摘要
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。
AIM： To prepare distinct human McAbs to TNF-α with high neutralizing potency using ribosome display technology. METHODS： The immunoglobulin heavy and light chain variable （VH, VL） genes were prepared from the peripheral blood lymphocytes in three arthritis patients by PCR. The genes encoding VH/K fragments were prepared by randomly combining VH and VL genes by SOE PCR. TNF-α binding fragments were selected over three cycles of ribosome display and the selected VH/Ks genes were cloned into pET22b（ ＋ ）/BL21 （DE3）, from which soluble VH/K fragments were prepared. The expressed products of selected clones were analyzed by ELISA. Then the positive clones were characterized. RESULTS： A human VH/K gene library with 6.7 × 10^12 numbers used for ribosome display was constructed. Among the 180 selected clones, two clones TRB21 and TRB409 exhibiting the highest ELISA signals were isolated. The analysis of the sequence of TRB21 and TRB409 showed that they were new human immunoglobulin V genes to TNF-α and they recognize TNF-α specifically and antagonize the cytolytic effect of TNF-α on 1929 cell. CONCLUSION： The selected VH/Ks to TNF-α will be useful for constructing engineering antibodies with high affin-ity against arthritis. Ribosome display is a rapid means of generating fully human antibody fragments in vitro.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2009年第2期145-149,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
天津市科技攻关资助项目(06YFGSH03600)
