Search

Baculovirus expression systems (BES) are valuable and popular tools for the production of recombinant proteins. Because insect cells have a posttranslational modifications similar to those of mammalian cells, such as glycosylation, phosphorylation, and protein folding, BES are used to produce proteins from higher eukaryotes, for example, canine interferons–a, human serine–threonine kinase 11, and human acidic and basic fibroblast growth factors (Liu et al., 2005; Martinez et al., 2005; Gouveia et al., 2007). As regards the glycosylation in insect cells, the N–glycosylation pathway has been studied with a view to production of human–like N–glycosylated proteins, but there are few reports of O–glycosylation. In this study, to investigate whether the mucin–type O–glycosylation occurs in silkworm, Bombyx mori, we performed recombinant protein expression of a mucin– like protein as a target using BES in silkworm. Mucins are heavily O–glycosylated glycoproteins found in secreted mucous and as transmembrane glycoproteins of the cell surface to serve as a last protective barrier against extracellular environmental factors like low pH or hydrolytic enzymes (Strouss et al., 1992). Mucins have serine and threonine rich domains called variable number of tandem repeat (VNTR) regions. Mucins may have hundreds of O–GalNAc glycans attached to serine or threonine residues in the VNTR regions and these abundant O–glycan chains may comprise 80% of the molecule by weight (Lan et al., 1987). Four main O–glycan core structures are well known in mammals (Daniel., 2009). The most common O–GalNAc glycan is Gal β 1–3GalNAc α–Ser/Thr (Fig. 1). It is termed a Core 1 or T–antigen and forms many longer, more complex structures. Another common structure is Core 2, contains a branching N–acetylglucosamine attached to core 1 (Fig. 1). Core1 and Core 2 O–GalNAc glycans are found in both glycoproteins and mucins from a variety of cells and tissues. Core 3 and Core 4 stuructures (Fig. 1) are found in a few tissues, such as colon (Podolsky., 1985), bronchi (Lamblin., 1984) and salivary glands (Wieruszesk et al., 1987). In insects, Core 1 type mucin has been observed (E Tian et al., 2008), but other type mucins are yet to be detected. Expression of Glycosylated Mucin–like Domain Using Baculovirus Expression System in Silkworm, Bombyx mori

INTRODUCTIONTransgenic organisms including cultured cell lines are one of powerful tool for analyzing the functions of specific genes of interest. At present, very efficient gene transfer methods such as lipofection, electroporation and the virus–mediated transformation are available (Carbonell

RNAi induction by the injection of long dsRNA into hemocoel has been tried to archive efficient gene knockdown in silkworm larvae or pupae, and severas reports successfully applying RNAi to silkworm have been brought out. However, effect of RNAi in silkworm is often insufficient and improvement of efficiency is desirable. On the other hand, the metabolic turnover or organ uptake of dsRNA injected into insects has not been sufficiently clarified so far. In this study, we report that silkworm larval hemolymph has dsRNA binding activity. We also show that the molecular entity of dsRNA binding activity of hemolymph is lipophorin, a major lipoprotein complex in insects. dsRNA binding activity of silkworm larval hemolymph was observed by electrophoretic mobility shift assay. dsRNA binding activity–associated protein was purified by affinity chromatography with immobilized dsRNA as a ligand. Amino acid sequence of the purified protein was determined by Edman degradation and the protein was revealed to be a silkworm homolog of Apolipophorin I, an unexchangeable apolipoprotein subunit of lipophorin. Lipophorin fraction prepared from hemolymph by density gradient ultracentrifugation also exhibited the similar dsRNA binding activity. We determined full– length sequence of Apolipophorin II/I precursor homolog of silkworm, whose structure was highly conserved among insects. Although physiological role of dsRNA binding activity is still unknown, the present study indicates potential effect by injection of dsRNA into silkworm individuals.

radiation has beenused as a tool for chromosomal cleavage to generatemutations, such as the sex–linked sable and sex–linkedzebra (Tazima, 1941; Hashimoto, 1948). These werecreated by the methods in which chromosomal dou-ble–strand breaks (DSBs) by ionizing radiation (IR)induced marker gene translocation from an autosomeinto the sex–chromosome, female specific W. Similarly,recessive mutations were frequently induced by IR inthe oocyte nuclei during meiosis (Murakami, 1966,1971a, b). In these experiments was irradiated 2,000 to15,000 rad (20 to 150Gy) of X–ray, which must haveinevitably broken DNA double strands causing chromo-somal arrangements, but, in many cases, was not lethalto the silkworm. The robust resistance of this species toγ–irradiation has also been reported (Miki, 1986). Weobserved previously that γ–irradiation at a larval stageled to the abnormally immature wing but not to anyother features of pupae (Takahashi