Development of a polyclonal antibody against the protein encoded by the metabolic syndrome-associated gene for...

The metabolic syndrome-associated gene (MSAG) encodes a polypeptide of MSAG composed of 110 amino acids and was observed to responds to high glucose conditions. However, scant studies have been documented since it was first reported in 2009. The function of MSAG and its underlying mechanisms remain unclear. To explore them, antibodies against MSAG are needed. Therefore, the current study aimed to generate a polyclonal antibody against MSAG and assess its applications. The pET-28a-mMSAG vector was constructed for prokaryotic expression. The recombinant MSAG was obtained and identified with a coverage of 100 %. Subsequently, a polyclonal antibody against MSAG was prepared by rabbit immunization with an antibody titer exceeding 1:128,000 as determined by ELISA. The prepared antibody was validated using MSAG knockdown mouse and HepG2 cells. The mRNA and protein levels of MSAG were lower in MSAG knockdown mouse livers and HepG2 cells. Additionally, MSAG mRNA and protein levels in mouse hepatic tissues were significantly increased following the intervention with a high carbohydrate diet as evaluated by RT-qPCR and western blotting using the prepared antibody. This reproducibly demonstrated that MSAG was involved in the regulation of glucose metabolism. Taken together, the rabbit anti-MSAG polyclonal antibody has been successfully generated in the current study, which lays the foundation for future studies to explore the functions of MSAG and its underlying mechanisms.