摘要
目的构建在哺乳动物细胞中表达的septin 8红色荧光蛋白融合表达载体(pmCherry-C2/septin 8),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cDNA文库中扩增获得septin 8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Western blotting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/septin 8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/septin 8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septin 8细胞内生物学功能打下了良好的基础。
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.
出处
《热带医学杂志》
CAS
2010年第2期120-122,136,F0002,共5页
Journal of Tropical Medicine
基金
长江学者和创新团队发展计划项目(No.IRT0731)
国家自然科学基金委员会-广东省人民政府自然科学联合基金重点项目(No.U0632004)
国家自然科学基金(No.30670828
30572151)
