DDX1影响猪流行性腹泻病毒复制的机制研究

doi:10.16431/j.cnki.1671-7236.2023.04.027

摘要/Abstract

摘要： 【目的】探究RNA解旋酶DEAD-box家族蛋白DDX1在猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)感染猪回肠上皮细胞(IPI-2I)期间发挥的作用,为揭示DDX1的生物学功能奠定基础。【方法】参照GenBank中猪源DDX1基因序列(登录号:XM_021087822.1),设计特异性引物PCR扩增DDX1基因全长并连接至pMD19-T克隆载体,双酶切鉴定成功后连接至pcDNA3.1表达载体;连接产物转化后筛选阳性菌落;培养IPI-2I细胞,取传代2次的细胞铺于6孔板内进行后续试验,将阳性菌落的重组质粒转染至6孔板培养24 h后收集蛋白质,Western blotting检测DDX1转染效率;以感染复数(MOI)为1.0的PEDV感染已转染pcDNA3.1-DDX1或PBS (对照)的IPI-2I细胞,建立PEDV感染细胞模型,PEDV感染12 h后分别收集细胞总蛋白质和RNA;间接免疫荧光试验检测细胞水平上PEDV N蛋白的表达,实时荧光定量PCR检测PEDV N基因mRNA表达水平,Western blotting检测PEDV N蛋白的表达水平;使用生物信息学软件预测DDX1蛋白的磷酸化位点;免疫沉淀试验检测PEDV感染IPI-2I细胞12 h后DDX1的磷酸化水平。【结果】PCR成功扩增得到大小为2 223 bp的DDX1目的基因条带。双酶切鉴定结果显示,成功构建pMD19-T-DDX1克隆载体和pcDNA3.1-DDX1真核表达载体。与PBS组相比,转染pcDNA3.1-DDX1的细胞DDX1蛋白表达水平极显著上升(P＜0.01),表明DDX1转染成功。间接免疫荧光试验结果显示,与PBS组相比,PEDV感染组中出现大量绿色荧光信号,表明PEDV感染IPI-2I细胞模型构建成功;与PBS组相比,PEDV N蛋白的荧光信号明显减少,PEDV N基因mRNA表达水平及蛋白表达水平均极显著降低(P＜0.01)。生物信息学分析发现,DDX1蛋白具有多个磷酸化位点,其中包括17个丝氨酸、12个苏氨酸及6个酪氨酸。免疫沉淀试验结果显示,与未感染组相比,PEDV感染组中DDX1磷酸化水平极显著上升(P＜0.01)。【结论】本研究成功构建了pcDNA3.1-DDX1真核表达载体,发现PEDV感染IPI-2I细胞12 h后,DDX1抑制了PEDV的复制,升高了磷酸化水平,说明DDX1在PEDV感染期间可能通过磷酸化抑制PEDV的复制。

关键词: DDX1; 猪流行性腹泻病毒(PEDV); 磷酸化

Abstract: 【Objective】 The purpose of this study was to explore the function of RNA helicase DEAD-box family protein DDX1 in porcine ileal epithelial cells (IPI-2I) infected by Porcine epidemic diarrhea virus (PEDV),and lay a foundation for revealing the biological function of DDX1.【Method】 According to the sequence of porcine DDX1 gene in GenBank (accession No.XM_021087822.1),specific primers were designed.The full length of DDX1 gene was amplified by PCR and ligated into pMD19-T cloning vector.After successful identification by double enzyme digestion,the DDX1 gene was ligated into pcDNA3.1 expression vector.After the transformation of the product,the positive colonies were screened.IPI-2I cells were cultured,and the cells that had been passaged twice were spread in a six-hole plate for follow-up experiment.The recombinant plasmids of the positive colonies were transfected into the six-well plate for 24 h.The protein was collected,and the transfection efficiency of DDX1 was detected by Western blotting.IPI-2I cells transfected with pcDNA3.1-DDX1 or PBS (control) were infected with PEDV with a multiple of infection (MOI) of 1.0 to establish a cell model of PEDV infection.Total protein and RNA were collected 12 h after PEDV infection.The expression of PEDV N protein at cell level was detected by indirect immunofluorescence assay,the expression of PEDV N gene mRNA was detected by Real-time quantitative PCR,and the expression of PEDV N protein was detected by Western blotting.Bioinformatics software was used to predict the phosphorylation site of DDX1 peotein,and immunoprecipitation test was used to detect the phosphorylation level of DDX1 after PEDV infection in IPI-2I cells for 12 h.【Result】 The target DDX1 gene band of 2 223 bp was successfully obtained by PCR amplification.The results of double digestion identification showed that pMD19-T-DDX1 cloning vector and pcDNA3.1-DDX1 eukaryotic expression vector were successfully constructed.The expression of DDX1 protein in the cells transfected with pcDNA3.1-DDX1 was extremely significantly higher than that in PBS group (P＜0.01),indicating that DDX1 transfection was successful.The results of indirect immunofluorescence assay showed that compared with PBS group,there were a large number of green fluorescence signals in PEDV infection group,indicating that the IPI-2I cell model infected by PEDV was successfully constructed.In IPI-2I cells transfected with pcDNA3.1-DDX1 recombinant plasmid,compared with PBS group,the fluorescence intensity of PEDV N protein significantly decreased,the mRNA and protein expression of PEDV N gene were extremely significantly decreased (P＜0.01).Bioinformatics analysis showed that DDX1 protein had multiple phosphorylation sites,including 17 serine,12 threonine and 6 tyrosine.The results of immunoprecipitation test showed that the level of DDX1 phosphorylation in PEDV infected group was extremely significantly higher than that in uninfected group (P＜0.01).【Conclusion】 In this study,the eukaryotic expression vector of pcDNA3.1-DDX1 was successfully constructed.It was found that 12 h after PEDV infection,DDX1 inhibited the replication of PEDV and increased the phosphorylation level,indicating that DDX1 protein might inhibit the replication of PEDV through phosphorylation during PEDV infection.

Key words: DDX1; Porcine epidemic diarrhea virus (PEDV); phosphorylation

中图分类号:

S852.65^＋1

霍明凯, 关飞虎, 邱润辉, 魏春燕, 于海涛, 朱嘉乐, 张辉. DDX1影响猪流行性腹泻病毒复制的机制研究[J]. 中国畜牧兽医, 2023, 50(4): 1556-1566.

HUO Mingkai, GUAN Feihu, QIU Runhui, WEI Chunyan, YU Haitao, ZHU Jiale, ZHANG Hui. Study on the Mechanism of DDX1 Inhibiting Porcine Epidemic Diarrhea Virus Replication[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1556-1566.

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