• 한국잠사곤충학회지
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• 제39권1호
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• pp.36-43
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• 1997

In order to express the FLP recombinase in B. mori cultured cell line, BmN-4, transient expression system using a heat shock protein gene (hsp70) promoter of Dorosophilla melnogaster was constructed. This vector was designated as pHsSV. Activity strength of the hsp70 promoter was compared with that of immediate early gene (IE-1) and polyhedrin gene of BmNPV employing the E. coli $\beta$-galactosidase gene as a reporter gene. The result showed that the pHs $\beta$-gal plasmid vector expressed the $\beta$-galactosidase at 2nd and 3rd day after the transfer of plasmid DNA into BmN-4 cells, which was similar to that of pIE1 $\beta$-gal vector, but different from that of a recombinant virus, vBm $\beta$-gal. For the construction of FLP recombinase transient expression vector, the FLP recombinase gene was cloned by polymerase chain reaction technique. To express the FLP recombinase, this gene was inserted into pHsSV plasmid vector, under the control of the hsp70 promotor, and tranfected in BmN-4 cells. The expressed FLP recombinase was estimated at 44kDa on a 12.5% SDS-PAGE.

• International Journal of Industrial Entomology and Biomaterials
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• 제4권1호
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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.

• International Journal of Industrial Entomology and Biomaterials
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• 제13권2호
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• pp.73-77
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• 2006

Combining ability and hybrid vigour analysis was carried out in hybrids between newly developed non-susceptible lines to BmDNV1 and popular bivoltine breeds for certain quantitative traits viz. Pupation rate, Cocoon yield, Cocoon weight, Cocoon shell weight and Cocoon shell ratio, Survival rate against BmIFV and BmNPV. General combining ability (GCA) effects revealed that among the lines CSR2DR was found good general combiner exhibiting significant GCA effects for six characters, out of seven traits evaluated. Among testers CSR28DR was found as good combiner exhibiting significant GCA effects for six traits. Out of 36 hybrids made between $resistant{\times}resistant,\;resistant{\times}susceptible\;and\;susceptible{\times}susceptible$ breeds, one hybrid $CSR21DR{\times}CSR28DR$ exhibited significant SCA effects for six traits. The selected hybrid $CSR21DR{\times}CSR28DR$ also exhibited significant positive heterosis and heterobeltiosis expressions for maximum traits and could be exploited as commercial silkworm hybrid resistant to important viral diseases.

• International Journal of Industrial Entomology and Biomaterials
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• 제2권1호
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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.

• 한국잠사곤충학회지
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• 제44권2호
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• pp.69-73
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• 2002

본 연구는 누에신 단백질의 대량발현을 통해 농업용 소재로서 이용하고자 하는 연구의 일환으로 누에 핵다각체병 바이러스 유래의 pBm10po1-Xa 벡터에 누에신 유전자를 도입하여 누에 배양세포(Bm5) 및 누에 생체에서 발현한 결과 누에신 전사체는 바이러스 접종 후 1일째부터 발현되기 시작하여 5일째에 최대로 발현되었음을 확인할 수 있었고, 누에신 단백질은 3일째부터 발현량이 증가하여 5일째까지 계속 지속적으로 발현되었으나 그 발현량은 전사체에서 처럼 많지 않음을 확인할 수 있었으며, 누에신 단백질의 발현량은 누에 세포에 비해 누에 생체에서 기대만큼 그 발현량이 많지 않았다. 또한 누에신의 베큘로바이러스 발현계 (baculovirus expression vector system, BEVS)를 이용하여 세포내 및 번역후 변형과정을 통하여 세포외로 분비된 누에신의 발현양상을 확인한 결과 세포내에 비해 세포외로 분비시 그 발현량이 현저히 줄어들었음을 확인할 수 있었다. 따라서 베큘로바이러스 발현계를 이용하여 외래 단백질을 생산할 경우 정확한 메카니즘은 밝혀지지 않았으나 강력한 프로모터에 의해 세포내 단백질 생산량은 많은데 비해 정확한 단백질 고차구조 형성을 도와주는 foldase 및 chaperon의 양은 한정되어져 있으므로 정확한 고차구조를 형성하여 세포외로 분비되는 생물학적 활성을 띠는 단백질은 매우 적은 양간이 발현됨을 확인할 수 있었다. 그러므로 추후 누에 신 단백질 대량생산을 위해서는 분비 프로세싱의 해명 및 번역 후 변형과정의 개선을 통한 발현계의 개량이 시급히 요구된다.

• International Journal of Industrial Entomology and Biomaterials
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• 제7권1호
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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.

• International Journal of Industrial Entomology and Biomaterials
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• 제12권1호
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• pp.7-14
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• 2006

Silkworm diseases are better prevented than cured. Disinfection and hygiene are the two important aspects in silkworm rearing to prevent the diseases. Suitable disinfectant is the primary need to disinfect the rearing house, its surroundings and appliances to eliminate the persistent pathogens from the rearing environment. In this direction, Serichlor, a new disinfectant in Indian Sericulture marketed as Serichlor-60 (contains 60,000 ppm of chlorine dioxide) and Serichlor-20 (contains 20,000 ppm of chlorine dioxide) has been evaluated for its germicidal effect against the pathogens of silkworm, viz., spores of Nosema bombycis, Bacillus thuringiensis, polyhedra of BmNPV and conidia of Beauveria bassiana both in vitro and in vivo. Results indicated that high concentration (2,500 ppm of chlorine dioxide) is required to kill all the pathogens at 100% level. The efficacy of the Serichlor was greatly enhanced by the addition of 0.5% slaked lime solution. 500 ppm of chlorine dioxide in 0.5% slaked lime solution was found effective against all the pathogens tested. This concentration of disinfectant was also found effective for disinfection of rearing house, rearing appliances and silkworm egg surface. The disinfectant is stable, non hazardous, least corrosive and most suitable for Indian Sericulture.

• 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.