| Infectious bursal disease (IBD) is an acute, highly contagious infectious disease caused by infectious bursal disease virus (IBDV). It can cause huge economic losses to the poultry industry by inducing severe immunosuppression that leads to an increased susceptibility to other pathogens subsequently beside the clinical disease directly resulting from the infection. Indirect enzyme-linked immunosorbent assay (iELISA) is the most often used method to detect antibody against IBDV by its advantages of highly sensitivity, specificity, easy operation and simple device. VP2, VP1 and VP2-VP1 recombinant proteins of IBDV encoded by the major structure-protein genes were expressed by using a prokaryotic expression system pET, and three iELISA kits were developed by using the recombinant proteins as the coating antigens, for persuit of development a method that will serve as the reliable detection mean of antibody titter against IBDV and the antibody response of the genetic engineering vaccine.The target fragments VP2 and VP1 were amplified respectively by PCR on an IBDV strain NN1172. The segments within VP2 and VP1 with better antigenicity, expression possibility and hydrophilicity were selected to generate the gene multimers of VP2-VP1 by the method of isocaudarner. The VP2, VP1 and VP2-VP1 were subcloned into a prokaryotic expression vector pET-32a to generate the recombinants ΔVP2-pET, ΔVP1-pET and ΔVP2-VP1-pET. After sequencing confirmation, the recombinants were transferred into E. coli strain BL21 and optimized the expression condition by the inducement with IPTG. The results of SDS-PAGE of the expressed products showed that the band sizes of VP2, VP1 and VP2-VP1 recombinant proteins were 69 kDa,114 kDa and 63 kDa respectively. The recombinant proteins were purified by Ni-NTA resin affinity chromatography and then were analyzed by Western blotting, the results indicated that the expressed proteins could react with specific antibody against IBDV.Three purified recombinant proteins VP2, VP1 and VP2-VP1 were used as the coating antigens to develop three iELISA kits e.g. VP2-ELISA, VP1-ELISA and VP2-VP1-ELISA. Specific sera of IBV, ReoV, ALV and NDV were detected by the developed kits and all result in negative reactions, showing the specificity of the iELISA kits. The coefficients of variations in the tests within batch and among batches were 2.2%-6.9% and 2.8%～14% respectively, showing iELISA kits had very good reproductivities. A total of 570 serum samples collected from the commercial chicken farms that were vaccinated with IBD inactivated vaccine, genetic engineering vaccine and live vaccine respectively, were detected by iELISA kits. The results could well reflect the patterns of antibody development in the birds.184 serum samples those OD450 values around the cutoff value were selected for parallel detection by the developed kits and two commercial kits A and B. By the comparison with the commercial kit A, the sensitivity, specificity and coincidence rates of the VP2-ELISA were 91.5%,93.3% and 92.4% respectively, those of the VP1-ELISA were 86.2%,91.1% and 88.6% respectively, while those of the VP2-VP1-ELISA were 89.4%,92.2% and 90.8% respectively. By the comparison with the commercial kit B, those of the VP2-ELISA were 92.6%, 95.5% and 94% respectively, those of the VP1-ELISA were 87.3%,93.2% and 90.2% respectively, while those of the VP2-VP1-ELISA were 90.5%,94.3% and 92.3% respectively.The results of the study demonstrated that the developed iELISA kits with the recombinant expressed IBDV major structural proteins as the coating antigens showed high reproductivities, sensitive and specific, and also had very good coincidence rates with two commercial kits. And the developed kits could be applied for detecting antibody against IBDV. |
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