宿主细胞erp57基因敲除抑制流感病毒复制的研究

Genetic knockout of erp57 gene in host cells inhibits influenza virus replication

ERp57是细胞内质网内的蛋白二硫键异构酶家族成员,可以催化蛋白二硫键的合成以促进蛋白折叠。为研究erp57基因敲除对流感病毒复制的影响,本研究通过CRISPR/Cas9技术构建了erp57基因的向导RNA(g RNA)敲除质粒p GEM-erp57g RNA,并将其与p Cas9-EGFP共转染至人肺腺癌细胞A549中,采用流式分选及限制性稀释的方法筛选单细胞克隆株,通过靶基因组测序及western blot检测,筛选获得了erp57基因敲除的A549细胞系。erp57基因敲除细胞系与野生型细胞生长动力学无显著差异。此外,将H1N1亚型流感病毒分别感染野生型和erp57基因敲除细胞系以测定erp57基因敲除对流感病毒复制水平的影响。结果显示流感病毒在erp57基因敲除细胞中的复制显著地受到了抑制,表明ERp57是流感病毒复制过程中的一个关键蛋白。本研究为开发新的抗流感病毒手段提供了相关的实验依据。

ERp57 is a member of the protein disulfide isomerase family in cellular endoplasmic reticulum which assists the formation of disulfide bond in the protein to promote protein folding. In order to study the roles of ERp57 plays in influenza virus replication, we first set up erp57 gene-deficient human lung adenocarcinoma cell line （A549） via clustered regularly interspaced short palindromic repeat （CR/SPR）/Cas9 system, i.e. pCas9-EGFP plasmid was co-transfected with erp57 gRNA-carrying recombinant plasmid into A549 cells, and the green fluorescence-positive cells were sorted by flow cytometry and further identified the erp57 gene knocked out by western blot from the monoclonal cell lines which were prepared by limited dilution. While the erp57 knockout cell line exhibited similar growth characteristics with wild type cells. However, when the wild type and erp57 knockout cells were infected with H1N1 influenza virus, it was found that the viral replication was significantly inhibited in erp57 knockout cells, suggesting that ERp57 positively impacted viral replication the cells. This study provides useful information for the development of new anti-viral strategies.