小鼠Wnt-1基因真核表达载体构建及其在大鼠成肌细胞中表达.docVIP

小鼠Wnt-1基因真核表达载体构建及其在大鼠成肌细胞中表达 王秋艳1，杜文娟2， 李继科3，杨艳波4， 于波2△ （1河北省秦皇岛港口医院，心内科，秦皇岛，河北省 066002 ；哈尔滨医科大学附属第二临床医学院, 2心内科，3心外科；4哈尔滨市第二医院，心内科， 哈尔滨， 黑龙江省 150086） ［摘要］目的：构建p-EGFP-C3-Wnt-1真核表达载体并转染体外培养大鼠成肌细胞，观察Wnt-1基因在其中的表达，为成肌细胞移植修复心肌梗死奠定基础。方法：利用RT-PCR方法从小鼠胚脑中扩增获得Wnt-1基因编码区全长序列，克隆到含有增强型绿色荧光蛋白报告基因的真核表达载体p-EGFP-C3上，形成重组载体p-EGFP-C3-Wnt-1。转化感受态大肠杆菌 DH5α菌株，进行菌落PCR初步筛选。扩增 DH5α后抽提质粒 DNA，进行质粒PCR鉴定，经Hind Ⅲ 和 KnpⅠ双酶切，电泳和DNA测序鉴定。大鼠成肌细胞原代培养及desmin免疫荧光染色鉴定。利用LipofectamineTM2000脂质体将重组质粒p-EGFP-C3-Wnt-1转染成肌细胞。结果：重组质粒p-EGFP-C3-Wnt-1构建成功；将其转染成肌细胞72 h后，观察到EGFP报告基因和目的基因Wnt-1表达。结论：构建小鼠胚脑Wnt-1基因的真核表达载体p-EGFP-C3-Wnt-1并成功转染成肌细胞，为进一步研究奠定基础。 ［关键词］Wnt-1；载体构建；成肌细胞；转染 Construction of mouse Wnt-1 gene eukaryotic expression vector and its expression in rat skeletal myoblasts WANG Qiu-yan1,DU Wen-juan2, LI Ji-ke3, YANG Yan-bo4, YU Bo2△ (1Department of cardiology , Qinhuangdao port hosipital of hebei province , 066002; 2Department of cardiology,3Department of cardiology surgery, The Second Clinical College, Harbin Medical University; 4Harbin the second hosipital, Harbin 150086, China) ［Abstract］AIM: To construct mouse Wnt-1 gene eukaryotic expression vector and detected its expression in rat skeletal myoblasts in vitro. Methods: Mouse Wnt-1 gene was cloned from the brain tissue of fetal mouses by RT-PCR and was inserted into an eukaryotic expression vector carrying enhanced green fluorescent protein, p-EGFP-C3 which constructed a new plasmid p-EGFP-C3-Wnt-1. E. colli strain DH5α was transfected with the new recombinant plasmid and then selected by PCR. Plasmid DNA extracted from the expanded DH5α was identifed by plasmid PCR and by cutting with Hind Ⅲ, KnpⅠnuclease and by DNA sequencing. Skeletal myoblasts were cultured and identified by desmin immunofluorescence stain. The identified recombinate p-EGFP-C3-Wnt-1 eukaryotic expression vector was transfected into rat skeletal myoblasts with LipofectamineTM2000 liposome. Results: The eukaryotic expression vector containing mouse Wnt-1 gene was constructed succes