膜活性肽Marmelittin的原核表达及抗肿瘤活性研究  

Prokaryotic expression and antitumor activity of membrane active peptide Marmelittin

作　　者：衣同辉[1] 王宏兰[1] 王洁[1] 张春晶[1] 谭佳音 潘洪明[2] 刘哲丞[1] YI Tong-hui;WANG Hong-lan;WANG Jie;ZHANG Chun-jing;TAN Jia-yin;PAN Hong-ming;LIU Zhe-cheng(School of Medical Technology,Qiqihar Medical University,Qiqihar 161006,Heilongjiang Province,China;School of Basic Medicine,Qiqihar Medical University,Qiqihar 161006,Heilongjiang Province,China)

机构地区：[1]齐齐哈尔医学院医学技术学院,黑龙江齐齐哈尔161006 [2]齐齐哈尔医学院基础医学院,黑龙江齐齐哈尔161006

出　　处：《中国临床药理学杂志》2020年第22期3696-3699,共4页The Chinese Journal of Clinical Pharmacology

基　　金：黑龙江省教育厅基本科研业务费基金资助项目(2016-KYYWF-0843)。

摘　　要：目的将膜活性肽Marmelittin与泛素蛋白Ub融合表达并测定其抗肿瘤活性。方法构建pCold-TF-Ub-Marmelittin质粒并转化到BL21(DE3)菌中进行诱导表达。筛选Marmelittin肽的最佳表达条件后并对Marmelittin肽进行纯化。圆二色光谱测定Marmelittin肽的二级结构, CCK-8实验和流式细胞术分析Marmelittin肽抗肿瘤活性及机制。结果 Marmelittin肽在表达菌为1.20 mmol·L^-1异丙基硫代-β-D-半乳糖苷(IPTG)诱导培养10 h时表达量最高。圆二色光谱测定Marmelittin肽为α-螺旋结构。CCK-8实验测定Marmelittin肽对宫颈癌细胞株HeLa半数抑制浓度(IC50)为(46.61±1.25)μg·mL^-1。流式细胞术检测表明Marmelittin肽通过诱导凋亡方式杀伤Hela细胞。结论用泛素蛋白Ub为促溶标签成功表达了Marmelittin肽并测定了其抗肿瘤活性,为后续抗肿瘤机制及应用研究提供了一定的基础。Objective The membrane active peptide Marmelittin was expressed with ubiquitin protein and its antitumor activity was determined. Methods The pCold-TF-Ub-Marmelittin plasmid was constructed and expressed in E.coli BL21(DE3). The optimal expression conditions was screening.The recombinant Marmelittin peptide was purified.The secondary structure of Marmelittin peptide was determined by circular dichroism spectroscopy. Antitumor activity and mechanism of Marmelittin peptide were analyzed by CCK-8 assay and flow cytometry. Results The expression of Marmelittin peptide was the highest when induced with 1.20 mmol·L^-1 isopropyl-β-D-thiogalactopyranoside(IPTG) for 10 h. The secondary structure of Marmelittin peptide was α-helix. The IC50 of Marmelittin peptide inhibits the proliferation of HeLa cells was(46.61±1.25) μg·mL^-1. Flow cytometry showed that Marmelittin peptide killed HeLa cells by inducing apoptosis. Conclusion Marmelittin peptide was successfully expressed with ubiquitin protein as a soluble label and its antitumor activity was determined,then provides a basis for further study on anti-tumor mechanism and application.

关 键 词：Marmelittin肽 泛素蛋白 原核表达 圆二色光谱 宫颈癌 

分 类 号：R97[医药卫生—药品]