PCIF1基因敲低细胞系的构建及其对猪流行性腹泻病毒复制影响的探究  

Construction of PCIF1 knockdown cell line and its effect on replication of porcine epidemic diarrhea virus

作　　者：房梦桃 刘众 吴琪 黄冬艳[1,2] 叶昱 万根[1,2] 宋德平 FANG Mengtao;LIU Zhong;WU Qi;HUANG Dongyan;YE Yu;WAN Gen;SONG Deping(College of Animal Science and Technology,Jiangxi Agricultural University,Nanchang 330045,China;Jiangxi Engineering Research Center for Animal Health Products,Nanchang 330045,China)

机构地区：[1]江西农业大学动物科学技术学院,江西南昌330045 [2]江西省动物疫病防控制剂工程研究中心,江西南昌330045

出　　处：《江西农业大学学报》2024年第5期1266-1274,共9页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金项目(31970611);江西省自然科学基金重点项目(20232ACB205014)。

摘　　要：【目的】旨在构建甲基转移酶——磷酸化C端结构域相互作用因子1(PCIF1)敲低细胞系,为探索PCIF1对猪流行性腹泻病毒(PEDV)复制及其分子机制研究提供试验基础。【方法】利用RNA干扰(RNAi)技术构建PCIF1基因敲低(PCIF1-KD)细胞系。首先构建PCIF1基因特异性靶向干扰载体——pcDNA3.1-Double U6-mCherry-sh PCIF1,后经脂质体介导转染至非洲绿猴肾细胞(Vero-81),并通过遗传霉素筛选、富集PCIF1-KD重组细胞,以RT-qPCR和Western blotting检测PCIF1基因的敲低效果,并验证PEDV在获得的重组敲低细胞上的增殖能力。【结果】(1)重组干扰质粒经测序显示基因序列正确,证明成功构建PCIF1干扰质粒;(2)RT-qPCR和Western blotting试验结果表明,重组Vero-81细胞中PCIF1被显著敲低,获得了稳定的PCIF1-KD重组细胞系sh-△PCIF1-Vero-81;(3)PEDV感染验证发现PEDV在sh-△PCIF1-Vero-81细胞的增殖水平被显著抑制,且在感染后24~36 h病毒mRNA表达水平和蛋白表达水平均被进一步抑制。【结论】利用RNAi技术成功构建了一株稳定敲低PCIF1基因的细胞系sh-△PCIF1-Vero-81,PCIF1基因的沉默对PEDV的复制具有显著的抑制作用,且抑制程度随着时间增加而增加。研究结果可为进一步探究PCIF1对PEDV的影响或相互作用的分子机制提供有力工具和参考。[Objective]The purpose of this study is to construct methyltransferase,phosphorylated Cterminal domain interacting factor 1(PCIF1)knockdown cell lines to efficiently investigate the effect and mechanism of PCIF1 on porcine epidemic diarrhea virus(PEDV)replication[.Method]In this study,a PCIF1 knockdown(PCIF1-KD)cell line was constructed using RNA interference(RNAi)technology.Initially,a PCIF1-specific targeting vector,pcDNA3.1-Double U6-mCherry-shPCIF1,was constructed,and tranfected into African green monkey kidney cells(Vero-81)mediated by lipofectamine.The PCIF1-KD recombinant cells were then screened by geneticin,and then indentified by RT-qPCR and Western blotting.Finally,the replication capacity of the porcine epidemic diarrhea virus(PEDV)on the obtained recombinant PCIF1-KD cells was verified by RT-qPCR and Western blotting.[Result]The recombinant interference plasmid was successfully generated,as demonstrated by sequencing.The results of RT-qPCR and Western blotting showed that PCIF1 was significantly knocked down in the recombinant Vero-81 cells,and thus a stable PCIF1-KD recombinant cell line,designated as sh-△PCIF1-Vero-81,was obtained.The level of PEDV replication was markedly suppressed in sh-△PCIF1-Vero-81 cells,and this inhibiting effect was magnified from 24 to 36 hours post-infection.[Conclusion]This study constructs a PCIF1-KD Vero-81 cell line,which shows substantial inhibition of PEDV proliferation in a time-dependent manner.The results of the study could provide a powerful tool to further investigate the effect of PCIF1 on PEDV or to elucidate the molecular mechanism of how PCIF1 interacts with PEDV.

关 键 词：磷酸化C端结构域相互作用因子1 猪流行性腹泻病毒 非洲绿猴肾细胞 RNA干扰 

分 类 号：S855.3[农业科学—临床兽医学]