大鼠MKK7真核表达质粒的构建及其在COS7细胞中的转染效率  

Construction of a eukaryotic expression plasmid containing rat MKK7 gene and the transfection efficiency in COS7 cells

作　　者：代春潇 张云山 赵小芳 王玉兰 

机构地区：[1]徐州医科大学解剖学教研室,江苏 徐州221004 [2]徐州医科大学遗传学教研室

出　　处：《徐州医科大学学报》2017年第4期211-215,共5页Journal of Xuzhou Medical University

基　　金：国家自然科学基金面上项目（81371300）;江苏省普通高校研究生科研创新计划项目（KYLXl5-1472）;江苏省普通高校研究生科研创新计划项目（KYLXl5-1473）

摘　　要：目的探索大鼠丝裂原活化蛋白激酶激酶7（mitogen activated protein kinase kinase7，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Pvpl6-AD中，构建出Pvpl6-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Pvpl6-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72h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 （MKKT） gene in vitro. Methods The full length of rat MKK7 cDNA was cloned into a eukaryotic expressive vector Pvpl6 - AD enco- ding enhanced green fluorescent protein （eGFP） and Flag label encoding DYKDDDDK polypeptide by using molecular biological methods to establish a recombinant Pvpl6 - Flag - MKK7 - eGFP plasmid. After enzymatic digestion and DNA sequencing, the resultant recombinant plasmid was transiently transfected into COS7 cells by liposome. Then, the expres- sion of rat MKK7 in COS7 cells was determined by fluorescence microscopy at various ratios of recombinant plasmid to li- posome and different time points. Results The eukaryotie expression plasmid of Pvp16 - Flag - MKK7 - eGFP was es- tablished 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 sta- tistical significance （ P 〈 0.05 ）. The protein expression level of MKK7 in COS7 cells became the highest after transfec- tion 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 cellu- lar differentiation, proliferation, apoptosis, inflammation, stress and other biological changes.

关 键 词：真核表达质粒 COS7 转染效率 脂质体 

分 类 号：Q343[生物学—遗传学]