This peptide has antibacterial activity against several Gram-positive and Gram-negative bacteria. 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 11%. 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.

Silkworm transgenesis is now a routine method leading to a satisfactory yield of transformed animals and the reliable expression of transgenes during multiple successive generations. However, the screening of G1 transgenic individuals from numerous progeny has proved to be difficult and time-consuming work. Previously, we characterized the promoter of heat shock protein 70 from Bombyx mori (bHsp70), which is ubiquitously expressed in all tissues and developmental stages. To investigate the utilization of the bHsp70 promoter to screen transgenic individuals, the EGFP marker gene was inserted into the piggyBac vector under the control of the bHsp70 promoter. Mixtures of the donor and helper vectors were micro-injected into 3,060 eggs of bivoltine silkworms (Keomokjam). EGFP fluorescence was observed in 17 broods of transgenic silkworms under a florescence stereomicroscope. Interestingly, this fluorescent marker protein was detected not only in parts of the embryo segments on the seventh day of the G1 embryonic developmental stage but it was also detected in a part of the body of G1 hatched larvae, in the middle silk gland of G1 fifth instar larvae, and in the wings of seven-day-old G1 pupae and G1 moths. Therefore, we suggest that the bHsp70 promoter can be used for the rapid and simple screening of transgenic silkworms.

To product the blue fluorescent protein (AmCyan) expressed cocoon, we were fused AmCyan cDNA to the heavy chain gene and injected the gene into a silkworm. AmCyan was one of the existing violet fluorochromes and originally derived from the fluorescent protein amFP486. AmFP486 was cloned from the sea anemone Anemonia majano (GenBank accession number AF168421), and belongs to the family of fluorescent proteins (FPs) isolated from coral reef organisms. The AmCyan fusion protein, each with N- and C- terminal sequences or the fibroin H-chain, were designed to be secreted into the lumen of the posterior silk glands. The expression of the AmCyan/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3 EGFP as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor and helper vectors were micro-injected into 300 eggs of bivoltine silkworms (Baegokjam). EGFP fluorescence was observed in 3 broods of transgenic silkworms under a florescence stereomicroscope. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the AmCyan gene expressed cocoon will be enable the production of the novel biomaterials based on the transgenic silk.

Screening for antimicrobial peptide genes in the immune-induced Antheraea yamamai larvae led to the identification of a novel antifungal moricin-like peptide (MLP10) gene. The complete MLP10 cDNA is comprised of 403 bp with 174 bp open reading frame encoding a 58 amino acid precursor that contains a putative 23-residue signal peptide, a 2-residue propeptide and a 33-residue mature peptide. The deduced amino acid sequence of MLP10 has 26∼52% identity to those of moricin-related peptides from lepidopteran insects. The MLP10 was highly expressed in E. coli BL21(DE3) by fusing with ketosteroid isomerase (KSI) to avoid the cell death during induction. The resulting expressed KSI-MLP10 fusion protein was in a insoluble form. Recombinant MLP10 was released by cleavage of the fusion protein with cyanogen bromide (CNBr). Subsequently, we purified pure active MLP10 by FPLC chromatography, and 5.2mg of MLP10 was obtained from 1L culture medium. The purified MLP10 was prevented the growth of candida albicans at 6.25 uM, and was also active against gram negative and gram positive bacteria. This potent antimicrobial activity suggests that MLP10 may play a role in the immune response of A. yamamai.

The antibiotic peptide PAJE (RWKIFKKPFKISIHL-NH2), designed incorporating the N-terminal α-helical segments of papiliocin and jelleine, is a 15-residue hybrid peptide that has a broad spectrum of activity against Gram-negative, positive bacteria and fungi. In this study, we successfully expressed bioactive PAJE in Escherichia coli cells that are highly sensitive to this peptide. For the efficient production of peptide, we synthesized gene encoding PAJE, and fused the sequence in-frame to ketosteroid isomerase (KSI) gene to construct an expression vector pET29b-PAJE-KSI, which was then used to transform E. coli BL21 (DE3). The fusion protein PAJE-KSI was expressed as inclusion body at high level (more than 30% of the total proteins). Recombinant PAJE was easily released by cleavage of the fusion protein with cyanogen bromide (CNBr). Subsequently, we purified the recombinant PAJE by FPLC chromatography. The purified PAJE displayed considerably antibacterial activity identical to that previously reported for chemically synthesized PAJE. The results indicated that successful expression of PAJE in E. coli cells and efficient procedure for purification may lead to a cost-effective platform for the mass production of PAJE.

To product the blue fluorescent protein (AmCyan) expressed cocoon, we were fused AmCyan cDNA to the heavy chain gene and injected the gene into a silkworm. AmCyan was one of the existing violet fluorochromes and originally derived from the fluorescent protein amFP486. AmFP486 was cloned from the sea anemone Anemonia majano (GenBank accession number AF168421), and belongs to the family of fluorescent proteins (FPs) isolated from coral reef organisms. The AmCyan fusion protein, each with N- and C- terminal sequences or the fibroin H-chain, were designed to be secreted into the lumen of the posterior silk glands. The expression of the AmCyan/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3 EGFP as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor and helper vectors were micro-injected into 300 eggs of bivoltine silkworms (Baegokjam). EGFP fluorescence was observed in 3 broods of transgenic silkworms under a florescence stereomicroscope. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the AmCyan gene expressed cocoon will be enable the production of the novel biomaterials based on the transgenic silk.

BmCecB1 are antimicrobial peptides from Bombyx mori and belongs to cecropin family. Antimicrobial peptides are important components of the innate immune systems in all living organism. This peptide has antibacterial activity against several Gram-positive and Gram-negative bacteria. 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 11%. 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.

Immune-inducible antimicrobial peptides were produced using transgenic silkworms that expressed Rel family transcription factor, truncated BmRelish1 (BmRelish1t) genes under the control of the BmA3 promoter using the piggyBac vector. BmRelish1t gene contains all domains of Bmrelish: a Rel homolog domain (RHD), nuclear localization signal (NLS), acidic and hydrophobic amino acids (AHAA) rich region except the Ankyrin repeat domain (ANK) and the death domain (DD). (1:1) Mixtures of the donor vector (pG-3xP3EGFP-BmA3BmRelish1t) and helper vector were micro-injected into 1,800 eggs of bivoltin silkworms, Baegokjam and EGFP-induced fluorescence was observed for 25 broods of transgenic silkworms under a florescence stereomicroscope. Analysis by real-time PCR indicated that transgenic silkworms expressing BmRelish1t recombinant proteins displayed higher mRNA expression levels of the Bombyx mori antimicrobial peptides such as lebocin, moricin, and nuecin than the normal silkworms. Moreover, transgenic silkworms expressing BmRelish1t showed antibacterial activity against Escherichia coli. We suggest that transgenic expression of BmRelish1t may find useful applications for the production of various antimicrobial peptides at the same time in transgenic silkworms.

Silkworm transgenesis is now a routine method leading to a satisfactory yield of transformed animals and the reliable expression of transgenes during multiple successive generations. However, the screening of G1 transgenic individuals from numerous progeny has proved to be difficult and time-consuming work. Previously, we characterized the promoter of heat shock protein 70 from Bombyx mori (bHsp70), which is ubiquitously expressed in all tissues and developmental stages. To investigate the utilization of the bHsp70 promoter to screen transgenic individuals, the EGFP marker gene was inserted into the piggyBac vector under the control of the bHsp70 promoter. Mixtures of the donor and helper vectors were micro-injected into 3,060 eggs of bivoltine silkworms (Keomokjam). EGFP fluorescence was observed in 17 broods of transgenic silkworms under a florescence stereomicroscope. Interestingly, this fluorescent marker protein was detected not only in parts of the embryo segments on the seventh day of the G1 embryonic developmental stage but it was also detected in a part of the body of G1 hatched larvae, in the middle silk gland of G1 fifth instar larvae, and in the wings of seven-day-old G1 pupae and G1 moths. Therefore, we suggest that the bHsp70 promoter can be used for the rapid and simple screening of transgenic silkworms.

It is true that the proper environmental risk assessments for many GM insects almost have not been executed in Korean research situation. Therefore, we tested the environmental risk assessment of GM silkworms if there is any difference between GM and non-GM silkworms by three. First, we measured their mobility in the breeding environment conditions with food and without food. Secondly, we measured their viability at the Korean artificial extreme environmental conditions (temperature, humidity, food) after escaping from their breeding environments. We observed the egg productivity and the hatchability between non-GM silkworm and transgenic silkworms with four different pair experiments. The mobility of non-GM silkworms and GM silkworms statistically did not differ and the egg production and hatchability was not also different. The hatchability by couple of GM female silkworm and non-GM male silkworm was lower than by non-GM male and female couple. We observed their viability (High Temp., wet and with food: p=0.0434; High Temp., wet and without food p=0.0430; High Temp., dry and with food: p=0.0005; High Temp., dry, without food: p=0.0479) between the GM silkworm and non-GM silkworm, and there was statistically different. Relatively, the viability of GM silkworm was lower than non-GM silkworms. We could not exactly test for viability of silkworm in low temperature conditions because of their hibernating. Although there was any difference in viability and hatchability between GM silkworm and non-GM silkworm, the all ability of GM silkworm was lower than non-GM silkworm. Conclusively, risk of GM silkworm was lower than non-GM silkworm.

Attacin is a well-studied glycine-rich antibacterial protein in insect immune response, which has limitary antibacterial effect to some Gram-negative bacteria. A cDNA encoding the attacin gene was screened and isolated from the immunized larvae of the swallowtail butterfly, Papilio xuthus. The complete P. xuthus attacin cDNA is 949 nucleotides encoding a 250 amino acid precursor that contains a putative 18-residue signal peptide, a common 42-residue propeptide sequence and a presumed 190-residue mature protein with a theoretical mass of 19904.01 and a pI of 9.13. The putative mature protein of P. xuthus attacin showed 48%~52% and 24%~30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. The attacin transcript was induced at significant level after injection with bacterial lipopolysaccharide (LPS). Recombinant attacin was highly expressed in E. coli BL21 (DE3) cells by fusing with an N-terminal S-tag/thrombin cleavage site configuration protein to avoid the cell death during induction. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC). After desalting and cleavage with thrombin, the recombinant attacin was released and showed considerably antibacterial activity against Gram-negative bacteria, E. coli ML 35. Our results proved that this protein family with a potent antibacterial activity may play a role in the immune response of butterflies.

Disulfide bond formation, reduction and isomerization are important posttranslational modification in proteins that occur in most, if not all, living organisms. In eukatoyes, disulfide bond in substrate proteins are primarily formes by ERO1 and PDI. ERO1, oxidized by molecular oxygen, acts as a specific oxidant of PDI, which then makes disulfide bonds in folding proteins oxidized directly. It means that ERO1 plays an essential role in setting the redox potential in the ER, and the regulation of Ero1p activity is critical to maintain redox homeostasis and proper ER folding activity. We have isolated and analysed a endoplasmic reticulum oxidoreductase (ERO1) from Bombye mori. It apperas that both an N-terminal CxxxxC motif and a C-terminl CxxCxxC motif are necessary for Ero1p fuction. In vivo, the result of the 5day of 5th instar larvae by RT-PCR and Real-Time PCR shows that posterior silkgland, skin and mid silkgland are revealed more than rhose of other tissues. The same result for tissue distribution of transcripts is appeared about ERO1 and PDI. In Bombyx mori, ERO1 is also supposed to correlate with PDI. Afterwards, more experiments are needed to figure out accurate interrelation between ERO1 and PDI.

Recently Transgenesis was achieved in Bombix mori. For stable and effective transgenesis in B.mori, B.mori cytoplasmic actin gene (BmA3) promoter was used to expression of marker gene, the green fluorescent protein(GFP). Green fluorescent protein expression for selection of transformants was visible in all larval, pupal, and adult tissues but, unexpectdly, was not detectable in embryos. So, it spend times and money on rearing of silkworm. Furthermore, the BmA3 promoter is predominantly active in the midgut, which makes it difficult to reliably identify transformants since autofluorescence of many insect foods can mask low-level fluorescence and only allows the detection of strongly expressing individuals with potentially multiple insertions. Therefore, we need more intensely promoter than BmA3 promoter for selected by expression of GFP in embryos and selected by reliable expression of GFP in larvae. We performed dot blot hybridization to develop strong promoter. Nine differentially expressed clones were isolated and we focused one clone of them which has high similarity with heat shock protein 70 gene from D.melanogaster. We named it as bHSP70 (Bombyx mori heat shock protein 70). Expression from the hsp70 promoter was strong and heat shock-dependent. And Drosophila hsp70 promoter appears useful for regulating expression of Exogenous DNA. So, we analyzed transcriptional activity of promoter with bHSP70 gene by using dual luciferase assay system. bHSP70 promoter has about 264 folds more intensely than BmA3 promoter. Also, when bHSP70 promoter treated heat shock(42℃), transcriptional activity incresed 2 times more than normal condition. Therefore, we suggest that bHSP70 promoter is more effective candidate for stable transformation and selection of transformants.

Peptidyl prolyl cis/trans isomerases (PPIases) catalyze the slow cis/trans isomeraization of proline peptide (Xaa-Pro) bonds in oligopeptides and accelerates slow, rate-limiting steps in the folding of several proteins. We studied the characterization of Cyclophilin A (bCyp A) isolated from Bombyx mori . The cDNA of bCyp A is 947 bp. There is a 5´-untranslated region of 91 nucleotides followed by an initiating ATG codon. The TAA termination codon occurs at nucleotide 588. Thus translation of the sequence from nucleotides 91 to 588 would produce a protein of 166 amino acids with a calculated molecular mass of 18.2kDa. The 'AATAAA' consensus polyadenylation signal and poly A tail are present in the 3´-untranslated region. To analysis of PPIase activity, we expressed the bCyp A protein in Sf9 cell by using baculovirus expression vector system (BEVS). SDS-PAGE and Western blot analysis showed that the molecular weights of intracelluar expressed protein was approximately 28.2 kDa. The PPIase activity assay was monitored by proteolytic cleavage of the chromophore p-nitroanilide byα-chymotrypsin. As substrate the synthetic tetrapeptide succinyl-Ala-Ala- Pro-Phe-p-nitroanilide was used.

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