蛋白激酶R的原核表达、纯化及多克隆抗体制备  

Prokaryotic expression,purification and polyclonal antibody preparation of protein kinase R

作　　者：张涛[1,2] 罗维玉[1,2] 姜水涛 朱鹏阳[2] 李呈军[2] 陈化兰[2] 孙晓林[1] 姜丽[2] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室农业部动物流感重点开放实验室,黑龙江哈尔滨150001

出　　处：《甘肃农业大学学报》2016年第1期7-12,共6页Journal of Gansu Agricultural University

基　　金：中央级公益性科研院所基本科研业务费专项(03022014001);国家自然基金青年基金(31402206)

摘　　要：【目的】构建蛋白激酶R的原核表达系统并制备其多克隆抗体.【方法】利用RT-PCR方法从人的A549细胞中扩增蛋白激酶R(PKR)基因,将其克隆到表达载体pGEX-6P-1中,经纯化后制备特展异性多克隆抗体.【结果】PKR基因经测序鉴定后转化宿主菌BL21(DE3)感受态细胞,经终浓度0.5mmol/L IPTG诱导表达GST-PKR融合蛋白,SDS-PAGE分析表明融合蛋白获得稳定表达.表达产物经Glutathione-sepharose 4B亲和层析初步纯化后,用凝血酶切除GST标签,而后经离子交换层析及分子筛在AKTA Explorer上进一步纯化,得到高纯度无标签的PKR蛋白.以纯化后的PKR蛋白为抗原免疫新西兰白兔制备多克隆抗体,斑点杂交试验检测其效价达到1∶50 000以上,Western-blot和间接免疫荧光试验(IFA)表明制备的多抗与PKR蛋白的反应具有高度特异性.【结论】PKR蛋白在大肠杆菌中成功表达,且制备的多克隆抗体可用于PKR蛋白的检测,从而为研究PKR在病毒感染过程中的作用提供了重要的工具.【Objective】Establishing prokaryotic expression system and preparing polyclonal antibody preparation of protein kinase R（PKR）.【Method】Using the total RNA of human A549 cells as template,the protein kinase R（PKR）gene was amplified by RT-PCR and cloned into the expression vector pGEX-6p-1and prepared as polyclonal antibody.【Result】After sequencing validation of PKR,the recombinant plasmid was transformed into E.coli BL21（DE3）and were induced with 0.5 mmol/L IPTG into GSTPKR fusion protein.SDS-PAGE analysis showed that the GST-PKR fusion protein was stably expressed.The expression product was purified by glutathione-sepharose 4B affinity chromatography,treated bythrombin to remove the GST tag,further purified by ion-exchange chromatography and molecular sieve using the AKTA Explorer system.The purified PKR protein with high purity and no tag was then used to vaccinate New Zealand rabbits four times to prepare polyclonal antibody.Dot blot hybridization assay showed that the polyclonal antibody titer was more than 1∶50 000 in the rabbit antiserum.The specificity of the polyclonal antibody against PKR was then validated by western-blot and indirect immunofluorescence assay（IFA）.【Conclusion】PKR protein was successfully expressed in E.coli,and the polyclonal antibody could be used to detect PKR protein,thus providing a necessary tool for researching the role of PKR in the virus infection process.

关 键 词：PKR 原核表达 蛋白纯化 多克隆抗体 

分 类 号：Q78[生物学—分子生物学]