鸡IFIT5对J亚群禽白血病病毒在DF-1细胞内复制的影响

doi:10.16431/j.cnki.1671-7236.2023.01.031

Abstract

Abstract: 【Objective】 Polyclonal antibody of interferon-inducible protein containing triangular tetrapeptide repeat 5 (IFIT5) in chickens was prepared to explore the effect of IFIT5 on the replication of J subgroup Avian leukemia virus (ALV-J) in chicken embryonic fibroblasts (DF-1).【Method】 Using cDNA obtained by reverse transcription of RNA extracted from spleen tissue cells in chickens infected with ALV-J as template, IFIT5 gene was amplified by PCR, and the purified PCR product and linearize vector were connected using recombinant enzymes to construct recombinant plasmids pET-28a-IFIT5 and pcDNA5-flag-IFIT5 and sequece.The correctly sequenced recombinant plasmid pET-28a-IFIT5 transformed Escherichia coli BL21 competent cells for induced expression.The fusion protein His-IFIT5 was purified and immuned 6-week-old BALB/c female mice to prepare polyclonal antibody.The specificity and titer of the antibody were determined by Western blotting and indirectly ELISA, respectively.The correctly sequenced recombinant plasmid pcDNA5-flag-IFIT5 was transacted in DF-1 cells, inoculated with ALV-J after 24 h, and the effect of overexpressed IFIT5 on ALV-J replication in DF-1 cells was detected by Western blotting.【Result】 IFIT5 gene with a size of 1 440 bp was successfully amplified, the recombinant plasmids pET-28a-IFIT5 and pcDNA5-flag-IFIT5 were successfully constructed.pET-28a-IFIT5 could express the soluble protein His-IFIT5 of about 56 ku.Western blotting results showed that the prepared polyclonal antibody of mouse anti-IFIT5 protein could specifically identify overexpressed IFIT5 protein in DF-1 cells with a titer of 1:32 000.pcDNA5-flag-IFIT5 could be highly expressed in DF-1 cells, and overexpressed IFIT5 in DF-1 cells could promote the replication of ALV-J.【Conclusion】 The polyclonal antibody of mouse anti-IFIT5 protein prepared in this study had high reactivity and specificity, overexpressed IFIT5 in DF-1 cells could promote the replication of ALV-J.The results provided a reference for in-depth study of the biological function of IFIT5 protein.

Key words: IFIT5 gene; J subgroup Avian leukemia virus (ALV-J); prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺9.3

ZHU Lilin, WANG Houkun, CHEN Xueyang, LIU Jing, LIANG Xiongyan, FANG Chun, YANG Yuying. Effect of Chicken IFIT5 on Replication of J Subgroup Avian Leukemia Virus Within DF-1 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 310-316.

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Effect of Chicken IFIT5 on Replication of J Subgroup Avian Leukemia Virus Within DF-1 Cells

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