| Tropical theileriosis is a tick-borne hemoprotozoan parasite disease caused by bovine Theileriaannulata, which parasitic in erythrocytes, macrophages and lymphocytes, and could cause anemia,continued fever. So far, there is no efficient drug against tropical theileriosis. The incidence of the diseaseshowed an increasing trend due to the special geographical environmental conditions and widelydistributing vector ticks in Xinjiang, it has seriously affected the development of the cattle industry.Therefore, it is particularly important to research high sensitivity and high-throughput detection technologybased on the screening of target gene of pathogen surface antigens during the prevention and control ofbovine tropical theileriosis. The present study contains three researches:（ⅰ）In this research, five pair of primers were designed and synthesized according to the sequence ofTams1gene of Theileria annulata published in GenBank. Three fragments（the complete gene-AE, thecomplete gene lacking N-terminal and C-terminal hydrophobic domain-BC, gene fragment without thesequences coding for N-terminal hydrophobic domains-BE）of Tams1gene were amplified, then constructprokaryotic expression recombinant plasmid by genetic engineering technology. The result indicated thatAE, BC and BE fragments of Tams1gene were successfully amplified and seven prokaryotic expressionrecombinant plasmid including pGEX-4T-2-BE， pGEX-4T-2-BC， pGEX-4T-2-AE〠pET-28a-BC，pET-28a-BE，pET-30a-BC and pET-30a-BE were constructed. Sequencing result showed that the higestidentity of cloned Tams1sequence and deduced amino acid was all being99%with the correspondingsequences published in GenBank, however, that of Gansu strain were92%and86%, respectively. Thisexperiment could lay the foundation for the study of the diagnosis of tropical theileriosis with purifiedantigen.（ⅱ）The expression of the target genes was induced with IPTG after all the recombinant plasmidswere transformed into competent cells of E.coli BL21（DE3）by genetic engineering technology, Thenscreen high soluble expression recombinant plasmid through analyzing and comparing the expressingquantity and form by SDS-PAGE. Finally, purified proteins were analysed by Western blotting after optimizing inducing conditions. The results showed that six fusion proteins were successfully obtained.SDS-PAGE demonstrated that GST-BC（53ku）and His-BE（32ku）fusion protein was effectivelyexpressed as soluble form in E.coli, which contains the pGEX-4T-2-BC without the hydrophobic region ofthe target gene）and pET-30a-BE（containing the C-terminal hydrophobic region of the target gene）plasmid, respectively. Western blotting showed that purified recombinant proteins GST-BC and His-BEhave favorable reactionogenicity. Therefore, it could be used as a diagnostic antigen.（ⅲ）An indirect ELISA detection method was successfully established using the purified His-BEprotein as antigen through optimalling reaction condition, and the sensitivity, specificity and repeatabilityof the method were evaluated through evaluation tests. Finally,250samples were detected by using thismethod. The results showed: the antigen concentration for coating was3μg/mL, and incubated forovernight at4℃, The plates were subsequently blocked with3%skim milk for1h，and then incubated for1h with diluted serum samples（1:100）. The diluted HRP-secondary antibody（1:12000）was added andincubated for1h at37℃，and then substrate of TMB solution was added for incubation20min at37℃todevelop color. The thresholds for indirect ELISA was0.221. The established indirect ELISA had certainsensitivity, specificity, no cross-reaction, and the coefficient of variation was less than10%. The methodwas used to detect250bovine suspected serum samples from Turpan, with the positive rate ofserum antibody39.2%and the coincidence rate was96.7%when compared with Western blotting detectionmethod. It could provide a theoretical basis for the prevention and treatment of Theileria annulata in thisregion. |
