抗人VEGF受体Ⅱ基因Ⅲ区单链抗体基因的构建和表达

Construction and Expression of Single Chain Fv Gene Against Domain Ⅲ of Human VEGF Receptor Ⅱ

采用RT PCR技术从分泌抗人血管内皮生长因子受体Ⅱ (kinaseinsertdomain containingreceptor,KDR)基因Ⅲ区单克隆抗体Ycom1D3的杂交瘤细胞中克隆出VH 和VL 可变区基因 ,通过重叠延伸拼接 (splice overlapextension)PCR方法在VH 和VL 基因之间引入柔性短肽 (Gly4Ser) 3 ,体外构建Ycom1D3单链抗体基因 (Ycom1D3 ScFv) ,将其克隆至pAYZ表达载体 ,在大肠杆菌中表达。SDS PAGE和Western blot分析结果表明 ,Ycom1D3 ScFv在E .coli 16C9中获得表达 ,重组蛋白的相对分子量为 3 0kD ,与预期结果一致。表达产物主要以不溶性包涵体形式存在 ,经过溶解包涵体 ,TALON金属亲合层析基质 (TALONmetalaffinityresin)纯化和体外复性过程 ,获得了高纯度的单链抗体片段。流式细胞分析结果证实该单链抗体可与人脐静脉内皮细胞结合 ,保留了鼠源单抗与KDR抗原的特异性结合活性。抗KDRⅢ单链抗体基因Ycom1D3

The genes encoding for the light and heavy chain variable regions (V H and V L) has been cloned by RT-PCR from a murine hybridoma that produced monoclonal antibody (mAb) Ycom1D3, which was against domain Ⅲ of human vascular endotheli al growth factor receptor Ⅱ (KDRⅢ) and were then connected to each other by a short peptide linker containing 15 amino acids (Gly 4Ser) 3 using splice-ov erla p extensive PCR. The recombinant Ycom1D3-ScFv gene was cloned into the expressi on vector pAYZ and induced to express in E.coli 16C9 SDS-PAGE and Wester n blot analysis showed that the recombinant Ycom1D3-ScFv gene was expressed in E.coli 16C9 and the relative molecular weight of the fusion protein is 30kD which was consistent with the theoretically predicted value. ScFv expression wa s in the form of an inclusion body and the purified fusion protein was obtained after a series of purification steps including cell breakage, inclusion body sol ubilization, TALON metal affinity chromatography and protein refolding. Flow cyt ometric analysis showed that the ScFv fragment can react with human umbilical ve in endothelial cells (HUVECs) which express KDR on the cell surface. In Conclusi on, Recombinant Ycom1D3-ScFv gene has been successfully constructed and express ed in E.coli 16C9, which could be useful in both diagnostic and therapeutic applications.