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延胡索酸水合酶(fumarate hydratase,FH)是三羧酸循环(tricarboxylic acid cycle,TCA)中的一个关键性酶。FH的基因突变将导致延胡索酸合酶缺乏症、遗传性平滑肌瘤和肾细胞癌(hereditary leiomyomatosis and renal cell carcinoma,HLRCC)等疾病。FH缺失导致HLRCC分子的确切生物学机制尚不明确。作者将FH基因片段克隆到原核表达载体上,在大肠杆菌中表达GST-FH(400-510)融合蛋白。该融合蛋白为抗原免疫小鼠获得抗体,通过免疫印迹实验证明了该抗体的特异性并发现FH在心脏和肝脏中的含量较高。通过免疫共沉淀实验发现,在大鼠肝脏组织中FH可能与多个蛋白形成蛋白复合物。
Fumarate hydratase (FH) is a key enzyme in tricarboxylic acid cycle (TCA). Mutations in FH result in diseases such as fumarate synthase deficiency, hereditary leiomyomatosis and renal cell carcinoma (HLRCC). The exact biological mechanism that led to the deletion of FH molecules in HLRCC remains unclear. The authors cloned FH gene fragment into prokaryotic expression vector and expressed GST-FH (400-510) fusion protein in E. coli. The fusion protein was used to immunize mice for antigen to obtain antibodies. The specificity of the antibody was confirmed by immunoblotting and the content of FH in heart and liver was higher. Co-immunoprecipitation experiments found that FH in rat liver tissue may form protein complexes with multiple proteins.