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目的:构建人源核糖体展示抗体库,筛选制备抗TNF-α的特异性、高亲和力三结构域抗体(VH/K)。方法:应用核糖体抗体库技术,从类风湿关节炎患者的外周血淋巴细胞中分离、构建核糖体展示VH/K基因库;体外转录翻译后,以TNF-α重组蛋白作纯化抗原,采用亲和富集法淘选TNF-α特异性抗体基因;将筛选获得的VH/K基因克隆并在原核系统pET22b(+)/BL21(DE3)中实现可溶性表达;表达产物经进行ELISA鉴定,并对阳性克隆的生物学特性进行初步研究。结果:成功构建了库容量约为6.7×1012核糖体展示VH/K抗体基因库,在180个筛选克隆中,其中两株抗体TRB21、TRB409显示出极高的ELISA值,序列分析表明两株克隆具有全新的人源抗以TNF-α抗体基因,他们不仅可以特异识别TNF-α而且可以抑制以TNF-α对L929细胞的细胞毒性。结论:人源抗TNF-α特异性抗体的获得,为进一步研制抗TNF-α的特异性、高亲和力基因工程抗体奠定了基础。核糖体展示技术为体外筛选制备全人源抗体提供了一种简便、有效的技术平台。
OBJECTIVE: To construct human ribosome display antibody library and screen for specific, high-affinity triple-domain antibody against anti-TNF-α (VH / K). METHODS: The ribosomal antibody library was used to isolate ribosomal VH / K gene from peripheral blood lymphocytes of patients with rheumatoid arthritis. After translation and translation, the TNF-α recombinant protein was purified and used as antigen And enriched method panning TNF-α-specific antibody gene; cloned VH / K gene cloned and purified in the prokaryotic expression system pET22b (+) / BL21 (DE3); the expression product was identified by ELISA, and The biological characteristics of positive clones were studied preliminarily. Results: The gene library of VH / K with a capacity of about 6.7 × 1012 ribosomes was successfully constructed. Of the 180 screening clones, two of the antibodies, TRB21 and TRB409, showed extremely high ELISA values. Sequence analysis showed that two clones With a new human anti-TNF-α antibody gene, they not only specifically recognize TNF-α but also inhibit the cytotoxicity of TNF-α against L929 cells. CONCLUSION: Obtaining of human anti-TNF-α specific antibody lays the foundation for further development of anti-TNF-α specific and high affinity genetically engineered antibody. Ribosome display technology for in vitro screening for the preparation of all-human antibodies provides a simple and effective technology platform.