人LIMK1基因荧光真核表达载体的构建及在人成骨肉瘤MG63细胞中的表达

Construction of fusion protein green fluorescent protein (EGFP)-LIMK1 and the expression in human osteogenic sarcoma MG63 cell

目的构建并表达人LIMK1与绿色荧光蛋白(EGFP-C1)的融合蛋白EGFP-LIMK1,并监测其在人成骨肉瘤MG63细胞内的表达及活性。方法利用基因重组技术将LIMK1与绿色荧光蛋白融合并构建真核细胞表达质粒,通过脂质体介导法导入人成骨肉瘤MG63细胞内,用荧光显微镜观察LIMK1基因表达情况,通过Western blot检测其在MG63细胞中的活性。结果双酶切鉴定证实LIMK1片段已克隆到pEGFP-C1载体的KpnⅠ和NotⅠ位点之间,测序结果证实了插入片段的正确性,转染MG63细胞后,利用荧光显微镜观察可见到绿色荧光蛋白的表达;Western blot检测证实LIMK1在MG63细胞中活性上调。结论成功构建了含有绿色荧光蛋白基因的LIMK1真核表达载体并检测到其在真核细胞MG63中的表达及活性。

Objective To construct the expressive human LIMK1 with green fluorescent protein （EGFP-C1） express fusion protein EGFP-LIMK1 in MC,63 cellline in order to monitor it＇s expression and activation in human MG63 cells. Methods Fuse LIMK1 with green fluorescent protein using gene reconstruct technology and construct eukaryotic expression plasmid, transfected human MG63 cells with lipofectamine,observe the expression of LIMK1 gene through fluorescent microscope, check it＇s activation in MG63 cells through Western-blot. Results Confirmed LIMK1 fragment has cloned in pEGFP-C1 vector through enzyme analysis with Kpn Ⅰ and NotⅠ ,and confirmed the correct of insert fragment through partial DNA sequence analysis, and the recombinant plasmid EGFP-LIMK1 was transfeeted into human MG63 with lipofectamine, the expression of green fluorescent protein was observed through fluorescent microscope. Conclusions Recombi-nant LIMK1 eukaryotie expression vector including green fluorescent protein gene and check out it＇s expression and activation in eukaryotic cell MG63.