Mouse VEGF/FGF-2 Vaccine Construction And Tumor Intervention And Ebola Virus-like Particle Construction

Background:The progression of tumor growth can be devided into two stages:a slow stage with avascularity and a fast stage with vascularity.During the avascularity the nourishment for tumor growth is supplied by the adjacent vessels and the size of tumor is usually limited to 1-2mm3.As the tumor growth,oxygen and nutrients supplied by adjacent vessels cannot meet the metabolic requirements,and sustained expansion of a tumor mass requires a new blood vessel formation to provide quick-proliferating tumor cells with an adequate supply in oxygen and nutrients.Since VEGF is one of the key regulators of angiogenesis,which is secreted by most cancer cell types,blocking the action of VEGF is a proven approach to treat multiple types of tumor diseases.VEGF potently increased vascular permeability,which allows tumor cells disseminate into the blood system.FGF-2 is another important pro-angiogenesis factor,which plays a key role in aquired resistance of anti-VEGF therapy.Moreover,accumulating literatures suggest the abnormal blood vessels contribute significantly to the formation of immunosuppressive tumor microenviroment.Objectives:To construct VEGF and FGF-2 vaccines,and assess the potentials of active immune strategy with the vaccines on suppressing tumor progression by induceing persistent specific antibodies to neutralize the over-expressing VEGF and FGF-2 in tumors.Methods:The peptide of VEGF and full-length FGF-2 sequence were inserted into hepatitis B core antigen(HBcAg)and bacteriophage Q? by gene recombinant methods.Together with the carrier protein,the chimeric proteins were co-expressed.Then the proteins were purified and characterized by ammonium sulfate precipitation method,sucrose density gradient centrifugation and SEC-HPLC,as well as electron microscope analysis.BALB/c mice were immunized with the vaccine candidates Q?-VEGF and Q?-FGF-2,then the serum specific antibody responses were detected with ELISA.In 4T1 cells grafted murine tumor model of breast cancer,the influences of the vaccination against VEGF and FGF-2 on tumor progression and lung metastasis were assessed by using a preventative immunization strategy.In another experiment using a treatment strategy of vaccination,the effects of the anti-VEGF vaccine on suppressing tumor growth and the influences on the tumor-killing efficacy of an experimental therapeutic HPV vaccine were evaluated.The tumor growth was dynamically monitored every two or three days.At endpoint of the experiments,the mice were sacrificed,and the lung and tumor mass was weighed.The numbers of tumor metastasis nodes in lung were counted.Further,splenocytes were isolated and IFN-y expressing cells were detected by ELISPOT,and the level of CD8+ cells were checked with flow cytometry.Results:The recombinant plasmids pHBcAg-VEGF,p?-VEGF,pHBcAg-FGF-2 and pQP-FGF-2 were constructed.The expression of the recombinant proteins were induced successfully in E.coli.The recombinat proteins HBcAg-VEGF and Q?-VEGF folded highly efficiently into the form of VLPs,and Q?-FGF-2 was expressed in the form of inclusion bodies.Mice immunized with the purified vaccine candidates produced persisitent and high tittered specific antibody responses.In the experiment in 4T1 grafted tumor model,active immunization of anti-VEGF and anti-FGF-2 vaccines using a preventive immunization strategy inhibited the growth and lung metastasis of 4T1 tumor,companied with the elevated level of IFN-?-expressing splenocytes.In TC-1 grafted mouse model of HPV-induced tumor,employing a therapeutic immunization strategy,anti-VEGF vaccine produced asynergistic effects with an experimental therapeutic HPV vaccine on suppressing tumor growth and increasing the population of CD8+ cells in splenocytes.Conclusion:CQ-expression the chimeric protein carriyng antigenic peptide with the carrier protein can be an effective way to form a chemeric VLP,for increasing the antigenicity of a specific antigen.The active immunization with ani-VEGF and anti-FGF-2 vaccines could elicit specific immune response and facilitate suppressing tumor growth and metastasis,and thus could be a promising approach of tumor immunotherapy.Background In 2014,the largest Ebola virus disease outbreak in the world affected large population.Through July 2015,a total of 27741 Ebola virus disease cases were reported from all affected countries.Due to its high fatality and infectivity Ebola virus is classified as category A pathogen by the NIH.Objective To co-expression Ebola virus proteins GP and VP40 in mammalian cells and generate Ebola virus-like particles.Methods According to amino acid sequences,the nucleotide sequences of Ebola-Zaire GP and VP40 genes were optimized based on the codon usage bias in mammalian cells.The synthesized genes were cloned into expression plasmids pcDNA3.1 or pBudCE4.1 which has two expression units,and then 293FT cells were transfected with the recombinant plasmids using lipofectamine2000.The expression of recombinant proteins was detected by western blot,and the VLPs were observed by electron microscope.Results Specific reactive bands recognized by anti-GP antibody were found in cells transfected with the recombinant plasmid carrying optimized GP gene.The expression of recombinant GP protein mediated by the plasmid pBudCE4.1/GP/VP40 which co-expressing GP and VP40 was significantly higher than that mediated by co-transfection with the plasmids pcDNA3.1/GP which expresses GP and pcDNA3.1/VP40 which expresses VP40.Besides,VP40 expression was detectable although the level was low.Classical Ebola virus-like particles were found under the observation with electron microscope.Conclusion Ebola virus proteins GP and VP40 were successfully expressed and assembled into virus-like particles in 293FT cells,which laid an important foundation for further studies on the development of a vaccine or virus detection reagent.