摘要
目的:构建小鼠Ccr2基因真核表达载体,转染RAW264.7,建立稳定转染Ccr2的RAW264.7细胞系。方法:应用RT-PCR从小鼠脾脏组织中扩增Ccr2mRNA全序列,插入真核表达载体pEGFP-N1中。用脂质体将重组质粒pEGFP-N1/Ccr2转染RAW264.7细胞,通过G418筛选建立Ccr2稳定转染的RAW264.7细胞系。经RT-PCR、Western blot检测、荧光显微镜和流式细胞仪FCM检测,了解Ccr2在RAW264.7细胞中的表达水平及细胞内定位。结果:成功构建了pEGFP-N1/Ccr2重组质粒,建立了稳定转染的RAW264.7细胞株。RT-PCR和Western blot检测证实,Ccr2基因在该细胞系中成功表达,荧光显微镜下观察到该基因定位于细胞膜上,FCM显示90%以上的细胞膜上均有CCR2的表达。结论:构建了Ccr2真核表达载体并建立了稳定转染的RAW264.7细胞株。为进一步研究MCP1/CCR2信号通路在结核杆菌诱导巨噬细胞凋亡中的作用奠定了基础。
AIM： To construction of eukaryotic expression vector of murine Ccr2 and establishment of its stable transfected RAW264.7 cell line.METHODS： The whole coding region of murine Ccr2 mRNA was amplified by PCR and was inserted into a vector pEGFP-N1.The recombinant pEGFP-N1/Ccr2 was transfected into RAW264.7 cells by Lipofectamine 2000 reagent.The stable transfected RAW264.7 cells were screened by G418-media.The expression of Ccr2 on the membrane of the stable transfected cells was identified by RT-PCR,Western blot and flow cytometry.The fusion protein CCR2-EGFP was located by a converted fluorescence microscope.RESULTS： The recombinant plasmid pEGFP-N1/Ccr2 was successfully constructed and the stably transfected RAW264.7 cells was established.Both RT-PCR and Western blot revealed the higher expression of Ccr2 in the stably transfected RAW264.7 cells.Under the fluorescence microscope,the CCR2 was located on the membrane of RAW264.7 cells.CONCLUSION： The recombinant pEGFP-N1/Ccr2 has been constructed successfully and has been stably expressed in RAW264.7 cells.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2010年第12期1214-1216,1219,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30660158)
宁夏自然科学基金(NZ0781)
宁夏医科大学重点项目(XZ200902)
