基于核糖体展示技术进行抗狂犬病毒人源抗体的制备

构建了核糖体展示人源抗狂犬病毒单链抗体(scFv)库,筛选制备特异抗狂犬病毒糖蛋白(RVGp)的稳定性人源抗体.应用核糖体抗体库技术,从经狂犬病毒Vero疫苗免疫的志愿者外周血淋巴细胞中分离、构建核糖体展示scFv基因库.体外转录翻译后,以RVGp重组蛋白作筛选抗原,采用亲和富集法淘选RVGp特异性scFv抗体基因.在原核系统pET22b(+)/BL21(DE3)中实现scFv抗体片段的可溶性表达,ELISA鉴定阳性克隆.然后对筛选的scFv进行稳定性改构,构建VH-Lc-VK稳定性抗体,并对其生物学活性进行初步研究.成功构建了库容量约为6.2×1012的核糖体展示scFv抗体基因库.在180个筛选克隆中,克隆RB24、RB71、RB109和RB156显示出较高的ELISA值,其基因序列分析结果显示,它们是全新的人源抗RVGp抗体.改构后的抗RVGp VH-Lc-VK抗体的稳定性明显改进,可特异识别RVGp并有效中和狂犬病毒,抑制狂犬病毒对靶细胞的感染.以上结果表明,人源抗RVGp特异性抗体的获得,为狂犬病的有效预防、诊断和治疗提供了新的途径,而且将为其他人源抗体的制备提供理论依据和技术基础.

Preparation of human antibody fragments against rabies virus based on ribosome display technology

To prepare human antibody to rabies virus with high neutralizing potency using ribosome display technology. The immunoglobulin heavy and light chain variable (VH, VL) genes were prepared with the peripheral blood lymphocytes from three volunteers immunized with rabies virus vaccine by PCR. The genes encoding scFv fragments were prepared by randomly combining VH and VL genes by SOE PCR. Rabies virus glycoprotein (RVGp) specific scFv genes were selected over five cycles of ribosome display. The isolated scFv genes were cloned into pET22b(+)/BL21(DE3), from which soluble scFv fragments were prepared. The expressed products of selected clones were analyzed by ELISA for isolating the positive clones. To improve the stability of obtained scFvs, VH-Lc-VK were constructed and characterized. A human scFv gene library with 6.2×10¹² numbers used for ribosome display was constructed. Among the 180 selected clones, four clones RB24, RB71, RB109 and RB156 which exhibited the highest ELISA signals were isolated. The analysis of their sequences showed that they were new human immunoglobulin V genes to RVGp. And the reconstructed VH-Lc-VK antibody fragments can recognize RVGp specifically and antagonize the cytolytic effect of rabies virus on Vero cells. The prepared VH-Lc-VK fragments to RVGp will be useful for preparing engineering antibodies with high affinity against rabies virus. Ribosome display is a rapid means of generating fully human antibody fragments in vitro.