【摘要】 目的　构建A型流感病毒核蛋白全套单链抗体库。　方法　利用全套噬菌体抗体表面展示技术 ,从A型流感病毒核蛋白免疫的BALB/c小鼠脾淋巴细胞中提取总RNA ,利用抗体可变区PCR混合引物进行全套抗体重、轻链基因的扩增。经重叠延伸反应 ,在体外随机装配成单链抗体。将其克隆到噬菌粒载体PCANTAB5E中 ,电转化TG1细菌 ,以辅助噬菌体M13 K0 7超感染噬菌体文库 ,构建全套ScFv抗体库。　结果　成功利用噬菌体表面展示技术 ,构建了A型流感病毒核蛋白全套单链抗体库 ,库容量达到 8 2× 10 7。　结论　构建全套A型流感病毒核蛋白全套单链抗体库 ,为利用亲和富集筛选技术 ,获得具有A型流感病毒核蛋白结合活性的完整重组噬菌体克隆奠定了基础。更多还原

【Abstract】 Objective To construct a library of repertoire immunoglobulin against nucleoprotein of influenza virus A. Methods Phage display technique were used for construction of library of repertoire immunoglobulin from BALB/c mouse immunized with nucleoprotein of influenza virus A. The heavy-chain and kappa light-chain variable region gene repertoire of immunoglobulin were amplified individually from the spleen cell mRNA by RT-PCR and joined by a DNA linker encoding peptide (GGGGS)3 as a single-chain Fv DNA fragment with overlap extension. These fragments were cloned into the phagemid pCANTAB5E and expressed as a fusion protein with phage M13 PVIII in E.coli TG1. In the presence of helper phage M I3K07. the ScFv fusion protein was displayed on the surface of recombinant phages. Result The repertoire single-chain antibody library against nucleoprotein of influenza virus A were successfully constructed. Conclusion construction of repertoire single-chain antibody library against nucleoprotein of influenza virus A have set up basis for obtaining the recombinant positive phage clone with the activity of binding nucleoprotein of influenza virus A.更多还原