小鼠Dazl基因融合蛋白表达载体的构建及转染  

Construction of eukaryotic expression vector pEGFP-C1-Dazl and its expression in 293 and NIH-3T3 cells

作　　者：王龙[1] 周莹[2] 胡玥[1] 于萌[1] 白耀富[1] 华进联[1] 

机构地区：[1]西北农林科技大学动物医学院陕西省干细胞工程技术研究中心农业部动物生物技术重点开放性实验室,陕西杨凌712100 [2]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《中国兽医学报》2013年第2期236-240,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30972097);教育部重点科研资助项目(109148);教育部优秀人才支持计划资助项目(NCET-09-0654);中国博士后特别科学基金资助项目(200801438)

摘　　要：根据NCBI数据库上公布的小鼠Dazl基因的mRNA序列设计引物,以小鼠睾丸组织RNA为模板,RT-PCR扩增小鼠Dazl基因编码区片段,并将其克隆到增强型绿色荧光蛋白表达载体pEGFP-C1中,构建重组融合蛋白表达载体pEGFP-C1-Dazl,单双酶切和测序验证正确。将pEGFP-C1-Dazl质粒转染293和NIH-3T3细胞,荧光显微镜下观察到融合表达的绿色荧光蛋白,且呈胞质表达;对照组转染pEGFP-C1,绿色荧光遍布整个细胞。Dazl蛋白的免疫荧光试验也证明重组载体转染后,Dazl基因和GFP共同定位于胞质部分。pEGFP-C1-Dazl融合蛋白表达载体的成功构建为进一步研究生殖特异性基因Dazl在小鼠和大型动物的表达特性奠定了一定的基础。To obtain mouse Dazl coding sequence （CDS）, primers were designed according to the mRNA sequence published in NCBI database. With mouse testis RNA as template,Dazl CDS was amplified by RT-PCR and cloned into pEGFP-C1 vector. Proved by enzyme digestion and sequencing,the recombinant plasmid was constructed,named pEGFP-C1-Dazl. The vector with pEGFP- C1 was then transfected into 293 and NIH-3T3 cells,analysed by real-time PCR and immunofluo- rescence staining. The results showed that Dazl-GFP fusion protein was expressed predominantly in cytoplasm while GFP was alone located in cells. The reconstruction of pEGFP-C1-Dazl could help to fur- ther understand the function of germ cell-specific gene Dazl in mice and livestock.

关 键 词：小鼠 生殖细胞 Dazl基因 绿色荧光蛋白 

分 类 号：S814.8[农业科学—畜牧学] Q78[农业科学—畜牧兽医]