国家自然科学基金项目(30700504);转基因生物新品种培育重大专项(2009ZX08009-083B)
MAPK蛋白激酶是一类重要的植物胁迫信号调控因子。为了研究小麦MAPK基因的功能,本实验克隆了小麦MAPK蛋白激酶基因TaMAPK2。为了制备TaMAPK2基因的多克隆抗体,TaMAPK2的非保守区段的DNA序列anti-MAPK2被构建到原核表达载体pET-28a-(+)上,表达融合蛋白His-antiMAPK2。在终浓度为1 mmol/L IPTG诱导1h的条件下,融合蛋白His-antiMAPK2表达量达到最大。通过蛋白标记亲和层析柱(HisTrapTMHP)得到纯化的His-antiMAPK2融合蛋白。利用新西兰大白兔制备了TaMAPK2基因的多克隆抗体,ELISA竞争抑制法检测抗体效价检测,效价为1:80000,能满足后续试验要求的效价值,为进一步分析TaMAPK2的蛋白定位、表达等提供基础。
MAPKs are of importance in stress signal transduction process in plant. In order to investigate the function of wheat MAPKs, a MAPK gene named TaMAPK2, was isolated from wheat. To obtain the polyclonal antibody of TaMAPK2, the non-conservation fraction of TaMAPK2 gene was constructed into the prokaryotic expression vector pET-28a-(+). Under the condition of 1 mmol/L of IPTG, the expression of the fusion protein His-antiMAPK2 was up to the peak. The fusion protein His-antiMAPK2 was purified by HisTrapTMHP and used to prepare antibody. The titer of the rabbit`s anti-serum was measured by ELISA method. The rabbit` antiserum with high titer (>80000) was obtained. The polyclonal antibodies can be used for further investigation, which establish the foundation for investigating the function of the MAPK2 gene in protein level.
刘 沛,徐兆师,晏月明,等.小麦TaMAPK2激酶基因的原核表达以及多克隆抗体制备[J].植物遗传资源学报,2011,12(1):92-95.
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