绵羊NYD-SP27基因真核表达载体的构建及表达

Construction and expression of ovine NYD-SP27 gene eukaryotic expression vector

为了构建绵羊NYD-SP27基因重组真核表达载体,进一步研究该基因的功能,试验根据GenBank中公布的绵羊NYD-SP27基因序列设计引物,克隆绵羊NYD-SP27基因,将其连接至真核表达载体pmcherry-n1中,在目的基因和红色荧光蛋白之间通过猪捷申病毒2A肽（P2A）连接以实现共表达,将重组质粒pmcherry-Flag-NYDSP-P2A转染至HEK-293FT细胞中,48 h后通过荧光显微镜可观察红色荧光,构建NYD-SP27真核表达载体成功。结果表明：重组质粒pmcherry-Flag-NYDSPP2A在HEK-293FT细胞中得以表达;P2A在NYD-SP27蛋白和红色荧光蛋白翻译过程中已起到自我剪切的作用。

To construct the recombinant eukaryotic expression vector and further study the function of ovine NYD-SP27 gene,the PCR primers were designed based on the sequence of ovine NYD-SP27 gene released in Gen Bank. Then the NYD-SP27 gene was cloned and inserted into eukaryotic expressing vector pmcherry-n1. To co-express red fluorescent protein gene and NYD-SP27 gene,both of them were linked by porcine teschovirus-1 2 A peptide（ P2 A）. Furthermore,the recombinant plasmid pmcherry-Flag-NYDSP-P2 A was transfected into HEK-293 FT cells. 48 hours later,red fluorescence was observed successfully by fluorescence microscopy,indicating that the eukaryotic expression vector of NYD-SP27 gene was constructed. The results showed that the recombinant plasmid pmcherry-Flag-NYDSP-P2 A can be expressed in HEK-293 FT cells,and P2 A has played the role of self splicing in the process of NYD-SP27 protein and red fluorescent protein translation. Above all,this study successfully obtained the eukaryotic expression vector of NYD-SP27 gene,which laid the foundation for further research on the gene function.