伪狂犬病病毒gE/gI/TK基因缺失对感染PK-15细胞的差异表达蛋白质组分析

Proteomic analysis of PK-15 cells infected with gE/gI/TK triple gene deleted pseudorabies virus

为了研究伪狂犬病病毒(pseudorabies virus,PRV)变异株3个主要毒力基因gE/gI/TK缺失对感染宿主细胞后蛋白表达的影响,探究PRV与宿主细胞相互作用的分子机制,将PRV SD-2017ΔgE/gI/TK株接种PK-15细胞,运用串联质谱标签(tandem mass tags,TMT)标记定量蛋白质组学技术,开展PRV感染细胞后差异表达蛋白分析,并通过Western blot进行数据验证。通过生物信息学分析发现,与亲本毒株PRV SD-2017感染组比较,SD-2017ΔgE/gI/TK感染组共鉴定到19个差异表达蛋白,其中7个蛋白上调,12个蛋白下调;与传统疫苗株Bartha-K/61比较,SD-2017ΔgE/gI/TK感染组共鉴定到176个差异表达蛋白,其中157个蛋白上调,19个蛋白下调。这些差异蛋白主要与紧密连接、RNA降解途径、核糖体生物发生、B细胞受体等信号通路有关,其中ATP2A1、KDM8、Eri1、XRCC6、DNM2和PYCR2等差异蛋白参与细胞凋亡、免疫反应和自噬相关功能。通过Western blot验证了MYLPF、ERI1、XRCC6和GAMT的表达情况与TMT比率一致,证明了该数据的可靠性。本研究为进一步探讨PRV变异株的致病和免疫机制奠定了理论基础。

In order to study the effect of three major virulence genes gE/gI/TK of pseudorabies virus(PRV)mutant on the protein expression of infected cells and explore the molecular mechanism of the interaction between PRV and host cells,PK-15cells were infected with PRV SD-2017ΔgE/gI/TK and the differentially expressed proteins of PRV infected cells were analyzed by tandem mass tags(TMT)labeling quantitative proteomics technology.The results were further verified by Western blot.Through bioinformatics analysis,it was found that compared with the parental strain PRV SD-2017,a total of 19differentially expressed proteins were identified in SD-2017ΔgE/gI/TK infected cells,of which 7proteins were up-regulated and 12proteins were downregulated.Compared with the traditional vaccine strain Bartha-K/61,176differentially expressed proteins were found in SD-2017ΔgE/gI/TK infected cells,of which 157proteins were up-regulated and 19proteins were down-regulated.These differential proteins were mainly related to signaling pathways such as tight junctions,RNA degradation pathways,ribosome biogenesis and B cell receptors.Among them,differential proteins such as ATP2A1,KDM8,Eri1,XRCC6,DNM2and PYCR2were involved in apoptosis,immune response and autophagy-related process.It was verified by Western blot that the expression of MYLPF,ERI1,XRCC6and GAMT was consistent with2(^)372 the TMT ratio,which proved the reliability of the proteomic analysis.This study provides a theoretical basis for further exploring the pathogenic mechanism and host immune response of PRV variants.