Screening And Functional Studies Of Host Molecules Interacting With Theileria Annulata Schizonts

Tropical theileriasis is a protozoal disease caused by Theileria annulata,and usually manifests clinical symptoms such as lymphadenopathy,hyperthermia,anemia and emaciation.Similar to cancer cells,the host monocytes / macrophages,dendritic cells and B lymphocytes infected with T.annulata schizont show unlimited proliferation,which is named as transformation of property when cultured in vitro.However,the host cells recover the normal growth and apoptosis after the treatment by anti-parasite drug BW720 c.Referring to T.annulata genome and proteome data and combining to the related literature,in this study,several membrane or secreted proteins of T.annulata schizont involved in host cell transformation were selected and used as bait protein to screen host interaction proteins by yeast two hybrid.For clarifying the roles of these screened host proteins in reulating host cell signal pathway,the gene expression or phosphorylation level of key molecules in signal pathway were detected after siRNA interference and BW720 c treatment for T.annulata schizont infected cell line TaNM1,which was the foundation for explainning the molecular mechanism how T.annulata regulate host cell transformation.The main results are as follows:1.pET30a-TaSP/SVSP2/EMM/Glu/CP/URP/Cyp1 prokaryotic expression vectors were successfully constructed.After the induction and purification,the recombinant proteins with a concentration of 0.3 mg/mL were obtained.To acquire the polyclonal antibody of the 7 proteins,the purified proteins were used to immuning New Zealand rabbits.Subcellular localization results showed the endogenous proteins of Cyp1,Glu and URP were mainly distributed in the schizont and few on the surface of schizont;SVSP2 was mainly near the nucleus in schizont,and EMM and CP were mainly distributed around schizont.The distribution of these proteins in host cells suggested that they might regulate cell transformation by interacting with host cell proteins.2.High purity of B lymphocytes were isolated from bovine PBMCs by immune magnetic beads,and the purity was identified as 95.3% through flow cytometry,which meet the requirement of constructing the single B lymphocyte cDNA library.The results showed the primary library capacity of ORF 1,2 and 3 were greater than 2.6 x 106 CFU,1.1 x 106 CFU and 2 x 106 CFU respectively,and the amplification base was more than 1.5 million CFU.The inserted fragments of ORF 1,2 and 3 were at the range of 1000?3500 bp,750?2250 bp and 600?3500 bp in size,suggesting that the constructed B lymphocytes library had a high quality and could be used for screening host proteins interacting with transformation related molecules.Meanwhile,the pGBKT7 recombinant bait plasmids were successfully constructed.The auto-activation and toxicity experiments proved all bait plasmids with no auto-activation and toxicity,which indicated the 7 bait plasmids could be used as a bait plasmid for yeast two hybrid screening.3.Through the functional domain prediction of TaCP and TaCyp1,and SWISS-Model homology modeling,the advanced structure models of TaCP and TaCyp1 proteins were successfully constructed,which showed the distribution relationship between functional domain and the secondary structure,and provided the basis for the prediction of protein interaction sites.In addiation,the expression of TaCP and TaCyp1 in yeast cells were successfully identified by western blot.Through yeast two hybrid screening,it was first demonstrated that CRBN and Ppp4 C could interact with TaCP,meanwhile MED21 and SEC31 A interact with TaCyp1,which provided effective target molecules for studying the interaction of CP and Cyp1 with host cells.4.Through GST-pull down,the interaction between MED21 and TaCyp1 was further verified.The results of MED21 inference and BW720 c treatment experiment for TaNM1 cells showed that after BW720 c treatment,the phosphorylation level of IκBα and IκBβ were significantly decreased,indicating the activity of NF-κB signaling pathway was decreased;for MED21 interference,NF-κB1 and NF-κB2 gene expression were obviously decreased,but P105 and P52 protein expression had no obvious change.As for its molecular mechanism,further study is still needed.