[1]任晓婷,安乐乐,王春林,等.温度与时间对BVDV Npro蛋白裂解活性的影响[J].江苏农业学报,2026,42(03):598-605.[doi:doi:10.3969/j.issn.1000-4440.2026.03.018]
　REN Xiaoting,AN Lele,WANG Chunlin,et al.Impact of temperature and time on the cleavage activity of BVDV Npro protein[J].,2026,42(03):598-605.[doi:doi:10.3969/j.issn.1000-4440.2026.03.018]
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温度与时间对BVDV Npro蛋白裂解活性的影响()

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
42
期数:
2026年03期
页码:
598-605
栏目:
畜牧兽医·水产养殖
出版日期:
2026-03-31

文章信息/Info

Title:
Impact of temperature and time on the cleavage activity of BVDV Npro protein
作者:
任晓婷1; 安乐乐1; 2; 王春林1; 刘泽煜1; 马晓霞1; 2
(1.西北民族大学生物医学研究中心/生物工程与技术国家民委重点实验室,甘肃兰州730030；2.西北民族大学生命科学与工程学院,甘肃兰州730100)
Author(s):
REN Xiaoting1; AN Lele1; 2; WANG Chunlin1; LIU Zeyu1; MA Xiaoxia1; 2
(1.Biomedical Research Center, Northwest Minzu University/Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission, Lanzhou 730030, China；2.College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730100, China)
关键词:
牛病毒性腹泻病毒; Npro; 原核系统; 自裂解
Keywords:
bovine viral diarrhea virus; Npro; prokaryotic system; self-cleavage
分类号:
S852.653
DOI:
doi:10.3969/j.issn.1000-4440.2026.03.018
文献标志码:
A
摘要:
为探究牛病毒性腹泻病毒(Bovine viral diarrhea virus, BVDV)Npro蛋白酶的自催化裂解活性及其关键调控因素,本研究通过构建含Npro自切割位点的重组融合表达载体pET-28a-Npro-C5-EGFP,采用大肠杆菌BL21(DE3)为工程菌,在不同温度(16 ℃、25 ℃、30 ℃、37 ℃)及不同诱导时间(2 h、4 h、6 h、8 h、10 h)条件下进行重组蛋白诱导表达,并利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)与蛋白质印迹法(Western blot)检测重组蛋白表达情况。结果表明,相对分子量4.54×104的Npro-C5-EGFP融合蛋白在诱导条件下可自主裂解产生相对分子量为1.85×104的Npro蛋白,且有完整的自催化活性。裂解率随诱导时间的延长呈显著线性升高趋势；随着温度的增加,裂解率呈先升高后降低的趋势,最适裂解温度为30 ℃。Npro蛋白含有潜在的活性中心(Cys69-His49催化二元体),具备执行生物功能的空间构象。本研究结果不仅揭示了BVDV Npro蛋白在原核系统表达后仍具有明显的自催化裂解活性,而且揭示了酶学行为受温度和诱导时间的协同调控。本研究结果为进一步研究BVDV Npro的自裂解特性及后续作为抗病毒药物筛选靶点提供了依据。
Abstract:
In order to explore the autocatalytic cleavage activity of Npro protease of bovine viral diarrhea virus (BVDV) and its key regulatory factors, the recombinant fusion expression vector pET-28a-Npro-C5-EGFP containing Npro self-cleavage site was constructed, and Escherichia coli BL21 (DE3) was used as engineering bacteria. The recombinant protein was induced to express at different temperatures (16 ℃, 25 ℃, 30 ℃, 37 ℃) and different induction times (2 h, 4 h, 6 h, 8 h, 10 h). The expression of recombinant protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The results showed that the Npro-C5-EGFP fusion protein with a relative molecular mass of 4.54×104 could be cleaved to produce Npro protein with a relative molecular mass of 1.85×104 under induction conditions, and had complete autocatalytic activity. The cleavage rate showed a significant linear growth trend with the extension of induction time. With the increase of temperature, the cleavage rate increased first and then decreased, and the optimum cleavage temperature was 30 ℃. The Npro protein contained a potential active center Cys69-His49 catalytic dyad with a spatial conformation that performed biological functions. This study not only revealed that the BVDV Npro protein has obvious autocatalytic cleavage activity after expression in the prokaryotic system, but also revealed that the enzymatic behavior is synergistically regulated by temperature and induction time. The results of this study provide a basis for further study of the self-cleavage characteristics of BVDV Npro and its subsequent use as a target for antiviral drug screening.

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备注/Memo

备注/Memo:
收稿日期：2025-05-11基金项目：甘肃省自然科学基金项目(23JRRA715)；中央高校基本科研业务费专项基金项目(31920250029)作者简介：任晓婷(2001-),女,山东淄博人,硕士研究生,主要从事病毒基因工程研究。(Tel)15964493398；(E-mail)15964493398@163.com通讯作者：马晓霞,(E-mail)maxiaoxia956@163.com
更新日期/Last Update: 2026-04-17