中国水产科学2018，Vol.25Issue(1)：53-59,7.DOI:10.3724/SP.J.1118.2018.17034
鲫卵黄脂磷蛋白的纯化鉴定及其夹心ELISA的建立
Purification and characterization of lipovitellin from crucian carp (Carassius carassius)and establishment of a sandwich ELISA
摘要
Abstract
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.关键词
鲫/卵黄脂磷蛋白/纯化/性质鉴定/ELISAKey words
Carassius carassius/lipovitellin/purification/characterization/ELISA分类
资源环境引用本文复制引用
张振忠,马淑伟,郑明驿,王军,汝少国..鲫卵黄脂磷蛋白的纯化鉴定及其夹心ELISA的建立[J].中国水产科学,2018,25(1):53-59,7.基金项目
国家自然科学基金项目(21607144). （21607144）
