Construction of a Stable Expression Cell Line of Human Phospholamban

doi:10.12116/j.issn.1004-5619.2020.400909

Abstract

Abstract: Objective

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-In^TM T-REx^TM 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.

Conclusion

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.

Key words: forensic pathology, forensic toxicology, phospholamban, human-derived cell line, aconitine, biochemistry, molecular biology

CLC Number:

DF795.1

Lan-qing CHEN, Yu-ning WANG, Dian-yi LI, Lu-yao XU, Lian-jie LI, Ze-hao LI, Hua LIU, Qian LIU. Construction of a Stable Expression Cell Line of Human Phospholamban[J]. Journal of Forensic Medicine, 2021, 37(5): 615-620.

Figures/Tables 5

Fig. 1 Gene sequencing results in successfully transfected cells

Fig. 2 Indirect immunofluorescence detection of PLN expression in hPLN-293 cells

	FITC	DAPI	FITC与DAPI重叠
阴性对照组
未加Dox诱导组
加入Dox诱导组

Fig. 3 PLN expression in hPLN-293 cells detected by Western blot

Tab. 1 Effects of Dox induction on the relative expression of PLN in hPLN-293 cells

组别	第1代细胞		第5代细胞		第10代细胞
组别	n	PLN相对表达量	n	PLN相对表达量	n	PLN相对表达量
未加Dox诱导	2	0.24	3	0.29±0.23	3	0.21±0.09
加入Dox诱导	2	1.24	3	1.66±0.15^1）	3	1.19±0.22^1）

Fig. 4 Effects of aconitine exposure on the phosphorylation level of Thr17 site of PLN in hPLN-293 cells detected by Western blot

	无毒组	染毒组
Thr17-P-PLN
PLN
GAPDH

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