Prokaryotic expression and polyclonal antibody preparation of TRaA in Japanese flounder Paralichthys olivaceus

Abstract:

To study the role of the thyroid hormone receptor TRaA involved in the process of the metamorphic development of Japanese flounder, we firstly cloned the TRαA gene, then ligated into the fusion expression vector pET30a and expressed in Escherichia coli DE3 (BL21) host cells. After induced for 4 h with 1 mmol/L Isopropyl b-D-Thiogalactoside, the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. The recombinant protein was denatured and purified by His-Bind resin, then renatured through gradient washing on His-bind resin column. After that, polyclonal antibody was prepared by immunizing New Zealand rabbits with purified protein. Dot blotting analysis showed the antibody with the titer of 1:200 000 reacted specifically to the expressed recombinant protein. Furthermore, a chromatin immunoprecipitation assay was performed to identify the specific binding between the antibody and TRαA in living cells of Japanese flounder. The result showed that thyroid hormone was involved in the alkaline phosphatase (ALP) gene transcriptional regulation through TRaA in vivo.