Expression And Preliminary Functional Analysis Of Theileria Annulata TaSDP Gene

Theileria annulata is one of tick-transmitted hemoprotozoan parasites and causes the tropicaltheileriosis. Bovine lymphocytes infected by schizonts of T. annulata or T. parva present the capabilityof immortalization and permanent proliferation in vitro culture system. In another word, T. annulata andT. parva have the feature of transforming host lymphocytes and are classified into Theileria group oftransforming host lymphocytes. Theileria parasites are the only eukaryotes known to transform anothereukaryotic cell,which is an ideal model for understanding the interaction between parasites and hosts.So far, some important signal pathways and molecules involved in host cell transformation process byTheileria have been revealed, such as: Tash AT( Theileria annulata macroschizont-specific AThook-containing protein), SVSP(subtelomere-encoded variable secreted protein), NF-κB, ISG15,TGF-β, PI-3K, E2 F and so on. T. parva Tp SCOP( T. parva schizont-derived cytoskeleton-bindingprotein)was shown to trigger the activation of NF-κB in host cells. T. annulata Ta SDP(T. annulataschizont-derived protein)protein is a homologue of Tp SCOP. In this study, we produced combinantproteins r Ta SDP with prokaryotic expression system and the antibodie against r Ta SDP from rabbits. Thelocalization of Ta SDP in the T. annulata schizont-infected lymphocytes was determined byimmunofluorescence using anti-Ta SDP antibodies under the confocal microscope. To examine if Ta SDPcan enhance the activation of NF-κB in HEK293 T cells, the Dual-Glo Luciferase Assay was conducted.In addition, optimum electroporation buffer and voltage were determined for testing the function ofTa SDP in T. annulata schizont-infected lymphocytes. This study developed the platforms of materialsand techniques for understanding the function of Ta SDP in host cell transformation process infuture.The major works were presented as following:1. Expression of Ta SDP gene of T. annulata and preparation of polyclonal antibody. The c DNA of T.annulata schizont was used as template for amplification of the Ta SDP gene by PCR. Following theamplification, the PCR products were inserted into expression vector for protein expression. The fusionprotein r Ta SP was mainly expressed in the form of inclusion body. After purification, the protein wasused to immunize rabbits to produce polyclonal antibody. The prepared antibody reacted with bothHis-Ta SDP fusion protein and the natural antigen. This study will provide an important biologicalmaterial for the research on the mechanism of T. annulata transforming cells.2. Localization of Ta SDP in T. annulata-infected lymphocytes. To investigate whether the Ta SDPprotein was expressed in T. annulata schizonts, indirect immunofluorescenceassay was used. T.annulata–infected lymphocytes were stained using purified anti-Ta SDP antibody and examined byconfocal microscopy. Tp SCOP was shown to localize on T. annulata schizont. Ta SDP can be used in thestudy as a marker of T. annulata schizont.3. Functional analysis of Ta SDP in activating NF-κB using HEK293 T cells. To examine if Ta SDPcan activate NF-κB in HEK293 T cells, the Dual-Glo Luciferase Assay was conducted. The studyshowed that Ta SDP expression in HEK293 T cells augmented NF-κB activity approximately 1.7-fold.Finally, we also optimized the electroporation parameters in C5 cells and obtainedoptimum electroporation buffer and voltage for testing the function of Ta SDP in T. annulataschizont-infected lymphocytes. This study developed the platforms of materials and techniques forunderstanding the function of Ta SDP in host cell transformation process using Dual-Glo LuciferaseAssay System in future.