抗GD2/抗CD16单链双特异性抗体的构建及在大肠杆菌中的表达

Construction and Expression of Anti-GD2/Anti-CD16 Single-chain Bispecific Antibody

利用重叠PCR(overlap-PCR)将抗GD2表面抗原GD2的单链抗体基因m-ScFv和抗CD16的单链抗体基因NM3E2融合在一起,得到新型的单链双特异性抗体基因n-m,双抗的联接肽序列为SerGly4Ser,在肽连的C端引入了6×his以利于纯化。该双特异性抗体基因的序列经测序验证后,连接表达载体pET-22b(+),转化大肠杆菌菌株BL21(DE3),诱导表达,凝胶成像系统扫描显示表达的外源融合蛋白约占菌体总蛋白的27%。表达蛋白分泌于胞质空间,经超声波破胞后收集上清,经Ni+亲和层析柱分离,纯度可达90%以上。经SDS-PAGE及Westernblotting鉴定表达蛋白的分子量为53KD,与预期的相符。

This study sought to construct a recombinant vector that expresses anti-GD2/anti-CD16 bispecific single-chain antibody（sc-BsAb）, and to assess its biological activities. The anti-GD2 gene and the anti-CD16 gene （NM3E2） were obtained using PCR amplification technique, and then the fusion gene was constructed by overlapping PCR. The sc-BsAb gene was subcloned into the pET-22b （±） plasmid from the pMD18-T easy vector by digestion with NcoI, Hind Ⅲ restriction endonucleases, whose sites exist in both the vectors. Then the combinant plasmids were transferred into E. coli BL21 （DE3）. The expression product in the periplasmic was analyzed by both SDS-PAGE and Western blot technique, then was purified with Ni^2＋-NTA superflow affinity chromatography. It was demonstrated that the linker in the sc-BsAb fusiori protein is SerGly4Ser. and the molecular is 53 KD.