卵黄蛋白原的纯化分析及汞、砷的快速生物检测
文献类型：学位论文
| 作者 | 时国庆 |
| 学位类别 | 博士 |
| 答辩日期 | 2003 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 江桂斌 |
| 关键词 | 卵黄蛋白原 汞 离子交换膜色谱 砷 生物检测 vitellogenin ion-exchange membrane chromatography mercury arsenic bioanalysis |
| 其他题名 | Purification and determination of vitellogenin in plasma and rapid bioanalytical techniques for Mercury and Arsenic detection in water samples |
| 中文摘要 | 本论文研究了典型环境内分泌干扰物生物标志物一卵黄蛋白原的纯化与分析方法以及两种重要环境内分泌干扰物汞和砷的快速生化检测技术。卵黄蛋白原是一种由雌激素诱导生成的高磷糖蛋白。正常情况下，雄性动物体内没有卵黄蛋白原，但是在雌激素或类雌激素的诱导下，雄性动物的肝脏中也可合成卵黄蛋白原，并分泌到血液中。所以，卵黄蛋白原被认为是一种理想的雌激素和类雌激素生物标志物。本文建立了一种从卵生脊椎动物血清中快速提取和纯化卵黄蛋白原的高效离子交换膜色谱方法，并对色谱分离条件进行了优化。当流动相流速为10mL／min时，对卵黄蛋白原的纯化可在3min内完成，蛋白纯度在95％以上。此方法被成功的用于四种不同种属的卵生脊椎动物卵黄蛋白原的纯化。将高效离子交换膜色谱纯化与高效凝胶过滤色谱分析结合，本论文建立了一种可快速检测血清卵黄蛋白原的二步色谱分析方法。对血清卵黄蛋白原的实际检测限为20μg／mL，加标回收率＞80%，RSD＜4.8％。该方法可用于检测化合物的中等或强雌激素效应。作为环境内分泌干扰物“黑名单”中的两种重要的金属物质，汞、砷在环境水体中的污染一直是人们关注I狗热点。本论文研究开发了一种基于汞离子对脉酶的抑制反应的可灵敏检测水样中汞化合物的检测条。该检测条的反应区有两层膜组成：上层膜是一片固定有尿素酶的醋酸纤维素膜，下层膜是一片含有尿素及固定剂的精密州试纸，颜色为黄色。检测原理是基于下层膜上黄色斑点的消失时间进行的。该黄色斑点的消失时间与样品中汞化合物的浓度成正比。在优化实验条件下，本检测条对于汞离子的检测限为0．Zng／L，检测区间为0.2-20Ong／mL。一些主要的重金属阳离子以及水样品中的其它基质对检测无影响。利用亚砷酸根对乙酰胆碱酯酶具有抑制作用的特点，我们还研究了一种基于酶膜反应器的流动注射分析系统。通过在样品溶液中加入2-PAM和EDTA以屏蔽有机磷类毒剂及重金属阳离子的影响，实现了对饮用水中亚砷酸根的特异性检测。本方法的检测限为38ng／mL，线性范围为50-100ong／mL。实验中研究的有机磷类毒剂、砷酸根及重金属阳离子对亚砷酸根的检测无干扰。 |
| 英文摘要 | The objectives of this dissertation are to develop a new method for the purification and determination of vitellogenin (Vtg), and to develop bioanalytical techniques for the measurement of mercury and arsenic. Vtg is a lipophospho-glycoprotein that is normally produced by females in response to estrogens. Generally, males itself couldn't synthesize Vtg since they have lower or negligible level of estrogen to reach the threshold required to induce Vtg production. But because of the exposure of estrogen or estrogen mimic chemicals, Vtg can also be synthesized in the liver of male fishes, and be exported into the blood. Thus, Vtg is generally agreed to be a good biomarker for the effect of estrogen or estrogen mimic chemicals. In this dissertation, a membrane chromatographic method has been developed for the rapid purification of Vtg from the plasma of oviparous vertebrates. The conditions for the purification are optimized. The time used by the proposed procedure is less then 3 minutes at 10 mL/min of the mobile phase and the purity of purified Vtg is higher that 95%. The feasibility of this method was tested to four kinds of animals from different species Combining high performance anion-exchange membrane chromatography purification with high performance gel-permeation chromatography (HP-GPC) analysis, a two-step chromatographic method was developed to the rapid determination of Vtg in fish plasma. The detection limit of Vtg is 20 u.g/mL. The spiked recovery and interassay variability were better than 80% and 4.8%. This method is adequate to detect moderate to strong estrogenic effects. As two important metals in the catalogue of endocrine disruptor chemicals, the pollution of mercuric and arsenic compounds in aquatic samples are of great concerned. In this paper, a sensitive dip-and-read test strip for the determination of mercury in aqueous samples based on the inhibition of urease reaction by the ion has been developed. The strip has a circular sensing zone that containing two layers: the top layer is a cellulose acetate membrane where urease is immobilized on it; the bottom layer is a pH indicator wafer that is impregnated with urea. The principle of the measurement is based on the time disappearance of a yellow spot on the pH indicator wafer. The elapsing time until the disappearance of the spot which depends on the concentration of mercury(II) ion is measured with a stopwatch. Under the experimental conditions, as low as 0.2 ng/mL mercury can be observed with the detection range from 0.2 to 200 ng/mL in water. Organomercury compounds give essentially the same response as inorganic mercury. Heavy-metal ions such as Ag(I), Cu(II), Cd(II), Ni(II), Zn(II), and Pb(II) as well as other sample matrixes basically do not interfere with the mercury measurement. Based on the inhibition of arsenite to the acetylcholinesterase (AChE), we developed a flow injection assay (FIA) for the determination of arsenic in drinldng water. The detection limit for the arsenite was 38 ng/mL and the liner range was 50-1000 ng/mL. Because of the addition of 2-PAM and EDTA, the method achieved higher selectivity for arsenite and those chemicals such as arsenate, heavy metal cations, organophosphate pesticide did not interfere with the determination. This FIA system could be developed as a portable device for the field analysis of arsenic. |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/34943]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 时国庆. 卵黄蛋白原的纯化分析及汞、砷的快速生物检测[D]. 北京. 中国科学院研究生院. 2003. |
入库方式： OAI收割
来源：生态环境研究中心
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