• 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.

• The Korean Journal of Pesticide Science
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• v.12 no.3
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• pp.283-290
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• 2008

Baculoviruses have been used to control some serious lepidopteran pests. However, their narrow target insect spectrum and slow efficacy are main limitations to be used in various applications. This study introduces a technique to overcome these limitations by inhibiting insect immune defence to enhance the viral pathogenicity. Polydnaviruses are an insect DNA virus group and symbiotic to some ichneumonid and braconid endoparasitoids. Cotesia plutellae bracovirus (CpBV) is a braconid polydnavirus and encodes several immunosuppressive genes. We selected seven CpBV genes and recombined them to wild type Autographa California multiple nucleopolyhedrovirus (AcNPV). A bioassay of these seven recombinants indicated that most recombinants had similar or superior efficacy to wild type AcNPV against beet armyworm, Spodoptera exigua, and diamondback moth, Plutella xylostella. Recombinant AcNPV with CpBV-ELP was the most potent in terms of lethal time by shortening more than 2 days compared to wild type AcNPV. This recombinant was further proved in its dose-dependent pathogenicity and its efficacy by spray application on S. exigua infesting cabbage cultivated in pots. We discussed the efficacy of CpBV-ELP recombinant AcNPV in terms of suppressing antiviral activity of target insects.

• Korean Chemical Engineering Research
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• v.44 no.1
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• pp.40-45
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• 2006

The baculovirus-insect Drosophila melanogaster S2 cell system combines advantages of conventional baculovirus system and non-lytic S2 cell system because baculoviruses can infect non-permissive cells such as mammalian and Drosophila S2 cells but cannot replicate themselves inside the cells. In the present work, we investigated effects of infection conditions on production of green fluorescent protein (GFP) as a target protein using this baculovirus-S2 cell system. Even though higher MOI and longer baculovirus contact time showed better GFP expression yield during the shorter period, overall protein yield could be lower during the longer period due to the relatively higher cell detachment and lysis (lower cell viability). In addition, maintaining high MOI will be not practical for large-scale cell culture. Therefore, instead of maintaining high MOI, we found that high initial cell number and concentrated (10X) baculovirus volume can confer comparable protein expression even under the moderate MOI condition. Also, we found that the post-infection time that is connected to state of cells after infection was an important factor for production yield.

• The Journal of Korean Society of Virology
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• v.28 no.3
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• pp.247-265
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• 1998

To determine the sequence and expression of the VP7 gene of Korean isolates (CAU-9), viral RNA was purified and used for cDNA amplification by RT-PCR. The VP7 cDNA was cloned, sequenced, and expressed using baculovirus expression system. The result showed that the sequence homologies CAU-9 compared with foreign isolated strains Wa, 417, TMC-II, 95B and SA11 were ranged from 74.0% to 95.1 % of nucleotide sequence and 35% to 43% of amino acid sequence, respectively. High homology of CAU-9 was observed in Japanease isolates 417 (nucleotide sequence homology was 95.1% and amino acid sequence homology was 43%). To express VP7 gene, the VP7 cDNA was cloned into pCR-Bac vector and inserted into the genome of baculovirus adjacent to the polyhedrin promoter by cotransfection of Spodoptera frugiperda (Sf9) insect cells with wild type baculovirus DNA. In antigenic analysis of Sf9 cells inoculated with the recombinant VP7, immunofluorescence assay revealed positive for viral antigens. In metabolic labeling of Sf9 cell lysates infected with recombinant baculoviruses, it was revealed that the protein of 34 kDa was expressed. The limited study of expressed VP7 protein inoculated with guinea pigs failed to elicit neutalizing antibody. As a results, the sequence analysis and expression of VP7 protein of rotavirus CAU-9 isolated from an infant in Korea could permit the conformation and development of virus like particles which may be useful in designing vaccine strategy.