[1]张越秀,张华伟,李连峰*,等.利用CRISPR/Cas9技术构建OAS2敲除的PK-15细胞系及敲除OAS2对CSFV复制的影响[J].中国预防兽医学报,2019,(11):1094-1098.[doi:0.3969/j.issn.1008-0589.201903029]
 ZHANG Yue-xiu,ZHANG Hua-wei,LI Lian-feng*,et al.Construction of OAS2 knockout PK-15 cell line and its effect on the replication of classical swine fever virus[J].Chinese journal of preventive veterinary medicine,2019,(11):1094-1098.[doi:0.3969/j.issn.1008-0589.201903029]
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利用CRISPR/Cas9技术构建OAS2敲除的PK-15细胞系及敲除OAS2对CSFV复制的影响()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年11
页码:
1094-1098
栏目:
病原生物学
出版日期:
2019-12-25

文章信息/Info

Title:
Construction of OAS2 knockout PK-15 cell line and its effect on the replication of classical swine fever virus
文章编号:
1008-0589(2019)11-1094-05
作者:
 

张越秀张华伟李连峰*仇华吉*

 (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,黑龙江 哈尔滨 150069)
Author(s):
 

ZHANG Yue-xiu ZHANG Hua-wei LI Lian-feng* QIU Hua-ji*

 (State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute,
Chinese Academy of Agricultural Sciences, Heilongjiang Province, Harbin 150069, China)
关键词:
CRISPR/Cas92’-5’寡腺苷酸合成酶2PK-15猪瘟病毒
Keywords:
 CRISPR/Cas9  OAS2  PK-15  CSFV
分类号:
S852.65
DOI:
0.3969/j.issn.1008-0589.201903029
文献标志码:
A
摘要:
为研究猪源2’-5’寡腺苷酸合成酶2(OAS2)对CSFV复制的影响,本研究利用CRISPR/Cas9系统构建敲除OAS2的PK-15细胞系(PK-OAS2-KO)。在构建PK-OAS2-KO细胞系时,设计2条分别靶向OAS2基因第1和第2外显子的特异性sgRNAs,将sgRNA插入LentiCRISPRv2载体获得重组质粒plentiCRISPRv2-sgRNA;将重组质粒与辅助质粒共转染HEK293T细胞,包装获得携带sgRNA的慢病毒后以MOI 1转导PK-15细胞,通过嘌呤霉素药物筛选及有限稀释法获得单克隆细胞株,经基因测序和western blot鉴定OAS2敲除效果。利用MOI 0.01 rCSFV-Rluc株(报告病毒)或CSFV Shimen株分别感染PK15细胞获得的PK-OAS2-KO细胞系,经荧光素酶活性检测和qRT-PCR证实敲除OAS2基因能够促进CSFV复制。综上,本研究利用CRISPR/Cas9基因编辑技术构建了OAS2基因敲除的PK-15细胞系,并发现OAS2具有抑制CSFV复制的作用,本研究为进一步研究OAS2抗CSFV作用的分子机制提供了依据。
 
Abstract:
To identify the role of OAS2 on CSFV replication, CRISPR/Cas9 system was employed to construct OAS2- knockout PK-15 (PK-OAS2-KO) cell lines. To establish PK-OAS2-KO cell lines, two specific single guide RNAs (sgRNAs) were designed to target exons 1 and 2 of OAS2 gene. And sgRNA was inserted into LentiCRISPRv2 to obtain the recombinant plasmid plentiCRISPRv2-sgRNA. HEK293T cells were transfected with plentiCRISPRv2-sgRNA and helper plasmids. The resulting lentiviruses were harvested and transduced into PK-15 cells at a multiplicity of infection (MOI) of 1. The transduced PK-15 cells were selected under puromycin and seeded on 96-well plates with serial dilutions to obtain a single-cell-derived colony. The knockout results were confirmed by sequencing analysis and western blot. Subsequently, PK-OAS2-KO cell lines were infected with rCSFV-Rluc (reporter virus) or CSFV Shimen strain at an MOI of 0.01, and the replication level of CSFV was evaluated by luciferase activities detection assay and real-time reverse transcription-PCR. The results showed that OAS2 gene knockdown promoted the replication of CSFV. In summary, this study established PK-OAS2-KO cell lines using CRISPR/Cas9 system and found that OAS2 inhibited CSFV replication, which provides foundations for further study of the molecular mechanism of OAS2 anti-CSFV effect.

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(本文编辑:李 爽;英文编辑:王玉娥)

更新日期/Last Update: 2019-12-09