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Expression of
pprI andpprA genes from deinococcus radiodurans in eukaryotic 293T cells- Journal of Radiation Research and Radiation Processing Vol. 34, Issue 2, Pages: 20206(2016)
- 作者机构：
1.南华大学公共卫生学院放射医学教研室 衡阳 421001；
2.南华大学生物化学与分子生物学教研室 衡阳 421001
- 作者简介：
- 基金信息：
DOI：10.11889/j.1000-3436.2016.rrj.34.020206
CLC：
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Xiao XIAO, Yun MA, Fangzhu XIAO, et al. Expression of
pprI andpprA genes from deinococcus radiodurans in eukaryotic 293T cells. [J]. Journal of Radiation Research and Radiation Processing 34(2):20206(2016)Xiao XIAO, Yun MA, Fangzhu XIAO, et al. Expression of
pprI andpprA genes from deinococcus radiodurans in eukaryotic 293T cells. [J]. Journal of Radiation Research and Radiation Processing 34(2):20206(2016) DOI： 10.11889/j.1000-3436.2016.rrj.34.020206.
摘要
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,及其产物在辐射调控网络中的相互作用和协同作用、提高真核细胞的辐射抗性提供了研究基础。
Abstract
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 1000 bp, 4800bp and 1 100 bp. No apparent frameshift mutation occurred as shown in the sequencing results. The fluorescent microscopy showed red and green phosphors; the Western blot results indicated protein expression at 65 kDa and 60 kDa. pDsRed1-N1-flag-,pprI, and pEGFP-N1-,pprA, were successfully constructed to express proteins for eukaryotic cells 293T, in vitro,. The results indicated that prokaryotic genes ,pprA, and, pprI, could co-express proteins in eukaryotic cells successfully, and laid a foundation for the interaction and synergism of ,pprA,pprI, and its products in the regulation network of radiation verified by DR, enhancing the radiation resistance of eukaryotic cells.
关键词
耐辐射奇球菌pprIpprA真核细胞脂质体2000
Keywords
Deinococcus radioduranspprIpprAEukaryotic cellsLipofectamine 2000
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