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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
Objective: To construct a soluble expression vector against bladder cancer recombinant immunotoxin BDI 1 PE38 / KDEL and to express and purify the soluble protein with anti-tumor activity. Methods: The gene fragment of recombinant human immunodeficiency virus BDI 1 PE38 / KDEL against bladder cancer was inserted into pTMFK, a co-expression vector containing FkpA gene of E. coli prolyl cis-trans isomerase to construct recombinant co-expression vector pTMFK IT. The recombinant plasmid was transformed into E. coli BL21 (DE3) Star to express the target protein and FkpA. The expressed product was purified by nickel ion metal chelate chromatography and anti-PE antibody affinity column chromatography. The antigen binding activity of the immunotoxin was detected by ELISA and the cell killing experiment was carried out by MTT method. Results: The co-expression vector of anti-bladder cancer immunotoxin gene and FkpA gene was successfully constructed and the target product with high purity was obtained. The purified expression product has good specific binding activity with BIU 87 bladder cancer cell membrane antigen, and has obvious in vitro specific killing effect on BIU 87 bladder cancer cells. CONCLUSION: The immunotoxin with significant specific killing activity on bladder cancer cells was obtained, which laid the foundation for its further application in the targeted therapy of bladder cancer