重组毒素rCCK_(96-104)PE38靶向结肠癌生物特性研究  

Characterization of Recombinant Toxin rCCK_(96-104)PE38 Targeting Colon Cancer

作　　者：张嵩[1] 柳溪林[2] 李萌[1] 常江[1] 高世奇[1] 胡盼[1] 柳增善[1] ZHANG Song LIU Xi-lin LI Meng CHANG Jiang GAO Shi-qi HU Pan LIU Zeng-shan(Key Laboratory of Zoonosis, Research Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130062 China-Japan Union Hospital of Jilin University, Changchun 130033)

机构地区：[1]吉林大学动物医学学院人兽共患病研究所教育部重点实验室,长春130062 [2]吉林大学中日联谊医院,长春130033

出　　处：《生物技术通报》2017年第4期247-252,共6页Biotechnology Bulletin

基　　金：国家青年科学基金项目(81401953);中国博士后科学基金面上项目(2014M561303);中国博士后科学基金特别资助项目(2015T80312);吉林省青年基金项目(20160520162JH);教育部博士点基金项目(20120061110078);吉林省科学技术厅计划项目(20120966)

摘　　要：旨在原核表达并纯化新型重组毒素rCCK_(96-104)PE38,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_(96-104)PE38的抗肿瘤能力。结果显示,扩增出的rCCK_(96-104)PE38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_(96-104)PE38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。This work aims to conduct the prokaryotic expression and purification of a novel recombinant toxin rCCK96-104PE38,and to verify the targeted killing effect on colon cancer cells. A reversed cholecystokinin（rCCK96-104）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 rCCK96-104PE38 was verified by in vitro cytotoxicity of coloncancer cells and vivo experiment with nude mice model. Results showed that the amplified gene of rCCK96-104PE38 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 rCCK96-104PE38 in high purity and with no tags,the vitro and vivo experimentsconfirmed that it had inhibitory ability on colon cancer.

关 键 词：重组毒素 结肠癌 胆囊收缩素 绿脓杆菌外毒素 

分 类 号：R735.35[医药卫生—肿瘤]