EGFP-Arg7融合蛋白表达及在HeLa细胞中的转导活性

Expression of EGFP-Arg7 Fusion Protein and Its Transduction Activity into HeLa Cells

目的构建pET-28a-EGFP-Arg7重组子,观察表达的融合蛋白EGFP-Arg7在细胞内的转导活性。方法构建EGFP-Arg7(阴性对照为EGFP)序列,与pET-28a连接后,转化E.coli BL21(DE3),IPTG诱导表达,并经Ni2+-NTA纯化。纯化产物作用于HeLa细胞后用荧光显微镜观察其转导活性。结果重组pET-28a-EGFP-Arg7经酶切鉴定和序列分析,证明构建正确。转化E.coli BL21(DE3)后,重组蛋白获得可溶性表达。纯化后的融合蛋白纯度达90%以上。重组蛋白EGFP-Arg7作用于HeLa细胞后用荧光显微镜可观察到强绿色荧光。结论 Arg7具有很好的转导活性,能携带与其连接的蛋白质穿过HeLa细胞膜。

Objective To construct an expression vector of pET-28a-EGFP-Arg7 and to investigate its transduction activity into cells. Methods EGFP-Arg7 gene was amplified by PCR and cloned into prokaryotic expression vector pET-28a. The constructed recombinant plasmid pET-28a-EGFP-Arg7 was transformed to E.coli BL21 （DE3） for the expression under the induction of IPTG. The expressed protein was purified by Ni2＋-NTA affinity chromatography and its transduction activity into the HeLa cells was detected under the fluorescence microscope. Results Both endonuclease restriction analysis and sequence analysis proved that the recombinant plasmid pET-28a-EGFP-Arg7 was constructed correctly. The recombinant fusion protein was expressed in a soluble form, with a purity of more than 90 % after the purification. It showed strong green fluorescence into the HeLa cells under the fluorescence microscope. Conclusion EGFP-Arg7 fusion protein has a good transduction activity and can take the linked protein through the HeLa cells.