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 in the spliceosome pathway,only the mRNA transcription of the protein O43143 was up-regulated,while the mRNA transcription of the other 33 proteins(A0A024R1K8,Q13435,Q13573,etc.)was down-regulated.In summary,Ats-1 protein probably involved in the regulation of the spliceosome,N-glycosylation,and protein export pathways in host cells.It may inhibit the transcription of some proteins in the spliceosome regulatory pathway,thereby potentially inhibiting spliceosome regulation.This study provides new directions and ideas for studying the regulatory mechanisms of the Anaplasma phagocytophilum effector protein Ats-1 in host cells.
NETL
NSTL
维普
万方数据
