Development of suganegaating technology for animalbiozeactor

通过注射逆转录病毒载体，在肌动蛋白启动子的控制下表达人促红细胞生成素，获得转基因鸡。促红细胞生成素在血清和白色中产生，但在蛋黄中不产生。样品用蓝色琼脂糖凝胶柱纯化约10次，回收率为50%。虽然血清衍生的促红细胞生成素的生物活性比蛋清衍生的或市售的动物细胞衍生的促红细胞生成素强30至40倍，这通过培养细胞系的促红细胞生成素依赖性增殖来指示，但纯化的样品显示出与源自蛋清白色和动物细胞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。虽然血清衍生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显示出更强的生物活性，但来自鸡蛋白色的促红细胞生成素的生物活性不受处理的影响，这表明糖基化的差异。为了研究输卵管促红细胞生成素糖链的作用机制，将三种半乳糖基转移酶和三种唾液酸转移酶克隆到表达载体中，利用昆虫细胞生产可溶性蛋白。在转染细胞的培养上清液中检测到所有蛋白质，现在检查其酶活性。此外，我们发现Siglecs，结合唾液酸的内源性哺乳动物凝集素，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，而细胞产生高得多的抗炎细胞因子白细胞介素-10，当细胞用各种炎症诱导材料刺激时，包括脂多糖、一种引起细菌性鲨鱼的强效内毒素和肽聚糖。

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.