中国医学装备2016，Vol.13Issue(8)：120-122,3.DOI:10.3969/J.ISSN.1672-8270.2016.08.038
构建原核表达质粒pGEX-4T-2-GFP并鉴定其在大肠杆菌中的表达
Construction of pGEX-4T-2-GFP prokaryotic expressing plasmid and its protein expression
摘要
Abstract
Objective:To construct prokaryotic expressing plasmid of GFP gene.Methods: The PCR product of GFP coding sequence, which was digested with EcoR I and BamH I restriction enzymes, was taped into the plasmid pGEX-4T-2. Then pGEX-4T-2-GFP was transformed into E.coli DH5α and plasmid DNA was extracted. The recombinant plasmid was sequenced and then the expression of GST-GFP fusion protein was induced in BL21. After that, the E.coli which expressed GST-GFP fusion protein was transferred into the dish of cultivating macrophages. Fluorescence microscope was utilized to observe the GFP expression and e.coli swallowed.Results: The recombinant plasmid was sequenced correctly. E. coli expressing GFP both in medium and swallowed by macrophages can be observed clearly with the fluorescence microscope.Conclusion: The recombinant prokaryotic expressing plasmid pGEX-4T-2-GFP was successfully constructed, which facilitated the research for phagocytic capacity of macrophage.关键词
绿色荧光蛋白/pGEX-4T-2载体/重组质粒/表达/荧光显微镜Key words
Green fluorescent protein/pGEX-4T-2 vector/Recombinant plasmid/Expression/Fluorescence microscope分类
医药卫生引用本文复制引用
陆琤,周晨辰,张鹏飞,张硌..构建原核表达质粒pGEX-4T-2-GFP并鉴定其在大肠杆菌中的表达[J].中国医学装备,2016,13(8):120-122,3.基金项目
国家自然科学基金(31400739)“PH结构域蛋白PLEKHQ1协调巨噬细胞迁移与激活的机制研究”；军事医学创新基金(2015CXJJ29)“PLEKHQ1调控细菌性脓毒败血症的功能和机制研究” （31400739）
