人源抗TLR4抗体IgG的制备及其中和特性分析  

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

作　　者：杨瑾[1] 唐奇[1] 熊四平[1] 蔡炳刚 朱旭辉[3] 王长军[3] 汪茂荣[2] 冯振卿[1] 朱进[3] 

机构地区：[1]南京医科大学病理系,卫生部抗体技术重点实验室,江苏南京210029 [2]中国人民解放军第八一医院全军肝病中心,江苏南京210002 [3]南京军区军事医学研究所,江苏南京210002

出　　处：《南京医科大学学报（自然科学版）》2015年第6期777-781,786,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家新药创制项目资助(2013ZX09J13110-05B);全军医学科学技术研究计划(08G023)

摘　　要：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 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 transfeeted 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.

关 键 词：TOLL样受体4 全人源抗体 中和抗体 

分 类 号：R392.11[医药卫生—免疫学]