Construction Of SSH Library And Identification Of Differential Genes Of Bovine Cells Transformed By Theileria Annulata Schizonts

Tropical theileriasis is a tick-borne hematic protozoosis, caused by Theileria annulata which parasitized in lymphocytes, macrophages and red blood cells of cattle. T. annulata and T. parva are the only known eukaryotes that can transform mammalian cells and this kind of transformation induceed by T. annulata is entirely reversible. When the parasite is killed by the medicine against Theileria Buparvaquone(BW720c), the Theileria-transformed cells will lose the characteristics of uncontrolled proliferation and undergo apoptosis. It is considered that T. annulata and is an ideal model for better understanding of the interactions between parasites and host cells. In the present study, a suppression subtractive hybridization(SSH) library was constructed using bovine lymphocytes transformed by Theileria annulata and PBMCs of healthy calves. It purposed to screen schizont genes of Theileria annulata and differential genes of bovine cells between T. annulata schizont transformed and uninfected cells via bioinformatics, Real-time PCR and Western blot analysis. Our studies established a foundation for further better understanding transformation mechanisms of this parasite, and for better understanding the molecular mechanisms of interactions between T. annulata and host cells. The major works were presented as following:1. A SSH cDNA library of bovine cells between transformed and untransformed by T. annulata was successfully constructed. A total of 364 effective differentially Expressed Sequence Tag(ESTs) was obtained via DNA sequencing, ranging from 350 to 1200 bp insert size. After comparing the sequences with BLASTn in the GenBank, these ESTs belonged to 192 gene sequences. Sixty of ESTs matched to T. annulata genes(11 protease genes, 10 membrane protein genes, 7 hypothetical protein genes, 5 apoptosis related protein genes, 5 ribosomal protein genes, 4 cell-cycle protein genes, 4 antigen protein genes and 14 other genes), and 131 were bovine genes(19 tumor related genes) and one unknown gene.2. The 13 full-length CDS sequences of T. annulata schizont genes were obtained from SSH Library by genes using cloning and sequencing techniques, and their proteins sequences and characteristics were predicted and analyzed by Signa lP3.0, TMHMM 2.0, Lasergene(protean)and Protfun bioinformatics softwares. The results revealed that Ta65, Ta129 and Ta513 proteins from the T. annulata schizont had many antigenic epitopes. These proteins had the potential as candidates of antigen for further study for developing diagnostic methods or vaccines. In addition, Ta129, Ta511 and Ta513 proteins might be associated with host cell signal transduction and transport function. These works established a good foundation for further research of T. annulata diagnosis and immunoprophylaxis.3. The Real-time PCR primers of five candidate reference genes, 18 S rRNA, GAPDH, ACTB, PRKG1 and TBP, were designed using Primer Express 3.0 software. And then the stabilities were evaluated by amplification efficiencies, primers specificity, standard curve, geNorm and NormFinder program using T. annulata transformed cells and PBMCs of uninfected calves as experiment materials. The results showed that five reference genes had better the primers specificity by the dissociation curve analysis, and the amplification efficiencies were ranged between 81.2% and 120.1%. Standard curve analysis results indicated that only 18 S rRNA and GAPDH exhibited good linear relationships under the condition of various dilution degrees. Moreover, geNorm analysis confirmed that 18 S rRNA was the most stable expressed gene. NormFinder analysis got the same results. On the basis of the above analysis results, we proposed that 18 S rRNA was the most stable expressed reference gene could be chosen during studying the transformation mechanism of T. annulata by signaling pathways analysis.4. Differential expressed genes from T. annulata transformed host cells were verified by Real-time PCR. The results showed that the mRNA abundance of TFG,HCLS1 and SENP5 from transformed cells were 2.74, 2.06 and 1.32 times that of non-transformed cells. The mRNA abundance of Ta65(TA08105) and Ta129(TA20380)from schizont were 2.10 and 2.08 times that of piroplasms/merozoites(stage of parasitized in RBC) of T. annulata.5. Using BW720 c treatment to kill the parasite in T. annulata transformed cells, we aimed to identify changes to host cell gene expression by Real-time PCR and Western blot. The results showed that HCLS1 and TFG gene transcription from T. annulata transformed cells were decreased significantly by the presence of the test group compared to control groups. Moreover, TFG protein expression was strongly reduced at 72 h of exposure of T. annulata transformed cells to BW720 c. In summary, TFG protein expression of T. annulata transformed cells is relevant with the T. annulata parasitization. The results helped to lay the groundwork for better understanding the interactions between T. annulata and host cells.