人源性抗精氨酸加压素Fab抗体的筛

发布时间：2018-05-11 18:46

本文选题：噬菌体抗体库 + 精氨酸加压素　；参考：《天津医科大学》2012年硕士论文

【摘要】：目前小分子激素的检测相对困难,这极大地影响了对复杂内分泌现象的认识和内分泌疾病的诊断。很多年来,我们对相关内分泌疾病只能通过间接推测作出诊断。如下丘脑、垂体疾病中尿崩症的诊断仅能以尿量、尿比重、尿渗透压等间接指标反映体内精氨酸加压素的水平,因此建立体内小分子激素的快速检测方法对于临床上疾病的诊治具有重要价值。由于这些激素的分子量小,半衰期短,传统制备抗体的方法如杂交瘤技术和机体免疫的方法制备抗体得到的抗体少,且工作繁琐,缺乏稳定性不宜大规模生产,因此寻找一种快速、简单制备精氨酸加压素抗体的方法是开展进一步研究的关键环节。噬菌体抗体库技术是在基因工程技术基础上发展起来的一种模拟自然免疫选择系统制备抗体的新技术。它将抗体的基因型和表现型统一于一体,使其识别抗原的能力和噬菌体的扩增能力偶联起来,具有强大的筛选能力。它不需人工免疫动物,绕过杂交瘤技术,用基因工程技术制备人源性抗体,较好地解决了人杂交瘤技术低效及人体排异反应的难题,在制备难以获得的抗体方面具有很大的潜力。近些年,噬菌体抗体库技术已广泛应用于生命科学的各个领域,但利用该技术制备小分子激素抗体的报道少见。本研究以精氨酸加压素为模型分子,探索利用噬菌体抗体库技术从构建的人源性天然噬菌体Fab抗体库中直接筛选精氨酸加压素特异性Fab抗体,并进行可溶性表达及免疫活性鉴定,建立小分子激素抗体的制备方法,为下一步的临床研究奠定了基础。方法：以纯化的精氨酸加压素为靶抗原,梯度稀释浓度包被到酶标板上,应用噬菌体抗体库技术,从库容为2.4×108的人源噬菌体抗体库中筛选抗精氨酸加压素Fab抗体。采用固相筛选法,经过5轮“吸附-洗脱-扩增”富集筛选。Phage-ELISA鉴定精氨酸加压素特异性噬菌体抗体,挑选结合活性最强的阳性克隆,制备噬菌体上清,感染大肠杆菌HB2151,经IPTG诱导高效表达,表达的可溶性抗体经SDS-PAGE、Western Blot检测鉴定抗体的表达情况,ELISA鉴定其抗原结合活性和特异性。结果：经过5轮富集筛选,最终筛选出6株具有较强特异结合活性的抗精氨酸加压素抗体。其中,阳性克隆C4展示了最强的结合活性。挑选阳性克隆C4在大肠杆菌HB2151中诱导可溶性表达,表达的抗体片段经SDS-PAGE电泳和Western Blot检测,在相对分子质量约为50kD附近有特异条带的出现,同预计的目的蛋白分子量大小一致,ELISA证实该抗体片段具有较好的抗原特异性和结合活性。结论：本研究从自建的人源性Fab噬菌体抗体库中筛选出抗精氨酸加压素抗体,并对抗体的有效性进行了初步鉴定,构建了精氨酸加压素天然抗体制备技术的平台,为将来更多小分子激素快速检测方法的建立奠定了良好的基础。
[Abstract]:At present, the detection of small molecular hormones is relatively difficult, which greatly affects the understanding of complex endocrine phenomena and the diagnosis of endocrine diseases. For many years, we diagnosed endocrine diseases only through indirect speculation. For example, the diagnosis of diabetes insipidus in hypothalamus and pituitary diseases can only reflect the level of arginine vasopressin by indirect indexes such as urine volume, specific gravity and osmotic pressure of urine. Therefore, the establishment of a rapid detection of small molecular hormones in vivo is of great value in the diagnosis and treatment of diseases. Because of the small molecular weight and short half-life of these hormones, the traditional methods of preparing antibodies, such as hybridoma and immune methods, have less antibodies, and the work is tedious, so the lack of stability is not suitable for mass production. Therefore, to find a rapid and simple method to prepare arginine vasopressin antibody is the key to further research. Phage antibody library (Phage antibody library) is a new technique which is developed on the basis of genetic engineering and simulates natural immune selection system to prepare antibodies. It unifies the genotype and phenotype of antibody, and makes the ability of antigen recognition and phage amplification coupling together, and has a strong screening ability. It does not need artificial immunity to animals, bypasses hybridoma technology, and uses genetic engineering technology to prepare human antibody, which solves the problem of low efficiency of human hybridoma technology and rejection of human body. There is great potential in the preparation of difficult-to-obtain antibodies. In recent years, phage antibody library technology has been widely used in various fields of life science, but the preparation of small molecular hormone antibodies by using this technique is rare. In this study, arginine vasopressin was used as model molecule to screen arginine vasopressin specific Fab antibody directly from human natural phage Fab antibody library by phage antibody library technology. The soluble expression and immunological activity were identified, and the preparation method of small molecular hormone antibody was established, which laid a foundation for clinical research in the next step. Methods: the purified arginine vasopressin was used as the target antigen and the gradient dilution concentration was coated on the enzyme marker. The anti-arginine vasopressin Fab antibody was screened from the library containing 2.4 脳 10 ~ 8 human phage antibody library by phage antibody library technique. The specific phage antibody of arginine vasopressin was identified by 5 rounds of "adsorption-eluation-amplification" enrichment screening. The phage supernatant was prepared by selecting the positive clones with the strongest binding activity. E. coli HB2151 was infected and highly expressed by IPTG. The expressed soluble antibody was detected by SDS-PAGEG Western Blot and its antigen-binding activity and specificity were identified by Elisa. Results: after 5 rounds of enrichment screening, 6 strains of anti-arginine vasopressin antibodies with strong specific binding activity were screened. Among them, the positive clone C4 showed the strongest binding activity. The positive clone C4 was selected to induce soluble expression in Escherichia coli HB2151. The expressed antibody fragment was detected by SDS-PAGE electrophoresis and Western Blot, and there were specific bands near the relative molecular weight of 50kD. In accordance with the predicted molecular weight of the target protein, Elisa confirmed that the antibody fragment had good antigen-specificity and binding activity. Conclusion: the anti-arginine vasopressin antibody was screened from the human Fab phage antibody library, and the effectiveness of the antibody was preliminarily identified, and a platform for the preparation of arginine vasopressin natural antibody was established. It has laid a good foundation for the establishment of more rapid detection methods of small molecular hormones in the future.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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