Screening Of ZWINT And Study Of Its Antiviral Innate Immune Mechanism

The innate immune system is the first line of defense against the invasion of foreign pathogens by organisms.It is a non-specific immune system established during the long-term evolution of the organism.It has the ability to detect invading pathogens and the subsequent activation of adaptive immunity.It plays an important role in the body's anti-infection process and plays an important defensive function.The identification of microbial pathogens is an essential element in initiating an innate immune response,and the conserved components called pathogen-associated molecular patterns(PAMPs),including viral pathogens,are recognized by pattern recognition receptors.After pattern recognition receptors(PRRs)are recognized,a series of downstream signal transductions are initiated,thereby generating an anti-viral innate immune response and protecting the host cells from pathogen infection.TBK1(TANK-binding kinase 1)is one of the serine/threonine protein kinases.It can be widely expressed in many tissues and plays an important role in the signal transduction of the RLR antiviral signal pathway.When the virus infects the body,components of the virus such as DNA and RNA are recognized by different pattern recognition receptors(such as TLR3,RIG-I/MDA5,cGAS,etc.).These pattern recognition receptors recruit different and specific linker proteins,such as TRIF,VISA/MAVS,MITA/STING,and activate TBK1 to promote the phosphorylation and dimerization of the interferon regulatory factor IRF3,which in turn promotes the production of type I interferons and initiates anti-viral innate immunity response.From this we can see that TBK1 is involved in so many different types of receptor-mediated antiviral innate immune signaling pathways,and its role can never be ignored.Therefore,if we want to explore the mo lecular mechanism of antiviral innate immunity more clearly,the research on the mechanism of TBK1's participation in the antiviral immune response will be a starting point for us to continue to explore and ponder.Based on the above researches,we used human TBK1 as the bait and 293 cell cDNA as the expression library to screen through the yeast two-hybrid screening system,and then through subsequent PCR,sequencing analysis,sequence Blast comparison,and after analyzing the sequence expression framework and its protein function,a total of 27 candidate proteins that can interact with TBK1,including ZWINT,were successfully screened and obtained.Subsequently,we successfully cloned the full length of these candidate genes into mammalian cell expression vector pRK5-Flag by molecular cloning techniques for preparation of subsequent cell transfection experiments and analytical studies.We will use ZWINT as the main research object for the study of immune mechanism.The results indicate that overexpression of ZWINT enhances the activation of IFN-? and IRF3 induced by Sendai virus(SeV)and promotes the dimerization of IRF3 mediated by TBK1 and VISA,which positively regulates the antiviral RLR signaling pathway.Knockdown ZWINT expression negatively regulates Sendai virus(SeV)-induced activation of the RLR signaling pathway.At the same time,ZWINT will also promote the polyubiquitination of TBK1.In summary,ZWINT positively regulates antiviral RLR innate immune response,providing a new regulato ry mechanism for antiviral natural immune signaling pathway.