【作者】 雷莉辉；

【导师】 陈德坤； 曲志娜；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2004， 硕士

【摘要】 己烯雌酚（diethylstilbestrol，DES）属于人工合成的雌激素，在人和动物体内会产生雌激素样效应。在养殖业中，DES能够显著促进动物的生长，提高饲料转化率，但由于DES对人体的致癌和致畸作用，世界各国均规定食品动物养殖中不得以任何途径和方式使用DES，动物及动物性食品中不得检出DES。DES残留免疫分析是DES残留监控中的重要手段之一。本研究属于DES残留免疫检测试剂盒研制的前期工作内容，也是必不可少的研究。研究取得如下结果：1.在室温条件下，将DES与丁二酸酐按一定比例溶解于无水吡啶中,搅拌反应生成棕黄色物质，将其分离纯化后对其进行高分辨质谱、红外光谱、核磁共振碳谱及氢谱等鉴定，试验结果表明，该棕黄色物质分子量为368，分子式为C22H24O5，含有酚羟基、羧基等关键基团，为所要制备的DES衍生物己烯雌酚半琥珀酸（DES-HS）。2.利用混合酸酐法将DES-HS与载体蛋白（BSA，OVA）进行偶联得到DES人工抗原。 合成的人工抗原经紫外扫描、SDS-PAGE 凝胶电泳鉴定证明DES-HS与载体蛋白偶联成功；并用UVP Bioimaging system 中的labworks 4.0 Image Acquisition and Analysis software 分析估算BSA与DES-HS的偶联率为1：30～32。OVA与DES-HS的偶联率为1:25～28。3.用合成的DES-HS-BSA结合物分别免疫家兔和Balb/c小鼠，采集免疫动物血清。应用琼脂双扩散试验和间接ELISA试验、阻断ELISA检测特异性DES抗体的产生，抗体效价及其与游离DES反应的特异性。结果表明：无论免疫兔还是免疫小鼠，其免疫血清均能够与DES-HS-OVA上的DES决定簇发生特异性结合反应；免疫兔血清效价不低于1：6000，免疫小鼠血清效价不低于1：12000；兔免疫血清能够与游离DES发生特异性反应。通过上述研究和分析，可得出如下的结论：建立了DES-HS制备方法；成功地制备出了DES的人工抗原；用DES-HS-BSA分别免疫兔和小鼠制备出了抗DES免疫血清；建立了DES抗体的间接ELISA检测方法。本研究为研制DES单克隆抗体及拥有我国自主知识产权的DES残留免疫检测试剂盒奠定了基础。更多还原

【Abstract】 Diethylstilbestrol(DES) is a synthetic estrogen. It can produce the effects like natural estrogens in the bodies of human beings and animals.In feeding, it was used illegally to promote growth and feed conversion, but it can lead to cancer and deformity. So it was ruled not to use DES in feeding animal by any way all over the world. Animal and animal products could not be detected DES. The immunoassay of residual DES is one of the critical means in monitor of DES residues. The study is necessary and piror to develop DES residual immunoassay reagent box . The main results are as follows: 1.At the room temperature,the certain ratio of DES and succinate anhydride was stirred in anhydrous pyridine, and got the brown product. The analysis results of HRMS, IR , 13C-NMR and 1H-NMR showed that the separated and pured is product was the derivative of DES wanted, called as DES-HS. Its molecular weight is 368, Its molecular formula is C22H24O5, It includes the critical structure such as -OH ,–COOH and so on.2.The artificial antigen of DES was synthesized by mixed anhydride method using DES-HS and bovine serum albumin(BSA) and ovabumin(OVA) respectively. The characterization of hapten-protein conjugates were examined ultraviolet spectrophotometry(UV) and sodium doclecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), the results showed that the binding is successful. The number of hapten molecules per molecules of protein estimated by UVP Bioimaging system of labworks 4.0 Image Acquisition and Analysis software was 30～32 and 25～28 respectively.3.BSA-hapten conjugate synthesized was used as immunogen to immunized rabbits and Balb/c mice respectively and the polyclonal antisera were all emerged in rabbits and Balb/c mice. The specificity of antisera was tested by agar double-diffusion method and indirect enzyme-linked immunosorbent assay(ELISA), blocked ELISA detected the antisera titer at the same time. The results showed that the immune antisera titer of rabbits were not below 1:6000,the immune antisera titer of Balb/c mice were not below 1:12000. Rabbits antisera can react with free DES.To sum up above all,the conclusions were drawn: the preparation method of DES-HS was built,the artificial antigen of DES were synthesized successfully,the polyclonal antisera of DES were prepared using the synthetic DES-HS-BSA conjugate; the indirect ELISA method was builded to detect DES antibody with prepared DES polyclonal <WP=7>antibody. It established the base of preparing DES monoclonal and developing DES residual immunoassay reagent box which owned the independent knowledge authority of our country.更多还原