SOX12基因在Vero细胞感染猪流行性腹泻病毒过程中的功能研究

doi:10.16431/j.cnki.1671-7236.2025.01.026

摘要/Abstract

摘要： 【目的】探究SOX12基因对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)复制的影响,以期为抗PEDV育种提供有效分子标记。【方法】本研究合成3条SOX12基因干扰序列(siRNA1、siRNA2、siRNA3)及其阴性对照(siNC)并转染至非洲绿猴肾细胞(Vero细胞),转染48 h后收集细胞,利用实时荧光定量PCR检测干扰效率。将有效的干扰片段和siNC分别转染至Vero细胞24 h后,用感染复数为0.05的PEDV感染细胞,分为4组:感染PEDV 12 h试验组(12 hpi-siRNA)、感染PEDV 12 h对照组(12 hpi-siNC)、感染PEDV 24 h试验组(24 hpi-siRNA)及感染PEDV 24 h对照组(24 hpi-siNC),利用实时荧光定量PCR检测PEDV N及SOX12基因的表达水平,通过Western blotting检测各组细胞PEDV N蛋白表达水平,利用组织半数感染量(50% tissue culture infective dose,TCID₅₀)检测PEDV的病毒滴度,并利用间接免疫荧光(indirect immunofluorescence assay,IFA)法检测各组细胞中PEDV复制情况。通过GeneMANIA网站预测SOX12基因的互作基因,利用实时荧光定量PCR检测抑制SOX12基因表达后其互作基因的表达变化。【结果】 SOX12基因3条干扰片段的干扰效率为30%~60%,其中siRNA3干扰效率最高,可达60%。与12 hpi-siNC和24 hpi-siNC组相比,12 hpi-siRNA和24 hpi-siRNA组PEDV N基因的转录和蛋白表达水平均显著降低(P＜0.05)。病毒滴度表型测定结果表明,12 hpi-siRNA和24 hpi-siRNA组的PEDV病毒滴度显著低于各自对照组(P＜0.05);IFA结果也表明,12 hpi-siRNA和24 hpi-siRNA组的PEDV复制明显少于对照组。基因互作预测及实时荧光定量PCR检测结果显示,与siNC组相比,干扰SOX12基因的表达后,其互作基因SOX17、DDX51、ZMIZ2、SSRP1、TAF6和ABCB8的表达水平均显著降低(P＜0.05)。【结论】本研究揭示了SOX12基因在PEDV感染Vero细胞过程中的调控作用,发现SOX12基因的下调表达能显著抑制PEDV复制和其互作基因SOX17、DDX51、ZMIZ2、SSRP1、TAF6和ABCB8的表达。

关键词: 猪流行性腹泻病毒(PEDV); SOX12基因; Vero细胞; 基因干扰

Abstract: 【Objective】 The aim of this study was to investigate the effect of SOX12 gene on the replication of Porcine epidemic diarrhea virus (PEDV),so as to provide the effective molecular marker for anti-PEDV breeding. 【Method】 Three SOX12 gene interference sequences (siRNA1,siRNA2 and siRNA3) and a negative control sequence (siNC) were synthesized and transfected into African green monkey kidney cells (Vero cells) in this study.At 48 h after transfection,the cells were collected,and the interference efficiency was detected by Real-time quantitative PCR.Effective interference fragments and siNC were transfected into Vero cells for 24 h,respectively.Then,the cells were infected with PEDV at a multiplicity of infection of 0.05,and divided into four groups:PEDV-infected assay group at 12 h (12 hpi-siRNA),PEDV-infected control group at 12 h (12 hpi-siNC),PEDV-infected assay group at 24 h (24 hpi-siRNA),and PEDV-infected control group at 24 h (24 hpi-siNC).Cell samples from each group were collected and their RNA was extracted.The expression of PEDV N and SOX12 genes was detected by Real-time quantitative PCR,and the expression of PEDV N protein was analyzed by Western blotting.The viral titer of PEDV was determined through 50% tissue culture infective dose (TCID₅₀) assay,and the replication of PEDV of cells in each group was detected using indirect immunofluorescence (IFA) method.The SOX12 interaction genes were predicted using GeneMANIA website,and the expression changes of these interaction genes were detected by Real-time quantitative PCR after inhibiting SOX12 gene expression. 【Result】 The interference efficiency of the three SOX12 interfering fragments was 30% to 60%,with siRNA3 showing the highest efficiency of 60%.Compared with 12 hpi-siNC and 24 hpi-siNC groups,the transcription and protein expression of PEDV N gene in 12 hpi-siRNA and 24 hpi-siRNA groups was significantly decreased,respectively (P＜0.05).The results of the viral titer phenotype determination indicated that the viral titers of PEDV in 12 hpi-siRNA and 24 hpi-siRNA groups were significantly lower than that in their control groups,respectively (P＜0.05).IFA results also showed that PEDV replication in 12 hpi-siRNA and 24 hpi-siRNA groups was significantly less than that in control group.The gene interaction prediction and Real-time quantitative PCR detection results revealed that compared with siNC group,the expression of interacting genes (SOX17,DDX51,ZMIZ2,SSRP1,TAF6 and ABCB8) were significantly decreased after interfering with SOX12 gene expression (P＜0.05). 【Conclusion】 This study revealed that the regulatory role of SOX12 gene in the process of PEDV infection in Vero cells,and found that the downregulation of SOX12 gene expression significantly inhibited PEDV replication and the expression of its interacting genes SOX17, DDX51,ZMIZ2,SSRP1,TAF6 and ABCB8.

Key words: Porcine epidemic diarrhea virus (PEDV); SOX12 gene; Vero cells; gene interference

中图分类号:

S852.65⁺1

向娇娇, 元娜, 李慧慧, 邵明珠, 赵福平, 张龙超, 王立贤, 石丽君, 陈斌. SOX12基因在Vero细胞感染猪流行性腹泻病毒过程中的功能研究[J]. 中国畜牧兽医, 2025, 52(1): 289-297.

XIANG Jiaojiao, YUAN Na, LI Huihui, SHAO Mingzhu, ZHAO Fuping, ZHANG Longchao, WANG Lixian, SHI Lijun, CHEN Bin. Function Study on SOX12 Gene in Vero Cells Infected with Porcine Epidemic Diarrhea Virus[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(1): 289-297.

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