The Cloning And Expression Of The Merozoite/Piroplasm Surface Antigen Of The Bovine Theileria Annulata In Xinjiang And Establishment Of Nested PCR On Theileria Annulata

Theileria annulata is a tick-borne protozoan parasite that multiplies in erythrocytes, causing mild hyperthermia and anemia. When infected calves are under stress, such as mixed infection with other parasites or viruses, they show severe symptoms and occasionally die.Theileriosis is one of the most economically important diseases of grazing cattle in Xinjiang area.To analyze gene sequence of the bovine Theileria annulata in Xinjiang, a pair of primers was designed, Tams1 gene fragment was amplified by PCR, And sequence of positive clones was showed that the gene fragment has a total length of 846 bp, which encoded 281 amine acids.The homology of the nucleotide sequence was 93.6 %~ 99.8 %,and homology of the Amino acid sequence was 88.6 %~99.6% compared with other 9 international prevalent strains.The phylogenetic tree was shown that the isolated Xinjiang strain is closely related to other strains from India,Mauritania,Turkey,Tunisia, but not to those from Bahrain and Sudan.The analysis of antigenic determinant was showed that the Xinjiang isolated strain have 13 antigenic determinants.Based on the study mentioned above, pGEX-4T-2/Tams1 expression plasmid was constructed successfully by inserting the Tams1 gene into pGEX-4T-2 vector.Expressed protein induced by TPTG was analyzed by SDS-PAGE and Western-blotting.The results showed that the 56KDa expressed fusion protein was recognized by positive serum against the Bovine Theileria annulata, showing that the fusion protein have strong reactogenicity.According to gene of cattle Theileria annulata encoding the 30 kDa major merozoite surface antigen (Tams1), two pairs of primers were designed to detect Theileria annulata by nested PCR assay.Results showed that Comparison of sequence results and Tams1 sequence embodied by GenBank showed that the homology Was 92%~99%. With this nested PCR method, the PCR assay was specificity to Theileria annulata and had good repetition and stability.98 cattle in Xinjiang samples have been detected for Theileria annulata and the positive results was 62.2%(61/98),higher than PCR and microscopic examination of blood smear that the positive results were 58.2%(57/98)and 35.7%(35/98).The results is that this nested PCR is feasible method used epidemiological survey for cattle Theileria annulata.