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目的 :构建全人源抗人Toll样受体4(Toll-like receptor 4,TLR4)抗体轻、重链表达载体,在293Free style细胞中表达并纯化,分析该重组全分子抗体的生物学活性。方法:设计引物扩增全人源抗人TLR4抗体可变区编码序列,将其分别克隆到真核表达载体p FUSE-CHIg-h G1和p FUSE-CLIg-hl中,共转染至293Free style细胞,表达产物用protein A亲和层析柱纯化。应用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)、Western blot、免疫共沉淀、质谱分析及蛋白芯片检测抗体的免疫学特性,并检测该抗体对人单核细胞淋巴瘤THP1细胞肿瘤坏死因子(tumor necrosis factor,TNF)-α表达的影响。结果:成功构建全人源抗人TLR4抗体真核表达载体,获得的全分子抗TLR4抗体保持了与抗原的结合活性,对THP-1细胞TNF-α表达的抑制率可达85.7%。结论:重组全人源抗TLR4 Ig G保持了与TLR4的结合特异性,并具有明显的中和作用,对炎症治疗具有潜在的应用价值。
OBJECTIVE: To construct a light and heavy chain expression vector of human full-fledged Toll-like receptor 4 (TLR4) antibody and to express and purify it in 293Free style cells. The biological activity of the recombinant full-antibody was analyzed. Methods: Primers were designed to amplify the full-length anti-human TLR4 antibody variable region coding sequence and cloned into the eukaryotic expression vector pFUSE-CHIg-hG1 and pFUSE-CLIg-hl respectively and co-transfected into 293Free style The cells were purified by protein A affinity chromatography. The immunological characteristics of the antibody were detected by enzyme-linked immunosorbent assay (ELISA), Western blot, co-immunoprecipitation, mass spectrometry and protein microarray. The anti-tumor activity of this antibody against human monocytic lymphoma THP1 cell tumor Effect of tumor necrosis factor (TNF) -α expression. Results: The eukaryotic expression vector of human anti-human TLR4 antibody was successfully constructed. All-molecule anti-TLR4 antibody retained the binding activity with antigen, and the inhibitory rate of TNF-α expression in THP-1 cells reached 85.7%. CONCLUSION: Recombinant human anti-TLR4 Ig G retains its binding specificity with TLR4 and has obvious neutralization effect, which has potential value in the treatment of inflammation.