目的:构建研究H-Ras12V突变蛋白的表达载体,并在体外进行表达和榆测.方法:从细胞中分离提取RNA,通过RT-PCR扩增获得人的正常H-Ras cDNA,测序鉴定.通过PCR介导的定向突变的方法,在其第12位氨基酸处引入一个错义突变G→V,连入测序载体进行鉴定.应用DNA重组技术,将获得的H-Ras12V cDNA插人真核表达载体pEGFPc2,使用限制性酶切反应鉴定.通过脂质体将重组质粒转染入Hela细胞,使用Western Blot对其融合蛋白进行检测.结果:经过Xho I和BamH I的双酶切以及测序鉴定证实成功构建了 pEGFP-H-Ras12V融合蛋白表达载体,克隆基因与GenBank登陆结果一致并成功实现其第12位氨基酸G→v的突变.Western Blot 证实融合蛋白特异性表达,并在转染的细胞系中观察到绿色荧光蛋白的表达.结论:成功构建了pEGFP-H-Ras12V融合蛋白的表达载体,并在体外鉴定EGFP-HRasl2V融合蛋白的表达.
Construction and expression of human H-Ras12V fusion protein expression vector
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.
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