抗A、B血型单链抗体的构建

发布时间：2018-09-17 14:34

【摘要】：单链抗体(Single chain FV, scFV)是一种应用最广泛的小分子重组抗体,它是由一段弹性连接链(linker)把抗体重链可变区VH与轻链可变区VL相连而成,具有亲代抗体的全部抗原结合特异性的最小功能结构单位,大小为完整抗体的六分之一。它有分子小、穿透力强、体内半衰期短、免疫原性低,可通过包涵体大量表达、易于基因操作,尤其易于构建抗体融合蛋白等优越性。结合现在肿瘤治疗个性化概念的出现,我们期望构建出个体病人特有的单链抗体库,并以此为基础筛选到针对自身肿瘤的抗体。以此为出发点,我们开展了单链抗体的研究,从首先通过抽取健康个体的外周血并得到个体的cDNA,作为基因扩增的模板,并参阅了之前大量的报道与抗体数据库,设计出涵盖人体所有抗体基因的引物。利用这些引物随机组合并进行PCR扩增,扩增出的重链与轻链片段,产生的scFV基因型非常丰富,其中也会包含一部分在机体正常情况下被免疫耐受等过程而失去功能的抗体。就此而言,单链抗体(scFV)产生的抗体类型较传统的抗体类型更加丰富,且易于基因操作。共设计了31条涵盖重链与轻链基因的引物,并进行了75个独立的PCR反应。并将得到的重链与轻链片段以一个(GGGGS)3的弹性linker连接到一起。同时,我们利用电转的方法来构建得到scFV单链抗体库,通过提高电转的效率来构建出更大的scFV单链抗体库。,最终,我们得到了2×106的scFV单链抗体库。构建的O型血个体的单链抗体用于筛选抗A,B血型的单链抗体,筛选是利用ELISA的方法,因此我们前期对红细胞膜的处理及ELSA实验中的包被条件进行了大量的实验摸索,期望得到高亲和力抗原包被的条件对包被条件通过酶标仪进行检测,结果证实细胞膜包被条件成功,可用于ELISA筛选。
[Abstract]:Single chain antibody (Single chain FV, scFV) is one of the most widely used recombination antibodies of small molecules. It is composed of a segment of elastic link chain (linker) that connects the heavy chain variable region VH with the light chain variable region VL. The smallest functional structural unit with the full antigen-binding specificity of the parental antibody, the size of which is 1/6 of the complete antibody. It has the advantages of small molecule, strong penetration, short half-life in vivo and low immunogenicity. It can be expressed in large quantities through inclusion bodies, easy to operate genes, and especially easy to construct antibody fusion protein. With the emergence of the concept of individualized tumor therapy, we hope to construct a single chain antibody library that is specific to individual patients and screen antibodies against autotumours on the basis of this library. From this point of view, we carried out the study of scFv. First, we extracted the peripheral blood of healthy individuals and obtained individual cDNA, as template for gene amplification. We also read a large number of previous reports and antibody databases. Primers covering all human antibody genes were designed. By random combination of these primers and PCR amplification, the heavy and light chain fragments were amplified, resulting in a very rich scFV genotypes, which also contained some antibodies which were incapacitated by immune tolerance and other processes under normal conditions. In this case, the type of antibody produced by scFv (scFV) is more abundant than that of traditional antibody type, and it is easy to operate gene. A total of 31 primers covering heavy and light chain genes were designed and 75 independent PCR reactions were performed. The obtained heavy chain and light chain fragment are connected together with an elastic linker of (GGGGS) 3. At the same time, we use electroporation to construct scFV single chain antibody library. By improving the efficiency of electroporation, we construct a larger scFV single chain antibody library. Finally, we get 2 脳 10 6 scFV single chain antibody library. The single chain antibodies of O blood type individuals were used to screen the single chain antibodies against Agna B blood group. The screening method was based on ELISA. Therefore, we have done a lot of experiments on the treatment of erythrocyte membrane and the conditions of encapsulation in ELSA experiment. It was expected that the conditions of high affinity antigen coating could be detected by enzyme labeling instrument. The results showed that the conditions of membrane coating were successful and could be used for ELISA screening.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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