小鼠Akt基因真核表达载体的构建及鉴定

作者:蒲静;俞晓丽;马会明;李昕;王蒙蒙; 刊名:宁夏医学杂志 上传者:薛红文

【摘要】目的构建含有小鼠Akt基因以及绿色荧光蛋白报告基因的真核表达载体pDoubleEx-EGFPAkt1。方法以小鼠卵巢RNA为模板,通过RT-PCR扩增Akt,并将其克隆到pDoubleEx-EGFP载体中。通过酶切和测序鉴定,构建真核表达载体pDoubleEx-EGFP-Akt1。之后转染人卵巢癌上皮细胞SKOV-3。结果经测序鉴定,构建的Akt基因序列与野生型Akt序列完全相符。将pDoubleEx-EGFP-Akt1转染进入人卵巢癌上皮细胞SKOV-3,观察到稳定转染后的细胞有明显绿色荧光蛋白表达。结论成功构建含有小鼠Akt基因的真核表达载体pDoubleEx-EGFP-Akt1,为进一步研究Akt基因的功能提供初步的实验基础。

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Doi:10. 13621/j. 1001 -5949. 2018. 07. 0577 ·论 著· 小鼠 Akt 基因真核表达载体的构建及鉴定 蒲 静,俞晓丽,马会明,李 昕,王蒙蒙 [摘要] 目的 构建含有小鼠 Akt 基因以及绿色荧光蛋白报告基因的真核表达载体 pDoubleEx - EGFP -Akt1。方法 以小鼠卵巢 RNA 为模板,通过 RT - PCR 扩增 Akt,并将其克隆到 pDoubleEx - EGFP 载体中。通过酶切和测序鉴定,构建真核表达载体 pDoubleEx - EGFP - Akt1。之后转染人卵巢癌上皮细胞 SKOV -3。结果 经测序鉴定,构建的 Akt 基因序列与野生型 Akt 序列完全相符。将 pDoubleEx - EGFP - Akt1 转染进入人卵巢癌上皮细胞 SKOV -3,观察到稳定转染后的细胞有明显绿色荧光蛋白表达。结论 成功构建含有小鼠 Akt 基因的真核表达载体 pDoubleEx - EGFP - Akt1,为进一步研究 Akt 基因的功能提供初步的实验基础。 [关键词] 真核表达载体; 构建; 转染 [中图分类号] R575 [文献标识码] A Construction and identification of eukaryotic expression vector of Akt gene in mouse PU Jing,YU Xiaoli,MA Huiming,LI Xin,WANG Mengmeng. Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education,Ningxia Medical University,Yinchuan 750004,China [Abstract] Objective To construct an eukaryotic expression vector pDoubleEx - EGFP - Akt1 containing mouse Akt gene and green fluorescent protein reporter gene. Methods Using RNA of mouse ovary as template,Akt cDNA was amplified by RT - PCR and cloned into vector pDoubleEx - EGFP. It was confirmed by enzyme digestion and sequencing before making recombinant plasmid pDou-bleEx - EGFP - Akt. The correct recombinant plasmid was transfected into SKOV - 3 cells. Results Akt gene sequence contained in pDoubleEx - EGFP - Akt1 recombinant plasmid was verified by enzyme digestion as well as sequencing results. After being transfected into SKOV -3 cells,it showed stable and highly transfection with green fluorescent prote

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