Construction Of Immune-Enhancing DNA Vaccine Of Fowl Pox And Its Immune Evaluation

In this research, we firstly examined chickens suspected of"mixed type"fowl pox with pathological methods. Eosinophilic dying A type inclusion body could be observed in the skin, trachea and laryngeal. Then virus was isolated and passaged onto the chorioallantoic membrane of 10-day-old embryonated chicken eggs for several times, the compact variola could be formed on CAM. The CAM-adapted virus was propagated on primary chicken embryo fibroblast cell (CEF) until cytopathic effect was noted. According to American standards FPV genome sequence published in GenBank, a pair of primers for 4b core protein gene was designed with Oligo 6.0 and Primer 5.0 software, sequence analysis of PCR product and regression of chicken test proved that the isolated pathogen was fowl poxvirus, named HH2008.According to published gene sequence (AF198100) in GenBank, a pair of primers for ORF 140 gene was designed. After idenfication and sequence analysis of the PCR product, it was subcloned into prokaryotic expression vector and designed pET30a-ENV, then expressed in E. Coli Rosetta system and ENV protein is approximately 42 ku as expected. The recombinant ENV protein was purified and used as immunogen to generate rabbit polyclonal antibody. Western-blot and ELISA indicated that the antibody could detect FPV ENV protein. More importantly, an indirect ELISA was developed with this anti-FPV140 antibody was capable of distinguishing FPV from other common avian pathogens.FPV ORF140 gene was then modified and inserted into an eukaryotic expression vector, pVAX1, to construct pVAX1-ENV. Furthermore, a flexible linker, which express (G₄S)₃ polypeptide was used to link ChIFN-γgene with FPV ORF140 gene and got a fusion gene eukaryotic expression plasmid (pVAX1-ENV-Linker-IFN). Transient transfection of these two plasmids in vitro showed that the pVAX-ENV and pVAX1-ENV-Linker-IFN could express in BHK21 cells demonstrated by their antibodies, respectively. Intramscular injection experiment of these two plasmids in vivo showed pVAX-ENV and pVAX-ENV-IFN were able to expressed too. It proved that the protein expressed by fusion gene could retain their bioactivities, and provided theoretical foundation for the following immunoprotection research.The experimental chicken were divided into 5 groups, E group inoculated with pVAX-ENV plasmid, E-I group inoculated with pVAX-ENV-IFN plasmid, V group inoculated with FP commericial vaccine, P group inoculated with empty pVAX vetor, and B group inoculate with PBS as control. Thereafter, lymphocyte proliferative assay, enzyme Linkered immunosorbent assay and flow cytometric technique were used to detect the immune response of different groups. Finally chickens in different groups were challenged with FPV HH2008 strain to compare their protection rate. The results showed that most of immune response of V, E and E-I groups were higher than those of B and P groups, especially the E-I groups. T, B lymphocyte proliferative function, the number of CD4⁺, CD8⁺ T cell, the special anti-FPV ENV antibody titer in immune groups were all higher than those of B, P groups. And the E-I and V groups were the most significant among these groups. The histopathological change of FPV target organs of each group were examined after challenge and the immunoprotection rates were calculated among every group. Above all, ChIFN-γwere inoculated with FPV antigen gene, it could improve the host immune response and higher protection rate could be observed against FPV chanllenge. Furthermore, FPV antigen genes and ChIFN-γfusion plasmid was firstly constructed and the results could provide solid scientific ground to the further study of ChIFN-γbioactivities, and the theoretical basis to the dicovery of new type immune-enhancing vaccine for virual diseases.