Effect Of Ubiquitin-like Activating Enzyme NAE On The Proliferation Of Influenza Virus And Preparation Of Monoclonal Antibodies Against NEDD8

NEDD8?neural-precursor-cell-expressed developmentally down-regulated 8?is a ubiquitin like small molecule protein involved in the regulation of cell growth,DNA damage,signal transduction,apoptosis,viability and development,etc.Recent studies have found that the influenza virus protein PB2 can be modified by Neddylation.This modification leads to a decrease in the stability of PB2 protein,thereby inhibiting the replication of influenza virus.It indicates that the Neddylation plays an antiviral role during influenza virus infection.Neddylation is the process by which NEDD8 is conjugated to its target proteins.This process is analogous to ubiquitination,although it relies on its own E1 and E2 enzymes.In this study,the knock-down?KD?cell lines of the NEDD8-activating enzyme?NAE?were constructed and infected with influenza virus strain A/PR/8/34?PR8?to investigate the proliferation of Influenza virus in the KD cell lines.This provide a theoretical basis for better elucidating the relationship between Influenza virus and Neddylation pathway.At the same time,we also prepared monoclonal antibodies against NEDD8 that laid a good foundation for further study of endogenous Neddylation modifications.The main results of this study are as follows:1.Increased proliferation of influenza virus in NAE knock-down cell linesThe NAE1-KD,UBA3-KD and corresponding control cell lines screened by Puromycin and then the cells were infected with PR8 virus at a dose of 1 MOI,respectively.The cell and supernatants were collected at different time points of 6 hours post-infection?hpi?,12 hpi,24hpi,and 30 hpi.Analyzed by Western blotting,virus titers were detected by titration and Quantitative Real-time PCR to calculate the copies of M segment viral RNAs?vRNA?.At each time point in the KD cell line,the expression levels of viral proteins?NP,NS1?,or the expression levels of vRNA of M segment,or viral titers(TCID₅₀/mL),were significantly higher than those in the control shRNA cells.It was shown that the decrease in the expression level of NAE1 in cells can significantly promote the replication of Influenza virus.2.Preparation of monoclonal antibodies against ubiquitin-like small molecule proteinNEDD8The full-length fragment of NEDD8 gene was amplified from A549 cells by RT-PCR and cloned into prokaryotic expression vector pET30a?+?.After sequencing and verification,it was transformed into BL21?DE3?and induced by Isopropyl?-D-1-thiogalactopyranoside?IPTG?.The results showed that the fusion protein was successfully expressed with a molecular mass of about 15 kDa.The expressed NEDD8 was purified and inoculated into6-week-old BALB/c mice to prepare monoclonal antibodies?mAb?.The hybridoma clones were screened and selected by Enzyme-linked immunosorbent assay?ELISA?and Western-blot analysis.Finally,three hybridoma cells were prepared as 4B7?1G11?1G3.The mAbs were successfully used in the co-immunoprecipitation assay?co-IP?by checking neddylated Cullin-1.The epitope of NEDD8 mAb 1G11 was specially located at the²⁹RVEE³² motif by identification with the truncated peptides.This study provides a practical tool for better elucidating the immune regulation of Neddylation in viral infection.In conclusion,this study primarily revealed the role of NAE activation enzyme in the process of influenza Neddylation modification,and successfully prepared NEDD8 monoclonal antibodies.It has important foundation for further studying the mechanism of immune regulation of influenza virus molecular pathogenesis.