人源化抗Siglec-9抗体Fab片段的制备及鉴定

摘要

Objective:The prokaryotic expression vector of human anti-Siglec-9 Fab fragment antibody was constructed and purified,while was identified. Methods:The variable and conserved regions of heavy chain and light chain were obtained by polymerase chain reaction respectively(PCR),which was combined by overlap extension PCR and was digested with restriction enzyme,and then it was transformed into Escherichia coli BL21 and was purified by His-trap Lambda Fab column and AKTA system. SDS-PAGE,ELISA and Western blot were used for the identification of human anti-Siglec-9 Fab fragment antibody. The effect of human anti-Siglec-9 Fab fragment antibody on regulating the mRNA expression of TNF-α,IL-1,IL-6,IL-8 was detected by real-time PCR. Results:Successfully obtained the chains of heavy and light, while constructed an activation human anti-Siglec-9 Fab fragment antibody which could specifically bind to Siglec-9 protein. The human anti-Siglec-9 Fab fragment antibody could specifically bind to Siglec-9 was confirmed by SDS-PAGE,ELISA and Western blot. The human anti-Siglec-9 Fab fragment antibody inhibited the mRNA expression of TNF-α,IL-1, IL-6,IL-8. Conclusion:Successful prokaryotic expression,purification,character analysis,and suppressed the mRNA expression of in-flammatory cytokines with the human anti-Siglec-9 Fab fragment antibody and lay the biology foundation for the further study.%目的:构建人源化抗Siglec-9抗体Fab段原核表达质粒,表达、纯化此抗体以及特性分析,并初步探讨此抗体的生物学功能.方法:利用聚合酶链式反应(PCR)分别获得抗体轻链和重链的可变区及恒定区,经重叠延伸PCR连接,酶切后与原核表达载体pETDUET-1重组,转入表达感受态Escherichia coli BL21 中,通过蛋白纯化系统AKTA和His-trap Lambda Fab纯化.使用SDS-PAGE、ELISA以及Western blot鉴定和分析.采用实时荧光定量PCR初步检测抗Siglec-9抗体Fab段对炎性细胞因子TNF-α、IL-1、IL-6、IL-8的mRNA表达量的影响.结果:成功获得抗体轻链和重链,构建人源化抗Siglec-9抗体Fab段原核表达系统,经 SDS-PAGE、Western blot、ELISA 检测证实抗 Siglec-9 抗体 Fab 段与 Siglec-9 抗原特异性结合.抗Siglec-9抗体Fab段可抑制炎性细胞因子TNF-α、IL-1、IL-6、IL-8的mRNA表达量.结论:人源化抗Siglec-9抗体Fab段进行了原核系统表达、纯化和鉴定,并初步验证可抑制炎性细胞因子的mRNA表达量,为进一步研究该抗体的生物学特性及应用奠定基础.