• Korean journal of applied entomology
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• v.36 no.4
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• pp.331-340
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• 1997

Hyalophora cecropia attacin-like antibacterial gene was isolated from Bombyx mori induced with nonpathogenic bacteria. It was expressed in Spodopfera frugiperda 9 (Sf9 cells using baculovirus expression vector system (BEVS), and examined its antibacterial activity. With a cDNA library constructed from fifthinstar B. mori injected with Escherichia coli(4 X IOhcellsllarva), differential screening was performed using naive and induced mRNA probes. BmInc6 clone was screened by partial nucleotide sequence and GenBank database analysis. A complete nucleotide sequence of Bmlnc6 cDNA was determined (GenBank, AF005384). Its insert size was 852 bp and had open reading frame that started translation at position 35 and stopped at 679. And its putative polyadenylational signal existed at 812 bp. The number of amino acid deduced from Bmlnc6 cDNA was 214 and hydropathy analysis showed that this peptide was hydrophilic. This peptide deduced by BmInc6 was named nuecin. When the nuecin gene was expressed in Sf9 cells using BEVS, about 950 bp of the transcripts was detected. In addition, SDS-PAGE analysis showed that the molecular weights of intracellular expressed protein and the mature protein secreted to culture media were approximately 23 and 20 kDa, respectively. The antibacterial activity of nuecin against E. coli and Bacillus subtilis was significantly high, demonstrating that nuecin had a wider antibacterial spectrum with gram negative and positive bacteria than attacin.

• Journal of Sericultural and Entomological Science
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• v.44 no.2
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• pp.64-68
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• 2002

The antibacterial protein gene, nuecin was expressed in Sf9 cells using baculovirus expression vector system (BEVS). The antibactetial activity of mature nuecin against Pectobacterium carotovorum subsp. carotovorum, Ralstonia solanacearum and Pseudomonas tolaasii was significantly high, demonstrating that nuecin had a wider antibacterial spectrum on gram negative and positive bacteria. The result appears to be superior to other antibacterial peptide, attacin. The nuecin was purified by SP-sepharose and Mono Q HR ion-exchange chromatography, and then by Superdex 200 HR 10/30 column. The purified nuecin is quite stable at 80$\^{C}$ and 100$\^{C}$ for several hours of incubation and in a wide pH range (pH 2-12).

• Journal of Mushroom
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• v.9 no.3
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• pp.91-95
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• 2011

Pleurotus ostreatus, the oyster mushroom, is one of the most important edible mushrooms. It is especially susceptible to bacterial blotch disease, which is caused by Pseudomonas tolaasii. In order to develop bacterial blotch disease-resistant transgenic mushroom, NUECIN cDNA, a gene for an antibacterial peptide cloned from Bombyx mori, was overexpressed in Pleurotus ostreatus. NUECIN cDNA was fused to the ${\beta}$-TUBULIN promoter of oyster mushroom and co-transformed with the pTRura3-2 vector into the uracil auxotrophic mutant strain. Twelve transformants containing the NUECIN gene were identified by genomic PCR and Southern blot analysis. NUECIN gene expression was confirmed by Northern blot analysis. Three transformants showed the transcriptional expression of the gene. However, we could not detect expression of the protein in the transformants. This study showed the possibility of transgenic mushroom development for disease resistance.

• 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.

• BMB Reports
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• v.41 no.5
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• pp.400-403
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• 2008

The insect baculovirus expression vector system (BEVS) is useful for producing biologically active recombinant proteins. However, the overexpressions of foreign proteins using this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we developed a versatile baculovirus expression and secretion system using Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion was found to improve the secretions and antibacterial activities of recombinant nuecin proteins. Thus, we conclude that bPDI gene fusion is a useful addition to BEVS for the large-scale production of bioactive recombinant proteins.

• Proceedings of the Korean Society of Life Science Conference
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• 2000.09a
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• pp.89-89
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• 2000

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2002.10a
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• pp.113-113
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• 2002

No Abstract, See Full Text

• International Journal of Industrial Entomology and Biomaterials
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• v.37 no.2
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• pp.49-54
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• 2018

To artificially enhance antimicrobial peptide expression in Bombyx mori, we constructed genetically engineered silkworms overexpressing Rel family transcription factor. The truncated BmRelish1 (BmRelish1t) gene contained a Rel homolog domain (RHD), nuclear localization signal (NLS), acidic and hydrophobic amino acid (AHAA)-rich region, and death domain (DD), but no ankyrin-repeat (ANK) domain. The BmRelish1t gene was controlled by B. mori cytoplasmic actin 3 promoter in the PiggyBac transposon vector. Chromosome analysis of G1 generations of a transgenic silkworm with EGFP expression confirmed stable insertion of BmRelish1t. BmRelish1t gene overexpression in transgenic silkworms resulted in higher mRNA expression levels of B. mori antimicrobial peptides such as lebocin(~20.5-fold), moricin(~8.7-fold), and nuecin(~17.4-fold) than those in normal silkworms.

• International Journal of Industrial Entomology and Biomaterials
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• v.26 no.2
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• pp.119-123
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• 2013

The insect baculovirus expression vector system (BEVS) is useful for producing biologically active recombinant proteins. However, the overexpression of heterologous proteins using this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we developed a versatile baculovirus expression and secretion system using Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion was found to improve the secretions and antibacterial activities of recombinant nuecin and enbocin proteins. Thus, we conclude that bPDI gene fusion is a useful addition to BEVS for the large-scale production of bioactive recombinant proteins.