高致病性禽流感H5N1 NP蛋白与NIK蛋白相互作用初步研究

发布时间：2018-01-16 23:31

本文关键词：高致病性禽流感H5N1 NP蛋白与NIK蛋白相互作用初步研究　出处：《军事医学》2017年04期 　论文类型：期刊论文

【摘要】：目的研究高致病性禽流感H5N1(A/goose/Jilin/hb/2003)核蛋白(NP)与NF-κB诱导激酶(NIK)是否存在蛋白质相互作用,探讨H5N1 NP对NIK诱导的NF-κB转录活性的影响。方法从人宫颈癌细胞系He La细胞的总RNA中经逆转录和PCR获得NIK的全长cDNA双链片段,分别以其和pcDNA3-Flag-NP载体为模板,p CMVMyc-N和pGEX-4T-1为载体,构建真核表达载体pCMV-Myc-NIK和原核表达载体pGEX-4T-1-NP(GST-NP),并通过免疫共沉淀及GST pull-down实验检测H5N1 NP和NIK之间是否存在相互作用,利用双荧光素酶报告基因系统检测H5N1 NP对NIK诱导的NF-κB转录活性的影响。结果免疫共沉淀及GST pull-down实验表明,构建的真核表达载体pCMV-Myc-NIK和原核表达载体pGEX-4T-1-NP(GST-NP)均能正确表达,H5N1 NP与NIK之间存在相互作用;报告基因结果显示,H5N1 NP蛋白能明显抑制NIK诱导的NF-κB转录活性。结论 H5N1 NP与NIK之间存在蛋白质相互作用,并抑制NIK诱导的NF-κB转录活性,为进一步研究H5N1的致病机制打下基础。
[Abstract]:Objective to study the nucleoprotein (NPN) and NF- 魏 B induced kinase (NIKK) of the highly pathogenic avian influenza A / A / J / J / J / J / 2003 nuclear protein (NPN) and NF- 魏 B induced kinases (NIKs). Whether there is a protein interaction. To investigate the effect of H5N1 NP on NF- 魏 B transcriptional activity induced by NIK. The full-length cDNA double strand fragment of NIK was obtained from the total RNA of La cells by reverse transcription and PCR. Its and pcDNA3-Flag-NP carriers were used as template CMVMyc-N and pGEX-4T-1 as carriers, respectively. The eukaryotic expression vector pCMV-Myc-NIK and the prokaryotic expression vector pGEX-4T-1-NPGST-NPwere constructed. The interaction between H5N1 NP and NIK was detected by immunoprecipitation and GST pull-down assay. Double luciferase reporter gene system was used to detect the effect of H5N1 NP on NF- 魏 B transcriptional activity induced by NIK. The constructed eukaryotic expression vector pCMV-Myc-NIK and prokaryotic expression vector pGEX-4T-1-NPGST-NP) can be expressed correctly. There was interaction between H5N1 NP and NIK. The results of the reporter gene showed that the H5N1 NP protein could significantly inhibit the NF- 魏 B transcriptional activity induced by NIK. Conclusion there is a protein interaction between H5N1 NP and NIK. The inhibition of NF- 魏 B transcriptional activity induced by NIK provides a basis for further study on the pathogenesis of H5N1.
【作者单位】：安徽大学健康科学研究院;军事医学科学院生物工程研究所;
【基金】：国家自然科学基金资助项目(31400046)
【分类号】：R373.1
【正文快照】： 禽流感是由A型禽流感病毒(influenza A virus,IAV)引起的一种高度接触性急性传染病,对养殖业的发展以及人类公共卫生具有严重危害。由于缺乏有效的预防和治疗手段,禽流感H5N1病毒已成为威胁全球健康的长期隐患。IAV属于正黏病毒科(Orthomyxoviridae),其基因组由8条负义单链RNA

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