| Infectious bursal disease (IBD) is an acute, highly contagious viral infection of chickens and turkeys. The bursa of Fabricius is a target organ for Infectious bursal disease virus (IBDV). B-lymphocytes are destroyed by IBDV infection, followed by-severe immunosuppression which increases susceptibility to other disease in young chickens at 3-12 weeks of age. Recently, the emergence of antigenic variant strains as well as very virulent ones has caused vaccination failures in vaccinated flocks. Thus, safer and more efficacious IBD vaccines must be studied for a new generation of vaccine.Marek's disease virus (MDV) vaccine virus is considered as one of the most potent vectors for recombinant live vaccines expressing foreign antigens related to vaccine-induced immunity against poultry diseases. It has clearly demonstrated MDV vaccine virus, CVI988/Rispens. is safety and effectiveness in field experiences. In this paper, we constructed and investigated the immune characterization of a recombinant MDV. rMDV. by inserting the IBDV-VP2 gene.1 Construction of VP2 Eukaryotic Expression Vector of IBDV and its Immunological CharacterizationThe VP2 gene of IBDV JS strain was amplified by RT-PCR, subcloned into pGEM-T easy vector, designated as pT-VP2. By sequencing, the amino acid sequence of VP2 gene was high homology to that of very virulent IBDV strains (96.2-99.2%). The evolution relationship between JS VP2 with HK46. OKYM or UK661 VP2 wasestablished by phylogenetic tree analysis, which indicated it is of very virulent IBDV strain. The VP2 gene of IBDV was cloned into pcDNA3.1/zeo (+) vector to obtain the pcDNA3.1-VP2. The transiently expression of VP2 in COS-1 cells was identified with the McAb specific to IBDV by immunflurescent assay.Chickens vaccinated with pcDNA3.1-VP2 were challenged with IBDV JS. The results showed that the specific anti-IBDV antibody could be detected in 14 days after twice immunizations. 67% of the chickens was protected from challenge with 100LD50 IBDV JS strain. These results suggested that the VP2 gene is essential in development of DNA vaccination against IBDV infection.2 Construction of transfer vector CVI988The TK and US10 genes of CVI988/Rispens strain were amplified respectively by PCR using primers synthesized according to the nucleotide sequence of the "virulent" GA strain of MDV serotype I published in GeneBank. Both genes were cloned into pUC18 plasmid to obtain the vector pUC18-US10 and pUC-TK. The expression cassette including eGFP gene controlled by CMV promoter and enhancer was cloned into pUC18-US10 and pUC18-TK vector to give rise to the transfer vector pUC18- US10-eGFP and pUC 18-TK-eGFP. A recombinant MDV (rCVI988-US10-eGFP) expressing eGFP in US10 ORF by homologous recombination was developed, whereas no recombinant MDV was obtained in TK ORF. The plague size and the growth characterization of rCVI988-US10-eGFP are similar to CVI988/Rispens. The recombinant virus reported here proved the US10 of MDV is one of the non-essential sites in the viral genome for viral growth in vitro.3 Construction and Immunological Characterization of Recombinant MDV Vaccine Strain CVI988/Rispens Expressing the Fusion Protein of IBDV-VP2A recombinant MDV CVT988/Rispens virus expressing the enhancer green fluorescent protein (eGFP) fused to the VP2 gene of very virulent IBDV JS strain was constructed by homologus recombination. The recombinant virus was designated as rMDV (rCVI988-eGFP-VP2). rMDV was stable in 31 passages detected by PCR and IFA analysis. Chickens vaccinated with rCVI988-eGFP-VP2 would be protected from challenge with 100LD50 of IBDV JS. The protection ratio of the chickens vaccinatedwith the 1000PFU, 2000PFU, 5000PFU of the rMDV were 50%, 60%, and 80%, respectively, compared with 100% in chickens vaccinated with conventional live IBDV vaccine for twice immunizations. The chickens vaccinated with 5000PFU of the rCVI988-eGFP-VP2 showed high protective level from IBDV JS strain challenge, compared to the control group (p<0.01), however, there was no diffe...
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