抗恶性黑素瘤单链抗体-鱼精蛋白融合蛋白表达载体的构建及评价

目的 构建抗MM单链抗体-鱼精蛋白融合蛋白表达载体,并验证其表达效率及功能.方法 通过PCR方法将鱼精蛋白截短片段序列与抗黑色素瘤细胞表面抗原单链抗体基因相连接;采用TaKaRa pMD 19-T载体试剂盒将PCR后获得的融合蛋白扩增产物进行T载体构建;构建GST融合蛋白表达载体以实现在大肠杆菌中该融合蛋白的可溶性表达,经两步亲和纯化后获得scFv-tP蛋白;采用凝胶阻滞电泳迁移率变动分析EMSA技术检测Anti-MM scFv-tP与siRNA的结合活性;荧光分子标记scFv-tP蛋白后分别采用流式细胞术检测和共聚焦显微镜观察其与LiBr细胞株进行细胞表面抗原结合活性验证;采用不同浓度FITC标记的siRNA与scFv-tP进行混合,分别与LiBr细胞(A)及DU145细胞(B)进行混合孵育,观察肿瘤细胞对携载siRNA的单链抗体的内化活性.结果 成功构建抗恶性黑素瘤单链抗体-鱼精蛋白片段融合蛋白(Anti-MM scFv-tP)表达载体;实现在大肠杆菌中该融合蛋白的可溶性表达,可获得较纯的scFv-tP蛋白;Anti-MM scFv-tP蛋白与siRNA有明显的结合与携载能力,可以有效与LiBr细胞表面特异性抗原进行结合,且有较强的内化活性.结论 本研究构建的抗MM单链抗体-鱼精蛋白融合蛋白表达载体可稳定表达,Anti-MM scFv-tP蛋白具有siRNA结合携载能力,可靶向识别结合进入LiBr细胞,为后续进一步研究该单链抗体融合蛋白靶向载体携载siRNA在体内外抑制恶性黑色素瘤恶性进展奠定坚实的基础.

Construction and verification of anti-MM scFv-tP fusion protein expression vector

Objective To construct an expression vector of anti-MM scFv-tP fusion protein and test its expression efficiency and function.Methods The truncated protamine (tP) gene sequence was added to the gene of single chain antibody against the specific antigen on the surface of malignant melanoma tumor cells using PCR.A GST-fusion expression vector was constructed and the soluable protein was expressed in the E.coli system.After cleavage and purification,the purified fusion protein was obtained.The binding activity of Anti-MM scFv-tP and siRNA was detected by EMSA.Flow cytometry and confocal microscopy were used to detect the cell surface antigen binding activity of the fusion protein.Results The expression vector of Anti-MM scFv-tP fusion protein was successfully constructed.The soluable protein could be expressed in the E.coli system,and the purified fusion protein was obtained.The anti-MM scFv-tP fusion protein retained siRNA binding ability and could directly target malignant melanoma (MM) LiBr cells.Conclusion The recombinant GST-Anti-MM-scFv-tp expression vector was successfully constructed.The fusion protein retains siRNA binding ability and can directly target LiBr cells to provide a reliable tool for further study.