【摘要】 目的 :构建抗膀胱癌重组免疫毒素BDI 1 PE38/KDEL的可溶性表达载体 ,并表达、纯化具有抗肿瘤活性的可溶性蛋白。方法 :将抗膀胱癌重组免疫毒素BDI 1 PE38/KDEL的基因片段 ,插入含有大肠杆菌脯氨酰顺反异构酶FkpA基因的共表达载体pTMFK中 ,构建重组共表达载体pTMFK IT。以重组质粒转化大肠杆菌BL2 1(DE3) Star,共表达目的蛋白与FkpA。表达产物以镍离子金属螯合层析柱及抗PE抗体亲和柱层析进行纯化。用ELISA检测免疫毒素的抗原结合活性 ,用噻唑蓝(MTT)法进行体外细胞杀伤实验。结果 :成功地构建了抗膀胱癌免疫毒素基因与FkpA基因的共表达载体 ,并获得纯度较高的目的表达产物。纯化后的表达产物与BIU 87膀胱癌细胞膜抗原具有良好的特异性结合活性 ,对BIU 87膀胱癌细胞有明显的体外特异性杀伤作用。结论 :获得了对膀胱癌细胞具有显著特异性杀伤活性的免疫毒素 ,为将其进一步应用于膀胱癌的靶向治疗研究奠定了基础更多还原

【Abstract】 AIM: To construct soluble expression vector of the recombinant immunotoxin against human bladder carcinoma and express and purify the immunotoxin with high biological activity. METHODS: The gene fragment of the immunotoxin containing the genes encoding the humanized single-chain antibody against human bladder carcinoma and the truncated and modified form of Pseudomonas exotoxin(PE) named PE38/KDEL was digested from the plasmid pABDIT and inserted into the expression vector pTMFK containing the FkpA gene. The recombinant plasmid was used to transform the E.coli strain BL21(DE3)-Star and the immunotoxin was co-expressed with the FkpA which served as the folding assistant factor. The immunotoxin was purified through Ni-NTA agarose and anti-PEAb Sepharose 4B columns. The binding activity of the immunotoxin to the antigen on BIU-87 cells was detected by ELISA. The specific cytotoxic effect of the immunotoxin against BIU-87 cells was assessed by MTT colorimetry. RESULTS: The immunotoxin was over-expressed in soluble form in E.coli. The immunotoxin showed good binding activity to the membrane antigen on BIU-87 cells. The result of MTT assay demonstrated that purified recombinant immunotoxin could specifically kill BIU-87 cells. CONCLUSION: The immunotoxin was obtained with specific cytotoxicity to human bladder cancer cells, which lays the foundation for the further application of the immunotoxin in the target therapy of human bladder carcinoma.更多还原