拟南芥WUSCHEL蛋白的原核表达、亲和纯化和多克隆抗体制备

Induced expression of Arabidopsis thaliana WUSCHEL in Escherichia coli, affinity protein purification and polyclonal antibody preparation

摘要:

本研究构建了带有His标签的拟南芥(Arabidopsis thaliana)WUSCHEL基因原核表达载体pET-31b(+)-WUS-His(6), 优化了大肠杆菌(Escherichia coli)诱导表达体系, 将亲和层析纯化后的WUS融合蛋白, 经尿素梯度透析复性溶解, 免疫新西兰大白兔, 成功制备了WUS蛋白多克隆抗体。通过琼脂糖免疫扩散检测确定了抗血清效价和特异性, 并以斑点杂交和Western blotting检验其灵敏性。结果表明, 成功构建的拟南芥WUS原核表达载体, 在E. coli中以0.5 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)28°C诱导表达10 h后, 融合蛋白得到高水平表达, 亲和纯化后目标蛋白纯度达96%以上, 所制备的多克隆抗体具有较高特异性和灵敏性, 可用来检测纳克级蛋白抗原。

Abstract:

We constructed a His-tagged prokaryotic expression vector of WUSCHEL gene of Arabidopsis thaliana, pET-31b(+)-WUS-His(6). The induction condition of the fusion protein expression in Escherichia coli was optimized. After purified by affinity chromatography, the recombinant WUS protein was resolved by renaturation of gradient urea dialysis, then used as antigen to immune rabbit to prepare polyclonal antibody. The rabbit anti-WUS antibody titer and specificity were analyzed and confirmed by agarose immunodiffusion testing; the antiserum sensitivity was assayed by dot blot and Western blotting. The results showed that the A. thaliana WUS prokaryotic expression vector was successfully constructed, and the optimized protein expression induction condition in E. coli was 0.5 mmol/L IPTG (isopropy-β-D-thiogalactoside) at 28°C for 10 hours. The purity of the affinity purified protein was higher than 96%, and the prepared polyclonal antibody was with high specificity and sensitivity, it was able to detect protein antigen at ng level.