| The Fowlpox virus(FPV) genome, containing 260 to 309 kbp of double-stranded DNA, is larger than most of other virus genomes. As FPV genome have many genes, its contains a lot of non-essential gene for replication, which can accommodate 25 to 30 kb exogenous gene. It is generally accepted that the FPV cause disease only in avian species and it is also generally accepted that FPV are unrestricted for entry to most mammalian cell types, but that they are restricted for productive replication in mammalian cells. Because all of these, FPV became a very safe vector for recombinant vaccines and showed its unique advantages as FPV-based expression vectors.In the past twenty years, live recombinant FPV vaccines as well as FPV vaccines have made great progress. A number of important poultry viral pathogens became early targets for FPV vaccines, notably: avian influenza virus, Newcastle disease virus, infectious bronchitis virus, MDV and infectious bursal disease virus. A major strength of the FPV as vectors is its large capacity for foreign gene insertion and choosing a potential site for foreigner integration is the first step of constructing a recombinant FPV virus. But now few research about the non-essential gene for the foreigner integration were reported in domestic. And it is also reported that inserting in the different non-essential gene for replication would influence the ability of the expression level of the foreigner integration. So it is very essential to screen and optimize some new non-essential region for replication. And it would lay the foundation for restructuring the FPV-based expression vectors.In this study, some non-essential areas for replication were chosen according to the FPV genome that was published. we considers the areas’ effect on FPV replication, virulence and biological characteristics of FPV. According to all of these affects, we choose two area in FPV genome assuming that its would not influence the ability of replication. To restructuring the FPV-based expression vectors, two DNA fragments in FPV054 and two DNA fragments between FPV ORF161 and ORF162 were amplified by PCR and cloned into pUC18 flanking the EGFP gene as the homologous arms to construct transfer recombinant plasmid, which was transfected into the pre-rFPV-gB(the recombinant FPV expressing gB gene of infectious bronchitis of chicken) infected chicken embryo fibroblast(CEF) to rescue the recombinant FPV(rFPV). After three round of plaque purification in CEF cells, we got the purified recombinant viruses of rFPV054-EGFP and rFPV161-EGFP. The rFPV were proved to be genetically stable when passed in CEF for 20 passages according to the EGFP expression and PCR detection of the foreigner integration. Moreover, the replication dynamics were compared for the rFPV and the parental virus which showed they had the similar replication curves. After observing the CEF cells infected by the r FPV for each generation, we can see continuous expression of EGFP in rFPV. At the same time, immunoblot detection of exogenous genes show further evidence of the sustained and steady expression of exogenous gene. Furthermore, negatively stained virus particles of purified rFPV054-EGFP, rFPV161-EGFP and rFPV-gB all possessed the characteristic morphology of mature FPV. Subsequently, ultrathin section transmission electron microscopy was performed to determine the different particle forms during virus morphogenesis in CEF cells. All of this show that rFPV054-EGFP and rFPV161-EGFP had no effect on FPV particle structure morphogenesis in CEF.In our study, the recombinants virus rFPV054-EGFP and rFPV161-EGFP expressing EGFP were obtained, suggesting that recombinant arms were available. And its also providing the basis for some of other protective antigen gene of some pathogens(poultry or mammals). Furthermore it provided a basis for the development of bivalent or multible valent rFPV vaccine. |
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