鲫(Carassius carassius)卵黄原蛋白(vitellogenin,Vtg)是检测水体环境雌激素活性常用的生物标志物.本研究利用凝胶过滤结合离子交换层析与选择性沉淀结合离子交换层析2种方法, 从鲫卵巢匀浆液中纯化得到了卵黄脂磷蛋白(lipovitellin, Lv), 经鉴定该蛋白含有糖、磷、脂基团, 天然分子量约为521 kD, SDS变性电泳显示分子量为117 kD和103 kD的2个亚基.Western blot结果显示,金鱼(Carassius auratus)Lv抗体和斑马鱼(Danio rerio)Lv抗体都能与鲫Lv发生很好的交叉反应.利用纯化的鲫Lv与金鱼Lv抗体和斑马鱼Lv抗体建立了2种夹心ELISA, 发现金鱼 Lv 和鲫 Lv 的结合曲线基本重合, 并且利用鲫 Lv 与金鱼 Lv 抗体建立的夹心 ELISA 工作范围为15.6~1000 ng/mL, 检出限约为6.8 ng/mL, 显著低于利用斑马鱼 Lv 抗体建立的夹心 ELISA, 结合此前研究者建立的鲫Vtg竞争ELISA, 为鲫Vtg指标的测定提供了可靠方法.%Crucian carp (Carassius carassius) vitellogenin (Vtg) is a commonly used biomarker for the detection of estrogenic activity in aquatic environments. In this study, gel filtration combined with ion-exchange chroma-tography and selective precipitation combined with ion-exchange chromatography were used to purify lipovitellin (Lv) from ovarian homogenates of crucian carp. Lv was identified as a phospholipogly coprotein with an apparent molecular weight of ~521 kD and separated into two major polypeptides corresponding to ~117 and ~103 kD by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The results of the western blot showed that both an-ti-goldfish(Carassius auratus)Lv antibodies and anti-zebrafish(Danio rerio)Lv antibodies had good cross-reac-tion with crucian carp Lv. Using anti-goldfish Lv antibody, anti-zebrafish Lv antibody, and purified crucian carp Lv, two sandwich enzyme-linked immunosorbent assays (ELISAs) were established. The results showed that the binding curves of goldfish Lv and crucian carp Lv basically overlapped. The sandwich ELISA developed using anti-goldfish Lv antibodies and purified crucian carp Lv had a working range form 15.6 to 1000 ng/mL and a de-tection limit of 6.8 ng/mL, which was significantly lower than that of the anti-zebrafish Lv antibody-based ELISA and other ELISAs previously reported for crucian carp Vtg. Therefore, we suggest that it is a reliable tool for the quantification of crucian carp Vtg.
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