【摘要】 旨在原核表达并纯化新型重组毒素rCCK_96-104PE38,验证其对结肠癌细胞的靶向杀伤作用。使用基因扩增技术将反向编译的胆囊收缩素CCK_96-104与绿脓杆菌外毒素PE38融合,构建原核表达载体。利用Ni-NTA亲和层析进行纯化。通过结肠癌细胞体外杀伤实验以及结肠癌裸鼠模型的治疗验证rCCK_96-104PE38的抗肿瘤能力。结果显示,扩增出的rCCK_96-104PE38序列正确,成功利用pET-28a原核表达系统实现了活性表达。纯化后的重组蛋白能够在体外诱导结肠癌细胞凋亡,并能抑制裸鼠模型体内的肿瘤生长。成功制备高纯度、无标签的rCCK_96-104PE38重组免疫毒素,体内、体外实验证实其具有抑制结肠癌的能力。更多还原

【Abstract】 This work aims to conduct the prokaryotic expression and purification of a novel recombinant toxin rCCK_96-104PE38,and to verify the targeted killing effect on colon cancer cells. A reversed cholecystokinin(rCCK_96-104)was conjunctto pseudomonas exotoxin（PE38）by gene amplified technology to construct a prokaryotic expression vector. Ni-NTA affinitychromatograph was used for purification. The anti-tumor ability of rCCK_96-104PE38 was verified by in vitro cytotoxicity of coloncancer cells and vivo experiment with nude mice model. Results showed that the amplified gene of rCCK_96-104PE38 was in correctsequence as designed,i.e.,the active expression was successfully achieved using prokaryotic expression system of pET-28 a.The purified recombinant protein induced the apoptosis of colon cancer cells in vitro,and inhibited the tumor growth in nude mice model.Conclusively,it was successful to produce the recombinant toxin rCCK_96-104PE38 in high purity and with no tags,the vitro and vivo experimentsconfirmed that it had inhibitory ability on colon cancer.更多还原