紧密连接蛋白CLDN4促进猪流行性腹泻病毒感染

doi:10.11843/j.issn.0366-6964.2025.06.026

摘要/Abstract

摘要：

紧密连接蛋白在多种病毒感染过程中起着重要作用，紧密连接蛋白4(claudin-4，CLDN4)是紧密连接蛋白家族中的主要成员之一，其在猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)感染中作用尚不明确。为探索PEDV感染对CLDN4表达的影响以及CLDN4在PEDV感染中的作用，本研究通过荧光定量PCR (qPCR)和Western blot检测了PEDV感染后CLDN4的转录水平与蛋白表达情况。其次，构建了CLDN4基因的真核表达质粒，设计了靶向CLDN4的特异性干扰RNA，通过qPCR、Western blot、间接免疫荧光(IFA)和PEDV病毒滴度测定检测了过表达和敲减CLDN4对PEDV复制的影响。最后，检测了过表达CLDN4对PEDV吸附与侵入阶段的影响。结果显示，PEDV感染可上调细胞中CLDN4的转录与蛋白表达水平，过表达CLDN4显著促进PEDV的复制，而干扰CLDN4表达抑制PEDV复制，并且CLDN4蛋白可促进PEDV的吸附与侵入。本研究表明CLDN4蛋白具有促进PEDV感染与复制的作用，为研究CLDN4的功能与解析PEDV的复制机制提供参考。

关键词: CLDN4, 猪流行性腹泻病毒, 促进, 感染

Abstract:

Tight junction proteins play a crucial role in various viral infections, with tight junction protein 4 (Claudin-4, CLDN4) being a major member of this family. The role of CLDN4 in porcine epidemic diarrhea virus (PEDV) infection remains unclear. To explore the impact of PEDV infection on CLDN4 expression and the role of CLDN4 in PEDV infection, we performed quantitative PCR (qPCR) and Western blot to assess the transcriptional and protein expression levels of CLDN4 following PEDV infection. Additionally, a eukaryotic expression plasmid for the CLDN4 gene was constructed, and specific interference RNA targeting CLDN4 was designed. The effects of overexpression and knockdown of CLDN4 on PEDV replication were analyzed using qPCR, Western blot, immunofluorescence assay (IFA), and PEDV viral titer measurements. Finally, we examined the effects of overexpression of CLDN4 on the adhesion and invasion phases of PEDV infection. Results showed that PEDV infection upregulates CLDN4 transcription and protein levels, while overexpression of CLDN4 significantly promotes PEDV replication, and interference with CLDN4 expression inhibits PEDV replication. Moreover, CLDN4 facilitates PEDV adhesion and invasion. This study suggests that CLDN4 plays a role in promoting PEDV infection and replication, providing a reference for investigating CLDN4 function and elucidating PEDV replication mechanisms.

Key words: CLDN4, porcine epidemic diarrhea virus, promote, infection

中图分类号:

S852.659.6

李程程, 赵永祥, 曹秋霞, 宋旭, 李宇鹏, 范宝超, 郭容利, 徐业芬, 李彬. 紧密连接蛋白CLDN4促进猪流行性腹泻病毒感染[J]. 畜牧兽医学报, 2025, 56(6): 2826-2835.

LI Chengcheng, ZHAO Yongxiang, CAO Qiuxia, SONG Xu, LI Yupeng, FAN Baochao, GUO Rongli, XU Yefen, LI Bin. Tight Junction Protein CLDN4 Promotes Porcine Epidemic Diarrhea Virus Infection[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2826-2835.

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引物/siRNA名称 Primer/siRNA names	序列(5'→ 3') Sequence	用途 Aapplication
CLDN4-F	CTAG$\underline{{\rm{TCTA}}}$GAGCCACCATGGCCTCCATGGGGCTAC	扩增CLDN4
CLDN4-R	CCG$\underline{{\rm{CTCGAG}}}$TCAGATTACAAGGACGACGATGACAAGCACGTAGTTGCTGGCAGC	基因
Q-CLDN4-F	CAGTGCAAGGTGTACGACTC	CLDN4
Q-CLDN4-R	TCATCCTCCAGGCAGTTTGT	基因定量
Q-PEDV-N-F	CGCAAAGACTGAACCCACTAACTT	PEDV-N
Q-PEDV-N-R	TTGCCTCTGTTGTTACTCGGGGAT	基因定量
Q-GAPDH-F	TCACTGCCACCCAGAAGACT	GAPDH
Q-GAPDH-R	ATGACCTTGCCCACACAGCCTT	基因定量
si-CLDN4-F1	CCCUCGUCAUCAUCAGCAUTT	siRNA干扰
si-CLDN4-R1	AUGCUGAUGAUGACGAGGGTT	序列1
si-CLDN4-F2	CGCACAGACAAGCCUUACUTT	siRNA干扰
si-CLDN4-R2	AGUAAGGCUUGUCUGUGCGTT	序列2
si-NC-F	UUCUCC GAACGUGUCACGUTT	干扰对照
si-NC-R	ACGUGACACGUUCGGAGAATT	序列

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