KR102272651B1 - Baculovirus with increased yield of foreign protein production - Google Patents

Abstract

The present invention relates to a novel baculovirus Bacmid for long-term overexpression of a foreign protein in its original form and to an increased production method of a foreign target protein using the same, wherein the baculovirus of the present invention has reduced pathogenicity. By using Bacmid, it is possible to express in a unique form of an exogenous useful protein with high prophylactic or therapeutic value, thereby increasing the biological activity or expression level. Therefore, useful proteins such as antigens for prophylactic or therapeutic purposes in insects and insect cells can be produced. It can be produced with low cost and high efficiency.

Description

Baculovirus with increased expression amount and expression time of foreign protein {Baculovirus with increased yield of foreign protein production}

The present invention relates to a novel baculovirus Bacmid for long-term overexpression of an exogenous protein in its original form and to an increased production method of the exogenous target protein using the same.

The Baculovirus Expression System (BES) is an expression system using the strong p10 gene promoter or polyhedrin gene promoter of Nucleopolyhedrovirus (NPV).

BES not only has very high expression efficiency of foreign proteins, but also has excellent biological and immunological activities of foreign proteins expressed by using insect cells, which are higher eukaryotic cells, so that E. coli, yeast (E. coli), yeast (E. coli) Yeast), mammalian cells (mammalian cells), etc. compared to other expression systems have many advantages compared to other aspects, it is receiving a lot of attention.

In the case of expressing foreign proteins in insect cells, proteins can be mass-produced to a level similar to that of the E. coli expression system, and post-translational modifications such as glycosylation, acylation, and disulfide bond formation ( Unlike the case where E. coli and yeast are used as host cells because the post-translational modification process is effectively performed, it is very useful industrially because it is possible to obtain a large amount of foreign protein having a biological activity similar to that of a native protein.

On the other hand, the recombinant protein production method using mammalian cells can obtain a protein with high therapeutic value as a human drug, but various hormones and growth regulators derived from mammals are required in large amounts for culturing and maintaining mammalian cells. There is a problem that a considerable cost is required.

However, in the case of using insect cells, it is known as a more economical protein production method because it is possible to produce recombinant proteins at low cost even after a post-translational modification process similar to mammalian cells.

In addition, it is widely used in the production and production of virus-like particles that require simultaneous expression of various proteins because multiple gene expression is possible most efficiently compared to other expression systems.

Currently, BES using insect cells is representative of two expression systems using Autographa californica NPV (AcNPV) and Bombyx mori NPV (BmNPV).

However, the polyhedral protein gene promoter mainly used in BES shows different expression levels depending on the type of useful protein, and most of the polyhedral protein gene promoters have the disadvantage that they are expressed at a lower level than the polyhedrin originally expressed by the polyhedral protein gene promoter. , it is pointed out as a technical limitation of BES.

In order to overcome this limitation, the vector, baculovirus genome, and cell line, which are major components of BES, are being improved. In particular, research on the improvement of relatively easy-to-manipulate vectors has been most actively conducted. come.

Most of the improvements were made using a very late gene promoter, such as the polyhedral protein gene promoter or the p10 gene promoter, which is a promoter that exhibits explosive transcriptional activity at the last stage of the virus's life cycle.

However, due to the characteristics of baculovirus as a pathogenic virus, when such a promoter is used, the host or host cell in which the useful protein is not sufficiently expressed leads to death, and the production of the useful protein and the post-translational modification of the produced protein are reduced. The problem is that it does not work as expected.

(001) Korean Patent Registration KR 10-0353475 (002) Korean Patent Registration KR 10-1812250

Accordingly, the present inventors removed the p10 gene, one of pathogenic factors, and the p10 gene promoter, which competitively shares transcription factors with the polyhedral protein gene promoter from the genome of baculovirus, thereby increasing the expression level of the target useful protein and the cell survival time. The effect was confirmed, and by additionally removing chitinase and cysteine proteolytic enzyme (v-cath), another pathogenic factor of baculovirus, the production of useful proteins and cell survival time are significantly increased. The effect was confirmed and the present invention was completed.

Accordingly, the present invention provides a recombinant baculovirus in which the p10 gene promoter and the p10 gene are deleted to increase the production and expression time of an exogenous target protein.

In addition, in the present invention, the p10 gene promoter, p10 gene, baculovirus chitinase and cysteine proteolytic enzyme (v-cath) are deleted so that the production and expression time of the exogenous target protein is increased by recombinant baculo. provide the virus.

In addition, the present invention comprises the steps of preparing a recombinant transfer vector in which the p10 gene promoter and the p10 gene are deleted; cloning the nucleotide sequence encoding the foreign target protein into the recombinant transfer vector; and introducing the cloned recombinant transfer vector and Bacmid into insects or insect cells.

In addition, the present invention comprises the steps of preparing a recombinant transfer vector in which p10 gene promoter, p10 gene, baculovirus chitinase and cysteine proteolytic enzyme (v-cath) are deleted; cloning the nucleotide sequence encoding the foreign target protein into the recombinant transfer vector; and introducing the cloned recombinant transfer vector and Bacmid into insects or insect cells.

The baculovirus of the present invention can be expressed in a unique form of an exogenous useful protein with high preventive or therapeutic value using Bacmid with reduced pathogenicity, thereby increasing the biological activity or expression level of insects and insect cells. useful proteins such as antigens for prophylactic or therapeutic purposes can be produced at low cost and with high efficiency.

1 is a schematic diagram illustrating the production of a BmBacmid-Δp10 system by homologous recombination in Bm5 cells.
2 is a photograph confirming the construction of BmBacmid-Δp10 and BmBacmid-Δpcc by PCR: (A) ORF1629 region PCR; (B) transposase gene PCR for helper plasmid detection; (C) Mini-F replicon region PCR; (D) p10 region PCR; (E) chitinase and cysteine protease gene region PCR.
3 is a photograph showing colonies formed on LB plates after electroporation of BmBacmid-Δp10 into DH10B cells.
Figure 4 is a schematic diagram of wild-type baculovirus BmNPV-K1 and modified Bacmids:
(A) BmNPV-K1; (B) BpBacmid; (C) BmBacmid-Δp10; (D) BmBacmid-Δpcc.
5 is a schematic diagram of the arrangement of chitinase and cysteine protease genes in the baculovirus genome.
6 is a schematic diagram illustrating the production of a BmBacmid-Δpcc system after modification of BmBacmid using a lambda recombination system.
Figure 7 is the recovery (curing) of pREDGm after the preparation of BmBacmid-Δpcc through the lambda recombination system.
After curing of pREDGm, colonies were cloned in LB plate A and LB plate B;
(A) LB plates containing kanamycin, chloramphenicol, tetracycline and gentamicin; (B) LB plates containing kanamycin, chloramphenicol and tetracycline.
8 shows the infection of Bm5 cells infected with rBm-p6.9v-EGFP-wt (wt), rBm-p6.9v-EGFP-Δp10 (Δp10), rBm-p6.9v-EGFP Δpcc) on days 1 to 12 of culture. It is a fluorescence micrograph.
9 is a graph showing the fluorescence intensity of EGFP:
Bm5 cells were harvested between 1 and 12 days post infection at MOI of 5 mice with each recombinant virus, fluorescence intensity of cell extracts was measured using a fluorescence spectrometer, bars represent mean±SE (n=3) , wt, rBm-p6.9v-EGFP-wt; Δp10, rBm-p6.9v-EGFP-Δp10; Δpcc, rBm-p6.9v-EGFP.
10 is a graph comparing BV growth curves of WT BmNPV-K1 and modified BmBacmids.
11 is a graph showing the survival rate of Bm5 cells infected with WT BmNPV-K1 or modified BmBacmid virus.

Hereinafter, the present invention will be described in detail.

The present invention has also improved the genome of baculovirus in order to remove genes that exist in baculovirus itself and affect recombinant protein expression efficiency and stability along with improvement of the expression vector, as well as show pathogenicity in the host. Together with the polyhedrin promoter, which is a major promoter used for recombinant protein expression, the p10 gene, which is known to belong to the very late promoter and share transcription factors, and its promoter were removed to increase the production of the recombinant protein and improve the survival rate of the host cell.

In addition, cysteine protease (V-cath), which reduces production through stability degradation by causing degradation of recombinant proteins, and chitinase, which acts as a pathogenic factor in the host, is known as an endoplasmic reticulum transport inhibitory gene By removing the (chitinase) gene, not only the production of the recombinant protein was increased, but also the production time of the recombinant protein was increased by delaying the death of virus-infected cells, and it was confirmed that the survival time could be greatly increased even in the 5th instar silkworm larvae. .

In addition, it is believed that a higher yield can be expected than before by extending the production time of the recombinant protein according to the increase in cell viability. In particular, the outer membrane type that surrounds the cell membrane and is released after assembly and the cell membrane after the expression of the structural protein. It can be a very useful tool for the production of virus-like particles (VLPs) of enveloped viruses.

The present invention provides a recombinant baculovirus in which the p10 gene promoter and the p10 gene are deleted to increase the production and expression time of an exogenous target protein.

The p10 gene promoter is represented by the nucleotide sequence of SEQ ID NO: 1.

The p10 gene is represented by the nucleotide sequence of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 3.

In addition, in the present invention, the p10 gene promoter, p10 gene, baculovirus chitinase and cysteine proteolytic enzyme (v-cath) are deleted so that the production and expression time of the exogenous target protein is increased by recombinant baculo. provide the virus.

The chitinase of the baculovirus is represented by the nucleotide sequence of SEQ ID NO: 4, and is represented by the amino acid sequence of SEQ ID NO: 5.

The cysteine proteolytic enzyme (v-cath) is represented by the nucleotide sequence of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 3.

In addition, the present invention comprises the steps of preparing a recombinant transfer vector in which the p10 gene promoter and the p10 gene are deleted; cloning the nucleotide sequence encoding the foreign target protein into the recombinant transfer vector; and introducing the cloned recombinant transfer vector and Bacmid into insects or insect cells.

In one embodiment of the present invention, the vector is Bac-to based on the BpGOZA bacmid used to construct an occlusion body positive recombinant BmNPV expressing a polyhedral protein using the p10 gene promoter. It is possible to construct a recombinant transfer vector BmBacmid-Δp10 in which the viral DNA of BpBacmid introduced with the -Bac system and the p10 gene promoter and its gene are removed from the sequence from the upstream region of the p10 gene promoter to the downstream region of the p10 gene.

In one embodiment of the present invention, the insect cells are BT1-Tn-5B1-4 cells, Hi5 cells, LD652Y cells, Sf9 cells, Sf21 cells, Kc1 cells, SL2 cells, Bm5 cells, BmN cells and mosquitoes (mosquito). It may be one selected from the group consisting of cells.

In one embodiment of the present invention, the insect may be one selected from the group consisting of silkworm moth (Bombyx mori), thief moth (Spodoptera frugiperda), cabbage silver pattern chestnut moth (Trichoplusia ni), and beehive moth (Galleria mellonella). .

In one embodiment of the present invention, the baculovirus may be Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nucleopolyhedrovirus (BmNPV).

In addition, the present invention comprises the steps of preparing a recombinant transfer vector in which p10 gene promoter, p10 gene, baculovirus chitinase and cysteine proteolytic enzyme (v-cath) are deleted; cloning the nucleotide sequence encoding the foreign target protein into the recombinant transfer vector; and introducing the cloned recombinant transfer vector and Bacmid into insects or insect cells.

In one embodiment of the present invention, in the vector, the baculovirus cysteine proteolytic enzyme gene and the chitinase gene are present between the open reading frame (ORF) of the lef-7 gene and the ORF of the gp64 gene, 41 to 122 They exist in opposite directions with respect to the bidirectional promoter of the short sequence of bp (Michale, 2011). Therefore, the cysteine proteolytic enzyme gene and the chitinase gene can be simultaneously removed using the lambda red recombination system, and the predicted promoter sequence of lef-7, a gene surrounding the chitinase gene, and the cysteine proteolytic enzyme gene BmBacmid-Δpcc can be produced by removing these genes after setting the removal position so as not to damage the poly A sequence of the gene gp64.

In one embodiment of the present invention, the insect cells are BT1-Tn-5B1-4 cells, Hi5 cells, LD652Y cells, Sf9 cells, Sf21 cells, Kc1 cells, SL2 cells, Bm5 cells, BmN cells and mosquitoes (mosquito). It may be one selected from the group consisting of cells.

In one embodiment of the present invention, the insect may be one selected from the group consisting of silkworm moth (Bombyx mori), thief moth (Spodoptera frugiperda), cabbage silver pattern chestnut moth (Trichoplusia ni), and beehive moth (Galleria mellonella). .

In one embodiment of the present invention, the baculovirus may be Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nucleopolyhedrovirus (BmNPV).

Hereinafter, the present invention will be described in more detail through examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

< Example 1> Materials and Methods

<1-1> Viruses and cell lines

Bm5 cells, a cell line derived from the ovary of Bombyx mori, were used for the experiment by adding 10% of fetal bovine serum (FBS) (Welgene, Korea) to TC-100 insect medium (Welgene, Korea) and culturing stationary in an incubator at 27°C. . Cells were counted through trypan blue staining, and subculture was performed at 2.0 × 106 cells per ^{T-25 flask for cell culture (25 cm 2 ) and redistributed every 4 days.} Insect virus was a wild type isolated from Korea, and BmNPV-K1 strain was inoculated into Bm5 cells and used in the experiment after propagation.

<1-2> recombination BmNPV making

Transform the recombinant transfer vector into DH10BmBac or DH10Bm-ΔP10 or DH10Bm-ΔPCC competent cells, and for DH10BmBac and DH10Bm-ΔP10, gentamicin (10 μg/mL), kanamycin (40 μg/mL), tetracycline (10 μg/mL) , LB plate treated with X-gal and IPTG, for DH10Bm-ΔPCC, chloramphenicol (30 μg/mL), gentamicin (10 μg/mL), kanamycin (40 μg/mL), tetracycline (10 μg/mL), X -Gal and IPTG-treated LB plates were used for cancer culture at 37°C for 24 hours or longer, and white colonies were selected and cultured, and extracted using PureLink® HiPure Plasmid Miniprep Kit (ThermoFisher, USA). To confirm the transfer of the target gene to att-Tn7 in the LacZ gene, PCR was performed using AccuPower® ProFi Taq PCR Premix (Bioneer Co., Daejeon, Korea) and the primers in Table 2 below.

primer SEQ ID NO: order M13F(-40) 8 5'-GTT TTC CCA GTC ACG AC-3' M13R(-40) 9 5’-CAG GAA ACA GCT ATG AC-3’

<1-3> recombination BmNPV sublimation

Purification of recombinant BmNPV was performed by plaque assay (O'Reilly et al., 1992). Bm5 cells were aliquoted into 2.0×10 ⁶ pieces in a 60 mm dish (SPL Co. Ltd., Pocheon, Korea), and the cell culture medium in which the production of recombinant BmNPV was confirmed ^{was diluted to 10 -4} ∼ 10 ^-6 and inoculated 4 It was incubated for a period of time. Thereafter, 5% SeaPlaque ^ⓡ Agarose (Lonza, Basel, Switzerland) was sterilized and dissolved, left still in a water bath at 45° C., and mixed with a cell culture medium to finally prepare 0.5% agarose. After 4 hours of virus inoculation, the inoculum was completely removed, and 3 mL of the prepared agarose gel was overlaid on the infected cells, incubated at 27°C, and the formation of plaques was observed after 3 days. The resulting plaque was separated using a micropipette, added to 100 μL of cell culture medium, vortexed, and the mixture was inoculated into a 24-well plate. Three days after inoculation, cells in which the production of recombinant virus was confirmed were collected, viral DNA was extracted, and PCR was performed to confirm the purity of the recombinant virus. In PCR to confirm the purity of the virus, insertion was confirmed using the primers in Table 1 at the location where the target gene was transferred.

<1-4> baculovirus dose

The titer of the recombinant virus whose purity was confirmed was measured using an end-point dilution assay (TCID50) method. After dispensing 2.0×10 ⁴ cells/90 μL of insect cells in a ^{96 well plate, the virus culture was diluted to 10 -5} , 10 ^-6 , 10 ^-7 , 10 ^-8 concentrations, and then the virus inoculum at each dilution concentration was inoculated into 12 wells by 10 μL and cultured at 27°C for 5 days, and pfu (plaque forming unit) per mL was calculated from the ratio of the number of infected wells to the number of uninfected wells using a phase contrast microscope.

<1-5> baculovirus DNA extraction

For isolation of viral DNA, cells infected with each recombinant virus were centrifuged at 1,000 × g for 5 minutes to collect BV present in the supernatant, and 20% PEG solution (20% Polyethylene Glycol 8000, 0.5M NaCl) was 1: The mixture was mixed in 1 Volume, left on ice for 30 minutes, and centrifuged at 10,000×g, 4° C. for 10 minutes to obtain precipitated BV. Then, 500 μL of lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 5% β-mercaptoethanol, 4% SDS, pH 8.0) was added, vortexed for 15 seconds, and left at room temperature for 10 minutes. Then, the supernatant was collected by centrifugation at 12,000 × g for 20 minutes, and phenol and isoamyl alcohol (IAA) treatment was performed. After adding 3 M sodium acetate to 1/10 of the final volume and double volume of 99% cold ethanol, it was left at -70°C for 30 minutes, and then centrifuged at 12,000×g for 15 minutes to obtain a DNA precipitate. After washing with 70% ethanol and drying, it was dissolved in sterile water and used for the experiment.

<1-6> SDS-PAGE and Western blot analysis

Bm5 cells inoculated with each recombinant BmNPV were centrifuged at 1,000 × g for 5 minutes to collect the precipitated cells and washed with PBS. Cell precipitate is suspended in 0.1% PBS-T buffer (PBS with 0.1% Triton X-100), and protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, USA) is added to make 1% of the final volume and placed on ice. After leaving for 30 minutes, add 5X SDS-PAGE loading sample buffer (312.5 mM Tris-HCl, 50% glycerol, 5% β-mercaptoethanol, 10% SDS, 0.1 % bromophenol blue, pH 6.8) and heat at 100°C for 10 minutes and centrifuged at 12,000 × g for 10 minutes to prepare a sample. SDS-PAGE samples were electrophoresed at a voltage of 125V on 10% and 12% SDS-polyacrylamide gels. After electrophoresis, the gel was stained with Coomassie brilliant blue solution (1 g coomassie brilliant blue, 450 mL methanol, 100 mL glacial acetic acid with 450 mL distilled water) at room temperature for 1 hour, and destaining solution (400 mL methanol, 100 mL). It was observed by decolorization with glacial acetic acid with 500 mL distilled water). For Western blot analysis, the SDS-PAGE gel was transferred to a nitrocellulose membrane (Pall Corp, New York, USA) at a voltage of 15 V for 1 hour, and for blocking, TBS-T (20 mM Tris, 150 mM NaCl, 0.1 % Tween) 20, pH 7.4) was treated with 5% skim milk for 1 hour. EGFP-specific anti-GFP antibody (Cell signaling, USA) was diluted in TBS-T and washed three times for 15 minutes each. Secondary antibody (anti-mouse IgG) was also diluted in TBS-T and washed three times for 15 minutes each. Finally, the membrane was treated with Western HRP substrate (Merck Millipore, Burlington, USA), and an image was taken with an Azure c300 Western Blot Chemiluminescent Blot Imaging System (Azure biosystem, Dublin, USA) to observe a specific band.

<1-7> Fluorescence microscopy

In order to confirm the expression of the fluorescent protein by recombinant BmNPV, it was observed using a fluorescence microscope Sundew MCX1600 (micros, Sankt Veit an der Glan, Austria). The green fluorescent protein EGFP showed the highest wavelength at 488 nm and the highest emission wavelength at 510 nm, and the blue light (Max 470 nm) emitted under a fluorescence microscope was converted into green light (510 nm) to observe fluorescence. After observation under a fluorescence microscope, the expressed fluorescence pictures were taken using INFINITY software program.

<1-8> Fluorescence photometry

Bm5 cells inoculated with EGFP-overexpressing recombinant BmNPV with 5 M.O.I. were collected at 24 hour intervals from day 1 to day 6, centrifuged at 1000 × g for 5 minutes, and washed with phosphate buffered saline (PBS). 1 mL of RIPA buffer was added to the precipitated cells, left on ice for 30 minutes, and then 2 mL of PBS was added. A minimum of 3 mL of the measurement sample was used, and the fluorescence intensity was measured with a K2™ fluorescence spectrometer (ISS inc., USA) in an excitation filter of 488 nm and an emission filter of 510 nm.

<1-9> p10 promoter and gene removal

Upstream of the p10 promoter for removal of the baculovirus p10 promoter and gene

For the 500bp region, 10 pmole of p10-5'-F and p10-5'-R of Table 2 below were added using AccuPower® ProFi Taq PCR Premix (Bioneer Co., Daejeon, Korea) using baculovirus DNA as a template. , The final volume of the reaction solution was adjusted to 20 μL using sterile water, and PCR was performed with a Thermal Cycler (TaKaRa, Kusatsu, Japan). Cloning into the pMD20-T vector (TaKaRa, Kusatsu, Japan) was performed in the same manner as in the previous experiment.

After ligation of the p10 promoter upstream 500 bp region cloned into pMD20-T vector (TaKaRa, Kusatsu, Japan) to pBlueScript II SK(+) vector using restriction enzymes (Kpn I/Hind III), transform into DH5α competent cells. and cultured in LB plates treated with amiciline. After selecting colonies and culturing them in LB medium to which ampiciline was added, the recombinant plasmid was extracted and named pBlue-p10-5'. After that, 10 pmole of p10-3'-F and p10-3'-R of Table 2 below were added to the 500 bp region downstream of the p10 gene, and the final volume of the reaction solution was adjusted to 20 μL using sterile water, followed by a Thermal Cycler (TaKaRa, Kusatsu, Japan) PCR was performed. Cloning into the pMD20-T vector (TaKaRa, Kusatsu, Japan) was performed in the same manner as in the previous experiment. The 500 bp region downstream of the p10 gene cloned into the pMD20-T vector (TaKaRa, Kusatsu, Japan) was ligated into the pBlue-p10-5' vector using restriction enzymes (Pst I/Spe I), and then transformed into DH5α competent cells. and cultured in LB plates treated with amiciline. After selecting colonies and culturing them in LB medium to which ampiciline was added, recombinant plasmids were extracted and named pBm101-ΔP10. All recombinant plasmids produced were finally confirmed in sequence through sequencing requested by COSMOgenetech (Daejeon, Korea).

500 ng of the prepared pBm101-ΔP10 and 100 ng of BpBacmid DNA (Lee, 2018) were mixed in 100 μL of TC-100 insect medium (Welgene, Korea), and 8 μL of Cellfectin II (Invitrogen, Carlsbad, USA), a transfection reagent, was added. After adding to the medium, each mixture was remixed and reacted at 27° C. for 30 minutes. After the reaction, Bm5 ^{cells seeded at 1×10 6} cell/mL in a 6 well plate were inoculated and co-transfected. From 3 days after inoculation, the infection pattern of recombinant BmNPV was confirmed using a phase-contrast microscope, and plaque assay was performed in the same manner as in the previous experiment to select baculovirus plaques in which polyhedron was not formed and used for the experiment.

primer SEQ ID NO: order p10-5'-F 10 5' - GGT ACC TGT CAT TTA TTA ATT TGG ATG A - 3' p10-5'-R 11 5' - AAG CTT TTA ACT ATA ATA TAT TGT GTT GGG T- 3' p10-3'-F 12 5' - CTG CAG ATG AAT CGT TTT TAA AAT AAC A - 3' p10-3'-R 13 5' - ACT AGT GAA GAA CAC ACG ATC ATG G - 3'

<1-10> Bacmid making

To remove the baculovirus chitinase gene and cystein protease (v-cath) gene, pREDGm was prepared by introducing a gentamicin resistance gene into pREDKI (addgene #51626) expressing red recombinase. After that, transform into DH10Bm-ΔP10 competent cells and use LB plates treated with gentamicin (10 μg/mL), kanamycin (40 μg/mL), X-gal, and IPTG. After selecting and culturing colonies, using SOB medium supplemented with L-arabinose 0.1 M for red recombinase expression through induction of the araBAD promoter at 30°C to OD600 = 0.6 abs, electro-competent cells were prepared. . Cm-F primer and Cm-R primer in Table 3 using AccuPower® ProFi Taq PCR Premix (Bioneer Co., Daejeon, Korea) from pIDC plasmid to destroy the ORF of the gene to be removed and insert the chloramphenicol resistance gene and promoter 10 pmole was added, and the final volume of the reaction solution was adjusted to 20 μL using sterile water, and then PCR was performed with a Thermal Cycler (TaKaRa, Kusatsu, Japan). The PCR product was cloned into pMD20-T vector (TaKaRa, usatsu, Japan) to construct pMD20-CmR. Then, 10 pmole of Chitinase-F primer and Cystein-R primer in Table 4 were added to the baculovirus DNA using AccuPower® ProFi Taq PCR Premix (Bioneer Co., Daejeon, Korea), and the final volume of the reaction solution was After adjusting to 20 μL using sterile water, PCR was performed. The PCR product was cloned into pMD20-T vector (TaKaRa, Kusatsu, Japan) to construct pMD20-CC. A vector was prepared using restriction enzymes (Aat II/Apa I) in pMD20-CC, and the chloramphenicol resistance gene and promoter sequence obtained by treatment with the same restriction enzyme from pMD20-CmR were ligated. After the ligation reaction, the cells were transformed into DH5α competent cells and cultured in LB plates treated with chloramphenicol. After selecting colonies and culturing them in LB medium to which chloramphenicol was added, recombinant plasmids were extracted and named pMD20-Partial-CC-CmR. Then, PCR was performed using the Chitinase-F primer and Cystein-R primer in Table 3 using AccuPower® Pfu PCR PreMix (Bioneer Co., Daejeon, Korea) in pMD20-Partial-CC-CmR. Transform 0.5 μg of the PCR product containing the chloramphenicol resistance gene and promoter between the partially cleaved chitinase gene and the cysteine protease gene obtained through PCR into DH10Bm-ΔP10 cells having pREDGm at a voltage of 2.8 kV by electroporation. did. After 4 hours of incubation at 37°C using SOC medium, using LB plates treated with chloramphenicol (5 μg/mL), X-gal and IPTG, blue colonies were selected after cancer culture at 37°C for more than 24 hours. ORF disruption of genes to be removed in Bacmid DNA and insertion of chloramphenicol resistance genes and promoters were retested by PCR using Chitinase-F and Cystein-R shown in Table 3.

primer SEQ ID NO: order Cm-F 14 5' - GAC GTC ATA GAT CTG GGC CAA CTT TTG G -3' Cm-R 15 5' - GGG CCC CAG GCG TTT AAG GGC ACC AAT AAC-3' Chitinase-F 16 5'-CTG CAG TTG AGC AAG TCG CCG TTA TCG GC-3' Cystein-R 17 5' - GAA TTC CGA CAA AAT CAC AAT CGA TCA TTT G - 3'

<1-11> Budded virus ( BV ) production test

Bm5 cells seeded at 1×10 ⁶ cell/mL in a 6 well plate were infected with each BV at an MOI of 5 for 1 hour and then replaced with a fresh medium. After that, cells from each well were collected at 1-day intervals until the 6th day and centrifuged at 1,000×g, 4°C for 5 minutes to obtain BV present in the upper layer. The BV titer was measured using an endpoint dilution assay (TCID50) method.

<1-12> Cell viability assay

Bm5 cells seeded at 1×10 ⁶ cell/mL in a 6 well plate were infected with each BV at an MOI of 5 for 1 hour and then replaced with a fresh medium. After that, cells from each well were collected at 1 day intervals until the 6th day, and cell counting and viability were measured through Trypan blue staining.

<1-13> Bioassay

^{Baculovirus BV was diluted to 1×10 5} pfu in 50 μL of TC-100 medium and inoculated into 10 4 instar silkworms using a 1 mL syringe. In the case of negative control, only TC-100 medium was injected and inoculated. . Thereafter, survival was observed and recorded at 1-day intervals to confirm the average survival time. The experiment was performed in total 3 repetitions.

< Example 2> A new method for increasing the production of recombinant proteins BmBacmid making

<2-1> A new method through gene deletion BmBacmid making

① p10 promoter and gene removed BmBacmid - Δp10 making

Based on BpGOZA, which is used to construct polyhedrin-expressing recombinant BmNPV using the p10 promoter, the viral DNA of BpBacmid (Lee, 2018) introduced with the Bac-to-Bac system and p10 from the sequence upstream of the p10 promoter After co-transfection into Bm5 cells with the recombinant transfer vector pBm101-ΔP10 in which the p10 promoter and gene were removed from the sequence up to the downstream region of the gene, the infection pattern was confirmed and BV was collected. Thereafter, plaque assay was performed and plaques in which polyhedron was not formed were selected through microscopic observation (FIG. 1). After that, the presence or absence of the p10 promoter and gene was re-tested through PCR (FIG. 2), and BmBacmid-Δp10 was produced. After transforming BmBacmid-Δp10 DNA into DH10B electro-competent cells, select blue colonies (FIG. 3) and culture them. Using them, electro-competent cells were prepared, transformed with pMON7124, a transposition helper plasmid, and then blue colonies were selected again. cultured through The presence of BmBacmid-Δp10 and helper plasmid through PCR assay for ORF1629 region (Fig. 2A), presence or absence of helper plasmid (Fig. 2B), Mini-F region (Fig. 2C), and p10 gene region (Fig. 2D) using cultured colonies It was retested and the Bacmid system for BmBacmid-Δp10 was completed (FIG. 4).

② p10 promoter and gene; cysteine protease (v- cath ) and chitinase Production of the removed BmBacmid-Δpcc

The cysteine protease gene and chitinase gene of baculovirus exist between the ORF of the lef-7 gene and the ORF of the gp64 gene, and exist in opposite directions centering on the bidirectional promoter of a short sequence of 41 to 122 bp (FIG. 5). . Therefore, the cysteine protease gene and the chitinase gene can be simultaneously removed using the lambda red recombination system, and the predicted promoter sequence of lef-7, the chitinase gene, and the polyA sequence of gp64, the cysteine protease gene, are not damaged. These genes were removed after setting the removal positions so that they do not exist. pREDGm expressing red recombinase was transformed into DH10Bm-ΔP10 competent cells, blue colonies were selected, and electro competent cells were prepared using the cultured colonies. Then, the bidirectional promoter and gene sequence of the cysteine protease gene and chitinase gene were removed through restriction enzyme (Aat II/Apa I) treatment, and PCR was performed from pMD20-Partial-CC-CmR prepared by inserting the chloramphenicol resistance gene and promoter sequence. After transforming the obtained product, select blue colonies cultured on LB plates treated with chloramphenicol (5 μg/mL), X-gal and IPTG, and reconfirm the ORF deletion of cysteine protease gene and chitinase gene through PCR (FIG. 2), BmBacmid-Δpcc production and selection were completed (FIG. 6). After that, plasmid curing of pREDGm was performed to complete the bacmid system. LB plate treated with chloramphenicol (5 μg/mL), X-gal and IPTG, gentamicin (10 μg/mL), chloramphenicol (5 μg/mL), In LB plates treated with X-gal and IPTG, colonies from which pREDGm that could not grow in gentamicin medium were removed were selected through duplicate assay (FIG. 7), and then reconfirmed by PCR (FIG. 2). Thereafter, electro-competent cells were prepared, transformed with pMON7124, a transposition helper plasmid, and then cultured by selecting blue colonies again. Using the cultured colonies, the presence of BmBacmid-Δpcc and helper plasmid was re-tested through PCR, and DH10Bm-Δpcc, a Bacmid system for BmBacmid-Δpcc, was completed (FIG. 6).

③ Optimal production of recombinant protein BmBacmid Selection

In order to test the effect of increasing the protein production efficiency of BmBacmid-Δp10 from which the p10 gene and p10 gene promoter were removed, and BmBacmid-Δpcc from which cysteine protease and chitinase were additionally removed, pBm-p6, which showed the highest expression level in overexpression vector selection, was tested. Recombinant virus was constructed using .9v-EGFP. Transform pBm-p6.9v-EGFP into DH10BmBacmid (Lee, 2018), DH10BmBacmid-Δp10 and DH10BmBacmid-Δpcc competent cells. Colonies were selected and cultured. After that, for additional pure separation, streak was performed on an LB plate of the same composition and cultured, and white colonies were selected again. In white colonies, expression of LacZα is impossible because the target gene sequence of the transfer vector is transferred to LacZα-att Tn7 of BmBacmid. If no metastasis occurs, blue colonies are formed due to normal expression of LacZα. Therefore, after culturing the finally selected white colonies, recombinant BmBacmid DNA was extracted, and to reconfirm whether the extracted DNA was recombinated by the target gene, whether or not recombination was performed through PCR using the M13 F and M13 R primer sites present in the LacZα sequence. was finally confirmed. When no metastasis occurs, a band of about 300 bp is formed, and when metastasis occurs, a band with a size of 300 bp added to the sequence between Tn7 R and Tn7 L is formed. A specific band was observed, confirming that recombinant BmBacmid was produced by normal metastasis. After that, each recombinant BmBacmid DNA was transferred into Bm5 cells, observed through a fluorescence microscope until virus infection appeared, and then the medium of the cells showing the infection pattern was collected, and the new Bm5 cells were subjected to mass proliferation of BV. Afterwards, each recombinant virus was named rBm-p6.9v-EGFP-wt, rBm-p6.9v-EGFP-Δp10, rBm-p6.9v-EGFP, and was used in the experiment after titer measurement.

In order to compare the effect of increasing EGFP expression for each of the various types of BmBacmid produced, recombinant BmNPV was inoculated into Bm5 cells, and fluorescence microscopy was performed at 1 day intervals from the 1st to the 12th day, and the cells were collected and the fluorescence photometry was performed. As a result of fluorescence microscopy, in the case of Bm5 cells infected with rBm-p6.9v-EGFP-wt, the largest number of EGFP-expressing cells could be identified on the 3rd day of infection, and then, as time passed, cell lysis by the virus occurred. In the case of Bm5 cells infected with rBm-p6.9v-EGFP-Δp10, the highest number of EGFP-expressing cells could be identified on the 4th day of infection, and cell lysis was observed after 1 day compared to rBm-p6.9v-EGFP-wt. Therefore, it was confirmed that the pathogenicity of the virus to Bm5 cells was reduced only by removing the p10 gene and the promoter of the p10 gene. In the case of Bm5 cells infected with rBm-p6.9v-EGFP, unlike other BmBacmid types, a very large number of EGFP-expressing cells could be identified on days 3-6. EGFP expression was confirmed in veterinary cells (FIG. 8).

Similar to the results of fluorescence microscopy, in the fluorescence measurement using Bm5 cells harvested at 1-day intervals, Bm5 cells infected with rBm-p6.9v-EGFP-wt showed the fastest and highest activity of EGFP until the second day of infection. , showed the peak of EGFP activity on the 3rd day, and after that, it was confirmed that the EGFP activity decreased rapidly following cell lysis ( FIG. 9 ). In the case of Bm5 cells infected with rBm-p6.9v-EGFP-Δp10, higher EGFP activity was confirmed compared to rBm-p6.9v-EGFP-wt, which was used as a control, in almost all days except for days 1-2, After the 4th day, it showed a rapid decrease in EGFP activity due to cell lysis, confirming the effect of increasing the cell viability of about 1 day. It is believed that by removing the factor competing with the polyhedrin gene promoter through the removal of the p10 gene promoter, the expression level increased by about 3 times due to the monopoly of the transcription factor of the polyhedrin gene promoter. It is believed that the effect of increasing the viability of the infected cells was shown ( FIG. 9 ). In the case of Bm5 cells infected with rBm-p6.9v-EGFP, a very high level of EGFP activity was confirmed, unlike other BmBacmid types, in the fluorescence measurement results, and even on the 12th day of infection, 3 of rBm-p6.9v-EGFP-wt The activity level was almost similar to that of the primary. On all days, EGFP activity was significantly higher than that of other BmBacmids, and in particular, the increase rate of EGFP activity peaked on the 5th day and the result on the 4th day of rBm-p6.9v-EGFP-Δp10 showed an increase rate of about 1.2 times. and rBm-p6.9v-EGFP-wt showed an increase rate of about 3.2 times in comparison with the results on the 3rd day, confirming the removal effect of cysteine protease and chitinase on the enhancement of protein expression efficiency. Because dissolution occurred, it showed an effect of increasing the survival rate of about 4 days compared to rBm-p6.9v-EGFP-wt.

Therefore, it was confirmed that BmBacmid was the most efficient for the production of exogenous useful proteins by confirming the highest EGFP expression and delay in cell death in the form of BmBacmid in which p10/cysteine protease/chitinase of BmNPV was removed. In the case of the existing AcMNPV p10/cysteine protease/chitinase-removed form of bacmid, the effect of increasing the expression level of EGFP was confirmed, but rather, a phenomenon in which cell viability decreased through the removal of p10 was reported (Hitchman et al., 2010). , In this study, the effect of increasing both EGFP expression level and cell viability through removal of the p10/cysteine protease/chitinase gene of BmNPV was presented for the first time (FIG. 9).

< Example 3> new BmBacmid Characterization

In order to increase the target protein production of the baculovirus expression vector system, the p10 gene promoter, p10 gene, cysteine protease gene, and chitinase gene were removed from BmBacmid. The finally completed BmBacmid-Δpcc exhibited an effect of about 3.2 times higher recombinant protein production and a 48 hour delay in the viability of virus-infected cells compared to recombinant BmNPV using the GOZA system similar to wild-type. Therefore, the possibility that these effects may have any effect on normal virus proliferation was further investigated. To this end, we tried to complete the construction of the BmBacmid-Δpcc system for recombinant protein expression by evaluating the primary viability of cells and larvae infected with the recombinant BmNPV produced through BmBacmid-Δpcc as well as the normal BV proliferation of BmBacmid-Δpcc.

<3-1> BmBacmid according to improvement BV production test

① BmBacmid BV impact on production

In order to compare the production of BV for each type of BmBacmid produced, recombinant BmNPV was inoculated into Bm5 cells, and the virus was collected at 24 hour intervals from 24 hours to 144 hours to measure the daily production of BV. In the case of BmNPV-K1, a wild strain baculovirus used as a control ^{, it exhibited a titer of about 2 × 10 8} pfu/mL through rapid BV production up to 72 hours after inoculation, and after that, the titer peaked at 96 hours. After that, it showed a gradually decreasing pattern (FIG. 10). In addition, in the case of recombinant BmNPV using the GOZA system, each recombinant BmNPV by BmBacmid-Δp10 and BmBacmid-Δpcc showed a slightly lower titer than the control group, but showed a rapid BV production up to 72 hours, and then gradually increased. was shown (FIG. 10). In the end, it was confirmed that the low pathogenicity of BmBacmid-Δpcc to host cells was not related to the production of BV and was an effect of the removal of the pathogenic gene by showing a titer at a level almost similar to that of the wild strain at 120 hours of infection.

<3-2> BmBacmid hospital history black

① Bm5 for cells hospital history black

In order to verify the effect of weakening pathogenicity and increasing cell viability of BmBacmid-Δpcc-VV expressing BmBacmid-Δp10, BmBacmid-Δpcc and Vankyrin-1, cells infected with BmNPV-K1, a wild strain baculovirus, and non-inoculated with baculovirus Comparative evaluation with cells was performed. Bm5 cells were inoculated with each virus, and cells were collected at 1 day intervals until the 7th day, stained with trypan blue, and the viability of Bm5 cells was measured using a hemocytometer. Bm5 cells, which are non-inoculated baculovirus cells, showed a survival rate of about 90% until the 6th day, and then showed a decrease in the survival rate from the 7th day due to nutrient depletion of the culture medium and lack of growth space (Fig. 11). Cells infected with BmNPV-K1 showed a survival rate of about 90-80% until the 3rd day of infection, followed by a sharp decline, and showed a survival rate of less than 20% on the 7th day (FIG. 11). In addition, the GOZA virus, which is almost similar to the wild strain baculovirus, also showed a pattern very similar to that of BmNPV-K1. On the other hand, BmBacmid-Δp10 showed a survival rate of 80% or more until the 6th day, which was later than those of these viruses, and then declined sharply. In particular, BmBacmid-Δpcc and BmBacmid-Δpcc-vv showed a high survival rate of about 85% on the 6th day of infection and a slight decrease on the 7th day, but still showed a high survival rate of 70% or more (FIG. 11). This result is a very high survival rate considering that the 7-day survival rate of the virus-inoculated cells is about 82%, and it was about 50% higher than that of BmNPV-K1. Therefore, this result indicates that a significant level of pathogenicity was lost through the removal of the p10 gene and chitinase, which are pathogenic genes of baculovirus. It is expected that a higher yield can be expected than before by extending the production time of the recombinant protein according to the increase in cell viability. In particular, virus-like particles of an enveloped virus that surround the cell membrane of cells and are released. It is a result that proves that it can be a very useful tool for like particle (VLP) production.

② For silkworm larvae hospital history black

To confirm the pathogenicity of BmBacmid-Δp10, BmBacmid-Δp10-VV, BmBacmid-Δpcc and BmBacmid-Δpcc-VV in silkworm larvae, the same viruses as in the above experiment were diluted ^{to 1 × 10 5 pfu in TC-100 medium.} Using an mL syringe, 10 5 instar silkworms were inoculated and the average survival time was measured. As a result, in the case of untreated silkworms that were not inoculated with the virus, all silkworms were cocooned and lysed on the 7th and 8th days. For silkworms infected with BmNPV-K1, an average of 120.0 hours, for silkworms infected with GOZA virus, 140.8 hours, and for BmBacmid-Δp10, 166.4. Time, 187.2 hours for BmBacmid-Δp10-VV, 201.6 hours for BmBacmid-Δpcc, and 193.6 hours for BmBacmid-Δpcc-VV, almost similar to the results in Bm5 cells, BmBacmid-Δpcc and BmBacmid-Δpcc-VV were very In the case of BmBacmid-Δpcc-infected silkworms with low pathogenicity and the longest survival time, the survival time was increased by about 82 hours compared to BmNPV-K1, resulting in the production of recombinant proteins or virus-like particles using cells as well as silkworm larvae. It was a result of reconfirming that it is a very efficient expression system (Table 4).

virus ST50(h) 95% confidence interval (h) Lower Upper Control silkworms 192.0 ^a* 187.2 196.8 BmNPV-K1 120.0 ^d 105.6 129.6 GOZA 140.8 ^c 139.2 144.0 BmBacmid-Δp10 166.4 ^b 144.0 182.4 BmBacmid-Δp10-VV 187.2 ^a 182.4 192.0 BmBacmid-Δpcc 201.6 ^a 196.8 206.4 BmBacmid-Δpcc-VV 193.6 ^a 182.4 201.6

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Baculovirus with increased yield of foreign protein production <130> PN1903-146 <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 110 <212> DNA <213> Unknown <220> <223> p10 promoter DNA seq <400> 1 gacctttaat tcaacccaac acaatatatt acagctaaat aagaattatt attaaattat 60 ttgtatatta attaaaatct tatactgtaa attacattt atttactatc 110 <210> 2 <211> 285 <212> DNA <213> Unknown <220> <223> p10 gene DNA seq <400> 2 atgtcaaagc ctaacgtttt gacgcaaatt ttagacgccg ttacggaaac taacacaaag 60 gttgacagtg ttcaaactca gttaaacggg ctggaagaat cattccagct tttggacggt 120 ttgcccgctc aattgaccga tcttaacact aagatctcag aaattcaatc catattgacc 180 ggcgacattg ttccggatct tccagactca ctaaagccta agctgaaaag ccaagctttt 240 gaactcgatt cagacgctcg tcgtggtaaa cgcagttcca agtaa 285 <210> 3 <211> 94 <212> PRT <213> Unknown <220> <223> p10 gene A.A. seq <400> 3 Met Ser Lys Pro Asn Val Leu Thr Gln Ile Leu Asp Ala Val Thr Glu 1 5 10 15 Thr Asn Thr Lys Val Asp Ser Val Gln Thr Gln Leu Asn Gly Leu Glu 20 25 30 Glu Ser Phe Gln Leu Leu Asp Gly Leu Pro Ala Gln Leu Thr Asp Leu 35 40 45 Asn Thr Lys Ile Ser Glu Ile Gln Ser Ile Leu Thr Gly Asp Ile Val 50 55 60 Pro Asp Leu Pro Asp Ser Leu Lys Pro Lys Leu Lys Ser Gln Ala Phe 65 70 75 80 Glu Leu Asp Ser Asp Ala Arg Arg Gly Lys Arg Ser Ser Lys 85 90 <210> 4 <211> 1659 <212> DNA <213> Unknown <220> <223> chitinase gene DNA seq <400> 4 atgttgtaca aattgttaaa cgttttgtgg ttggtggtcg ccgtttccaa cgcgattccc 60 ggcacgccgg tgatcgattg ggccgaacgc aattatgcgc tcgtaaaaat aaattacgag 120 gccaccgctt acgaaaattt aataaagctc aaagaacaag tcgacgttca cgtcagttgg 180 aacgtatgga acggcgacat tggcgacata gcgtacgtgt tctttgacga gcagcaggta 240 tggaaaggcg acgccgacag taaaagggct accattaatg ttattgtgag cgggcaattt 300 aacatgcgtg tcaaactttg caatgaggac ggctgttcca taagcgatcc cgtgttggtc 360 aaaatcgcag acaccgacgg cggtcatctg gcgccgctcg aatacacatg gctggaaaac 420 aacaaacccg gcagaagaga ggataaaatt gtcgctgcgt actttgtcga gtggggtgtg 480 tacgggcgca gctttcccgt agacaaagtt cccttgccaa atttatcgca cttgttgtac 540 ggtttcatac ccatctgcgg cggcgatgga ataaacgacg ccctcaaaac aatacccgga 600 agttttgaag ctctgcaacg atcgtgcagg ggacgcgaag atttcaaagt tgccatccac 660 gatccgtggg ccgccgtaca aaaaccccaa aagggcgtgt ccgcttggaa cgaaccgtac 720 aaaggcaatt ttggacaatt gatggcggcg aaattagcaa acccacatct aaaaattctt 780 ccttcaatag gaggctggac tctgtcggac ccattctatt tcatgcacga cgttgaaaaa 840 agaaacgttt ttgtagagtc ggttaaggaa tttttgcaag tgtggaaatt ttttgatggt 900 gtagacgtcg attgggaatt tccgggcggc aaaggggcta acccgtcgct gggcgatgcg 960 gagcgtgacg ccaaaacata cattctgttg ttggatgagc tgcgcgaaat gctagacgac 1020 ctcgaagtgc aaaccggcag ggtttacgaa ttaacaagcg ctataagcgc gggctacgac 1080 aagattgccg tggtaaacta cgccgaagcg caaaagtcat tagacaaaat atttctcatg 1140 acttacgatt ttaaaggggc ttggtcaaac acggatttgg gctaccaaac aacagtctac 1200 gcgccaagtt ggaactcgga agagctgtac actacacatt acgctgtcga tgcgttactg 1260 gaacaaggcg tcgatcccaa caaaataatt gtgggcgtcg ccatgtacgg ccgcggctgg 1320 accggcgtaa caaattatac gaatggcaat tatttttccg gcactggcaa cgggccggtg 1380 tcgggcacgt gggaggacgg tgttgtagat tatcgtcaaa ttcaaaaaga tctcaacaat 1440 tatgtgtaca cgtttgacag cgccgctcaa gcgtcgtacg ttttcgataa aagtaaaggc 1500 gatttgattt cgtttgacag cgtcgactct gtgttaggaa aagttaaata tgtcgaccga 1560 aataaattgg gcggcttgtt tgcttgggag attgatgccg ataacggcga cttgctcaac 1620 gcgatgaacg cacagtttaa acttagagat gaactgtaa 1659 <210> 5 <211> 552 <212> PRT <213> Unknown <220> <223> chitinase gene A.A. seq <400> 5 Met Leu Tyr Lys Leu Leu Asn Val Leu Trp Leu Val Val Ala Val Ser 1 5 10 15 Asn Ala Ile Pro Gly Thr Pro Val Ile Asp Trp Ala Glu Arg Asn Tyr 20 25 30 Ala Leu Val Lys Ile Asn Tyr Glu Ala Thr Ala Tyr Glu Asn Leu Ile 35 40 45 Lys Leu Lys Glu Gln Val Asp Val His Val Ser Trp Asn Val Trp Asn 50 55 60 Gly Asp Ile Gly Asp Ile Ala Tyr Val Phe Phe Asp Glu Gln Gln Val 65 70 75 80 Trp Lys Gly Asp Ala Asp Ser Lys Arg Ala Thr Ile Asn Val Ile Val 85 90 95 Ser Gly Gln Phe Asn Met Arg Val Lys Leu Cys Asn Glu Asp Gly Cys 100 105 110 Ser Ile Ser Asp Pro Val Leu Val Lys Ile Ala Asp Thr Asp Gly Gly 115 120 125 His Leu Ala Pro Leu Glu Tyr Thr Trp Leu Glu Asn Asn Lys Pro Gly 130 135 140 Arg Arg Glu Asp Lys Ile Val Ala Ala Tyr Phe Val Glu Trp Gly Val 145 150 155 160 Tyr Gly Arg Ser Phe Pro Val Asp Lys Val Pro Leu Pro Asn Leu Ser 165 170 175 His Leu Leu Tyr Gly Phe Ile Pro Ile Cys Gly Gly Asp Gly Ile Asn 180 185 190 Asp Ala Leu Lys Thr Ile Pro Gly Ser Phe Glu Ala Leu Gln Arg Ser 195 200 205 Cys Arg Gly Arg Glu Asp Phe Lys Val Ala Ile His Asp Pro Trp Ala 210 215 220 Ala Val Gln Lys Pro Gln Lys Gly Val Ser Ala Trp Asn Glu Pro Tyr 225 230 235 240 Lys Gly Asn Phe Gly Gln Leu Met Ala Ala Lys Leu Ala Asn Pro His 245 250 255 Leu Lys Ile Leu Pro Ser Ile Gly Gly Trp Thr Leu Ser Asp Pro Phe 260 265 270 Tyr Phe Met His Asp Val Glu Lys Arg Asn Val Phe Val Glu Ser Val 275 280 285 Lys Glu Phe Leu Gln Val Trp Lys Phe Phe Asp Gly Val Asp Val Asp 290 295 300 Trp Glu Phe Pro Gly Gly Lys Gly Ala Asn Pro Ser Leu Gly Asp Ala 305 310 315 320 Glu Arg Asp Ala Lys Thr Tyr Ile Leu Leu Leu Asp Glu Leu Arg Glu 325 330 335 Met Leu Asp Asp Leu Glu Val Gln Thr Gly Arg Val Tyr Glu Leu Thr 340 345 350 Ser Ala Ile Ser Ala Gly Tyr Asp Lys Ile Ala Val Val Asn Tyr Ala 355 360 365 Glu Ala Gln Lys Ser Leu Asp Lys Ile Phe Leu Met Thr Tyr Asp Phe 370 375 380 Lys Gly Ala Trp Ser Asn Thr Asp Leu Gly Tyr Gln Thr Thr Val Tyr 385 390 395 400 Ala Pro Ser Trp Asn Ser Glu Glu Leu Tyr Thr Thr His Tyr Ala Val 405 410 415 Asp Ala Leu Leu Glu Gln Gly Val Asp Pro Asn Lys Ile Ile Val Gly 420 425 430 Val Ala Met Tyr Gly Arg Gly Trp Thr Gly Val Thr Asn Tyr Thr Asn 435 440 445 Gly Asn Tyr Phe Ser Gly Thr Gly Asn Gly Pro Val Ser Gly Thr Trp 450 455 460 Glu Asp Gly Val Val Asp Tyr Arg Gln Ile Gln Lys Asp Leu Asn Asn 465 470 475 480 Tyr Val Tyr Thr Phe Asp Ser Ala Ala Gln Ala Ser Tyr Val Phe Asp 485 490 495 Lys Ser Lys Gly Asp Leu Ile Ser Phe Asp Ser Val Asp Ser Val Leu 500 505 510 Gly Lys Val Lys Tyr Val Asp Arg Asn Lys Leu Gly Gly Leu Phe Ala 515 520 525 Trp Glu Ile Asp Ala Asp Asn Gly Asp Leu Leu Asn Ala Met Asn Ala 530 535 540 Gln Phe Lys Leu Arg Asp Glu Leu 545 550 <210> 6 <211> 972 <212> DNA <213> Unknown <220> <223> Cysteine protease gene DNA seq <400> 6 atgaacaaaa ttttgtttta tttgtttgtg tacgccgttg taaagagcgc ggcctacgat 60 cctttgaaag cgcctaatta ttttgaagaa tttgttcatc gattcaacaa aaattatagt 120 agcgaagttg aaaaattgcg aagattcaaa attttccaac acaatttaaa tgaaattatc 180 aataaaaacc aaaacgattc ggccaaatat gaaataaaca aattctcgga tttgtccaaa 240 gacgaaacta tcgcaaaata cacaggtctg tctttgccta ctcagactca aaatttttgc 300 aaggtcatac tcttagacca gccgccgggt aaagggcccc ttgaatttga ctggcgtcgt 360 ctcaacaaag tcactagcgt aaaaaatcag ggcatgtgtg gcgcctgctg ggcgtttgcc 420 actctggcta gtttggaaag tcaatttgca atcaaacata accagttgat taatctgtcg 480 gagcagcaaa tgatcgattg tgattttgtc gacgctggct gtaacggcgg cttgttgcac 540 acagcgttcg aagccatcat taaaatgggc ggcgtacagc tggaaagcga ctatccatac 600 gaagcagaca ataacaattg ccgtatgaac tccaataagt ttctagttca agtaaaagat 660 tgttatagat acattaccgt gtacgaggaa aaacttaaag atttgttacg ccttgtcggc 720 cctattccta tggccataga cgctgccgac attgttaact ataaacaggg tattataaaa 780 tattgtttcg acagcggtct aaaccatgcg gttcttttag tgggttatgg tgttgaaaac 840 aacattccat attggacctt taaaaacact tggggcacgg attggggaga ggacggattt 900 ttcagggtac aacaaaacat aaacgcctgt ggtatgagaa acgaacttgc gtctactgca 960 gtcatttatt aa 972 <210> 7 <211> 323 <212> PRT <213> Unknown <220> <223> Cysteine protease gene A.A. seq <400> 7 Met Asn Lys Ile Leu Phe Tyr Leu Phe Val Tyr Ala Val Val Lys Ser 1 5 10 15 Ala Ala Tyr Asp Pro Leu Lys Ala Pro Asn Tyr Phe Glu Glu Phe Val 20 25 30 His Arg Phe Asn Lys Asn Tyr Ser Ser Glu Val Glu Lys Leu Arg Arg 35 40 45 Phe Lys Ile Phe Gln His Asn Leu Asn Glu Ile Ile Asn Lys Asn Gln 50 55 60 Asn Asp Ser Ala Lys Tyr Glu Ile Asn Lys Phe Ser Asp Leu Ser Lys 65 70 75 80 Asp Glu Thr Ile Ala Lys Tyr Thr Gly Leu Ser Leu Pro Thr Gln Thr 85 90 95 Gln Asn Phe Cys Lys Val Ile Leu Leu Asp Gln Pro Pro Gly Lys Gly 100 105 110 Pro Leu Glu Phe Asp Trp Arg Arg Leu Asn Lys Val Thr Ser Val Lys 115 120 125 Asn Gln Gly Met Cys Gly Ala Cys Trp Ala Phe Ala Thr Leu Ala Ser 130 135 140 Leu Glu Ser Gln Phe Ala Ile Lys His Asn Gln Leu Ile Asn Leu Ser 145 150 155 160 Glu Gln Gln Met Ile Asp Cys Asp Phe Val Asp Ala Gly Cys Asn Gly 165 170 175 Gly Leu Leu His Thr Ala Phe Glu Ala Ile Ile Lys Met Gly Gly Val 180 185 190 Gln Leu Glu Ser Asp Tyr Pro Tyr Glu Ala Asp Asn Asn Asn Cys Arg 195 200 205 Met Asn Ser Asn Lys Phe Leu Val Gln Val Lys Asp Cys Tyr Arg Tyr 210 215 220 Ile Thr Val Tyr Glu Glu Lys Leu Lys Asp Leu Leu Arg Leu Val Gly 225 230 235 240 Pro Ile Pro Met Ala Ile Asp Ala Ala Asp Ile Val Asn Tyr Lys Gln 245 250 255 Gly Ile Ile Lys Tyr Cys Phe Asp Ser Gly Leu Asn His Ala Val Leu 260 265 270 Leu Val Gly Tyr Gly Val Glu Asn Asn Ile Pro Tyr Trp Thr Phe Lys 275 280 285 Asn Thr Trp Gly Thr Asp Trp Gly Glu Asp Gly Phe Phe Arg Val Gln 290 295 300 Gln Asn Ile Asn Ala Cys Gly Met Arg Asn Glu Leu Ala Ser Thr Ala 305 310 315 320 Val Ile Tyr <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> M13F(-40) <400> 8 gttttcccag tcacgac 17 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> M13R(-40) <400> 9 caggaaacag ctatgac 17 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> p10-5'-F <400> 10 ggtacctgtc atttattaat ttggatga 28 <210> 11 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> p10-5'-R <400> 11 aagcttttaa ctataatata ttgtgttggg t 31 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> p10-3'-F <400> 12 ctgcagatga atcgttttta aaataaca 28 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> p10-3'-R <400> 13 actagtgaag aacacacgat catgg 25 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Cm-F <400> 14 gacgtcatag atctgggcca acttttgg 28 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Cm-R <400> 15 gggccccagg cgtttaaggg caccaataac 30 <210> 16 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Chitinase-F <400> 16 ctgcagttga gcaagtcgcc gttatcggc 29 <210> 17 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Cystein-R <400> 17 gaattccgac aaaatcacaa tcgatcattt g 31

Claims (10)

The p10 gene promoter, p10 gene, chitinase and cysteine proteolytic enzyme (v-cath) are all deleted to improve the viability of host cells, thereby increasing the production of foreign target proteins by recombinant baculovirus recombinant silkworm. Nuclear polyhedrosis virus (Bombyx mori nucleopolyhedrovirus, BmNPV). delete preparing a recombinant baculovirus transfer vector in which the p10 gene promoter, the p10 gene, the gene encoding chitinase and the gene encoding cysteine proteolytic enzyme (v-cath) are all deleted;
cloning the nucleotide sequence encoding the foreign target protein into the transfer vector; and
and introducing the recombinant transfer vector and Bacmid prepared by cloning into Bm5 insect cells, which are host cells,
The baculovirus is characterized in that the silkworm nuclear polyhedrosis virus (Bombyx mori nucleopolyhedrovirus, BmNPV),
A method for mass production of foreign target proteins by improving the survival rate of host cells. delete delete delete delete delete delete delete

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