The Study On Antitumor Effect Of Recombinant Nucleic Vaccine Containing HN Gene Of Newcastle Disease Virus

Tumor is a kind of disease which threaten human health seriously. Becauseof its specific biological behaviour, operation, radiotherapy and chemotherapy cannot increase survival quality of patients and prolong their life. With thedevelopment of molecular biology and Immunology, we realized that tumor is akind of disease related gene, abnormality of normocellular growth control andmutation of apoptosis regulatory gene can result in alteration of function, thentumor cell get the ability of metastasis and infiltration, proliferate minute primarylesion gradually. In this study, the HN gene of the NDV was fused with humanIL-18 gene, the fused gene and apoptin VP3 gene from Chicken anemia viruswere recombined eukaryotic expression vector pIRVP3IL-18HN, then tumor cellsBGC-823 were transfected with recombinant plasmid pIRVP3IL-18HN, studylethal effect and cell death of tumor cell. HN gene has activity of neuraminidase,hydrolyze sialic acid in the surface of tumor cell, expose surface antigen of tumorcell, strengthen identification and killing ability of tumor cell through immunesystem. HN can induce IFN, increase recognization of tumor cell by CTL, correctfunctional defect of immune system partly. VP3 gene can induce apoptosis oftumor cell, its function does not depend on p53 and bcl-2, VP3 gene can notinduce normal cell. As a kind of cytokine, IL-18 can be induced and generateIFN-γ, it can exert anti-tumor effect through increasing activity of NK cell and Tcell. In this study, the HN gene of the NDV was fused with human IL-18 gene, thefused gene and apoptin VP3 gene from Chicken anemia virus were cloned intoeukaryotic expression vector pIRESneo and the recombinant plasmid that carriedHN gene, apoptin VP3 gene and human IL-18 gene was constructed. Therecombinant plasmid was amplified in Ecoli and extracted from its host rupturedby alkaline lysis, indirect immunofluorescence, southern blot, Western blot andRT-PCR were used to detect the expression of the foreign genes in therecombinant plasmid and expression of the recombinant plasmid in the tumor cells.The results of indirect immunofluorescence, southern blot, western blot andRT-PCR showed that these three foreign gene were cloned into eukaryoticexpression vector pIRESneo together , and foreign gene carried by recombinantplasmid could express efficiently in tumor cells BGC-823.In this study, tumor cells BGC-823 were transfected with recombinantplasmid pIRVP3IL-18HN, then AO/EB staining, EM observation, genomic DNAelectrophoresis, DCFA staining to detect the Reactive oxygen species level,Rhodamine123 staining to detect the mitochondrial trans-memebrane potentialwere used to observe the death mode of BGC-823 induced by recombinantplasmid pIRVP3IL-18HN transfection and which pathway is involved in theapoptosis process. The results showed that recombinant plasmid pIRVP3IL-18HNtransfection caused nucleic condensation and localization, chromatinmargination ,the decline or loss of mitochondrial trans-membrane potential,fragmentation of genomic DNA, the infection ultimately induced BGC-823 cellapoptosis and the apoptosis induce by recombinant plasmid pIRVP3IL-18HN ismainly through mitochondrial pathway. Furthermore the transfection ofrecombinant plasmid pIRVP3IL-18HN increased the expression of MHC-I on thesurface of BGC-823tumor cells. We analyze that it may be a major mechanism ofits anti-tumor function.C57BL/6 mice model bearing H22 hepatoma was construced bytransplanting H22 cells into the right hind limb of the mice and the combinativeantitumor effect on H22 hepatoma of HN gene, VP3 gene and IL18 gene wasobserved through this model. Tumor suppression rate is 46.28%, it is higher thanthat of other therapy groups pIRHN and pIRVP3. EM observation of tumor cellsshowed nucleic condensation and localization, chromatin margination,transfectedtumor cells showed typical apoptosis. The result of pathohistology showedvacuolization of transfected tumor cells. The detection of the subset of Tlymphocyte showed that recombinant plasmid pIRVP3IL-18HN could increase thequantity of CD4+and CD8+ T lymphocyte. The result of CTL showed thatrecombinant plasmid pIRVP3IL-18HN could increase the special killing of Tlymphocyte. We can suppose that recombinant plasmid pIRVP3IL-18HN increaseits anti-tumor function through the recovery of cellular immunity. In this study,recombinant plasmid pIRVP3IL-18HN showed significant anti-tumor functionthan that of pIRHN and pIRVP3. According to the above results, we suggested thecombination of HN gene,VP3 gene and IL-18 gene has a potent antitumor efficacy,moreover they play a synergism role in anti-tumor mechanism.