Screening And Validation Of MDCK Cells For H3N2 Influenza Virus Response Proteins

Influenza is an acute respiratory infection caused by influenza virus.It spreads rapidly and is prone to cause large-scale outbreaks and epidemics.Vaccination is the main measure to prevent the spread of influenza virus.In recent years,with the acceleration of global influenza prevention and control capacity building and the increase in the quality requirements of influenza vaccine products,foreign influenza vaccines based on different types of cell matrix have been approved for marketing,and domestic influenza vaccines still rely entirely on chicken embryo For production,the current MDCK cell matrix influenza vaccine is the main direction of domestic vaccine research and development,but there are few studies on the cell mechanism of virus infection in the development of cell matrix influenza vaccine.In this study,MDCK cells and H3N2 influenza virus strain(NYMCX-223A)were used as the research objects.Using proteomics,immunofluorescence staining,real-time fluorescence quantitative PCR,and Western blot analysis,MDCK cells were screened for H3N2 influenza virus strain The differentially expressed proteins in the process laid the foundation for an in-depth understanding of the interaction mechanism between MDCK cells and influenza A virus,and obtained the following research results:1.A total of 374 differentially expressed proteins were identified by iTRAQ quantitative proteomics in uninfected and infected H3N2 influenza virus with MOI =1 for 12 hours in MDCK cells.Among them,51 differentially expressed proteins with increased expression levels were expressed There are 323 differentially expressed proteins whose amount is down-regulated.2.Through statistical analysis,GO enrichment and KEGG enrichment analysis of iTRAQ quantitative proteomics data,four differentially expressed proteins involved in influenza A virus infection were screened out,namely GSK3 B,RSAD2,VDAC1 and MEK2.Quantitative PCR confirmed that the trend of MEK2 mRNA expression level was the same as its protein expression trend,and speculated that MEK2 protein may be a key mechanism involved in regulating the replication and proliferation of influenza virus in cells.3.Infection of MDCK cells with H3N2 influenza virus can activate ERK signaling pathway,while the expression level of MEK2 protein gradually decreases with the extension of infection time,and its phosphorylation level gradually increases with the extension of infection time.Cells may activate the ERK signaling pathwaythrough MEK2-ERKs cascade activation.In this study,we determined the response protein screening of MDCK cells infected with H3N2 influenza virus with MOI = 1 for 12 h.ITRAQ quantitative proteomics was used to screen out the differentially expressed proteins during the infection process.Through statistics,GO enrichment,and KEGG signaling pathway Analysis and real-time fluorescence quantitative PCR further screened out MEK2 protein as the response protein of MDCK cells infected with H3N2 influenza virus.The protein quantity and phosphorylation level of MEK2 protein during infection were detected by Western blot.The results showed that the phosphorylation level of MEK2 protein The increase may be the molecular mechanism of H3N2 influenza virus infected MDCK cells to activate the ERK signaling pathway,which provides an important experimental basis for further research on the role of ERK signaling pathway in regulating the replication and proliferation of H3N2 influenza virus in MDCK cells.