小麦TaSIM蛋白的原核表达与纯化及Western blotting检测

Prokaryotic Expression, Purification and Western blotting Detection of TaSIM Protein in Wheat

为了获得TaSIM(Salinity-induced R2R3-MYB)基因的纯化蛋白,以便研究TaSIM转录因子的DNA结合特异性。本研究以含有pET28a-TaSIM原核表达载体的大肠杆菌BL21为材料,用0.5 mmol/L IPTG,在37℃诱导TaSIM融合蛋白表达2 h,收集上清液,采用Ni-NTA柱纯化含有His标签的TaSIM融合蛋白,SDS-PAGE电泳检测结果表明获得了分子量大小约为33 kD的TaSIM融合蛋白。采用Western blotting检测TaSIM融合蛋白的表达,以鼠抗血清和辣根过氧化酶标记的山羊抗小鼠Ig G为抗体,结果表明TaSIM融合蛋白能够和抗体特异结合,说明诱导的蛋白是TaSIM融合蛋白。研究结果为进一步利用凝胶阻滞方法检验TaSIM蛋白与顺式作用元件的结合提供实验基础。

In order to obtain a purified protein of the TaSIM(salinity-induced R2 R3-MYB)gene,it is convenient to further study the DNA binding specificity of the TaSIM transcription factor.Escherichia coli BL21 containing pET28 a-TaSIM prokaryotic expression vector was used as material,the TaSIM fusion protein was induced for 2 h at 37℃with 0.5 mmol/L IPTG,and the supernatant was collected.The His-tagged TaSIM fusion protein was purified using a Ni-NTA column,the results of SDS-PAGE electrophoresis showed that the TaSIM fusion protein with a molecular weight of about 33 kD was obtained.The expression of TaSIM fusion protein was detected by Western blotting.Mouse anti-serum and horseradish peroxidase(HRP)-labelled goat anti-mouse Ig G are used as antibodies.The results showed that the TaSIM fusion protein could specifically bind to the antibody,indicating that the induced protein was TaSIM fusion protein.The results provide an experimental basis for further testing of the binding of TaSIM proteins to cis-acting elements using electro mobility shift assay.