免疫共沉淀联合质谱技术筛选蓝舌病病毒NS4蛋白的互作蛋白

doi:10.16431/j.cnki.1671-7236.2024.02.034

摘要/Abstract

摘要： 【目的】筛选蓝舌病病毒(Bluetongue virus,BTV)NS4蛋白颉颃干扰素(interferon,IFN)信号通路的内源性互作蛋白,以便进一步研究NS4抑制IFN信号转导的作用机制。【方法】在前期仙台病毒(Sendai virus,SeV)诱导IFN信号通路基因表达研究基础上,利用免疫共沉淀方法从转染NS4融合eGFP标签表达载体pcDNA3.1-NS4-eGFP和pcDNA3.1-eGFP空载体的HEK-293T细胞中钓取NS4互作蛋白,对免疫沉淀物进行SDS-PAGE分析,免疫共沉淀洗脱液进行质谱鉴定及生物信息学分析,利用实时荧光定量PCR检测候选蛋白的编码基因表达特征。【结果】通过免疫共沉淀及质谱鉴定分析和数据库比对,共获得189个差异表达蛋白。生物信息学分析结果显示,候选蛋白主要参与蛋白转录、翻译、病毒感染及免疫调控。亚细胞定位分析结果显示,差异表达蛋白主要定位于细胞核、细胞质以及胞质-胞核,筛选出4个与BTV NS4蛋白存在互作的候选蛋白:多聚嘧啶束结合蛋白1(PTBP1)、细胞周期依赖性蛋白激酶9(CDK9)、N-端豆蔻酰化酶1(NMT1)和Y盒结合蛋白1(YBX1)。实时荧光定量PCR结果显示,与对照组相比,SeV刺激72 h后,试验组PTBP1、NMT1、YBX1、CDK9基因mRNA表达量均显著或极显著上调(P＜0.05;P＜0.01)。【结论】BTV NS4蛋白颉颃IFN信号通路与PTBP1、NMT1、YBX1、CDK9蛋白表达上调有关,本试验结果为进一步研究BTV NS4蛋白颉颃宿主天然免疫应答的分子机制提供靶标参考。

关键词: 蓝舌病病毒(BTV); 免疫共沉淀; 质谱分析; NS4蛋白

Abstract: 【Objective】 The purpose of this study was to screen the endogenous interaction protein of NS4 protein of Bluetongue virus (BTV) against interferon (IFN) signaling pathway, in order to further investigate the mechanism of NS4 inhibition of IFN signaling.【Method】 On the basis of previous studies on the gene expression of IFN signaling pathway induced by Sendai virus (SeV), NS4 interacting proteins were extracted from HEK-293T cells transfected with NS4 fusion eGFP label expression vector pcDNA3.1-NS4-eGFP and empty vector pcDNA3.1-eGFP by co-immunoprecipitation method.The immunoprecipitates were analyzed by SDS-PAGE, and the immunocoprecipitated eluents were identified by mass spectrometry and bioinformatics.Real-time quantitative PCR was used to detect the expression characteristics of candidate protein-coding genes.【Result】 A total of 189 differentially expressed proteins were obtained by co-immunoprecipitation, mass spectrometry analysis and database comparison.The results of bioinformatics analysis showed that the candidate proteins were mainly involved in protein transcription, translation, viral infection and immune regulation.The results of subcellular localization analysis showed that the differentially expressed proteins were mainly located in the nucleus, cytoplasm and cytoplasm-nucleus, and four candidate proteins interacting with BTV NS4 protein were screened out, which were polypyrimidine bunch-binding protein 1 (PTBP1), cell cycle dependent protein kinase 9 (CDK9), N-terminal myristylase 1 (NMT1) and Y-box binding protein 1 (YBX1).Real-time quantitative PCR results showed that compared with control group, the mRNA expressions of PTBP1, NMT1, YBX1 and CDK9 genes in experimental groups were significantly or extremely significantly up-regulated after 72 h of SeV stimulation (P＜0.05 or P＜0.01).【Conclusion】 The antagonistic IFN signaling pathway of BTV NS4 protein was related to the up-regulation of PTBP1, NMT1, YBX1 and CDK9 proteins, which provided a target reference for further research on the molecular mechanism of BTV NS4 protein antagonizing host innate immune response.

Key words: Bluetongue virus (BTV); co-immunoprecipitation; mass spectrometry; NS4 protein

中图分类号:

S852.65⁺9.4

马鲜平, 陈玉娟, 罗世美, 卓晓静, 杨义彬, 刘祎毅, 蔡旭研, 唐艺匀, 陈畅昶, 魏小蓉, 易华山. 免疫共沉淀联合质谱技术筛选蓝舌病病毒NS4蛋白的互作蛋白[J]. 中国畜牧兽医, 2024, 51(2): 779-789.

MA Xianping, CHEN Yujuan, LUO Shimei, ZHUO Xiaojing, YANG Yibin, LIU Yiyi, CAI Xuyan, TANG Yiyun, CHEN Changchang, WEI Xiaorong, YI Huashan. Screening of the Interacting Proteins with BTV NS4 by Co-immunoprecipitation Combining with Mass Spectrometry[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 779-789.

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