Prokaryotic Exression Of Structural Protein VP3 Gene Of IBDV And Development Of Differential Diagnosis Methods

The VP3 gene of structural protein was obtained from Infectious bursal disease virus (IBDV)by RT-PCR amplification .The VP3 gene was subcloned into the expression vector pPROEX-Htato construct a recombinant plasmid named VP3/PA. E.coli DH5αwas transformed withVP3/PA to express the fusion protein. The results of SDS-PAGE and Western blot indicatedthat the VP3 protein was expressed at a high level(33.9%). The recombinant fusion protein wasabout 33kDa, and it had immunological reactive activity.The VP3 protein expressed in the E.coli was used in ELISA to develop differentialdiagnostic methods. The optimal coating quality of VP3 antigen was 2ug/ml, and selectedsealing liquid contained 0.5% equine serum PBST. The serum samples, which were tested, werediluted 1:100 , followed by a reactive time of 60 minutes. The total 92 serum samples fromSPF were detected under optimal conditions. The critical standard was obtained, and S/P≥0.24is considered positive and S/P≤0.17 as negative. To be established, ELISA methods have highersensitivity through experiments in field chickens and a 96% agreement rate was obtained incomparison with the commercial AGP.Chickens experimentally infected with wild-type IBDV-GX were diagnosed positive byELISA. Chickens experimentally VP2 sub vaccinated were negative by ELISA, but they werepositive in different types of viruses. ELISA tests of specificity and sensitivity in comparisonwith a commercial AGP indicated that the ELISA is highly specific, sensitive and swift for largescale sero-epidemiological studies. The differential diagnosis methods would be very useful infurther eradication programs for IBDV.