pAPN基因敲除的IPEC-J2介导的TGEV感染特征分析

doi:10.11843/j.issn.0366-6964.2024.08.013

摘要/Abstract

摘要：

旨在探究猪氨基肽酶N(porcine aminopeptidase N，pAPN)基因敲除的猪空肠上皮细胞系(intestinal porcine epithelial cell line J2，IPEC-J2)介导猪传染性胃肠炎病毒(transmissible gastroenteritis virus，TGEV)感染的特征，为深入了解pAPN基因在TGEV感染过程中的作用机制提供理论依据。研究分为pAPN基因敲除IPEC-J2组(IPEC-J2-KO组)、野生型IPEC-J2组(IPEC-J2-WT组)和未接种TGEV的野生型IPEC-J2组(Mock组)3组，每组设置3个重复。首先通过实时荧光定量PCR(quantitative real-time PCR，qPCR)确定IPEC-J2接种TGEV毒株后收取细胞样品的最佳时间节点；其次，对IPEC-J2-KO进行了脱靶效应检测；然后，通过qPCR、蛋白免疫印迹(western blot，WB)、间接免疫荧光分析(indirect immunofluorescence assay，IFA)和50%组织细胞感染量(50% tissue culture infective dose，TCID₅₀)对接种TGEV的IPEC-J2-KO、IPEC-J2-WT及Mock进行感染特征分析；最后，通过WB检测IPEC-J2-KO、IPEC-J2-WT和Mock中NF-κB p65及其磷酸化蛋白pp65的表达情况。qPCR结果显示，接毒24 h是收集细胞样本以评估TGEV对IPEC-J2影响的最佳时间节点；脱靶分析结果显示，在IPEC-J2-KO中未检测到脱靶效应；病毒感染特征分析结果显示，与IPEC-J2-WT相比，IPEC-J2-KO内病毒拷贝数、病毒滴度均极显著降低(P＜0.001)，与Mock相比，IPEC-J2-KO内病毒拷贝数、病毒滴度均无显著差异(P>0.05)，且IPEC-J2-KO内未检测到TGEV-N蛋白的表达；此外，与Mock相比，接种TGEV后，IPEC-J2-WT组NF-κB p65的磷酸化水平极显著上调(P＜0.001)，而IPEC-J2-KO组无显著差异(P>0.05)。本研究表明, IPEC-J2-KO可有效抵抗TGEV的感染，接种TGEV未影响IPEC-J2-KO中先天免疫相关信号通路中转录因子NF-κB的活性。该研究为IPEC-J2作为TGEV感染特征研究的细胞模型提供了理论依据，为阐明pAPN基因在TGEV入侵宿主细胞的机制及抗病猪新品种的研究奠定了基础。

关键词: 猪氨基肽酶N, IPEC-J2, TGEV, NF-κB

Abstract:

The aim of this study was to explore the characteristics of porcine transmissible gastroenteritis virus (TGEV) infection mediated by porcine aminopeptidase N (pAPN) gene knockout intestinal porcine epithelial cell line J2 (IPEC-J2) and to provide theoretical basis for further understanding the mechanism of pAPN gene in the process of TGEV infection. The study was divided into 3 groups, pAPN gene knockout IPEC-J2 group (IPEC-J2-KO group), wild-type IPEC-J2 group (IPEC-J2-WT group), and wild-type IPEC-J2 group not inoculated with TGEV (Mock group), with 3 replicates set up in each group. Firstly, quantitative real-time PCR (qPCR) was used to determine the best time point for collecting cell samples following inoculation with TGEV strain. Secondly, the off-target effect of IPEC-J2-KO was detected. Then, the infection characteristics of IPEC-J2-KO, IPEC-J2-WT inoculated with TGEV and Mock were analyzed by qPCR, western blot (WB), indirect immunofluorescence assay (IFA), and 50% tissue culture infective dose (TCID₅₀). Finally, the expression of NF-κB p65 and its phosphorylated protein pp65 in Mock, IPEC-J2-WT and IPEC-J2-KO were detected by WB. The results of qPCR showed that 24 hours after exposure was the best time point to collect cell samples to evaluate the impact of TGEV on IPEC-J2; The off-target analysis results showed that no off-target effects were detected in IPEC-J2-KO; The results of virus infection characteristic analysis showed that the virus copy number and virus titer in IPEC-J2-KO were significantly lower than those in IPEC-J2-WT (P < 0.001). Compared with the Mock, there was no significant difference in virus copy number and virus titer in IPEC-J2-KO (P>0.05), and no expression of TGEV-N protein was detected in IPEC-J2-KO. In addition, compared with the Mock, after inoculation with TGEV, the phosphorylation level of NF-κB p65 was significantly increased in the IPEC-J2-WT group (P < 0.001), while there was no significant difference in IPEC-J2-KO group (P>0.05). This study results showed that IPEC-J2-KO can effectively resist TGEV infection, and TGEV did not affect the activity of the transcription factor NF-κB in the innate immunity-related signaling pathway in IPEC-J2-KO. This study provided the evidence that IPEC-J2 could serve as a cell model for the study of TGEV infection characteristics, and laid the foundation for elucidating the mechanism of pAPN gene in TGEV invading host cells and researching new disease-resistant pig varieties.

Key words: porcine aminopeptidase N, IPEC-J2, TGEV, NF-κB

中图分类号:

S813.1

夏振涛, 王楠, 王婉洁, 周期律, 黄雷, 牟玉莲. pAPN基因敲除的IPEC-J2介导的TGEV感染特征分析[J]. 畜牧兽医学报, 2024, 55(8): 3395-3407.

Zhentao XIA, Nan WANG, Wanjie WANG, Qilü ZHOU, Lei HUANG, Yulian MU. Characteristics Analysis of TGEV Infection Mediated by IPEC-J2 with Knockout of pAPN Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3395-3407.

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潜在脱靶位点 Potential off-target sites	序列(5′→3′) Sequence
g3 On-target	TACCTCACTCCCAACGCGGATGG
g5 On-target	GGGGGAACTTGCCGACGACCTGG
g3-OT1	aggCTCACTCCCAACGCtGATGG
g3-OT2	TcCCgCAtTCCCAtCGCGGATGG
g3-OT3	TAtCTCcCTtCCAACGCGGgAGG
g3-OT4	cACCTCcCcCCCAAaGCGGACGG
g3-OT5	TACCaCtCTCCCAACaCtGAAGG
g3-OT6	TtCCTCACgCCCAACGCctAGGG
g3-OT7	TACCTggCTCCCAACGaGGtGGG
g3-OT8	TACCTCAggagCAACGCGGATGG
g5-OT1	GtGaGAAaTTGCCGAaGACCAGG
g5-OT2	aGGcGgACTTGCCGAaGACCAGG
g5-OT3	GGGGcAtCcaGCCGACGACCCGG
g5-OT4	GcGGGAACagGCCGgCGACCTGG
g5-OT5	GGGtGAACgTGCCaAaGACCTGG
g5-OT6	GcaGGAACTTGCtGACGgCCAGG
g5-OT7	tGGGGAACTTGCCGgCGAaaAGG
g5-OT8	GGGGGcACTTGCtGACagCCTGG

引物 Primer	引物序列(5′→3′) Primers sequence
g3-OT1	F: AGAGTTTTGAAGGCCTGGTG	R: TGAAGCTGATGGCACCTTTT
g3-OT2	F: CGCAGGGACAGCTTTTAAGA	R: AGACAAATGGACCCTGGAGA
g3-OT3	F: CCAAGGAATCTCAGACTGGC	R: TCAGAATTTGGCAGCCCTTT
g3-OT4	F: ACATTGAGCTTGTCTGGACC	R: GTCCTGAGCTGGTACGAAAG
g3-OT5	F: GTCACAGGGTTACCTCCTCT	R: GACAAAGATGGAGTGGAGGC
g3-OT6	F: AGGTCAAGATGCTGGACTCT	R: AGTCCATGAAAGGGAGTCCA
g3-OT7	F: CGTTTTACACCCATGGTCCA	R: CTTCCAGTGAGAGCAACTGG
g3-OT8	F: GAACAGCAGAGTCAGAGCTC	R: CCCAAAAGCTCTCTCTCGAC
g5-OT1	F: AAGACTTCATGCCTCAGCTG	R: GGTTTGGGGAGAAGTCACTG
g5-OT2	F: CGAAGGAGAAGAACCAGTGG	R: CCTTCATCAAGACCATCCGG
g5-OT3	F: GTGAGAGGATGGCGAACC	R: GATGCTGAGGGTTGAGTGAG
g5-OT4	F: GCAATGTTCTCGGCCAAAC	R: ACCAGAAGCTTCTCTCTCCA
g5-OT5	F: GTTGTCAGACTCCAACCTGG	R: GCACAGGGTGGTCACATTAA
g5-OT6	F: TGTGCTACTACAACGTGCTG	R: CACGCTGAACACGTGGTAG
g5-OT7	F: GCTTTCCTCACCGGATGAAT	R: AGTATCCTTGTCTGGCCAGT
g5-OT8	F: GCCTATCCCTTCCTTCTTGC	R: TCACAGTGAATTGGACTGCC

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