玉米ZmICS1和ZmSARD1的原核表达及多克隆抗体制备

Prokaryotic expression and antibody preparation of maize ZmICS1 and ZmSARD1

【目的】将玉米异分支酸合成酶1(ZmICS1)及系统获得抗性缺陷1(ZmSARD1)进行原核表达和纯化,并免疫家兔获得多克隆抗体,为深入研究ZmICS1和ZmSARD1在玉米抵抗病原菌胁迫中的功能奠定基础。【方法】克隆玉米ZmICS1和ZmSARD1基因CDS区,连接到原核表达载体pET-28a,转化大肠杆菌表达菌株Rosett-gami2(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达His_(×6)-ZmICS1和His_(×6)-ZmSARD1重组蛋白,以透析纯化后的重组蛋白为抗原免疫家兔,制备多克隆抗体,并进行验证和检测。【结果】成功构建了原核表达载体pET-28a-ZmICS1和pET-28a-ZmSARD1,于37℃、0.84 mmol/L IPTG在大肠杆菌表达菌株Rosett-gami2(DE3)中诱导出His_(×6)-ZmICS1和His_(×6)-ZmSARD1重组蛋白。纯化蛋白免疫家兔,所得ZmICS1抗体能够检测到3 ng的His_(×6)-ZmICS1,ZmSARD1抗体能检测到低至0.3 ng的His_(×6)-ZmSARD,且分别能特异性地检测玉米B73原生质体过表达的ZmICS1和ZmSARD1蛋白。【结论】成功制备了玉米ZmICS1、ZmSARD1多克隆抗体,可用于玉米内源ZmICS1和ZmSARD1蛋白含量的检测。

【Objective】Maize isochorismate synthase 1(ZmICS1)and system acquired resistance deficient 1(ZmSARD1)were expressed and purified in E.coli and polyclonal antibodies were prepared by immunizing rabbits as antigens to provide basis for understanding function of ZmICS1 and ZmSARD1 in resistance to pathogens in maize.【Method】The coding regions(CDS)of maize ZmICS1 and ZmSARD1 were cloned and inserted into the prokaryotic expression vector of pET-28a and the expression vectors were transformed into E.coli Rosett-gami2(DE3)strain.The recombinant proteins were induced by IPTG and purified by dialysis before being used to immunize rabbits subcutaneously to prepare polyclonal antibodies,and the antibodies were verified and detected by western blot.【Result】Prokaryotic expression vectors of pET-28a-ZmICS1 and pET-28a-ZmSARD1 were successfully constructed.The proteins of ZmICS1 and ZmSARD1 were induced in E.coli Rosett-gami2(DE3)at 37℃with 0.84 mmol/L IPTG.The prepared antibodies were able to detect 3 ng of purified His_(×6)-ZmICS1 protein,0.3 ng of purified His_(×6)-ZmSARD1 protein,and the expressed ZmICS1 and ZmSARD1 proteins in maize B73 protoplasts that were transformed with the ZmICS1 and ZmSARD1 expression vectors.【Conclusion】Polyclonal antibodies against maize ZmICS1 and ZmSARD1 were successfully prepared for detection of ZmICS1 and ZmSARD1 proteins.