Genome And Proteome Analysis Of Fowlpox Virus282E4Strain Vector Construction And Recombinant HIV Vaccine Research

Fowlpox virus (FPV) is a representative type of the avipoxvirus genus of the Poxviridae. It could only infect poultry and lead to disease. Fowlpox virus can not infect humans and other mammals, but it can express exogenous DNA effectively, and it replicates in the cytoplasm of infected cell, the study about fowlpox as vector become the current hot spot. In order to design a safer and more effective FPV expression vector system, an in-depth understanding of FPV virulence, pathogenic mechanism, host range and immune escape is needed. In this study, the FPV282E4was constructed as vector to express HIV-1multi-epitope to do background analyze of genome and protein composition, construction of neotype expression vector, vaccine screening of recombinant fowlpox virus and experimental immune research.In this study, Illumina high-throughput sequencing technologies were used to sequence the whole genome of FPV282E4strains. The sequences data were assembled, predicted and annotated using the software for de novo prediction and functional annotation after assembly the sequencing data. The results showed that the whole genome of FPV282E4was308826bp, the GC%content was around28%, and313Coding sequence (CDS) were predicted and carry out the equality synteny analysis of the nucleic acid of FPV282E4, FWPV and CNPV. Analysis of functional annotation indicates that FPV has the function of nucleotide metabolism, transport, transcription, replication, recombination and repair, and modification after translation, partner and signal transduction which lay a theoretical foundation for the further study of the fowlpox virus. Based on the results, we did full spectrum proteomic analysis of FPV282E4by LC/ESI-MS/MS, and we have identified76proteins and proceeded functional annotation which showed a results that were similar to the whole genome sequencing. The results provided basis research for FPV infection and pathogenic mechanism.Based on the FPV282E4whole-genome sequencing data and the problems such as low recombinant efficiency and long screen cycle that occurred during construction of fowlpox virus, we have chosen5CDSs zone of recombinant non-essential genes as homologous recombination arm to construct the fowlpox virus vector through inserting three gene-expression-box which contains PE/L early/late promoter and termination signal; and then made red, green and blue fluorescent protein to be the report gene to validate to expression activity of recombinant virus and exogenous gene. The result of fluorescent protein expression showed that three express boxes can express exogenous genes independently; and through the detection of morphology and biological characteristics, we have constructed three rFPV successfully of which the gene deletion has no impact on the morphous and replication of recombinant fowlpox virus and there was no difference among of three rFPV.Base on the well abtain of expression vector, and the construction of immunogen of HIV-1multi-epitope(MEGNp24), CpG motif and CTB genes (CCMp24), we got an rFPV-CCMp24through plaques screening and purification, and then, the recombinant virus was identified by PCR, RT-PCR and WB methods. Results showed that the rFPV-CCMp24was successful constructed, and replication efficiency of recombinant virus was concordance with wild stain FPV, it could express exogenous genes in vitro and possess reactionogenicity. Then we proceeded combined immunization in mice with the rFPV-CCMp24and the recombinant Fowlpox virus rddVTT-CCMp24to analyze the immunogenicity, then comprehensively analyze the immune effect through detecting the specific HIV-1antibodies, lymphocyte stimulation indices, lymphoid cells secrete IFN gamma and CD3+/CD8+, CD4+/CD8+to retrieval effective immunization strategy based on virus vector. The results showed that immunization in mice with rFPV-CCMp24only and with combination of rFPV-CCMp24and rddVTT-CCMp24would show the advantage in induction of cellular and humoral immunity. This study lays the foundation for the further study of rFPV live vector vaccine development and the pre-clinical experiment.