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目的：构建小鼠热休克蛋白GP96（GP96）基因真核表达载体并在真核细胞293T中表达。方法用RT-PCR法从小鼠脾脏扩增出GP96的成熟肽基因片段，先克隆于pMD19-T载体，再亚克隆到真核表达载体pcDNA3.1中。以脂质体法将重组质粒转入293T细胞，免疫印迹法鉴定GP96的蛋白表达。结果限制性内切酶酶切和测序结果表明，扩增出GP96基因完全正确；免疫印迹法检测表明，转染的重组质粒能在293T细胞中高效表达。结论构建的pcDNA3.1-GP96重组载体在293T细胞系中高效表达，为深入研究GP96作为DNA疫苗在病毒性心肌炎中的作用及机制打下基础。“,”Objective To construct an eukaryotic vector expressing heat shock protein GP96 (GP96) gene and detect its expression in human embryonic kidney 293T cells. Methods The GP96 full length gene was amplified by RT-PCR using spleen cDNA from BALB/c mice. It was then cloned into the pMD19-T vector before subclone into an eukaryotic vector pcDNA3.1. The recombinant vectors were transfected into HEK 293T cells by lipofectamine 2000. The quantity of GP96 was determined by Western blotting. Results The resultant GP96 gene was correct according to restriction enzyme digestion and sequencing. The recombinant vector was highly expressed in 293T cells. Conclusion The high expression of GP96 protein in 293T cells provide experimental basis for investigating the role of GP96 in viral myocarditis as a DNA adjuvant and potential mechanism.