小鼠Uncv基因克隆及真核表达

Cloning and eukaryotic expression of mouse Uncv gene

目的克隆小鼠的Uncv基因并在真核细胞表达。方法采用RT-PCR方法扩增小鼠皮肤组织中Uncv基因编码区,以真核表达质粒pcDNA 3.1-Flag为载体,构建Uncv真核表达质粒,将重组载体转染Hela细胞并用Western blot法检测基因表达。结果构建Uncv基因真核表达载体pcDNA 3.1-Flag/Uncv,重组质粒在Hela细胞中有效表达约95×103的融合蛋白。结论成功构建真核表达载体pcDNA 3.1-Flag/Uncv,并且在真核细胞中有效表达,为研究Uncv基因生物学功能奠定基础。

Objective To clone the full length cDNA of Uncv gene in mice and to express the gene in eukaryotic cells. Methods RT-PCR assay was applied to clone the full length coding region of the Uncv gene and constructed its expression plasmid pcDNA 3. 1-Flag/Uncv. The recombinant plasmid was transfected into HeLa cells and the fusion protein was identified by Western blot analysis. Results The complete coding sequence was obtained and cloned into the pcDNA 3.1-Flag vector. The recombinant pcDNA 3.1-Flag/Uncv plasmid was transiently expressed in HeLa cells. HeLa cell clones expressing fusion protein with molecular weight of about 95 x 103 were obtained. Conclusions A recombinant eukaryotic expression plasmid of Uncv has been successfully constructed and expressed in HeLa cells. It may provide a foundation for further biological studies of Uncv gene.