Production, Characterization and Application of Recombinant Gonadotropin (FSH and LH)

重组促性腺激素（FSH 和 LH）的生产、表征和应用

基本信息

批准号：
07556124
负责人：
MORI Junichi
金额：
$ 1.09万
依托单位：
KITASATO 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 1996
项目状态：
已结题

项目摘要

Production of biologically active recombinant porcine FSH (rpFSH) free from cognate pituitary glycoprotein hormone LH was performed. The rpFSH was synthesized by a baculovirus vector-insect cell system using two cDNAs ecoding glycoprotein alpha and FSH beta subunits. Its antigenicity was same as that of pFSH prepared from pituitary. Glycosylation of rpFSH was observed by treatment of tunicamycin but the molecular weight of each subunit was lower than that of pituitary-darived FSH (pFSH), due to the absence of trimming of terminal sugar and addition of sialic acid in insect cells. RpFSH was secreted in the culture media at about 1 mg/l and purified in 6 fractions, due to the heterogeneity of sugar group, by S-Sepharose and Con-A column chromatographies. The biological activity of rpFSH was examined in progesterone secretion from porcine granulosa cells and germinal vesicle break down (GVBD) of porcine cocytes. RpFSH showed adcquate activity of progesterone sacretion, though some fractions rich in sugar group showed lower activity than that of pFSH.RpFSH gave higher GVBD activity than that of pFSH at low concentration as 1 ng/ml. These results demonstrated that baculovirus vector-insect cell system provided biologically active rpFSH.Moreover, rpFSH induced ovulation of pregnant mare serum gonadotropin (PMSG) primed and hypophysectomized immature female rats, with associated increase in tissue-type plasminogen activator (tPA).

生产不含同源垂体糖蛋白激素 LH 的具有生物活性的重组猪 FSH (rpFSH)。 rpFSH 由杆状病毒载体昆虫细胞系统使用编码糖蛋白α和FSHβ亚基的两个cDNA合成。其抗原性与垂体制备的pFSH相同。通过衣霉素处理观察到 rpFSH 的糖基化，但由于昆虫细胞中没有末端糖的修剪和唾液酸的添加，因此每个亚基的分子量低于垂体来源的 FSH (pFSH)。由于糖基的异质性，RpFSH 在培养基中以约 1 mg/l 的浓度分泌，并通过 S-Sepharose 和 Con-A 柱色谱纯化为 6 个级分。在猪颗粒细胞的黄体酮分泌和猪母细胞的生发囊泡分解（GVBD）中检查了 rpFSH 的生物活性。 RpFSH显示出足够的孕酮分泌活性，尽管一些富含糖基的级分显示出比pFSH低的活性。在1ng/ml的低浓度下，RpFSH比pFSH具有更高的GVBD活性。这些结果表明，杆状病毒载体-昆虫细胞系统提供了具有生物活性的rpFSH。此外，rpFSH诱导怀孕马血清促性腺激素（PMSG）引发和垂体切除的未成熟雌性大鼠的排卵，并伴随组织型纤溶酶原激活剂（tPA）的增加。