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利用能与CA125结合的间皮素最小活性片段IAB与跨膜肽TAT和凋亡肽BH3连接后,插入至质粒pET32a(+)中,构建成免疫毒素IAB-TAT-BH3融合表达载体pET32a(+)-IAB-TAT-BH3。将此重组质粒及对照组质粒pET32a(+)-IAB-BH3分别转化E.coli BL21(DE3),表达后经western blot验证,获得了相应的可溶性蛋白表达产物。表达菌的裂解上清经镍柱亲和层析、超滤脱盐后,分别收集到高纯度且浓度均大于250μg/ml的融合蛋白。经MTT检测,这两种融合蛋白针对细胞表面表达有受体蛋白CA125的癌细胞并具有抑制其增殖的作用。其中IAB-TAT-BH3与对照IAB-BH3相比,具有更强抑制细胞增殖的作用。IAB-TAT-BH3这一新型免疫毒素的表达、纯化及初步鉴定,为CA125阳性癌细胞的靶向治疗研究奠定了基础。
The IAB-TAT-BH3 fusion expression vector pET32a (+) was constructed by ligating the transmembrane peptide TAT and apoptotic peptide BH3 into the plasmid pET32a (+) using the minimal mesothelin fragment IAB capable of binding to CA125. ) -IAB-TAT-BH3. The recombinant plasmid and control plasmid pET32a (+) - IAB-BH3 were transformed into E. coli BL21 (DE3), respectively, and then verified by western blot to obtain the corresponding soluble protein expression product. The lysis supernatant of the expression bacteria was separated by nickel affinity chromatography and desalted by ultrafiltration, and the fusion proteins of high purity and concentration higher than 250 μg / ml were collected respectively. According to MTT assay, the two fusion proteins were targeted to cancer cells expressing receptor protein CA125 on cell surface and had the effect of inhibiting their proliferation. Among them, IAB-TAT-BH3 has a stronger inhibitory effect on cell proliferation than the control IAB-BH3. The expression, purification and preliminary identification of IAB-TAT-BH3, a novel immunotoxin, laid the foundation for the targeted therapy of CA125-positive cancer cells.