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Development of suganegaating technology for animalbiozeactor

动物生物反应器甘蔗化技术的开发

基本信息

批准号：
18560752
负责人：
NISHIJIMA Ken-ichi
金额：
$ 2.43万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18560752/
关键词：
Transgenic avian Erythronoietin Glycosylation Glvcosyltransferase Lectin

项目摘要

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.

在肌动蛋白启动子的控制下，注射逆转录病毒载体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蛋清中产生，但不在蛋黄中产生。样品用蓝色琼脂糖柱纯化约10倍，回收率为50%。虽然血清来源的促红细胞生成素的生物活性是蛋清来源的或商业上可获得的动物细胞来源的促红细胞生成素的30-40倍，但纯化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。红细胞生成素样品用唾液酸酶和β-半乳糖苷酶消化。血清来源的促红细胞生成素和动物细胞来源的促红细胞生成素在这些酶处理后显示出较强的生物活性，但蛋清来源的促红细胞生成素的生物活性不受处理的影响，表明糖基化作用的差异。为了探讨促红细胞生成素在输卵管中增加糖链的机制，将三种半乳糖转移酶和三种唾液酸转移酶克隆到表达载体中，在昆虫细胞中表达可溶性蛋白。在转基因细胞的培养上清液中检测到所有蛋白质，现在正在检测其酶活性。此外，我们还发现内源性哺乳动物凝集素Siglecs对巨噬细胞具有抗炎活性：表达Siglecs的巨噬细胞株产生较少的促炎细胞因子，如肿瘤坏死因子-α和白介素6，而当细胞受到各种炎症诱导物质刺激时，产生的抗炎细胞因子-白介素10要高得多，包括脂多糖，一种能引起细菌性鲨鱼的内毒素，以及肽多聚糖。