Role of pAPN and NEU3 in TGEV Infection on Pig
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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pAPN和NEU3两个基因。经过病毒感染试验，测定敲除pAPN和NEU3两个目标基因的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pAPN和NEU3两个目标基因的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pAPN和NEU3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 pAPN和NEU3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pAPN和NEU3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。 Abstract:Objective Role of aminopeptidase gene pAPN and sialic acid neuraminidase gene NEU3 in the transmissible gastroenteritis virus (TGEV) infection on pigs was investigated. Methods Being the main receptor of TGEV, pAPN was removed from pAPN and NEU3 in ST cells to verify its supposed key function on the disease. The CRISPR gene editing technique was applied to clip the target gene in ST cells prior to an artificial TGEV infection test. The resulting changes on the infection, virus copy number, cytopathic improvement, and fibronectin were monitored. Results Compared with control, the ST cells free of pAPN and NEU3 significantly attenuated TGEV infection-induced cytopathies and the virus copy number. In addition, at a same TGEV titer the mRNA immune responders induced by the knockdown ST cells were significantly lower than the wild-type counterparts. Conclusion It was confirmed that the removal of pAPN and NEU3 inhibited the TGEV infection in pigs with reduced viral induced cytopathies. Thus, an antiviral therapy and a guideline for breeding resistant pigs could be developed by targeting these two key genes in the ST cells. -
Key words:
- pAPN /
- NEU3 /
- TGEV /
- Titer of virus
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图 2 pAPN和NEU3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 4 pAPN和NEU3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pAPN和NEU3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pAPN和NEU3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 
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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′
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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′
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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
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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
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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
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