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:苏维词

【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.

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Doi:10.13621/j.1001—5949.2018.07.0577 ‘论小鼠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 ·577· 著· 【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— Aktl。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—Aktl。之后转染人卵巢癌上皮细胞SKOV一3。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—Aktl转染进入人卵巢癌上皮细胞SKOV一3,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—Aktl,为进一步研究hkt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如曙,YU Xiaoli,MA Huirning,U Xin,WANG Mengraeng.Key laboratory ofFertility Preservation and Maintenance ofMinistry ofEducation,lⅥngxia Medical Un/vers/ty,Yinchuan 750004,China [Abstract]Objective To construct an eukaryotie expression vector pDoubleEx—EGFP—Aktl 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一3 cells.Results Akt gene sequence contained in pDoubleEx—EGFP—Aktl recombinant plasmid was verified by enzyme digestion鹪well as sequencing rosdts.After being transfected iato SKOV一3 cells。it showed stable and highly transfection with green fluorescent protein expression.Conclusion pDoubleEx—EGFP —Akt recombinant plasmid has been constructed succ

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