文章摘要
杨 瑾,唐 奇,熊四平,蔡炳刚,朱旭辉,王长军,汪茂荣,冯振卿,朱 进.人源抗TLR4抗体IgG的制备及其中和特性分析[J].南京医科大学学报,2015,(6):777~781,786
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
投稿时间:2015-03-17  
DOI:10.7655/NYDXBNS20150604
中文关键词: Toll样受体4  全人源抗体  中和抗体
英文关键词: TLR4  full human IgG  neutralize mAb
基金项目: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)
作者单位
杨 瑾 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 
唐 奇 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 
熊四平 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 
蔡炳刚 中国人民解放军第八一医院全军肝病中心,江苏 南京 210002 
朱旭辉 南京军区军事医学研究所,江苏 南京 210002 
王长军 南京军区军事医学研究所,江苏 南京 210002 
汪茂荣 中国人民解放军第八一医院全军肝病中心,江苏 南京 210002 
冯振卿 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 
朱 进 南京军区军事医学研究所,江苏 南京 210002 
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中文摘要:
      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。
英文摘要:
      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.
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