生物活性重组人神经生长因子的生产、纯化及特性鉴定

Production, purification and characterization of biologically active recombinant human nerve growth factor.

作者信息

Iwane M, Kitamura Y, Kaisho Y, Yoshimura K, Shintani A, Sasada R, Nakagawa S, Kawahara K, Nakahama K, Kakinuma A

机构信息

Biotechnology Research Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.

出版信息

Biochem Biophys Res Commun. 1990 Aug 31;171(1):116-22. doi: 10.1016/0006-291x(90)91364-x.

DOI:10.1016/0006-291x(90)91364-x

PMID:2393385

Abstract

The human NGF gene was isolated and inserted downstream from murine leukemia virus LTR in a plasmid having dihydrofolate reductase cDNA. The expression plasmid was introduced into CHO cells. Selection of the transformants for the resistance to methotrexate gave a CHO cell line which produced human NGF at a level of 4 mg/L in the culture medium. The recombinant human NGF was purified to near homogeneity from the culture supernatant. The NH2-terminal amino acid sequence, the COOH-terminal amino acid (Ala), and the amino acid composition of the human NGF were identical to those deduced from the nucleotide sequence of the human NGF gene. The recombinant human NGF was composed of 120 amino acid residues. Three disulfide linkages were determined to be Cys15-Cys80, Cys-58-Cys108, and Cys68-Cys110; the locations were identical to those in the mouse 2.5S NGF molecule. The specific biological activity of the recombinant human NGF was comparable with that of authentic mouse 2.5S NGF as determined by stimulation of neurite outgrowth from PC12 cells.

摘要

人神经生长因子（NGF）基因被分离出来，并插入到含有二氢叶酸还原酶cDNA的质粒中鼠白血病病毒长末端重复序列（LTR）的下游。将该表达质粒导入中国仓鼠卵巢（CHO）细胞。通过选择对甲氨蝶呤具有抗性的转化子，得到了一种CHO细胞系，该细胞系在培养基中产生人NGF的水平为4mg/L。重组人NGF从培养上清液中纯化至近乎同质。人NGF的氨基末端氨基酸序列、羧基末端氨基酸（丙氨酸）以及氨基酸组成与从人NGF基因核苷酸序列推导出来的一致。重组人NGF由120个氨基酸残基组成。确定三个二硫键为Cys15-Cys80、Cys58-Cys108和Cys68-Cys110；其位置与小鼠2.5S NGF分子中的相同。通过刺激PC12细胞的神经突生长测定，重组人NGF的比生物活性与天然小鼠2.5S NGF相当。