胰淀素人源性Fab抗体的筛选及鉴定
发布时间：2018-12-21 21:33
【摘要】：目的胰淀素也称胰岛淀粉样多肽(IAPP),它与胰岛素共同存在于胰岛B细胞的分泌颗粒中,与胰高血糖素、胰岛素共同调节人体血糖平衡。目前研究表明胰淀素的异常分泌及早期形成的可溶性低聚物在局部堆积对于B细胞有毒性作用,可引起B细胞凋亡,最终导致胰岛分泌功能的渐进性衰竭。该激素自从1988年被发现以来,其生理和病理意义愈发被关注,然而由于胰淀素的体内含量低且分子量较小,其mRNA含量仅为胰岛素的0.2-0.3%,目前采用单克隆抗体免疫荧光方法检测,其方法复杂,抗体缺乏稳定性和特异性,用传统制备抗体的方法如杂交瘤技术和抗原免疫的方法制备抗体得到的抗体少,且工作繁琐。给研究这一激素在体内的变化规律带来了困难。因此寻找一种快速和具有高度特异性的方法提取和纯化胰淀素抗体是研究工作的关键,我们采用噬菌体表面展示技术的方法,试图制备人源化的胰淀素抗体,为体外大规模生产抗体提供可行的方法。 方法用固相法将抗原胰淀素包被到酶联板上,应用噬菌体抗体库技术,筛选特异性的抗体。收集每轮洗脱的噬菌体并进行扩增,再用于下一轮筛选。同样的“吸附一洗脱-扩增”的富集反应共进行了5轮。在第5轮筛选后得到的抗胰淀素噬菌体抗体克隆中,选取数十个克隆扩增,提取其质粒,用Nhe I和SpeⅠ双酶切,去除gⅢ基因,自身环化构建其可溶性表达噬菌粒,转化至大肠杆菌BL21(DE3)pLysS中,IPTG诱导高效表达,并用SDS-PAGE鉴定所得蛋白是否为目的蛋白,ELISA鉴定其抗原结合活性和特异性。 结果通过对人天然Fab段噬菌体抗体库的5轮筛选,尽管从第一轮到第五轮洗涤次数由1次增加到5次,最后增加到10次,从固相洗脱下来的噬菌体滴度却显示了明显的增加趋势,回收的噬菌体产出率共增加了约443倍,说明特异性抗胰淀素的Fab噬菌体抗体得到了富集。构建的可溶性Fab抗体表达载体,在IPTG的诱导下得到了高效表达,生成含有可溶性的Fab抗体的上清。经SDS-PAGE蛋白电泳,在相对分子质量约为47KD处可见一蛋白条带；转膜后HRP标记的羊抗人FabIgG抗体进一步显色,证实了目的蛋白人源特异性Fab片段的表达；ELISA鉴定证明有3个克隆可与胰淀素抗原特异性结合,且与BSA无交叉反应。 结论噬菌体抗体库技术的出现为人天然抗体的制备开辟了新的途径,该技术便捷有效,可不经免疫,绕过杂交瘤技术,直接从抗体库中筛选特异性抗体,可解决杂交瘤技术的低效问题,并可在体外改善抗体的性能。本工作特异性抗胰淀素Fab抗体的制备及鉴定方法的建立,为进一步的研究胰淀素的生理和病理功能奠定了基础。
[Abstract]:Objective amylin, also known as islet amyloid polypeptide (IAPP), co-exists with insulin in the secretory granules of islet B cells and regulates the blood glucose balance with glucagon and insulin. Recent studies have shown that the abnormal secretion of amylin and the early accumulation of soluble oligomers have toxic effects on B cells, which can induce apoptosis of B cells and eventually lead to progressive failure of pancreatic islet secretory function. Since it was discovered in 1988, the physiological and pathological significance of the hormone has become more and more concerned. However, because of the low content and molecular weight of amylin in vivo, its mRNA content is only 0.2-0.3 of insulin. At present, monoclonal antibody immunofluorescence method is used, its method is complex, the antibody is lack of stability and specificity, the traditional methods of preparing antibody, such as hybridoma technique and antigen-immunized method, the antibody preparation method is less, and the work is tedious. This makes it difficult to study the changes of the hormone in the body. Therefore, to find a rapid and highly specific method to extract and purify amylin antibody is the key of the research. We use phage display technology to prepare human amylin antibody. To provide a feasible method for mass production of antibodies in vitro. Methods Antigen amylin was coated on enzyme linked plate by solid phase method and specific antibody was screened by phage antibody library technique. The phage eluted in each round was collected and amplified, and then used for the next round of screening. The same "adsorption-elution-amplification" enrichment reaction took place for a total of five rounds. After the fifth round of screening, dozens of clones were selected to amplify and extract their plasmids. The plasmids were digested with Nhe I and Spe 鈪，
本文编号：2389494
[Abstract]:Objective amylin, also known as islet amyloid polypeptide (IAPP), co-exists with insulin in the secretory granules of islet B cells and regulates the blood glucose balance with glucagon and insulin. Recent studies have shown that the abnormal secretion of amylin and the early accumulation of soluble oligomers have toxic effects on B cells, which can induce apoptosis of B cells and eventually lead to progressive failure of pancreatic islet secretory function. Since it was discovered in 1988, the physiological and pathological significance of the hormone has become more and more concerned. However, because of the low content and molecular weight of amylin in vivo, its mRNA content is only 0.2-0.3 of insulin. At present, monoclonal antibody immunofluorescence method is used, its method is complex, the antibody is lack of stability and specificity, the traditional methods of preparing antibody, such as hybridoma technique and antigen-immunized method, the antibody preparation method is less, and the work is tedious. This makes it difficult to study the changes of the hormone in the body. Therefore, to find a rapid and highly specific method to extract and purify amylin antibody is the key of the research. We use phage display technology to prepare human amylin antibody. To provide a feasible method for mass production of antibodies in vitro. Methods Antigen amylin was coated on enzyme linked plate by solid phase method and specific antibody was screened by phage antibody library technique. The phage eluted in each round was collected and amplified, and then used for the next round of screening. The same "adsorption-elution-amplification" enrichment reaction took place for a total of five rounds. After the fifth round of screening, dozens of clones were selected to amplify and extract their plasmids. The plasmids were digested with Nhe I and Spe 鈪，
本文编号：2389494
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