Guo Ai-hua, Liu Zhi-feng, Sun Xue-gang, Li Hai-yu, Deng Peng, Jiang Yong
Department of Pathophysiology/Key Laboratory of Functional Proteomics of Guangdong Province, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2006 May;26(5):545-8.
To explore the reasons for the low intracellular transduction efficiency of a previously constructed His-TAT-Flag recombinant protein and establish a more efficient transduction system.
The Flag tag of pET14b-His- Tat-Flag vector was deleted with PCR mutant kit, and enhanced green fluorescent protein (EGFP) coding sequence was inserted into the new pET14b-His-TAT recombinant vector. Enzyme digestion and DNA sequencing were performed for identification of pET14b-His-TAT-EGFP vector, which was then transformed into E. coli BL21(DE(3)). After IPTG induction, the recombinant protein of His-TAT-EGFP was isolated and analyzed with SDS-PAGE. Purified His-TAT-EGFP recombinant protein was added to ECV304 cells and the fluorescence was observed to evaluate the transduction efficiency.
pET14b-His-TAT vector and pET14b-His-TAT- EGFP vector were successfully constructed, which was identified with enzyme digestion and DNA sequencing. His-TAT-EGFP fusion protein was expressed and purified successfully and showed cellular transduction activity.
The prokaryotic expression vector has been successfully constructed by modifying pET14b-His-TAT-Flag, and the expressed and purified recombinant protein of His-TAT-EGFP possesses high efficiency of intracellular transduction activity.
探究先前构建的His-TAT-Flag重组蛋白细胞内转导效率低下的原因，并建立一个更高效的转导系统。
使用PCR突变试剂盒删除pET14b-His-Tat-Flag载体的Flag标签，并将增强型绿色荧光蛋白（EGFP）编码序列插入新的pET14b-His-TAT重组载体。对pET14b-His-TAT-EGFP载体进行酶切和DNA测序鉴定，然后将其转化到大肠杆菌BL21(DE(3))中。IPTG诱导后，分离His-TAT-EGFP重组蛋白并用SDS-PAGE进行分析。将纯化的His-TAT-EGFP重组蛋白加入ECV304细胞中，观察荧光以评估转导效率。
成功构建了pET14b-His-TAT载体和pET14b-His-TAT-EGFP载体，经酶切和DNA测序鉴定。His-TAT-EGFP融合蛋白成功表达并纯化，且具有细胞转导活性。
通过对pET14b-His-TAT-Flag进行改造，成功构建了原核表达载体，表达并纯化的His-TAT-EGFP重组蛋白具有高效的细胞内转导活性。
