苯二氮卓类药物广谱单克隆抗体及沙拉沙星单链抗体的制备基础兽医学专业论文.docxVIP

摘 摘 要 喹诺酮类药物和苯二氮卓类药物在畜牧养殖过程中的大量使用或滥用，不可避 免地造成在动物性食品中的药物残留。本研究旨在制备苯二氮卓类药物的广谱性单 克隆抗体和沙拉沙星的单链抗体，为研究建立免疫分析方法和研制相关检测产品奠 定基础。 在实验中，首先对氯硝西泮进行结构改造，将其分子中的=N02还原为-NH2从 而合成半抗原，然后采用戊二醛法将其与牛血清白蛋白偶联制备免疫原。用该免疫 原免疫Balb／c小鼠，采用间接竞争ELISA方法测定血清效价及特异性。取小鼠脾 细胞与SP2／0骨髓瘤细胞进行融合从而制备杂交瘤细胞。将杂交瘤细胞注入小鼠腹 腔，收集腹水并进行纯化得到单克隆抗体。经间接竞争ELISA法测定，该单克隆抗 体能识别五种苯二氮卓类药物(氯硝西泮、硝西泮、氟硝西泮、地西泮、奥沙西泮)， 交叉反应率为24～100％，检测限为O．2～1．5 ng／mL。将五种药物分别添加至空白猪 组织中(肌肉、肝脏、肾脏)，经提取、ELISA检测，添加回收率为74．5～96．5％， 变异系数低于15．4％。 另一方面，从分泌抗沙拉沙星单克隆抗体的杂交瘤细胞中提取总RNA，反转 录得到cDNA，再通过聚合酶链式反应扩增得到单链抗体的重链可变区(VH)和轻 链可变区(VI。)基因片段。然后将VH和VI。进行拼接，得到单链抗体基因(ScFv)。 经测序鉴定后，将ScFv基因插入到表达载体pMALTM．C5X中得到重组表达载体， 将其转化入Rosetta—gami(DE3)菌株中，表达ScFv抗体。结果表明，该ScFv抗体 是以可溶性表达的形式存在。经过纯化后得到的抗沙拉沙星ScFv抗体对沙拉沙星 的识别能力与母源单克隆抗体相似。 本研究制备的苯二氮卓类药物广谱单克隆抗体和沙拉沙星单链抗体可以用于 动物性食品中两类兽药的残留检测，对保障动物源性食品安全有重要意义。 关键词：苯二氮卓类；氯硝西泮；单克隆抗体；沙拉沙星：单链抗体；残留 万方数据 Preparation Preparation of the generic monoclonal antibody for Benzodiazepine drugs and the single chain fragment viarable antibody for Sarafloxacin Candidate：Shan Wenchong Major：Basic Veterinary Medicine Advisors：Wang Jianping Abstract The wide uses and illegal uses of quinolone drugs and benzodiazepine drugs in food producing animals may inevitably lead to their residues in animal derived foods．The purpose of this study was to prepare the broad-specific monoclonal antibody of benzodiazepines and the ScFv antibody of sarafloxacin，which Can build the foundation for the development of immunoassays and related detection products． During the experiments，the molecule of clonazepam Was modified by transforming the-N02 group to-NH2 group to synthesize the hapten，and the hapten was coupled to bovine serum albumin to prepare the immunogen．The immunogen was used to immunize Balb／c mice，and the serum titer and specificity were determined by indirect competitive ELISA method．111e mouse spleen cells were fused with SP2／0 myeloma cells to prepare the hybridoma cells．The cells were inj ected into the mice abdominal cavity,and the ascites were collected and purified