• International Journal of Industrial Entomology and Biomaterials
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• 제2권2호
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• pp.155-160
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• 2001

We have generated a novel baculovirus genome which can be maintained in Escherichia coli that facilitates the rapid and efficient generation of recombinant baculovirus expression vectors. To make $Occ^{+}$ recombinant expression vectors, polyhedrin gene under the control of p10 promoter was inserted to bAcGOZA and this genome was designated bApGOZA. As in bAcGOZA, bApGOZA lacks a portion of the essential ORF1629 gene, but includes a mini-F replicon and selectable kanamycin-resistance marker, This occasion-producing activity of bApGOZA can be used very conveniently for its oral infectivity to insect larvae in mass production of foreign protein and insecticides.

• International Journal of Industrial Entomology and Biomaterials
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• 제6권1호
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• pp.49-55
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• 2003

We describe the generation of transgenic silkworm that carrying the chimeric fibroin light chain (L-chain) gene. Previously, we have cloned the complete fibroin L-chain gene from the silkworm Baekok-Jam, Bombyx mori, and the complete fibroin gene from the oak silkworm, Antheraea yamamai. The 444 bp repetitive sequence of A. yamamai fibroin gene was inserted into the exon 6 of B. mori fibroin L-chain gene to produce chimeric fibroin L-chain gene. The chimeric fibroin L-chain gene was cloned into the polyhedrin gene site of Autographa californica nuclear polyhedrosis virus (AcNPV) to yield a recombinant baculovirus as a fibroin gene targeting vector, One-day-old fifth instar female silkworm larvae were injected with the recombinant baculovirus and then mated with normal male moths. Genomic DNA from their progenies was extracted and screened for the desired targeting event by using PCR and Southern blot analysis. The analysis showed that the chimeric fibroin gene had intergrated into the L-chain gene on the genome by homologous recombination and was transmitted through generations. The transgenic silkworm carrying the chimeric fibroin gene were approximately 43.2% in $F_2$ generation, and the silkworms synthesized the fusion protein in cocoons layer.

• 한국미생물·생명공학회지
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• 제24권1호
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• pp.72-76
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• 1996

To investigate the expression efficiency of new transfer vector of Bombyx mori nuclear polyhedrosis virus (BmNPV), Escherichia coli lacZ gene was inserted into new transfer vector pBmKSK1, under the control of polyhedrin promoter and expressed in BmN-4 cells and larvae of silkworm, Bombyx mori. The recombinant virus containing lacZ gene was isolated from BmN-4 cells coinfected with transfer vectro pBmKSK1-LacZ and wild type BmNPV genome, and analysed by Southern blotting. The expression of ${\beta}$-galactosidase was characterized by SDS-PAGE, Western blotting and ${\beta}$-galactosidase activity assay. The results showed that the level of expression in silkworm larvae was higher than that of BmN-4 cells.

• Journal of Microbiology and Biotechnology
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• 제15권3호
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• pp.466-471
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• 2005

An Escherichia coli strain with high phytase activity was screened from pig excreta. The phytase gene, appA, was amplified by PCR technique. To obtain large amounts of appA phytase, the appA gene was subcloned into the baculovirus transfer vector pVL1393 under the control of the Polyhedrin promoter. The recombinant baculovirus harboring the appA gene was obtained after co-transfection and screening. The early $5^{th}$ instar larvae of silkworm were infected with the recombinant virus. Using this system, the appA phytase was overproduced up to 7,710 U per ml hemolymph. SDS-PAGE analysis revealed the baculovirus-derived appA phytase to be approximately 47 kDa in size. The optimal temperature and pH of the expressed phytase were $60^{\circ}C$ and pH 4.5, respectively. The enzymatic activity was increased by the presence of 1 mM $Ca^{2+}$, 1 mM $Mn^{2+}$, or $0.02\%$ Triton X-100.

• Journal of Microbiology and Biotechnology
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• 제2권2호
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• pp.122-128
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• 1992

Post-translational modifications of the nucleocapsid protein of bovine coronavirus (Quebec strain) were investigated. Coronavirions were radiolabelled in vivo with inorganic $[^{32}P]$orthophosphate and analysed by SDS-PAGE, followed by autoradiography. A single polypeptide with a migration rate of 55 KDa was identified by metabolic phosphate labelling, demonstrating that the nucleocapsid protein of bovine coronavirus was a phosphoprotein. A gene encoding the nucleocapsid protein was inserted immediately downstream from the polyhedrin promoter of Autographa californica nuclear polyhedrosis baculovirus. Spodoptera frugiperda cells infected with this recombinant baculovirus synthesized a 55 KDa polypeptide, as demonstrated by immunoprecipitation with anti-nucleocapsid monoclonal antibody. The recombinant nucleocapsid protein synthesized in Spodoptera cells could also be labelled by $[^{32}P]$orthophosphate. Phosphoamino acid analysis showed that both serine and threonine residues were phosphorylated in authentic, as well as in recombinant nucleocapsid proteins, with a relative phosphorylation ratio of 7:3. Our studies demonstrated that the nucleocapsid protein of bovine coronavirus was a serine and threonine-phosphorylated protein and that Spodoptera insect cells were able to properly phosphorylate the relevant foreign proteins.

• International Journal of Industrial Entomology and Biomaterials
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• 제1권2호
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• pp.149-153
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• 2000

We have constructed a recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), containing green fluorescent protein (GFP) gene from the jellyfish, Aequorea victoria, and transferred it into the domestic silkworm Bombyx mori larvae for the production of visible transgenic silkworm of living organism. When one day-old fifth instar female larvae were injected with the recombinant AcNPV of 1x10$^{5}$ plaque forming units, the bright glow of GFP was detected in the recombinant AcNPV-infected larvae and in the newly hatched larvae of the next generation. Our findings demonstrate that the viral replication was detected in the silkworm treated with the recombinant ACNPV and the gfp gene was expressed under the transcriptional control of the polyhedrin gene promoter, Furthermore, the gfp gene was transmitted to the next generation, suggesting that this system can be applied for the development of transgenic silkworms.

• International Journal of Industrial Entomology and Biomaterials
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• 제4권1호
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• pp.43-49
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• 2002

We have cloned cDNAs encoding toxin from the spider, Araneus ventricosus, and constructed a recombinant baculovirus expressing the insecticidal toxin. The cDNAs encoding toxin were cloned from the cDNA library of A. ventricosus. Sequence analysis of the cDNAs encoding the toxin of A. ventricosus revealed that the 240 bp cDNA for AvTox-1 and 192 bp cDNA for AvTox-2 have an open reading frame of 80 and 64 amino acid residues, respectively. The deduced protein sequence of the toxin genes of AvTox-1 and AvTox-2 was aligned to that of the snack Anemonia sulcata and scorpion Centruroides limpidus limpidus, respectively. Northern blot analysis indicated that AvTox-2 toxin gene showed a fat body-spe-cific expression pattern at the transcriptional level. Furthermore, we have explored the possibility of improving baculovirus by incorporating the A. vontricosus toxin gene into Bombyx mori nuclear polyhedrosis virus genome under the control of polyhedrin promoter, The AvTox-2 toxin gene was expressed as approximately 5.8 kDa band in the recombinant baculovirus-injected silkworm larvae. Bioassays with the recombinant virus expressing AvTox-2 on 5th instar silkworm larvae demonstrated a decrease in the time to kill $(LT_{50} days)$ compared to wild-type BmNPV-Kl $(LT_{50} 6.72 days)$ in the injection of 10 viruses. These results indicate that A. ventricosus toxin is a novel member of the spider toxin family, suggesting that the toxin gene can be used in recombinant baculoviruses to reduce insect feeding damage and increase the speed of insect kill.

• Molecules and Cells
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• 제28권1호
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• pp.19-24
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• 2009

The goal of this study was to create a novel baculovirus expression system that does not require recombinant virus purification steps. Transfection of insect cells with transfer vectors containing barnase under control of the Cotesia plutellae bracovirus (CpBV) promoters ORF3004 or ORF3005 reduced cell growth. Co-transfection with bApGOZA DNA yielded no recombinant viruses and nonrecombinant backgrounds. To further investigate the detrimental effects of barnase on insect cells, two recombinant bacmids harboring the barnase gene under control of the CpBV promoters, namely bAcFast-3004ProBarnase and bAcFast-3005ProBarnase, were constructed. While no viral replication was observed when only the recombinant bacmids were transfected, recombinant viruses were generated when the bacmids were co-transfected with the transfer vector, pAcUWPolh, through substitution of the barnase gene with the native polyhedrin gene by homologous recombination. Moreover, no non-recombinant backgrounds were detected from unpurified recombinant stocks using PCR analysis. These results indicate that CpBV promoters can be used to improve baculovirus expression vectors by means of lethal gene expression under the control of these promoters.

• International Journal of Industrial Entomology and Biomaterials
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• 제19권1호
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• pp.199-204
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• 2009

The newly cloned Bacillus thuringiensis cry1-5 gene showed high activity to both Plutella xylostella and Spodoptera exigua, while cry1Ac only showed high activity against P. xylostella but low to S. exigua. Through the alignment of amino acid sequences between Cry1Ac and Cry1-5, we found 12 different residues in domain I (6 residues) and domain II (6 residues). In this study, the modified cry1Ac gene, which is constructed according to a crop-preferring codon usage, was used as a template to construct mutant B. thuringiensis cry1Ac genes based on cry1-5 gene through multi site-directed mutagenesis. Total 63 various mutant cry genes were obtained at 12 positions randomly. Among them, ten mutant cry genes, whose domain I was totally converted and domain II was randomly, were selected to express in baculovirus expression system as a polyhedrin fusion form. The recombinant proteins were 95 kDa in size and were stably activated as 65 kDa by trypsin. The expressed mutant Cry proteins were applied to bioassays against P. xylostella and S. exigua. All mutants showed high insecticidal activity both to P. xylostella and S. exigua similar to cry1-5. These results suggest that these mutant cry genes might be expected of desirable cry genes for introduction to transgenic crops.

• 한국미생물·생명공학회지
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• 제25권4호
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• pp.359-366
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• 1997

The usefulness of host range expanded recombinant viruses for economical viral insecticide and expression vector system has been studied. Host range expanded recombinant viruses, RecS-B6 and RecB-8, constructed by cotransfection of Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica NPV (AcNPV), and a host range expanded AcNPV recombinant, Ac-BH, constructed by substitution of the 0.6Kb fragment of the BmNPV helicase gene were compared. The restriction enzyme digestion patterns showed that RecS-B6 and RecB-8 had expanded host ranges by genomic recombination and were more similar to genome of AcNPV than that of BmNPV. SDS-PAGE and PCR analysis showed that the polyhedrin gene of RecS-B6 and RecB-8 was derived from BmNPV genomic DNA. The morphology of polyhedra of recombinant viruses showed a slight difference between the two host cells, Sf and BmN cells, indicating that the morphology of polyhedra was influenced by host cells. The bioassay data for insect larvae showed that Ac-BH, compared to wild type viruses, had superior pathogenicity against Bombyx mori larvae but inferior pathogenicity against Spodoptera exigua larvae. Although the pathogenicity was lower than that of wild type viruses in both larvae, RecS-B6 showed the pathogenicity in both larvae. These results suggested that Ac-BH was a less useful economical insecticide than random genomic recombinant virus RecS-B6.