Gene Cloning, Eukaryotic Expression Vector Construction of Chicken PRDX1 Gene and Its Effect on Differentiation of Preadipocytes

Purpose To clone the chicken peroxiredoxin 1 (PRDX1) gene and investigate its effect on the differentiation of chicken preadipocytes, laying the foundation for further analysis of PRDX1 function.

Methods RT-qPCR was used to detect the expression of PRDX1 in different tissues of AA white feathered broiler chickens; ELISA was used to detect the PRDX1 enzyme activity at 0 and 48 hours of adipogenic differentiation in an immunized chicken preadipocyte cell line (ICP1); PCR was used to clone the coding sequence of the chicken PRDX1 gene, and then, the eukaryotic expression vector was construct and transfect into ICP1, after 24 hours, ICP1 differentiation was induced by sodium oleate, and the accumulation of lipid droplets in ICP1 was detected by oil red O staining; RT-qPCR and Western-blot were used to detect the mRNA and protein expression levels of lipid related genes overexpressing PRDX1. The functional bioinformatics analysis of PRDX1 was performed.

Results The expression level of PRDX1 was the highest in ovaries of AA white feather broilers, and the lowest in perigastric fat. After ICP1 differentiation 48 hours, the enzyme activity of PRDX1 decreased extremely significantly. The pCMV-HA-PRDX1 eukaryotic expression vector was successfully constructed. Overexpression of PRDX1 decreased the accumulation of lipid droplets in ICP1 cells, and the expression levels of lipid-related genes C/EBPα, PPARγ, FABP4 and FAS showed a downward trend. PRDX1 gene encoded 199 amino acids, the relative molecular weight was 22.31 ku, PRDX1 could interact with TXN, SRXN1, SOD2 and other proteins, PRDX1 had high similarity among different species.

Conclusion The eukaryotic expression vector of PRDX1 is successfully cloned and constructed. Overexpression of PRDX1 can inhibit the differentiation of chicken preadipocytes.