鸡DDX41基因克隆表达、细胞定位及其在禽腺病毒4型感染调控天然免疫中的作用

doi:10.3969/j.issn.1004-1524.2023.05.06

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (5): 1028-1036.DOI: 10.3969/j.issn.1004-1524.2023.05.06

鸡DDX41基因克隆表达、细胞定位及其在禽腺病毒4型感染调控天然免疫中的作用

刘琪(), 曹影丽, 魏宁波, 杨侃侃, 梁月巧, 宋祥军, 邵颖, 涂健, 祁克宗()

兽医病理生物学与疫病防控安徽省重点实验室,安徽省动物性食品质量与生物安全工程实验室,安徽合肥 230036

收稿日期:2022-06-13 出版日期:2023-05-25 发布日期:2023-06-01
作者简介:刘琪(1997—),女,山东寿光人,硕士研究生,主要从事兽医病理学研究。E-mail: 2455289830@qq.com
通讯作者: *祁克宗,E-mail: qkz@ahau.edu.cn
基金资助:
国家自然科学基金(31972642)

Cloning, expression, cellular localization of chicken DDX41 gene and its role in regulation of natural immunity by avian adenovirus type 4 infection

LIU Qi(), CAO Yingli, WEI Ningbo, YANG Kankan, LIANG Yueqiao, SONG Xiangjun, SHAO Ying, TU Jian, QI Kezong()

Anhui Key Laboratory of Veterinary Pathobiology and Disease Control, Anhui Province Animal Food Quality and Biosafety Engineering Laboratory, Hefei 230036, China

Received:2022-06-13 Online:2023-05-25 Published:2023-06-01

摘要/Abstract

摘要：

为获得鸡DDX41(chDDX41)的原核表达蛋白,探讨chDDX41的细胞定位及其在禽腺病毒4型(FAdV-4)感染引起的天然免疫中的作用。根据GenBank中chDDX41基因序列,设计特异性引物,以LMH细胞cDNA为模板扩增,将扩增序列连接至pET-32a、pCAGGS-HA、pCMV-3×Flag和pmCherry-C1空载体,构建重组质粒。将pET-32a-chDDX41转化至大肠埃希菌BL21(DE3)感受态细胞中,经IPTG诱导表达。利用激光共聚焦显微镜观察chDDX41在鸡肝癌细胞(LMH)和鸡巨噬细胞(HD11)中的亚细胞定位;利用实时荧光定量PCR(qRT-PCR)检测在LMH细胞中过表达chDDX41对IFN-β等细胞因子表达量的影响。结果表明,克隆得到1 854 bp的chDDX41基因,共编码617个氨基酸。SDS-PAGE结果显示,重组蛋白pET-32a-chDDX41主要以可溶性蛋白的方式在上清液中表达,经Western Blot鉴定,在90 ku左右有特异性条带,与预期结果一致;chDDX41在LMH和HD11细胞中主要定位在细胞核;FAdV-4感染过表达chDDX41基因后的LMH细胞,发现IFN-β、IL-6、IL-1β和IL-8等细胞因子在mRNA水平上的表达量上调,表明chDDX41参与了FAdV-4引起的天然免疫应答。研究结果可为进一步研究DDX41基因功能及其在天然免疫中的分子机制提供参考。

关键词: chDDX41, 原核表达, 细胞定位, 禽腺病毒4型, 天然免疫

Abstract:

To obtain the prokaryotic expression protein of chicken DDX41 (chDDX41) and to investigate the subcellular localization of chDDX41 and its in natural immunity induced by avian adenovirus type 4 (FAdV-4) infection, based on the sequence of chDDX41 gene in GenBank, specific primers were designed to amplify and ligation of amplified sequences to pET-32a, pCAGGS-HA, pCMV-3×Flag and pmCherry-C1 empty vectors using LMH cell cDNA as template to construct recombinant plasmids. The pET-32a-chDDX41 was transformed into Escherichia coli BL21 (DE3) receptor cell with IPTG-induced expression. The subcellular localization of chDDX41 in chicken liver cancer cells (LMH) and chicken macrophage cell line (HD11) cells was observed by laser confocal microscopy, and the effect of overexpression of chDDX41 on cytokine expression in FAdV-4-infected LMH cells was examined by real-time fluorescence quantitative PCR (qRT-PCR). The result showed that chDDX41 gene was of 1 854 bp in size, encoding 617 amino acids. SDS-PAGE showed that the recombinant protein pET-32a-chDDX41 was mainly expressed as soluble protein in the supernatant, and the specific band appeared at around 90 ku was identified by Western Blot, which was consistent with the expected results. chDDX41 was mainly localized in the nucleus in LMH and HD11 cells; FAdV-4 infection of LMH cells after overexpression of the chDDX41 gene revealed an upregulation of the expression of cytokines such as IFN-β, IL-6, IL-1β and IL-8 at the mRNA level, indicating that chDDX41 was involved in the natural immune response induced by FAdV-4. The result provided a basis for further study of the gene function of DDX41 and its specific molecular mechanism in natural immunity.

Key words: chDDX41, prokaryotic expression, cellular localization, avian adenovirus type 4, natural immunity

中图分类号:

S855.3

刘琪, 曹影丽, 魏宁波, 杨侃侃, 梁月巧, 宋祥军, 邵颖, 涂健, 祁克宗. 鸡DDX41基因克隆表达、细胞定位及其在禽腺病毒4型感染调控天然免疫中的作用[J]. 浙江农业学报, 2023, 35(5): 1028-1036.

LIU Qi, CAO Yingli, WEI Ningbo, YANG Kankan, LIANG Yueqiao, SONG Xiangjun, SHAO Ying, TU Jian, QI Kezong. Cloning, expression, cellular localization of chicken DDX41 gene and its role in regulation of natural immunity by avian adenovirus type 4 infection[J]. Acta Agriculturae Zhejiangensis, 2023, 35(5): 1028-1036.

图/表 10

表1 PCR扩增的特异性引物

Table 1 Specific primers for PCR

引物名称Primer name	引物序列Primer sequence(5'→3')
chDDX41-F	ATGGAGCCGGGCGCGGAGCGGAAGA
chDDX41-R	CTAGAAGTCCATAGAGCTGTG
pET-32a-chDDX41-F	gctgatatcggatccgaattcATGGAGCCGGGCGCGGAG
pET-32a-chDDX41-R	ttgtcgacggagctcgaattcCTAGAAGTCCATAGAGCTGTGGGC
pCAGGS-HA-chDDX41-F	gttccagattacgctgaattcATGGAGCCGGGCGCGGAG
pCAGGS-HA-chDDX41-R	acctcgatgagctcgaattcCTAGAAGTCCATAGAGCTGTGGGC
pCMV-3×Flag-chDDX41-F	caagcttgcggccgcgaattcATGGAGCCGGGCGCGGAG
pCMV-3×Flag-chDDX41-R	cctctagagtcgactggtaccCTAGAAGTCCATAGAGCTGTGGGC
pmCherry-chDDX41-F	tcagatctcgagctcaagcttATGGAGCCGGGCGCGGAG
pmCherry-chDDX41-R	ggatcccgggcccgcggtaccCTAGAAGTCCATAGAGCTGTGGGC
qchIFN-F	CCTCAACCAGATCCAGCATT
qchIFN-R	GGATGAGGCTGTGAGAGGAG
qβ-actin-F	CAGACATCAGGGTGTGATGG
qβ-actin-F	TCAGGGGCTACTCTCAGCTC
qchIL-6-F	CTGTGGTGTGCTCAGAATCCA
qchIL-6-R	GACTTCAGATTGGCGAGGAG
qchIL-8-F	ATTCAAGATGTGAAGCTGAC
qchIL-8-R	AGGATCTGCAATTAACATGAGG
qchIL-1β-F	CAGCACCTCAGCGAAGAG
qchIL-1β-R	CTGTGGTGTGCTCAGAATCCA
qchMx-1-F	GTTTCGGACATGGGGAGTAA
qchMx-1-R	GCATACGATTTCTTCAACTTTGG
qchMYD88-F	AGCGTGGAGGAGGACTGCAAGAAG
qchMYD88-R	CCGATCAAACACACACAGCTTCAG
qchIRF7-F	GCCTGAAGAAGTGCAAGGTC
qchIRF7-R	CTCTGTGCAAAACACCCTGA
qchTBK1-F	GTTTGCTATTGAGGAAGAGACAAC
qchTBK1-R	CCATTTTCCCGGAGATGATTCATC
qchMDA5-F	TGAAAGCCTTGCAGATGACTTA
qchMDA5-R	GCTGTTTCAAATCCTCCGTTAC
qchNF-κB-F	CATTGCCAGCATGGCTACTAT
qchNF-κB-R	TTGAAGGGGTTGTTATTGGTG
qchPKR-F	TGCTTGACTGGAAAGGCTACT
qchPKR-R	TCAGTCAAGAATAAACCATGTGTG

图1 chDDX41基因扩增产物 M,DL2000 marker;1,chDDX41基因PCR产物;2,阴性对照。

Fig.1 PCR product of chDDX41gene M, DL2000 marker; 1, PCR product of chDDX41 gene; 2, Negative control.

图2 重组质粒的酶切鉴定 M,DNA marker(A,1 kb plus DNA ladder;B-C,DL5000)。1,pET-32a(+)-chDDX41重组质粒酶切产物;2,pmCherry-chDDX41重组质粒酶切产物;3,pCMV-3×Flag-chDDX41重组质粒酶切产物;4,pCAGGS-HA-chDDX41重组质粒酶切产物。

Fig.2 Enzyme digestion identification of recombinant plasmids M:DNA marker(A, 1 kb plus DNA ladder; B-C, DL5000). 1, pET-32a(+)-chDDX41 recombinant plasmid digested product; 2, pmCherry-chDDX41 recombinant plasmid digested product; 3, pCMV-3×Flag-chDDX41 recombinant plasmid digested product; 4, pCAGGS-HA-chDDX41 recombinant plasmid digested product.

图3 pET-32a-chDDX41重组蛋白SDS-PAGE验证结果 M,10~250 ku marker;1~2, 15 ℃ pET-32a空载体未诱导、诱导产物;3~4, 15 ℃ pET-32a-chDDX41重组蛋白未诱导、诱导产物;5~6, 15 ℃诱导19 h pET-32a-chDDX41重组蛋白的包涵体、上清液。

Fig.3 SDS-PAGE verification of pET-32a-chDDX41 protein M, 10-250 ku marker; 1-2, Not induced and induction products of pET-32a empty vector at 15 ℃; 3-4, Not induced and induction products of pET-32a-chDDX41 recombinant protein at 15 ℃; 5-6, Induction of inclusion bodies and supernatants of pET-32a-chDDX41 recombinant protein at15 ℃for 19 h.

图4 pET-32a-chDDX41重组蛋白的纯化 M,10-250 ku marker;1,pET-32a-chDDX41重组蛋白在15 ℃的诱导产物;2,15 ℃诱导19 h pET-32a-chDDX41重组蛋白的包涵体;3,15 ℃诱导19 h pET-32a-chDDX41重组蛋白的上清;4~5,流穿液;6~7,洗涤液;8~10,洗脱液。

Fig.4 Purification of pET-32a-chDDX41 recombinant protein M, 10-250 ku marker; 1, Induction product of pET-32a-chDDX41 recombinant protein at 15 ℃; 2, Inclusion body of pET-32a-chDDX41 recombinant protein induced at 15 ℃ for 19 h; 3, Supernatant expression of pET-32a-chDDX41 recombinant protein induced at 15 ℃ for 19 h; 4-5, Flow-through fluid; 6-7, Detergent; 8-10, Elution solution.

图5 pET-32a-chDDX41重组蛋白Western-blot验证 1,特异性条带;M,10~250 ku marker。

Fig.5 Western-blot validation of pET-32a-chDDX41 recombinant protein 1, Specific band; M, 10-250 ku marker.

图6 chDDX41在LMH细胞中的定位

Fig.6 Subcellular localization of chDDX41 in LMH cells A-C, pmCherry-chDDX41; D-F, FITC-chDDX41; H-J, AF594-chDDX41.

图7 chDDX41在HD11细胞中的亚细胞定位

Fig.7 Subcellular localization of chDDX41 in HD11 cells A-C, pmCherry-chDDX41; D-F, FITC-chDDX41.

图8 过表达chDDX41对细胞因子表达量的影响 1,转pCMV-3×Flag-N空载质粒的LMH细胞;2,转pCMV-3×Flag-chDDX41质粒的LMH细胞。

Fig.8 Effect of overexpression of chDDX41 gene on cytokine expression 1, LMH cell transformed with empty plasmid pCMV-3×Flag-N; 2, LMH cell transformed with plasmid pCMV-3×Flag-chDDX41.

图9 siRNA干扰影响FAdV-4复制 A,siRNA引物筛选。B,FAdV-4-Hexon蛋白表达;1,对照;2,转pCMV-3×Flag-chSTING质粒的LMH细胞;3,同时过表达chDDX41基因和用siRNA敲低chSTING基因的LMH细胞。

Fig.9 siRNA interference affects FAdV-4 replication A, Screening of siRNA primers. B, FAdV-4-Hexon protein expression; 1, Control; 2, LMH cell transformed with plasmid pCMV-3×Flag-chSTING; 3, LMH cell overexpressing chDDX41 gene and knocking down chSTING gene simultaneously.

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鸡DDX41基因克隆表达、细胞定位及其在禽腺病毒4型感染调控天然免疫中的作用

Cloning, expression, cellular localization of chicken DDX41 gene and its role in regulation of natural immunity by avian adenovirus type 4 infection

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