Development Of An Indirect ELISA For Diagnosis Of Brovine Theileriosis Sergenti By Expressing Fusion P33 In E.coli

The p33 of T. sergenti with good antigenicity and reactivity is one of a main surface protein . In this test, the p33 gene isolated from Korean strain T. sergenti (the identity of p33 between Korean strain and Chinese strain is 99.6%) has been expressed in E.coli DH5a and obtained a lot of the fusion p33 that it is used antigen of indirect ELISA to detect the antibody against p33 antibody in the bovine infected with T.sergenti in China. Both the depurated p33 and the recombinant p33 could recognize the specific antibody against p33 in the T. sergenti-infected bovine in China in the Western blot analysis, but MBP could not. This results indicated the recombinant p33 could be used as a diagnostic antigen in indirect ELISA.The various factors and conditions for indirect ELISA were explored, and the optimal reaction conditions were determined. It was shown that the optimal concentration of p33 recombinant protein for coating of plate was 1.5μg/ml, the optimal coating condition of recombinant protein was 4℃ overnight, the dilution of serum sample was 1:100, the working concentration of HRP-labeled goat anti-bovine IgG was 1:4000, serum sample for detecting and goat anti-bovine IgG should be incubated at 37℃C for 1h before terminated with the stopping solution. The optimal confining solution and confining time were 3% skimmed milk of PBST, 37℃ for 1h respectively. The albumen concentration in PBST was 3% skimmed milk. The substrate for indirect ELISA was incubated at RT for 25 min before reading the OD₄₀₅. The threshold for indirect ELISA was 0.35.The blocking test for the reaction between the positive serum of T.sergenti and the recombinant protein was carried out by using fusion p33 expressed in E.coli, the results indicated that fusion p33 could block the reaction completely. It was also confirmed that there was no cross reaction by the crossing test. Meanwhile, the indirect ELISA had ideal repeatability while it was done both in the same reaction plate and the different reaction plate. The indirect ELISA had good specificity for the detection of 60 serum samples from bovine when compared with LAT simultaneously. It was found that the negative, positive and total coincidence of the developed indirect ELISA were 90.0%,100% and 96.7% respectively. In addition, 104 bovine serum samples collected from Hunchun, .Dunhua and Helong city of Yanbian canton were detected by the indirect ELISA and found the positive rate is 72.3% and 60.4% respectively from 56 bovine serum samples of Hunchun city and 48 bovine serum samples of Helong city.In the domestic field of T.sergenti, this test firstly successfully apply Korean strain fusion p33 as an antigen to developed indirect ELISA to detect Chinese strain antibody. It should afford a rapid, sensitive and specific means of serodiagnosis in monitoring T.sergenti infection or epidemiology investigation in the field.