大容量人天然噬菌体单链抗体库构建.doc

大容量人天然噬菌体单链抗体库构建[摘要] 目的：构建大容量和具有良好多样性的人天然噬菌体单链抗体库。方法：从人外周血分离淋巴细胞，提取mRNA后用RT-PCR技术采用新设计的引物扩增VH 和VL 基因片段，两者分别与Linker连接后，采用改进的重叠延伸PCR法将VH基因和VL基因连接成人单链抗体（scFv)基因（VH-Linker-VL片段），继而连接到噬菌粒载体pCANTAB5E中，连接产物通过电转化方法转入大肠杆菌TG1，构建噬菌体scFv抗体库。结果：所有VH、VL亚类基因都得到了扩增和有效的连接，经电转化E. coli TG1和噬菌体的超感染，构建了总库容达到了6×108的单链抗体库。结论：成功地构建了一个多样性良好的人源天然噬菌体抗体库, 可用于制备具有应用前景的人源抗体。 [关键词] 单链抗体(scFv)；噬菌体抗体库；重叠延伸拼接 [中图分类号] R392-33 [文献标识码]A[文章编号]1673-7210（2008）05（a）-011-03 Construction of a large human natural phage single-chain antibody library SUN Chun-yan （Guangdong Food and Drug Vocational College,Guangzhou510520，China） [Abstract] Objective: To construct a human natural phage single -chain antibody (scFv) library with diversity.Methods:Total RNA was extracted from the peripheral blood lymphocytes of the non-immunized healthy donors, VH and VL genes were amplified by RT-PCR and were assembled to form scFv by overlap PCR and cloned into phagemid pCANTAB5E, and then transformed into E. coli TG1 by electroporation to construct a human natural phage scFv antibody library. Results:VH gene families and VL gene families were successfully amplified. A large human antibody library containing 6×108 clones was created after rescuing the recombinant phagemids from the transformed E. coli TG1 cells.Conclusion:A human scFv antibody library is successfully constructed finally. In the future, we can try to obtain antibody by panning purposely using related antigens. [Key words] Single-chain antibody (scFv); Phage display library; Overlapping PCR 噬菌体表面展示抗体库技术是目前可用于制备人源单克隆抗体的新技术。它使用RT-PCR方法把人免疫球蛋白全套可变区基因扩增出来后重组到原核表达载体中，并通过与噬菌体外壳蛋白形成融合蛋白使抗体片段表达于噬菌体表面，用淘洗的方法筛选特异性的可变区基因，可以方便高效地进行抗体的高通量筛选，为人源单克隆抗体的制备开创了一条简便、快速的生产路线。该技术的关键环节是构建人源性噬菌体抗体库，以大容量、良好多样性抗体库作为技术平台开发治疗性人源单克隆抗体，其前景诱人[1,2]。本实验探索了组装单链抗体基因的新方法，保证了抗体库的多样性，构建了大容量人天然噬菌体单链抗体库，为治疗性人源单克隆抗体的筛选奠定了基础。 1材料与方法 1.1实验材料 1.1.1 噬菌粒、菌株噬菌粒载体pCANTAB5E、辅助噬菌体M13K07和大肠杆菌E. coli TG1购自Amersham Biosciences。 1.1.2 主要试剂高保真TaqDNA聚合酶、T4 DNA连接酶、限制性内切酶SfiⅠ和NotⅠ、核酸分子