Molecular Cloning Of Major Surface Protein (P33) Of Theileria Sergenti And Establishment Of PCR Diagnosis Method Of Theileria Sergenti

Theileriosis is one of blood protozoal disease, caused by Theileria family, Theileria genus, Theileria sergenti. It multiplies in cattle intererythrocytes. A pair of synthetic oligonucleotide primers which designed according to the gene encoding P33 major surface protein of Theileria sergenti, were used to amplify the parasite's DNA from the blood of T.sergenti-infected cattle .The 866bp PCR product was purified then ligated with the plasmid pGEM-T Easy vector. The recombinant plasmid was transformed into competence E.coli BL-21 and positive clones were selected by digesting the recombinant plasmid with restriction enzyme and PCR amplifying. Sequence comparison was analyzed with the software DNAstar after nucleotide sequencing. The results showed that P33 nucleotide acid sequence homology of China strain had 99.7%, 99.4%, 86.5%, 82.3% to Japanese strain(D11047). Korean strain(AF521557). Theileria buffeli strain (AF236096), and Theileria orientalis strain(AB008369).A 499bp PCR product were amplified by using a pair of specific primers which were designed based on the Theileria sergenti gene sequence of China strain. To check the stability, 5 samples were selected randomly and treaded as the method established. The identical results showed this method was safe enough. Sequence of PCR product had 100% homology to the China strain .The special test and sensitive test showed that there was no cross reaction between Theileria sergenti and other parasites, such as Anaplasma marginale.Babesia bovis and Theileria annulata. There was detectable amplification of DNA from the PCR product of these positive samples were as same as the model. The lowest DNA concentration of Theileria sergenti that could be detected was 18fg/ml.This assay could be used to diagnosis early orinapparent infection and epidemiological investigation, etc. Using established PCR diagnosis method to detect 75 random samples, 72% samples (45/75) were positive but 42.7% samples(32/75) were microscopicaly positive.It will be useful for further research on Theileria sergenti in molecular biology.