown that.

  On the other hand, even when pMVPXXX, which is the vp39 promoter alone, is used, the proportion of aggregation is reduced to some extent, but there are some cases where the expression level sufficient to withstand comparative studies cannot be obtained (FIGS. 9B, 10B, and 12B). It was found that the protein is susceptible to foreign proteins to be expressed and the expression host. In addition, as a whole trend, the soluble ratio is pM01 <pMVPXXX <pMVPLR, and this is the inverse correlation with the expression start time shown in FIG. 3, that is, the earlier the expression start time, the lower the aggregation ratio. It was thought to do.

  Therefore, we tried a multiple regression analysis of a very general least square method and linear model. In other words, the other genes (4 species) and the expression host (2 species) were used as dummy variables based on the expression of PDE4C expressed in acupuncture, and 10% expression time of each baculovirus transfer vector and independent variable (total) 7 variables), and the solubility coefficient as a dependent variable, the correlation coefficient matrix was obtained. Further, the partial correlation coefficient and the like obtained therefrom are shown in the following table.

  Since dummy variables were used, the comparison of numerical values was performed using standardized partial regression coefficients. As a result, ASB6, UGDH, and BCKDHA had a negative value with a p value of <0.05, indicating that these are proteins that tend to aggregate. Also, the 10% expression period was negative at a p value <0.05, indicating that there was an inverse correlation between the 10% expression period and aggregation. That is, it is considered that the earlier the onset of expression, the less the aggregation.

  On the other hand, regarding the expression host, when pupae were used as a reference, larvae and BmN cells had negative values, but no significant difference was observed (p value> 0.05).

  Summarizing the above results, it was found that the amount of foreign proteins expressed by the conventional polh promoter was insoluble, and the expression was not stable with the vp39 promoter alone. Compared with these, when the promoter of the present invention is used (the above pMVPLR), the expression is generally stable, and the proportion of soluble expression is increased, or the soluble expression amount is increased. It was shown that intracellular quality control can be expected by early expression while ensuring.

Example 5
Preparation of baculovirus transfer vector using other HRs Primer 13 (5'-agccatgggacatcatcgtttgattgtgttttacac-3 ') and primer 14 (5'-tgctagcaccgaactcgttttacgagtagaatt-) 3 ′) was used to obtain a PCR fragment (hereinafter referred to as “HR1”). Similarly, a PCR fragment (hereinafter referred to as “HR5”) was obtained using primer 15 (5′-tgccatggaaattgaactggctttacgagtagaattc-3 ′) and primer 16 (5′-agctagcatgacatcatattttgattgtgttttacacg-3 ′).

  The HR1 and HR5 fragments were digested with restriction enzymes NcoI and NheI, respectively, and the HR1 and HR5 fragments were excised. On the other hand, pMVPXX produced in Example 1 (3) was digested with restriction enzymes NcoI and NheI, and the cut surface was dephosphorylated with alkaline phosphatase. PMVPXX linearized with each of the HR1 and HR5 fragments was ligated to transform E. coli. The transformant clones (pMVP1R and pMVP5R) were selected by plasmid purification and nucleotide sequence confirmation using primer 8 (5′-agatcttaagattgttgccgttataaatatggac-3 ′).

  HRs conserved palindromic sequences were also synthesized. That is, 1 μM each of synthetic DNA 3 (5′-catggtttacaagtagaattctactcgtaaag-3 ′) and synthetic DNA 4 (5′-caaatgttcatcttaagatgagcatttcgatc-3 ′) is mixed in 50 mM NaCl, heat-denatured, and slowly cooled to form double-stranded DNA. I let you. Furthermore, the 5 'end was phosphorylated by T4 polynucleotide kinase.

  On the other hand, pMVPXX produced in Example 1 (3) was digested with restriction enzymes NcoI and NheI, and the cut surface was dephosphorylated with alkaline phosphatase. PMVPXX linearized with each of the HR1 and HR5 fragments was ligated to transform E. coli. A clone of this transformant (pMVP1-palindromes) was selected by plasmid purification and nucleotide sequence confirmation using primer 8 (5′-agatcttaagattgttgccgttataaatatggac-3 ′).

  It can be seen that even when foreign proteins are introduced into the three types of baculovirus transfer vectors thus obtained, the expression start time or the rate of aggregation changes in the same manner as in the above examples.

Example 6
Preparation of anti-chymase antibody molecule by recombinant baculovirus Hybridoma 2D11G10D strain (this is FERM P-18700 as of February 8, 2002) that produces an antibody against human chymase (Katakura Kogyo: JP-A-2002-3000886) National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Postal Code 305-8565, 1st, 1st, 1st East, Tsukuba City, Ibaraki Prefecture, Japan 6): Katakura Industry: JP 2003-235556 A A PCR fragment containing a heavy chain gene was obtained by PCR using primer 17 (5′-gaGCGGCCGCATGGCTTGGGTGTGGACCTTG-3 ′) and primer 18 (5′-gaGATATCTCATTTACCAGGAGAGCGGGAG-3 ′) using the genomic DNA of FIG. Similarly, a PCR fragment containing a light chain gene was obtained by PCR using primer 19 (5′-gaGCGGCCGCATGGACATGAGGACCCCTGC-3 ′) and primer 20 (5′-gaGATATCCTAACACTCATTCCTGTTGAAGCTC-3 ′).

  The PCR fragment containing the heavy chain and light chain genes obtained above was digested with restriction enzymes NotI and EcoRV, respectively, and the heavy chain and light chain gene fragments were excised. On the other hand, pMVPLR prepared in Example 1 and pM01, which is a conventional plasmid, were digested with restriction enzymes NotI and EcoRV, and the cut surface was dephosphorylated with alkaline phosphatase. Escherichia coli was transformed by ligating each of the heavy chain and light chain gene fragments with linearized pMVPLR and pM01. The clone of this transformant was selected by purifying the plasmid and confirming the base sequence using the primer 8 (5′-agatcttaagattgttgccgttataaatatggac-3 ′).

  Recombinant viruses were prepared in the same manner as in Example 2 for the four types of plasmids obtained above. Subsequently, an infection experiment was carried out in the same manner as in Example 4 (1) except that the heavy and light chain recombinant viruses were mixed and infected to obtain a culture supernatant of BmN cells and a larva body fluid. These samples were subjected to ELISA in which chymase was immobilized, and antibodies that normally bind to chymase (hereinafter referred to as “anti-chymase antibody 2D”) were obtained. The production amount of this anti-chymase antibody 2D was measured with an anti-mouse IgG rabbit antibody (ALP label: Nacalai Tesque) and Blue-Phos (KPL). As the standard antibody, a monoclonal antibody obtained by purification from the culture supernatant of the 2D11G10D strain was used.

  The production amount of anti-chymase antibody 2D was found to be the highest expression level when pMVPLR was used for both heavy and light chains (FIG. 13). In particular, in the culture supernatant, the increase in the expression level was remarkable as compared with pM01 which is the conventional technique.

Example 7
Properties of Anti-Chymase Antibody Molecules Produced with Recombinant Baculovirus Of the larval body fluids obtained in Example 6, the plasmids used for expression were those of heavy chain and light chain of pM01 and pMVPLR, and Protein A-SepharoseFF ( (GE Healthcare) was used to purify antibody molecules by the method recommended by the manufacturer, followed by ultracentrifugation analysis (Katakura Industry) by the sedimentation rate method. For ultracentrifugation analysis, analysis by SEDFIT (NIH) was performed using an analytical ultracentrifuge ProteomeLab XL-A (Beckman Coulter). The purified recovery amount and the ELISA in Example 6 were converted to the amount per head and are shown in Table 6 together with the results of ultracentrifugation analysis. Moreover, electrophoresis (reduction SDS-PAGE) was performed on the anti-chymase antibody 2D expressed and purified using each plasmid.

From the result of electrophoresis (FIG. 14), it was found that there was not much difference in the purity of each purified antibody molecule. However, as a result of ultracentrifugation analysis, it was found that many aggregates were formed when the plasmid used for recombinant virus production produced antibody molecules with pM01, that is, a conventional polyhedrin promoter. In addition, when an antibody molecule is produced with a conventional polyhedrin promoter, the amount of the purified antibody molecule is larger than the amount of the active antibody molecule confirmed by ELISA and can be purified with Protein A-Sepharose FF but has no activity. However, it was found to account for about 50%. On the other hand, when pMVPLR, that is, the promoter of the present invention was used, the formation of aggregates was suppressed, and the ELISA measurement value per head and the purified recovery amount were consistent. In addition, the production per head increased. From this, it was shown that when the promoter of the present invention was used, the amount of antibody molecule secreted increased, and the quality control within the cell was sufficiently received.

  Although the preferred embodiments of the structure and method according to the present invention have been described above, various modifications can be made to the above-described structure, method, steps and temporal order of these methods without departing from the concept, spirit and scope of the present invention. It will be easily understood by those skilled in the art that various changes and modifications are possible. For example, similar results can be obtained using other HRs. In addition, the expression level and the expression time may be changed by changing the number of HRs conserved palindromic sequences or adding a region for expressing the ie-1 gene product or the like upstream thereof. Furthermore, multiple genes may be coexpressed by inserting multiple promoters into one plasmid, or individual recombinant viruses may be expressed by multiple coinfections to form complexes. It can also be said that it is effective to form a complex by shifting the expression time in combination with the conventional last promoter.

  According to the present invention, a stably transformed recombinant baculovirus including the baculovirus transfer vector described above can be obtained. This recombinant baculovirus can advance the expression time of a protein or peptide and enable protein expression in an appropriate amount, so that it can produce a foreign protein or polypeptide with relatively little aggregation. It is possible to obtain a foreign protein having the desired physiological activity.

  Therefore, the recombinant baculovirus of the present invention can be advantageously used for the production of proteins used as medicines and reagents.

It is drawing which shows the structure of pM000001 used by this invention. This has a sequence around the porh of baculovirus and can introduce a foreign gene inserted into MCS by homologous recombination into the viral genome. It is drawing which shows the outline | summary of construction | assembly of this invention recombinant baculovirus. It is drawing which shows the relationship between the time after recombinant baculovirus infection, and the production amount of GFP. It is drawing which shows the structure of the promoter periphery of pM01-DestCH, pMVPXXX-DestCH, and pMVPLR-DestCH. It is drawing which shows the time-dependent expression of PDE4C in a cocoon when using each promoter of pM01, pMVPXXX, and pMVPLR. In the figure, A shows the result for the soluble fraction, and B shows the result for the precipitated fraction. It is drawing which shows the time-dependent expression of PDE4C in a larva when each promoter of pM01, pMVPXXX, and pMVPLR is used. In the figure, A shows the result for the soluble fraction, and B shows the result for the precipitated fraction. It is a figure which shows time-dependent expression of SPHK1 in a larva when each promoter of pM01, pMVPXXX, and pMVPLR is used. In the figure, A shows the result for the soluble fraction, and B shows the result for the precipitated fraction. It is drawing which shows the time-dependent expression of ASB6 in a larva when each promoter of pM01, pMVPXXX, and pMVPLR is used. In the figure, A shows the result for the soluble fraction, and B shows the result for the precipitated fraction. It is an electrophoresis photograph (BCIP-NBT dyeing | staining image) which shows the influence of each baculovirus transfer vector in expression of PDE4C. In the figure, A shows the result when expressed in pupae, and B shows the result when expressed in larvae. It is an electrophoresis photograph (BCIP-NBT dyeing | staining image) which shows the influence of each baculovirus transfer vector in expression of SPHK1. In the figure, A shows the result when expressed in pupae, and B shows the result when expressed in larvae. It is an electrophoresis photograph (BCIP-NBT dyeing | staining image) which shows the influence of each baculovirus transfer vector in the expression of ASB6. In the figure, A shows the result when expressed in larvae, and B shows the result when expressed in BmN cells. It is an electrophoresis photograph (BCIP-NBT stained image) showing the influence of each baculovirus transfer vector when using BmN cells. In the figure, A shows the results when UGDH is expressed, and B shows the results when BCKDHA is expressed. It is a figure which shows the expression level of the active antibody molecule | numerator in a BmN culture supernatant and a larva body fluid when expressing the heavy chain and light chain of anti-chymase antibody 2D using the promoter of pM01 and pMVPLR. It is a figure which shows the electrophoresis result of the anti- chymase antibody 2D refine | purified after making it express using the promoter of pM01 or pMVPLR.

Claims (11)

Baculovirus transfer vector comprising the following constitutions b) and c) from left to right:
b) Enhancer DNA region including baculovirus HR3 c) DNA region including promoter region derived from baculovirus vp39 gene A baculovirus transfer vector comprising the following a) to e) from left to right:
a) viral DNA sequence of baculovirus encoding the 5′-end or 3′-end flanking sequence of the baculovirus nonessential gene site b) an enhancer DNA region including baculovirus HR3 c) a promoter region derived from the baculovirus vp39 gene Including DNA region d) DNA region encoding desired protein or polypeptide e) Baculovirus viral DNA sequence encoding 3 'end or 5' end flanking sequence of baculovirus non-essential gene site A baculovirus transfer vector comprising the following components a ′) to e ′) from left to right:
a) a DNA sequence containing Tn7L or Tn7R b) an enhancer DNA region containing baculovirus HR3 c) a DNA region containing a promoter region derived from the baculovirus vp39 gene d) a DNA region encoding the desired protein or polypeptide e ') DNA sequence containing Tn7R or Tn7L   The baculovirus transfer vector according to claim 1, further comprising a DNA region including a cloning restriction enzyme cleavage site into which a DNA sequence encoding a desired protein or polypeptide can be inserted downstream of the promoter region of c). . A recombinant baculovirus obtained by introducing a foreign gene with the baculovirus transfer vector according to any one of claims 1 to 4 . The recombinant baculovirus according to claim 5 , wherein the foreign gene is an antibody gene. A recombinant baculovirus expression system comprising lepidopteran insect cells infected with the recombinant baculovirus according to claim 5 or 6 . The lepidopteran insect cells are Spodoptera frugiperda, Bombyx mori, Bombyx mandarina, Heliothis virescens, Heliothis zea, Mamestra The baculovirus expression system according to claim 7 , wherein the baculovirus expression system is a cell selected from the group of lepidopteran insects consisting of Mamestra brassicas, Estigmene acrea or Trichoplusia ni. The baculovirus expression system according to claim 7 , wherein the lepidopteran insect cell is any one of a cultured cell, a larva or a moth of Bombyx mori. A method for producing a recombinant protein and / or polypeptide comprising the recombinant baculovirus expression system according to any one of claims 7 to 9 . A method for producing an antibody comprising the recombinant baculovirus expression system according to any one of claims 7 to 9 .

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