Preparation Of Polyclonal Antiserum Against Goldfish Vitellogenin And Its Application In Studies Of Endocrine Disrupting Chemicals

In the past decades, scientific interest in the possible impact of exposure to endocrine disrupting chemicals(EDCs) on human or wildlife has grown dramatically. Some in vivo screening systems selected fish as model(such as medaka, fathead minnow and zebrafish) have been developed. Nevertheless there is no fish screening system has been developed in our country so far. Therefore, in this paper, we selected goldfish as screening model, which widely distribute in our country; and on the basis of preparation of polyclonal antiserum against goldfish vitellogenin(Vtg), developed the technique of western-blot for the Vtg qualitative detection. We firstly raised the goldfish environmental estrogen screening system: the Vtg induction as a main biomarker,γ-GTP and LDH as supplementary biomarkers. This screening system also been successfully used in the screening experiments for some species of pesticide.Vtg from estradiol-treated goldfish plasma was isolated and purified by gel filtration and ion-exchange chromatography. By the phosphor-, lipo- and glycol-protein staining methods, we recognized this protein as Vtg. The molecular weight of Vtg is about 440kDa detected by native-PAGE. In SDS-PAGE, the Vtg broke into 2 main subunits, which are 84 and 60 kDa. Making use of purified Vtg, we prepared the polyclonal antiserum against goldfish Vtg, and developed the western-blot for Vtg detection, which detection limit is 0.5 mg L-1(polyclonal antiserum 1: 2000 diluted).Male goldfish received 0.05mg g-1BW 17β-estradiol intraperitoneal injection, after 14 d's induction, the blood was collected from caudal vessel. The results of western-blot showed that 17β-estradiol could induce male...