小鼠frataxin抗体的制备及应用

Production and application of polyclonal antibody against mouse frataxin

摘要:

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。

Abstract:

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by reduced expression levels of the frataxin gene (FXN) due to expansion of triplet nucleotide GAA repeats in the first intron of FXN. FXN is a mitochondrial protein which plays an important role in the regulation of intracellular iron trafficking, biogenesis of iron-sulfur cluster and heme, and removal of reactive oxygen species. Our previous work showed that tissue-specific expression of FXN in cerebellum and heart generates two novel isoforms. In order to find the isoforms in mouse tissues, we tried to obtain a polyclonal antibody against mouse Fxn with high specificity and sensitivity. Thus, the recombinant plasmid pET24(+)-mFxn was constructed to express his-tagged Fxn in BL21(DE3) cells. The expressed protein is a mature form with 130 amino acids (aa, 14.38 kDa) without the N-terminal signal peptide (77 aa), purified on Ni-NTA column and further dialyzed with Centrifugal Filtration Device. The polyclonal antibody against Fxn was produced by immunizing rabbits with highly purified protein. The collected antiserums were preliminarily purified by precipitation with (NH4)2SO4. Western blotting analysis and cell immunofluorescence showed that the obtained antibody was able to detect both purified and endogenous Fxn. It also worked well in immunoprecipitation with mouse tissues. This is the first time, to our knowledge, to report that mouse Fxn was used as immunogen to generate antibody with high specificity and sensitivity. This work provides a powerful tool for our further research on mouse Fxn isoforms.