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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2019, Vol. 39 Issue (1): 46-54    DOI: 10.13523/j.cb.20190106
    
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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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     
Received: 28 March 2018      Published: 28 February 2019
ZTFLH:  Q78  
Corresponding Authors: Duo-liang RAN,Hong-yan CHEN,Qing-wen MENG     E-mail: xjrdl7@163.com;chenhongyan@caas.cn;mqw@hvri.ac.cn
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Sai-bao LIU
Ya-fang LI
Hui WANG
Wei WANG
Duo-liang RAN
Hong-yan CHEN
Qing-wen MENG
Cite this article:

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.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190106     OR     https://manu60.magtech.com.cn/biotech/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'
Table 1 The oligo sequences of Tpl2-sgRNA
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'
Table 2 The primer to identify target site
Fig.1 Design strategy of sgRNA plasmid for Tpl2 knockout
Fig. 2 Tpl2 sgRNA sequencing (a)Tpl2-sgRNA1 sequencing (b)Tpl2-sgRNA2 sequencing
Fig.3 Plasmid transfected MDCK cells (a)Microscopy under fluorescent field of the control group(10×) (b)Microscopy under fluorescent field of the experimental group(10×) (c)The control flow analysis results (d)The experimental group flow analysis results
Fig.4 The targeted knockout effect of Tpl2-sgRNA1 measured by Polyacrylamide gel electrophoresis M:DL 500 ;1-15:Sample to be tested; WT:Untransfected cells; Water: Negative control
Fig.5 Tpl2-sgRNA1-6# sequencing results
Fig.6 Tpl2-sgRNA1-6# F10 sequencing results
Fig.7 WT MDCK and Tpl2 knockout MDCK cell growth curve
Fig.8 Observation of cell morphology (a)Observation of MDCK morphology(10×) (b)Observation of MDCK-Tpl2-/- morphology(10×)
Fig.9 The cell DNA stained by Hochest
Fig.10 Check the tumorigenicity of MDCK-Tpl2-/- cells (a)Negative control(MDCK cell group) (b)MDCK-Tpl2-/- cell group (c)Positive control(HeLa cell group)
Fig.11 Comparison of HA titers after infected with AIV
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