利用CRISPR/Cas9技术构建流感病毒高产细胞系MDCK-Tpl2~(-/-)
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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。
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 12 h,24 h,48 h,72 h,and the TCID50 titer at 72 h 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 12 h,24 h,48 h,and 72 h,the hemagglutination titer of MDCK-Tpl2~(-/-)cell culture supernatant increased by 1. 78-2. 5 times. At72 h 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.
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 12 h,24 h,48 h,72 h,and the TCID50 titer at 72 h 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 12 h,24 h,48 h,and 72 h,the hemagglutination titer of MDCK-Tpl2~(-/-)cell culture supernatant increased by 1. 78-2. 5 times. At72 h 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.
引文
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[2]Nerome K,Ishida M. The multiplication of an influenza C virus in an established line of canine kidney(MDCK)cells. Journal of General Virology,1978,39(1):179-181.
[3]Barrett P N,Mundt W,Kistner O,et al. Vero cell platform in vaccine production:moving towards cell culture-based viral vaccines. Expert Review of Vaccines,2009,8(5):607-618.
[4]Bonjardim C,Ferreira P E. Interferons:signaling,antiviral and viral evasion. Immunology Letters,2009,122(1):1-11.
[5]Hamamoto I,Takaku H,Tashiro M,et al. High yield production of influenza virus in Madin Darby canine kidney(MDCK)cells with stable knockdown of IRF7. PLo S One,2013,8(3):e59892.
[6]Liu A L,Li Y F,Qi W,et al. Comparative analysis of selected innate immune-related genes following infection of immortal DF-1cells with highly pathogenic(H5N1)and low pathogenic(H9N2)avian influenza viruses. Virus Genes,2015,50(2):189-199.
[7]Yi E,Oh J,Giao N Q,et al. Enhanced production of enveloped viruses in BST-2-deficient cell lines. Biotechnology&Bioengineering,2017,114(10):2289-2297.
[8] Luo W,Zhang J,Liang L,et al. Phospholipid scramblase 1interacts with influenza A virus NP,impairing its nuclear import and thereby suppressing virus replication. PLo S Pathogens,2018,14(1):e1006851.
[9]Makris A,Patriotis C,Bear S E,et al. Genomic organization and expression of Tpl-2 in normal cells and moloney murine leukemia virus-induced rat T-cell lymphomas:activation by provirus insertion. Journal of Virology,1993,67(7):4283-4289.
[10]Kuriakose T,Tripp R A,Watford W T. Tumor progression locus2 promotes induction of IFNλ,interferon stimulated genes and antigen-specific CD8+T cell responses and protects against influenza virus. PLo S Pathogens,2015,11(8):e1005038.
[11]国家药典委员会.中华人民共和国药典2010版.北京:中国医药科技出版社,2010.Chinese Pharmacopoeia Commission. Pharmacopoeia of People’s Republic of China:2010 Edition. Beijing:China Medical Science Press,2010.
[12] Mussolino C,Cathomen T. RNA guides genome engineering.Nature Biotechnology,2013,31(3):208-209.
[13]Onions D,Egan W,Jarrett R,et al. Validation of the safety of MDCK cells as a substrate for the production of a cell-derived influenza vaccine. Biologicals,2010,38(5):544-551.
[14]Medema J K,Meijer J,Kersten A J,et al. Safety assessment of Madin Darby canine kidney cells as vaccine substrate.Developmental Biology,2006,123:243-250.
[15]Gregersen J P,Schmitt H J,Trusheim H,et al. Safety of MDCK cell culture-based influenza vaccines. Future Microbiology,2011,6(2):143-152.
[16]陈超,池晓娟,白庆玲,等.甲型流感病毒感染过程中干扰素介导的天然免疫应答机制.生物工程学报,2015,31(12):1671-1681.Chen C, Chi X J, Bai Q L, et al. Mechanisms underlying interferon-mediated host innate immunity during influenza A virus infection. Chin J Biotech,2015,31(12):1671-1681.