抗鼠CTLA-4单链抗体的构建与表达及其免疫学活性的鉴定

Construction,Expression and Immunologic Activity of Murine Anti-CTLA-4 ScFv

目的构建具有免疫学活性的抗小鼠细胞毒性T淋巴细胞相关抗原4(CTLA-4)单链抗体ScFv(single chain fragment of variety region),为进一步将其应用到动物体内奠定基础。方法采用RT-PCR技术,从分泌抗小鼠CTLA-4单克隆抗体(mAb)的杂交瘤细胞中扩增mAb的VH、VL基因,并进一步将其组装成VH-Linker-VL型的ScFv片段。将ScFv片段亚克隆至pGMET载体,测序正确后将其克隆至真核分泌型表达载体pSect2-GFP,将pSect2-GFP-ScFv纯化质粒转染中国仓鼠卵巢细胞(CHO)并进行表达,同时经Zeocin筛选稳定表达株。用Western blot方法检测ScFv融合蛋白的表达,采用ELISA检测ScFv的亲和活性。结果经Zeocin筛选2周后,CHO细胞株可稳定表达GFP-ScFv蛋白。Western blot法证实GFP-ScFv蛋白在细胞上清和细胞裂解液中均有表达,大小约55kD。ELISA检测表明,CHO培养上清中的GFP-ScFv蛋白经浓缩初纯后能与重组小鼠CTLA-4纯化抗原结合,并具有抑制亲本单抗与其结合的能力。结论成功构建了抗小鼠CTLA-4的ScFv真核表达载体,并能有效表达出具有免疫学活性的ScFv融合蛋白。

Objective To construct and express the single chain fragment of variety region （ScFv） of monoclonal antibody against murine CTLA-4 and analyze the immunologic activity of recombinant ScFv protein. Methods The VL and VH genes were cloned by RT-PCR from anti-murine CTLA-4 mAb excreted by hybridoma cells. VH-linker-VL fragment （ScFv） was constructed and subcloned into vector pGMET and then cloned into vector pSect2-GFP. The purified pSect2-GFP-ScFv plasmid was transfected into CHO cells and selected by Zeocin for two weeks. The expression and activity of GFP-ScFv protein were assayed by Western blot and ELISA. Results CHO cells could stably express GFP-ScFv protein after Zeocin selection. SDS- PAGE and Western blot analysis revealed that the GFP-ScFv was expressed both in culture supernatant and cell lysis. Indirect ELISA results indicated that the expressed ScFv could bind specifically to recombinant protein of murine CTLA-4, and the epitope recognized by the ScFv was the same as parental mAb of CTLA-4. Conelsuion Eukaryotic expression vector encoding murine anti-CTLA-4 ScFv was successfully constructed and it could express ScFv with immunologic activity efficiently.