Preliminary Study On Modulation Of MAP2 In RLH Signaling Pathways

Objective:Pathogens can invade the body,causing infection and even infectious diseases,among the various pathogenic microorganisms,bacteria and virus are the most harmful pathogens.Viral infection in human is widespread,such as in third world countries,almost all adults infected with herpes simplex virus;others such as viral upper respiratory infection is also common,almost everyone has a naturally occurring body's immune response,not for a certain kind of allogeneic substances(including microorganisms),but against all invasion allogeneic substance,that is the innate immune response.In the early stages of virus invasion and infection(especially the specific immune response has not formed),the inherent immune response plays a major role to prevent the virus invasion,kill and remove the virus,terminate infection.Our previous studies have found that MAP2 interacts with MAVS which is the core protein in RLH antiviral innate immune signaling pathway,and MAP2 is involved in the regulation of antiviral immune responses.Therefore,we used cell immunofluorescence and other experimental techniques to explore the possible mechanisms of MAP2 in antivirus regulation.Method:1?Tumor cell lines named Hela was used in experimental studies as the carrier;2?Applied DNA recombination technology as the core of the biotechnology to build the plasmids used in our experiments;The target gene fragments used in the experiment were searched and confirmed on NCBI,and primers were synthesized by Biotechnology Company;The polymerase Chain Reaction(PCR)was used to amplify the specific DNA fragment;DNA electrophoresis was used to examine the target fragment and the product was recovered using the agarose gel DNA recovery kit,the target fragment was digested with double digestion,and then using the PCR product recovery kit to recover the target product,after confirmation by electrophoresis,the recovered product was ligated with the vector and then transformed,after the sequencing,the plasmids extracted by using plasmid midi preparation kit.3?The genes were overexpressed in Hela cell line by cell transfection and the RLH antiviral innate immune signaling pathway was activated by related plasmids or virus.4?Using immunofluorescence staining and confocal microscopy to observe the effects of MAP2 on mitochondria and MAVS in different cases.5?The effect of NO donor and NO precursor on cell mitochondria was observed by confocal microscopy.Results:1?Immunofluorescence assay was used to verify that MAVS was localized inmitochondria;2?MAP2 and MAVS co-overexpression can change the distribution of MAVS,that MAVS aggregates in the cells;3?if only just overexpressing MAP2,the distribution of mitochondria could not change,so MAP2 did not directly affect the mitochondrial distribution;4 ? If co-overexpressed MAP2 and MAVS in cells,the mitochondria occurred aggregation;5 ? MAP2 overexpression could change the distribution of mitochondria only when cells' RLH antiviral innate immune signaling pathway was activated by different ways,that is the same aggregation phenomenon as before;6?The MAP1 and MAVS co-overexpression could not alter the distribution of MAVS in cells;7?the mitochondria also occurred aggregation after NO donor and NO precursor treatments.Summary:1?MAP2 interacts with MAVS and affects the distribution of mitochondria only when RLH antiviral innate immune signaling pathway is activated.It is speculated that MAP2 may regulate antiviral immune response through its impact on MAVS and mitochondria distribution;2?After NO donor and NO precursor treatments,the mitochondria also appeared the aggregation phenomenon,so MAP2 may affect MAVS and mitochondria distribution through regulating the production of NO.