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氨基肽酶(pAPN)和唾液神经氨酸酶(NEU3)敲除对TGEV病毒侵染的影响

李兆龙 丰志华 张冰晨 方舟 梁旺旺 陈文志

李兆龙,丰志华,张冰晨,等. 氨基肽酶(pAPN)和唾液神经氨酸酶(NEU3)敲除对TGEV病毒侵染的影响 [J]. 福建农业学报,2021,36(6):506−514
引用本文: 李兆龙,丰志华,张冰晨,等. 氨基肽酶(pAPN)和唾液神经氨酸酶(NEU3)敲除对TGEV病毒侵染的影响 [J]. 福建农业学报,2021,36(6):506−514
LI Z L, FENG Z H, ZHANG B C, et al. Role of pAPN and NEU3 in TGEV Infection on Pig [J]. Fujian Journal of Agricultural Sciences,2021,36(6):506−514
Citation: LI Z L, FENG Z H, ZHANG B C, et al. Role of pAPN and NEU3 in TGEV Infection on Pig [J]. Fujian Journal of Agricultural Sciences,2021,36(6):506−514

氨基肽酶(pAPN)和唾液神经氨酸酶(NEU3)敲除对TGEV病毒侵染的影响

基金项目: 福建省农业科学院对外合作项目(DEC201911);福建省农业科学院自由探索科技创新项目(ZYTS2019017)
详细信息
    作者简介:

    李兆龙(1973−),男,博士,副研究员,主要从事畜禽肠道微生物及分子育种的研究(E-mail:lizhaolong522@163.com

  • 中图分类号: S 858

Role of pAPN and NEU3 in TGEV Infection on Pig

  • 摘要:   目的   氨基肽酶( pAPN )是猪的传染性胃肠炎病毒(TGEV)侵染的主要受体,验证pAPN和唾液酸神经氨酸酶(NEU3)双基因对猪的传染性胃肠炎病毒(TGEV)入侵机制的影响。   方法   应用CRISPR-Cas9基因编辑技术,敲除猪睾丸细胞(ST)的pAPNNEU3两个基因。经过病毒感染试验,测定敲除pAPNNEU3两个目标基因的ST细胞对病毒的侵染变化以及病毒拷贝数的变化、细胞病变改善,同时监测病毒侵染后纤连蛋白的变化。   结果   与对照组相比,敲除pAPNNEU3两个目标基因的ST细胞,明显减轻TGEV侵染引起的细胞病变,TGEV的拷贝数也出现明显下降。此外,同样滴度的TGEV侵染pAPNNEU3双基因敲除ST细胞后,诱导ST细胞的免疫应答物IFNβ的mRNA水平明显低于野生型ST细胞组。   结论   pAPNNEU3双基因的敲除,明显降低了ST细胞中的TGEV病毒拷贝数,同时也减少病毒引起的细胞病变,这个结果证实细胞中的pAPNNEU3双基因可作为将来养猪生产中抗病毒治疗及抗病品种选育的靶基因。
  • 图  1  试验设计

    Figure  1.  Experimental design

    图  2  pAPNNEU3双基因敲除ST细胞电泳结果

    注:1:DNA Marker;2~3:阴性克隆;4~5:阳性克隆。

    Figure  2.  PCR of ST cells after pAPN and NEU3 knockdown

    Note: First band: 5000 DNA marker; 2nd and 3rd bands: negative clones; 4th and 5th bands: positive clones.

    图  3  pAPNNEU3双基因敲除测序结果

    Figure  3.  Sequencing on ST cells after pAPN and NEU3 knockdown

    图  4  pAPNNEU3的敲除及免疫印迹印证(pAPN, NEU3和GAPDH)

    Figure  4.  Western blot on ST cells after pAPN and NEU3 knockdown

    图  5  TGEV感染pAPNNEU3双基因敲除ST细胞和野生型ST细胞结果

    Figure  5.  TGEV infection on pAPN-and-NEU3-knockdown and wild-type ST cells

    图  6  不同滴度的TGEV感染pAPNNEU3双敲除ST细胞和野生型细胞

    Figure  6.  Morphological changes on knockdown and wild-type ST cells induced simultaneously by TGEV of different titers

    表  1  试剂及来源

    Table  1.   Names and suppliers of reagents applied

    试剂 Reagents来源 Sources
    MiniBEST通用DNA提取试剂盒5.0 TaKaRa 9765
    TaKaRa小型DNA片段纯化试剂盒Ver.4.0 TaKaRa 9761
    无内毒素质粒小提试剂盒 康为-cw2106
    MiniBEST通用RNA提取试剂盒 TaKaRa 9767
    PrimeScriptTM常温反应试剂 TaKaRa DRR047A
    SYBR® Premix DimerEraserTM(Perfect Real Time) TaKaRa RR091A
    SYBR® Premix Ex Taq II(Tli RNaseH Plus),ROX Plus TaKaRa RR82WR
    1640 RPMI粉 life technology
    RPMI 1640培养基 Gibco 1598911
    DMEM/F12(高糖) Hyclone 11966-025
    DMEM Gibco 11960-044
    T4 DNA连接酶 TaKaRa 2011B
    TaKaRa Ex Taq® TaKaRa RR001C
    PrimeSTAR® Max DNA聚合酶 TaKaRa R045A
    Premix TaqTM(Ex TaqTM Version 2.0) TaKaRa RR003A
    RNA酶 全式基因-j11030
    FastDigest Sac I ThermoFD1134
    FastDigest Bam HI Thermo FD0055
    FastDigest EcoRI Thermo FD0274
    兔抗抗磷酸化-STAT6抗体 DZ-23319
    兔抗抗-STAT6抗体 CST 9362s
    IRF3(D614C)XP Rabbit mAb CST 11904
    磷酸化-IRF-3(Ser396)(4D4G)兔抗 CST 4947
    下载: 导出CSV

    表  2  sgRNA名称及序列

    Table  2.   Names and sequences of sgRNA

    名称 Name序列 Sequences
    pAPN sgRNA-1F: 5′CACCGGGGCATCCTCCTCGGCGTGG3′
    pAPN sgRNA-1R: 5′AAACCCACGCCGAGGAGGATGCCCC3′
    pAPN sgRNA-2F: 5′CACCGCTACCGCAGCGAGTACATGG3′
    pAPN sgRNA-2R: 5′AAACCCATGTACTCGCTGCGGTAGC3′
    pAPN sgRNA-3F: 5′CACCGCCTCATGTTCGACCGCTCCG3′
    pAPN sgRNA-3R: 5′AAACCGGAGCGGTCGAACATGAGGC3′
    pAPN sgRNA-4F: 5′CACCGCGACAAACTCCGCTCAGCGC3′
    pAPN sgRNA-4R: 5′AAACGCGCTGAGCGGAGTTTGTCG3′
    NEU3 sgRNA1-F: 5′CACCGATGCTCAGTCATCGGCGTGA3′
    NEU3 sgRNA1-R: 5′AAACTCACGCCGATGACTGAGCATC3′
    NEU3 sgRNA2-R: 5′AAACTGGGCAGTACGTATAACTACC3′
    NEU3 sgRNA2-F: 5′CACCGGTAGTTATACGTACTGCCCA3′
    下载: 导出CSV

    表  3  验证引物

    Table  3.   Primers for verification

    名称 Name序列 Sequences
    pAPN F:5′ATGGCCAAGG GATTCTACAT3′
    pAPN R:5′AACCAAGTATCTCGTGTCGATT3′
    NEU3 F:5′ATGGGAAACTCGCCATCAAAAA3′
    NEU3 R:5′TTCTTTCTGTTCTTCTACGCAACT3′
    下载: 导出CSV

    表  4  实时定量PCR引物

    Table  4.   Primers for RT-PCR reaction system

    基因名称 Name of genes序列 Sequences
    h-GAPDH-F CCCTGAGCTGAACGGGAAGCTCAC
    h-GAPDH-R CTTGCTGTAGCCAAATTCGTTGCT
    h-IFNβ-F AGGACAGGATGAACTTTGAC
    h-IFNβ-R TGATAGACATTAGCCAGGAG
    h-Spike protein-F TAATAGCAGT TGTTTCTGCT
    h-Spike protein-R GCATTGCTGT ACTCTTTGTA
    下载: 导出CSV

    表  5  干扰素mRNA

    Table  5.   Real time PCR assay for the expression of IFNβ

    时间
    Time/h
    双基因敲除细胞
    Double knockout/拷贝数
    野生型细胞
    Wild type cells/拷贝数
    20 1 4 1 1103000 1103900 1102700
    40 2 3 3 1290030 1203009 1203007
    60 2 2 3 1322000 1322004 1322043
    80 3 5 5 1103005 1103008 1103009
    100 3 3 3 1140001 1143006 1143003
    120 1 2 3 1120000 1102300 1102307
    下载: 导出CSV

    表  6  纤突蛋白的mRNA

    Table  6.   The T vector containing the TGEV spike protein gene fragment

    时间
    Time/h
    双基因敲除细胞
    Double knockout/拷贝数
    野生型细胞
    Wild type cells/拷贝数
    20 41 43 45 1423341 1322356 1421345
    40 68 67 60 1367893 1323567 1309876
    60 62 69 62 1656431 1665789 1632454
    80 43 50 54 1456445 1433421 1432098
    100 31 35 32 1432223 1432554 1478909
    120 49 35 36 1444009 1422112 1423115
    下载: 导出CSV
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出版历程
  • 收稿日期:  2020-09-15
  • 修回日期:  2021-02-08
  • 网络出版日期:  2021-04-20

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