嗜吞噬无形体效应蛋白Ats-1调控宿主细胞剪接体转录机制的初步分析  

iTRAQ-based screening of differentially expressed proteins in host cells induced by Anaplasma phagocytophilum effector protein Ats-1

作　　者：李芮芮 史梦华 张妍 何洪琴 王雨璐 陈佳 王勇[1,2,3] 陈创夫 LI Rui-rui;SHI Meng-hua;ZHANG Yan;HE Hong-qin;WANG Yu-lu;CHEN Jia;WANG Yong;CHEN Chuang-fu(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;Collaborative Innovation Center for Sheep Health Breeding and Zoonoses Prevention and Control,Shihezi 832003,China;Key Laboratory of the Corps for Animal Disease Prevention and Control,Xinjiang Production and Construction Corps,Shihezi 832003,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]绵羊健康养殖与人兽共患病防控协同创新中心,新疆石河子832003 [3]动物疾病防控兵团重点实验室,新疆石河子832003

出　　处：《中国预防兽医学报》2024年第1期39-48,共10页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金项目(31760738)。

摘　　要：为研究嗜吞噬无形体效应蛋白Ats-1对宿主细胞内蛋白表达的影响,并初步分析Ats-1对宿主细胞剪接体的调控机制,本研究将嗜吞噬无形体的Ats-1基因克隆后连接至pcDNA3.1质粒,构建pcDNA3.1-Ats-1重组质粒,经PCR和测序鉴定正确后利用脂质体LipofectamineTM3000将其转染HEK293T细胞,继续培养至24 h、48 h时经western blot鉴定,结果显示,在48 ku(全长蛋白)和35 ku(成熟蛋白)出现特异性条带,表明Ats-1蛋白在细胞内可正确表达;且相较于24 h,48 h Ats-1蛋白在细胞内的表达量显著增加(P<0.05)。将重组质粒pcDNA3.1-Ats-1和空质粒pcDNA3.1分别转染HEK293T细胞,48 h后收获细胞并裂解取裂解液,利用同量异位标签定量蛋白质组学方法(iTRAQ)筛选Ats-1表达后宿主细胞内差异显著表达的蛋白,并采用基因本体论(GO)分析差异显著表达蛋白的功能,利用京都基因与基因组百科全书(KEGG)数据库分析差异显著表达蛋白参与的信号通路。从剪接体通路选取34个差异表达蛋白通过q RT-PCR验证。i TRAQ结果显示,与空质粒转染细胞组相比,重组质粒pcDNA3.1-Ats-1转染细胞组共鉴定到852个差异显著表达的蛋白,其中表达显著上调蛋白406个,表达显著下调蛋白446个。GO功能分析结果显示,差异显著表达蛋白主要定位在膜、胞内膜结合细胞器、膜组分,参与大分子生物合成过程,有核酸和RNA结合活性。KEGG信号通路分析结果显示,差异显著表达蛋白显著富集于剪接体、N-糖基化、蛋白输出和RNA运输等信号通路。q RT-PCR结果显示,剪接体通路富集的差异显著表达蛋白仅O43143蛋白的m RNA转录水平上调,其他33个蛋白(A0A024R1K8、Q13435、Q13573等)m RNA的转录水平均显著下调,与iTRAQ的分析结果一致。综上所述,本研究首次证实Ats-1蛋白表达后可能参与宿主细胞中剪接体、N-糖基化和蛋白输出信号通路的调控,抑制宿主细胞中剪接体调控通路中部分蛋白的转录,�The study aimed to investigate the differential protein expression after the expression of the Anaplasma phagocytophilum effector protein Ats-1 in host cells,as well as to preliminarily analyze the regulatory mechanisms of Ats-1.In this study,pcDNA3.1-Ats-1 was constructed to express Ats-1.The recombinant plasmid pcDNA3.1-Ats-1 and vector plasmid pcDNA3.1 were separately transfected into HEK293T cells for 48 hours,and the cell lysates were collected.The differentially expressed proteins in host cells after Ats-1 expression were screened using the Isobaric Tag for relative and absolute quantification techniques(iTRAQ).The gene ontology(GO)analysis was used to analyze the functions of significant differentially expressed proteins,and the Kyoto Encyclopedia of Genes and Genomes(KEGG)database was used to analyze the signaling pathways involved in differentially expressed proteins.Thiry-four differentially expressed proteins were selected from the spliceosome pathway and validated by qRT-PCR.Western blot results showed specific bands at 48ku(full-length protein)and 35ku(mature protein),indicating successful expression of Ats-1 protein in the cells.Moreover,the expression of Ats-1 protein in the cells significantly increased at 48 hours compared to 24 hours.iTRAQ results showed that compared to the vector plasmid transfected cell group,the pcDNA3.1-Ats-1 transfected cell group identified a total of 852 differentially expressed proteins,including 406 up-regulated proteins and 446 down-regulated proteins.GO results showed that significant differentially expressed proteins were mainly localized in the membrane,intracellular membrane-bound organelles,membrane components,and involved in macromolecule biosynthesis processes with nucleic acid and RNA binding activities.KEGG results showed that significant differentially expressed proteins were significantly enriched in pathways such as spliceosome,N-glycosylation,protein export,and RNA transport.qRT-PCR results showed that among the differentially expressed proteins enriched

关 键 词：Ats-1蛋白 ITRAQ 差异表达蛋白 剪接体 

分 类 号：S852.6[农业科学—基础兽医学]