DEAD-box解旋酶21对猪传染性胃肠炎病毒复制的调控作用

doi:10.7668/hbnxb.20194747

摘要/Abstract

摘要：

旨在探究DEAD-box解旋酶21(DDX21)对猪传染性胃肠炎病毒(TGEV)复制的调控作用。首先通过蛋白免疫印迹(Western Blot)分析了TGEV 感染对 DDX21表达的影响;进一步构建了猪DDX21真核表达质粒以及建立稳定敲低DDX21的PK-15细胞系,运用荧光定量PCR(RT-qPCR)、蛋白免疫印迹、间接免疫荧光(IFA)和半数细胞培养物感染量(TCID₅₀)探究外源过表达DDX21及敲低 DDX21 对 TGEV体外复制的调控作用;构建一系列DDX21截短突变体真核表达质粒,鉴定了DDX21调控TGEV增殖的关键功能域。Western Blot分析显示,在TGEV WH-1株感染早期,PK-15细胞内源性DDX21蛋白的表达水平显著上调; RT-qPCR、Western Blot、IFA和TCID₅₀试验显示,超表达DDX21可以显著提高TGEV N 基因的mRNA水平、N蛋白的表达及病毒滴度,且呈剂量依赖性,其601—784 aa区域是其影响TGEV复制的关键;与阴性对照相比,在相应DDX21敲低细胞系中TGEV的增殖显著被抑制,而回补试验逆转了DDX21敲低细胞系中TGEV的滴度。该研究首次揭示了猪DDX21对TGEV增殖的促进作用并鉴定了其调控TGEV复制的关键结构域,为今后研究DDX21蛋白的功能及TGEV的致病机理提供了理论依据。

关键词: 猪传染性胃肠炎病毒, DEAD-box解旋酶21, 宿主蛋白, 病毒复制

Abstract:

To investigate the regulatory effect of DEAD-box helicase 21(DDX21)on the replication of Transmissible gastroenteritis virus(TGEV).Firstly,Western Blot was utilized to analyze the effect of TGEV infection on DDX21 expression.Furthermore,we constructed eukaryotic expression plasmids of porcine DDX21 and established knockdown stable cell lines.RT-qPCR,Western Blot,Indirect immunofluorescence(IFA)and TCID₅₀ assays were used to investigate the regulatory effect of DDX21 on TGEV replication in vitro.Western Blot analysis showed that the protein levels of DDX21 were significantly up-regulated in PK-15 cells at the early stage of TGEV infection.RT-qPCR,Western Blot,IFA and TCID₅₀ experiments showed that over-expression of DDX21 significantly increased the mRNA level and protein of TGEV N in a dose-dependent manner.And the amino acids 601—784 aa of DDX21 were critical for promoting TGEV replication.Otherwise,the titer of TGEV was significantly down-regulated in DDX21 knockdown cell lines,whereas the titer of TGEV in DDX21 knockdown cell lines was reversed under the rescue experiment.This study revealed for the first time that DDX21 promotes the proliferation of TGEV and identified the key domain of DDX21 in regulating TGEV replication,which provided a theoretical a basis for future research on the function of DDX21 protein and the pathogenesis of TGEV.

Key words: Transmissible gastroenteritis virus, DEAD-box helicase 21, Host protein, Viral replication

谢立兰, 尹杰, 黄冬蛾, 李么明. DEAD-box解旋酶21对猪传染性胃肠炎病毒复制的调控作用[J]. 华北农学报, 2024, 39(4): 215-222. doi: 10.7668/hbnxb.20194747.

XIE Lilan, YIN Jie, HAUNG Donge, LI Yaoming. Regulatory Role of DEAD-box Helicase 21 on Transmissible gastroenteritis virus Replication[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 215-222. doi: 10.7668/hbnxb.20194747.

http://www.hbnxb.net/CN/Y2024/V39/I4/215

图/表 7

表1 本研究所用的PCR引物

Tab.1 The PCR primers used in this study

引物名称Primer name	引物序列Primer sequence	备注Notes
GAPDH-F	ACATGGCTCCAAGGAGTAAGA	RT-qPCR
GAPDH-R	GATCGAGTTGGGGCTGTGACT
JC-TGEV-N-F	CCCGTGGTCGGAAGAATAATAA	RT-qPCR
JC-TGEV-N-R	GGTACAAAGTCTCTCGGACATAAG
DDX21-F	GGCAAGTCAAGTAAGCAGAGA	RT-qPCR
DDX21-R	GCGATCAATCTGTCCTCCATAG
DDX21-FF	GGAGAATTCATGCCGGGGAAACTTCGTAGT	构建重组质粒HA-DDX21wt及截短
DDX21-FR	TCTCTCGAGTTACTGTCCAAACGCTTTGC	突变体质粒ΔGUCT、DEAD和GUCT
DDX21-600R	TCTCTCGAGTTACTCAGCTGACTGCTTGAAG
DDX21-430R	TATCTCGAGTTAATCCCCGATAACTGCTGCC
DDX21-601F	GGAGAATTCATGAAGCTGATAGAGGAGAAGG
PL548F	CAAACTTAAGCATGTTGT	构建回补突变体质粒HA-DDX21m
PL548R	TCTCAGCTGACTGCTTGA
PL548F1	ATGTGTCGCCTATAAAAAAGATTCAGAAGAC
PL548R1	TTATAGGCGACACATAAAATCTCT

图1 TGEV感染调控细胞内源性DDX21的表达 A.Western Blot检测细胞内源性DDX21蛋白的表达(+表示接毒,-表示未接毒);B.Image J 分析图 A的灰度值(+/-)。不同小写字母表示相同时间不同组别差异显著(P<0.05)。

Fig.1 TGEV infection regulates the expression of endogenous DDX21 A.The change of DDX21 protein expression levels in TGEV-infected cells(+means virus-infected,-means uninfected);B.Gray values were analyzed by Image J soft(+/-).The difference in small letters between different groups at the same time is significant(P<0.05).

图2 HA-DDX21wt真核表达质粒的构建及Western Blot 检测质粒的表达 A.重组质粒HA-DDX21wt的酶切鉴定:M1.DL2000 Marker;M2.DL15000 Marker;B.Western Blot 检测重组质粒的表达。

Fig.2 Construction of eukaryotic expression plasmid HA-DDX21wt and detection of its expression by Western Blot A.Identification of recombinant plasmid HA-DDX21wt by restriction enzyme digestion:M1.DL2000 Marker;M2.DL15000 Marker;B.Western Blot analysis of recombinant plasmid expression.

图3 过表达DDX21促进TGEV的复制 A~C.TCID₅₀(A)、实时荧光定量(B)和Western Blot(C)检测DDX21对TGEV增殖的影响;D.DDX21调控TGEV增殖呈剂量依赖性;不同的小写字母代表差异显著(P<0.05)。图4—6同。

Fig.3 Over expression of DDX21 positively regulates the replication of TGEV A—C.TCID₅₀(A),Real-time Fluorescence Quantification(B)and Western Blot(C)were used to detect the effect of DDX21 on TGEV proliferation;D.DDX21 regulates TGEV proliferation in a dose-dependent manner;different small letters indicate the significant difference at P<0.05.The same as Fig.4—6.

图4 敲低DDX21的表达抑制TGEV的复制 A.Western Blot验证敲低DDX21的表达效果;B~C.TCID₅₀和qRT-PCR试验检测TGEV的复制。

Fig.4 Knockdown of DDX21 expression inhibits replication of TGEV A.The expressions of DDX21 were measured by Western Blot; B—C.The replication of TGEV were detected by TCID₅₀ assay(B)and qRT-PCR assay(C),respectively.

图5 回补突变体质粒的构建及功能分析 A.突变碱基标注;B.回补突变体质粒测序的峰图(部分);C.回补试验。

Fig.5 Construction and functional analysis of the rescue plasmid A.Mutation bases;B.Sequencing map(partial)of the complement plasmid;C.Rescue experiment.

图6 DDX21不同结构域对TGEV增殖的影响 A.DDX21截短突变体质粒的示意图;B.TCID₅₀检测DDX21不同结构域对TGEV增殖的影响。

Fig.6 Effect of different DDX21 domains on TGEV proliferation A.Schematic showing the DDX21 deletion mutants;B.TCID₅₀ assay was used to detect the effects of DDX21 domains on TGEV proliferation.

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