Development of suganegaating technology for animalbiozeactor

在肌动蛋白启动子的控制下，通过注射逆转录病毒载体获得了表达人促红细胞生成素的转基因鸡。血清和蛋清产生促红细胞生成素，蛋黄不产生促红细胞生成素。样品部分纯化约10倍，采用蓝葡糖柱，回收率50%。虽然血清来源的促红细胞生成素的生物活性比蛋清来源的或市售的动物细胞来源的促红细胞生成素强30到40倍，这是通过培养细胞系的促红细胞生成素依赖性增殖来证明的，纯化后的样品显示出与蛋清和动物细胞来源的促红细胞生成素相似的活性。然后用唾液酸酶和β-半乳糖苷酶消化促红细胞生成素样品。血清来源的促红细胞生成素以及动物细胞来源的促红细胞生成素在这些酶处理后表现出更强的生物活性，而蛋清的促红细胞生成素的生物活性不受处理的影响，表明糖基化的差异。为了研究糖链加入促红细胞生成素的机制，将3种半乳糖基转移酶和3种唾液基转移酶克隆到表达载体中，通过昆虫细胞产生可溶性蛋白。在转染细胞的培养上清液中检测到所有蛋白质，现在检查酶活性。此外，我们发现Siglecs，内源性哺乳动物凝集素结合唾液酸，对巨噬细胞具有抗炎活性：表达Siglecs的巨噬细胞系产生较少量的促炎细胞因子，如肿瘤坏死因子-α和白细胞介素-6，而当细胞受到各种炎症诱导物质（包括脂多糖，一种有效的内毒素引起的细菌鲨鱼）和肽聚糖刺激时，细胞产生大量的抗炎细胞因子白细胞介素-10。

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.