Construction and identification of an apoptosis detection system based on firefly luciferase reporter gene.

Luping Che; Yonghua LI; Bin Yang; Zhikai XU; Ying Liao; Xusheng Qiu; Lei Tan; Yingjie Sun; Cuiping Song; Chan Ding; Gang Yao; Jinquan Wang; Chunchun Meng

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Construction and identification of an apoptosis detection system based on firefly luciferase reporter gene.

Author: Luping CHE ¹ ; Yonghua LI ¹ ; Bin YANG ¹ ; Zhikai XU ¹ ; Ying LIAO ² ; Xusheng QIU ² ; Lei TAN ² ; Yingjie SUN ² ; Cuiping SONG ² ; Chan DING ² ; Gang YAO ¹ ; Jinquan WANG ¹ ; Chunchun MENG ²
Author Information

1. College of Veterinary Medicine, Xinjiang Agricultural University, Urumgi 830052, Xinjiang Uygur Autonomous Region, China.
2. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
Publication Type:Journal Article
Keywords: Caspase-3; apoptosis; luciferase
MeSH: Apoptosis; Genes, Reporter; Luciferases, Firefly; Transfection
From: Chinese Journal of Biotechnology 2019;35(8):1557-1565
CountryChina
Language:Chinese
Abstract: To construct a eukaryotic expression plasmid containing the luciferase reporter gene (Fluc) to quickly detect apoptosis. Four amino acids, Asp-Glu-Val-Asp (DEVD), the recognize motif of Caspase-3, were introduced into the middle of the Fluc-C and N fragment. Meanwhile, four amino acids, Asp-Glu-Val-Gly (DEVG), were selected as a negative control. Subsequently, the recombinant gene was cloned into the N and C terminal end of the split intein, and named as pFluc-DEVD and pFluc-DEVG. Then the plasmids were transfected into cells and renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency. Then the apoptosis level was detected by the double luciferase reporter gene and the Western blotting analysis. The results showed that when apoptosis occurred, the content of firefly luciferase expressed in the pFluc-DEVD plasmid transfected group was about 3 times higher than pFluc-DEVG plasmid transfected group. Furthermore, Western blotting detection indicated that the Fluc level was significantly increased in pFluc-DEVD transfected group when pre-treated by apoptosis stimulants. The activation degree of Caspase-3 was closely related to the expression of Fluc, and had a significant statistical difference. These results confirmed that firefly luciferase protein expressed by pFluc-DEVD plasmid can be cleaved by the intracellular Caspase-3 enzyme, and this plasmid can accurately reflect the cell apoptosis level, which provides a useful method for quantitative detection of apoptosis.