抗CD5单链抗体的基因构建、蛋白纯化及活性鉴定

摘要

目的:构建高表达的抗CD5单链抗体(scFv anti-CD5)工程菌,为开展以抗CD5单链抗体为构件的肿瘤免疫治疗研究奠定基础.方法:从Genbank中获得抗CD5单克隆抗体H65的重链可变区(VH)和轻链可变区(VL)的基因序列,用连接肽的编码序列连接,用重叠PCR技术获得目的基因,酶切后插入pET22b(+)构成重组质粒.测序正确的重组质粒转入E.coli BL21(DE3)诱导表达,产物经Ni-NTA柱纯化后复性,流式细胞术和Western blotting检测活性.结果:获得序列正确的重组质粒,工程菌表达量高,产物复性后具有识别CD5抗原的活性.结论:成功构建了高表达scFv anti-CD5的工程菌,为scFv anti-CD5的应用奠定了基础.%Purpose To construct a genetic engineering E. coli strain with high expression level of scFv anti-CD5 and to lay the foundation for tumor immunotherapy using scFv anti-CD5 as a component.Methods VH and VL fragment of H65 (an anti-CD5 mAb)is acquired from Genbank, and then joined with a linker coding sequence, and the gene via overlapping PCR is constructed. The gene is digested and inserted into pET22b( + ) to form a recombinant plasmid. The E. coli BL21 (DE3) bear the plasmid with right sequence is induced with IPTG and the product is purified with Ni-NTA column, renatured, and subjected to activity analysis via FCM and Western blotting. Results The recombinant plasmid and the engineering E. coli strain with high expression level of scFv anti-CD5 were acquired. The expression products were able to recognize CD5 antigen after being renatured. Conclusion The acquisition of engineering E. coli strain with high expression level of scFv anti-CD5 would contribute to further application of scFv anti-CD5.