Purification of gonadotropin II from a teleost fish, the hybrid striped bass, and development of a specific enzyme-linked immunosorbent assay.
Purification of gonadotropin II from a teleost fish, the hybrid striped bass, and development of a specific enzyme-linked immunosorbent assay.
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从硬骨鱼、杂交条纹鲈中纯化促性腺激素 II，并开发特定的酶联免疫吸附测定。
DOI：
10.1006/gcen.1997.6966
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发表时间：
1997
影响因子：
2.7
通讯作者：
Y. Zohar
中科院分区：
文献类型：
--
作者：
E. Mañanós;P. Swanson;J. Stubblefield;Y. Zohar
高纯度的促性腺激素II（GtH II），简称条纹鲈GtH II（stbGtH II），其α和β亚基是从性成熟杂交条纹鲈的垂体中制备的。用乙醇提取垂体糖蛋白，并通过 Sephadex G-100 上的凝胶过滤色谱、DE-52 上的离子交换色谱 (IEC) 和 Superdex 75 上的快速高效液相色谱 (FPLC) 纯化完整的 stbGtH II。通过反相高效液相色谱 (rpHPLC) 上的特征洗脱和体外类固醇生成活性来监测纯化过程中 GtH 的存在。通过凝胶过滤、IEC 和 rpHPLC 从垂体乙醇提取物中纯化 stbGtH II α 和 β 亚基，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (SDS-PAGE)、rpHPLC 和 N 端氨基酸测序评估其身份。通过 SDS-PAGE 测定，完整 stbGtH II 及其 α 和 β 亚基的分子量分别为 34.5、14.8 和 20.4 kDa。 stbGtH II β 亚基用于产生特异性抗体，并使用完整的 stbGtH II 作为标准曲线开发了竞争性酶联免疫吸附测定法。该测定的灵敏度为 156 pg/ml（15.6 pg/孔），测定内和测定间变异系数（50% 结合）分别为 7.7% (n = 16) 和 8.7% (n = 10)。通过测量成熟条纹鲈雌性注射 GnRHa ([d-Ala6,Pro9-NEt]-mGnRH, 100 microg/kg BW) 后血浆 GtH II 水平的变化来进行测定的生理学验证。注射后 12 小时观察到血浆中 GtH II 的最大峰值（22.5 +/- 3. 01 ng/ml），而对照鱼中的 GtH II 水平（约 4 ng/ml）保持不变。通过连续稀释多种鲈形物种的血浆和垂体获得的位移曲线与标准曲线平行，表明该测定可用于多种鱼类的 GtH II 测量。
A highly purified gonadotropin II (GtH II), referred to as striped bass GtH II (stbGtH II), and its alpha and beta subunits were prepared from pituitaries of sexually mature hybrid striped bass. Pituitary glycoproteins were extracted with ethanol and intact stbGtH II purified by gel-filtration chromatography on Sephadex G-100, ion-exchange chromatography (IEC) on DE-52, and fast-performance liquid chromatography (FPLC) on Superdex 75. The presence of GtHs during the purification procedure was monitored by characteristic elution on reversed-phase high-performance liquid chromatography (rpHPLC) and in vitro steroidogenic activity. The stbGtH II alpha and beta subunits were purified from the pituitary ethanol extract by gel-filtration, IEC, and rpHPLC, and their identities assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), rpHPLC, and N-terminal amino acid sequencing. Molecular weights of intact stbGtH II and its alpha and beta subunits, determined by SDS-PAGE, were 34.5, 14.8, and 20.4 kDa, respectively. The stbGtH II beta subunit was used to produce specific antibodies, and a competitive enzyme-linked immunosorbent assay was developed using intact stbGtH II for the standard curve. The sensitivity of the assay was 156 pg/ml (15.6 pg/well) and the intra- and interassay coefficients of variation (at 50% binding) were 7.7% (n = 16) and 8.7% (n = 10), respectively. Physiological validation of the assay was performed by measuring changes of plasma GtH II levels in mature striped bass females, after injection of GnRHa ([d-Ala6,Pro9-NEt]-mGnRH, 100 microg/kg BW). A maximum surge of GtH II in plasma was observed at 12 hr postinjection (22.5 +/- 3. 01 ng/ml), whereas GtH II levels in control fish (around 4 ng/ml) remained unchanged. Displacement curves obtained with serial dilutions of plasma and pituitaries from a number of perciform species were parallel to the standard curve, indicating that this assay can be used for GtH II measurements in a variety of fish species.