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目的 探索大鼠丝裂原活化蛋白激酶激酶7(mitogen activated protein kinase kinase 7,MKK7)基因在体外细胞实验中的表达调控规律.方法 利用分子生物学方法将大鼠MKK7全长cDNA序列克隆到编码增强型绿色荧光蛋白(enhanced green fluorescent protein, eGFP)和Flag(编码DYKDDDDK亲水性多肽)标签的真核表达载体Pvp16-AD中,构建出Pvp16-Flag-MKK7-eGFP 重组质粒.通过酶切及DNA测序的方法验证重组质粒序列的正确性后,采用脂质体介导方法将重组质粒瞬时转染COS7细胞,通过荧光显微镜观察质粒与脂质体不同比例情况下以及确定最高转染效率的比例后不同时间段大鼠MKK7基因表达情况.结果 成功构建重组真核表达质粒Pvp16-Flag-MKK7-eGFP.酶切及DNA测序方法确定重组质粒中大鼠MKK7序列与原序列一致.重组质粒与脂质体的比例为1∶3时,重组质粒在COS7细胞中的转染效率最高,差异具有统计学意义(P<0.05).质粒转染后72 h时,COS7细胞中MKK7蛋白表达量相对最高,差异具有统计学意义(P<0.05).结论 大鼠MKK7重组真核表达载体的构建及其在COS7细胞中转染效率的探索工作为研究大鼠MKK7基因在体外引起细胞分化、增殖、凋亡、炎症反应和应激反应等一系列生物学改变奠定基础.“,”Objective To investigate the expression of rat mitogen activated protein kinase kinase 7 (MKK7) gene in vitro.Methods The full length of rat MKK7 cDNA was cloned into a eukaryotic expressive vector Pvp16-AD encoding enhanced green fluorescent protein (eGFP) and Flag label encoding DYKDDDDK polypeptide by using molecular biological methods to establish a recombinant Pvp16-Flag-MKK7-eGFP plasmid.After enzymatic digestion and DNA sequencing, the resultant recombinant plasmid was transiently transfected into COS7 cells by liposome.Then, the expression of rat MKK7 in COS7 cells was determined by fluorescence microscopy at various ratios of recombinant plasmid to liposome and different time points.Results The eukaryotic expression plasmid of Pvp16-Flag-MKK7-eGFP was established successfully.The sequence of rat MKK7 in the recombinant plasmid was consistent with its original sequence.The expression of MMK7 in COS7 cells reached the peak at the ratio of recombinant plasmid to liposome of 1∶3, with statistical significance (P<0.05).The protein expression level of MKK7 in COS7 cells became the highest after transfection for 72 h, with statistical significance (P<0.05).Conclusions The established eukaryotic recombinant vector of rat MKK7 and related transfection results provide experimental foundation for investigating the impacts of MKK7 in cellular differentiation, proliferation, apoptosis, inflammation, stress and other biological changes.