人p52Shc1蛋白的真核表达及其活性鉴定

Eukaryotic expression and identification of human p52Shc1

[目的]构建带his标签的人p52Shc1基因真核表达载体,并根据其生物学活性进行鉴定。[方法]采用PCR技术以Shc1 cDNA基因为模板扩增Shc1基因,将其插入pEnter载体构建pEnter-p52shc1重组质粒,将重组质粒与空载体分别转入293T细胞,利用Western Blot检测p52Shc1表达情况,并使用cck-8法绘制细胞生长曲线。[结果]通过双酶切获得2条与目的基因大小相符的片段,测序结果显示符合率为100%,Western Blot结果显示在52 kDa大小处出现清晰单一条带,cck-8法绘制的细胞生长曲线显示,转染重组质粒pEnter-p52Shc1的细胞生长速率明显大于转染pEnter空载的细胞(P<0.01)。[结论]成功构建带his标签的人p52Shc1基因真核表达载体并在293T细胞中成功表达,转染重组质粒并表达p52Shc1蛋白的293T细胞的增值速率明显提高。

[Objective]To construct the eukaryotic expression vector of human p52Shc1 gene labeled with his tag and to identify it according to its biological activity. [Methods]Human p52Shc1 coding region was amplified from human p52Shc1 cDNA by PCR and cloned into pEnter vector to construct a recombinant plasmid of pEnter-p52Shc1. The recombinant plasmid pEnter-p52Shc1 and pEnter were transfected into 293 T cells respectively by the mediation of Jet reagent and the expressions were detected by Western Blot. Then the cell growth curve was drawn by cck-8 assay. [Results]Two fragments consistent with the size of the target gene were obtained by double enzyme digestion. Sequencing showed that the sequence coincidence rate was100%. Western Blot results showed that a single band was found at 52 kDa,and the cell growth curve of cck-8 showed that the growth rate of transfected plasmid pEnter-p52Shc1 was significantly faster than the control cell（ P 〈0. 01）. [Conclusion]The eukaryotic expression vector of his-p52Shc1 was successfully constructedand expressed in 293 T cells. The cell proliferation rate of 293 T cells transfected with pEnter-p52Shc1 was significantly increased.