抗BLyS单链抗体的构建、纯化和初步活性鉴定

发布时间：2018-06-20 16:59

本文选题：BLyS + 单链抗体　；参考：《天津大学》2012年硕士论文

【摘要】：B淋巴细胞刺激因子（B lymphocyte stilulator，BLyS）是一种主要由巨噬细胞和树突状细胞产生的肿瘤坏死因子，广泛参与了机体免疫，对外周B淋巴细胞的生存、成熟和分化，生发中心的形成，浆细胞抗体的分泌等生命过程有着至关重要的作用。BLyS的不正常表达与人类多种疾病相关，如免疫缺陷病，自身免疫性疾病和B细胞淋巴瘤等。研究表明自身免疫性疾病和血液系统恶性肿瘤患者体内的BLyS水平明显升高，而且BLyS信号的阻断对此类疾病的发展有显著的治疗作用。单链抗体（ScFv）既具备抗原结合得特异性，又具有分子量小、穿透力强免疫原性低的特点，在免疫学和生物医学领域有着广阔的应用前景。因此抗BLyS单链抗体具有治疗自身免疫性疾病和B淋巴瘤的潜在作用。本课题研究方法：运用计算机模拟技术设计抗BLyS单链抗体的基因序列（VHL和MHL），用重叠PCR等方法构建重组质粒，借助原核表达系统表达并纯化目的蛋白，通过ELISA技术初步检测抗体的活性。主要研究成果：（一）扩增了两种抗体的重链和轻链可变区，并用重叠PCR方法构建了两种单链抗体基因片段，并且分别添加了Myc标签和Fc标签，VHL-Myc大小为780bp，MHL-Fc大小为1500bp。（二）构建了VHL-Myc-pET28a和MHL-Fc-pET28a表达载体，对蛋白表达的诱导条件进行了优化，获得了较高水平的表达。蛋白大量表达后利用Ni亲和柱从包涵体中纯化出两种目的蛋白。（三）体外活性检测结果显示VHL-Myc能够与特异性BLyS结合，并且这种结合是剂量依赖的。
[Abstract]:B lymphocyte stimulating factor B lymphocyte stilulatorus is a tumor necrosis factor produced mainly by macrophages and dendritic cells. It is widely involved in the immune system, the survival, maturation and differentiation of peripheral B lymphocytes, and the formation of germinal centers. The abnormal expression of BLyS is related to a variety of human diseases, such as immune deficiency disease, autoimmune disease and B cell lymphoma. Studies have shown that the BLyS level in patients with autoimmune diseases and malignant tumors of the blood system is significantly increased, and the blocking of BLyS signals has a significant therapeutic effect on the development of these diseases. ScFv has the characteristics of low molecular weight, low immunogenicity and low molecular weight, so it has a wide application prospect in immunology and biomedicine. Therefore, anti-BLyS scFv has a potential role in the treatment of autoimmune diseases and B-lymphoma. Methods: the gene sequences of BLyS single chain antibody (BLyS) were designed by computer simulation. The recombinant plasmids were constructed by overlapping PCR, and the target protein was expressed and purified by prokaryotic expression system. The activity of antibody was detected by Elisa. The main results were as follows: (1) the heavy chain and light chain variable regions of the two antibodies were amplified, and two scFv gene fragments were constructed by overlapping PCR, and the size of VHL-Myc was 780bpMHL-Fc and the size of VHL-Fc was 1500bp. respectively. (2) VHL-Myc-pET28a and MHL-Fc-pET28a expression vectors were constructed. Two kinds of target proteins were purified from inclusion bodies by Ni affinity column. (3) the results of in vitro activity test showed that VHL-Myc could bind to specific BLyS in a dose-dependent manner.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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