Screening And Preliminary Identification Of The Interaction Proteins Of Neospora Caninum NcAMA1 Protein And Vero Cells

Neosporosis has a worldwide distribution,and the current global infection rate was 17.14%.The abortion caused by neosporidiosis has caused great losses to the global animal husbandry economy.The main clinical symptoms of the disease are abortion,low birth weight and dyskinesia,etc.,N.caninum is an apicomplexan parasite with a wide range of hosts,including cattle,horses,sheep,goats and other livestock.The neck protein secreted by its unique clava organelles and the NcAMAI protein form movement combination(MJ)that plays a key role in the invasion of the parasites.its unique clava organelles clava neck protein secretion and NcAMA1 protein movement combination(MJ)in insect body plays a key role in the process of invasion.In this study,the AMA1 protein of N.caninum was used as the research object to explore the host protein interacting with NcAMA1 protein.To screen the proteins that interact with the NcAMA1 protein in the host cell,In this experiment,the primers were designed based on the NcAMA1 gene published in GenBank,and the NcAMA1 gene was amplified from the N.caninum cDNA to construct a yeast two-hybrid bait vector pGBKT7-NcAMA1.The feasibility of the bait carrier was verified by the detection of self transcription activity and toxicity,and the expression of the bait carrier in yeast strains was also verified.The cDNA library of Vero cells was screened by yeast two-hybrid technique.To construct the prokaryotic expression vector of NcAMA1 gene and the selected host cell protein Tmed2.After prokaryotic expression in vitro,two kinds of proteins were obtained,and the interaction between NcAMA1 protein and Tmed2 protein could be verified again by pull-down.Finally,RNA interference technology and antibody blocking test were used to detect whether Tmed2 had an impact on the invasion of N.caninum.The results showed that the NcAMA1 gene was successfully amplified from the cDNA of N.caninum.The verified yeast two-hybrid bait vector pGBKT7-NcAMA1,which had no self transcription activity and no cytotoxicity to yeast strain,could successfully express NcAMA1 protein in yeast strain.The yeast two-hybrid technique was successfully used to screening and NcAMA1 protein interactions in the host protein filament protein(FLNA)and across a membrane transport protein 2(Tmed2)emp24 domain from Vero cell cDNA library.The prokaryotic expression vectors pGEX-4T-1-Tmed2 and pET28a-NcAMA 1 were successfully constructed.Tmed2 and ncamaal proteins were expressed in vitro,and the interaction between Tmed2 and NcAMA1 protein was verified by pull-down technique.After the treatment of Vero cells with RNA interference and antibody blocking test,the invasion rate of the of N.caninum has increased,which indicates that the host cell protein Tmed2 screened in this time may play an inhibitory role in the invasion process of N.caninum.Providing a certain scientific basis for exploring the invasion process of N.caninum.