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PubMed2006

Expression, purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor.

To date, many recombinant proteins have been expressed in Bombyx mori cells or silkworm larvae, apart from in pupae. Silkworm pupae may be more suitable for the expression of heterologous proteins as a bioreactor. If maintained at an appropriate temperature, silkworm pupae could be inoculated with recombinant baculovirus for the expression of a protein of interest. In this study, human granulocyte-macrophage colony-stimulating factor was successfully expressed in silkworm pupae using B. mori nucleopolyhedrovirus, purified and characterized with respect to its physico-chemical properties. The target protein expressed had an apparent molecular mass of 29 kDa and an isoelectric point of 5.1. The protein was purified using three chromatographic steps with a final recovery of 10.3%. Finally, approximately 3.5mg of the protein was obtained with a biological activity of up to 8.4 x 10(6) cfu mg(-1). The results of this study suggest that silkworm pupae represent a convenient and low-cost bioreactor for the expression of heterologous proteins.

Animals↗
PubMed2006

Generation of hybrid transgenic silkworms that express Bombyx mori prolyl-hydroxylase alpha-subunits and human collagens in posterior silk glands: Production of cocoons that contained collagens with hydroxylated proline residues.

Prolyl 4-hydroxylase (P4H) is a heterotetramer enzyme consisting of alpha-subunits (P4Halpha) and beta-subunits (P4Hbeta), and is required for collagen biosynthesis. Previously, we generated transgenic silkworms that produced human type III collagen fragments (mini-collagens) in the posterior silk gland (PSG). However, prolyl 4-hydroxylation did not occur on the mini-collagens, because in spite of an abundant expression of P4Hbeta in PSGs, P4Halpha expression was quite low there, thus resulting in an insufficient activity of P4H. In this study we aimed at generating hybrid transgenic silkworms whose PSGs are capable of producing mini-collagens and enough P4H for their prolyl 4-hydroxylation. Isolated PSGs were bombarded with fibroin L-chain gene promoter-driven vectors containing Bombyx mori P4Halpha (BmP4Halpha) cDNAs and were transplanted into the hemolymphatic cavity. The P4H activity in the PSG cells significantly increased, indicating that the expressed BmP4Halpha formed active tetramers with endogenous BmP4Hbeta. Using germ-line transgenesis technology, silkworms were generated that synthesized BmP4Halpha in PSG cells. The P4H activity in the transgenic silkworms was 130-fold higher than that of wild-type counterparts. Finally, we generated hybrid transgenic silkworms that expressed cDNAs of both BmP4Halpha and mini-collagen in PSG cells. They spun cocoons that contained mini-collagens whose appropriate proline residues had been adequately hydroxylated.

Animals↗
PubMed2005

Serial analysis of gene expression in the silkworm, Bombyx mori.

The silkworm Bombyx mori is one of the most economically important insects and serves as a model for Lepidoptera insects. We used serial analysis of gene expression (SAGE) to derive profiles of expressed genes during the developmental life cycle of the silkworm and to create a reference for understanding silkworm metamorphosis. We generated four SAGE libraries, one from each of the four developmental stages of the silkworm. In total we obtained 257,964 SAGE tags, of which 39,485 were unique tags. Sorted by copy number, 14.1% of the unique tags were detected at a median to high level (five or more copies), 24.2% at lower levels (two to four copies), and 61.7% as single copies. Using a basic local alignment search tool on the EST database, 35% of the tags matched known silkworm expressed sequence tags. SAGE demonstrated that a number of the genes were up- or down-regulated during the four developmental phases of the egg, larva, pupa, and adult. Furthermore, we found that the generation of longer cDNA fragments from SAGE tags constituted the most efficient method of gene identification, which facilitated the analysis of a large number of unknown genes.

Animals↗
PubMed1990

Expression of a silkworm eclosion hormone gene in yeast.

Recombinant silkworm eclosion hormone was produced for the first time in yeast which was transformed with a shuttle plasmid containing a construct coding a signal peptide and the mature sequence of the silkworm eclosion hormone. Successfully transformed yeast processed recombinant silkworm eclosion hormone I (EH-I) and transported it to periplasm at the concentration of 60 micrograms per liter of culture. The biological activity of the purified recombinant silkworm eclosion hormone exhibited the ED50 value of 0.2 ng which is the same as that of the authentic hormone isolated from the silkworm brain.

Amino Acid Sequence↗
PubMed2003

Changes in growth and lipid profiles of silk gland, mid-gut biochemical composition of silkworm, Bombyx mori L. on exposure to prostaglandin F2alpha.

The growth of the silkworm is influenced by the outside and inside environment. Among them, the category of various endocrine hormone of inside is the main factors that adjust the characters such as growth and propagate. In this experiment, we applied different dosage of prostaglandin to the fourth and fifth instar silkworm to observe the effects of prostaglandin F2alpha (PGF2alpha) on silk gland growth, mid-gut biochemical constituents and the lipid profiles of silkworm larva, Bombyx mori L. The weight of the posterior silk gland increased significantly (P < 0.001) by 20-24% after treatment with PGF2alpha. The increase in the lipid profiles except lipase activity suggests that the silk gland had more synthetic activity that might reflect in active spinning of silkworm larva. The changes of total proteins, free amino acids and alkaline phosphatase in mid-gut of control and PGF2alpha treated silkworm, B. mori L. indicate that PGF2alpha favored stimulatory effect on physiology of digestion, absorption and transportation of nutrients which might influence on the growth and development of larva.

Animals↗
PubMed1998

Cloning and characterization of the TATA-binding protein of the silkworm Bombyx mori.

The TATA-binding protein is a general transcription factor required by all three eukaryotic nuclear RNA polymerases. In order to study the function of this protein in the transcription of tRNA genes in the silkworm Bombyx mori, we have cloned TBP cDNA from a silkworm cDNA library. As in most other eukaryotes, TBP in silkworms is encoded by a single copy gene and contains a highly conserved C-terminal domain that includes a basic region and two direct repeats. In the less conserved N-terminal domain, silkworm TBP exhibits characteristics such as a glutamine-rich stretch and three imperfect Pro-Met-Thr-like repeats that are also found in Drosophila and human TBP. Silkworm TBP expressed in Escherichia coli and purified to apparent homogeneity binds the TATA element of the wild-type adenovirus major late promoter with nanomolar affinity.

Amino Acid Sequence↗
PubMed2006

Isolation and characterization of neutral lipids of desilked eri silkworm pupae grown on castor and tapioca leaves.

The neutral lipid of desilked eri silkworm pupae (Samia cynthia ricini) grown on two different host plants, castor (Ricinus communis Linn.) and tapioca (Manihot utilizsima Phol.) leaves, was extracted with hexane. The oil content in pupae was estimated to be in the range of 18-20% (dry basis). The pupal oil was found to be enriched with alpha-linolenic acid (ALA) with palmitic acid as the second major fatty acid. The level of ALA in the oil of silkworm pupae was found to be significantly higher (P < 0.001) when grown on tapioca (58.3%) as compared to those grown on castor (42.9%). Other chemical parameters such as percent free fatty acid, peroxide value, phosphorus content, percent unsaponifiable matter, and composition of sterols were also determined in both of the oils and compared. Reversed-phase high-performance liquid chromatography analysis of triacylglycerol molecular species showed that the pupal oil is rich in molecular species with equivalent carbon numbers (ECN) C36, C40, C42, C44, and C48. There was a significantly higher level (P < 0.001) of trilinolenin (C36) in the oil of tapioca-based silkworm as compared to castor-based silkworm pupae. Regiospecific analysis of the oil showed a higher level of ALA at the sn-2 position of silkworm pupae grown on tapioca (60.2%) as compared to those grown on castor (47.3%) oil. Thus, the presence of a large amount of ALA and their predominance at the sn-2 position make the eri pupal oil highly nutritious, provided that the oxidative stability is ensured.

Animals↗
PubMed1996

Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori.

A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori. The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced. Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region displaying significant homology to the putative catalytic region of a group of bacterial beta-1,3 glucanases and beta-1,3-1,4 glucanases. Silkworm GNBP was also shown to have amino acid sequence similarity to the vertebrate lipopolysaccharide receptor CD14 and was recognized specifically by a polygonal anti-CD14 antibody. Northern blot analysis showed that GNBP was constitutively expressed in fat body, as well as in cuticular epithelial cells of naive silkworms. Intense transcription was, however, rapidly induced following a cuticular or hemoceolien bacterial challenge. An mRNA that hybridized with GNBP cDNA was also found in the l(2)mbn immunocompetent Drosophila cell line. These observations suggest that GNBP is an inducible acute phase protein implicated in the immune response of the silkworm and perhaps other insects.

Acute-Phase Proteins↗
PubMed2004

The genome sequence of silkworm, Bombyx mori.

We performed threefold shotgun sequencing of the silkworm (Bombyx mori) genome to obtain a draft sequence and establish a basic resource for comprehensive genome analysis. By using the newly developed RAMEN assembler, the sequence data derived from whole-genome shotgun (WGS) sequencing were assembled into 49,345 scaffolds that span a total length of 514 Mb including gaps and 387 Mb without gaps. Because the genome size of the silkworm is estimated to be 530 Mb, almost 97% of the genome has been organized in scaffolds, of which 75% has been sequenced. By carrying out a BLAST search for 50 characteristic Bombyx genes and 11,202 non-redundant expressed sequence tags (ESTs) in a Bombyx EST database against the WGS sequence data, we evaluated the validity of the sequence for elucidating the majority of silkworm genes. Analysis of the WGS data revealed that the silkworm genome contains many repetitive sequences with an average length of <500 bp. These repetitive sequences appear to have been derived from truncated transposons, which are interspersed at 2.5- to 3-kb intervals throughout the genome. This pattern suggests that silkworm may have an active mechanism that promotes removal of transposons from the genome. We also found evidence for insertions of mitochondrial DNA fragments at 9 sites. A search for Bombyx orthologs to Drosophila genes controlling sex determination in the WGS data revealed 11 Bombyx genes and suggested that the sex-determining systems differ profoundly between the two species.

Animals↗
PubMed1995

Silkworm TFIIIA requires additional class III factors for commitment to transcription complex assembly on a 5S RNA gene.

We find striking similarities in promoter structure and requirements for template commitment on 5S RNA and tRNA genes from silkworms. The promoters are nearly the same size (approximately 160 bp) and include flanking as well as internal sequences. To analyze the factor requirements for 5S RNA transcription complex assembly in a completely homologous system, we have isolated a silkworm fraction that is highly enriched for the 5S RNA-specific transcription factor, TFIIIA. Using this fraction, together with the other silkworm fractions, TFIIIB, TFIIIC, TFIIID and RNA polymerase III, we demonstrate that the requirements for 5S RNA transcription complex assembly are very similar to those previously established for a tRNA(C)(Ala) gene. Specifically, no individual factor fraction is sufficient for commitment of silkworm 5S RNA genes to transcription complex assembly. Rather, combinations of at least three factor fractions are required. Our observation that more than one subset of factors is competent for commitment suggests that silkworm 5S RNA genes further resemble tRNA(C)(Ala) genes in their ability to use multiple pathways for transcription complex formation.

Animals↗
PubMed2005

Silkworm pathogenic bacteria infection model for identification of novel virulence genes.

Silkworms are killed by injection of pathogenic bacteria, such as Staphylococcus aureus and Streptococcus pyogenes, into the haemolymph. Gene disruption mutants of S. aureus whose open reading frames were previously uncharacterized and that are conserved among bacteria were examined for their virulence in silkworms. Of these 100 genes, three genes named cvfA, cvfB, and cvfC were required for full virulence of S. aureus in silkworms. Haemolysin production was decreased in these mutants. The cvfA and cvfC mutants also had attenuated virulence in mice. S. pyogenes cvfA-disrupted mutants produced less exotoxin and had attenuated virulence in both silkworms and mice. These results indicate that the silkworm-infection model is useful for identifying bacterial virulence genes.

Animals↗
PubMed1988

Transcriptional properties of BmX, a moderately repetitive silkworm gene that is an RNA polymerase III template.

We analyzed the transcriptional properties of a repetitive sequence element, BmX, that belongs to a large gene family (approximately 2 x 10(4) copies) in the genome of the Bombyx mori silkworm. We discovered BmX elements because of their ability to direct transcription by polymerase III in vitro and used them to test the generality of the properties of previously identified silkworm polymerase III control elements. We found that the signals that act in cis to control BmX transcription strongly resemble those that direct transcription of other silkworm polymerase III templates. As with silkworm tRNA and 5S RNA genes, transcription of BmX requires sequence signals located both upstream and downstream from the site of transcription initiation. The critical upstream sequences are structurally as well as functionally similar in the three kinds of templates. The downstream control region of BmX resembles the corresponding part of a silkworm alanine tRNA gene in that it provides a large (greater than 100 base pairs) region that influences transcription factor binding. Moreover, the factor-binding regions of both tRNA(Ala) and BmX genes are remarkable in that under certain conditions, key elements within them (the B boxes, for example) appear dispensable. This behavior can be understood if, in both of these templates, the downstream control region acts as a large target for interaction with a multifactor complex.

Animals↗
PubMed1992

The properties of monoclonal antibody against sepiapterin reductase from fat body of the silkworm, Bombyx mori.

A specific monoclonal antibody prepared for the 29-kDa a subunit of silkworm fat body sepiapterin reductase (SPR) was able to recognize the subunit in crude extract of fat body after SDS treatment. Although SPR from the silkworm fat body has biochemical properties similar to those reported for SPR from mammalian sources, especially rat erythrocytes, the antibody failed to recognize the 28-kDa subunit of rat erythrocyte SPR. This result indicates that SPR from silkworm fat body has a different amino-acid sequence from that of the rat erythrocyte enzyme. Sepiapterin reductase activity has not been found in crude extract of fat body from the silkworm mutant lemon. Although the antibody recognized only 29-kDa protein in the crude extract of silkworm fat body from normal strain after SDS-treatment, the antibody recognized only an approximately 80-kDa protein in the crude extract of the lemon mutant after SDS-treatment.

Alcohol Oxidoreductases↗
PubMed1990

[Allergen skin tests to insects, chironomid, caddis fly and silkworm moth, in patients with nasal allergy].

The present study was undertaken to assess the importance of insects as allergens causing nasal allergy. We performed intradermal skin tests with extracts from three insects, chironomid, caddis fly, and silkworm moth, on patients with nasal allergy and on volunteers among medical students. Twenty-two (27.5%) of 80 nasal allergic patients showed positive reactions to chironomid allergen and 9 (11.3%) had positive reactions to caddis fly allergen and 18 (22.5%) to silkworm moth allergen. These positive rates were as frequent as those to Candida or ragweed. Medical students were divided into two groups, symptomatic and asymptomatic, according to the presence or the absence of nasal allergy. Three (11.5%) of 26 symptomatic group showed positive reactions to chironomid and one (3.8%) to caddis fly and 5 (19.2%) to silkworm moth. While even 4 (8.3%) of 48 asymptomatic group showed positive reactions to chironomid, 2 (4.8%) to caddis fly, and 6 (12.5%) to silkworm moth. These results suggested that these insects, chironomid, caddis fly, and silkworm moth, are relatively common than we expected and suspected as important allergens in nasal allergy.

Adolescent↗
PubMed1999

Transgenic Silkworm of Anti-NPV Ribozyme.

The plasmid pGL2Rz including ribozyme gene was linearized and introduced into early eggs of silkworm (G(0)) by gene gun. The luciferase activity in blood of the G(1) generation was detected, then the resistant silkworm was selected by NPV infection from G(2) generation. The transgenic silkworm resistant against NPV 10 times more than control ones was got at the G(4) generation. PCR and Southern blotting proved that the ribozyme gene was integrated into the genome of silkworm multicopily. The expression of ribozyme was also detected by RT-PCR in pupa. The results showed that the transgenic silkworm strain of anti-NPV ribozyme has been got.

Journal Article↗
PubMed1999

[Expression of Schistosoma japonicun fatty acid binding protein gene in silkworm cells and larvae].

AIM: To express the fatty acid binding protein (Sj14FABP) gene of Schistosoma japonicun in the silkworm cells and larvae. METHODS: A 600 bp DNA fragment containing Sj14FABP gene was cloned into baculovirus transfer vector of pBacPAK His1 to construct recombinant transfer vector Sj14-pBac PAK His1. Coinfection was accomplished with this vector and Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA in BmN cells. The recombinant virus of Bm-Sj14 was screened using dot-blotting. The BmN cells and silkworm larvae were infected with Bm-Sj14 to express Sj14FABF gene. Western blotting and ELISA were used to identify the antigenicity of the recombinant protein. RESULTS: Sj14FABP gene was successfully expressed in the BmN cells and silkworm larvae infected with Bm-Sj14. The product was a 18 kDa fusion protein. The yield in BmN cells was about 100 micrograms/1 x 10(6) cells and 33 micrograms/ml cell supernatant. In silkworm larvae, the product yield was 4 mg/ml haemolymph as well as 4.6 mg/g silkworm tissue. The recombinant protein could be recognized by Western blotting and ELISA using the sera from mice immunized with SWAP. CONCLUSION: Sj14FABP gene has been successfully expressed in BmNPV system and the product has high antigenicity.

Animals↗
PubMed2003

Influence of form and quantity of chromium on the development and survival of two silkworm (Bombyx mori L.) races.

Growth inhibitory activity has proven important in Qiufeng x Baiyu and Qingsong x Haoyue silkworm larvae. The consumption of mulberry leaves was reduced in both silkworm races while Qiufeng x Baiyu larvae showed the higher reduction in leaf consumption. From the results obtained, it was revealed that even at low concentrations of 400 mg/L of either Cr(III) or Cr(VI) ions, growth of Qiufeng x Baiyu and Qingsong x Haoyue was significantly depressed. Depression in relative growth index (RGI) and high death rate in both silkworm races indicated that the different concentrations of the two ions used caused their growth inhibitions. Through linear regression analysis, the EC50 (concentration of the compound that caused 50% reduction) was interpolated for both tested compounds. EC50 (mg/L) of Cr(III) ions in Qiufeng x Baiyu and Qingsong x Haoyue were 800 and 600 respectively. EC50 (mg/L) of Cr(VI) ions in Qiufeng x Baiyu and Qingsong x Haoyue was 600 and 316 respectively. According to the analysis of relative growth index, and an analysis of linear regression technique for measuring the growth of the silkworm races, it was indicated that the form of Cr affected growth rates, growth inhibition responses of the larvae, and toxicological effects. Thus, form and quantity of Cr accumulating in silkworms reared with contaminated leaves are likely to influence their population dynamics.

Animals↗
PubMed2006

[Investigation and analysis of the bacteria community in silkworm intestine].

Majority of the environmental microorganisms are still unculturable. To shed a light on the bacteria community in the silkworm intestine more completely, culture-dependent and culture-independent methods were used to perform the investigation. As the latter, 16s rRNA gene was amplified and a library was constructed by using the meta-genomic DNA extracted from the bacteria in the silkworm intestine as template. Restriction fragment length polymorphism (RFLP) method was used to screen recombinants originated from different bacteria, and the nucleotide sequences were determined. A BLAST searching in the GenBank and an analysis on phylogenesis were performed. The taxonomy of these bacteria and their physiological function to silkworm were speculated. Results showed that the bacteria in silkworm intestine are mainly belong to the genera Arthrobacter, Lactobacillus, Pseudomonas, Escherichia, Micrococcus, Bacillus, and Staphylococcus. They may play important roles to the host silkworm in the utilization of their food mulberry and disease prevention. Both the culture-dependent and culture-independent methods have their advantages and disadvantages, and they are complemented with each other.

Animals↗