三种连接子多肽对抗癌融合蛋白翻译后剪切效果比较研究  

A Study of Three Linker Peptides' Cleavage Efficiency at The Post-translational Level

作　　者：李红艳[1,2] 周志明[2] 潘强[1] 杨冬梅[1] 顾锦法[2] 沈佳妮[1] 王世兵[1] 章康健[2] 

机构地区：[1]浙江理工大学生命科学学院,新元医学与生物技术研究所,杭州310018 [2]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海200031

出　　处：《生物化学与生物物理进展》2014年第6期558-566,共9页Progress In Biochemistry and Biophysics

基　　金：浙江理工大学项目(1204807-Y);浙江理工大学科研启动基金(11610032611303);国家重点基础研究发展计划(973)(2010CB529901;2011CB510100);浙江省自然科学基金(LY13H080005);上海自然科学基金(13ZR1446300)资助项目~~

摘　　要：IL-24和Smac为靶向癌症的多基因治疗研究中重要的候选基因.在构建IL-24和Smac双基因表达载体过程中,选择一个稳定高效的连接子非常关键.利用三个不同的连接子多肽序列IETD、EEED、F2A构建三个真核表达质粒pcDNA3.1(+)-IL-24-IETD-Smac、pcDNA3.1(+)-IL-24-EEED-Smac、pcDNA3.1(+)-IL-24-F2A-Smac,并联合5-氟尿嘧啶(5-FU)处理研究各连接子介导的融合蛋白剪切效果.上述实验结果表明,在三种IL-24-linker-Smac双基因表达载体中,F2A连接子剪切效果最佳且与caspase活性成正相关.IETD、EEED连接子介导的融合蛋白都有一定的剪切,但IETD/EEED多肽影响了上游IL-24蛋白的半衰期,加速了剪切后IL-24蛋白的泛素化降解.本研究为构建靶向癌症应用的双基因或多基因载体提供了设计参考.IL-24 and Smac are the important candidate genes for the targeting multi-genes therapy of cancer. In the construction of IL-24 and Smac dual gene expression vector, a stable and efficient linker is critical. We use three different linkers such as IETD, EEED and F2A to obtain three eukaryotic expression plasmids which are pcDNA3.1（＋）-IL-24-IETD-Smac, pcDNA3.1（ ）-IL-24-EEED-Smac, pcDNA3.1（ ）-IL-24-F2A-Smac, and then combined with 5-fluorouracil （5-FU） treatment to study the cleavage efficiency of the three linker peptides. The results showed that among three IL-24-linker-Smac dual gene expression vectors, the F2A linker is superior to IETD and EEED linkers. In addition, its cleavage efficiency is positively correlated with activated caspases. Moreover, the fusion protein mediated by both IETD and EEED linker also can be cleaved,but their upstream IL-24 protein with 4 additional amino acid residues derived from IETD or EEED linker was detected with obvious degradation regulated by the ubiquitin-proteasome system. This study will provides a reference for the construction of dual-gene or muti-gene expression vector in cancer therapy.

关 键 词：连接子多肽 IETD EEED F2A caspase活性 

分 类 号：Q2[生物学—细胞生物学] Q7