重组毒素rCCK96-104PE38靶向结肠癌生物特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2017.04.032

生物技术通报 ›› 2017, Vol. 33 ›› Issue (4): 247-252.doi: 10.13560/j.cnki.biotech.bull.1985.2017.04.032

• 研究报告 • 上一篇

重组毒素rCCK_96-104PE38靶向结肠癌生物特性研究

张嵩¹, 柳溪林², 李萌¹, 常江¹, 高世奇¹, 胡盼¹, 柳增善¹

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

收稿日期:2016-12-21 出版日期:2017-04-25 发布日期:2017-04-25
作者简介:张嵩,男,硕士研究生,研究方向：动物性食品安全;E-mail：442557139@qq.com
基金资助:
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）

Characterization of Recombinant Toxin rCCK_96-104PE38 Targeting Colon Cancer

ZHANG Song¹, LIU Xi-lin², LI Meng¹, CHANG Jiang¹, GAO Shi-qi¹, HU Pan¹, LIU Zeng-shan¹

1. Key Laboratory of Zoonosis,Research Ministry of Education,College of Veterinary Medicine,Jilin University,Changchun 130062;
2. China-Japan Union Hospital of Jilin University,Changchun 130033

Received:2016-12-21 Published:2017-04-25 Online:2017-04-25

摘要/Abstract

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。

关键词: 重组毒素, 结肠癌, 胆囊收缩素, 绿脓杆菌外毒素

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 conjunct to pseudomonas exotoxin（PE38）by gene amplified technology to construct a prokaryotic expression vector. Ni-NTA affinity chromatograph was used for purification. The anti-tumor ability of rCCK_96-104PE38 was verified by in vitro cytotoxicity of colon cancer cells and vivo experiment with nude mice model. Results showed that the amplified gene of rCCK_96-104PE38 was in correct sequence as designed,i.e.,the active expression was successfully achieved using prokaryotic expression system of pET-28a. 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 experiments confirmed that it had inhibitory ability on colon cancer.

Key words: recombinant toxin, colon cancer, cholecystokinin, pseudomonas exotoxin

张嵩, 柳溪林, 李萌, 常江, 高世奇, 胡盼, 柳增善. 重组毒素rCCK_96-104PE38靶向结肠癌生物特性研究[J]. 生物技术通报, 2017, 33(4): 247-252.

ZHANG Song, LIU Xi-lin, LI Meng, CHANG Jiang, GAO Shi-qi, HU Pan, LIU Zeng-shan. Characterization of Recombinant Toxin rCCK_96-104PE38 Targeting Colon Cancer[J]. Biotechnology Bulletin, 2017, 33(4): 247-252.

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重组毒素rCCK_96-104PE38靶向结肠癌生物特性研究

Characterization of Recombinant Toxin rCCK_96-104PE38 Targeting Colon Cancer

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重组毒素rCCK96-104PE38靶向结肠癌生物特性研究

Characterization of Recombinant Toxin rCCK96-104PE38 Targeting Colon Cancer

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重组毒素rCCK_96-104PE38靶向结肠癌生物特性研究

Characterization of Recombinant Toxin rCCK_96-104PE38 Targeting Colon Cancer