PEF1在肠道病毒71型复制中的作用研究

Effects of PEF1 on the replication of enterovirus 71

目的探讨PEF1(penta-EF-hand domain containing 1)对肠道病毒71型(enterovirus 71,EV71)复制和感染的影响。方法采用小干扰RNA(small interfering RNA,siRNA)方法,敲低人恶性胚胎横纹肌肉瘤细胞(rhabdomyosarcoma,RD)中PEF1的表达,通过RT-PCR、蛋白免疫印迹(western blot,WB)实验检测siRNA的效率。通过RT-PCR、WB和空斑实验检测并比较对照细胞和PEF1敲低细胞中EV71的复制水平。利用免疫荧光检测EV71感染后PEF1在细胞中的定位。采用CRISPR-Cas9技术构建PEF1敲除的RD细胞系,并通过测序和WB实验验证敲除效果,同时使用WB实验检测PEF1敲除的细胞中EV71的复制。结果siRNA能够显著降低RD细胞中PEF1的表达。EV71在PEF1敲低的细胞中的复制水平低于对照细胞。EV71感染后细胞内PEF1的表达水平并未发生改变,但是在细胞内的定位发生了改变。PEF1敲除细胞中EV71的复制水平也显著降低。结论敲低或敲除PEF1的表达能抑制EV71的复制,EV71感染并不影响PEF1的表达但改变其在细胞内的定位。

Objective To investigate the effect of penta-EF-hand domain containing 1 (PEF1) on the replication of enterovirus 71 (EV71). Methods Small interfering RNA (siRNA) method was used to knock down the expression of PEF1 in rhabdomyosarcoma (RD) cells. RT-PCR and western blot (WB)were performed to detect the efficiency of siRNA. RT-PCR, WB and plaque assay were conducted to detect and to compare the replication levels of EV71 in control cells and PEF1 knock-down cells. After EV71 infection, the localization of PEF1 in the infected cells was detected by immunofluorescence. The PEF1 knock-out RD cells were constructed by CRISPR-Cas9 technology, and the knock-out effect was verified by sequencing and WB. The replication of EV71 in PEF1 knock-out cells was detected by WB. Results siRNA can significantly reduce the expression of PEF1 in RD cells. The virus replication level in PEF1 knock-down cells was dramatically reduced compared with RD control cells. EV71 infection didn’t affect the expression of PEF1, but affected the localization of PEF1. EV71 replication was significantly decreased in PEF1 knock-out cells. Conclusions Knock-down and/or knock-out the expression of PEF1 inhibited the replication of EV71. EV71 infection did not affect the expression of PEF1 but changed its localization in cells.