• Journal of Sericultural and Entomological Science
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• v.38 no.2
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• pp.144-149
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• 1996

To develope the baculovirus expression vector system (BEVS) using Spodoptera exigua nuclear polyhedrosis virus (SeNPV), we characterized the polyhedrin of SeNPV. The SeNPV polyhedra was irregular and composed of the major protein molecular weight of 30 kDa determined by electronmicroscopy and SDS-AGE analysis, respectively. The nucleotid suquences of 876 bases including the coding region of polyhedrin gene was determined and it was revealed that the polyhedrin gene is located within Xho I 3.0Kb and Nco I 6.0 Kb by Southern blot analysis, respectively. Also, the Xho I 3.0 Kb and the Nco I 6.0 Kb fragments were cloned and restriction enzyme map of these clones were determined.

• Korean journal of applied entomology
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• v.36 no.4
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• pp.341-350
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• 1997

We have now constructed a novel recombinant baculovirus producing fusion protein with Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin and Bacillus thuringiensis(Bt) cryIA(c) crystal protein. The fusion protein expressed by the recombinant baculovirus in insect cells was characterized. The N-terminal of cryIA(c) gene of Bt subsp. kurstaki HD-73 was introduced under the control of polyhedrin gene promoter of AcNPV, by fusion in the front of intact polyhedrin gene or by insertion into the HindIII site in polyhedrin gene. The recombinant baculoviruses were named as BtrusI or BtrusII, respectively. Although single transcript from the fusion protein gene was apparently observed. BtrusI was produced the two proteins, 92 kDa fusion protein and only polyhedrin. In addition, fusion protein produced by BtrusI did not form polyhedra. Interestingly, however, the cells infected with BtrusII did not show a 33 kDa polyhedrin band as a cells infected with BtrusI. Cells infected with BtrusII were only produced fusion protein, but the polyhedra formed by fusion protein was not observed. To determine the insecticidal toxicity of fusion protein, therefore, Sf9 cells infected with BtrusI were inoculated to Bombyx mori larvae. Sf9 cells infected with BtrusI that expressed the fusion protein caused larval mortality although the insecticidal toxicity was low. In conclusion, our results clearly demonstrated that the fusion protein with polyhedrin and Bt cryIA(c) crystal protein have a insecticida toxicity.

• Journal of Sericultural and Entomological Science
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• v.38 no.1
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• pp.36-41
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• 1996

Recombinant baculoviruses having expanded host range were selected by coinfection of Autographa california NPV and Bombyx mori NPV into Sf-9 and BmN-4 insect cell lines. In order to determine the polyhedra morhplogy of RecS-A6, one of a recombinant baculovirus, polyhedra of RecS-A6 produced in insect cells were observed by phase contrast microscope and scanning electron microscope. The results revealed that the recombinant baculovirus had a various polyhedra morphology which was different from its parental viruses, suggesting that the various morhpology of recombinant baculovirus with an expanded host range was due to the genetic recombination of viral genome. To analyze the genomic recombinantion of the recombinant baculoviruses, genomic DNAs of two parent viruses and RecS-A6 were digested with restriction endonuclease and subjected to agarose gel electrophoresis. Southern blot analysis revealed that RecS-A6 has two polyhedrin gene of AcNPV and BmNPV in a viral genome. Polyhedral protein of recombinant baculovirus was analysed by SDS-PAGE. The result showed that molecular weight of polyhedral protein of RecS-A6 containing two polyhedrin gene of AcNPV and BmNPV was as the 31 kDa band of AcNPV and 30 kDa band of BmNPV.

• Journal of Sericultural and Entomological Science
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• v.34 no.2
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• pp.20-25
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• 1992

The location of the polyhedrin gene of Bmbyx mori nuclear polyhedrosis virus(BmNPV) was determined by using a cloned polyhedrin gene from the Autographa californica nuclear polyhedrosis virus(AcNPV) as a hybridization probe. The 7.4 Kb PstⅠ fragment DNA of Bm-NPV was cloned to plasmid pUC19 vector. A fragment containing this gene was mapped and sequenced in its entire polyhedrin reading frame. Nucleotide sequences comparison of the polyhedrin of the BmNPV to that of previously reported by Ⅰatrou(1985) revealed that the sequence varied in 10 base, Comparison of the amino acid sequence of the two structured gene revealed that coding sequence varied 74 valine to isoleucine, 76 aspargine to serine and 155 methionine to valine.

• Korean journal of applied entomology
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• v.38 no.2
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• pp.139-144
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• 1999

We have now constructed a novel recombinant baculovirus producing fusion protein with Autogrqha c.uliforrzica nuclear polyhedrosis virus (AcNPV) polyhedrin and green fluorescent protein (GFP). The fusion protein expressed by the recombinant baculovirus in insect cells was characterized. The GFP gene was introduced under the control of polyhedrin gene promoter of AcNPV, by fusion in the front or back of intact polyhedrin gene. The recombinant baculoviruses were named as Ac-GFPPOL or Ac-POLGFP. respectively. As expected, the 56 kDa fusion protein was expressed in the recombinant virus-infected cells. Interestingly. however, the fluorescence of GFP in the cells infected with Ac- POLGFP was only detected within the nuclei. and that was observed as polyhedra-like granular particles. In the microscopy of cells infected with Ac-GFPPOL, furthermore, GFP was detected in both cytoplasm and nuclei although most of GFP were present within the nuclei. However, fusion protein produced by recombinant virus did not form polyhedra although the fusion protein was fused with polyhedrin and GFP. It is suggested that difference of GFP location in the infected cells appear to be involved in the region of polyhedrin in the fusion protein, and the polyhedrin in the fusion protein might be responsible for the polyhedra-like granular particles present within nuclei.

• Korean journal of applied entomology
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• v.32 no.1
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• pp.51-60
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• 1993

The polyhedrin gene-containing DNA fragment of Hyphantria cunea nuclear polyhedrosis virus (HcNPV) was localized by southern hybridization with Autographa california CPA EcoRI-I fragment (7.3 kb), Bombyx mori NPV PatI-F fragment (7 kb) and synthetic oligonucleotide(30-mer) as probes. the PstI-L(5.3 kb) fragment of HcNPV was cloned to E. coli and the plasmid of the fragment was named as pHcP-L(8.0 kb). The pHcP-L was physically mapped and subcloned to E. coli as pHcP-L1(4.7 kb), pHcP-L2(7.1 kb), pHcP-L3(5.3 kb), pHcP-L4(4.2 kb) and pHcP-L5(4.5 kb).

• Journal of Sericultural and Entomological Science
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• v.37 no.1
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• pp.46-51
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• 1995

In order to develope baculovirus expression vector system, we constructed new transfer vector of nuclear polyhedrosis virus of the silkworm, Bombyx mori. The promoter region containing only adenine of translation start codon of polyhedrin gene was cloned by polymerase chain reaction technique. And the 5' and 3' leader regions of polyhedrin gene was sequentially cloned. The polyhedrin coding gene was deleted from the ＋2 to the ＋597 position. As the result, we constructed new transfer vector which has EcoRI, SacI and KpnI sites for the cloning sites of foreign gene. New transfer vector was named as pBmKSKl. Escherichia coli $\beta$-galactosidase gene as foreign gene was inserted into pBmKSKl, under the control of the polyhedrin promoter and expressed in B. mori cells. The result showed that the new transfer vector pBmKSK1 is functional.