Generation of transgenic silkworms for production of erythropoietin in Bombyx mori

성지연,Min-Jung Kim,Hyo-Sun Kim,Sun-Ah Kim,Hyung-Wook Jeon,Seung-Hyun Sung,김근종,Dong Sang Suh 한국유전학회 2011 Genes & Genomics Vol.33 No.3

There have been many attempts to generate various essential proteins using transformed E. coli systems. However, prokaryote systems are not equipped with the protein maturation mechanisms necessary to generate eukaryotic proteins. In this sense, among the eukaryotes, silkworms have major merits in overcoming the difficulties. Such protein maturation mechanisms are available in silkworms. In this study, a transgenic silkworm producing rhEPO in the cocoon was generated and purified. Specifically, we constructed a transgenic silkworm using a vector system that could be controlled to the next generation. To accomplish this, we microinjected the system into eggs laid during the preblastoderm stage. The rhEPO was then purified from transgenic silkworm cocoons using a Con A affinity column. The biological activity of rhEPO isolated from the cocoon of transgenic silkworms was then assessed in a cell culture system using an EPO-dependent cell line,F-36E. Next, PCR analysis was used to demonstrate that stable gene expression can occur in the embryos of the silkworm,Bombyx. mori. Transgenic silkworms were then selected and observed to ensure that the transgenic silkworm was maintained and transmitted to their progeny. The rhEPO was subsequently purified from the transgenic silkworm cocoon and the electrophoretic pattern of the purified rhEPO revealed a protein band with a molecular weight of approximately 20kDa. A total of 3 mg of rhEPO was eluted from 10 g of cocoons. The proliferation of F36E cells for 25 ng/ml rhEPO was 1.32, while the proliferation for 2.5 IU/ml hEPO was 1.32. The proliferation of these cells could be induced by commer-cial hEPO, as well as by rhEPO from transgenic silkworm cocoons. An in vivo test of mice treated with rhEPO revealed relatively high RBC values when compared to normal mice. These results indicated that purified glycosylated EPO from transgenic silkworms had biological activities. Overall, the transgenic silkworm technique will be very useful for the large scale production of proteins for diagnostic and therapeutic purposes.

Sensory Profiles of Protein-Fortified Kefir prepared Using Edible Insects (Silkworm Pupae, Bombyx mori) : A Preliminary Study

( Young-ji Kim ),( Jung-whan Chon ),( Kwang-young Song ),( Dong-hyeon Kim ),( Hyunsook Kim ),( Kun-ho Seo ) 한국유가공기술과학회 2017 Journal of Dairy Science and Biotechnology (JMSB) Vol.35 No.4

Bombyx mori (silkworm pupae) is a unique and biologically significant insect, which is a recognized source of high quality protein that provides all the essential amino acids required for human health. Recently, many studies have focused on various biomedical applications of B. mori proteins. The purpose of this study was to manufacture protein-fortified kefir containing different concentrations of B. mori powder according to pH and sensory evalua-tions. The value of the protein-fortified kefir increased but the pH decreased with increasing incubation time, indicating that the amount of B. mori powder did not affect and pH. Addition of B. mori powder also did not affect the sensory properties of overall acceptability, texture, and color compared to control group without addition of B. mori powder. However, flavor and taste were affected by increasing the amounts of B. mori powder, with a significant difference in both flavor and taste between the control and treated groups (both p<0.05). There was no significant difference in overall acceptability, texture, and color. Fur-ther studies are needed for producing kefir as a dietary supplement utilizing the functional properties of B. mori.

The innate immune response transcription factor Bombyx mori Relish1 induces high-level antimicrobial peptides in silkworm

( Seong-wan Kim ),( Seong-ryul Kim ),( Tae-won Goo ),( Kwang-ho Choi ) 한국잠사학회 2018 International Journal of Industrial Entomology Vol.37 No.2

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.

집누에(Bombyx mori)와 멧누에(Bombyx mandayina)의 種間交雜에 있어서 卵殼構造 및 Chorion 蛋白質

金鍾吉,盧時甲 한국잠사학회 1994 한국잠사곤충학회지 Vol.36 No.1

Chorion(egg-shell) morphology of the F1 hybrid between Bombyx mori and Bombyx mandarina has been observed by scanning electron microscope and chorion protein was analyzed by electrophoresis. The chorion surface structure of F1, hybrids in the lateral (flat) region was similar to that of maternal line. The F1, hybrids chorion was found to have basically a three layer structure. The middle and inner layer were very much like those of the Bombyx mandarina and Bombyx mori. There were many conic pillar structures in the outer layer of the F1, hybrid, which was similar to Bombyx mandarina. This conic pillar structure had a thin cover layer was more clear in the dorsal and ventral side of the F1, hybrid chorion. The conic pillar structure of Bombyx mandarina was found to be dominant in F1 hybrid chorion irrespective of their maternal line. Major components of chorion protein were analyzed by two-dimensional electrophoresis and found to have isoelectric points in the range of pH 4.0-6.5 and molecular weight 10 to 50 kd. F1 hybrid chorion protein components related directly to those of the maternal line. The conic pillar structure was dominat characteristic and it was present in all F1 hybrid.

The innate immune response transcription factor Bombyx mori Relish1 induces high-level antimicrobial peptides in silkworm

김성완,김성열,구태원,최광호 한국잠사학회 2018 International Journal of Industrial Entomology Vol.37 No.2

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.

Isolation of a New Microsporidian sp. (NIK-5hm) forming Spores within the Haemocytes of Silkworm, B. mori L.

( T. Selvakumar ),( B. Nataraju ),( K. Chandrasekharan ),( S. D. Sharma ),( M. Balavenkatasubbaiah ),( P. Sudhakara Rao ),( V. Thiagarajan ),( S. B. Dandin ) 한국잠사학회 2005 International Journal of Industrial Entomology Vol.11 No.1

While observing silkworm larval samples received from field, microsporidian spores formed within the haemocytes of silkworm haemolymph were observed. The spores of microsporidian sp. were purified and characterized for morphological characters viz., size, shape as well as serological affinity with different Nosema spp. (M11 and M12). The infectivity of the isolated spores to silkworm was also studied. The microsporidian sp. was found to be highly pathogenic to silkworm, B. mori. The isolated microsporidian sp. was designated as NIK-5hm, which formed ovocylindrical spore in the haemocytes of silkworm and differed in spore size (length, 4.55 μm & width, 2.10 μm) and shape from Nosema bombycis (NIK-1s), NIK-2r (Nosema sp. Mysore [3.6 & 2.8 μm]), NIK-3h (Nosema sp. M11 [3.8 & 1.8 μm]), NIK-4m (Nosema sp. M12 [5.0 & 2.1 μm]) and Lb㎳ (Nosema sp. in Lamerine breed of silkworm [4.36 & 2.14]). In immonological test (Latex agglutination test), the isolated microsporidian spores did not react with antibody sensitized latex particles of N. bombycis, M11, M12 and Lb㎳ and thus are different type of microsporidian sp., parasitic to silkworm, Bombyx mori L.

Sensory Profiles of Protein-Fortified Kefir prepared Using Edible Insects (Silkworm Pupae, Bombyx mori) : A Preliminary Study

Kim, Young-Ji,Chon, Jung-Whan,Song, Kwang-Young,Kim, Dong-Hyeon,Kim, Hyunsook,Seo, Kun-Ho Korean Society of Dairy Science and Biotechnology 2017 Journal of Dairy Science and Biotechnology (JMSB) Vol.35 No.4

Bombyx mori (silkworm pupae) is a unique and biologically significant insect, which is a recognized source of high quality protein that provides all the essential amino acids required for human health. Recently, many studies have focused on various biomedical applications of B. mori proteins. The purpose of this study was to manufacture protein-fortified kefir containing different concentrations of B. mori powder according to pH and sensory evaluations. The value of the protein-fortified kefir increased but the pH decreased with increasing incubation time, indicating that the amount of B. mori powder did not affect and pH. Addition of B. mori powder also did not affect the sensory properties of overall acceptability, texture, and color compared to control group without addition of B. mori powder. However, flavor and taste were affected by increasing the amounts of B. mori powder, with a significant difference in both flavor and taste between the control and treated groups (both p<0.05). There was no significant difference in overall acceptability, texture, and color. Further studies are needed for producing kefir as a dietary supplement utilizing the functional properties of B. mori.

The innate immune response transcription factor Bombyx mori Relish1 induces high-level antimicrobial peptides in silkworm

Kim, Seong-Wan,Kim, Seong-Ryul,Goo, Tae-Won,Choi, Kwang-Ho Korean Society of Sericultural Science 2018 International Journal of Industrial Entomology Vol.37 No.2

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.

Differential Level of Host Gene Expression Associated with Nucleopolyhedrovirus Infection in Silkworm Races of Bombyx mori

( Govindaraj Lekha ),( Esvaran Vijayagowri ),( Sasibhushan Sirigineedi ) 한국잠사학회 2014 International Journal of Industrial Entomology Vol.29 No.2

The variation in the level of immune response related gene expression in silkworm, Bombyx mori following infection with Bombyx mori nucleopolyhedrovirus (BmNPV) was analyzed at different time intervals. The occlusion bodies of BmNPV orally inoculated to the two most divergent silkworm races viz., Sarupat (resistant to BmNPV infection) and CSR2 (susceptible to BmNPV infection) were subjected to oral BmNPV inoculation. The expression profile of gp41 gene of BmNPV in the Sarupat and CSR2 races revealed that the virus could invade the midguts of both susceptible and resistant races. However, its multiplication was significantly less in the midgut of resistant race, while, in the susceptible race, the viral multiplication reached maximum level within 12 h. These findings indicate that potential host genes are involved in the inhibition of viral multiplication within larval midgut. The immune response genes arylphorin, cathepsin B, gloverin, lebocin, serpin, Hsp 19.9, Hsp 20.1, Hsp 20.4, Hsp 20.8, Hsp 21.4, Hsp 23.7, Hsp 40, Hsp 70, Hsp90 revealed differential level of expression on NPV infection. The gloverin, serpin, Hsp 23.7 and Hsp 40 genes are significantly up-regulated in the resistant race after NPV infection. The early up-regulation of these genes suggests that these genes could play an important role in baculovirus resistance in the silkworm, B. mori.

Isolation of a New Microsporidian sp. (NIK-5hm) forming Spores within the Haemocytes of Silkworm, B. mori L.

Selvakumar T.,Nataraju B.,Chandrasekharan K.,Sharma S. D.,Balavenkatasubbaiah M.,Sudhakara Rao P.,Thiagarajan V.,Dandin S. B. Korean Society of Sericultural Science 2005 International Journal of Industrial Entomology Vol.11 No.1

While observing silkworm larval samples received from field, microsporidian spores formed within the haemocytes of silkworm haemolymph were observed. The spores of microsporidian sp. were purified and characterized for morphological characters viz., size, shape as well as serological affinity with different Nosema spp. (M$_{11}$ and M$_{12}$). The infectivity of the isolated spores to silkworm was also studied. The microsporidian sp. was found to be highly pathogenic to silkworm, B. mori. The isolated microsporidian sp. was designated as NIK-5hm, which formed ovocylindrical spore in the haemocytes of silkworm and differed in spore size (length, 4.55 $\mu$m & width, 2.10 $\mu$m) and shape from Nosema bombycis (NIK-ls), NIK-2r (Nosema sp. Mysore [3.6 & 2.8 $\mu$m]), NIK-3h (Nosema sp. M$_{11}$ [3.8 & 1.8 $\mu$m]), NIK-4m (Nosema sp. M$_{12}$ [5.0 & 2.1 $\mu$m]) and Lb$_{ms}$ (Nosema sp. in Lamerine breed of silkworm [4.36 & 2.14]). In immonological test (Latex agglutination test), the isolated microsporidian spores did not react with antibody sensitized latex particles of N. bombycis, M$_{11}$, M$_{12}$ and Lb$_{ms}$ and thus are different type of microsporidian sp., parasitic to silkworm, Bombyx mori L.