蛋白同化激素多残留检测免疫学分析方法的基础研究

项目名称： 蛋白同化激素多残留检测免疫学分析方法的基础研究

项目编号： No.U1204310

项目类型： 联合基金项目

立项/批准年度： 2013

项目学科： 免疫学

项目作者： 姜金庆

作者单位： 河南科技学院

项目金额： 30万元

中文摘要： 蛋白同化激素用于治疗骨质疏松症，蛋白质缺陷型疾病和烧伤等，还可以提高运动员和竞技动物比赛成绩，增强食源性动物瘦肉生产率，但其残留物引发严重的内分泌失调、神经问题和遗传毒性。本项目依据动物代谢试验和药代动力学理论测定蛋白同化激素残留部位、代谢物种类、存在形式和消除规律；采用荧光光谱、三维荧光光谱、同步荧光光谱和紫外吸收光谱研究蛋白质与半抗原相互作用，筛选“母核”化合物；通过偶联方法、结合位点、载体蛋白和偶联率的对比实验，制备人工抗原，并进行UV、IR、PAGE和抗血清鉴定；细胞融合技术筛选杂交瘤细胞株，运用间接ELISA、间接竞争ELISA和交叉反应实验制备类特异性单克隆抗体；以反转录PCR、原核转化和核糖体展示为基础的单链抗体技术制备基因重组抗体分子，批量表达类特异性靶标蛋白。该研究为建立食源性动物蛋白同化激素多残留免疫学分析方法奠定基础，并且为解决我国食品安全的可持续研究提供科学依据。

中文关键词： 蛋白同化激素；类特异性；人工抗原；免疫原检测；基础研究

英文摘要： Anabolic steroids are used for the treatment of osteoporosis, protein-deficient diseases and burns, which can also enhance the competition performance for athletes and athletic animals, and increase lean productivity and feed conversion ratio of food-producing animals, but their residues cause serious endocrine disorders, neurological problems, and genetic toxicity. Based on the animal metabolism studies and pharmacokinetic theory, this project aimed to determine the residue targets of anabolic hormone, metabolite types and forms, and the elimination ways. The mutual reaction between hapten and carrier proteins was studied by fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, synchronous fluorescence spectroscopy and UV absorption spectra, and therefore the “general nucleus” compounds were selected. Through comparison tests of different coupling methods, binding sites, carrier proteins, and conjugation rates, the artificial antigen was synthesized, which was then identified by UV, IR, PAGE and antiserum. Cell fusion technology was employed to screen hybridoma cell lines, and indirect ELISA, indirect competitive ELISA and cross-reactivity experiments were used to prepare class-specific monoclonal antibody. Based on reverse transcription-PCR, prokaryotic transformation and ribosome display, th

英文关键词： anabolic steroids；group specificity；artificial antigen；immunoassay；basic research