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

doi:10.3969/j.issn.1004-1524.2023.05.06

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (5): 1028-1036.DOI: 10.3969/j.issn.1004-1524.2023.05.06

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S855.3

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.

Figures/Tables 10

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

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

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.

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.

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.

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

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

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

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.

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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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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