Screening Of Candidate Molecular Interact With P104in Theileria Annulata By Yeast Two Hybrid System

The tick-borne parasite, Theileria annulata is the causative agents of lymphoproliferative diseases andTropical Theileriosis of cattle, which cause significant economic losses in large parts of Asia and Africa.Accoding to the latest report, T. annulata predominantly infects macrophages/monocytes and B-cells. Itpossess the unique capacity of transforming their host cells, inducing uncontrolled proliferation andresistance to apoptosis. The invasion of T. annulata is mediated by a set of molecules distributed on theparasite surface and within specialised apical secretory organelles. The rhoptry is an unusual secretoryorganelle in parasite which was thought to be related to secretory lysosomes in higher eukaryotes. P104secreted from these organelles p104provide a window to discover the mechanism of invasion andtransformation.1. hiTAIL-PCR cloning T. annulata p104gene flanking sequencesUsing hiTAIL-PCR amplificated the flanking fragments of p104from cDNA of TaIM cell line. Jointedflanking fragment and middle sequence formatted a complete sequence of p104. Analysised by BLAST, itwas95%similar with T. annulata Ankara.2. Prokaryotic expression and Subcellular localization of p104Expressed the470-650amino acids of p104. Purificaed the expression protein and generatedpolyclonal antibody by immune rabbit. The indirect immunofluorescence assay (IIF) was performed in thisstudy to identify the subcellular localization of p104protein in T. annulata schizonts and T. annulataschizonts-infected bovine lymphocytes. The results showed that the proteins were predominantly present onthe membrane of Theileria annulata schizonts, and only a small amount was present in the cytoplasm ofhost cell. Which make a big possible to interact with other protein in bovine lymphocytes.3. Construction Yeast Two-hybrid cDNA library of TaIM cell line and construction of p104bait plasmidA Yeast Two-hybrid cDNA library was constructed by TaIM cell line, the result showing the capacityof the library was2.4×107cfu with a recombination rate of100%and an average size of insert of1000bp.Meet the requirements of the kit. Amplificated the fragment of p104by RT-PCR, cloned the fragment to abait plasmid. The result showing it was successes generated without autoactivation and toxicity.4. Screening of cDNA library by yeast two hybrid system and confirmation of yeast interacting clonesFourteen specific clones were obtained by yeast two hybrid system. Two proteins of them, Bos taurus lectin galactoside-binding, soluble1(Galectin1) and Bos indicus heat shock protein70(HSP70) showedstrongest interaction and were further confirmed. The interaction domains were determined by yeasttwo-hybridization retransformation experiment, as a result, the interaction domain of Galectin1is between91-183amino acid, and HSP70is between435-500amino acid.