一个植物半胱氨酸蛋白酶多克隆抗体的制备及其应用

半胱氨酸蛋白酶 (cysteine proteinase,CysP)是一类重要的蛋白酶家族,广泛参与植物多种生理过程。为了分析植物CysP的特性,本实验首先通过RT-PCR技术从本氏烟中扩增获得了一个编码CysP的基因(NbCysP)序列并连接至pEASYTM-T5 Zero载体,测序验证后亚克隆至原核表达载体pGEX-6P1,命名为pGEX-NbCysP;将其导入大肠埃希菌BL21plysS中诱导表达;重组表达的NbCysP融合蛋白经过亲和层析纯化,免疫兔子制备多克隆抗体,Western印迹分析显示,该抗体能和重组NbCysP蛋白发生强烈的免疫学反应且条带单一,表明所获得的CysP抗体具有良好的特异性。上述结果为进一步鉴定植物CysP的功能特性奠定了基础。

Preparation and application of multiclonal antibody against plant cysteine proteinase

Cysteine proteases (CysPs) are a family of important proteases that are involved in a wide range of plant physiological processes. To characterize such proteinase, a gene encoding CysP was amplified by RT-PCR from Nicotiana benthamiana plants and ligated into pEASYTM-T5 Zero vector. The insertion was sequenced and then sub-cloned into the prokaryotic expression plasmid pGEX-6P1, named as pGEX-NbCysP. The plasmid was transformed into Escherichia coli BL21plysS for inducible expression. The recombinant NbCysP fusion protein was purified with affinity chromatography and used for producing polyclonal antibody by immunizing rabbits. In immunoblotting assays, the polyclonal antibody could react strongly with the recombinant NbCysP protein as the presence of a single band, indicating that the antibody was specific against the proteinase. These results laid a foundation for further characterization of its function.