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

摘要/Abstract

摘要： 【目的】制备鸡干扰素诱导蛋白含三角形四肽重复5（IFIT5）多克隆抗体，以探究IFIT5对J型禽白血病病毒（ALV-J）在鸡胚成纤维细胞（DF-1）内复制的影响。【方法】以感染ALV-J的鸡脾脏组织细胞提取的RNA反转录获得的cDNA作为模板，对IFIT5基因进行PCR扩增，利用重组酶将纯化后的PCR产物和线性化载体进行连接，构建重组质粒pET-28a-IFIT5和pcDNA5-flag-IFIT5并测序。将测序正确的重组质粒pET-28a-IFIT5转化大肠杆菌BL21感受态细胞进行诱导表达，表达的融合蛋白His-IFIT5纯化后免疫6周龄BALB/c雌鼠制备多克隆抗体，通过Western blotting鉴定抗体的特异性，利用间接ELISA测定抗体的效价。将测序正确的重组质粒pcDNA5-flag-IFIT5转染DF-1细胞，24 h后接种ALV-J，通过Western blotting检测DF-1细胞中过表达IFIT5对ALV-J复制的影响。【结果】试验成功扩增到大小为1 440 bp的IFIT5基因，并成功构建重组质粒pET-28a-IFIT5和pcDNA5-flag-IFIT5。pET-28a-IFIT5可以表达约56 ku的可溶性蛋白His-IFIT5，Western blotting结果显示，制备的鼠抗IFIT5蛋白多克隆抗体能特异性识别DF-1细胞中过表达的IFIT5蛋白，效价为1：32 000。pcDNA5-flag-IFIT5能在DF-1细胞中高效表达，且在DF-1细胞中过表达IFIT5可以促进ALV-J复制。【结论】本研究制备的鼠抗IFIT5蛋白多克隆抗体具有较高的反应性和特异性；在DF-1细胞中过表达IFIT5可以促进ALV-J复制。本试验结果为深入研究IFIT5蛋白的生物学功能提供参考。

关键词: IFIT5基因; J型禽白血病病毒(ALV-J); 原核表达; 多克隆抗体

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

中图分类号:

S852.65⁺9.3

朱丽霖, 王后坤, 陈雪阳, 刘晶, 梁雄燕, 方春, 杨玉莹. 鸡IFIT5对J亚群禽白血病病毒在DF-1细胞内复制的影响[J]. 中国畜牧兽医, 2023, 50(1): 310-316.

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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