鸟苷酸结合蛋白GBP1和GBP2抑制猪轮状病毒体外复制

doi:10.11843/j.issn.0366-6964.2024.01.023

摘要/Abstract

摘要： 本研究旨在探究鸟苷酸结合蛋白1（guanylate-binding protein-1，GBP1）、鸟苷酸结合蛋白2（guanylate-binding protein-2，GBP2）的全长、片段、GTPase酶活性对猪轮状病毒（porcine rotavirus，PoRV）复制的影响。以猪肾细胞cDNA和GBP1、GBP2质粒为模板，PCR扩增出GBP1、GBP2基因的全长和截断体（G、M、E、ME区），克隆到pCDNA3.1（+）真核表达载体上。在恒河猴细胞（MA104）细胞上过表达或沉默GBP1、GBP2基因，探究对PoRV复制的影响。设置不同的时间点，筛选GBP1、GBP2对PoRV复制产生影响作用的时间点。应用泛GTPase酶抑制剂（CID-1067700）探究GBP1、GBP2的GTPase酶活性对PoRV的作用。通过免疫印迹法Western blot、荧光定量PCR、病毒毒价测定等方法检测GBP1、GBP2蛋白的表达情况，并研究其抗病毒功能。结果表明：成功构建pCDNA3.1（+）-GBP1-HIS和pCDNA3.1（+）-GBP2（47aa-592aa）、（131aa-592aa）-HIS重组质粒，经验证能够表达。PoRV在蛋白和mRNA水平上能显著促进GBP1、GBP2基因表达上调。过表达GBP1、GBP2基因能显著抑制PoRV复制，沉默GBP1、GBP2基因的表达能显著促进PoRV复制。成功构建并表达了GBP1、GBP2蛋白的G、M、E、ME结构域截断体。过表达截断体基因发现GBP1、GBP2的G区域抑制PoRV的复制。通过泛GTPase抑制酶活性，发现GBP1、GBP2抑制PoRV复制需要依赖GTPase酶活性。GBP1、GBP2蛋白能够抑制PoRV的复制，该抑制作用依赖GBP蛋白GTPase酶活性区域。研究结果为PoRV寻找新的药物靶点和研发新型疫苗提供理论依据。

关键词: 猪轮状病毒, GBP1, GBP2, 抗病毒, 结构域

Abstract: The aim of this study was to investigate the effects of the full length, fragmentation and GTPase activity of guanylate-binding protein-1 (GBP1) and guanylate-binding protein-2 (GBP2) on the replication of porcine rotavirus (PoRV). The full-length and truncations (G, M, E, ME regions) of GBP1 and GBP2 genes were amplified by PCR using porcine kidney cell cDNA and GBP1 and GBP2 plasmids as templates and cloned into pCDNA3.1(+) eukaryotic expression vector. Overexpression or silencing of GBP1 and GBP2 genes on rhesus monkey cells (MA104) cells was performed to explore the effect on PoRV replication. Different time variables were set to screen the time points at which GBP1 and GBP2 exerted their effects on PoRV replication. Apply pan-GTPase enzyme inhibitor (CID-1067700) to probe the effect of GTPase enzyme activity of GBP1 and GBP2 on PoRV. The expression of GBP1 and GBP2 proteins were detected by Western blot, quantitative PCR and viral TCID₅₀ assays, and their antiviral functions were investigated. The results showed that pCDNA3.1 (+)-GBP1-full-length-HIS and pCDNA3.1(+)-GBP2(47aa-592aa), (131aa-592aa)-HIS recombinant plasmids were successfully constructed and verified to be expressd. PoRV significantly promoted up-regulations of GBP1 and GBP2 at protein and mRNA expressions. Overexpression of GBP1 and GBP2 significantly inhibited PoRV replication, and silencing the expression of GBP1 and GBP2 genes significantly promoted PoRV replication. The pCDNA3.1 (+)-GBP1/GBP2-G, M, E, ME-HIS structural domain truncations of GBP1 and GBP2 proteins were successfully constructed and verified to be expressed. Overexpression of the truncator gene revealed that the G region of GBP1 and GBP2 exhibited an inhibitory effect on PoRV replication. It was verified by pan-GTPase inhibitory enzyme activity that GTPase enzyme activity is required for GBP1 and GBP2 to inhibit PoRV replication. The results suggest that the GBP1 and GBP2 proteins are able to inhibit PoRV replication at replication phase of the virus, and the inhibitory effect dependents on its G region containing the GTPase enzyme active sites. The results of the study provide a theoretical basis for finding new drug targets for PoRV and developing novel vaccines.

Key words: porcine rotavirus, GBP1, GBP2, antiviral, structural domain

中图分类号:

S852.659.4

陈姝宇, 朱雪蛟, 周金柱, 陶然, 张雪寒, 索朗斯珠, 贡嘎, 李彬. 鸟苷酸结合蛋白GBP1和GBP2抑制猪轮状病毒体外复制[J]. 畜牧兽医学报, 2024, 55(1): 245-257.

CHEN Shuyu, ZHU Xuejiao, ZHOU Jinzhu, TAO Ran, ZHANG Xuehan, Suolangsizhu, Gongga, LI Bin. Inhibition of Porcine Rotavirus in vitro Replication by Guanylate-binding Proteins GBP1 and GBP2[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 245-257.

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