| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥300,000)
Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2006: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Transgenic chickens were produced by the injection of retroviral vector to express human erythropoietin under the control of actin promoter. Erythropoietin was produced into serum and egg white but not into yolk. Samples were partially purified by approximately ten times using blue-sepharose column with 50% recovery. Although serum-derived erythropoietin had thirty to forty times stronger bioactivity compared with egg white-derived or commercially available animal cell-derived erythropoietin, which was indicated by erythropoietin-dependent proliferation of a cultured cell line, purified sample showed a activity similar to erythropoietin derived from egg white and animal cells. Erythropoietin samples were then digested by sialidase and β-galactosidase. While serum-derived erythropoietin, as well as that of animal cell origin, showed a stronger bioaclivityafer the treatment of these enzymes, the bioactivity of erythropoietin from egg white was not affected by the treatment, indicating the differences in glycosylation. To examine the mechanisms to add sugar chains to erythropoietin in oviduct, three galactcsyltransferases and three sialyltransferses were cloned into expression vector to produce soluble proteins by insect cells. All proteins were detected in the culture supernatants of transfected cells, which are now examined for enzymatic activity. Furthermore, we found that Siglecs, endogenous mammalian lectins to bind sialic acids, had anti-inflammatory activity toward macrophages: macrophage cell line that expressed Siglecs produced lower amounts of proinflammatory cytokines such as tumor necrosis factor-α and interleukin-6, while the cells produced much higher amounts of anti-inflammatory cytokine interleukin-10, when cells were stimulated with various inflammation-inducing materials including lipopolysaocharides, a potent endotoxin causing bacterial shark, and peptidoglycans.
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