Carp (Cyprinus carpio) vitellogenin:: purification and development of a simultaneous chemiluminescent immunoassay
Carp (Cyprinus carpio) vitellogenin:: purification and development of a simultaneous chemiluminescent immunoassay
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DOI：
10.1016/s1095-6433(02)00348-3
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发表时间：
2003-03-01
影响因子：
2.3
通讯作者：
Hara, A
中科院分区：
文献类型：
--
作者：
Fukada, H;Fujiwara, Y;Hara, A
用羟基磷灰石柱层析和凝胶过滤法从卵黄发生母鲤（Cyprinus carpio）血清中分离纯化卵黄蛋白原（Vg）。Vg的表观分子量为490 kDa，在还原条件下进行SDS-PAGE后，出现两条对应于190和156 kDa的条带。这些条带在Western印迹中使用抗鲤鱼卵黄脂磷蛋白（抗-Lv）的抗血清进行免疫反应，所述卵黄脂磷蛋白是来源于Vg的蛋黄蛋白。鲤鱼Vg的氨基酸组成与鲤科鱼类相似。利用纯化的鲤鱼Vg和抗Lv抗体建立了鲤鱼Vg荧光免疫分析法（CLIA）。其可测范围为1.95 ~-1000 ng/ml。卵黄发生母血清CLIA的稀释曲线与纯化Vg的标准曲线平行。批内和批间变异系数分别小于5%。此外，该方法与其他雌性鲤科动物（鲫鱼和日本？）的血清有交叉反应。在鱼饲料实验中，在含有大豆（20%）的鱼粉组的所有鱼中检测到Vg，但在鱼粉组的几乎所有鱼中未检测到Vg。这表明，大豆为基础的饮食可能会诱导养殖鲤鱼血清中的Vg的生产。(C)2002年爱思唯尔科学公司All rights reserved.
Vitellogenin (Vg) was purified from the serum of vitellogenic female carp (Cyprinus carpio) by hydroxylapatite column chromatography and gel filtration. Vg had an apparent molecular mass of 490 kDa and appeared as two bands corresponding to 190 and 156 kDa after SDS-PAGE under reducing conditions. These bands were immunoreacted in Western blotting using antiserum against carp lipovitellin (anti-Lv) which is an egg yolk protein derived from Vg. The amino acid composition of carp Vg was similar to previous reports of cyprinids. The chemiluminescent immunoassay (CLIA) for carp Vg was developed to quantify serum Vg using purified carp Vg and anti-Lv. Its measurable range was from 1.95 to- 1000 ng/ml. The dilution curve in the CLIA of vitellogenic female serum was parallel to the standard curve of purified Vg. The coefficient variations of intra- and inter-assay were less than 5%, respectively. Furthermore, the assay had cross-reactivity with the sera of other female cyprinids (crucian carp and Japanese dace). In fish diets-experiments, Vg was detected in all fish in the fish meal containing soybean (20%) group, but was not detected in almost all of the fish in the fish meal-group. This suggests that a soybean based-diet may induce Vg production in the serum of cultivated carp. (C) 2002 Elsevier Science Inc. All rights reserved.