Construction Of MDA5^-/- Mice And Study On Innate Immune Response Of STING^-/- Mice To H9N2 Subtype AIV

As a member of the RLRs protein family,MDA5 specifically recognizes long double-stranded RNA,to activate signaling pathways in vivo,promotes type I interferon expression,and plays an important role in the natural immune system.Using CRISPR/Cas9 technology,a sequence of the first exon region of the mouse MDA5 gene,in vitro transcripts containing sg RNA and Cas9 plasmids into fertilized eggs of C57BL/6 inbred mice,eight F0 primary mice were MDA5 knockout homozygous mice;MDA5^-/-mice with wild-type（WT）mice and then homozygous by full homo cage mating.Genomic PCR analysis showed four homozygous mice in F2 generation mice.Further RT-PCR results showed no MDA5 gene expression in all organs of MDA5 knockout mice,indicating the successful construction of MDA5 knockout mouse model based on CRISPR/Cas9 technology,and providing valuable models for further investigation of MDA5 in natural immune response mechanisms and antiviral infection.Stimulator of interferon gene（STING）is an important adaptor protein involved in the body.The cytosolic DNA receptor c GAS forms signaling pathway together with the downstream adaptor protein STING is the main pathway for signaling activation by STING.The c GAS-STING signaling pathway mainly recognizes DNA viruses,but in recent years it has been found that RNA virus infection can activate the mt DNA-c GAS-STING signaling pathway.In this study,STING in the natural immune response against RNA virus infection by H9N2 virus infection.Six 6-week-old and WT mice were divided into two groups:6immunization group and 6 unimmunized group;vaccinated with H9N2 inactivated vaccine by intradermal multipoint injection for 21 days after immunization;14 days after immunization and unimmunized group were vaccinated with ck/HLJ/3/00 strain by nasal infection.Three mice from the four groups on day 3and day 6 after challenge were selected to collect blood by orbital blood sampling.HI for STING^-/-mice and WT mice;lung tissue was used for HE staining and some for quantitative PCR detection of downstream signaling molecules,inflammatory factors,interferon and antiviral proteins of STING.Results:HI antibody titer:the serum antibody levels in STING^-/-immunization group on days 3 and 6 were lower than those in WT group;Virus titer of lung tissue:STING^-/-Inimmune challenge mice were higher than WT mice;Quantitative PCR results:lower m RNA expression of STING proteins（c GAS/DDX41/MAVS/Mx1）and factors（IRF3/NF-B/IL-1β）and IFN-β/ISG15 than in WT group except for inflammatory factor IL-10.The results show that after STING knockdown,STING-mediated natural immune response is suppressed,downstream signaling is blocked,unable to inhibit influenza virus replication,and weakened antiviral infection.