Expression Of Infectious Bursal Disease Virus (IBDV) Polyprotein In Bombyx Mori And The Study Of Its Immunogenicity

Infectious bursal disease virus (IBDV) can cause acute infectious disease with the character of immunosuppression and large scale of loss due to higher susceptibility to other pathogens and immunization defeat. Classic method of inactive vaccine and low-virulence vaccine have been confronted with the threat of variant strains and very virulent strains of IBDV isolated since 1980's. The scale of poultry industry in China is enlarging increasingly, however, the epidemic profile of IBDV is very complicated in China and only a few IBDV vaccines with independence intellectual property right are acquirable. As an access to the contradiction, we made the program of expressing IBDV viral proteins with baculovirus-silkworm expression system. The study should help us to make sure that (1) whether silkworm is fit for expressing IBDV proteins as bioreactor and (2) whether the polyprotein or VP2 protein is a more suitable antigen for subunit vaccine against IBDV.A strain of IBDV, designed as JD1, was isolated from bursa of Fabricius of the sick chicken flocks in Jiande of Zhejiang province, attenuated through passage on chicken fibroblast and the whole segment A of its genome was cloned in our lab. cDNA of polyprotein(VP2/4/3) gene and VP2 gene were cloned with the cloned genomic segment A as template. The two fragment of polyprotein gene and VP2 gene were inserted into Bombyx mori nuclear polyhedrosis virus (BmNPV)-based vector pBacPAK-8, respectively. Recombinant vectors and linearized genome DNA of mutant baculovirus Bm-BacPAK6 were used to co-transfect cultured Bombyx mori cell (BmN) and recombinant baculovirus (rBV) BacPAK-A and BacPAK-VP2 without contamination of parental virus were obtained after three rounds of plaque purification.Total DNA of BmN cells infected by rBVs was extracted, spoted on a piece of NC membrane with the control of negative and positive DNA, and a positive hybridization signal was detected by a DIG-labeled IBDV specific DNA probe. Total DNA of cells infected by BacPAK-A, which has successively on BmN cell for 15 times, were extracted for PCR and Southern blotting. The results indicated that the polyprotein gene and VP2 gene have successfully inserted into the genome of baculovirus Bm-BacPAK6 through homologous recombinant and the recombinant BacPAK-A isstable within at least 15 passages in vitro.rBVs BacPAK-A and BacPAK-VP2 were injected into the haemocoel of fifth-instar silkworm (Bombyx mori} larvae for infection and expression. Haemolymph was harvested from 3 days post infection (PI) as the infection symptom began to appear and most of infected silkworm were on the point of death till 6 days PI, haemolymph of normal silkworm was also collected for negative control. The results of ELISA, SDS-PAGE and Western blotting analysis for the haemolymph samples suggested that polyprotein gene and VP2 gene were expressed in larvae with characteristic of immunoreactivity and the expression reached the highest level 5-6 days PI.The polyprotein- and VP2-contained silkworm haemolymph harvested in 5-day PI and normal haemolymph were emulsionized respectively with mineral oil as vaccines. Chickens were inoculated subcutaneously with 1-time or 5-time dose of polyprotein vaccine for the examination of vaccine safety. There's no reaction among the chickens since the inoculation and no abnormality in the autopsy 5 days post inoculation.chickens were divided into 6 groups randomly for inoculation with emulsions prepared with polyprotein, VP2, vormal haemolymph and commercial IBDV vaccine D78, respectively, as the primary at 14-day old and the boost at 28-day old, and the rest two groups were set for challenge control and normal control. Blood of the immunized chickens was collected periodically for antibody titration until the chickens were challenged with virulent IBDV BC6/85 25 days post the boost. Chickens were killed 5 days post challenge and autopsied. Results indicated that the polyprotein vaccine can resist the challenge of virulent IBDV (BC6/85 strain) more efficient than VP2 vaccine, as...