新型基因工程抗体scFab的构建、表达及活性检测

发布时间：2018-03-01 03:12

  本文关键词： 苏丹红I 单链Fab抗体 包涵体 酶联免疫吸附测定　出处：《华东师范大学》2009年硕士论文　论文类型：学位论文

【摘要】： 抗体是机体免疫系统针对侵入体内的抗原物质所产生的一种特异性免疫球蛋白,抗体研究的发展经历了多克隆抗体、单克隆抗体和基因工程抗体三个阶段。基因工程抗体是将抗体基因或基因片段通过基因工程技术进行重组,并在不同表达系统中产生的抗体或抗体片段,目前基因工程抗体包括人-鼠嵌合抗体、改型抗体、双特异性抗体、小分子抗体等。 所谓小分子抗体是指相对分子质量较小但具有抗原结合功能的抗体分子片段,包括Fab、Fv、scFv等,其中Fab和scFv是目前小分子抗体研究的重点。 小分子抗体具有制备方法简单、免疫原性弱、穿透力强等优点,因此在肿瘤导向治疗和病毒性疾病的治疗,以及放射免疫成像检查肿瘤等方面得到了广泛的应用。 本课题是在结合小分子抗体Fab和scFv优缺点的基础上,拟克隆抗苏丹红Ⅰ单克隆抗体的编码基因Lc和Fd,并将二者在原核表达载体pET24a内用人工合成的Linker连接,构建成基因工程抗体scFab的表达载体,最后通过原核表达,期望能得到一种兼具scFv和Fab优点的新型基因工程抗体scFab。本论文主要包括以下几个部分: 1.从分泌抗苏丹红Ⅰ单克隆抗体的杂交瘤细胞中通过PCR技术获得Lc和Fd基因,克隆至T-A克隆载体后,进行克隆鉴定和DNA测序。DNA序列BLAST和编码蛋白质序列分析表明所得到的抗体基因符合小鼠的IgG基因特征。 2.经过对已有载体的改造,重新设计了可以直接用于scFab原核表达的载体;同时,在上述对抗体分析的基础上,选取合适Lc和Fd基因片段,在其两端引入酶切位点后与人工合成的Linker相连,一起克隆到经过改造的载体pET24a(+)中,构建成表达载体pET24a-scFab,成功转染大肠杆菌E.coliBL21(DE3)。 3.IPTG诱导scFab在E.coli BL21(DE3)表达,SDS-PAGE分析和Western Blot鉴定,显示scFab蛋白以包涵体的形式表达,经过对其表达条件进行优化后,获得大量表达的scFab抗体蛋白包涵体。 4.尿素溶解scFab包涵体,以含谷胱甘肽氧化还原系统的复性液对其进行复性,透析浓缩得到复性抗体,间接ELISA和竞争ELISA检测结果表明复性抗体对苏丹红Ⅰ具有很高的结合活性和结合特异性。 论文结论:本研究成功构建了一种新型基因工程抗体scFab,为新型基因工程抗体研究奠定了初步的理论和实验基础。
[Abstract]:Antibody is a kind of specific immunoglobulin produced by the body's immune system against the antigen substances invading the body. The development of antibody research has experienced the development of polyclonal antibody. Monoclonal antibody and genetic engineering antibody are three stages. Genetic engineering antibody is the antibody or fragment produced in different expression systems by recombination of antibody gene or gene fragment through genetic engineering technology. At present, genetic engineering antibodies include human-mouse chimeric antibody, modified antibody, bispecific antibody, small molecule antibody and so on. The so-called small molecular antibody refers to the relatively small molecular weight of antibody fragments with antigen-binding function, including FV FV scFv, among which Fab and scFv are the focus of the study of small molecular antibodies. Because of the advantages of simple preparation, weak immunogenicity and strong penetration, small molecular antibodies have been widely used in tumor-oriented therapy, treatment of viral diseases, and radioimmunoimaging. On the basis of combining the advantages and disadvantages of small molecule antibody Fab and scFv, the encoding genes L c and FD of monoclonal antibody against Sudan I were cloned and ligated with synthetic Linker in prokaryotic expression vector pET24a. The expression vector of genetic engineering antibody (scFab) was constructed. Finally, by prokaryotic expression, we hope to obtain a novel genetic engineering antibody scFab. which has the advantages of both scFv and Fab. This thesis mainly includes the following parts:. 1. LC and FD genes were obtained by PCR from hybridoma cells secreting monoclonal antibody against Sudan 鈪，

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