SARS-CoV-2非结构蛋白Nsp2上调A549细胞泛素水平并被泛素化修饰研究

Research on SARS-CoV-2 Nonstructural Protein Nsp2 Up-regulating Ubiquitin Level in A549 and Modified by Ubiquitination

目的 新型冠状病毒(SARS-CoV-2)因其强大的感染力肆虐全球,因此深入研究病毒与宿主的相互作用是揭示SARS-CoV-2致病机制的重要途经。方法 利用同源重组方法 将SRAS-CoV-2的非结构蛋白Nsp1~Nsp16构建到真核表达载体上,并通过Western blot检测Nsp1~Nsp16重组质粒的蛋白表达情况。接着使用Western blot检测Nsp2对细胞总泛素水平的影响,并通过细胞免疫荧光检测Nsp2在细胞中的分布情况。最后通过免疫共沉淀技术对Nsp2与泛素之间的相互作用关系进行检测。结果 Western blot检测显示Nsp1~Nsp16重组质粒能够在A549细胞中正常表达;筛选发现非结构蛋白Nsp2能在细胞质中上调细胞总泛素表达水平;免疫共沉淀检测进一步发现Nsp2能与泛素蛋白发生相互作用。结论 成功构建了SARS-CoV-2非结构蛋白Nsp1~Nsp16真核细胞表达载体,并发现非结构蛋白Nsp2可以增加细胞总泛素表达水平并被泛素化修饰。提示Nsp2通过泛素途径参与到SARS-CoV-2与宿主的相互作用,有助于深入了解病毒与宿主的作用机制。

Objective As SARS-CoV-2 spreads around the world,an in-depth study of virus-host interaction is essential for exploring SARS-CoV-2 pathogenesis.Methods The SRAS-CoV-2 nonstructural protein Nsp1-Nsp16 was cloned by PCR and homologous recombination assay into a eukaryotic expression vector,and Nsp1-Nsp16 protein expression was detected by Western blot.Then,the influence of Nsp2 exerted on total ubiquitin levels was determined by Western blot,and the distribution of Nsp2 in the cells was found by immunofluorescence.Finally,co-immunoprecipitation was used to detect the interaction between Nsp2 and ubiquitin.Results Western blot analysis revealed that Nsp1-Nsp16 could express smoothly in A549 cells.Meanwhile,it was shown that Nsp2 increased total ubiquitin expression in the cytoplasm.Co-immunoprecipitation experiments results declared that Nsp2 could interact with ubiquitin protein.Conclusion We successfully construct the expression vector of nonstructural protein Nsp1-Nsp16,and find that Nsp2 can increase the total ubiquitin level of cells and be ubiquitinated.This study demonstrates that Nsp2 is involved in the interaction between SARS-CoV-2 and host via the ubiquitin pathway,which lays a foundation for further research on the mechanism of SARS-CoV-2 and host.