PKR基因表达高效阻断Marc-145细胞系建立及其对PRRSV增殖的影响

Establishment of Marc-145 cell lines with PKR gene knockdown and its effect on the proliferation of PRRSV

研究猪繁殖与呼吸障碍综合征(porcine reproductive and respiratory syndrome,PRRS)在Marc-145细胞蛋白激酶R(interferon-induced,double-stranded RNA-activated kinase,PKR)下调表达状态下的感染特性。以Macr-145细胞PKR基因序列设计3个shRNA,并设阳性对照,合成后插入pSilencer^(TM)2.1-U6载体构建重组质粒,与Macr-145细胞PKR+EGFP标签融合表达的重组质粒共转染HEK293细胞,以筛选能下调表达PKR的shRNA,并将该shRNA重组质粒转染Marc-145细胞,潮霉素B抗性筛选并克隆纯化获得PKR下调表达的细胞系,感染重组病毒PRRSV XZ06a-EGFP,通过检测病毒结构蛋白M与EGFP表达、观察细胞病变等,研究此状态下感染特性。结果显示筛选获得致使PKR下调表达的shRNA,以及稳定表达shRNA、PKR表达降低90%以上的Marc-145细胞系,重组病毒感染后48h,M蛋白与EGFP表达均受到抑制,且与对照相比,感染后72~96 h,未观察到典型的细胞病变效应,说明在PKR表达受到抑制时,病毒增殖受到明显抑制。本研究通过建立PKR下调表达细胞系与PRRSV感染试验,初步阐明PKR在PRRSV增殖时所发挥的作用,为进一步开展PRRSV与细胞抗病毒固有免疫互作机制研究莫定了基础。

The proliferation characteristics of PRRSV under the condition of down-regulated expression of protein kinase R(PKR)of Marc-145 cell were studied.The corresponding DNA of three designed shRNAS and one positive control shRNAs targeting coding region of PKR gene were synthesized and cloned into pSilencer^(TM) 2.1-U6 vector to construct recombinant plasmids,then these four plasmids were co-transfected into HEK293 cells with a recombinant plasmid expressing PKR fused with EGFP tag,respectively,to screen the shRNA that can down-regulate the PKR expression,and the celines expressing the screened shRNAs were constructed through plasmid transfection and purification of cell colonies resistant to hygromycin B.The cell lines were infected with the recombinant virus PRRSV XZ06a-EGFP containing EGFP gene,and expression of structural protein M and EGFP were detected and CPE was monitored to investigate the virus proliferation characteristics.The shRNAs that could knockdown the expression of PKR were screened through the detection of the expression of PKR after plasmid co-transfection into HEK293 cells,and the cell line expressing shRNAs stably and reducing the cellular PKR expression by more than 90%were obtained.At 48 h post the recombinant virus infection,the expression of M protein and EGFPwere inhibited sharply,and none of typical CPE was observed during 72-96h post infection compared to the controls,indicating that when the expression of PKR is knockdown,the proliferation of the virus is significantly inhibited.This study established a PKR knockdown expression cell line and clarified the role of PKR in PRRSV infection in Marc-145 cells,which laid a foundation for further research on PRRSV and cellular antiviral innate immune interaction mechanism.