• International Journal of Industrial Entomology and Biomaterials
• /
• 제34권1호
• /
• pp.11-15
• /
• 2017

We constructed the fibroin H-chain expression system to produce enhanced cyan fluorescent proteins (ECFP) in transgenic silkworm cocoon. Fluorescent cocoon could be made by fusing ECFP cDNA to the heavy chain gene and injecting it into a silkworm. The ECFP fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the ECFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworms. The EGFP fluorescence became visible in the ocelli and in the central and peripheral nervous system on the seventh day of embryonic development. A mixture of the donor and helper vector was micro-injected into 1,020 Kumokjam, bivoltin silkworm eggs. We obtained 6 broods. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the ECFP fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제6권1호
• /
• pp.49-55
• /
• 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.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제37권2호
• /
• pp.49-54
• /
• 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.

• 생명과학회지
• /
• 제21권12호
• /
• pp.1726-1731
• /
• 2011

본 연구의 목적은 누에형질전환체를 이용하여 재조합단백질 대량생산 시스템을 개발하는 것으로서, 본 실험에서는 hSCF유전자를 이용하여 누에에서 재조합단백질을 생산하였다. 실험에 사용된 piggyBac 전이벡터는 hSCF 유전자의 발현 조절을 위해 초파리 유래의 dHsp70 promoter를 사용하였고, EGFP marker유전자는 3xP3 promoter로 발현을 조절하였다. 총 1,020 개의 누에알에 microinjection 하여 G1 세대에서 22 bloods의 형질전환체를 선발하였고, 선발된 누에형질전환체는 초기배 단계의 눈과 신경조직, 유충과 번데기 그리고 성충의 눈에서 GFP 형광을 관찰 할 수 있었다. hSCF 재조합단백질의 발현은 Western blot 분석으로 확인 할 수 있었고, inverse PCR 분석을 통해서 누에 게놈에 전이벡터가 삽입된 것을 확인할 수 있었다. 지금까지의 실험 결과에서 hSCF 재조합 단백질이 누에에서 생산될 수 있음을 확인 할 수 있었다. 비록 누에에서 생산된 hSCF 재조합단백질의 생리활성에 대한 실험이 추후에 요구되지만, 이러한 실험결과는 piggyBac 전이벡터와 microinjection 법으로 누에에서 고부가가치의 재조합단백질을 대량생산 할 수 있음을 보여 주었다고 할 수 있겠다. 따라서 누에를 유용물질 생산을 위한 생체반응기로서 활용할 수 있을 것으로 기대된다.

• 한국잠사곤충학회지
• /
• 제52권2호
• /
• pp.117-122
• /
• 2014

선행연구를 통하여, 형질전환 누에를 제작하는 원천기술을 개발하고 이를 통해 녹색 형광실크를 생산하는 형질전환 누에를 제작한 바 있다. 본 연구에서는 선행연구에서 개발된 녹색 형광실크 형질전환 누에와 국립농업과 학원이 보유하고 있는 누에 유전자원 중 유색견(絹) 누에품종을 이용하여 형질전환 누에와 유색견 누에품종 간 교차교배를 통한 잡종강세를 이용하여 고치 생산량 및 고치의 색채 등이 향상된 녹색 형광실크 형질전환 누에 1대 잡종(F1) 계통을 개발하였다. 본 연구를 통해 선발된 실용형질이 우수한 농가보급형 녹색 형광실크 형질전환 누에 1대 잡종(F1) 계통은 기존 형질전환 누에의 녹색 형광단백질 뿐 아니라 유색견 누에 특유의 천연 연록색도 함께 갖고 있어 별도의 염색이 필요 없는 차별화된 고급 패션의류나 벽지 등의 고품질 소재로 활용이 가능할 것으로 기대된다.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제25권1호
• /
• pp.111-114
• /
• 2012

In previous studies we have shown that a sw17255 gene was expressed in hemocyte-specific tissues of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). It was verified that the sw17255 core promoter region contains elements that regulate the expression of this gene in hemocyte tissue; the selected promoter region spans nucleotides -1 to -2,112 upstream of the start codon. Each of the luciferase reporter gene expression vectors under the control of 4 different kinds of promoter candidates, (-2,112/-1), (-1,640/-1), (-1,169/-1) and (-579/-1), and the control reporter plasmid DNA, were introduced into B. mori larval coelom by direct injection using a syringe. The promoter candidate [E] (-579/-1) showed more than 1.67 fold transcriptional activity compared to control promoter activity. Higher productivity of an expressed gene in the transgenic silkworm by this promoter combination could be achieved in the near future. The foreign recombinant protein could be easily harvested from the blood of the transgenic silkworm.

• Journal of Microbiology and Biotechnology
• /
• 제17권12호
• /
• pp.1944-1948
• /
• 2007

Silkworm hemolymph (SH), prepared from fifth-instar larvae of Bombyx mori and heat-treated at $60^{\circ}C$ for 30 min, was used to improve cell viability and the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in transgenic Oryza sativa L. cell suspension cultures. Even though SH could not elevate cell viability at the concentrations up to 3% (v/v), addition of 0.3% (v/v) SH to a culture medium enhanced the production of hCTLA4Ig by 36.8% over an SH-free medium. Moreover, the production period of hCTLA4Ig could be shortened in a 0.3% (v/v) SH-added medium compared with that in an SH-free culture. As a result, addition of 0.3% (v/v) SH improved the productivity of hCTLA4Ig significantly in transgenic rice cell cultures.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제31권2호
• /
• pp.85-89
• /
• 2015

This peptide has antibacterial activity against several Gram-positive and Gram-negative bacteria. Bombyx mori cecropinB1(BmCecB1) is antimicrobial peptides from Bombyx mori and belongs to cecropin family. Antimicrobial peptides are important components of the innate immune systems in all living organism. To produce the BmCecB1 antimicrobial peptide, we constructed transgenic silkworm that expressed BmCecB1 gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 600 eggs of bivoltin silkworms, Baegokjam. In total, 49 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 22 broods, and we selected 2 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 9%. We identified 9 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a BmCecB1 as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that BmCecB1 possesses high antibacterial activities against Gram-negative bacteria.

• KSBB Journal
• /
• 제28권2호
• /
• pp.86-91
• /
• 2013

To date, various strategies have been studied to increase specific productivity in Chinese hamster ovary (CHO) cell cultures. Also, albumin-fusion platform is being applied to other important bioactive peptides with short half-lives. Here, we investigated the effects of silkworm gland hydrolysate (SGH) on the production of albumin-erythropoietin (Alb-EPO) in transgenic CHO cells. The viable cell density of CHO cells was increased by 13% in the medium containing 1 mg/mL SGH higher than in the control medium without SGH. In addition, the production of Alb-EPO was also 1.26- fold enhanced by reducing the early apoptosis of CHO cells. In conclusion, SGH could be used as a useful supplement for the enhancement of recombinant protein production.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제7권2호
• /
• pp.139-144
• /
• 2003

Over 350 million people worldwide are chronic carriers of hepatitis B virus (HBV). Chronic viral infections of the liver can progress to cirrhosis, which may ultimately lead to hepatic failure or the development of hepatocellular carcinoma. There are two antiviral drugs on the market approved for clinical management of chronic HBV infections; interferon-alpha and the nucleoside analog lamivudine. However, they showed adverse side-effects. In the rational drug design for such therapies we would like to utilize antiviral drugs that inhibit the HBV replication in the liver. Investigation of natural extracts of silkworm exhibiting antiviral potential was held in the functional HBV polymerase activity and the release of virion particle in the HepG2.2.15 cell lines. HBV-producing transgenic mouse fed with silkworm DNJ molecule was shown as an inhibitor of serum HBV particles. We could represent this DNJ molecule as an antiviral potential complementing conventional therapies after preclinical tests against WHBV-infected animal model, woodchuck.