TaMAPK5蛋白的抗体制备及其在小麦与叶锈菌互作过程中的表达特征

Antibody Preparation of TaMAPK5 Protein and Its Expression Characteristics in the Interaction Between Wheat and Puccinia triticina

为了深入探究TaMAPK5在小麦与叶锈菌互作过程中发挥的功能,克隆了TaMAPK5基因的CDS区,构建了原核表达载体pET28a-TaMAPK5,转化到大肠杆菌表达菌株BL21(DE3)中,对目的蛋白诱导表达的异丙基硫代半乳糖苷(IPTG)最佳浓度、诱导时间和诱导温度进行了探索,并通过Ni-NTA亲和层析对目的蛋白进行了纯化,将纯化后的重组蛋白免疫新西兰兔制备TaMAPK5多克隆抗体。结果表明,TaMAPK5的CDS区全长为1110 bp,TaMAPK5重组蛋白在16℃经终浓度为0.050 mmol/L的IPTG诱导48 h效果最好。将该重组蛋白作为抗原免疫新西兰兔,成功制备了可以特异性识别TaMAPK5的多克隆抗体,抗体效价为1∶51200。Western Blot结果表明,在小麦与叶锈菌不亲和组合中TaMAPK5受叶锈菌侵染诱导表达。成功表达、纯化了TaMAPK5重组蛋白并制备了其多克隆抗体,揭示了TaMAPK5蛋白可能正调控小麦抵抗叶锈菌侵染反应。

In order to further explore the function of TaMAPK5 in the interaction between wheat and Puccinia triticina,the CDS region of TaMAPK5 gene was cloned,and the prokaryotic expression vector pET28a-TaMAPK5 was constructed and transformed into E.coli BL21(DE3).The optimal concentration,induction time and induction temperature of isopropylβ-D-thiogalactoside(IPTG)induced expression of the target protein were explored,and the target protein was purified by Ni-NTA affinity chromatography.The purified recombinant protein was used to immunize New Zealand rabbits to prepare TaMAPK5 polyclonal antibody.The results showed that the full length of the CDS region of TaMAPK5 was 1110 bp,and the TaMAPK5 recombinant protein was induced with IPTG at a final concentration of 0.050 mmol/L and incubated at 16℃for 48 h.The recombinant protein was used as an antigen to immunize New Zealand rabbits,and a polyclonal antibody capable of specifically recognizing TaMAPK5 was successfully prepared.The antibody titer was 1∶51200.The results of Western Blot showed that TaMAPK5 was induced by P.triticina infection in wheat and P.triticina incompatible combinations.The recombinant protein TaMAPK5 was successfully expressed and purified,and its polyclonal antibody was prepared.It was revealed that TaMAPK5 protein may positively regulate wheat resistance to leaf rust infection.