Investigation Of AIM2 Inflammasome In EV71 Induced Encephalitis And Studies On The Baculovirus As The Live Virus Vaccine Vector Of SARS-CoV-2

This thesis contains two parts,in which we studied the AIM2 Inflammasome in Encephalitis Caused by EV71 Infection and the feasibility of the Baculoviruse as the live virus vaccine vector of SARS-CoV-2.First Part:In this section,we mainly used EV71 to infect its susceptible cells of human rhabdomyoma cells（RD）and human neuroblastoma（SK-N-SH）,quantitative real time polymerase chain reaction（qRT-PCR）,Western-Blot and Enzyme Linked Immunosorbent Assay（ELISA）were performed to detect the expression of absent in melanoma 2（AIM2）inflammasome and its downstream genes of the pathway.Flow cytometry was used to detect pyroptosis caused by EV71 infection.Purified EV71 virus were intracranially injected to the C57 wild type（WT）and AIM2^-/-newly born 24h mouse,respectively.Record the clinical score according to the symptom caused by EV71.Fixing the brain tissue in mouse after infection 7 days,dew-axing to the water,HE staining was used to observe the pathological changes.The expression of VP1 and AIM2 in mouse brain were detected by Immunohistochemical（IHC）staining.qRT-PCR were performed to detect the expression of VP1,Caspase1 and IL-1βin mouse brain tissue.Finally,identified the primary neuron cells,and observed the morphological changes of primary neuron cells infection with EV71.The virus titer of primary neuron cells supernatants in WT and AIM2^-/-were calculated by TCID₅₀in 48 hours.The expression of AIM2,Caspase1 and IL-1βwas detected by qRT-PCR,flow cytometry also were performed to detect the expression of pyroptosis.The results showed that AIM2 inflammasome and its pathway were activated by EV71 in RD cells,SK-N-SH cells and primary neuron cells.We established a infection model of EV71 in mouse primary neuron cells.More highly titer of EV71 in AIM2^-/-mouse primary neuron cells than WT proved that AIM2 inflammasome can restrict the replication of EV71 virus in mouse primary neuron cells.Compared with WT groups,more severe inflammatory infiltrates and more highly levels expression of VP1 in AIM2^-/-groups indicating AIM2inflammasome can restrict the replication of EV71 in mouse brain.All in all,these results provide a theoretical basis for how the host can resist virus infection,the mechanism of virus immune escape and the design of antiviral drugs.Second Part:In this study,we mainly constructed two baculoviruses,one of which was to clone the S protein of human SARS-CoV-2 into the underside of the p10promoter,which is S protein was expressed using the BAC to BAC eukaryotic expression system and displayed on the surface of the virus;the other was CMV promoter replaced the original p10 and Polh promoters,and clone the S protein of human SARS-CoV-2 into the underside of the CMV promoter,exogenous S protein was expressed by CMV promoter using baculovirus effective transduction into mammalian cells.BALB/c female mouse were immunized by hypodermic injection add intraperitoneal injection of the recombinant virus four times ones two weeks.The expression of specific SARS-CoV-2 Ig G in serum was detected by ELISA 10 days after the last immunization.After splenic lymphocyte cells were isolated,IFN-γand IL-4were detected to determine the type of specific cellular immune response by flow cytometry.Finally,the titer of neutralizing antibody in mouse serum was detected by SARS-CoV-2 pseudovirus.The results showed that the recombinant baculoviruses constructed by the two methods could induce specific cellular immunity and humoral immune reaction than control group.In high levels of specific anti-SARS-CoV-2 S protein Ig G antibody were produced by the recombinant baculoviruses.The level of neutralizing antibody had a significant increased in recombinant baculoviruses groups than control groups.These results indicated that recombinant baculovirus expressing S protein can be used as a potential live virus vaccine vector.