| 摘要: |
| 【目的】探究牛病毒性腹泻病毒（bovine viral diarrhea viru,BVDV）NS5A的3个结构域与核糖体内部进入位点（internal ribosome entry site，IRES）的相互作用。【方法】采用PCR法扩增Ⅰ型BVDV病毒NS5A基因的3个结构域DomainⅠ、DoaminⅡ和Domain Ⅲ片段，分别连接到pET-28a(+)载体上，在大肠杆菌中诱导表达融合蛋白，对表达产物进行Western Blot鉴定，并用镍柱法进行纯化。PCR扩增IRES DNA，转录获得IRES RNA。使用生物层干涉法(BLI)生物传感器(Gator Bio.)分子互作分析仪器对NS5A 3个结构域与IRES RNA的相互作用进行分析。【结果】成功表达并纯化了DomainⅠ、DoaminⅡ和Domain Ⅲ结构域重组蛋白，其质量浓度依次为76.2，96.8和57.7 μg/mL。体外转录获得了IRES RNA，其质量浓度为2 056.2 μg/mL。互作分析结果表明，NS5A的3个功能结构域中，只有DomainⅡ与IRES具有明显的相互作用，且这种互作存在快结合快解离的现象，其与IRES的亲和力为7.93×10-6 μmol/L。【结论】BVDV NS5A蛋白是调控IRES起始翻译的重要蛋白，其主要作用部位为DomainⅡ。 |
| 关键词: NS5A蛋白；牛；核糖体内部进入位点 牛病毒性腹泻病毒复制 |
| DOI：10.13207/j.cnki.jnwafu.2025.08.013 |
| 分类号: |
| 基金项目:国家自然科学基金项目（32360874）；甘肃省自然科学基金项目(23JRRA715); 西北民族大学引进人才科研项目(xbmuyjrc202225) |
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| Interaction between the NS5A domain of bovine viral diarrhea virus and the internal entry site of ribosome |
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GAO Mingyang, YANG Xuanye, HU Xinyan, WANG Jinqian, WU Yuhu, ZHOU Jianhua
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Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission,Biomedical Research Center,School of Life Sciences and Engineering,Northwest Minzu University,Lanzhou,Gansu 730010,China
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| Abstract: |
| 【Objective】This research aimed to explore the interaction between three domains of bovine viral diarrhea (BVDV) NS5A and internal ribosome entry site (IRES).【Method】The three domains of NS5A gene of typeⅠBVDV virus,Domain Ⅰ,Domain Ⅱ and Domain Ⅲ,were amplified by PCR and connected to pET-28a(+) vector,respectively.The fusion protein was induced in Escherichia coli,and identified by Western Blot,and the protein was purified by nickel column method.IRES DNA was amplified by PCR,IRES RNA was obtained through transcription by T7 in vitro,and the interactions between three domains and IRES RNA were analyzed using Biolayer Interferometry (BLI) biosensor (Gator Bio.).【Result】The recombinant proteins of Domain Ⅰ,DomainⅡ and Domain Ⅲ were successfully constructed and purified with concentrations of 76.2,96.8 and 57.7 μg/mL,respectively.IRES RNA was obtained through transcription in vitro with a concentration of 2 056.2 μg/mL.The results of interaction analysis showed that among the three functional domains of NS5A,only DomainⅡhad obvious interaction with IRES,which showed the feature of fast binding and fast dissociation,with an affinity of 7.93×10-6 μmol/L to IRES.【Conclusion】BVDV NS5A protein is an important protein regulating the initiation of IRES translation,and its main action site is DomainⅡ. |
| Key words: NS5A protein bovine internal ribosome entrysite (IRES) bovine viral diarrhea viru(BVDV) replication |
