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

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.