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采用分子筛和离子交换两步层析法分离纯化了真鲷(Pagrosomus major)的卵黄原蛋白(VTG),并采用非变性和变性聚丙烯酰胺凝胶电泳测定了真鲷VTG的分子量,发现真鲷VTG在非变性条件下的分子量大约为450kDa;在SDS变性条件下,两条亚基的分子量大约分别为170kDa和120kDa。Western-blot检测结果表明这两条亚基都能够与真鲷VTG多克隆抗血清产生免疫反应,而真鲷VTG的170kDa亚基是检测真鲷VTG的特异性条带,且最低检出浓度为0.1μg L~(-1)。鼠抗真鲷VTG多克隆抗血清对真鲷VTG具有良好的特异性,这一结果为采用真鲷为模式生物检测海洋环境雌激素奠定了实验基础。
The vitellogenin (VTG) of Pagrosomus major was isolated and purified by molecular sieve and ion exchange two-step chromatography. The molecular weight of VTG of Pagrosomus was determined by non-denaturing and denaturing polyacrylamide gel electrophoresis. The molecular weight of VTG under non-denaturing conditions is about 450 kDa; the molecular weights of both subunits are about 170 kDa and 120 kDa, respectively, under SDS denaturing conditions. The results of Western-blot showed that both of the two subunits could immunoreact with the polyclonal antiserum of VTG from sea bream, while the 170 kDa subunit of VTG from sea bream was the specific band for detecting VTG of sea bream, and the lowest detection concentration was 0.1 μg L -1. The anti-seabream VTG polyclonal antiserum has good specificity for the VTG of sea bream, which laid the experimental foundation for the use of seabream as a model organism for the detection of estrogen in the marine environment.