Development of suganegaating technology for animalbiozeactor

动物生物反应器甘蔗化技术的开发

• 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

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.

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类红细胞生成素，从而产生转基因鸡。红细胞生成素被生产成血清和蛋清，而不是蛋黄。使用蓝色隔离柱以50％的恢复，通过大约十倍将样品部分纯化。尽管血清衍生的促红细胞生成素具有三十至40倍的生物活性，与蛋清白色衍生或商业上可用的动物细胞衍生的促红细胞生成素相比，这是通过培养的细胞系的红细胞生成素依赖性依赖性细胞素的纯化样品表明的，但纯化的样品表现出类似于Erythropoietin orighthropoietin derypoiietin dery egg White cell egg White and Alter Animal of Egg and Alter Animal and Antile and Antile。然后通过唾液酸酶和β-半乳糖苷酶消化红细胞生成素样品。尽管血清衍生的促红细胞生成素以及动物细胞起源的生物可伴性疗法对这些酶的治疗较强，但蛋白促红细胞生物素的生物活性并未受到治疗的影响，表明糖基化的差异。为了检查将糖链添加到产卵中的促红细胞生成素的机制，将三个Galaccsylansferases和三个硫糖酶替代酶克隆到表达载体中，以通过昆虫细胞产生固体蛋白质。在翻译细胞的培养上清液中检测到所有蛋白质，现在检查了酶活性。此外，我们发现siglecs，内源性哺乳动物讲座结合唾液酸，具有抗炎活性与巨噬细胞的抗炎活性：表达siglecs的巨噬细胞系产生了较低量的促炎细胞因子，例如肿瘤性细胞，例如Tumor Necrisosis-α和Interleflim cyerfl量，而含量较高，而这些细胞含量较高。白细胞介素10，用各种感染材料刺激细胞，包括脂多糖，导致细菌鲨鱼的潜在内毒素和胡椒糖糖。

项目成果

Siglec-9 enhances IL-10 production in macrophages via tyrosine-based motifs

• DOI: 10.1016/j.bbrc.2008.02.111
• 发表时间: 2008-05-09
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Ando, Munetoshi;Tu, Wenjie;Iijima, Shinji
• 通讯作者: Iijima, Shinji

トランスジェニックニワトリによるヒトエリスロポエチンの生産

转基因鸡生产人类促红细胞生成素

• 发表时间: 2007
• 作者: 児玉大介;西宮大祐;西島謙一;飯島信司
• 通讯作者: 飯島信司