Septin8红色荧光蛋白融合载体的构建及表达

目的构建在哺乳动物细胞中表达的septin8红色荧光蛋白融合表达载体（pmCherry—C2／septin8），并观察其在细胞内的表达和定位情况。方法采用PCR的方法从人脑胶质细胞瘤eDNA文库中扩增获得septin8基因编码区序列，将其克隆至红色荧光蛋白载体pmCherry—C2上，重组质粒经PCR、酶切以及序列分析正确无误后转染NIH3T3细胞，并利用Westernblotting和细胞免疫化学技术分析重组蛋白在细胞内的表达和定位。结果重组pmCherry—C2／septin8融合蛋白在NIH3T3细胞中得到高量表达且主要分布于细胞浆。结论成功构建了pmCherry—C2／septin8融合表达载体，该载体能在哺乳动物细胞NIH3T3中表达，为进一步研究septin8细胞内生物学功能打下了良好的基础。

Construction and Expression of Setpin 8-red Fluorescent Fusion Protein Vector

Objective To construct eukaryotic cell expression plasmid of pmCherry-C2 fusion protein and analysis its expression and intracellular localization in NIH3T3 cell. Methods Septin 8 coding region was amplified by PCR from human glioma cells cDNA library and subcloned into pmCherry-C2 vector. After comfirmed by PCR, enzyme digestion and DNA sequencing,the construct was then transfected into NIH3T3 cells and analyzed by fluorescence microscopy. Results pmCherry-C2/septin 8 fusion protein was highly expressed in NIH3T3 cell. Fluorescence microscope analysis revealed that the fusion protein is mainly distributed in the cytoplasm. Conclusion The pmCherry-C2/septin 8 fusion protein plasmid has been successfully constructed and expressed in NIH3T3 cell, which laid the foundation for the functional analysis of septin 8 in the future.