Objective: To construct the fusion gene eukaryotic expression vector pEGFP-N1-SUMO-3, and to study the expression and localization of GFP-SUMO-3 fusion protein in human prostate cancer cell line LNCaP. Methods: The encoding region of SUMO-3 was obtained by PCR, and cloned into eukaryotic expression vector pEGFP-N1. Then the pEGFP-N1-SUMO-3 plasmid was transfected into LNCaP cells by liposome. The expression of the fusion protein was detected by Western blotting, and the subcellular localization was observed by fluorescence microscope. Results: The recombinant plasmid pEGFP-N1-SUMO-3 was identified by BamH I and Xho I double digestion. As expected, it showed two bands of 4 700 and 312 bp. The recombinant plasmid also showed right sequence by the full length sequencing. The pEGFP-N1-SUMO-3 plasmid was transfected into prostate cancer LNCaP cells. A specific protein expression band at molecular weight of 39 000 was detected with using GFP-antibody by Western blotting method. When observed by fluorescence microscope, the GFP-SUMO-3 fusion protein was mainly located in the nucleus of LNCaP cells. Conclusion: The recombinant plasmid pEGFP-N1-SUMO-3 is successfully constructed, and GFP-SUMO-3 fusion protein is successfully expressed in mammalian cells and mainly located in the nucleus.
|ジャーナル||Journal of Jilin University Medicine Edition|
|出版ステータス||Published - 2011 11 28|
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