真鲷血浆中卵黄蛋白原的柱后荧光衍生检测及其在环境雌激素生物标志物中的应用
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摘要
卵黄蛋白原(Vtg)通常只存在于性成熟的雌鱼里,但是当雄鱼或幼鱼暴露于环境雌激素时,其体内也能产生Vtg。因此,Vtg被认为是一种理想的环境雌激素的生物标志物。Vtg的检测方法主要是特异性的免疫分析方法,方法灵敏,选择性强;但耗时长,操作繁琐。因此,建立快速、简便、可靠的Vtg检测方法是深入开展Vtg作为环境雌激素标志物研究的关键前提。目前对Vtg的研究主要集中在淡水鱼类,对海水鱼类的研究较少。随着环境污染的加剧,大量污染物被排入大海,使沿海污染日益严重,因此有必要开展海洋鱼类Vtg的研究,用于监测海洋环境雌激素的污染。本论文选择海鱼真鲷为研究动物,从血浆中纯化获得Vtg的标样;建立了非特异性测定Vtg的分析新方法;并在新方法的基础上,开展真鲷血浆中Vtg作为环境雌激素标志物的研究。本研究的主要内容和结果包括以下几个方面:
1.真鲷Vtg的诱导、纯化及初步鉴定
采用17β-雌二醇(E2)腹腔注射,使真鲷在7 d内产生大量Vtg;利用阴离子交换(DEAE Sepharose Fast Flow树脂)低压液相色谱系统,一步纯化Vtg。从色谱图和电泳谱图可知,该蛋白只存在于E2诱导后的真鲷血浆中和雌鱼对照血浆中,而不存在于雄鱼对照血浆中,说明该蛋白是雌性特异性蛋白,能够被E2诱导产生。该蛋白经Native-PAGE电泳,结果表明真鲷有两种形式的Vtg,分子量分别为570 kDa和360 kDa;该蛋白经SDS-PAGE后,可形成分子量为180 kDa、115 kDa和70 kDa的3条蛋白带。磷、脂、糖蛋白分析证实,所获取的Vtg是一类含磷、含脂、含糖的大分子蛋白。对真鲷腹腔注射4-壬基酚,发现4-壬基酚也能诱导真鲷体内Vtg的产生。
2.柱后荧光衍生-高效液相色谱法测定鱼血浆中的Vtg
利用高效阴离子交换Mini Q柱分离和柱后邻苯二甲醛(OPA)荧光衍生,建立了快速测定血浆中Vtg的方法。对柱后衍生反应条件进行了优化,最终确定衍生反应的条件为:衍生液pH值为10.5;衍生液中硼砂浓度为40 mmol/L;OPA浓度为0.4 mg/mL;反应温度为50℃;2-巯基乙醇的浓度为0.5μL/mL。方法的线性
Vitellogenin (Vtg) is usually produced in sexually active female fish. However, Vtg can be detected in male and juvenile fish exposed to estrogen or estrogen mimic chemicals. Therefore, Vtg is generally considered to be a good biomarker for environmental estrogens. Immunoassays are the most commonly used approaches for Vtg determination. These antibody-based methods have considerable advantages of sensitivity and selectivity, however, at the same time suffer from a long process incubation period and troublesome wash steps. Therefore, development of a novel, rapid, easy and accurate method for Vtg determination is necessary and important. Studies so far about Vtg as a biomarker for environmental estrogens were mostly limited to freshwater fish. Due to the increasing pollution in marine environment, Vtg of marine fish receives great concern. In this study, Pagrosomus major was chosen as an experimental animal. Vtg standard had been purified from plasma of Pagrosomus major. An analytical method for rapid determination of Vtg in fish plasma had been developed. Based on this, a study about Vtg in plasma of juvenile Pagrosomus major as a biomarker for environmental estrogens had been carried out. The detailed information is as follows.
1. Induction, purification and initial characterization of Vtg from Pagrosomus major
Vtg was largely induced in Pagrosomus major by one intraperitoneal injection of 17β-estradiol (E2). With anion exchange liquid chromatography, Vtg was effectively purified by a column filled with DEAE Sepharose Fast Flow resin. It was found that Vtg was present in plasma of E2 treated Pagrosomus major and control female fish, and absent from plasma of control male fish, which suggested that the protein was a female specific protein and could be induced by E2. The results of Native-PAGE showed that the Vtg of Pagrosomus major had two forms, with molecular weights around 570 kDa and 360 kDa, respectively. After being separated by SDS-PAGE, the
1.真鲷Vtg的诱导、纯化及初步鉴定
采用17β-雌二醇(E2)腹腔注射,使真鲷在7 d内产生大量Vtg;利用阴离子交换(DEAE Sepharose Fast Flow树脂)低压液相色谱系统,一步纯化Vtg。从色谱图和电泳谱图可知,该蛋白只存在于E2诱导后的真鲷血浆中和雌鱼对照血浆中,而不存在于雄鱼对照血浆中,说明该蛋白是雌性特异性蛋白,能够被E2诱导产生。该蛋白经Native-PAGE电泳,结果表明真鲷有两种形式的Vtg,分子量分别为570 kDa和360 kDa;该蛋白经SDS-PAGE后,可形成分子量为180 kDa、115 kDa和70 kDa的3条蛋白带。磷、脂、糖蛋白分析证实,所获取的Vtg是一类含磷、含脂、含糖的大分子蛋白。对真鲷腹腔注射4-壬基酚,发现4-壬基酚也能诱导真鲷体内Vtg的产生。
2.柱后荧光衍生-高效液相色谱法测定鱼血浆中的Vtg
利用高效阴离子交换Mini Q柱分离和柱后邻苯二甲醛(OPA)荧光衍生,建立了快速测定血浆中Vtg的方法。对柱后衍生反应条件进行了优化,最终确定衍生反应的条件为:衍生液pH值为10.5;衍生液中硼砂浓度为40 mmol/L;OPA浓度为0.4 mg/mL;反应温度为50℃;2-巯基乙醇的浓度为0.5μL/mL。方法的线性
Vitellogenin (Vtg) is usually produced in sexually active female fish. However, Vtg can be detected in male and juvenile fish exposed to estrogen or estrogen mimic chemicals. Therefore, Vtg is generally considered to be a good biomarker for environmental estrogens. Immunoassays are the most commonly used approaches for Vtg determination. These antibody-based methods have considerable advantages of sensitivity and selectivity, however, at the same time suffer from a long process incubation period and troublesome wash steps. Therefore, development of a novel, rapid, easy and accurate method for Vtg determination is necessary and important. Studies so far about Vtg as a biomarker for environmental estrogens were mostly limited to freshwater fish. Due to the increasing pollution in marine environment, Vtg of marine fish receives great concern. In this study, Pagrosomus major was chosen as an experimental animal. Vtg standard had been purified from plasma of Pagrosomus major. An analytical method for rapid determination of Vtg in fish plasma had been developed. Based on this, a study about Vtg in plasma of juvenile Pagrosomus major as a biomarker for environmental estrogens had been carried out. The detailed information is as follows.
1. Induction, purification and initial characterization of Vtg from Pagrosomus major
Vtg was largely induced in Pagrosomus major by one intraperitoneal injection of 17β-estradiol (E2). With anion exchange liquid chromatography, Vtg was effectively purified by a column filled with DEAE Sepharose Fast Flow resin. It was found that Vtg was present in plasma of E2 treated Pagrosomus major and control female fish, and absent from plasma of control male fish, which suggested that the protein was a female specific protein and could be induced by E2. The results of Native-PAGE showed that the Vtg of Pagrosomus major had two forms, with molecular weights around 570 kDa and 360 kDa, respectively. After being separated by SDS-PAGE, the
引文
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