【作者】 陈小旋；

【导师】 关雄； 龚振明；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 盐酸克伦特罗（Clenbuterol hydrochloride）又称为“瘦肉精”、“营养重分配剂”,是一种人工合成的β-肾上腺素受体激动剂。该药物是最广泛使用的支气管扩张剂,用于人类和动物的哮喘疾病的治疗。另一方面,它具有促进动物生长、增加肌肉含量、降低胭体脂肪蓄积、提高瘦肉率的作用,曾被作为饲料添加剂广泛地应用于畜牧业生产。由于克伦特罗可残留在人和动物体内并造成中毒,因此已在世界范围内禁用。 目前对非法使用的克伦特罗（CL）的检测主要依赖于色谱技术和免疫分析技术。这两种方法需要专业人员操作,操作复杂,费时,成本比较高,不便于在屠宰现场检测。本实验利用杂交瘤技术制备了3株可分泌盐酸克伦特罗抗体的单克隆抗体,对其进行纯化鉴定;在此基础上建立了克伦特罗金标检测试纸条,从而达到快速、简单、方便检测克伦特罗的目的。 首先利用重氮法将CL和卵清白蛋白OVA、牛血清白蛋白BSA偶联产生免疫抗原BSA-CL和包被抗原OVA-CL。通过紫外扫描和蛋白电泳分析鉴定,发现偶联前后载体蛋白OVA、BSA的吸收峰发生改变,电泳结果显示偶联物的分子量大于载体蛋白的分子量,初步判定偶联成功。 以BSA-CL免疫Balb/C小鼠。三免后,以OVA-CL包被酶标板,用间接非竞争ELISA法检测小鼠血清,血清效价最高达1:1600。四免后取效价最高的小鼠的脾脏细胞,利用50%PEG1000将其与SP2/0细胞进行细胞融合,制备杂交瘤细胞。经过对培养上清的筛选和杂交瘤细胞的克隆化,最终得到3株能分泌盐酸克伦特罗抗体的杂交瘤细胞株:CL1-A3、CL2-C9和CLA-D8。用扩大培养所得的杂交瘤细胞诱导小鼠产生腹水,其效价分别为1.56x10^-5、7.8×10^-6和6.25x10^-5。经双向免疫扩散实验鉴定,3株单克隆抗体蛋白均为IgG,其亚类型为IgCl。辛酸-硫酸铵沉淀法纯化单抗腹水后,测得其蛋白含量分别为3.51mg/mL、6.50mg/mL和5.57mg/mL。用SDS-PAGE进行抗体纯度鉴定,电泳图谱均显示了IgG抗体及其重链和轻链条带,分离的抗体纯度在90%以上。选取其中纯化的单抗CL2-C9进行间接ELISA实验,结果表明其最小检出限为0.01mg/kg。将CL2-C9和结构类似物沙丁胺醇进行交叉反应实验,二者的交叉反应率为2.5%,说更多还原

【Abstract】 Clenbuterol hydrochloride is a sympathomimetic beta2- adrenergic agonist, also named "leanness enhancer", "repartitioning agent" in animal industry. It’s widely used for the treatment of asthma in both humans and animals. On the other hand, it has a powerful effect of promoting animal growth with increased accretion of skeletal muscle mass and decreased accretion of body fat. For this reason, it was widely used as feed additive in animal production. Accumulative residue of clenbuterol in animal products can cause symptoms of acute poisoning in people, therefore its addition to animal feeds has been forbidden in many countries for security reason.Illegal use of CL is mainly detected by chromatography and immunoassay at present. Both methods are complicated, inconvenient, costly, and need professional skills, which causes detection can not be performed in shambles. In this study, three clenbuterol monoclonal antibodies are prepared by cell fusion technology, and test strips of immunochromatographicy are developed. Thereby, detection of CL becomes fast, simple and convenient.CL is coupled with Ovalbumin (OVA) or Bovine Serum Albumin (BSA) by diazotization, producing the immune antigen (BSA-CL) and the coating antigen (OVA-CL). They are identified with UV scanning and SDS-PAGE. In the results, absorbent wavelength of carrier proteins (OVA, BSA) has been changed after conjugation, and we can know from electrophoresis that molecular weight of conjugant is larger than that of carrier proteins. From these, we can know CL has been conjugated to carrier proteins.Immunization of Balb/C mice with the conjugant is carried out by hypodermic injection. Antisera of mice are detected by indirect enzyme-linked immunosorbent assay (ELISA) with更多还原