| Infectious bursal disease (IBD) caused by infectious bursal disease virus, is a highly contagious viral disease of young chicken, which causes considerable economic losses to the poultry industry. IBD is characterized by muscle bleeding and bursal swelling or atrophy accompanied by hemorrhage, and3-6week old chickens are the susceptible targets. It is able to induce immune suppression, which leads to vaccination failure and occurrence of other disease. Agar gel precipitation (AGP) test and enzyme-linked immunosorbent assay (ELISA) are the main methods for diagnosing IBD in China. As advantages of specificity, sensitivity and high-throughput, the usage of ELISA is more and more widespread. But currently, there is still lack of a practical native ELISA kit for the detection of IBD. In this study, based on the first half of the VP2gene, an indirect ELISA method was established, which was able to detect serum antibodies against IBDV.1. The expressing of recombinant protein IBDV VP2According to the sequence of IBDV VP2in Genbank, two pairs of primers were designed targeting VP2(all) and VP2(up). The target fragments of1350bp and750bp were amplified by RT-PCR and cloned into pMD18-T vector respectively. Then, they were subcloned into the downstream of T7promoter of pET-28a (+) vector. The recombinant plasmids were transformed into E.coli BL21. Prior to the production of large-scale recombinant protein, a small scale optimization was carried out to estimate the optimum conditions for expression. An incubation temperature of37℃for6h with1mM of IPTG was the optimal condition for expression of VP2(up) protein. It was estimated that the VP2(up) protein constituted about30.1%of the total expressed protein. And the results of SDS-PAGE indicated that the size of VP2(up) was27kDa and the most of the protein was expressed in insoluble form. The VP2(all) was also successfully expressed in insoluble form following with lactose (10μg/mL) in E.coli BL21. It was about45kDa in size and constituted about11%of the total expressed protein.The concentrations of purified VP2(all) and VP2(up) protein were1.7mg/mL and2.5mg/mL respectively. The results of western blotting showed that both the recombinant proteins could react well with the positive serum against IBDV2. The establishment of an indirect ELISA for the detection of antibodies against IBDVThe purified VP2(up) protein (1μg/mL) was selected as the coating antigen and the optimal dilution of serum was1:100, which were determined by checkerboard titration. The incubation time of the serum and the HRP-conjugated antibody were both set to30min, and the optimal dilution of HRP-conjugated antibody was selected as1:2500. If OD450of the tested serum is less than or equal to0.6xODP+0.4×ODN (ODP is the mean of positive control serum, ODN is the mean of negative control serum), it is determined as negative. Otherwise, it is judged as positive. The test results of H5, H7, H9subtypes of avian influenza virus and Newcastle disease virus antiserum through this method show these four serums all are negative. So this method has a good specificity.A total of156clinical serum samples were detected parallel by VP2-ELISA and IDEXX ELISA kit. Compared to IDEXX ELISA kit, the coincidence rate, sensitivity and specificity of the VP2-ELISA were94.23%(147/156),95.65%(110/115) and90.24%(37/41) respectively. In summary, the indirect ELISA based on the recombinant protein VP2(up) is highly sensitive and specific. Therefore, this assay can be used to detect the antibody of chicken serum against IBDV and to evaluate the quality of IBDV vaccine. Otherwise the results of it are good for the formulation of immunization program. |
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