Characterization And Preliminary Application Of Tu88Gene From Theileria Uilenbergi

Theileriosis of small ruminants in China is mainly caused by Theileria uilenbergi and T. luwenshuni.The disease distributed mainly in the northwest of China, constituting a severe restriction for thedevelopment of the small ruminant livestock industry. The pathogenesis of the disease is believed to beclosely associated with not only schizonts but also the proliferation of the intraerythrocytic piroplasms.A positive clone Tu88was identified by immunoscreening T. uilenbergi merozoite cDNA library inprevious study. In order to enrich the genomic information of Theileria, Characterization andpreliminary application of Tu88gene and its encoded protein were explored.Bioinformatics software was used to determine the open reading frame and the correspondingprotein sequence, predict protein isoelectric point and molecular weight, secondary structure, thelocation of the signal peptide, transmembrane, linear B-cell epitopes, and the protein subcellularlocation. The Tu88gene was found to be a potential antigenic gene with signal peptides, transmembranedomain and antigenic determinants. The gene which was amplified from genomic DNA of T. uilenbergimerozoites with specific primers targeting a fragment of Tu88gene was consistent with that obtainedfrom cDNA library of T. uilenbergi merozoites. It indicated that there were no introns in Tu88gene. Nosimilar gene was amplified from genomic DNA of T. luwenshuni, T. ovis, A. ovis and Babesia ovis,indicating Tu88is T. uilenbergi specific. Partial Tu88gene was cloned into pET-30(a) vector andexpressed in vitro. The recombinant protein was purified and evaluated using western blot. The resultshowed that the Tu88protein was specific to T. uilenbergi positive sera. No cross reaction was found insera from animals infected with T. luwenshuni, Babesia sp. China or Anaplasma ovis as well as negativesheep serum. To estimate the copy number of the Tu88gene, Southern bot analysis was carried out.Genomic DNA of T. uilenbergi was digested with different restriction enzymes. DNA was sizefractionated on a0.8%agarose gel and then transferred onto a nylon membrane. Probing and furtherprocedures were done according to the manufacturer’s protocol of the DIG-labeling and detection kitwith CSPD-star. All digests demonstrated the presence of a single band whereas no signal wasdetectable in genomic DNA of ovis PBMC, indicating that Tu88is a single-copy gene in the genome ofT. uilenbergi. The Tu88ORF (except the stop codon) was amplified from the genome of T. uilenbergiwith the primer set Tu EGFP For/Tu EGFP Rev with the internal sites of KpnⅠand Bam HⅠrespectively. By digestion with restriction enzymes KpnⅠand Bam HⅠ, the PCR products was thensubcloned into eukaryotic expression vector pEGFP-N1that digested with the same enzymes. Therecombinant plasmid named pEGFP-N1-Tu88was constructed and transiently transfected into CHOcells using LipofectamineTM2000Transfection Reagent to examine the behavior of the individuallyexpressed protein form in a heterologous eukaryotic expression system in terms of their location. Theobservation result through the inverted fluorescence microscope and Western blot analysis verified thatTu88gene was expressed successfully in CHO cells. Tu88protein was mainly localized in cytoplasm ofCHO cells by confocal microscopy. Using the recombinant protein rTu88, an indirect ELISA wasestablished. Analyses of the antibody response dynamics of three experimentally infected animals demonstrated that the persistence of the Tu88-specifc antibodies lasted more than100days.The above data showed the biological characteristics of Tu88gene and also suggested its potentialapplications in the diagnosis and control of Theileria. However, its function remained to be furtherstudied. It is speculated that it may be a candidate molecule through parasite-host interactions whichplay an important role when the parasites invade host cells, or a candidate molecule for new diagnosismethods and the subunit vaccine research which could stimulate the host cells to produce primaryantibody after the parasite invade.