人p52Shc1蛋白的真核表达及其活性鉴定

发布时间：2018-10-08 08:27

【摘要】：[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(P0.01)。[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。
[Abstract]:[objective] to construct the eukaryotic expression vector of human p52Shc1 gene with his tag and to identify its biological activity. [methods] the Shc1 gene was amplified by PCR and inserted into pEnter vector to construct pEnter-p52shc1 recombinant plasmid. The recombinant plasmid and empty vector were transferred into 293T cells respectively. P52Shc1 expression was detected by Western Blot. Cck-8 method was used to draw cell growth curve. [results] two fragments were obtained by double enzyme digestion. The result of sequencing showed that the coincidence rate was 100%. The results of Western Blot showed that there was a clear cell growth curve drawn by single band cck-8 method at the size of 52 kDa. The growth rate of cells transfected with recombinant plasmid pEnter-p52Shc1 was significantly higher than that without pEnter transfection (P0.01). [conclusion] the eukaryotic expression vector of human p52Shc1 gene with his tag was successfully constructed and expressed in 293T cells. The proliferation rate of 293T cells transfected with recombinant plasmid and expressed p52Shc1 protein was significantly increased.
【作者单位】：南方医科大学检验与生物技术学院抗体工程研究所;
【基金】：国家自然科学基金面上项目(“羊口疮病毒ORFV118蛋白调控宿主细胞凋亡的分子机制研究”,No.31672536) 广州市科技计划项目(“产前快速诊断自动化设备及其创新型试剂的开发”,No.201604010046;“系列全自动管式化学发光免疫检测试剂盒及配套设备的产业化”,No.201604040003)
【分类号】：R3416

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