小鼠FasL基因真核表达载体的构建及在HEK293细胞中的表达

Construction of the eukaryotic expression vector of mouse FasL gene and its expression in HEK293 cells

目的构建含有小鼠Fas Ligand(FasL)基因的重组真核表达载体,并检测FasL蛋白在其稳定转染的HEK293细胞中的表达情况,为构建表达小鼠Fas L的树突状细胞(DC)模型,深入研究DC联合T细胞防治移植物抗宿主病(GVHD)的新方法奠定基础。方法从小鼠脾脏中提取RNA并逆转录为cDNA,以该cDNA为模板扩增FasL基因,插入pcDNA3.1(+)载体中构建pcDNA3.1(+)-FasL重组质粒,利用脂质体将其转染HEK293细胞,用G418筛选稳定表达细胞系,Western blot确定重组蛋白的表达。结果通过酶切及测序证实FasL序列正确插入表达载体pcDNA3.1(+),Western blot检测证实转染重组质粒的细胞能正确表达FasL蛋白,G418筛选后能够得到稳定表达FasL的抗性细胞株即FasL-HEK293。结论重组质粒pcDNA3.1(+)-FasL和稳定表达FasL的HEK 293细胞成功构建,为后续FasL-DC细胞的研究打下了坚实基础。

Objective To construct the eukaryotic expression vector containing Fas ligand （FasL） gene and detect the pro- tein express in its stable transfected HEK293 cell line, which lay the foundation for the establishment of stable dendritic ceil model expressing FasL, so as to further research the new method for prevention of graft versus host disease （GVHD） with dendritic cell and T cells. Methods RNA were extracted from mouse spleen and copied into cDNA. The coding sequence of FasL was amplified from the cDNA and cloned into pcDNA3.1 （＋） vector. The recombinant expression vector pcDNA3.1 （＋） -FasL was transfected into HEK293 cells by lipofectamine, and the stable cell strains were screened with G418. Then, the protein expression of FasL in the sta- ble cell line was detected by Western blot. Results Restriction endonuclease digestion and sequencing showed that the recombinant eukaryote expression vector containing FasL was constructed successfully. The result of Western blot confirmed the exact expression of FasL in the transfected HEK293 cells, the resistant cell strain which express FasL stably could be constructed after G418 screening. Conclusion The new recombinant expression vector pcDNA3.1 （＋）-FasL and the HEK 293 cells which could express FasL stably were constructed successfully, which lay the solid foundation for follow-up study of FasL-DC cell.