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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (1): 46-54    DOI: 10.13523/j.cb.20190106
技术与方法     
利用CRISPR/Cas9技术构建流感病毒高产细胞系MDCK-Tpl2 -/-*
刘赛宝1,2,李亚芳1,2,王辉1,2,王伟2,冉多良1,**(),陈洪岩2,**(),孟庆文2,**()
1 新疆农业大学动物医学学院 乌鲁木齐 830000
2 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 黑龙江省实验动物与比较医学重点实验室 哈尔滨 150069
Construction of Influenza Virus High-producing Cell Line MDCK-Tpl2 -/- with CRISPR / Cas9
Sai-bao LIU1,2,Ya-fang LI1,2,Hui WANG1,2,Wei WANG2,Duo-liang RAN1,**(),Hong-yan CHEN2,**(),Qing-wen MENG2,**()
1 College of Veterinary Medicine,Xinjiang Agricultural University, Urumqi 830000,China
2 State Key Laboratory of Veterinary Biotechnology,Heilongjiang Provincial Key Laboratory of Laboratory Animals and Comparative Medicine,Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences,Harbin 150069, China
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摘要：

Tpl2是调节I型(IFNα/β)和II型(IFNγ)IFN的关键调控因子,对病毒感染期间产生有效免疫应答至关重要。为了建立支持流感病毒高效繁殖的新型MDCK细胞系,利用CRISPR/Cas9基因编辑技术构建Tpl2基因敲除的MDCK细胞,绘制其细胞生长曲线;根据《中国药典》第三部,对细胞进行形态检查、内外源因子和致瘤性检测;流感病毒接种细胞(MOI=0.1),测定12h、24h、48h、72h细胞培养上清的血凝滴度及72h TCID50滴度。结果：靶基因组测序结果显示,获得一株稳定敲除Tpl2的MDCK细胞(MDCK-Tpl2 -/-);其与亲本细胞生长速率无显著差异,均为贴壁、上皮样细胞形态;细胞内细菌、真菌、支原体、病毒及致瘤性检测均为阴性;病毒接种后12h、24h、48h、72h,MDCK-Tpl2 -/-细胞培养上清的血凝效价提高了1.78~2.5倍。病毒接种后72h,MDCK-Tpl2 -/-细胞培养上清病毒的TCID50滴度提高了2.8倍。结果表明,利用Tpl2缺陷型MDCK细胞系可以提高流感病毒产量,为提升疫苗质量奠定基础。

关键词： Tpl2;  CRISPR/Cas9;  MDCK-Tpl2 -/-细胞;  细胞质控;  禽流感病毒    
Abstract:

Tpl2 is the key regulator of the regulation of I (IFNα/β) and II (IFNγ) IFN, which is essential for the effective immune response during virus infection. In order to establish a new MDCK cell line supporting the efficient reproduction of influenza virus, CRISPR/Cas9 gene editing technology was used to construct Tpl2 knockout MDCK cells, and its cell growth curve was drawn. Tests for characteristics, extraneous agents, endogenous agents and tumorigenicity are performed on cells according to Chinese Pharmacopeia Book III. Influenza virus was inoculated into cells (MOI=0.1), the hemagglutination titers of the cell culture supernatants at 12h, 24h, 48h, 72h, and the TCID50 titer at 72h were determined. The target genome sequencing results shows that a MDCK cell (MDCK-Tpl2 -/-) knocked out of Tpl2 stably is obtained;The Tpl2 knock out cell line is similar to wild type cells in growth characteristics, and the test of epithelial cell morphology, intracellular bacteria, fungi, mycoplasma, virus and tumorigenicity are negative. After inoculating virus for 12h, 24h, 48h, and 72h, the hemagglutination titer of MDCK-Tpl2 -/- cell culture supernatant increased by 1.78-2.5 times. At 72h after virus inoculation, the TCID50 titer of the MDCK-Tpl2 -/- cell culture supernatant virus increased 2.8 times.The results showed that Tpl2-deficient MDCK cell line could increase the production of influenza virus and lay a foundation for improving vaccine quality.

Key words: Tpl2    CRISPR/Cas9    MDCK-Tpl2 -/- cell line    Cell quality control    Avian influenza virus
收稿日期: 2018-03-28 出版日期: 2019-02-28
ZTFLH:  Q78  
基金资助: * 兽医生物技术国家重点实验室自主课题(SKLVBP2017010);国家自然科学基金(30771615);“十二五”农村领域国家科技计划资助项目(2011AA100305)
通讯作者: 冉多良,陈洪岩,孟庆文     E-mail: xjrdl7@163.com;chenhongyan@caas.cn;mqw@hvri.ac.cn
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刘赛宝
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引用本文:

刘赛宝,李亚芳,王辉,王伟,冉多良,陈洪岩,孟庆文. 利用CRISPR/Cas9技术构建流感病毒高产细胞系MDCK-Tpl2 -/-*[J]. 中国生物工程杂志, 2019, 39(1): 46-54.

Sai-bao LIU,Ya-fang LI,Hui WANG,Wei WANG,Duo-liang RAN,Hong-yan CHEN,Qing-wen MENG. Construction of Influenza Virus High-producing Cell Line MDCK-Tpl2 -/- with CRISPR / Cas9. China Biotechnology, 2019, 39(1): 46-54.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190106    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I1/46

sgRNA Oligonucleotide sequence
Tpl2-sgRNA1 F:5'-CCGGGATCGGTCAGATACGGAACCG-3'
R:5'-AAACCGGTTCCGTATCTGACCGATC-3'
Tpl2-sgRNA2 F:5'-CCGGGTCCGATGTAATAGACATTA-3'
R:5'-AAACTAATGTCTATTACATCGGAC-3'
表1  Tpl2-sgRNA寡核苷酸序列
Primer Primer sequence Product(bp)
p-Tpl2-sgRNA1 F:5'-AGAGGTGCCTTGGTTGTC-3' 113
R:5'-ATTCCTGTGGTCGGTGTC-3'
P-Tpl2-sgRNA2 F:5'-GAGTACATGAGCACTGGAAGT-3' 188
R:5'-AGCAGAGATTCTGAATGCTCC-3'
表2  靶位点鉴定引物
图1  Tpl2基因敲除质粒 sgRNA 的设计
图2  Tpl2 sgRNA 测序结果
图3  质粒转染MDCK细胞
图4  聚丙烯酰胺凝胶电泳检测Tpl2-sgRNA1的靶向敲除效果
图5  Tpl2-sgRNA1-6#测序结果
图6  Tpl2-sgRNA1-6# F10代测序结果
图7  野生型及Tpl2敲除MDCK细胞生长曲线
图8  细胞形态观察
图9  细胞DNA荧光染色
图10  MDCK-Tpl2-/-细胞的致瘤性检查
图11  流感病毒接种细胞后血凝效价比较
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