hGITR_(aa1-165)-IgG1Fc真核表达质粒的构建及其表达

Vector construction and eukaryotic expression of the human GITR_(aa1-165)-IgG1Fc

目的:构建含有人糖皮质激素诱导的肿瘤坏死因子受体(GITR)胞外段基因的真核表达质粒hGITRaa1-165-IgG1Fc-pcDNA3.1(+),检测hGITRaa1-165-IgG1Fc融合蛋白在真核细胞COS-7中的表达。方法:以pGEMT-GITR质粒为模板,通过PCR扩增获得人GITR胞外段的基因hGITRaa1-165,将该目的基因连接至IgG1Fc-pcDNA3.1(+)质粒,构建hGITRaa1-165-IgG1Fc-pcDNA3.1(+)真核表达质粒。将该重组质粒采用脂质体法转染COS-7细胞,通过SDS-PAGE和蛋白质印迹法检测hGITRaa1-165-IgG1Fc融合蛋白在真核细胞COS-7中的表达。结果:成功构建真核表达质粒hGI-TRaa1-165-IgG1Fc-pcDNA3.1(+),并在其转染的COS-7细胞培养上清中检测到hGITRaa1-165-IgG1Fc融合蛋白的表达。结论:成功表达hGITRaa1-165-IgG1Fc融合蛋白,为进一步研究GITR的生物学功能奠定了基础。

Objective： To constructed recombinant eukaryotic plasmid hGITRaa1-165-IgG1Fc-pcDNA3.1（＋）,then to express the fusion protein hGITRaa1-165-IgG1Fc in COS-7 cells.Methods： The hGITRaa1-165 gene was obtained by PCR amplification from the pGEMT-GITR vector and inserted into the vector hIgG1Fc-pcDNA3.1（＋）.The COS-7 cells were transfected in the mediation of liposome mixed with hGITRaa1-165-IgG1Fc-pcDNA3.1（＋）vector,the fusion protein hGITRaa1-165-IgG1Fc in the culture supernatant was analyzed by SDS-PAGE and Western blots. Results： The hGITRaa1-165-IgG1Fc fusion protein was analyzed in the culture supernatant in the COS-7 cells which were transfected with the hGITRaa1-165-IgG1Fc-pcDNA3.1（＋） vector. Conclusion： The fusion protein hGITRaa1-165-IgG1Fc was successfully expressed in COS-7 cells,providing research foundation for biology of hGITR.