人源抗TLR4抗体IgG的制备及其中和特性分析

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pFUSE-CHIg-hG1和pFUSE-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 IgG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?

Preparation and identification of characteristics of a full human anti-TLR4 IgG

To construct a full human anti-toll-like receptor 4 (TLR4)IgG expression vector,and to express and purify it in 293 free style cells,as well as analyze the biological activity of anti-TLR4 antibody. Methods:We designed a primer for amplify mAb variable region. VH and VL gene were cloned into pFUSE-CHIg-hG1 and pFUSE-CLIg-hl expression vectors,respectively,and both transfected into 293 freestyle cells. The IgG was purified by protein A column and the immune specificity of the mAb was detected by enzyme-linked immunosorbent assay(ELISA),Western blotting assay,Co-IP,mass spectrometry(MS)and Biacore. Then,the interaction of this antibody with TNF-a expression in THP1 cell was detected. Results:The results demonstrated that the full human anti-TLR4 IgG was successfully produced. This mAb effectively recognized TLR4 protein and inhibited the expression of THF-a in THP1 cells with the inhibition rate of 85.7%. Conclusion:The reconstructive full human anti-TLR4 IgG could recognize TLR4 protein and has obvious neutralizing effect,and may be potentially utilized for inflammation therapy.