宿主细胞转录因子E2相关因子2对EIAV复制影响的初步研究  

Preliminary study of the effect of host cell transcription factor E2-related factor 2 on EIAV replication

作　　者：马官芹 王岩[1] 林跃智[1] 王晓钧[1] MA Guan-qin;WANG Yan;LIN Yue-zhi;WANG Xiao-jun(Equine Infectious Diseases and Lentiviral Diseases Research Group,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/马传染病和慢病毒病研究创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2022年第2期119-125,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金面上项目(31672533);黑龙江省自然科学基金面上项目(C2016064、C2017077、C2017076)。

摘　　要：转录因子E2相关因子2(Nrf2)是细胞重要的调节抗氧化应激的转录因子,其活性状态与病毒感染引起的炎症及免疫清除相关。为研究Nrf2对马传染性贫血病毒(EIAV)复制的影响,本研究通过CRISPR在线设计工具,靶向于Nrf2设计2条特异性sgRNAs,经程序性退火后连接到LentiCRISPRv2-GFP载体中构建重组质粒并分别经测序鉴定正确后,转染HEK293T细胞,24 h后通过western blot筛选沉默Nrf2基因效率最高的sgRNA,结果显示,2条sgRNAs均具有较高的沉默效率。将重组质粒LentiCRISPRv2-GFP-sgRNA-N1转染HEK293T细胞,利用流式细胞仪筛选表达GFP的单细胞克隆,并扩大培养后,经western blot和测序鉴定,筛选到2株不表达Nrf2的细胞,这2株细胞基因组均缺失一个碱基T,导致Nrf2基因发生移码突变而无法表达。选择其中一株细胞,将该细胞连续传代,选择第10代(G10)、15代(G15)、20代(G20)细胞经western blot鉴定其遗传稳定性,结果显示各代次细胞均未检测到Nrf2基因的表达,表明该敲除细胞系可稳定遗传,将其命名为HEK293T-Nrf2^(KO)细胞。将EIAV感染性克隆CMV_(3-8)及Nrf2基因回补的感染性克隆CMV_(3-8)+pVR_(1012)-flag-Nrf2分别转染HEK293T-Nrf2^(KO)细胞,24 h后收获细胞和上清,通过western blot分别检测细胞和上清中的病毒蛋白Gag及利用反转录酶活性试验(RT)检测上清中病毒的复制。western blot结果显示,与CMV_(3-8)转染的HEK293T细胞相比,转染CMV_(3-8)的HEK293T-Nrf2^(KO)细胞和上清中病毒蛋白Gag表达均上调约1.5倍,而转染CMV_(3-8)+pVR_(1012)-flag-Nrf2HEK293T-Nrf2^(KO)细胞和上清中的病毒蛋白Gag表达下调约50%;RT的检测结果与western blot的检测结果一致。结果表明Nrf2基因可以抑制EIAV在HEK293T细胞中的表达。通过将相关质粒转染HEK293T细胞包装EIAV假病毒,并将其分别感染HEK293T和HEK293T-Nrf2^(KO)细胞24 h后,分别检测病毒的荧光素酶信号,结果显示,与对照组相比,感染假病毒的细胞病Transcription factor E2-related factor 2(Nrf2)is an important transcription factor for the regulation of cellular resistance to oxidative stress,and its activity status is closely related to inflammation and immune clearance caused by viral infection.To investigate the effect of Nrf2 on equine infectious anemia virus(EIAV)replication,two specific sgRNAs targeting Nrf2 were designed by CRISPR online design tool,ligated into LentiCRISPRv2-GFP vector after programmed annealing to construct recombinant plasmids and identified correctly by sequencing,respectively.After transfected into HEK293T cells,the sgRNAs with the highest efficiency of silencing Nrf2 gene were screened by western blot after 24 hours.The results showed that both sgRNAs had high silencing efficiency.The recombinant plasmid LentiCRISPRv2-GFP-sgRNA-N1 was transfected into HEK293T cells,and single cell expressing GFP was screened using flow cytometry and identified by western blot and sequencing.After expanded culture,2 cell lines were screened which did not express Nrf2,both of which had a single T deletion in their genome,resulting in the Nrf2 gene code-shifting mutation.One of the cells was selected,and the genetic stability of the cells was identified by western blot in the 10th(G10),15th(G15)and 20th(G20)generations,and no Nrf2 gene expression was detected in each generation of cells,indicating that the Nrf2 gene knockout cell line was stable,then it was named as HEK293T-Nrf2^(KO) cells.EIAV infectious clone CMV_(3-8) and Nrf2 gene back-complemented infectious clone CMV_(3-8)+pVR_(1012)-flag-Nrf2 were transfected into HEK293T-Nrf2^(KO) cells respectively,and the cells and supernatant were harvested 24 hours after transfection.The viral protein Gag in the cells and supernatant were detected by western blot and the viral replication in the supernatant was detected using reverse transcriptase activity assay(RT),respectively.The western blot results showed that compared with CMV_(3-8)-transfected HEK293T cells,viral protein Gag expression was upregul

关 键 词：NRF2 CRISPR/Cas9 敲除细胞系 马传染性贫血病毒 

分 类 号：S852.65[农业科学—基础兽医学]