抗CML单链免疫毒素载体的构建、表达及活性鉴定

Vector construction,expression and characterization of a single chain immuno-toxin against CML cells

目的:构建单链免疫毒素hscFv-ETA'的原核表达载体,经表达后鉴定其对CML细胞的特异性杀伤作用。方法:采用亚克隆技术,首先将hscFv与截短的假单胞菌外毒素基因ETA'通过酶切及连接反应,在载体pWW20上构建hscFvETA'单链免疫毒素基因;再将其亚克隆入pET32a(+)原核表达载体中,转化BL21(DH3)宿主菌,IPTG诱导表达;SDS-PAGE观察其表达水平并建立目的蛋白的纯化方法,Western blot鉴定纯化的目的蛋白,FCM鉴定融合蛋白hscFv-ETA'与细胞结合的活性以及诱导细胞凋亡的能力。结果:限制性酶切图谱和序列分析证实,在pET32a(+)原核表达载体上成功地构建了hscFvETA'单链免疫毒素载体;该基因在pET32a(+)载体中获得高效表达,表达产物的相对分子质量与预期值相符;经Western blot鉴定证实纯化后的重组免疫毒素hscFv-ETA'融合蛋白是正确的;经FCM证实hscFv-ETA'蛋白能特异性结合CML细胞并诱导细胞凋亡。结论:已成功构建并原核表达单链免疫毒素hscFv-ETA',证明该蛋白能发挥特异性结合并诱导CML细胞凋亡。

Objective：To eonstruct a prokaryotie expressing vector for recombinant immunotoxin which fused a humanized anti- CML cells scFv gene with a truncated pseudomonas exotoxin A （ETA＇） gene, and examine expression of hscFv-ETA＇ in E. coli BI21 （DH3）. Methods： Firstly, the gene fragment encoding hscFv was amplified by PCR and was cloned into pWW20 vector, which con- tains a truncated form of pseudomonas exotoxin. Then the hseFv-ETA＇ gene was subcloned into the pET32a（ ＋ ） E. coli expression vec- tor for produetion of the recombinant immunotoxin. The recombinant plasmids were transformed into E. coli BL21 （ DH3 ） and induced by IPTG. After purification hy affinity chromatography, the relative molecular weight and the binding activity of the chimeric protein were determined by SDS-PAGE. The binding ability and apoptosis induction ability of hscFv-ETA＇ to CML cells were confirmed by FCM analysis. Results： The construetion of hscFv-ETA＇ gene in pET32a（ ＋ ） vector was proved by enzyme digestion and sequencing. hscFv-ETA＇ was highly expressed in pET32a（ ＋ ） vector. Western blot results showed hscFv-ETA＇ could bind to CIVIL cell antigen. The molecular mass of the product was identical to the expected value. FCM analysis results demonstrated that hscFv-ETA＇ ean bind to and induce apoptosis of CML cells. Conclusion： A new recombinant immunotoxin, hscFv-ETA＇, is successfully constructed and expressed in E. eoli BL21 （DH3）. hscFv-ETA＇ was confirmed to bind to CML cells and induces apoptosis of CML cells specifically.