中文摘要：

为构建鼠类朊蛋白Shadoo基因重组真核表达质粒，并分析类朊蛋白Shadoo在鼠神经瘤细胞（Neuro-2a）内表达及定位。采用PCR方法把Flag标签序列插入到Shadoo阅读框122与123氨基酸位点之间，构建Shadoo—Flag融合基因片段，把Shadoo—Flag片段插入到真核表达质粒pcDNA3．1，构建重组真核表达pcDNA3．1-Shadoo—Flag，质粒酶切、测序鉴定正确后，脂质体法转入Neuro-2a细胞，检测Shadoo基因表达及表达部位。结果重组真核表达质粒pcDNA3．1-Shadoo—Flag转入Neuro-2a细胞，能够正确表达出相对分子质量为12000的Shadoo蛋白，并定位于细胞膜。

英文摘要：

To construct the recombinant eukaryotic expression plasmid pcDNA3. 1-Shadoo-Flag, Shadoo ORF with Flag epitope tag （8 aa） inserted between 122-123 of ORF by PCR method, pcD-NA3.1-Shadoo-Flag was cloned with Shadoo-Flag through HindⅢ and BamH Ⅰ . pcDNA3.1-Sha-doo-Flag was verified by double enzyme digestion and sequencing. Shadoo expression level and lo- cation in Neuro-2a were investigated after the constructed eukaryotic expression plamid was trans- fected to Neuro-2a cell line by lipofectamin method. The constructed eukaryotic expression plamid was transfected to Neuro-2α cell line by lipofectamin method,and fused protein in eukaryotic cell was detected by immunocytochemical staining as well as Western blot. Recombinant eukaryotic ex-pression plasmid pcDNA3. 1-Shadoo-Flag was successfully contructed. Shadoo fusion protein was expressed in Neuro-2α cell line and Shadoo is located on the membrane of Neuro-2α cell. It estab-lishs the foundation for future research on Shadoo gene function in TSE.