抗人B7-2单链抗体基因的构建及表达研究

发布时间：2018-06-18 19:57

本文选题：B7-2 + 单链抗体　；参考：《苏州大学》2009年硕士论文

【摘要】： 鼠源性单抗在人体使用后可产生人抗鼠抗体(Human Anti-Mouse Antibody, HAMA)反应,而且分子量大,不能有效进入病灶部位,临床使用受到限制。单链抗体(single chain antibody, ScFv)是由一条连接肽将IgG分子重链和轻链的可变区连接而成,减少了免疫原性,分子小,实体组织穿透力强,血浆半衰期短,因而在临床疾病的诊断和治疗中具有重要的应用价值。在成功建立稳定分泌鼠抗人B7-2杂交瘤细胞株基础上,扩增并克隆出该单克隆抗体的重链(VH)和轻链(VL)可变区基因。通过重叠延伸PCR(SOE-PCR)方法,在VH和VL可变区基因之间引入连接肽(Gly4Ser)3,体外构建抗人B7-2单链抗体(B7-2 ScFv)基因。为便于表达产物的纯化,在抗人B7-2 ScFv的C端增加了6×His tag序列。将其克隆至表达载体pET32a并在大肠杆菌中进行原核表达。SDS-PAGE和Western-blot分析结果表明,抗人B7-2 ScFv在BL21(DE3)中获得高效表达,表达量占菌体总蛋白30%以上,表达产物以不溶性包涵体形式存在。经过溶解包涵体,体外复性过程和镍柱亲和层析,纯化了抗人B7-2 ScFv。本研究在国内首次获得了高纯度的抗人B7-2 ScFv,为今后抗人B7-2ScFv的活性和功能研究以及抗肿瘤药物的开发奠定了基础。
[Abstract]:Human anti-mouse antibody human anti-mouse Antibody (Hama) reaction can be produced after human use of murine monoclonal antibody, and its molecular weight is too large to enter the lesion effectively, so its clinical use is restricted. Single chain antibody (scFv) is a linked peptide that connects the variable regions of the heavy and light chains of IgG molecules, reducing immunogenicity, small molecules, strong penetration of solid tissues, and short plasma half-life. Therefore, it has important application value in the diagnosis and treatment of clinical diseases. On the basis of the successful establishment of stable secreted mouse hybridoma cell lines against human B7-2, the variable region genes of heavy chain (VH) and light chain (VLV) of the monoclonal antibody were amplified and cloned. By means of overlapping extension of PCR- SOE-PCR, the conjugated peptide Gly4Serv3 was introduced between VH and VL variable region gene to construct anti-human B7-2 scFv3 single chain antibody (scFv3) gene in vitro. In order to purify the expressed product, 6 脳 his tag sequence was added to the C-terminal of anti-human B7-2 scFv. The recombinant plasmid pET32a was cloned into E. coli and expressed in E. coli. SDS-PAGE and Western-blot analysis showed that anti-human B7-2 scFv was highly expressed in BL21DDE3, and the expression amount was more than 30% of the total bacterial protein. The expression product existed as insoluble inclusion body. The anti-human B7-2 scFv was purified by dissolution of inclusion body, in vitro renaturation process and affinity chromatography of nickel column. In this study, high purity anti-human B7-2 scFvwas obtained for the first time in China, which laid a foundation for the study of the activity and function of anti-human B7-2ScFv and the development of anti-tumor drugs in the future.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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