Knockout Of INF-? And BST-2 And Analysis Of The Innate Immune Responses Against Aiv Infection In MDCK Cells

Avian influenza(AI)caused by avian influenza virus(AIV),is deadly infectious diseases of birds,which not only causes great economic losses to the international poultry industry,but also has a significant impact on human public health.MDCK cells,as susceptible cells for influenza virus,are commonly used for disease pathogenesis research and vaccine development.By using the CRISPR/Cas9 system to perform gene editing in host cells,which might improve the cell's ability to increase toxicity,achieve continuous and stable proliferation of the virus in the cell-based culture system.The innate immune system plays an important role in the body's resistance to virus invasion,IFN-?1 induces and activates the host natural immune signaling pathway during the infection of MDCK cells with avian influenza virus,which interferes with multiple processes of virus proliferation.IFN-?1 released extracellularly can further inhibit the spread of avian influenza virus between cells.BST2 inhibits the retrovirus by connecting the cell membrane to the virus envelope,thus preventing the virus from budding after infecting the cell.It can also cause the up-regulation of pro-inflammatory cytokines and type I IFN,triggering an enzymatic reaction to further prevent virus replication and spread.In this work,we constructed CRISPR/Cas9 gene editing system to edit MDCK cells,IFN-?1 and BST2 gene was knocked out.By detecting the key factors in the natural immune signal transmission pathway of MDCK cells responding to AIV,analyzing and constructing sg RNA library and editing the gene library of cells.The growth ability and viral proliferation level of different monoclonal cell lines obtained were examined.The main results of this study are as follows:(1)CRISPR/Cas9 system was successfully constructed,using this technique to knock out the IFN-?1 gene in MDCK cells,and confirmed 19 positive cell clones.PCR and target fragment sequencing results showed that large fragments deletion or random insertion occurred at the targeted cleavage sites,indicating that IFN-?1 gene was performed in MDCK cells.By selecting clone1,clone3,clone10,clone14 and clone16 with fast cell growth rate,and using these cells to conduct a comparative test on the proliferation of AIV,the cell clone1 showed the best virus proliferation effect.The cell clone was used to obtain the ability of H9 subtype AIV JS02 strain to continuously proliferate,and HA titer gradually increased and maintained at the highest proliferation level.(2)The BST2 gene in MDCK cells was edited,and the single cell clone was obtained by screening.The MDCK-BST2-KO clone cells were confirmed by PCR.The results of CCK8 test showed that the cell clone has the same growth capacity as the original cells.The expression levels of BST2 and IFN-?were lower in MDCK-BST2-KO cells.BST2 overexpression and complement expression cell lines were obtained by lentiviral transfection,and Indirect immunofluorescence assay showed that the expression of BST2 in different cells was different under the effect of H9 subtype AIV,the HA titer was highest in MDCK-BST2-KO cells after the virus continued to proliferate,it showed that BST2 has a limiting effect on H9 subtype AIV.(3)RT-PCR detected 84 gene expression changes related to the immune response of MDCK cells against AIV invasion,which showed that 35 genes were significantly upregulated within 24 hours after infection,and 28 genes reached the strongest level at 12 hours.Gene ontology and KEGG pathway analysis showed that the most important pathways involved in AIV-infected MDCK cells are NF-?B and TNF signaling pathways,cytokine-cytokine receptor interactions,and PAMPs and others.By editing the gene library of MDCK cells and screening single cell clones,it was found that when genes in the cellular antiviral immune pathway were knocked out,there were differences in cell growth capacity and viral proliferation effect.In summary,by studying the interaction mechanism between the host immune system and viruses,genome editing in host cells might probably achieve continuous and stable proliferation of the virus in the cell-based culture system.This experiment provided a reliable basis and new ideas for better understanding the host antagonism effect on the virus,constructing and screening high-quality vaccine producing cell lines.