Expression of VP1 gene and ELISA detection of antibodies against duck hepatitis virus

Abstract:

[objective] We developed an indirect enzyme-linked immunosorbent assay(ELISA) by the recombinant VP1protein of duck hepatitis virus (DHV) expressed in Escherichia coli to detect antibodies against DHV. [Methods] The VP1 gene of DHV was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pMD18-T and pET-32a vectors to get a prokaryotic expression vector pET-32a -VP1. DHV VP1 gene was expressed and analyzed. A method of enzyme-linked immunosorbent assay (ELISA) was developed and studied. [Results] We obtained the recombinant plasmid pET-32a-VP1 with correct sequence and orientation. DHV VP1 gene was expressed in E. coli BL21(DE3) at a high level and had good immunoreactivity by SDS-PAGE and western blot. The optimum working concentration of antigen was 5.0 mg in 100 mL per well, the working concentration of serum samples was 1∶100 dilution and the critical value was OD450≥0.302 for the ELISA assay. The rate of coincidence of ELISA and virus neutralization test(VN) was 97.5% by detecting 80 serum samples. The method was specific, sensitive and could be applied for antibody detection of maternal antibody and the rule of antibody growth and decline in immunizing ducklings. [Conclusion] The ELISA method developed by the purified recombinant protein could be used to detect antibodies against DHV.