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Published on: March 5, 2013
Construction of a Stable Expression Cell Line of Human Phospholamban
Lan-Qing Chen1,2, Yu-Ning Wang1, Dian-Yi Li1
1Department of Forensic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Objectives:
To construct a cell line that can stably express human phospholamban(PLN) and initially explore its application in the study of myocardial toxicity mechanism.
Methods:
FastCloning method was used to insert the open reading frame sequence of target gene PLN into eukaryotic expression vector pcDNA5/FRT/TO(hereinafter referred to as pDFT) to construct the pDFT-PLN-Flag plasmid. The Flp-InTM T-RExTM 293 cells were generated by cotransfection of the constructed plasmid and pOG44 plasmid to express the target gene. Successfully recombined monoclonal cell lines were screened by hygromycin B resistance. Western blot and indirect immunofluorescence (IFA) were used to examine the expression of the target protein in recombinant cells. After the cell line was exposed to aconitine, it was verified by Western blot to detect changes in PLN protein phosphorylation.
Results:
After PCR amplification of the recombinant plasmid and DNA electrophoresis, the length of the amplified product is the same as the known PLN gene fragment, which is consistent with the open reading frame (ORF) sequence of the human PLN gene after sequencing. IFA and Western blot showed that the constructed proliferation cell line can stably express high levels of human PLN under induction and regulation. Preliminary results showed that the phosphorylation level of Thr17-PLN decreased after two hours of exposure to 1 μmol/L aconitine.
Conclusions:
This human cell line can stably express PLN and can be used to study the mechanism of action of aconitine on the cell at molecular level.
