大泷六线鱼卵黄原蛋白的纯化鉴定及其ELISA检测方法的建立

Purification and Characterization of Vitellogenin from Fat Greenling(Hexagrammos otakii) and Establishment of An ELISA

海洋环境雌激素污染日益加剧,亟需建立可靠的生物检测技术,鱼类卵黄原蛋白(vitellogenin, Vtg)是国际上推荐的环境雌激素生物标志物。本研究采用凝胶过滤与离子交换层析技术从17β-雌二醇(E2)肌肉注射后的雄性大泷六线鱼(Hexagrammos otakii)血浆中纯化获得Vtg。经鉴定大泷六线鱼Vtg是分子量约为340 kDa的糖磷脂蛋白,SDS-PAGE显示其亚基分子量约为170 kDa。利用纯化的Vtg免疫新西兰大白兔制备了多克隆抗血清,并且Western blot结果证实抗血清对Vtg具有很好的特异性。以纯化的Vtg与兔抗Vtg多克隆抗体建立了检测大泷六线鱼血浆Vtg的间接竞争酶联免疫吸附反应(ELISA)。该检测方法的工作范围为15.62~1 000 ng/mL,组内与组间差异分别为6.85%与6.79%,可用于大泷六线鱼血浆Vtg的准确定量。本研究建立的大泷六线鱼Vtg ELISA具有较高的敏感度、特异性与精确度,为中国海洋环境雌激素的生物监测提供了可靠工具。

Vitellogenin(Vtg)is an important biomarker for screening environmental estrogens.In this study,Vtg was purified from the plasma of fat greenling(Hexagrammos otakii)injected with 17β-estradiol(E2)by gel filtration followed by anion-exchange chromatography.The purified protein was subjected to native polyacrylamide gel electrophoresis(Native-PAGE)and stained positively with methyl green,Sudan black B,and Schiff’s reagent,indicating that it was a phospholipoglycoprotein.The purified Vtg had an apparent molecular weight of 340 kDa and resolved in a main band of 170 kDa and two additional minor bands of 117 and 98 kDa in SDS-PAGE.The purified Vtg was used to immunize rabbits by subcutaneously injection to prepare anti-Vtg polyclonal antibody.Western blot result revealed that the prepared antibody reacted with plasma from E2-treated male fat greenling and purified Vtg,but no cross-reaction was observed in the plasma from control male fat greenling,indicating that the antibody was highly specific to fat greenling Vtg.Using the purified Vtg and polyclonal antibody,indirect competitive ELISA for quantifying Vtg concentrations of plasma in fat greenling was developed.The competitive ELISA had a working range from 15.62 to 1 000 ng/mL,and the intra-and inter-assay coefficients of variations were 6.85%and 6.79%,respectively.These results confirmed that the competitive ELISA could accurately quantify Vtg concentrations of plasma in fat greenling.The competitive ELISA of fat greenling developed in this study has high sensitivity,specificity and precision,and thus provides a useful tool for monitoring marine environmental estrogens.