绿色荧光蛋白融合人核糖核酸酶抑制因子表达载体pEGFP-C1-hri的构建及鉴定

Construction and identification of expression vector for human ribonuclease inhibitor fused to green fluorescent protein

目的构建表达绿色荧光蛋白融合人核糖核酸酶抑制因子的表达载体pEGFP-C1-hri,为探讨人核糖核酸酶抑制因子抗肿瘤作用的分子机制打下基础。方法用亚克隆法,将目的片段从表达载体pGEX-6p-1-hri克隆到pEGFP-C1上,用双酶切筛选得到阳性重组质粒pEGFP-C1-hri,用脂质体法将其瞬时转染到小鼠黑色素瘤细胞B16中,在荧光显微镜下检测其表达。结果pEGFP-C1-hri中插入了hri序列,绿色荧光高效表达于B16细胞浆中。结论pEGFP-C1-hri表达载体已成功构建。

Objective To construct the expression vector of human ribonuclease inhibitor fused to green fluorescent protein which was capable of expression in ceils and to provide a useful tool for investigation the molecular mechanism of antitumor effects from hri. Methods Hri in pGEX-6p-1-hri vector was subcloned into the green fluorescent protein vector pEGFP-C1. The recombinant plasmid was then transfected into mouse B16 Melanoma Ceils, followed by observation of the cells by fluorescent microscope. Results It was identified by enzyme digestion successfully that construction of the recombinant plasmid. The expression of EGFP-hri fusion protein was confirmed by fluorescent microscope. Conclusions pEGFP-hri fusion gene vector is successfully constructed and the fusion protein can be expressed in cytomatrix of B16 cells