Expression of biologically active bovine luteinizing hormone in Chinese hamster ovary cells.
Expression of biologically active bovine luteinizing hormone in Chinese hamster ovary cells.
复制标题
生物活性牛黄体生成素在中国仓鼠卵巢细胞中的表达。
DOI：
10.1073/pnas.82.21.7280
复制
发表时间：
1985
影响因子：
11.1
通讯作者：
Nilson,JH
中科院分区：
文献类型：
--
作者：
Kaetzel,DM;Browne,JK;Wondisford,F;Nett,TM;Thomason,AR;Nilson,JH
通过在二氢叶酸还原酶(DHFR)缺陷的中国仓鼠卵巢细胞系DUXB11稳定转化克隆中表达α和黄体生成素β亚基基因，获得了具有生物活性的牛黄体生成素(LH)。经细胞总RNA电泳后，印迹分析显示有预期大小的α和LHβ亚基mRNAs的表达(分别约为910和770个核苷酸)。此外，几个克隆系中促性腺激素mRNAs的存在与否与培养液中牛的一个或两个亚基的出现直接相关。来自三个克隆的培养液分泌两个亚基的显著免疫反应水平，也刺激了绵羊黄体细胞孕酮的释放，这表明分泌的黄体生成素被组装成具有生物活性的糖基化二聚体。免疫沉淀和NaDodSO4/PAGE对其中一个克隆CHODLH20分泌的[35S]蛋氨酸标记蛋白进一步证实了α/β二聚体的存在，其表观亚基分子量分别为20,500和16,000，仅略高于垂体α和LHβ亚基。
Biologically active bovine luteinizing hormone (LH) has been obtained through expression of the alpha- and LH beta-subunit genes in stably transformed clones of DUXB11, a Chinese hamster ovary cell line deficient in dihydrofolate reductase (DHFR). Expression of alpha-and LH beta-subunit mRNAs of the expected sizes (approximately 910 and 770 nucleotides, respectively) were revealed by blot analysis after electrophoresis of total cellular RNA. Furthermore, presence or absence of the gonadotropin mRNAs in several clonal lines was directly correlated with the appearance of one or both bovine LH subunits in the culture medium. Media from three clones secreting significant immunoreactive levels of both subunits also stimulated the release of progesterone in ovine luteal cells, suggesting that the secreted LH was assembled into a biologically active and glycosylated dimer. Immunoprecipitation and NaDodSO4/PAGE of [35S]methionine-labeled proteins secreted from one of the clones, CHODLH20, further confirmed the presence of an alpha/beta dimer with apparent subunit molecular weights of 20,500 and 16,000, only slightly higher than those of pituitary alpha and LH beta subunits.