小麦TaABI5-D3基因原核表达及多克隆抗体制备

Prokaryotic expression of wheat TaABI5-D3 gene and polyclonal antibody preparation

摘要:

碱性亮氨酸拉链转录因子脱落酸不敏感蛋白5(abscisic acid-insensitive 5,ABI5)是脱落酸介导的种子萌发的关键调控因子，而TaABI5基因与小麦穗发芽密切相关。TaABI5的多拷贝基因成员TaABI5-D3具有编码完整TaABI5蛋白的能力。本研究通过构建原核表达载体pET-28a-TaABI5-D3，并在大肠杆菌中进行表达，最终得到纯化的His-TaABI5-D3重组蛋白。该重组蛋白以包涵体的形式存在，最佳表达条件为16℃、0.6 mmol/L IPTG、150 r/min过夜诱导12 h。进一步将His-TaABI5-D3重组蛋白进行纯化后免疫Balb/c小鼠制备多克隆抗体，Western blotting结果显示抗体的特异性完好。该研究结果为进一步研究TaABI5蛋白在小麦籽粒中的功能奠定了基础。

Abstract:

Abscisic acid insensitive 5 (ABI5) is a basic leucine zipper transcription factor regulating ABA-mediated seed germination, and TaABI5 is closely related to the pre-harvest sprouting in wheat. Studies have shown that TaABI5-D3, one of multiple copies of TaABI5 gene, encodes the intact TaABI5 protein. In this study, we constructed the prokaryotic expression vector pET-28a-TaABI5-D3 for expression of TaABI5-D3 in Escherichia coli and obtained the purified recombinant protein His-TaABI5-D3. This protein existed in the form of inclusion bodies, with the best expression induced by incubation with 0.6 mmol/L IPTG at 16 ℃, 150 r/min overnight (for 12 h). Subsequently, the purified His-TaABI5-D3 was injected into Balb/C mice for the preparation of polyclonal antibodies. Western blotting analysis indicated that the polyclonal antibody had relatively high specificity, laying foundations for clarification of the function of TaABI5 protein in wheat.