Abstract

A transfer vector, defined as pAcUW31-VP2, was constructed by insertion BamHI fragment (1.1 kbp) of VP2 cDNA of infectious bursal disease virus (IBDV) strain CJ-801 into BamHI cloning site of baculovirus expression vector (pAcUW31). A contransfection was carried out with the purified pAcUW31-VP2 DNA and wildtype baculovirus (AcMNPV.LacZ) DNA that was linearized by Bsu361 digestion. Plaque-assays were conducted, and 10 white plaques were picked for further purification after dual staining with X-gal and neutral red. A VP2 cDNA probe was prepared. Dot hybridization analysis of the 10 putative recombinant viral DNA using the probe indicated that 7/10 were positive. One of the recombinants (1/7) was amplified in sf9, and the viral DNA was extracted from infected cells. Southern blot hybridization results demonstrated that the BamHI fragment of VP2 was inserted into the baculovirus genome. Direct immunofluorescence technique confirmed that the protein of interest was present in the cytoplasm and nucleus of the sf9 infected with the recombinants. The result indicated that the recombinant protein could react with the specific antibody. The molecular weight of the expressed protein was about 40 kDa determined by SDS-PAGE. In western blotting experiment, the protein did not react with the specific antibody, while in dot-ELISA, the native expression product can react with it.

To evaluate the protective properties of the recombinant protein, five groups (10 birds each) of SPF chickens were vaccinated with the expression protein and challenged with the virulent IBDV strain CJ-801. The results showed that the chickens vaccinated were partially protected against the infection with the virulent IBDV. Failures to obtain complete protection maybe account for an insufficient amount of recombinant protein used for immunization.