Dissertation
Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Cow

Cloning and Prokarvotie Expression of Tasp Gene of Theileria annulata in E.coli and Development of PCR Method

Author ZhengJinFeng
Tutor LiKui
School Henan Agricultural University
Course Veterinary
Keywords Theirelia annulata TaSP gene cloning expressing PCR detection method
CLC S858.23
Type Master's thesis
Year 2013
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Theileria annulata, also termed Tropical theileriosis, is a tick-borne protozoan parasite thatcause symptoms of high fever, bleeding, anemia and enlargement of superficial lymph node.Tropical theileriosis is distributed over a wide geographic area and it is one of the most seriousparasitosis endangering cattle. Theileria annulata sporozoites surface protein (TaSP) has goodimmunogenicity and its sequence is highly conserved thus it can be used as ideal antigen for thediagnosis and prevention of Theileria annulata.In this study, a pair of primers was designed according to TaSP gene sequence of Theileriaannulata in Genbank and the conserved fragment of TaSP gene was cloned from the genome ofwhole blood of Subei County Theileria annulata infected cattle by PCR. The size of this genefragment is396bp and encodes312amino acids. Sequence analysis showed that the nucleotidesequence identity is83%-94%, compared with the other9prevalent strains posted in GenBank. Theanalysis on its genetic evolution showed that the Subei isolate is closely related to the isolates fromTurkey, Lintan, Inner Mongolia, Morocco, India, Mauritania, Sudan, Ningxia and Germany, but notto those from Xinjiang and Tunisia. On this basis, the TaSP gene was inserted into pGEX-4T-1toconstruct the expression vector of pGEX-4T-1/TaSP. Expressed protein induced by TPTG wasanalyzed by SDS-PAGE and Western-blotting. The results showed that about41KDa expressedfusion protein was recognized by positive serum against the Bovine Theileria annulata, indicatingthat the fusion protein have strong reactogenicity.The secondary structure and B cell epitopes of TaSP protein of Theirelia annulata waspredicted by the molecular biology software of DNA-star Protean, the secondary structure waspredicted by the methods of Gamier-Robson. Kyte-Doolittle, Emini, Karplus-Schulz andJameson-Wolf program were used to predict its hydrophilicity, possibility of an epitope, flexibilityand antigenicity index. The results showed that B cell epitopes of TaSP were probably within oradjacent to10-19,26-36,49-57,73-86,125-130and the reliability index were higher. Moreover, allthese predicted epitopes were rich in random coils Therefore, B cell epitopes may be present inthese area. This study would be helpful for the identification of B cell epitopes and research on reverse vaccinology of TaSP protein.To establish a PCR method of Theirelia annulata,a pair of primers was designed to specificallyamplify a393bp high immunization fragment based on TaSP gene conserved region.Thespecificity assay on this method showed that the specific PCR product can only be obtained in T.annulata genome among the nine samples. The sensitivity result showed that the minimum dose ofT. annulata that could be detected by PCR assay was.150blood samples were detected by PCRassay and microscopic examination of Giemsa-stained blood smears respectively,which indicatedthat the PCR assay was sensitive and specific and, therefore, suitable for the diagnosis of T.Annulata.

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