Citation
- Authors: Cao X. et al.
- Year: 2023
- Journal: Fish Shellfish Immunol 108548
- Applications: in vitro / DNA / jetOPTIMUS
- Cell type: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
Method
A recombinant eukaryotic plasmid with CpPrx5 ligated was constructed in pEGFP-C1 vector and CpMafK ligated was constructed in DsRed-N1 vector (Prx5-pEGFPC1-F, Prx5-pEGFPC1-R, Mafk-pDsRed-F, Mafk-pDsRed-R, Table 1.1). The localization of target protein was observed in HEK-293T cells. HEK- 293T cells were provided by KeyGEN bioTECH. The frozen HEK-293T was resuscitated from liquid nitrogen and placed in a sterile incubator at 37 °C and 5% CO2. The cell culture solution was prepared as follows: DMEM (GIBCO, USA) + 10% FBS (Excell Biology, Shanaghai, China) + 1% penicillin-streptomycin solution (KeyGEN BioTECH, Jiangsu, China). The cells were transfected after passaging. jetOPTIMUS® (Yeasen, Shanghai, China) was chosen as the transfection reagent. The nucleus was stained with DAPI (Sigma, USA) staining solution. Finally, a laser confocal microscope was used to observe the distribution of red and green fluorescence.
Abstract
Cristaria plicata is one of the more important freshwater pearl bivalves in China, which is susceptible to pathogen infection, and greatly impacts the ability of breeding pearls. Nrf2/ARE signaling pathway and its downstream target gene Prx5 have endogenous antioxidant functions to protect cells from oxidative damage. The full-length cDNA of Prx5 was cloned from C. Plicata, which was 1420 bp, encoding a total of 189 amino acids and had two conserved cysteine residues (Cys78 and Cys179). The amino acid sequence of CpPrx5 was highly similar to Prx5 of other species. Real-time fluorescence quantitative PCR showed that CpPrx5 was distributed in various tissues of mussels, and the highest expression was in hepatopancreas. The expression of CpPrx5 up-regulated in hepatopancreas and gills after LPS, PGN and Poly:I:C stimulation. The recombinant plasmid DE3-PGEX-4T-1-CpPrx5 was expressed in Escherichia coli BL21 and showed antioxidant activity. With the increase of CpPrx5 protein concentration, the superhelical form of DNA was protected. The expression of CpPrx5 was up-regulated after interference CpKeap1 and down-regulated after interference CpNrf2. Gel block assay showed that CpNrf2 and CpMafK proteins blocked CpPrx5 promoter. Subcellular localization showed that CpPrx5 was located in 293T nucleus and cytoplasm and CpMafK was located in 293T nucleus. GST-Pull down verified that CpMafK and CpPrx5 could bind in vitro. These results indicated that Prx5 had antioxidant function and could protects DNA from oxidative damage, and participated in transcriptional regulation by combining with the transcription factor MafK. In addition, MafK could combine with Nrf2 to regulate the downstream target gene Prx5.