小鼠survivin基因重组真核表达载体的构建及鉴定

目的：应用质粒pEGFP-N1构建含小鼠survivin基因的重组真核载体。方法：采用Oligo核酸软件对Genbank上所发表的小鼠survivin mRNA序列进行分析,自行设计一对分别含有HindⅢ、BamHⅠ酶切位点的survivin基因上下游引物,利用PCR扩增出该基因的全序列cDNA,并将其定向克隆入pEGFP-N1的多克隆位点,构建pEGFP-N1/survivin重组真核表达载体。然后通过卡那霉素抗性筛选、双酶切及PCR鉴定,选取鉴定正确的克隆测序。结果：双酶切与测序结果表明目的基因序列克隆正确,成功构建了含有小鼠survivin基因的重组真核表达载体。结论：重组真核表达载体pEGFP-N1/survivin构建成功,为下一步研究sur-vivin在未成熟树突状细胞中诱导分化与致耐受作用奠定基础。

Construction and Identification of the Recombinant Eucaryotic Plasmid Encoding Survivin Gene of Mice

Objective: To construct and identify the recombinant eukaryotic plasmid pEGFP-N1/survivin encoding the survivin gene of mice with the eukaryotic plasmid pEGFP-N1. Method: The survivin gene sequence of mice publicated on Genbank was analyzed by using Oligo nucleic acid software; Morever, a couple of primer containing Hind Ⅲ and BamH Ⅰ was designed. The full length survivin cDNA was obtained by PCR and cloned directedly into multiple cloning sites of pEGFP-N1, then the recombinant eucaryotic plasmid pEGFP-N1/survivin was constructed. The recombinant plasmid was selected for Kanamycin resistance and then confirmed by digestion with restriction endonucleases Hind Ⅲ and BamH Ⅰ, PKDCR and sequencing the perfect cloning colony. Result: The consequence of enzyme digestion analysis with Hind Ⅲ, BamH Ⅰ and DNA sequencing confirmed that the recombinant plasmid of pEGFP-N1/survivin was correctly constructed. Conclusion:The expression vector pEGFP-N1/survivin was constructed succesfully, which laid foundation for the further study on whether the survivin played an important role in inducing cell differentiation and causing immunological tolerance during the development process of immature dendritic cell.