The Research Of The Construction And Immunity Of The Recombinant Fowl-pox Therapeutic Cancer Vaccine

Since the fowl pox virus can infect the mammalian cells with a high infection rate and adoptive infection , the virus was studied as a hopeful expression vector. The recombinant expression plasmid of pUTA2VP3HNIL18 was constructed by inserting VP3 gene and HNIL18 fused geng into the downstream of conbined promoter(ATI-P7.5×20), so did VP3 gene and HN gene, then we obtained respectively the other two recombinant expression plasmid of pUTA2VP3 and pUTA2HN. The above all recombinant expression plasmid and fowpox virus 282E4 strain were co-transfected into chick embryo fibroblast cells, then the homologous recombination occurred. The recombinant fowpox virus vFVHN, vFVVP3, vFV3 were screened by using BrdU and identified by RT-PCR, western blot and indirect immunofluorescence. The results of RT-PCR, western blot and indirect immunofluorescence showed that foreign gene carried by recombinant fowpox virus could express efficiently in tumor cells, and HN located in cell membrane, while VP3 located in the nucleus. AO/EB staining, genomic DNA electrophoresis, DCFA staining to detect the Reactive oxygen species level, Rhodamine123 staining to detect the mitochondrial trans-memebrane potential were used to observe the death mode of HCT-8 induced by recombination fowpox virus infection and which pathway is involved in the cell death process. The results showed that recombination fowpox virus infection caused nucleic condensation and localization, the decline or loss of mitochondrial trans-membrane potential, fragmentation of genomic DNA and the upregulation of p53 in HCT-8 cells, the infection ultimately induced HCT-8...