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目的:克隆并表达双链RNA调节的蛋白激酶(PKR)基因,以纯化分离与PKR相互作用的蛋白质。方法:设计特异性引物,PCR扩增分别得到FLAG-PKR-HA和HA-PKR-FLAG基因片段,克隆至表达载体pSG5中。将重组质粒通过脂质体法转染PKR稳定沉默(PKRkd)的HeLa细胞,Western blot检测PKR及标签表达情况;最后,利用HA、FLAG串联亲和纯化系统(TAP)纯化分离与PKR相互作用的蛋白,Western blot技术及银染SDS-PAGE验证PKR蛋白复合物的纯化和分离情况。结果:成功构建了PKR融合蛋白表达载体及FLAG、HA亲和纯化系统,SDS-PAGE和银染结果显示TAP系统分离得到了PKR蛋白带和两条可能与PKR相互作用的蛋白质片段。结论:成功纯化和分离了与PKR相互作用的蛋白质,为进一步研究奠定了基础。
OBJECTIVE: To clone and express the double-stranded RNA-regulated protein kinase (PKR) gene to purify and isolate the protein that interacts with PKR. Methods: Specific primers were designed and PCR amplified FLAG-PKR-HA and HA-PKR-FLAG gene fragments were cloned into the expression vector pSG5. The recombinant plasmids were transfected into PKR stably silenced (PKRkd) HeLa cells by lipofectamine and the PKR and tagged expression were detected by Western blot. Finally, HA and FLAG tandem affinity purification system (TAP) were used to purify and separate PKR-interacting Protein, Western blot and silver staining SDS-PAGE to verify the purification and isolation of PKR protein complexes. Results: The PKR fusion protein expression vector and FLAG, HA affinity purification system were successfully constructed. The results of SDS-PAGE and silver staining showed that PKR protein bands and two protein fragments which may interact with PKR were isolated by TAP system. Conclusion: The successful purification and isolation of proteins interacting with PKR have laid the foundation for further research.