Screening Interacting Proteins For HCV NS5A Domains ? Protein By GST Pull Down Coupled With LC-MS/MS

Part one Constuction and Expression Of recombinant plasmid pGEX-4T-1-Conl-NS5A-35-215Objective To construct the recombinant plasmid pGEX-4T-1-Conl-NS5A-35-215,induce its expression in DE3 and purify the fusion protein.Methods Primers were designed to code NS5A-35-215 sequence by PCR with p3×FLAG-CMV14-Conl-NS5A plasmid as template.Enzyme digestion,then recycle the digested NS5A-35-215 sequence and pGEX-4T-1 vector.The PCR products were cloned into the SmaI and NotI sites of pGEX-4T-1 vector.The recombinant plasmid pGEX-4T-1-Conl-NS5A-35-215 was transformed into DH5a,cultured with LB culture plate,the positive colonies were picked and the recombinant plasmids were extracted.To screen and identify the correct recombinant plasmid by colony PCR and enzyme digestion and plasmid DNA sequencing.The recombinant plasmid was transformed into DE3 to induce and purify the GST-Conl-NS5A-35-215 fusion protein.Results the pGEX-4T-1-Conl-NS5A-35-215 plasmid was successfully constructed and high efficiency expressed in DE3,the GST fusion protein was induced and purified.Conclusion the pGEX-4T-1-Conl-NS5A-35-215 plasmid was successfully construc-ted,which lays the foundation to study the function and interacting proteins of Conl-NS5A-35-215.Part two Screening interacting proteins for HCV NS5A domains I protein by GST pull down coupled with LC-MS/MSObjective To explore the roles of HCV NS5A domains I Protein and its involvement in virus replication mechanism.Method the pGEX-4T-1-Conl-NS5A-35-215 plasmid was constructed,and the expression of GST-Conl-NS5A-35-215 fusion protein was induced and purified.GST pull down coupled with LC-MS/MS were performed to screen the Conl-NS5A-35-215 associated proteins in cytosolic extracts from Huh7.5.1 cells.The GO(Gene Ontology)annotation and pathway enrichment of Con1-NS5A-3 5-215 interacting proteins were analyzed by using OmicsBean online software.Result 547 potential Conl-NS5A-35-215 interacting proteins were identified.GO(Gene Ontology)annotation analysis shows that these interacting proteins function in regulation of response to stress,apoptotic process,lipid metabolic process,Golgi vesicle transport,regulation of protein ubiquitination,protein transport.In particular,KEGG(Kyoto Enyoolpedia of genes and Genomes)pathway enrichment analysis reveals that these interacting proteins involve in several important pathways,including Proteasome,Proteasome,Biosynthesis of amino acids,Fatty acid degradation,RNA transport.Conclusion NS5A domain I is involved in many biological processes,such as the regulation of intracellular transport,stress response,protein homeostasis and so on.By GST pull down experiments,the interaction of NS5A-Conl-35-215 with ACBD3 and VPS35 were verified,which provided a clue for further study of NS5A involvement in HCV replication.