关键词: Rac1, 重叠区扩增法, 基因定点突变, 融合蛋白

Abstract: Construct plasmids contains Rac1 and Rac1 mutant gene by improved Overlap extension and observe the expression of EGFP-Rac1 in hepatocyte LO2. Rac1 gene was cloned by Reverse Transcription-Polymerase Chain Reaction, Rac1 mutant gene was generated by site-directed mutagenesis which was carried out by improved overlap extension. Both Rac1 gene and Rac1 mutant gene were inserted into the pEGFP-C1 vector, three recombinant plasmids: pEGFP-C1-Rac1, pEGFP-C1-Rac1V12 and pEGFP-C1-Rac1N17(constitutively active mutation Rac1V12 as Gly at codon 12 of Rac1 CDS is mutated into Val, and dominant negative mutation Rac1N17 as Thr at codon 17 is mutated into Asn.)were constructed. All kinds of plasmids were transfected into LO2 cells, Expression of EGFP was examined by fluorescence microscope, and exogenous EGFP-Rac1 fusion protein was determined by Western blotting. Three recombinant plasmids were verified by double digestion and gene sequencing. Exogenous EGFP-Rac1 fusion protein was highly expressed in cells transfected by three recombinant plasmids. Three recombinant plasmids which can express EGFP-Rac1 protein in LO2 cell were successfully constructed.

Key words: Rac1, Overlap extension, Site-directed mutagenesis, Fusion protein