Studies On Immunization Of Recombinant Plasmid Expressing Chicken Prolactin And NDV F Gene

Prolactin is one of the most key hormone in the broody. F protein of NDV is the most important immunogenic protein in F-based vaccines. The breeding performance or egg production and immune performance were increased through active immunization against prolactin and Newcastle disease F gene. In this study, gene clone, cell culture, gene immunization and Western blot were applied to construct the PRL and F recombinant eukaryotic plasmid to provide the availability of development on the PRL and F gene vaccine.1. Study on prokaryotic expression and immunity of prolactin and F gene of Newcastle disease virusThe prolactin and F gene of newcastle disease virus (NDV) was truncated and cloned into a prokaryotic expression vector pGEX-6P-1, and then were induced to express by0.5mM IPTG after transduction into Ecoli. BL21. A fusion protein GST-PRL and GST-F with molecular weight of54and86kDa were expressed in E.coli. ICR mice were immunized with the purified protein. The polyclone antibody was produced, the purified GST-PRL and GST-F protein showed a strong reaction with the PRL and F-positive sera in Western-blotting assay.2. Construction and identification of recombinant plasmids expressing prolactin and F gene of Newcastle disease virusIn this study, PRL and F gene were engineered with the codon usage optimized for mammalian cell expression. Recombinant pVAX-PRL, pVAX-F and pVAX-PRL-F were constructed. Positive clones were identified by restriction enzyme ananlysis and further confirmed by sequencing. To investigate the in vitro expression and authenticity of the PRL and F gene, HEK-293A cells were transfected with pVAX-PRL, pVAX-F and pVAX-PRL-F, and Western blot was performed at48h post-transfection. The PRL and F specific protein bands with expected molecular sizes could be detected in lysates of cells transfected with recombinant plasmids, but not in lysates of cells transfected with the empty vector. These results provide a good basis to further application of the recombinant plasmids.3. Analysis of immunogenicity of recombinant plasmids expressing PRL and F of NDV in chickenIn this study, five recombinant plasmids were developed as potential vaccine in a chicken model. Five groups of lays (30chickens per group) were inoculated intramuscularly three times at3-week intervals with the recombinants expressing PRL (pVAX-PRL), F (pVAX-F) and PRL-F (pVAX-PRL-F) protein. Two additional groups were injected with pVAX and PBS as control. The results showed that chicken immunized with pVAX-PRL-F showed a highest antibody at3weeks post third inoculation. Chicken with pVAX-PRL and pVAX-PRL-F showed a shorter nested day compared to the control and pVAX group by0.8and13.8days, respectively (P<0.05), a higher egg production of300days by8.6and16.4, respectively (P<0.05). The effect of prolactin responses in chicken vaccinated with the recombinant plasmids pVAX-PRL and pVAX-PRL-F was higher than the control and pVAX groups at38and42weeks post third inoculation. It was also found that lymphocytes of chicken immunized with pVAX-PRL-F was primed for significant higher levels of NDV specific IFN-y and IL-4responses upon stimulation with purified NDV antigen in vitro than that of chiken immunized with other recombinant plasmids. However, only chicken immunized with recombinant plasmids pVAX-PRL-F produced stronger lymphocyte proliferation responses and developed significantly higher titers of antibodies to F protein compared to chicken immunized with other recombinant plasmids. All these results suggest that PRL and NDV F recombinant plasmid immunization can promote the performance of egg production and immunity in chicken, which provide a good foundation for the development of gene vaccine.