人源抗伊维菌素单链抗体的筛

发布时间：2018-05-08 13:52

本文选题：噬菌体抗体库技术 + 伊维菌素　；参考：《扬州大学》2010年硕士论文

【摘要】： 伊维菌素是由阿维链霉菌发酵合成的一种大环内酯类多组分抗生素,被有效用作农药、兽药及人抗寄生虫药物。它能打开谷氨酸控制的Cl-通道,导致膜超极化,使肌肉细胞丧失收缩能力,从而导致虫体死亡。长期以来,伊维菌素被认为是安全的抗寄生虫药物,但是近年的研究结果表明,它的残留能降低牧草产量,影响水生群落,对环境平衡有重大威胁。伊维菌素的广泛应用和潜在危害的并存,使得建立一种简单高效的伊维菌素残留检测方法很有必要。目前建立的伊维菌素检测方法主要有中空纤维支撑液膜萃取法和液相色谱质谱-质谱法。这些方法需要昂贵的仪器及繁琐的前处理步骤,相比之下,免疫检测法较为快速、经济。现已有关于伊维菌素单克隆和多克隆抗体的报道,还未见其单链抗体的报道。本研究在参照国内外相关研究的基础上,以期从人源噬菌体抗体库中筛选出抗伊维菌素的单链抗体,并对其进行可溶性表达、蛋白结构功能分析,为环境及农产品中伊维菌素的安全监控提供材料,为研究抗原抗体反应提供理论基础。噬菌体展示技术是一种制备高亲和力抗体的有力工具,抗体库技术则为筛选高亲和力抗体提供了良好的平台。展示抗体的基因型与表现型的有效统一,使其不仅在农药残留检测及抗体制备等方面具有广泛应用前景,还能有效地应用于基因工程改造。研究目的和意义:应用噬菌体抗体库技术,筛选人源抗伊维菌素单链抗体,以期应用于伊维菌素的安全监控。对筛选出的抗体进行可溶性表达、纯化后鉴定其免疫活性。对抗体的结构功能进行分析,为研究抗原抗体结合反应提供理论基础。研究方法:以伊维菌素-牛血清白蛋白为包被抗原,应用噬菌体抗体库技术,从库容约为108的人源噬菌体抗体库中筛选出抗伊维菌素单链抗体。采用固相消减筛选法,共进行4轮“吸附-洗脱-富集”。对结合活性较强的阳性克隆,制备噬菌体上清,转化E.coli HB2151,以IPTG进行诱导,表达的可溶性抗体过镍亲和层析柱纯化,建立竞争抑制ELISA检测方法,进行灵敏度测定。应用计算机软件及生物信息学站点对抗体的结构功能进行分析。研究结果:经过四轮富集筛选,从人源抗体库中筛选出7株不同的阳性克隆,hsIVM8展示了最高的亲和力。IPTG成功诱导表达hsIVM8,SDS-PAGE显示其分子量约为28 kD,伊维菌素对纯化抗体的抑制中浓度IC50为4.11μg/mL,线性检测范围为0.1-5μg/mL。研究成果:从人源噬菌体抗体库中成功筛选到抗伊维菌素的单链抗体,有望应用于环境和农产品中伊维菌素的安全监控。对抗体蛋白进行的结构功能分析为研究抗体性质及与抗原反应机理提供理论基础。
[Abstract]:Ivermectin is a macrolide multicomponent antibiotic synthesized by Streptomyces avelicus. It is effectively used as a pesticide, veterinary drug and human antiparasitic drug. It can open the Cl- channel controlled by glutamate, lead to membrane hyperpolarization, make muscle cells lose the ability of contraction, and lead to the death of insect body. For a long time, ivermectin has been considered as a safe antiparasitic drug. However, recent studies have shown that Ivermectin residues can reduce forage yield, affect aquatic communities and pose a serious threat to environmental balance. The extensive application of ivermectin and the coexistence of potential hazards make it necessary to establish a simple and efficient method for the detection of Ivermectin residues. At present, Ivermectin detection methods are mainly hollow fiber supported liquid membrane extraction and liquid chromatography-mass spectrometry. These methods require expensive instruments and cumbersome pre-processing procedures, whereas immunoassay is faster and more economical. The monoclonal and polyclonal antibodies against ivermectin have been reported, but no single chain antibodies have been reported. The aim of this study was to screen the single chain antibody against ivermectin from human phage antibody library and analyze its soluble expression and protein structure and function. It provides materials for the safety monitoring of Ivermectin in environment and agricultural products, and provides a theoretical basis for the study of antigen-antibody reaction. Phage display is a powerful tool for preparing high affinity antibodies, and antibody library provides a good platform for screening high affinity antibodies. The effective unification of genotypes and phenotypes of antibodies not only has a wide application prospect in pesticide residue detection and antibody preparation, but also can be effectively used in genetic engineering. Objective and significance: to screen human single chain antibody against ivermectin by phage antibody library technology, and to apply it to the safety monitoring of ivermectin. The antibody was expressed in a soluble way and its immunological activity was identified after purification. The analysis of the structure and function of antibody provides a theoretical basis for the study of antigen-antibody binding reaction. Methods: Ivermecin-bovine serum albumin (BSA) was used as coating antigen and phage antibody library technique was used to screen Ivermectin single chain antibody from human phage antibody library containing about 108. Four rounds of "adsorption-elution-enrichment" were carried out by solid phase subtractive screening. The phage supernatant was prepared and transformed into E.coli HB2151. The expressed soluble antibody was purified by nickel affinity chromatography. A competitive inhibition ELISA detection method was established and the sensitivity was determined. The structure and function of antibody were analyzed by computer software and bioinformatics site. Results: after four rounds of enrichment screening, Seven different positive clones of hsIVM8 were screened from the human antibody library and showed the highest affinity. IPTG successfully induced the expression of hsIVM8 SDS-PAGE showed that its molecular weight was about 28 kD. the median inhibitory concentration of ivermectin on purified antibody was 4.11 渭 g / mL, and the linear detection range was 0.1-5 渭 g / mL. Results: single chain antibodies against Ivermectin were successfully screened from human phage antibody library, which is expected to be applied to the safety monitoring of Ivermectin in environment and agricultural products. The analysis of the structure and function of antibody protein provides a theoretical basis for the study of antibody properties and reaction mechanism with antigens.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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