敲低内源性PERK基因对犬细小病毒复制的影响

Effects of knockdown of endogenous PERK gene on replicationof canine parvovirus

利用新型CRISPR interference(CRISPRi)技术进行蛋白激酶R样内质网激酶(protein kinase-like endoplasmic reticulum kinase, PERK)目的基因PERK的敲低。首先,利用慢病毒包装系统经嘌呤霉素筛选后构建稳定表达dCas9-KRAB蛋白的MDCK细胞株。然后,设计靶向PERK转录起始位点(transcription start site, TSS)的特异性小向导RNA(small guide RNA,sgRNA),向该稳转细胞株转染表达sgRNA的质粒,通过qPCR检测PERK的mRNA表达水平;同时,内质网应激激活剂衣霉素(tunicamycin, Tu)处理该细胞后,通过qPCR检测PERK通路下游GRP78、GRP94、ATF4、GADD34和CHOP等基因mRNA的表达水平。最后,将犬细小病毒2型(canine parvovirus type 2,CPV-2)接种于PERK敲低的MDCK细胞株,通过绝对定量PCR测定不同时间点CPV-2的复制水平。结果表明,敲低PERK基因表达后,CPV-2的复制水平显著下降,说明PERK参与调控CPV-2的复制。以上结果为进一步探究内质网未折叠蛋白反应(endoplasmic reticulum unfolded protein response, UPR^(ER))对CPV-2复制的影响及病毒与宿主相互作用的分子机制研究奠定了基础。

The novel CRISPR interference(CRISPRi)technique was used to knock down the gene of the protein kinase-like endoplasmic reticulum kinase(PERK).First of all,a MDCK cell strain stably expressing dCas9-KRAB protein was constructed by using the lentiviral packaging system and screened by puromycin,and the specific small guide RNA(sgRNA)targeting the PERK transcription start site(TSS)was designed.Then,the mRNA expression level of PERK was detected by qPCR after transfection of plasmids expressing sgRNA.At the same time,the mRNA expression levels of PERK pathway downstream genes(such as GRP78,GRP94,ATF4,GADD34 and CHOP)were detected by qPCR after stimulating the cells with the endoplasmic reticulum stress activator tunicamycin(Tu).Finally,the PERK knockdown MDCK cells were inoculated with canine parvovirus type 2(CPV-2),and the replication level of CPV-2 at different time points was determined by absolute quantitative PCR.The results showed that the replication level of CPV-2 was significantly decreased after knockdown of PERK,indicating that PERK was involved in the regulation of CPV-2 replication.The above results lay a foundation for further exploration of the effect of endoplasmic reticulum unfolded protein response(UPR^(ER))on CPV-2 replication and the molecular mechanism of virus-host interaction.