Abstract： AIM:To construct the human H-Ras12V fusion protein expression vector and to observe its expression in vitro.METHODS:H-Ras cDNA was amplified with RT-PCR from cell line and was identified by DNA sequencing.Then,a missense mutation,12 G→V,was performed by PCR.Mediated Site-Directed Mutagcnesis and was identified by sequence analysis.The HRasl2v cDNA was inserted into the eukaryotic expression vector pEGFP-C2 by recombination DNA techniques,and Was conformed by restrictive enzymes digestion analysis.The constructed pEGFPH-Ras12V was transfected into Hela cell line and its expression Was detected by Western Blot.RESULTS:Restrictive enzymes digestion(Sal I/BamH I)and DNA sequencing revealed that the expression vector pEGFP-H-Rasl2V had been constructed saccessfully.The sequence of encoding Ras Was exactly the same as the human Ras sequence on GenBank except the point mutation we introduced.Western Blot demonstrated that the fusion protein EGFP-H-Ras12V expressed specifically and correctly in Hela cell and the green fluorescence W&S detected under fluorescence microscope.CONCLUSION:The fusion protein expression vector pEGFP-H-Ras12V has been constructed successfully and the EGFP-H-Ras12V fusion protein could be expressed in vitro.