[pcDNA3.0-RKIP重组质粒的构建及其在PC12细胞中的表达]
[Construction of the recombinant plasmid of pcDNA3.0-RKIP and expression in PC12 cells].
作者信息
Lin Tao, Zuo Hong-yan, Wang De-wen, Peng Rui-yun, Zhou Pei-lan, Wang Shui-ming, Gao Ya-bing, Wang Shao-xia
机构信息
Institute of Radiation Medicine, Academy of Military Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
出版信息
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2011 Apr;27(4):399-401, 404.
AIM
To construct the eukaryotic expression vectors of RKIP plasmid and detect its expression in PC12 cells.
METHODS
The coding sequence of RKIP was generated by nested-PCR using total RNA extracted from the root ganglion neurons of rats. RKIP gene was cloned into the eukaryotic expression vector pcDNA3.0. After restriction enzyme analysis and sequence identification, the recombinant plasmid was transfected into PC12 cells with non-liposome mediated method by Vigofect. The expression of RKIP was detected by Western blot.
RESULTS
The results of enzyme analysis and sequencing both identified DNA sequence of recombinant plasmid pcDNA3.0-RKIP correctly. The expression of RKIP increased obviously after transfection into PC12 cells.
CONCLUSION
The eukaryotic expression plasmid of pcDNA3.0-RKIP was constructed successfully and it can be sustainly expressed in PC12 cells. This provides experimental basis for further study on the neurological function of RKIP.
目的
构建RKIP质粒的真核表达载体并检测其在PC12细胞中的表达。
方法
采用巢式PCR从大鼠脊神经节神经元提取的总RNA中扩增出RKIP编码序列。将RKIP基因克隆至真核表达载体pcDNA3.0。经酶切分析及序列鉴定后，采用Vigofect非脂质体介导法将重组质粒转染至PC12细胞。通过蛋白质免疫印迹法检测RKIP的表达。
结果
酶切分析及测序结果均正确鉴定了重组质粒pcDNA3.0-RKIP的DNA序列。转染至PC12细胞后，RKIP的表达明显增加。
结论
成功构建了pcDNA3.0-RKIP真核表达质粒，且其可在PC12细胞中持续表达。这为进一步研究RKIP的神经功能提供了实验依据。
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