拟南芥ERA-1基因的原核表达及多克隆抗体的制备
Expression of Arabidopsis ERA-1 gene in E.coli and preparation of polyclonal antibody against ERA-1
【目的】原核表达纯化拟南芥叶绿体ERA-1蛋白，免疫家兔以制备抗ERA-1的多克隆抗体。【方法】将拟南芥ERA-1基因的cDNA序列克隆到大肠杆菌原核表达载体上，将重组质粒转化到大肠杆菌BL21 (DE3)中进行表达，亲和层析纯化重组ERA-1蛋白并用其免疫家兔，获得的抗血清并用免疫印迹实验检测多克隆抗体的效价及特异性。【结果】从大肠杆菌包涵体中获得了足量的重组ERA-1蛋白，对家兔进行了免疫，获得的的抗血清1:5000稀释后能够检测到10 ng的重组蛋白，并能在植物叶片总蛋白中特异地检测到目的蛋白。【结论】成功制备了ERA-1蛋白的多克隆抗体，可以用于今后对ERA-1基因功能的研究。
【Objective】The overall object of this study is to get recombinant Arabidopsis ERA-1 protein and prepare polyclonal antibody against Arabidopsis ERA-1, a small chloroplast GTPase. 【Method】ERA-1 cDNA was cloned into prokaryotic expression vector and transformed into E.coli BL21(DE3). Affinity chromatography was carried out to purify the recombinant ERA-1. Purified recombinant ERA-1 was used to immunize rabbits to obtain anti-ERA-1 antiserum. Western blotting was performed to assay the sensitivity and the specificity of the polyclonal antibody. 【Result】 We got sufficient amount of purified recombinant ERA-1 from E.coli inclusion body to immumize rabbits. The anti-ERA-1 antiserum, when diluted to 1:5000, can detect 10 ng of recombinant ERA-1 and can specifically detect endogenous ERA-1 in total Arabidopsis leaf protein preparations.【Conclusion】The prokaryotic expression of ERA-1 was successful and the anti-ERA-1 polyclonal antibody can be applied to future study on protein function of Arabidopsis ERA-1.
付世英、刘夏燕
分子生物学生物工程学
生物化学与分子生物学拟南芥ERA-1原核表达蛋白纯化多克隆抗体制备
Biochemistry and molecular biologyArabidopsis ERA-1prokaryotic expressionprotein purificationpolyclonal antibody preparation
付世英,刘夏燕.拟南芥ERA-1基因的原核表达及多克隆抗体的制备[EB/OL].(2015-11-26)[2025-07-21].http://www.paper.edu.cn/releasepaper/content/201511-651.点此复制
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