敲除RIG-I基因流感病毒高产MDCK细胞系的建立及功能研究
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摘要
利用CRISPR/Cas9技术构建RIG-I基因敲除MDCK细胞株,并验证其生物学功能。试验设计并构建了2个靶向RIG-I基因的导向RNA(sgRNA)表达载体,与pCAG-Cas9-EGFP表达载体共转染MDCK细胞,采用PAGE胶基因型检测、定点测序和Western blot筛选细胞株;通过荧光定量PCR检测H9N2亚型禽流感病毒(AIV)感染后MAVS、IRF3、IFN-β、IL-6、Mx1 mRNA表达水平和病毒基因拷贝数,测定TCID_(50)。结果表明:获得两株稳定敲除RIG-I基因的MDCK细胞(MDCK-RIG-I~(-/-)),Western blot检测RIG-I蛋白不表达;荧光定量PCR结果显示病毒感染后MDCK-RIG-I~(-/-)的MAVS、IRF3、IFN-β、IL-6、Mx1 mRNA表达水平显著降低,表明RIG-I基因敲除后RIG-I-Ⅰ型干扰素信号通路受阻;病毒基因拷贝数、TCID_(50)测定结果显示,差异最高可分别达到野生型MDCK的2.16倍、3.19倍,表明RIG-I基因敲除后流感病毒复制增加。该研究利用CRISPR/Cas9技术建立了敲除RIG-I基因流感病毒MDCK细胞株,为研究RIG-I天然免疫应答机制奠定基础;该技术方法和策略也为流感疫苗生产及产业更新换代提供候选细胞株。
The RIG-I knockout MDCK cell line was constructed using CRISPR/Cas9 technology and detected its biological function. Two targeting vectors(sgRNA) targeting RIG-I gene were designed and constructed, and MDCK cells were co-transfected with pCAG-Cas9-EGFP expression vector. The cell lines were detected through genotype detection,gene sequencing and western blot and infected by H9 N2 subtype avian influenza virus(AIV), and the mRNA expression levels of MAVS, IRF3, IFN-β, IL-6, Mx1 were detected by real-time PCR. The H9 N2 AIV viral gene copy numbers were also detected by real-time PCR, TCID_(50) was used to determine the level of virus replication.The results showed that two MDCK cells(MDCK-RIG-I~(-/-)) with stable knockout RIG-I gene were obtained, and western blot showed that RIG-I protein was not expressed. The results of real-time PCR showed that the mRNA levels of MAVS, IRF3, IFN-β, IL-6 and Mx1 of MDCK-RIG-I~(-/-)cell line decreased significantly after virus infection, indicating the Type Ⅰ interference signal pathway that mediated by RIG-I was blocked after RIG-I gene knockout. The results of viral gene copy number and TCID_(50) showed that the difference was up to 2.16 times and 3.19 times of wild-type MDCK, indicating that the replication of influenza virus was increased after RIG-I gene knockout. In this study, a MDCK cell line of RIG-I gene knockout was established by CRISPR/Cas9 technology, laid a foundation for studying the natural immune response mechanism of RIG-I. This technical method and strategy could provide candidate cell lines for influenza vaccine production and industrial upgrading.
The RIG-I knockout MDCK cell line was constructed using CRISPR/Cas9 technology and detected its biological function. Two targeting vectors(sgRNA) targeting RIG-I gene were designed and constructed, and MDCK cells were co-transfected with pCAG-Cas9-EGFP expression vector. The cell lines were detected through genotype detection,gene sequencing and western blot and infected by H9 N2 subtype avian influenza virus(AIV), and the mRNA expression levels of MAVS, IRF3, IFN-β, IL-6, Mx1 were detected by real-time PCR. The H9 N2 AIV viral gene copy numbers were also detected by real-time PCR, TCID_(50) was used to determine the level of virus replication.The results showed that two MDCK cells(MDCK-RIG-I~(-/-)) with stable knockout RIG-I gene were obtained, and western blot showed that RIG-I protein was not expressed. The results of real-time PCR showed that the mRNA levels of MAVS, IRF3, IFN-β, IL-6 and Mx1 of MDCK-RIG-I~(-/-)cell line decreased significantly after virus infection, indicating the Type Ⅰ interference signal pathway that mediated by RIG-I was blocked after RIG-I gene knockout. The results of viral gene copy number and TCID_(50) showed that the difference was up to 2.16 times and 3.19 times of wild-type MDCK, indicating that the replication of influenza virus was increased after RIG-I gene knockout. In this study, a MDCK cell line of RIG-I gene knockout was established by CRISPR/Cas9 technology, laid a foundation for studying the natural immune response mechanism of RIG-I. This technical method and strategy could provide candidate cell lines for influenza vaccine production and industrial upgrading.
引文
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[7] KANDASAMY M, SURYAWANSHI A, TUNDUP S, et al.RIG-I signaling is critical for efficient polyfunctional T cell responses during influenza virus infection[J]. PLoS Pathog, 2016, 12(7):1-24.
[8] KIM D, KIM S, KIM S, et al. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq[J]. Genome Res, 2016, 26(3):406-415.
[9] CHENG Y, HUANG Q, JI W, et al. Muscovy duck retinoic acid-induced gene I(MdRIG-I)functions in innate immunity against H9N2 avian influenza viruses(AIV)infections[J]. Vet Immunol Immunop, 2015, 163(3-4):183-193.
[10] SUN B, YU X, KONG W, et al. Production of influenza H1N1 vaccine from MDCK cells using a novel disposable packed-bed bioreactor[J]. Appl Microbiol Biot, 2013, 97(3):1063-1070.
[11] ITSUKI H, HIROSHI T, MASATO T, et al. High yield production of influenza virus in madin darby canine kidney(MDCK)cells with stable knockdown of IRF7[J].PLoS One, 2013, 8(3):e59892.
[12] LIU A L, LI Y F, QI W, et al. Comparative analysis of selected innate immune-related genes following infection of immortal DF-1 cells with highly pathogenic(H5N1)and low pathogenic(H9N2)avian influenza viruses[J]. Virus Genes, 2015, 50(2):189-199.
[2] ZHANG F, WEN Y, GUO X. CRISPR/Cas9 for genome editing:Progress, implications and challenges[J]. Hum Mol Genet, 2014, 23(1):40-46.
[3] LI D, QIU Z, SHAO Y, et al. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system[J]. Nat Biotechnol, 2013(31):681-683.
[4] CHEN Y, CAO J, XIONG M, et al. Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9[J]. Cell Stem Cell, 2015(17):233-244.
[5] OGBOMO H, MICHAELIS M, GEILER J, et al. Tumor cells infected with oncolytic influenza A virus prime natural killer cells for lysis of resistant tumor cells[J]. Med Microbiol Immunol, 2010, 199(2):93-101.
[6] PANG I K, PILLAI P S, IWASAKI A. Efficient influenza A virus replication in the respiratory tract requires signals from TLR7 and RIG-I[J]. Proc Natl Acad Sci,2013, 110(34):13910-13915.
[7] KANDASAMY M, SURYAWANSHI A, TUNDUP S, et al.RIG-I signaling is critical for efficient polyfunctional T cell responses during influenza virus infection[J]. PLoS Pathog, 2016, 12(7):1-24.
[8] KIM D, KIM S, KIM S, et al. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq[J]. Genome Res, 2016, 26(3):406-415.
[9] CHENG Y, HUANG Q, JI W, et al. Muscovy duck retinoic acid-induced gene I(MdRIG-I)functions in innate immunity against H9N2 avian influenza viruses(AIV)infections[J]. Vet Immunol Immunop, 2015, 163(3-4):183-193.
[10] SUN B, YU X, KONG W, et al. Production of influenza H1N1 vaccine from MDCK cells using a novel disposable packed-bed bioreactor[J]. Appl Microbiol Biot, 2013, 97(3):1063-1070.
[11] ITSUKI H, HIROSHI T, MASATO T, et al. High yield production of influenza virus in madin darby canine kidney(MDCK)cells with stable knockdown of IRF7[J].PLoS One, 2013, 8(3):e59892.
[12] LIU A L, LI Y F, QI W, et al. Comparative analysis of selected innate immune-related genes following infection of immortal DF-1 cells with highly pathogenic(H5N1)and low pathogenic(H9N2)avian influenza viruses[J]. Virus Genes, 2015, 50(2):189-199.