小麦WDHN2蛋白的多克隆抗体制备及其应用

Preparation and application of polyclonal antibody against WDHN2 protein from Triticum aestivum

脱水素(dehydrin, DHN)在植物响应非生物胁迫过程中发挥重要功能。本研究采用PCR技术,从已构建的重组质粒T-WDHN2中扩增小麦(Triticum aestivum)脱水素WDHN2的编码序列,将其插入到含6×His的原核表达载体pET-28a(+)上,转化至大肠杆菌BL21 (DE3)进行诱导表达, SDS-PAGE和Western blot检测6×His-WDHN2融合蛋白分子量为25 kDa左右。在28℃,最佳诱导IPTG浓度为0.75 mmol·L^(-1),最佳诱导表达时间为3 h。利用镍离子亲和层析获得纯化的WDHN2融合蛋白,融合蛋白对60℃条件下α-淀粉酶具有一定的酶活性保护作用。以原核表达的WDHN2的6×His融合蛋白为抗原,免疫Balb/c小鼠,制备WDHN2多克隆抗体。该多克隆抗体不但能识别WDHN2抗原,而且能识别小麦不同干旱程度诱导表达的WDHN2蛋白,这为深入研究WDHN2蛋白功能奠定基础。

Dehydrin(DHN) plays an important role in plant response to abiotic stress. In this study, PCR was used to amplify the coding sequence of Triticum aestivum dehydrin WDHN2 from the constructed recombinant plasmid T-WDHN2, and it was inserted into the prokaryotic expression vector pET-28a(+) containing 6×His and transformed into Escherichia coli BL21(DE3) for induced expression. SDS-PAGE and Western blot showed that the molecular weight of the fusion protein 6×His-WDHN2 was about 25 kDa. At 28℃, the optimal IPTG concentration was 0.75 mmol·L^(-1), and the optimal expression time was 3 h. The fusion protein WDHN2 was purified by nickel affinity chromatography. The fusion protein had a protective effect on α-amylase activity at 60℃. We immunized Balb/c mouse with recombinant 6×His fusion protein of WDHN2 as antigen and prepared polyclonal antibody against WDHN2 protein. Western blot showed that the polyclonal antibody of WDHN2 could recognize not only WDHN2 antigen, but also WDHN2 protein in leaves of wheat induced by different drought stress level. This research laid a foundation for further study of the function of WDHN2 protein.