CD163单等位基因表达的iPAMs细胞系的构建及其介导PRRSV感染的特征分析

doi:10.16431/j.cnki.1671-7236.2023.07.003

摘要/Abstract

摘要： 【目的】试验旨在利用CRISPR/Cas9基因编辑技术获得CD163（cluster of differentiation 163）单等位基因表达的永生化猪肺泡巨噬细胞系（immortalized porcine alveolar macrophages，iPAMs），并探究其介导猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus，PRRSV）感染的特征，为深入研究CD163基因在PRRSV感染中的作用机制奠定基础。【方法】在猪CD163基因外显子1区域设计8条向导RNA （single guide RNA，sgRNA），并将其连接到pX458载体中，通过T7E1酶切试验检测不同sgRNA载体的活性；将活性较高的3条sgRNAs表达载体电转染到iPAMs中，通过流式细胞术分选单克隆细胞，对获得的单克隆细胞进行基因型鉴定、蛋白表达检测、脱靶分析以及PRRSV感染分析。【结果】在设计的8条sgRNAs中有3条基因编辑效率在28%以上。基因型鉴定结果表明，50株片段敲除的单克隆细胞中有4株符合预期的CD163单等位基因表达的iPAMs，效率为8%。蛋白表达检测和脱靶分析结果显示，CD163单等位基因表达的iPAMs中CD163蛋白表达量显著下降（P<0.05），且在预测位置未发生脱靶现象。PRRSV感染分析显示，CD163单等位基因表达的iPAMs可以正常感染PRRSV，病毒拷贝数和PRRSV-N蛋白表达量与野生型细胞无显著差异（P>0.05）。【结论】本研究利用CRISPR/Cas9编辑系统构建了可以正常感染PPRSV的CD163单等位基因表达的iPAMs，为阐明CD163在猪繁殖与呼吸综合征传染病中所起的作用以及培育抗病猪新品种奠定了基础。

关键词: CD163; 猪繁殖与呼吸综合征病毒(PRRSV); CRISPR/Cas9; 永生化猪肺泡巨噬细胞系(iPAMs)

Abstract: 【Objective】 This study was to obtain an immortalized porcine alveolar macrophages (iPAMs) with CD163 (cluster of differentiation 163) monoallelic expression by using CRISPR/Cas9 gene editing technique, and explore the characteristics of their mediating Porcine reproductive and respiratory syndrome virus (PRRSV) infection, which laid the foundation for further study on the mechanism of CD163 gene in PRRSV infection.【Method】 Eight single guide RNA (sgRNA) in exon 1 of porcine CD163 gene were designed and introduced into pX458 vector, and the editing efficiency of different sgRNA vectors was detected by T7E1 assay.Three highly active sgRNA vector was electrotransfected into iPAMs, and the monoclonal cells were isolated by flow cytometry.Then genotypic identification, protein expression detection, off-target analysis and PRRSV infection analysis were performed on the obtained monoclonal cells.【Result】 The editing rate of 3 of the 8 sgRNAs was more than 28%.The results of genotype identification showed that 4 iPAMs cell lines with expected CD163 monoallelic expression were selected, with an efficiency of 8%.The results of protein expression detection and off-target analysis showed that the expression of CD163 protein significantly decreased in CD163 single allele expression cell line (P<0.05), and there was no off-target event at the predicted site.PRRSV infection analysis showed that CD163 monoallelic expression cell lines could be infected with PRRSV normally, and there was no significant difference in virus copy number and the expression of PRRSV-N protein between the CD163 monoallelic expression cells and wild-type cells (P>0.05).【Conclusion】 In this study, an iPAMs cell lines normally infecting PPRSV with CD163 monoallelic expression was constructed using CRISPR/Cas9 editing system, which laid a foundation for elucidating the role of CD163 in PRRSV infection and breeding new disease-resistant pig breeds.

Key words: CD163; Porcine reproductive and respiratory syndrome virus(PRRSV); CRISPR/Cas9; immortalized porcine alveolar macrophages (iPAMs)

中图分类号:

S828.9

王慧, 冯保亮, 向光明, 黄雷, 刘志国, 李奎, 牟玉莲. CD163单等位基因表达的iPAMs细胞系的构建及其介导PRRSV感染的特征分析[J]. 中国畜牧兽医, 2023, 50(7): 2617-2628.

WANG Hui, FENG Baoliang, XIANG Guangming, HUANG Lei, LIU Zhiguo, LI Kui, MU Yulian. Construction of iPAMs with CD163 Monoallelic Expression and Characterization of Their Mediating PRRSV Infection[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2617-2628.

参考文献

[1] TIAN K, YU X, ZHAO T, et al.Emergence of fatal PRRSV variants:Unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark[J].PLoS One, 2007, 2(6):e526.
[2] MENG X J, PAUL P S, HALBUR P G, et al.Phylogenetic analyses of the putative M (ORF6) and N (ORF7) genes of Porcine reproductive and respiratory syndrome virus (PRRSV):Implication for the existence of two genotypes of PRRSV in the U.S.A. and Europe[J].Archives of Virology, 1995, 140(4):745-755.
[3] DUAN X, NAUWYNCK H J, PENSAERT M B.Effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to Porcine reproductive and respiratory syndrome virus (PRRSV)[J].Archives of Virology, 1997, 142(12):2483-2497.
[4] 牛军伟, 郭龙军, 谷伟红, 等.稳定表达猪繁殖与呼吸综合征病毒受体CD163的HEK293细胞系的建立[J].中国预防兽医学报, 2015, 37(3):163-166. NIU J W, GUO L J, GU W H, et al.Establishment of HEK293 cell lines stably expressing Porcine reproductive and respiratory syndrome virus receptor CD163[J].Chinese Journal of Preventive Veterinary Medicine, 2015, 37(3):163-166.(in Chinese)
[5] DELPUTTE P L, VANDERHEIJDEN N, NAUWYNCK H J, et al.Involvement of the matrix protein in attachment of Porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages[J].Journal of Virology, 2002, 76:4312-4320.
[6] VANDERHEIJDEN N, DELPUTTE P, NAUWYNCK H, et al.Effects of heparin on the entry of Porcine reproductive and respiratory syndrome virus into alveolar macrophages[J].Advances in Experimental Medicine and Biology, 2001, 494:683-689.
[7] ANDERSEN C B, TORVUND-JENSEN M, NIELSEN M J, et al.Structure of the haptoglobin-haemoglobin complex[J].Nature, 2012, 489(7416):456-459.
[8] VAN GORP H, VAN BREEDAM W, VAN DOORSSELAERE J, et al.Identification of the CD163 protein domains involved in infection of the Porcine reproductive and respiratory syndrome virus[J].Journal of Virology, 2010, 84(6):3101-3105.
[9] WHITWORTH K M, ROWLAND R R, EWEN C L, et al.Gene-edited pigs are protected from Porcine reproductive and respiratory syndrome virus[J].Nature Biotechnology, 2016, 34(1):20-22.
[10] XU K, ZHOU Y, MU Y, et al.CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance[J].eLife, 2020, 9:e57132.
[11] KRISTIANSEN M, GRAVERSEN J H, JACOBSEN C, et al.Identification of the haemoglobin scavenger receptor[J].Nature, 2001, 409(6817):198-201.
[12] FABRIEK B O, POLFLIET M M, VLOET R P, et al.The macrophage CD163 surface glycoprotein is an erythroblast adhesion receptor[J].Blood, 2007, 109(12):5223-5229.
[13] MORENO J A, MUÑOZ-GARCÍA B, MARTÍN-VENTURA J L, et al.The CD163-expressing macrophages recognize and internalize TWEAK:Potential consequences in atherosclerosis[J].Atherosclerosis, 2009, 207(1):103-110.
[14] WANG T Y, LIU Y G, LI L, et al.Porcine alveolar macrophage CD163 abundance is a pivotal switch for Porcine reproductive and respiratory syndrome virus infection[J].Oncotarget, 2018, 9(15):12174-12185.
[15] LI B, FANG L, XU Z, et al.Recombination in vaccine and circulating strains of Porcine reproductive and respiratory syndrome viruses[J].Emerging Infectious Disease, 2009, 15(12):2032-2035.
[16] CONCORDET J P, HAEUSSLER M.CRISPOR:Intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens[J].Nucleic Acids Research, 2018, 46(W1):W242-W245.
[17] 刘思远, 卢丹, 唐中林.CRISPR/Cas9技术及其在猪基因编辑中的应用[J].畜牧兽医学报, 2020, 51(3):409-416. LIU S Y, LU D, TANG Z L.Research progress on CRISPR/Cas9 and its application in pig genome editing[J].Acta Veterinaria et Zootechnica Sinica, 2020, 51(3):409-416.(in Chinese)
[18] BEERLI R R, BARBAS III C F.Engineering polydactyl zinc-finger transcription factors[J].Nature Biotechnology, 2002, 20(2):135-141.
[19] MUSSOLINO C, MORBITZER R, LVTGE F, et al.Anovel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity[J].Nucleic Acids Research, 2011, 39(21):9283-9293.
[20] ZHANG F, WEN Y, GUO X.CRISPR/Cas9 for genome editing:Progress, implications and challenges[J].Human Molecular Genetics, 2014, 23(R1):R40-R46.
[21] HWANG W Y, FU Y, REYON D, et al.Heritable and precise zebrafish genome editing using a CRISPR-Cas system[J].PLoS One, 2013, 8(7):e68708.
[22] BASSETT A R, TIBBIT C, PONTING C P, et al.Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system[J].Cell Reports, 2013, 4(1):220-228.
[23] DICKINSON D J, GOLDSTEIN B.CRISPR-Based methods for caenorhabditis elegans genome engineering[J].Genetics, 2016, 202(3):885-901.
[24] NIU Y, SHEN B, CUI Y, et al.Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos[J].Cell, 2014, 156(4):836-843.
[25] WANG K, OUYANG H, XIE Z, et al.Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system[J].Science Reports, 2015, 5:16623.
[26] ZHENG Q, LIN J, HUANG J, et al.Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity[J].Proceedings of the National Academy of Sciences, 2017, 114(45):E9474-E9482.
[27] XIANG G, REN J, HAI T, et al.Editing porcine IGF2 regulatory element improved meat production in Chinese Bama pigs[J].Cellular and Molecular Life Sciences, 2018, 75(24):4619-4628.
[28] CALVERT J G, SLADE D E, SHIELDS S L, et al.CD163 expression confers susceptibility to Porcine reproductive and respiratory syndrome viruses[J].Journal of Virology, 2007, 81(14):7371-7379.
[29] WHITWORTH K M, LEE K, BENNE J A, et al.Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos[J].Biology of Reproduction, 2014, 91(3):78.
[30] CHEN J, WANG H, BAI J, et al.Generation of pigs resistant to highly pathogenic-Porcine reproductive and respiratory syndrome virus through gene editing of CD163[J].International Journal of Biological Sciences, 2019, 15(2):481-492.
[31] WANG H, SHEN L, CHEN J, et al.Deletion of CD163 exon 7 confers resistance to highly pathogenic Porcine reproductive and respiratory viruses on pigs[J].International Journal of Biological Sciences, 2019, 15(9):1993-2005.
[32] GUO C, WANG M, ZHU Z, et al.Highly efficient generation of pigs harboring a partial deletion of the CD163 SRCR5 domain, which are fully resistant to Porcine reproductive and respiratory syndrome virus 2 infection[J].Frontier in Immunology, 2019, 10:1846.
[33] MA H, LI R, JIANG L, et al.Structural comparison of CD163 SRCR5 from different species sheds some light on its involvement in Porcine reproductive and respiratory syndrome virus-2 infection in vitro[J].Veterinary Research, 2021, 52(1):97.
[34] XU H, LIU Z, ZHENG S, et al.CD163 antibodies inhibit PRRSV infection via receptor blocking and transcription suppression[J].Vaccines (Basel), 2020, 8(4):592.
[35] STOIAN A M M, ROWLAND R R R, BRANDARIZ-NUÑEZ A.Mutations within scavenger receptor cysteine-rich (SRCR) protein domain 5 of porcine CD163 involved in infection with Porcine reproductive and respiratory syndrome virus (PRRS)[J].Journal of General Virology, 2022, 103(5):10.
[36] 徐长江, 王晓朋, 徐奎, 等.利用CRISPR/Cas9编辑系统构建pAPN基因敲除的IPI-2I细胞系[J].中国畜牧兽医, 2021, 48(7):2282-2290. XU C J, WANG X P, XU K, et al.Establishment of pAPN gene knockout IPI-2I cell lines mediated by CRISPR/Cas9 system[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(7):2282-2290.(in Chinese)
[37] BURKARD C, LILLICO S G, REID E, et al.Precision engineering for PRRSV resistance in pigs:Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function[J].PLoS Pathogens, 2017, 13(2):e1006206.