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

制备了不含同源垂体糖蛋白激素LH的具有生物活性的重组猪卵泡刺激素（rpFSH）。利用编码糖蛋白α和FSH β亚基的两个cdna，在杆状病毒载体-昆虫细胞系统中合成了rpFSH。其抗原性与垂体制备的pFSH相同。tunicamycin可使rpFSH糖基化，但由于在昆虫细胞中没有末端糖的修饰和唾液酸的加入，rpFSH各亚基的分子量低于垂体驱动的FSH （pFSH）。RpFSH以1 mg/l左右的浓度在培养基中分泌，由于糖组的异质性，通过S-Sepharose和Con-A柱层析，RpFSH分为6个部分。研究了rpFSH在猪颗粒细胞孕酮分泌和猪卵母细胞生发囊泡分解（GVBD）中的生物活性。RpFSH具有较好的孕酮分泌活性，但部分富糖组的活性低于pFSH。1 ng/ml低浓度时，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诱导怀孕母马血清促性腺激素（PMSG）启动和垂体切除的未成熟雌性大鼠排卵，并伴有组织型纤溶酶原激活物（tPA）的增加。

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).