A Small Study On The Function Of TUBA1 Gene In Toxoplasma Gondii Strain ME49 And Its Effect On Autophagy Of N2a Cells

Toxoplasma gondii(T.gondii)is a specific intracellular parasitic protozoa with infectivity at all stages of its life cycle.And cats are the only final hosts of T.gondii,which can be widely parasitized in humans and other thermostatic animals.T.gondii has great harm to human health and animal husbandry development,and it is a public health issue of world concern.According to the different immune status of the host,the infection and clinical results of T.gondii are also quite different.Although often manifested as invisible infection,serious cases will die because of dissemination and damage of the central nervous system and the whole body.T.gondii has neurotropic characteristic and can cause Toxoplasma encephalitis and other brain diseases,but its pathogenesis is still the focus of current research.In our previous study,we selected Drebrin(Dbn1),the differentially expressed protein in the brain of infected mice,as bait protein,and extracted six T.gondii proteins interacting with Dbn1 by Co-immunoprecipitation and Pull-down,including α-tubulin(TUBA1)that is an exosome protein.According to the structure of exosome and its characteristics of easily penetrating the blood-brain barrier,the mechanism of interaction mechanism between exosome and parasite invasion in Toxoplasma encephalopathy needs further study.In order to study the biological function of TUBA1,in this study,the pGEX-6p-1 prokaryotic expression vector was used to construct pGEX-6p-1-TUBA1 recombinant plasmid.Then mice were immunized with TUBA1 recombinant protein to prepare mouse polyclonal antibodies.Meanwhile,the eukaryotic expression plasmid pcDNA3.1(+)-HA-TUBA1 containing HA tag was constructed and transfected into neuroblastoma cell(N2a).Then the autophagosome was observed by transmission electron microscopy and fluorescence microscopy.And the expression of LC3I/II was detected by WB.The results showed that the prokaryotic and eukaryotic expression plasmids of TUBA1 were successfully constructed,and the polyclonal antibodies were obtained.And the expression and distribution of TUBA1 in T.gondii were detected by immunofluorescence.Therefore,this provides technical support for the follow-up study of the biological function of TUBA1 and its role in the pathogenesis of T.gondii.After transfection of eukaryotic expression plasmid pcDNA3.1(+)-HA-TUBA1 into N2a cells for 48 hours,the N2a cells shrank and synapse decreased.Furthermore,characteristic autophagosome was observed and the expression of LC3I/II was up-regulated,which proved that Tg TUBA1 protein promoted the occurrence of autophagy in N2a cells.