Identification Of The Interaction Of Neospora Caninum AMA1 And RON2 Proteins And Their Immunoprotection

Neospora caninum(N.caninum)is a an apicomplexan parasite and leads to neosporosis in cattle.Neosporosis is globally distributed,and causes huge economic losses to breeding industry,especially the dairy industry.There are no specific drugs or vaccines against neosporosis at present.N.caninum tachyzoites′ invasion to the host cells is a key process for the parasite to break through the defense lines of the host.The process mainly includes recognizing and adhering to the host cell to form a sliding junction Moving junction invasion in order to proliferate intracellularly.The invasion process depends on the interaction of multiple proteins between the parasite and the host cell.Therefore,studying the invasion mechanism of N.caninum can contribute to discover invasion-related genes and host targets.Recent studies have shown that the complex of Toxoplasma gondii RON2/4/5/8 and apical membrane antigen-1(AMA1)is the key structure of MJ.N.caninum AMA1 can interact with many proteins,but whether N.caninum AMA1 can interact with RON2 needs further study.Therefore,this study aimed to detect the whether there was an interaction between N.caninum AMA1 and RON2 by GST-Pull down and bimolecular fluorescence complementary technology,and localization of N.caninum AMA1 and RON2 were determined by using immunofluorescence technology(IFA).Finally,the immunoprotections of N.caninum AMA1 and RON2 against N.caninum were assessed in mice and goats,we hope this work can provide a reference for the development of new drugs or vaccine targets against N.caninum.Identification of the Nc AMA1-Nc RON2 interaction by GST-Pull down: The purified recombinant p GEX-4T-Nc RON2 protein and recombinant p ET-28a-Nc AMA1 protein were used for GST-Pull down.Results showed that the band of recombinant p ET-28a-Nc AMA1 protein can be observed in the elution buffer by western blot,however,recombinant p ET-28a-Nc AMA1 protein didn’t exist in the elution buffer which only containing GST tag protein.These data indicate that Nc AMA1 can interact with Nc RON2 in vitro.Identification of Nc AMA1-Nc RON2 interaction by bimolecular fluorescence complementary: Bimolecular fluorescence complementary vectors p Nc AMA1-HA-KN151 and p Nc RON2-Myc-LC151 were constructed,red fluorescence,emitted by the interacting protein,can be detected in the Nc AMA1 and Nc RON2 co-transfection group by laser confocal microscopy.This data further confirmed the interaction of Nc AMA1 and Nc RON2 intracellularly.The subcellular localization of Nc AMA1 and Nc RON2 protein: green fluorescence of Nc AMA1 protein observed and this protein was localized on the top of the parasite under the laser confocal microscope.Nc RON2 protein,which was labeled with orange-red fluorescence,was localized at the junction of VERO cell membrane and N.caninum,and had co-localization with Nc AMA1 by laser confocal microscopy.This data proved the interaction of Nc AMA1 and Nc RON2 during N.caninum invasion process.As invading-related antigens,Nc AMA1 and Nc RON2’s research on their interaction during invasion process can benefit for studying their biological functions and relationships.Invasion assay of Nc AMA1 and Nc RON2: N.caninum tachyzoites were incubated with anti-AMA1 or/and RON2 polyclonal antibodies,and invasion rate and the proliferation of this parasite were detected.Results showed that the invasion rate of N.caninum was significantly reduced,the best effect was obtained when the dilution ratio of polyclonal antibody was 1:50,which was 36% in the AMA1 polyclonal antibody group,39% in the RON2 polyclonal antibody group,and 41% in the AMA1+RON2 polyclonal antibody group(p<0.001),and AMA1+RON2 combined treatment had a best effect on blocking the parasite invasion,but intracellular parasite numbers in each group showed little difference.Immunoprotective of Nc AMA1 and Nc RON2 in mice: Mice were immunized with recombinant Nc AMA1 and Nc RON2 proteins,and results found that antibody titers and cytokines in sera were greatly increased in immunized mice(p<0.001),especially in Nc AMA1+Nc RON2-immunized mice.Then immunized mice were challenged with N.caninum tachyzoites,survival rate and parasite burden in tissues were monitored.Results showed that survival rates of positive group and other treatment group were 0,but Nc AMA1+Nc RON2 immunization can increase the survival rate to 25%.Meanwhile,the Nc AMA1+Nc RON2 immunization obviously decreased the parasite burden in brain and alleviated the pathological lesions,indicating that the combination of these antigens had a better immunoprotective effect than single antigen.These data imply the potential of Nc AMA1 and Nc RON2 as vaccine candidates.Immunoprotective of Nc AMA1 and Nc RON2 mixed immunization in goat: Goat were immunized with recombinant Nc AMA1 and Nc RON2 proteins,and results found that antibody titers and cytokines in sera were greatly increased in immunized goat(p<0.001),The amount of worms in the brain tissue of goat was significantly reduced with N.caninum reduction rate was 56%(p<0.01),and the pathological changes of heart,liver,spleen,lung,kidney,and brain tissues were significantly reduced,indicating that the mixed immunization of Nc AMA1+Nc RON2 protein has a certain immunoprotective effect in goat.In summary,this study identified the interaction between N.caninum AMA1 and RON2 during invasion using GST-Pull down,bimolecular fluorescence complementary technology,and subcellular localization.In addition,N.caninum AMA1 and RON2 involved in the invasion process by invasion assay.N.caninum AMA1 and RON2 proteins have better immunoprotections against N.caninum in both mice and goats,providing a theory for preventing and controlling N.caninum infection.