百脉根Rac1蛋白多克隆抗体的制备与应用

Polyclonal Antibody Preparation and Application of Lotus japonicus Rac1 Protein

Rop基因在豆科植物与根瘤菌共生互作过程中发挥重要作用。该研究以模式豆科植物百脉根根系cDNA为模板,扩增得到百脉根的1个Rop基因(Rac1),将其连接到原核表达载体pET28a,转化获得Rac1基因的大肠杆菌BL21(DE3)工程菌。优化Rac1蛋白诱导表达条件,亲和吸附法纯化蛋白,制备Rac1多克隆抗体,并应用该抗体检测Rac1过表达转基因植株中Rac1蛋白的表达水平。结果显示:(1)经双酶切和测序鉴定,成功构建pET28aRac1原核表达载体。(2)Rac1蛋白的最佳诱导表达条件为:IPTG浓度0.1mmol/L、温度20℃、时间6h,重组蛋白以可溶形式高效表达;纯化的Rac1蛋白经SDS-PAGE检测,目的条带大小为25kD左右,且条带清晰、单一无杂带。(3)Western blotting显示,制备的多克隆抗体能特异识别其对应的抗原,且效价较高。(4)通过农杆菌介导的毛根转化法获得Rac1过表达植株的阳性毛根,提取阳性毛根总蛋白,Western blotting分析显示过表达植株中Rac1蛋白表达量显著高于空载体对照,从翻译水平证实过表达载体构建的有效性。该研究制备的Rac1多克隆抗体能够高效特异地检测来源于百脉根体内的Rac1蛋白,这将为进一步开展Rac1在共生信号转导途径中的生物学功能研究提供有利工具。

Rop gene plays an important role in the process of symbiotic interaction between legume and rhi- zobium. A Rop gene Racl was amplified from the root cDNA of model legume Lotusjaponicus and ligated ：o the prokaryotic expression vector pET28a. The engineering bacterium carrying Racl gene in E. coli BL21 （DE3） was obtained. The expression conditions of Racl protein was optimized, and the protein was ourified by affinity chromatography. Racl protein expression levels of overexpression plant were detected by using the prepared anti-Racl polyclonal antibody. The results showed： （1） the prokaryotic expression vector of pET28a-Racl was constructed successfully by double enzyme digestion and DNA sequencing. （2） The optimal expression condition was induction temperature at 20 ℃, time at 6 h, and IPTG concentration it 0.1 mmol/L. The recombinant protein was high-efficiency expressed in the form of soluble protein. The purified Racl protein was obtained by affinity chromatography and detected by SDS-PAGE. The band size was about 25 kD, and the bands were clear and single. （3） Western blotting analysis showed that the poly- clonal antibody could specifically react with the corresponding antigen, and the titer was high. （4） Agrobacterium mediated transformation of hairy roots method was used to obtain the positive hairy roots of Racl overexpression plant. The total protein of positive hairy roots was extracted and detected by West- ern blotting. The results showed that the expression of Racl protein in the Racl overexpression plant was significantly higher than that in the empty vector control, which proved the construction of overexpression vector was effective from translation level. The results showed that the preparation of Racl potyclonal an- tibody could specificity detect the native Racl protein in Lotus japonicus, which will provide a powerful tool for the further study of the biological function of Racl in the symbiosis signal transduction pathway.