坦布苏病毒感染诱导雏鸭体内未折叠蛋白反应

doi:10.3864/j.issn.0578-1752.2021.04.016

摘要/Abstract

摘要：

【目的】检测坦布苏病毒在雏鸭体内诱导未折叠蛋白反应的信号通路（PERK、IRE1和ATF6）,为揭示坦布苏病毒致病机制提供理论基础。【方法】取1日龄SPF雏鸭,腹腔接种坦布苏病毒（JS804株）,于接种后12、24、36和48 h从对照组和攻毒组各取5只剖杀,分别取肝脏、心脏和脑组织,利用组织总RNA提取试剂盒提取各个组织样品总RNA,反转录获得cDNA。根据未折叠蛋白反应的3条信号通路,选取不同通路中的标志性分子,设计合成特异性引物,利用荧光定量PCR方法检测靶基因。以GAPDH为内参基因,采用相对定量法（2^-ΔΔCt）,分析靶基因的表达水平。【结果】雏鸭肝脏中坦布苏病毒含量最高,心脏次之,脑最低。对未折叠蛋白反应标志性分子GRP78的检测结果显示,脑和肝脏中GRP78表达量持续升高,并在攻毒后36 h达到顶峰（4.21倍和10.14倍）,心脏中GRP78表达量仅在攻毒后36 h短暂升高（1.32倍）。PERK信号通路标志性分子ATF4表达水平在肝脏和脑中分别从攻毒后24 h和36 h持续升高至攻毒后48 h,并在攻毒后36 h达到顶峰（2.71倍和6.02倍）,心脏中ATF4的表达量则仅在攻毒后36 h时升高（1.57倍）。IRE1信号通路标志性分子XBP1s在肝脏中的表达量升高最为显著（9倍）,而脑中EDEM的表达量升高最为显著（3.87倍）且持续时间最长（从攻毒后12 h至攻毒后48 h）。与对照组相比,ATF6信号通路标志性分子GRP94和XBP1u均出现升高现象,虽然两种蛋白在不同组织中表达量变化的时间点和趋势不同,但均在攻毒后36 h出现峰值。【结论】首次报道了坦布苏病毒感染可在雏鸭体内激活未折叠蛋白反应的3条信号通路,本研究将有助于深入研究坦布苏病毒与宿主之间的相互作用机制。

关键词: 坦布苏病毒, SPF雏鸭, 内质网应激, 未折叠蛋白反应, 信号通路

Abstract:

【Objective】 The aim of this study was to determine the signal pathways (PERK, IRE1 and ATF6) of unfolded protein response induced by tembusu virus in ducklings, so as to provide a theoretical basis for elucidating the pathogenesis of TMUV. [Method] 1-day-old SPF ducklings were infected intraperitoneally with TMUV (JS804). Ducklings inoculated in the same manner with equal volume of RMPI-1640 were used as negative control. Five ducklings from each group were euthanized at 12, 24, 36 h and 48 h post infection, and their brains, livers and hearts were collected. The total RNAs were extracted from collected tissues by using total RNA extraction kit. Then the total RNAs were reverse transcribed into cDNA. Specific target genes representing the three known pathways of unfolded protein response were selected, and the primers were designed based on the published GenBank sequence. The relative expression of target genes was quantified by real time PCR. Real time PCR data were analyzed using the comparative Ct method (2^-ΔΔCt). GAPDH was chosen as a reference gene for internal control. 【Result】 In three organs from infected ducklings, it was observed that the viral titers were the highest in the liver, lower in the heart, and the lowest in the brain. The unfolded protein response was characterized by upregulated expression of GRP78. The relative expression of GRP78 in the brain and liver were persistently upregulated and reached a peak at 36 h post infection (4.21 fold and 10.14 fold, respectively). GRP78 expression in the heart was upregulated transiently at 36 h post infection (1.32 fold). ATF4 expression represented the activation of PERK pathway. The ATF4 expression in the liver and brain were persistently upregulated from 24 h and 36 h post infection to 48 h post infection respectively, and peaked at 36 h post infection (2.71 fold and 6.02 fold, respectively). However, the upregulation of ATF4 expression in the heart was observed at 36 h post infection (1.57 fold). The activation of IRE pathway was characterized by XBP1s. In the liver, the expression level of XBP1s increased most significantly (9 fold). In the brain, the expression level of EDEM enhanced most significantly (3.87 fold) and persistently upregulated from 12 to 48 h post infection. Comparing to negative control, the expression of ATF6 pathway marker GRP94 and XBP1u were upregulated in three tissues, which reached a peaked at 36 h post infection, although expression profiles of GRP94 and XBP1u were different at indicated time points. 【Conclusion】 It was the first report that TMUV infection induced three branches of unfolded protein response in ducklings, and these results might be helpful for understanding the interaction between tembusu virus infection and host response.

Key words: Tembusu virus, SPF ducklings, endoplasmic reticulum, unfolded protein response, signaling pathway

赵冬敏,黄欣梅,章丽娇,刘青涛,杨婧,韩凯凯,刘宇卓,李银. 坦布苏病毒感染诱导雏鸭体内未折叠蛋白反应[J]. 中国农业科学, 2021, 54(4): 855-863.

ZHAO DongMin,HUANG XinMei,ZHANG LiJiao,LIU QingTao,YANG Jing,HAN KaiKai,LIU YuZhuo,LI Yin. The Induction of Unfolded Protein Response in Tembusu Virus Infected Ducklings[J]. Scientia Agricultura Sinica, 2021, 54(4): 855-863.

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引物名称 Primers	序列 Sequences	UPR途径 UPR pathway
GRP78-F	5′-TGTGCCAGCCTACTTCAA-3′	所有途径 All pathways
GRP78-R	5′-GATGTTCTTCTCACCCTCTC-3′	所有途径 All pathways
ATF4-F	5′-GATCCCTCAATGACAACCCCTTCC-3′	PERK
ATF4-R	5′-GCGCGTGGCAGCTGTCTTATTC-3′	PERK
EDEM-F	5′-GGTGACCCGCCTTTGTATGTTA-3′	IRE1
EDEM-R	5′-AGCTTGCAGTTGCCAGTTGTATCT-3′	IRE1
XBP1s-F	5′-GCTGAGTCCGCAGCAGG-3′	IRE1
XBP1s-R	5′-CTGCCATCAGAATCCATGTG-3′	IRE1
GRP94-F	5′-CTCTCAACGTTTAACCCAGTCTCC-3′	ATF6
GRP94-R	5′-TGTTTTGTCATCTTCATTTTCCTT-3′	ATF6
XBP1-F	5′-GGAGGAGGAGAACCAGAAGC-3′	ATF6
XBP1-R	5′-CAGAACATCCAAACCCAAGC-3′	ATF6
TMUVE-F	5′-GTGAGATCTTACTGCTATGAG-3′	TMUV核酸 TMUV nucleic acid
TMUVE-R	5′-ACTTGGCACATGTCTGTATGC-3′	TMUV核酸 TMUV nucleic acid
GAPDH-F	5′-CACAGCCACACACGAAGACA-3′	内参基因 Internal reference genes
GAPDH-R	5′-CCTTAGCCAGCCCCAGTAGA-3′	内参基因 Internal reference genes