【作者】 谷峰；

【导师】 薛飞群；

【作者基本信息】 中国农业科学院 ， 临床兽医学， 2003， 硕士

【摘要】 克伦特罗临床上作为呼吸促进药物，主要用于治疗人兽哮喘。在畜牧生产中将克伦特罗作为生长促进剂使用具有明显提高肉品瘦肉率、降低脂肪含量的作用。克伦特罗在被机体吸收、转化、代谢的过程中，虽然大部分被代谢或排泄，但仍有相当量残留在肝脏、肌肉、视网膜等组织中。此残留对人畜的健康造成巨大威胁和危害，并由此在国内外引发多起食物中毒事件。研究克伦特罗药物残留检测相关技术及开展有克伦特罗样作用的安全药物的研究显得十分有意义。 本论文从研究药物残留检测相关技术与寻找安全药物两方面为导向，其研究内容包括克伦特罗偶联物的制备与鉴定、抗克伦特罗单克隆抗体的研制和鼠β₂肾上腺素能受体基因的克隆及表达。 因为克伦特罗等药物为小分子，它们只具有反应原性而无免疫原性，所以此类药物与大分子的偶联成功是产生抗体的关键，同时有关半抗原偶联物的鉴定工作少见报道，此研究以期积累鉴定药物偶联物的经验，更好作好药物免疫分析工作。在本论文中，在制备好克伦特罗的偶联物后，利用红外光谱、紫外光谱、FPLC、SDS-PAGE及免疫鉴定，确定偶联成功。本文对偶联物鉴定的几种方法作一比较与评价。通过实验，得出FPLC是以上几种鉴定偶联物的最佳方法。 抗克伦特罗单克隆抗体的研制采用常规单克隆抗体制备技术，其中包括动物免疫与血清效价的测定、骨髓瘤细胞的培养、饲养细胞的制备、免疫脾细胞的获得、细胞融合与阳性克隆的筛选、阳性克隆的单克隆化等步骤。在经过以上步骤以后，获得了分泌抗克伦特罗单克隆抗体的细胞并对初步获得的抗体进行了效价测定。单克隆抗体的研制成功将有助于克伦特罗检测试剂盒的研制。同时，有助于克伦特罗样品的前处理。 安全药物有两种思路：建立药物筛选模型筛选生物体内易分解的活性分子，从受体的抗体着手制备抗受体的抗体以及抗克伦特罗单抗的抗独特型抗体。而所有研究的前提是获得β₂肾上腺素能受体基因和抗克伦特罗单克隆抗体，所以此研究的基础工作之一是完成β₂肾上腺素能受体基因的克隆与表达工作。 鼠β₂肾上腺素能受体基因的克隆及表达实验内容包括：PCR扩增与产物回收、回收产物与载体连接、连接产物的转化、蓝白斑筛选、阳性克隆的鉴定与基因测序、序列的分析比较。通过鉴定与序列比较，确定该基因鼠β₂肾上腺素能受体基因被克隆。在完成鼠p／肾上腺素能受体基因的克隆后，将其进行原核表达。将表达的蛋白进行SDS－rxGE电泳以检测基因的表达情况。这些为寻找安全药物及受体药物分析奠定了基础。 本实验的研究意义在于：为药物残留检测及食品安全提供技术支持；为寻找安全药物奠定基础。更多还原

【Abstract】 Clenbuerol is a potent respiratory stimulant used in human and animal medicine. This drug is , however , used illegally as a growth promoter in production animals ,because its use leads to carcass which are fat-free and which have an extraordinary development of muscular mass. While much of the Clenbuterol will be excreted or metabolized, there is still a considerable amount retained by the animals especially in the liver, muscles, and retina. As a results , the residues rendered a great danger to consumer and animal ,furthermore ,several food poisoning accidents related to the consumption of illicit β 2 agonist in liver was happened in China and other country. There is a great demand about techniques related to the detection of residue and safety drug which have Clenbuerol positive effect and no lexicological characteristics.According to this, the content in this paper is included as follow : Synthesis and Identification of Clenbuterol -carrier protein conjugate, the Preparation of Monoclonal Antibody against Clenbuterol and Cloning & expression of (32 Adrenergic Receptor Gene of Mouse.For the reason that small moleculars liking Clenbuterol have their immunoreactivity but no immunogenicity, thus they couldn’t induce animal to produce antibody until they have been conjugated to macromolecular ,such as bovine serum albumin, human serum albumin. Unfortunately ,little study has been done to make sure that small molecular ,hapten ,has been conjugated with carrier protein. As a result, no effective method has been put forward . In this paper, some work have been done and tried to solve this problem . A conjugate prepared by the direct coupling of diazotized Clenbuterol to bovine serum albumin was identified by IR, UV , SDS-PAGE v FPLC and immunological experiment . Compared with each other, a brief assessment of each analytical method is given. A conclusion was draw that among all the analytical methods, FPLC is the best one.Monoclonal antibody was prepared by classical hybridomas technique . The whole process included the preparation of antigen(enough forimmunization and screen),planning a screening assay, developing the screening assay, immunization the mouse, preparation the sp2/0 cell, plasma cells in spleen ,feeding cell, the fusion of the sp2/0 cell and plasma cells in spleen cell , screening to get positive clone and cloning ,colony growth. After the whole operation , positive clone which could secrete antibody against Clenbuterol was obtained, furthermore the antibody concentration was measured. Monoclonal antibody could use as standard reagents in the residue immunoassay and reagents in the treatment of sample.There are two way to obtain safety drug , on the one hand, when a pharmaceutical screening model has been established , easy-to-catabolism bioactive molecular could be approached . on the other hand, antibody against β sadrenoceptor, which is binding with the receptor just as β 2 adrenergic stimulant, may have the same bioactivity, anti-idiotypic antibodies against anti-Clenbuterol Monoclonal Antibody could Mimics the bioactive ofβ2 adrenergic stimulant. Above all, the basis of these study is that the β 2 adrenergic receptor gene of mouse must have been cloned and the monoclonal antibody against Clenbuterol must been prepared successfully.β2 Adrenergic receptor Gene of Chinese Kunming mouse was amplified by PCR and it was ligated with pUCm-T vector by T4 ligase . The ligation product was then transfered into competent E.coli DH5α . After screening with α-complementarity, positive clone was identified by PCR ,restrict enzyme characterization and DNA sequencing . Furthermore ,the gene was blast in NCBI . A conclusion was draw that β2 adrenergic receptor gene of mouse had been cloned. Then,β2 Adrenergic receptor gene was expressed in E.Coli and fusion protein was detection by SDS-PAGE .All in all , The meaning all work in this paper is that providing technique associated with residue detection , lay a basis for the screening safety drug .更多还原