Expression Of TeRON2 And TeAMA1 Proteins And Identification Of Their Interaction In Theileria Equi

Theileria equi is a tick-borne parasitic parasite of the erythrocytes and reticuloendothelial cells of equine animals,and belongs to the category of equine pear-shaped worms together with Babesia caballi.The infection is mainly caused by the vector ticks when feeding on the blood of equine animals,and the widespread distribution of ticks in China increases the difficulty of preventing and treating the disease.The invasion of host cells is the basis for the establishment of an infection system by the protozoa,so the ability to effectively block the invasion of host cells by T.equi is the key to preventing infection by the disease.It has been found that the interaction between AMA1 and RON2 proteins during the invasion of host cells by Toxoplasma gondii and Plasmodium,and the resulting moving junction(MJ)is a key structure for host cell invasion.However,it has not been reported whether AMA1 and RON2 proteins can interact with each other as in the case of other T.equi.In this study,we firstly screened and amplified RON2 and AMA1 gene fragments,constructed eukaryotic and prokaryotic recombinant proteins of Te RON2 and Te AMA1,respectively,and identified the interaction relationship between them by protein interaction technique,and finally predicted their interaction sites by molecular docking technique.The main results of the study are as follows.(1)The RON2 gene sequence(Gen Bank accession number MT939257)of T.equi Xinjiang strain was successfully obtained,which is most closely related to the evolution of the American WA strain of T.equi and has a high conserved gene sequence.The molecular mass of prokaryotic Te RON2 fusion recombinant protein was 26 k Da and that of eukaryotic Te RON2 fusion recombinant protein was 37 k Da,with polyclonal antibody potency 1:409600.bioinformatics analysis showed that Te RON2 protein is a basic,unstable transmembrane protein with the proportion of α-helix in the secondary structure exceeding 50%.It is poorly hydrophilic and weakly antigenic.It has similar tertiary spatial specific structure and amino acid sequence with other acrosomal protozoan RON2 proteins,and may interact with other invasive proteins.(2)The AMA1 gene sequence(Gen Bank accession number MW346657)of T.equi Xinjiang strain was successfully obtained,and the genetic evolutionary analysis based on AMA1 gene showed that T.equi Xinjiang strain was the closest to T.equi American WA strain in terms of evolutionary relationship,and the nucleic acid and amino acid sequences were highly conserved.The molecular mass of Te AMA1 recombinant protein was about 60 k Da and 55 k Da,and the potency of the prepared polyclonal antibody was 1:819200.Protein antigenicity analysis showed that Te AMA1 protein is a hydrophilic type I transmembrane protein with more than 50% irregularly folded secondary structure,multiple hydrophilic,flexible and surface accessible regions with sites for antigenic epitope formation and consistent with predicted B-cell antigenic epitopes.The AMA1 protein of T.equi and other parietal complex protozoa have high similarity in tertiary structure and may have interaction with other invasion-related proteins.(3)GST-pull down results showed that His-Te RON2 recombinant protein was detected by Western blotting in the eluate of His-Te RON2 and GST-Te AMA1 co-incubation.Immunoprecipitation results showed that GFP-Te AMA1 and mcherry-Te RON2 eukaryotic recombinant proteins were successfully constructed.mcherry-Te RON2 and GFP-Te AMA1 proteins were detected in immunoprecipitation complexes of murine anti-GFP monoclonal antibody and rabbit anti-mcherry monoclonal antibody,respectively.The bimolecular fluorescence results showed that the successful construction of VN155-Te AMA1 and VC155-Te RON2 expression vectors and the observation of green fluorescence in the cotransfected group indicated that Te AMA1 interacted with Te RON2 inside and outside the cells.Four sets of potential action sites were predicted from Te AMA1 and Te RON2 by molecular docking,corresponding to five amino acids in Te AMA1 and four amino acids in Te RON2,namely: TRY43-PRO178,TRP53-MET198,GLY45-GLY149,ASN153GLU196-SER190.In summary,in this study,we obtained the RON2 and AMA1 genes for the first time,successfully expressed Te RON2 and Te AMA1 prokaryotic and eukaryotic recombinant proteins,and successfully identified the interaction between Te AMA1 and Te RON2 by three experimental techniques,namely GST-Pull down,immunoprecipitation and bimolecular fluorescence complementation,and predicted four groups of potential interactions by molecular This provides a theoretical basis and reference for exploring the composition of the mobile junction complex,a key structure for the invasion of host cells by T.equi,and for the subsequent development of vaccines and novel drug targets.