Infectious Bursal Disease Virus Particle Vaccine Research And Its Immune Efficacy Evaluation

Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). Infectious bursal disease virus (IBDV) belongs to genus Avibirnavirus of family Birnaviridae. It is a non-enveloped virus with icosahedral symmetry that contains two segments of doublestranded RNA. Segment A codes for VP2, VP4 and VP3, while segment B encodes for VP1, an RNA-dependent RNA polymerase. Two neutralizing epitopes are present on the VP2 protein and are considered to be major host protective antigens. Chickens are highly susceptible to the virus infection between 3 and 6weeks after hatching, when the bursa of Fabricius (BF) reaches maximum development. IBDV infection results in lymphoid tissuedepletion and the final destruction of the bursa, which is the predominant feature of its pathogenicity. The studies were divided into three parts as follows:1 Cloning and sequence analysis of IBDV VP2 geneTaken bursal organization from diseased chickens and pathological observations have obvious pathological changes.The disease material are grounded into virus suspension which have a higher antigen titer by agar diffusion assay. VP2 genes of infectious bursal disease virus (IBDV) were amplified by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis showed that the VP2 gene have 1452 nucleotides and encode 484 amino acids. The VP2 gene has the same heptapepetide motif (S-W-S-A-S-G-S) and critical amino acid residues A222, Q253, 1256, D279, A284,1294 and S299 in the variable region of VP2, suggesting that posses the molecular characteristics of virulent IBDV.2 Cloning and sequence analysis of chIL-15 genechIL-15 gene was amplified by RT-PCR from spleen lymphocytes total RNA stimulated with ConA. Sequence analysis showed that the chIL-15 gene have 579 nucleotides and encode 193 amino acids, sharing 98.6%-100% and 99.4%-100% holomogy with the chIL-15 gene of the published. 3 Fusion expression and identification of the VP2 gene and chIL-15 in insect cellsVP2 gene of infectious bursal disease virus (IBDV) were amplified from bursae of Fabricius and then cloned into PH site of the pFastBacDaul donor plasmid, the positive recombinant plasmid was named as pFastDaul-VP2.chIL-15gene was cloned into P10 site of the pFastBacDaul donor plasmid, and the positive recombinant plasmids were named as pFastDaul-IL15 and pFastDaul-VP2-IL15. The recombinant donor plasmids were constructed and transformed into competent E.coli DH10Bac cells. After the selection, the recombinant expression bacmids Bacmid-VP2. Bacmid-IL15 and Bacmid-VP2-IL15 were obtained and used to transfect insect cell Sf9. The protein band of approximate 47 kDa was detected in the western blotting, and the protein fluorescence can be observed by inverted fluorescence microscope.4 Immunogenicity of recombinant proteinsIn this study, respectively IBDV-VP2 protein and the fusion protein of VP2 and IL-15 for the preparation of oil emulsion vaccine antigen. Immunization of 14 days non-immune chickens and studies the immunogenicity of IBDV VP2 protein and its influenced by IL-15. Also set up the wild-type baculovirus control group and blank control group. And the purchase of commercial IBDV vaccine to IL-15 as adjuvant to study the adjuvant effect of IL-15. Measured antibody titer by indirect ELISA, and protection against IBDV. The results show that the VP2 protein has good immunogenicity and can induce immune chickens produce specific neutralizing antibodies and provide immune protection. The neutralizing antibodies can be maintained and kept in a long time by IL-15.