文档介绍：hGITRaa1165IgG1Fc真核表达质粒的构建及其表达
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作者:崔大伟王胜军陈君仝佳陈建国,王海生杨先知史烨许化溪邵启祥
【摘要】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(+),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(+)质粒,构建hGITRaa1165IgG1Fc(+)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hGITRaa1165IgG1Fc(+),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。
【关键词】糖皮质激素诱导的肿瘤坏死因子受体; COS
7细胞; 真核表达
[Abstract] Objective: To constructed binant eukaryotic plasmid hGITRaa1165IgG1Fc(+), then to express the fusion protein hGITRaa1165IgG1Fc in COS7 : The hGITRaa1165 gene was obtained by PCR amplification from the pGEMTGITR vector and inserted into the vector hIgG1Fc(+). The COS7 cells were transfected in the mediation of liposome mixed with hGITRaa1165IgG1Fc(+)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(+) vector. Conclusion: The fusion protein hGITRaa1165IgG1Fc was essfully expressed in COS7 cells, providing research foundation for biology of hGITR.
[Key words] glucocorticoidinduced tumor necrosis factor receptor(GITR); COS7 cells; eukaryotic expression
人类糖皮质激素诱导的肿瘤坏死因子受体(human glucocorticoid
induced tumor necrosis factor receptor,hGITR),也被称为TNFRSF18。天然CD4+CD25+Treg细胞组成性高表达GITR,而静止T细胞低水平表达,其膜表达量会随着T细胞的激活而增加[1,2]。GITR与其配体(GITRL)的相互作用与自身免疫疾病、慢性感染、抗肿瘤免疫等相关。GITR与GITRL结合后,可呈现增强效应T细胞的活化和取消Treg细胞的抑制功能的特点[3]。因此,关于GITR与GITRL相互作用的机制及其在肿瘤免疫,机体炎症及某些自身免疫疾病中的作用有待于更深入的研究。本研究成功克隆人GITR胞外区基因,构建GITRaa1165IgG1Fc融合蛋白的真核表达载体,并在COS7细胞中获得功能性表达,为进一步研究GITR的生物学功能奠定了良好的基础。
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