• International Journal of Industrial Entomology and Biomaterials
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• v.4 no.1
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• pp.57-62
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• 2002

Cationic liposomes complexed with DNA have been extensively utilized for the delivery of reporter or therapeutic genes both in culture and in vivo. We investigated and determined the optimum conditions of a cationic liposome, composed of dimethyldioctadecy-lammonium bromide (DDAB) and dioleoyl phosphati-dylethanolamine UOPE), mediated a reporter plasmid expressing luciferase into insect cell lines (Sf-21 and Bm-N) and silkworm larvae. Together the data demonstrated that Bombyx mori nuclear polyhedrosis virus (BmNPV) genomic DNA (128 kb) was successfully transfected into Bm-5 cells using this liposome. These results suggest that DDAB/DOPE liposome will be useful as delivery agents for gene transfer to insect cells both in vitro and in vivo.

• Journal of Sericultural and Entomological Science
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• v.37 no.2
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• pp.181-185
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• 1995

To enhance expression of foreign gene by the novel expression vector, pBmKSK1, of Bombyx mori nuclear polyhedrosis virus, E. coli $\beta$-galactosidase gene expressing recombinant virus was infected in BmN-4 cells and various concentrations of silkworm hemolymph were added to the recombinant virus-infected BmN-4 cells containing fetal bovine serum. The expression efficiency of foreign gene was determined by $\beta$-galactosidase activity in the culture media. The results showed that the silkworm hemolymph was effective to expression of foreign gene in the BmN-4 cells, suggesting that the silkworm hemolymph could be substituted for fetal bovine serum in the BmN-4 cells to enhance expression of foreign gene.

• International Journal of Industrial Entomology and Biomaterials
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• v.18 no.1
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• pp.8-12
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• 2009

A study has been made to know the effect of a phytoecdysteroid 'Sampoorna' on uniform maturation of silkworms during spinning and its effect on diseased silkworms infected by major silkworm disease viruses, Bombyx mori nuclear polyhedrosis virus (BmNPV) and Bombyx mori infectious flacherie virus (BmIFV). In the present investigation, the effect of the phytoecdysteroid "Sampoorna" on Grasserie disease caused by BmNPV have shown an average cocoon melting of 11.91% with a disease incidence of 5.83%. The values of 't' test for different treatments of BmNPV indicated low survival rate and cocoon traits were drastically reduced. Another major disease Flacherie caused by BmIFV has shown considerable levels of larval disease incidence (22-32%) and cocoon melting (3-7.67%) with an average melting of 12.95% and 20.24% disease incidence. There is a drastic reduction in survival rate, cocoon yield and other economic traits. The control batches were indicated negligible values for disease incidence and cocoon melting with Sapoorna application and without the inoculation of the two disease-causing viruses. The application of Sampoorna on already infected batches with major pathogens triggered high mortality and disease incidence and melting percentage was also significantly increased with reduced economic traits. Hence, it is suggested that application of Sampoorna in infected batches should be done only in the extreme conditions of rearing. Application of Sampoorna on healthy batches led to uniform maturation and improvement in productivity with the added advantage of better quality cocoons and labour saving.

• International Journal of Industrial Entomology and Biomaterials
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• v.2 no.1
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• pp.19-26
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• 2001

Transformed Spodoptera frugiperda (Sf9) cells expressing baculovirus very late factor (VLF-1) were constructed by using Autograha nuclear polyhedrosis virus (AcNPV) immediate earthy gene (ie1). Neomycin-resistance gene as a selectable marker was introduced under the control of AcNPV ie1 promoter, and Bombyx mori nuclear polyhedrosis (BmNPV-K1) vlf-1 gene was introduced under the control of the Drosophila heat shock protein gene (hspr70) promoter to yield dual expression plasmid with two independent transcription units. It was transfected into Sf9 cells and cell clones expressing vlf-1 were selected by G4l8 treatment. Genomic DNA from transformed cells was isolated and integration of AcNPV iel harboring vlf-1 was confirmed by PCR using AcNPV iel-specific primers and Southern blot analysis. The transformed cells expressing VLF-1 in an infection-independent manner expressed foreign gene product of recombinant baculovirus in the earlier stage of infection compared with control Sf9 cells. These results suggest the possible to develop highly efficient transformed insect cells for baculovirus expression vector system.

• Journal of Sericultural and Entomological Science
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• v.22 no.2
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• pp.8-12
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• 1981

Practical application of silkworm artificial diet is very desirable to save labour in sericultural industry as the problem of labour shortage is becoming serious in Korea. However, silkworms reared on the artificial diet are more susceptible to viruses than those reared on mulberry leaves because of the lower anti-viral activity of gut juice of silkworms grown on artificial diet compared with that of silkworms grown on mulberry leaves. In this study, authors investigated the varietal difference of silkworm larvae, Bombyx mori L., reared on artificial diet which contained 20 percent of dried mulberry powder, in the susceptibility to nuclear polyhedrosis virus (NPV). The results showed that there is no difference in susceptibility to NPV among tested varieties when high concentration of NPV was admitted to silkworm larvae, but varietal difference appeared in lower concentration admitted. Among 7 hybrids tested, Hansaeng 1${\times}$Hansaeng 2 was most resistant to NPV with an $LC_{50}$ of 2.7${\times}$10$\^$6/ and Jam 111${\times}$Jam 112 was also more resistant comparatively than other hybrids.

• Journal of Sericultural and Entomological Science
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• v.44 no.2
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• pp.69-73
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• 2002

For the practical use of nuecin protein, we tried to overexpress nuecin using Bm5 insect cell and Bombyx mori. We inserted nuecin cDNA into pBm10po1-Xa vector derived from B. mori nuclear polyhedrosis virus (BmNPV), and expressed in Bm5 cells and B. mori respectively. SDS-PAGE and Northern blot analysis showed an expressed of the protein when baculovirus expression vector system (BEVS) was used. The amount of intracellular protein is abundant, but the amount of extracellular protein is poor. The results suggest that the biologically active nuecin protein produced by using BEVS is poor because incresed level of misfolded nuecin by the strong promoter, polyhedrin and p 10 of BEVS, decrease the level of free chaperons and foldases by binding with them.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.685-691
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• 1998

Baculovirus Hyphantrin. cunea nuclear polyhedrosis virus (HcNPV) insecticide containing the insecticidal protein (ICP) gene from Bacillus thuringiensis subsp. kurstaki HD1 was constructed using a lacZ-HcNPV system. The ICP ($\delta$-endotoxin) gene was placed under the control of the polyhedrin gene promoter of the HcNPV. A polyhedrin-negative virus was derived and named ICP-HcNPV insecticide. Then, the insertion of the ICP gene in the ICP-HcNPV genome was confirmed by Southern hybridization analysis. Polyacrylamide gel electrophoresis (PAGE) analysis of the Spodoptera frugiperda cell extracts infected with the ICP-HcNPV showed that the ICP was expressed in the insect cells as 130 kDa at 5 days post-infection. The ICP produced in the cells was present in aggregates. When extracts from the cells infected with the ICP-HcNPV were fed to 20 Bombyx mori larvae, the following mortality rate was seen; 8 larvae at 1 h, 10 larvae at 3 h, and 20 larvae at 12 h. These data indicate that the B. thuringiensis ICP gene was expressed by the baculovirus insecticide in insect cells and there was a high insecticidal activity. The biological activities of the recombinant virus ICP-HcNPV were assessed in conventional bioassay tests by feeding virus particles and ICP to the insect larvae. The initial baculovirus insecticide ICP-HcNPV was developed in our laboratory and the significance of the genetically engineered virus insecticides is discussed.

• International Journal of Industrial Entomology and Biomaterials
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• v.7 no.1
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• pp.1-4
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• 2003

Sericulture is a traditional agro-industry, which involves mulberry cultivation and silkworm rearing, has made great contributes to the human civilization. With the development of national economy and modem technology, mulberry and silkworm are being used to develop products with functionality besides the traditional cocoon production in China. In this paper, we brief the current developing situation of sericultural byproducts with functionality in the following aspects. (1) Functional products from silkworm larvae: silkworm powder, white muscardine silkworm, isolation and purification of anti-bacterial proteins from the larvae and production of medically valuable substances by Bombyx mori nuclear polyhedrosis virus (BmNPV) vector. (2) Utilization of silkworm feces: for pillow and for isolation of chlorophyll etc. (3) Production of valuable Chinese traditional medicine like Cordyceps sinensis with pupae, functional utilization of pupa protein and chitin. (4) Silk as additives to cosmetics, silk food and medical materials. (5) Functional utilization of mulberry: cultivation of edible fungus on mulberry shoots as medium, mulberry fruit drinks, mulberry tea, etc. The prospect of sericultural byproduct industry in China is also discussed.

• The Journal of Korean Society of Virology
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• v.27 no.1
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• pp.29-37
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• 1997

To use recombinant viruses with wider host range as viral insecticides, we investigated the characteristics and pathogenicity of host range expanded recombinant viruses in insect cells. We compared host range expanded recombinant viruses, RecS-B6 and RecB-8, constructed by cotransfection of Autographa californica nuclear polyhedrosis virus (AcNPV) and Bombyx mori NPV (BmNPV), to host range expanded AcNPV, Ac-BH, by substitution of the 0.6 Kb fragment of the BmNPV helicase gene. Restriction endonuclease profiles of RecS-B6 and RecB-8 DNAs were different from those of parent viruses. Nucleotide sequence analysis of the 0.6 Kb region in the putative helicase gene of RecS-B6 and RecB-8 showed that their structures were identical to the counterpart region of BmNPV. Comparison of viral replication of these recombinant viruses in Sf-21 and BmN-4 cells showed that Ac-BH, compared to wild type viruses, replicated well in BmN-4 cells but poorly in Sf-21 cells. In contrast, RecS-B6 and RecB-8 replicated relatively well in both cells compared to parent viruses. These results may imply that random genomic recombinant viruses, RecS-B6 and RecB-8, possess better potential as viral pesticides than helicase-mediated recombinant virus, Ac-BH.

• International Journal of Industrial Entomology and Biomaterials
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• v.6 no.1
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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.