Study On The Functional Mechanism Of CBL Protein Regulating The Replication Of H9N2 Subtype Avian Influenza Virus

Avian influenza is an acute and highly exposed infectious disease caused by the avian influenza virus（AIV）,which can cause a variety of symptoms from the respiratory system to severe systemic sepsis.According to the type of pathogen,it is divided into three categories:high pathogenicity,low pathogenicity and non-pathogenicity,in which H9N2 subtype is mainly low pathogenic avian influenza.Because of its milder symptoms and low mortality after infection,it is easily overlooked in production and can easily cause rapid transmission and infection.The H9N2 subtype AIV has brought huge impacts and losses to poultry production,and it also has a certain impact on human health.Therefore,the antiviral study of the functional protein against H9N2 subtype AIV replication and the molecular mechanism of AIV infecting cells are vital for the development of the poultry industry and human health.In this study,the eukaryotic expression recombinant vector FLAG-CBL was constructed,and the CBL protein was overexpressed in A549 cells and the endogenous CBL protein was knocked down to prove that CBL protein could inhibit the replication of H9N2 subtype AIV at an early stage.Also,CBL pdrotein was prokaryotic expressed and purified,and inoculated A549 cells.The similar conclusion of inhibition on AIV early replication was confirmed again.Subsequently,illumina high-throughput sequencing technology was used to perform RNA-seq sequencing on mouse-derived macrophage cells（RAW 264.7）infected with different dosage of H9N2 subtype AIV,and the transcriptome sequencing results were analyzed to find the key differentially expressed genes and their biological functions,which revealed the potential molecular mechanisms of the pathogenicity of H9N2 AIV.The main research contents include:1.Eukaryotic expression of CBL protein inhibits early replication of H9N2subtype AIVThis study was to investigate the effect of casitas B-lineage lymphoma（CBL）on the replication of H9N2 subtype AIV.The CBL gene fragment was amplified by PCR and linked into FLAG-N vector.The expression of FLAG-CBL recombinant vector and the expression of viral NP protein were verified by western blot.The levels of NP,NS and HA genes were detected by quantitative PCR.Afterwards,the TCID₅₀ method was used to detect the virus titer.Additionally,RNAi technology was used to detect the effect of CBL on HA gene expression level and the expression level of viral protein NP.Also,western blot was used to detect the effect of CBL on cell apoptosis and autophagy after virus infection.The results of double enzyme digestion Eco RⅠand HindⅢand gene sequencing showed that the eukaryotic expression vector FLAG-CBL was successfully constructed.The results of western blot confirmed that the expression of NP protein was decreased at 6 h after H9N2 subtype AIV infection in A549 cells transfected with FLAG-CBL recombinant vector.Furthermore,the m RNA levels of NP,NS and HA genes were significantly decreased at 6 and 12 h in A549 cells transfected with FLAG-CBL recombinant vector.Virus titer determination results again verified that the virus titer was significantly reduced at 6 h,suggesting that CBL protein could inhibit the replication of H9N2 virus at early stage.After knockdown of endogenous CBL protein in A549 cells with si RNA,HA gene level and NP protein level of H9N2 subtype AIV increased significantly at 12,24 and 36 h,which suggested that the decrease of CBL protein might be beneficial to AIV replication.Western blot results showed that the ratio of apoptosis protein Bcl-2/Bax and autophagy protein LC3-Ⅱ/LC3-Ⅰdid not significant changes,compared with the control group.These results indicated that CBL protein might inhibit H9N2 subtype virus amplification in A549 cells in the early stage,which provided an important experimental basis for further study of the antiviral mechanism of CBL protein,and provided an important idea for anti-AIV drug developments.2.Prokaryotic expression of CBL protein inhibits early replication of H9N2subtype AIVThe aim of this study was to investigate the effect of prokaryotic expression of CBL protein on the replication of H9N2 subtype AIV,and the apoptosis and autophagy of infected cells.The CBL gene fragment was amplified by PCR and linked into p ET-28a vector.The expression of p ET-28a-CBL recombinant vector and the expression of viral NP protein were verified by western blot.The effect of purified CBL protein on the viability of A549 cells detected by CCK8,the levels of HA genes were detected by quantitative PCR,and virus titer determination results was used to detect changes in the virulence titer of the virus supernatant.Also,western blot was used to detect the ratio of apoptosis protein Bcl-2/Bax and autophagy protein LC3-Ⅱ/LC3-Ⅰ.The results of Bam HⅠ,HindⅢdouble enzyme digestion and gene sequencing showed that the prokaryotic expression vector p ET-28a-CBL was successfully constructed,and successfully induced expression and purified.CCK8results confirmed that the purified CBL protein has no effect on cell viability,western blot results confirmed that the expression of viral NP protein in A549 cells incubated with CBL protein was reduced when H9N2 subtype AIV was infected for 6,12 and 24h.Fluorescence quantitative PCR results confirmed that the expression level of virus HA gene was significantly reduced at 6,12 and 24 h.The virus titer determination results also confirmed that the virus titer had a significant downward trend at 6,12and 24 h.These results showed that CBL protein could inhibit the proliferation of H9N2 virus to a certain extent.Western blot proved that detected the ratio of apoptosis protein Bcl-2/Bax was increased at 12 h,but the ratio of autophagy protein LC3-Ⅱ/LC3-Ⅰhad no significant difference.These results indicated that CBL proteins might inhibit the replication of H9N2 virus on A549 cells and the apoptosis of A549cells after infection with AIV at an early stage,which provides a good foundation for in-depth study of the mechanism of CBL protein against viral replication.3.Study on the molecular basis of H9N2 subtype AIV infecting murine macrophage cellsIn the research of the first two chapters,we founded that the CBL protein can inhibit the early replication of H9N2 subtype influenza virus,in order to explore the immune-related pathways and molecules activated by H9N2 subtype avian influenza virus（AIV）after infection of mouse-derived macrophages（Murine RAW 264.7）.A/chicken/Shandong/LY1/2017（H9）was used to infect the RAW 264.7 cells,and transcriptome sequencing was performed on the group infected with different MOI viruses and the uninfected control group.Data analysis results showed that in the H9N2-MOI-1 group,there were 10552 differentially expressed genes,in which 7707genes were up-regulated and 2,845 genes were down-regulated.In the H9N2-MOI-0.1group,there were 2238 differentially expressed genes,in which 1992 genes were up-regulated and 246 genes were down-regulated.Subsequently GO functional analysis showed that most of the genes regulated by H9N2 subtype AIV infection were concentrated in the extracellular matrix,cell membrane components and organelles,and were functionally involved in virus infection,biological stimulation,cellular biological regulation and metabolism.The results of KEGG analysis showed that CBL was found to be significantly up-regulated in the ubiquitin-mediated proteolysis signaling pathway,suggesting that it was involved in various biological processes after virus infection.Furthermore,the main immune-related pathways activated by H9N2 subtype AIV infection includeed signal pathways such as MAPK,Toll-like receptors,cytokines,etc,indicating that these signal pathways had a close role in the process of H9N2 AIV interacting with the host cells.Finally,eight related up-regulated or down-regulated genes were selected for verification by fluorescence quantitative PCR,and the verification results were consistent with the transcriptome sequencing results,indicating that the transcriptome sequencing results were credible.In short,this study analyzed the genome-wide differential gene expression profiles in cells infected with H9N2 subtype avian influenza virus,and revealed the related immune pathways involved in the replication of H9N2 subtype AIV.In summary,the results of the overexpression of CBL protein and incubation of prokaryotic CBL protein in A549 cells infected with H9N2 subtype AIV proved that CBL could inhibit H9N2 virus replication at an early stage,and also has a certain inhibitory effect on cell apoptosis.Additionally,the infection of H9N2 subtype AIV to mouse-derived macrophages could activate relevant immune pathways and might play a certain role in inhibiting virus replication.At the same time,it was found in the ubiquitination pathway that CBL was significantly up-regulated after infection with H9N2 subtype AIV,indicating that it was involved in the regulation of immune response.In this study,H9N2 subtype AIV was infected on cells of different species to do related research,which laid the foundation for further research on the functional mechanism of CBL protein regulating the replication of H9N2 subtype avian influenza virus in the future.