一个植物半胱氨酸蛋白酶多克隆抗体的制备及其应用  

Preparation and application of multiclonal antibody against plant cysteine proteinase

作　　者：彭琪琪 羊健[2] 廖乾生[1] 张恒木[2] PENG Qiqi;YANG Jian;LIAO Qiansheng;ZHANG Hengmu(College of Life Scienees,Zhejiang Sci-Tech University,Hangzhou 310018,China;Institute of Virology and Biotechnology,Zhejiang Academy of Agricultural Scienees,Hangzhou 310021,China)

机构地区：[1]浙江理工大学生命科学学院,浙江杭州310018 [2]浙江省农业科学院病毒与生物技术研究所,浙江杭州310021

出　　处：《浙江农业学报》2018年第6期881-885,共5页Acta Agriculturae Zhejiangensis

基　　金：国家自然科学基金(3150160)

摘　　要：半胱氨酸蛋白酶(cysteine proteinase,Cys P)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Cys P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Cys P的基因(NbCys P)序列并连接至p EASYTM-T5 Zero载体,测序验证后亚克隆至原核表达载体p GEX-6P1,命名为p GEXNb Cys P;将其导入大肠埃希菌BL21plys S中诱导表达;重组表达的Nb Cys P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Nb Cys P蛋白发生强烈的免疫学反应且条带单一,表明所获得的Cys P抗体具有良好的特异性。上述结果为进一步鉴定植物Cys P的功能特性奠定了基础。Cysteine proteases（ Cys Ps） are a family of important proteases that are involved in a wide range of plant physiological processes. To characterize such proteinase,a gene encoding Cys P was amplified by RT-PCR from Nicotiana benthamiana plants and ligated into p EASYTM-T5 Zero vector. The insertion was sequenced and then subcloned into the prokaryotic expression plasmid p GEX-6 P1,named as p GEX-Nb Cys P. The plasmid was transformed into Escherichia coli BL21 plys S for inducible expression. The recombinant Nb Cys P 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 Nb Cys P 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.

关 键 词：半胱氨酸蛋白酶 原核表达 多克隆抗体 

分 类 号：Q78[生物学—分子生物学]