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弗里德赖希共济失调(Friedreich ataxia,FRDA)是一种常染色体隐性遗传性疾病,由frataxin(FXN)第一个内含子中GAA重复扩增导致其表达量减少所致。FXN是一种线粒体蛋白,可以调节细胞中铁的代谢,参与铁硫簇和血红素的合成,去除氧化应激等。前期研究发现FRDA病人受累组织有特异性表达的FXN亚型蛋白。为了检测小鼠不同组织中Fxn亚型蛋白的表达,首先要获得具有高度特异性和灵敏性的小鼠Fxn抗体。利用PCR技术扩增小鼠Fxn基因,通过酶切、连接、转化等常规分子克隆方法构建重组原核表达质粒pET24(+)-mFxn,经转化大肠杆菌BL21(DE3),表达可溶性含有his6标记的融合蛋白。该蛋白不含Fxn氨基端77个氨基酸的信号肽,含有130个氨基酸,理论分子量为14.38 kDa。表达的蛋白经Ni-NTA柱和连续梯度离心纯化,获得目的蛋白作为抗原;免疫新西兰大白兔制备抗血清并用硫酸铵沉淀初步纯化得到多克隆抗体。经Western blotting和免疫荧光分析测试,所获得的抗体能够特异性识别细胞内源Fxn,也可应用于组织的免疫沉淀和免疫荧光。这是首次报道利用鼠源Fxn作为免疫原制备的具有高度特异性和灵敏性的Fxn抗体,为深入研究小鼠frataxin亚型蛋白的存在和功能奠定了基础。
Friedreich ataxia (FRDA) is an autosomal recessive inherited disease caused by repeated GAA amplification in the first intron of frataxin (FXN) resulting in a decrease in its expression. FXN is a mitochondrial protein that regulates iron metabolism in cells, participates in the synthesis of iron-sulfur clusters and heme, and removes oxidative stress. Previous studies found that FRDA patients specifically affected the expression of FXN subtype protein. In order to detect the expression of Fxn subtypes in different tissues of mice, it is first necessary to obtain mouse Fxn antibodies with high specificity and sensitivity. The Fxn gene was amplified by PCR. The recombinant prokaryotic expression plasmid pET24 (+) - mFxn was constructed by restriction enzyme digestion, ligation and transformation. The fusion protein was transformed into E. coli BL21 (DE3) protein. This protein contains no signal peptide of 77 amino acids at the amino terminus of Fxn and contains 130 amino acids with a theoretical molecular mass of 14.38 kDa. The expressed protein was purified by Ni-NTA column and continuous gradient centrifugation, and the target protein was obtained as antigen. Immunized New Zealand white rabbits were used to prepare antiserum and polyclonal antibody was purified by ammonium sulfate precipitation. After Western blotting and immunofluorescence analysis, the obtained antibodies can specifically recognize endogenous Fxn, and can also be applied to tissue immunoprecipitation and immunofluorescence. This is the first report of a highly specific and sensitive Fxn antibody prepared by using murine Fxn as an immunogen, which lays a foundation for further study on the existence and function of mouse frataxin subtype protein.