| 双链RNA依赖的蛋白激酶(PKR)的真核表达、纯化及鉴定 |
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| 引用本文： | 关振宏,李影,张茂林,段铭. 双链RNA依赖的蛋白激酶(PKR)的真核表达、纯化及鉴定[J]. 安徽农业科学, 2009, 37(28) |
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| 作者姓名： | 关振宏 李影 张茂林 段铭 |
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| 作者单位： | 吉林大学人兽共患病教育部重点实验室,人兽共患病研究所,吉林长春,130062;吉林农业大学动物科学技术学院,吉林长春,130118 |
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| 基金项目： | 国家自然科学基金资助项目，吉林大学农学部引进人才启动基金项目 |
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| 摘 要： | [目的]获得真核表达的Flag-PKR融合蛋白,以用于流感病毒相关的PKR信号通路调控机制研究。[方法]以RT-PCR方法扩增PKR目的基因,将其克隆至真核表达载体pcDNA3.0-Flag中。将所构建的重组质粒pcDNA3.0-Flag-PKR转染293T细胞,利用Flag抗体偶联的琼脂糖凝胶(anti-Flag M2-Agarose)纯化Flag-PKR融合蛋白,所得产物进行SDS-PAGE、Western blot及体外活性鉴定。[结果]重组质粒转染293T细胞,Western blot显示表达出的融合蛋白能够被抗Flag的抗体特异性识别,其相对分子量约为69 kDa,与Flag-PKR融合蛋白大小相符;体外磷酸化试验表明,Flag-PKR融合蛋白能够发生自动磷酸化。[结论]在293T细胞中表达、纯化了有活性的Flag-PKR融合蛋白,为PKR信号通路的研究奠定了基础。
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| 关 键 词： | 流感病毒 双链RNA依赖的蛋白激酶 真核表达 纯化 |
Eukaryotic Expression,Purification and Identification of Double-stranded RNA-Dependent Protein Kinase(PKR) |
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| GUAN Zhen-hong et al. Eukaryotic Expression,Purification and Identification of Double-stranded RNA-Dependent Protein Kinase(PKR)[J]. Journal of Anhui Agricultural Sciences, 2009, 37(28) |
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| Authors: | GUAN Zhen-hong et al |
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| Affiliation: | GUAN Zhen-hong et al(Key Laboratory of Zoonosis,Education Ministry,Institute of Zoonosis,Jilin University,Changchun,Jilin 130062) |
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| Abstract: | [Objective]The research aimed to obtain the Flag-PKR fusion protein expressed in eukaryotic cells in order to elucidate PKR signal pathway regulation mediated by influenza virus.[Method]The cDNA of PKR was obtained using RT-PCR method and cloned to eukaryotic expression vector pcDNA3.0-Flag.The recombinant plasmid pcDNA3.0-Flag-PKR was transfected into 293T cells and the products were purified by anti-Flag M2-Agarose.The expression product was identified by SDS-PAGE,Western blot and activity assay.[Result]T... |
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| Keywords: | Influenza virus Double-stranded RNA-dependent protein kinase(PKR) Eukaryotic expression Purification |
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