Study on the production of N-Glycolylneuraminic acid-deficient animals using gene targeting techniques

利用基因打靶技术生产N-甘醇神经氨酸缺陷动物的研究

• 批准号: 07557155
• 负责人: KAWASAKI Toshisuke
• 金额: $ 12.67万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557155/
• 关键词: carbohydrate antigen; CMP-NeuAc hydroxylase; N-Glycolylneuraminic acid; HD antigen; gene targeting; 遺伝子クローニング; ノックアウトマウス; クローニング

N-Glycolylneuraminic acid (NeuGc) is derived from NeuAc by hydroxylation of a methyl moiety in the N-acetyl residue. This hydroxylation occurs by the conversion of CMP-N-acetylneuraminic acid (CMP-NeuAc) to CMP-N-glycolylCMP-NeuGc, which is carried out by an electron transport system involving NADH-dependent cytochrome b5 reductase, cytochrome b5, and CMP-NeuAc hydroxylase. CMP-NeuAc hydroxylase is the key enzyme in the hydroxylation of CMP-NeuAc, and functions at the final step of the electron transport system. We have previously purified CMP-NeuAc hydroxylase from the mouse liver, and isolated a cDNA for this enzyme which contains an ORF encoding a protein of 577 amino acids. All mammals examined except human have NeuGc and therefore NeuGc is known to be an epitope of one of major carbohydrate antigens in human. Recently, it is reported that NeuGc is detected in the sialic acid-containing medicines such as human erythropoietin produced in CHO cells.In this study, we plan to make NeuGc-deficient animals using gene-targeting technique in order to produce NeuGc-free tsialic acid-containing medicines.

n -糖基神经氨酸（NeuGc）是由NeuAc在n -乙酰基残基上的甲基片断羟基化而得到的。这种羟基化通过cmp - n -乙酰神经氨酸（CMP-NeuAc）转化为cmp - n -糖基cmp - neuc发生，这是一个涉及nadh依赖性细胞色素b5还原酶、细胞色素b5和CMP-NeuAc羟化酶的电子传递系统进行的。CMP-NeuAc羟化酶是CMP-NeuAc羟化的关键酶，在电子传递系统的最后一步起作用。我们已经从小鼠肝脏中纯化了CMP-NeuAc羟化酶，并分离出该酶的cDNA，该酶含有一个编码577个氨基酸的蛋白的ORF。除人类外，所有被检测的哺乳动物都有NeuGc，因此NeuGc已知是人类主要碳水化合物抗原之一的表位。最近，有报道称NeuGc在含唾液酸的药物中被检测到，如由CHO细胞产生的人促红细胞生成素。在本研究中，我们计划利用基因靶向技术制造neugc缺陷动物，以生产不含neugc的含草酸药物。

项目成果

T.Kawano et al: "Molecular cloning of CMP-N-acetylneuraminic acid hydroxylase from mouse liver" J.Biol.Chem.270(27). 16458-16463 (1995)

T.Kawano 等人：“来自小鼠肝脏的 CMP-N-乙酰神经氨酸羟化酶的分子克隆”J.Biol.Chem.270(27)。

S.Koyama et al: "A naturally occurring 46-amino acid deletion of cytidine monophospho-N-acetylneuraminic acid hydroxylase leads to a change in the intracellular distribution of the protein" Glycoconjugate J.13. 353-358 (1996)

S.Koyama 等人：“胞苷单磷酸-N-乙酰神经氨酸羟化酶天然存在的 46 个氨基酸缺失导致蛋白质的细胞内分布发生变化”Glycoconjugate J.13。

S.Koyama, T.Yamaji, H.Takematsu, T.Kawano, Y.Kozutsumi, A.Suzuki & T.Kawasaki: "A naturally occurring 46-amino acid deletion of cytidine monophospho-N-acetylneuraminic acid hydroxylase leads to a change in the intracellulardistribution of the protein" Gly

S.Koyama、T.Yamaji、H.Takematsu、T.Kawano、Y.Kozutsumi、A.Suzuki

T.Kawano, S.Koyama, H.Takematsu, Y.Kozutsumi, H.Kawasaki, S.Kawashimai, T.Kawasaki, & A.Suzuki: "Molecular cloning of CMP-N-acetylneuraminic acid hydroxylase from mouse liver" J.Biol.Chem.270(27). 16458-16463 (1995)

T.Kawano、S.Koyama、H.Takematsu、Y.Kozutsumi、H.Kawasaki、S.Kawashimai、T.Kawasaki、

T.Yamaji et al: "Neutral glycosphingolipids induce cell-cell aggregation of a variety ofhematopoietic cell lines." Eur.J.Biochem.247(1). 21-29 (1997)

T.Yamaji 等人：“中性鞘糖脂诱导多种造血细胞系的细胞间聚集。”