Screening And Identification Of Theileria Annulata Proteins Interacting With Bovine TFG Proteins

Tropical theileriosis is a tick-borne disease caused by Theileria annulata.This disease can be transmitted by Hyalomma anatolicum and Hyalomma detritum.Theileria annulata is a type of parasite that parasitizes the blood of its host and it causes leukocyte proliferation in cattle.Tropical theileriosis caused by T.annulata is highly contagious,with high morbidity and mortalityTFG(Tropomyosin-receptor kinase fused gene)is a significant proto-oncogene that regulates cell size,apoptosis and cell proliferation,and its activation can induce cancer.To further understand the role of Theileria annulata schizonts in regulating host cell transformation,the TFG gene was inserted into an empty vector of pGBKT7,and the yeast two-hybrid approach was used to screen out the proteins interacting with TFG from the Theileria annulata cDNA library.1.Construction of bait plasmid pGBKT7-TFG Design primers based on the nucleotide sequence of the TFG gene in GenBank,and insert target gene fragment TFG into the pGBKTt7 vector to create the recombinant plasmid pGBKT7-TFG.The results of the double enzyme digestion PCR analysis and base sequencing demonstrated that the vector was successfully constructed.2.Identification of the self-activation and toxicity of the pGBKT7-TFG Self-activation and toxicity of bait plasmid must be tested before yeast two-hybrid selection.The recombinant pGBKT7-TFG bait plasmid and pGBKT7 empty vector were transformed into Y2H Gold yeast cells,respectively,and the transformants were spread on the corresponding amino acid-deleted agar plates.The self-activation and toxicity of bait plasmid were judged according to the bacterial colonies’colour,size and number of spots.The recombinant bait plasmid showed no toxic effect on yeast cells without a self-activation of the reporter gene.3.Yeast two-hybrid screening for the Theileria annulata protein that interacts with bovine TFG The pGBKT7-TFG recombinant plasmid was combined with the Theileria annulata cDNA library.It was transform into Y2H Gold yeast competent medium and co-transform ants were evenly spread on DDO On the/X/A agar plate,the blue colonies grown on it were picked and spread on the QDO/X/A agar plate and passaged once for more stringent screening.The yeast plasmid was extracted from the blue colony obtained above,and PCR detection was performed with the library plasmid-specific detection primers pGADT7-F/R.After sequencing and identification,the unframe shifted library plasmid and the pGBKT7-TFG bait plasmid were co-transformed to In Y2H Gold yeast cells.The transformants were plated QDO/X/A agar plates,and the interaction between the bait plasmid and the library protein was further studied.The experimental results show that:two Theileria annulata proteins that interact with TFG proteins were successfully screened,namely TA11965 and TA21130.4.Verifying interaction between the bait protein and the capture protein by CO-IP(co-immunoprecipitation).Construct eukaryotic expression plasmids:TA1 1965-Flag,TA21 130-Flag,TFG-HA.Then,Then,Western Blotting technology was used to identify the expression of HEK293T cells after single and co-transfection,and CO-IP was used to verify the protein interaction.The experimental results showed that the Theileria annulata TA11965 protein screened by yeast two-hybrid could interact with the TFG protein,while the Theileria annulata TA21130 protein did not interact with the TFG protein.5.Sequence analysis and sample detection of Theileria annulata TA11965 protein The structure,domain,signal peptide,transmembrane region and antigenic epitope of Theileria annulata TA11965 protein were analyzed by using bioinformatics technology.Twenty-two samples of fresh blood collected from Jingyuan Cattle farm in Guyuan City,Ningxia were tested,and five positive samples were detected.In conclusion,this experiment screened out a Theileria annulata protein,TA11965 protein,which interacts with bovine TFG protein by the yeast two-hybrid method.The interaction between these proteins was verified by CO-IP.It will provide new clues to understand the molecular mechanism of Theileria annulata in regulating host cell transformation.