拟南芥DBB 1 a（double B-box 1a）蛋白的表达、纯化及Western blotting检测

PCR扩增拟南芥（Arabidopsis thaliana）DBB1a cDNA的保守区段（GenBank登录号：AT2G21320）,转化到冷诱导表达载体pCold TF上,构建pCold-DBB1a重组质粒,转化大肠杆菌DH5a.15℃下IPTG诱导表达融合蛋白,并通过SDS-PAGE检测.证实目的蛋白以可溶形式在约20 kD处高效表达,与预期蛋白大小相吻合.表达蛋白经Ni琼脂糖凝胶亲和层析纯化,SDS-PAGE及Western blotting检测证实纯化后获得高纯度融合蛋白,这为进一步研究DBB1a功能奠定了基础.

Expression,Purification and Western Blotting Detection of Arabidopsis DBB1a (double B-BOX1a)

To gain the recombination expression plasmid, cDNA sequence of DBBla was obtained by PCR（ GenBank accession number： AT2G21320） ; The sequence was then cloned into expression vector pCold TF to gain the recombination plasmid, which was then transformed into the host bacteria DH5a. The recombination protein expression was induced by IPTG at the temperature of 15℃ and detected by SDS-PAGE. It indicated that the pCold-DBBla fusion protein was soluble protein with a relative molecular mass 20 kD, which consistent fit with the molecular mass deduced from gene coding frame. Ni＋ -NTA agarose was used to purify the fusion protein. The produced fusion protein DBBla was confirmed by SDS-PAGE and Western blotting, which indicated that the recombinant DBB1 a protein had high specificity, which paved a way for further studies on DBBla.