DMSO增强原核表达TAT-EGFP（Apoptin）穿膜效应的研究  

Expression of TAT - EGFP （Apoptin） and its Enhancement of Penetrating Ability into Caski Cells after Treated by DMSO

作　　者：王琥[1] 钟春燕[1] 柳长柏[1] 

机构地区：[1]三峡大学分子生物学研究所,宜昌443002

出　　处：《实用医学进修杂志》2009年第3期154-159,共6页Journal of Practical Training of Medicine

基　　金：国家自然科学基金项目（20774094/B040203）.

摘　　要：目的：构建pET15b-TAT-EGFP和pET15b-TAT-Apoptin,观察表达的融合蛋白TAT-EGFP在细胞内的定位及TAT-Apoptin的诱导Caski凋亡活性。方法：人工合成编码TAT蛋白转导区的DNA片段,插入载体pET15b后再连接EGFP和Apoptin基因,组成pET15b-TAT-EGFP（Apoptin）重组子。转化大肠杆菌,1mMIPTG诱导TAT-EGFP（Apoptin）融合蛋白表达,His亲和层析柱纯化。培养的Caski细胞经过10%DMSO处理1h后,加入融合蛋白孵育1h,观察TAT-EGFP进入细胞的情况。同时用TUNEL检测TAT-Apoptin诱导Caski凋亡的情况。结果：成功地构建了高表达pET15b-TAT-EGFP（Apoptin）重组子,纯化了分子质量约为30KD的融合蛋白TAT-EGFP和17KD的TAT-Apoptin融合蛋白,并在体外培养的Caski细胞经10%DMSO处理后证实TAT-EGFP融合蛋白穿透生物膜的能力明显增强。结论：通过对TAT-EGFP融合蛋白的表达纯化及活性分析,证实了DMSO能够增强TAT的蛋白转导作用,为肽类及生物大分子药物进入组织细胞内发挥治疗作用提供了理论基础。Objective： To construct an expression vector of pET15 b - TAT - EGFP（Apoptin） , to purify the TAT- EGFP（Apoptin） fusion protein and to investigate its penetrating efficiency after treated by DMSO. Methods： A synthesized DNA fragment encoding TAT and EGFP（Apoptin） fragment was inserted into pET15b. The recombinant vector was transformed into E. coli BL21 and induced with IPTG. The expressed fusion protein was added into cultured Caski cells in vitro. The penetrating ability was detected using indirect fluorescence assay, the bioactivity of Apoptin was assayed by TUNEL. Results： pET15b- TAT- EGFP were expressed. TAT - EGFP fusion protein could go across the cytc ,embrane highy with DMSO treatment into Caski cells compared without DMSO treatment. TAT- Apoptin fusion protien could penetrate into the cytosol and induce Caski cells with a higher efficiency treated by DMSO. Conclusions： This experiment demonstrates the possibility for TAT deliver protein more efficiently after treated by DMSO into cells, which provides a theoretical basis for peptide and macromolecular medicines transducted into cells to treat various diseases more widely.

关 键 词：细胞膜穿透肽 TAT 融合蛋白 二甲基亚砜 转导 

分 类 号：R457.1[医药卫生—治疗学] R392.11[医药卫生—临床医学]