抗KDR单链抗体的构建、表达及生物学活性鉴定

Prokaryotic expression and characterization of an anti-KDR single chain antibody

目的: 构建并表达抗人血管内皮生长因子受体KDR单链抗体(scFv)蛋白, 并测定其生物学活性。方法: 设计合成抗KDR单抗 (mAb)Ycom1D3VH、VL引物, 通过重叠延伸拼接 (splicingoverlapextensive, SOE)PCR, 在VH 和VL基因间引入柔性短肽(Gly4Ser)3, 构建抗KDRscFv基因并进行序列分析。将其克隆入pAYZH原核表达载体, 在大肠杆菌中诱导表达, 并以ELISA及免疫荧光结合实验测定重组蛋白的生物学活性。结果: 序列分析表明, 抗KDRscFv基因的全长为 729bp, 编码 243个氨基酸。将重组体表达产物进行SDS PAGE及Westernblot分析显示, 融合蛋白的相对分子质量(Mr)约为 30 000, 同预期的结果一致。表达产物主要以不溶性包涵体的形式存在, 表达量占菌体总蛋白的 20%。表达产物经变性、纯化及体外复性后, 纯度达 90%以上。ELISA和竞争性免疫荧光结合实验显示, 重组小分子scFv可与可溶性KDR抗原及表达KDR受体的人脐静脉内皮细胞特异性结合, 保留了亲本mAb的抗原结合活性。结论: 成功地构建了抗KDRscFv基因, 并在大肠杆菌中功能性表达, 为靶向诊断、治疗及进一步基因工程改造奠定了基础。

AIM: To construct and express a single chain antibody (scFv) against human vascular endothelial growth factor (VEGF) receptor KDR and characterize its biological activity. METHODS: The restriction enzyme sites were added to the previously cloned V H and V L genes of mAb Ycom1D3 against KDR by PCR. The anti-KDR scFv gene was constructed by the splicing overlap extensive (SOE) PCR and then inserted into fusion expression vector pAYZH. The recombinant protein was expressed in E.coli 16C9 and purified with His-tag affinity chromatography. The specificity of the purified scFv was examined by ELISA and FACS. RESULTS: DNA sequencing indicated that the cloned scFv gene consisted of 729 bp, encoding 243 amino acids. After induction in low-phosphate medium of AP5, a new protein band with relative molecular mass (M r) of 30 000 appeared on gel of SDS-PAGE and on nitrocellulose membrane of Western blot, which was consistent with the theoretically predicted value. Anti-KDR scFv was expressed in the form of inclusion body, which accounted for 20% of total bacterial protein. ELISA and competitive immunofluorescence binding test showed that the anti-KDR scFv had the same binding activity as mAb Ycom1D3 and that it could block VEGF/KDR interaction effectively. CONCLUSION: Recombinant anti-KDR scFv gene has been successfully constructed and expressed in E.coli 16C9, which lays the foundation for its diagnostic and therapeutic application.