小麦TaMAPK2激酶基因的原核表达及多克隆抗体制备  

Prokaryotic Expression and Polyclonal Antibody Preparation of the Wheat TaMAPK2 Gene

作　　者：刘沛[1,2] 徐兆师[2] 晏月明[1] 李连城[2] 陈明[2] 马有志[2] 

机构地区：[1]首都师范大学生命科学院,北京100048 [2]中国农业科学院作物科学研究所/国家农作物基因资源和基因改良国家重大科学工程/农业部作物遗传育种重点开放实验室,北京100081

出　　处：《植物遗传资源学报》2011年第1期92-95,102,共5页Journal of Plant Genetic Resources

基　　金：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和2009ZX08002-008B)

摘　　要：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,本试验克隆了小麦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1mmol/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 important in stress signal transduction process of plant.In order to investigate the function of wheat MAPKs,a MAPK gene named TaMAPK2,was isolated from wheat.To obtain the polydonal antibody of TaMAPK2,the non-conservation fraction of TaMAPK2 gene was constructed into prokaryotic expression vector pET-28a-（＋）.Under the condition of 1 mmol/L of IPTG,the expression of the fusion protein His-anti MAPK2 was up to the peak.The fusion protein His-antiMAPK2 was purified by HisTrapTMHP and used to prepare antibody.The titer of the rabbits 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.

关 键 词：小麦 MAPK蛋白激酶 原核表达 蛋白纯化 多克隆抗体 

分 类 号：Q943.2[生物学—植物学]