Development Of An Inderect ELISA For Detecting Antibody Against Theileria Annulata TaSP Recombinant Protein

Theileria annulata as a hematic protozoosis causes serious harm to bovine, which infcted in macrophagocyte, lymphocyte and red blood cell of host, and led to acute death. Tropical theileriosis is an epidemic deisease in worldwide, brings tremendous influence for the countries, especially caused significant losses every year in developing countries. There are three theileriosis infected cattle in China, including Theileria annulata, Theileria sinensis and Theileria sergenti, but the most harmful for host is Theileria annulata. The protozoosis disease spans in Xingjiang, Inner Mongolia and other 11 regions in China, causing massive economic losses. The ticks as vector for Theileria annulata is Hyalomma, but Hyalomma deteitum as mainly vector in most regions in China, Hyalomma anatolicum is cause agent of theileriosis in Xinjiang province. Therefore, in this study, we developed an inderect ELISA for detecting antibody against Ta SP in Theileria annulata based on Theileria annulata sporozoites surface protein(Ta SP) as coating antigen after optimizing the conditions,purifing the recombinant Ta SP protein. The work is as following:1 According to the detecting results of expressed recombinant Ta SP protein by PCR, the positive results were sequenced with about 400 bp. The results indicated that the gene was 396 bp.Comparing Gan Su stain to Yin Du stain and Su Dan stain, the homologies of the amino acids of Tasp were97%. The recombinant protein was induced at different time and purified. The result of Western blotting showed that The immunogenicity of purified recombinant protein with anti- Theileria annulata serum was strong..And there is no cross reaction with the serums from T.sinensis, B.bovis, B.bigemina, T.sergrnti, T.luwenshuni, T.uilenbergi.2 We developed an inderect ELISA for detecting Theileria annulata using Ta SP recombinant protein as the coating antigen. The optimal coating dilution of the antigen was 3μg/ml; the best dilution of sera and HRP-secondary antibody was 1:50 and1:10000, respectively by optimized of every condition. The assay we established only reacted with anti- Theileria annulata serum, not with B.bovis, B.bigemina, T.sergrnti, T.sergrnti, T.luwenshuni,and T.uilenbergi. To further assess the assay, we detected 140 sera using the indirect ELISA, with 98.5% consistent with the result of nest-PCR. So it has the specificity of 95.7%, and the sensitivity of 100 %.