TAT-EGFP融合蛋白的表达及在PC12细胞的转导活性

目的:构建pET28a-TAT-EGFP重组子,观察表达的融合蛋白TAT-EGFP在细胞内的转导活性.方法:人工合成编码TAT蛋白转导区的DNA片段,插入载体pET28a后再连接EGFP基因,组成pET28a-TAT-EGFP重组表达子.转化大肠杆菌,IPTG诱导TAT-EGFP融合蛋白表达,组氨酸亲和层析柱纯化.将融合蛋白加入培养的PC12细胞,观察荧光进入细胞的情况.结果:成功地构建了高表达pET28a-TAT-EGFP重组子,纯化了分子质量约为29 ku的融合蛋白TAT-EGFP,并在体外培养的PC12细胞中证实TAT-EGFP融合蛋白穿透生物膜的能力.结论:通过对TAT-EGFP融合蛋白的表达纯化及活性分析,证实了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.

Expression of TAT-EGFP fusion protein and its transduction activity into PC12 cells

AIM: To construct an expression vector of pET28a-TAT-EGFP, to purify the TAT-EGFP fusion protein and to investigate its transduction efficiency. METHODS: A synthesized DNA fragment encoding TAT protein transduction domain was inserted into pET28a vector, then EGFP gene was ligated to construct the expression vector pET28a-TAT-EGFP. The recombinant vector was transformed into E. coli BL and induced with IPTG. The expressed fusion protein was added into cultured PC12 cells in vitro. The transduction efficiency was detected using indirect fluorescence assay. RESULTS: pET28a-TAT-EGFP was successfully constructed. About 29 ku fusion protein was expressed. TAT-EGFP fusion protein could go across the cytomembrane into PC12 cells in vitro. CONCLUSION: This experiment demonstrates the possibility for TAT directly to deliver protein into cells, which provides a theoretical basis for peptide and macromolecular medicines transducted into cells to treat various diseases.