绿色荧光蛋白融合人核糖核酸酶抑制因子逆转录病毒载体pLNCX-EGFP-C1-hri的构建及鉴定

构建表达重组绿色荧光蛋白融合人核糖核酸酶抑制因子的逆转录病毒载体pLNCX-EGFP-c1-hri,为探讨人核糖核酸酶抑制因子抗肿瘤作用机制打下基础.用亚克隆法,将目的片段egfp-c1-hri从表达载体pEGFP-C1-hri克隆到pLNCX上,用酶切筛选得到阳性克隆后,用脂质体法将其转染到小鼠黑色素瘤细胞B16中,用800 mg/L G418筛选2周后,在荧光显微镜下检测其表达.双酶切鉴定得到pLNCX-EGFP-C1-hri阳性克隆,荧光显微镜显示绿色荧光在B16细胞质中高效表达.成功构建逆转录病毒表达载体pLNCX-EGFP-C1-hri.

Construction and identification of retroviral vector expressing human ribonuclease inhibitor fused to green fluorescent protein

To construct a retroviral vector of human ribonuclease inhibitor(hri) fused to green fluorescent protein,which is capable of expression in cells and to provide a useful tool for investigation,the molecular mechanism of antitumor effects from hri.egfp-c1-hri from the green fluorescent protein vector pEGFP-C1-hri was subcloned into the retroviral vector pLNCX,and the vector was identified by enzyme digestion.And then transfected into rat B16 melanoma cells by LipofectaminTM2000.After 2 weeks of selection of 800 mg/L G418,the recombinated protein of B16 melanoma cells was identified by fluorescent microscope.Restriction enzyme digestion proved the construction of the recombinant plasmid correct.The fluorescent microscope showed green fluorescent B16 melanoma cells.The retroviral vector of pLNCX-EGFP-C1-hri is successfully constructed,and the fusion protein can be expressed in cytomatrix of B16 cells correctly.