期刊论文

Expression of pprI and pprA genes from deinococcus radiodurans in eukaryotic 293T cells

中文

辐射研究与辐射工艺学报

1000-3436

2016

34

2

020206-1-020206-5

10.11889/j.1000-3436.2016.rrj.34.020206

国家自然基金项目（81272993）； 湖南省教育厅资助科研项目（13C805）； 湖南省科技厅重点项目（2015JC3080）； 湖南省研究生科研创新项目（CX2013B398）； 衡阳市科技局资助项目（2014KJ10）资助~~；

本校为第一机构

以pGADT-7-pprA、pet28a-pprI载体为模板,构建pEGFP-N1-pprA、 pDsRed1-N1-flag-pprI真核表达载体,脂质体2000介导将两个重组载体共转入293T细胞。双酶切及琼脂糖凝胶电泳显示在4 700、1 000 bp处与4 800、1 100 bp处出现目的条带。测序结果显示构建序列与模板序列一致,氨基酸序列100%正确。荧光显微镜下见到红色和绿色荧光;Western blot结果显示在不同检测水平65 kDa及60 kDa大小处有融合蛋白表达。结果提示pEGFP-N1-pprA、pDsRed1-N1-flag- pprI真核表达载体构建成功,并在离体293T细胞中共同表达蛋白。证明原核基因pprI、pprA能够在真核细胞中共表达,为后续实验验证DR菌高抗性基因pprA、pprI及...

To construct fluorescence expression plasmids pEGFP-N1-pprA and pDsRed1-N1-flag-pprI, pGADT-7-pprA and pet28a-pprI plasmid constructed at an earlier laboratory stage was used as the template, and Lipofectamine 2000 was employed to transfect two recombinant vectors into 293T cells. A fluorescent microscope was used for Fluorescence observation, and Western blot was employed to examine the expression of the fusion protein. Double digestions and the agarose gel electrophoresis showed that target bands appeared at 4 700 bp and 1 000 bp, 4 800 bp and 1 100 bp. No apparent frameshift mutation occurr...