The Preparation and Function Identification of a Novel Anti-Fumarate Hydratase Antibody

Abstract

Fumarate hydratase(FH) is the key enzyme to be involved in tricarboxylic acid cycle(TCA).Defects of fumarase have been found to predispose to an autosomal dominant tumor predisposition syndrome hereditary leiomyomatosis and renal cell carcinoma(HLRCC).The exact molecular mechanisms linking defective FH to HLRCC are not known.The authors amplified FH(400-510) gene fragment and inserted into pGEX-4T-2 vector for the construction of a recombinant plasmid pGEX-4T-2/FH(400-510),which was therefore transfected into E.coli BL21 cells.The FH fusion protein was expressed in E.coli BL21 cells and purified with GST-glutathione affinity column.The FH fusion protein was used as an antigen to immunize the BALB/c mouse.The authors characterized the newly developed polyclonal antibody by Western blot and found that FH had higher expression in liver and heart than in brain.In the rat liver,FH protein complex was investigated with immunoprecipitation.