抗TNF-α多价抗体的构建及体内外生物学活性研究

发布时间：2018-01-09 01:22

本文关键词：抗TNF-α多价抗体的构建及体内外生物学活性研究　出处：《中国协和医科大学》2005年博士论文　论文类型：学位论文

【摘要】：肿瘤坏死因子α(TNF-α)是上世纪70年代发现的一个细胞因子，最初发现它能引起某些特定的肿瘤组织的出血性坏死，现在已经知道它除了具有抗肿瘤作用外，还在机体的免疫、炎症和病理中发挥重要的作用。TNF-α的拮抗剂包括中和性的抗体已被证明是急性脓毒血症、类风湿关节炎，局部性回肠炎和牛皮癣等急慢性炎症性疾病的有效治疗剂。美国FDA已批准了Etanercept、Infliximab和Adalimumab这三种抗TNF-α的生物治疗剂，其中后两种为抗TNF-α的单克隆抗体。抗体工程技术经历了鼠源杂交瘤抗体、人-鼠嵌合抗体、人源化抗体和人源抗体的发展阶段，而上世纪80年代基因工程技术的兴起使构建基因工程的小分子抗体如Fab、scFv及单域抗体等成为现实，这些小分子抗体由于分子量小、穿透性强、易于构建并可在大肠杆菌等原核系统中功能性的表达而具有广阔的应用前景。本研究通过基因工程的方法构建了具有中和活性的抗TNF-α的单链抗体，对其生物学活性进行了研究，并在此基础上对单链抗体进行了多价化，对多价抗体的体内外生物活性进行了研究，为开发新的抗TNF-α治疗剂打下了基础。一、抗TNF-α单链抗体TNF-scFv的构建及体外活性研究单链抗体是一种将抗体的重链可变区(VH)和轻链可变区(VL)通过一段连接肽连接起来而形成的重组蛋白。本研究利用连接肽(GGGGS)_3将抗TNF-α的单克隆抗体Di62的VH和VL连接起来，设计成为完整的单链抗体的氨基酸序列，然后根据设计的氨基酸序列以大肠杆菌偏爱的密码子将其反向翻译成编码的DNA序列。根据编码的DNA序列，合成了22个寡核苷酸片段并用装配PCR的方法将这22个寡核苷酸片段拼接成的完整的单链抗体编码基因，在编码基因的两端加上了克隆所需要的酶切位点。将此编码基因克隆进载体pTCM-2，通过阳性克隆的鉴定和序列测定，构建了单链抗体TNF-scFv的表达载体，命名为pTS。将载体pTS转化大肠杆菌BL21(DE3)star，，利用0.4mM的IPTG诱导目
[Abstract]:Tumor necrosis factor- 伪 (TNF- 伪) is a cytokine found in -30s, which was initially found to cause hemorrhagic necrosis in certain tumor tissues. It is now known that in addition to its antitumor effects, it also plays an important role in the body's immunity, inflammation and pathology. TNF- 伪 antagonists, including neutralizing antibodies, have been shown to be acute sepsis. An effective treatment for acute and chronic inflammatory diseases such as rheumatoid arthritis, local colitis and psoriasis. The United States FDA has approved Etanercept. Infliximab and Adalimumab are three biotherapeutic agents against TNF- 伪. The latter two are monoclonal antibodies against TNF- 伪. The antibody engineering technology has experienced the development of murine hybridoma antibody, human-mouse chimeric antibody, humanized antibody and human antibody. In -20s, the rise of genetic engineering technology made the construction of small molecular antibodies such as FabscFv and single-domain antibodies become a reality. These small molecular antibodies have high penetration due to their small molecular weight. It is easy to construct and can be expressed in E. coli and other prokaryotes. In this study, a neutralizing single-chain antibody (scFv) against TNF- 伪 was constructed by genetic engineering. The biological activity of the antibody was studied, and on the basis of this, the single-chain antibody was polyvalent, and the biological activity of the polyvalent antibody in vivo and in vitro was studied. It lays a foundation for the development of a new anti-TNF- 伪 therapeutic agent. 1. Construction and in vitro activity of anti-TNF- 伪 scFv TNF-scFv Single chain antibody (scFv) is a recombinant protein formed by linking the heavy chain variable region (VH) and the light chain variable region (VLH) of the antibody via a linked peptide. The VH and VL of Di62, a monoclonal antibody against TNF- 伪, were linked together. The amino acid sequence of scFv was designed to be complete, and then reverse translated into the encoded DNA sequence according to the designed amino acid sequence with the preferred codon of Escherichia coli. According to the encoded DNA sequence. Twenty-two oligonucleotide fragments were synthesized and the 22 oligonucleotide fragments were spliced into a complete single-chain antibody coding gene by assembling PCR. The encoding gene was cloned into the vector pTCM-2 and identified and sequenced by positive cloning. The expression vector of scFv TNF-scFv was constructed and named pTS.The vector pTS was transformed into Escherichia coli BL21DE3DE-star. the target was induced by 0.4mm IPTG.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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