转录因子XBP1的融合表达、纯化及多克隆抗体的制备

Expression and Purification of XBP-1 Fusion Protein and Preparation of Polyclonal Antibody Against Protein

人X盒结合蛋白 1(XBP 1)为一种转录因子 ,与多种肿瘤的发生、发展有密切关系 .XBP 1有2种剪切形式 ,即XBP 1S和XBP 1U .将这 2种剪切形式中的一段相同编码序列 (编码 82～ 14 7位氨基酸 )重组于谷胱甘肽S转移酶 (GST)融合蛋白表达载体pGEX KG中 ,构建成重组质粒pGST XBP 1(82～ 14 7位氨基酸 ) .将该重组质粒转化E .coliDH5α后 ,表达GST XBP 1(82～ 14 7位氨基酸 )融合蛋白 ,经谷胱甘肽 Sepharose 4B亲和层析获得纯化的融合蛋白 .用此融合蛋白免疫家兔制备多克隆抗体 .利用制备的抗体分别用Western印迹和免疫细胞化学检测XBP 1的 2种剪切形式在哺乳动物细胞中的表达 .结果表明 ,该抗体对XBP 1的 2种剪切形式均具有反应原性 ,效价高 ,特异性好 ,可以用于进一步研究XBP

X-box binding protein-1(XBP-1) is a transcription factor that plays an important role in regulating proliferation of cancer cells.XBP-1 has two splicing variants that were designated XBP-1S and XBP-1U.The coding sequences(82～147 residues) of XBP-1S and XBP-1U were cloned into the expression vector pGEX-KG, which directs expression of glutathione S-transferase(GST) fusion protein.The GST-XBP-1(82～147 residues) fusion protein expression vector was constructed. The recombinant plasmid was then transformed into E.coli DH5α. The GST-XBP-1(82～147 residues) fusion protein was expressed and then purified by glutathione-Sepharose 4B affinity chromatography. The polyclonal antibody against GST-XBP-1 (82～147 residues) fusion protein was prepared by immunizing rabbit with the purified protein. The ectopic expression of XBP-1S and XBP-1U in 293T cells were confirmed by Western blotting and immunocytochemistry with the antiserum. The polyclonal antibody recognized both of the splicing variants of XBP-1 with high titer and specificity.These results suggest that the polyclonal antibody can be used for further studies of XBP-1 function.