| Background Gastric cancer is a common malignancy in many countries of the world, especially in China, for which the routine therapies are ineffective. Tumor vaccine is an effective method for cancer therapy that was demonstrated by a lot of preclinical and clinical research. MG7-Ag, discovered by our institute, is a new marker of gastric cancer. The epitope of MG7-Ag was found to locate in a sugar chain, suggesting that the MG7-Ag was a kind of carbohydrate antigen. Using phage display technology, the mimicry peptides of MG7-Ag were identified. In vitro experiment showed that the mimic peptide could mimic the primary antigen effectively, suggesting that it is a competent candidate for vaccine. Using these mimicry peptides, we have constructed the protokaryotic expression vector of the fusion gene of MG7-Ag mimotope gene and carrier protein HBcAg. Using Salmonella typhimurium expressing the fusion gene as an oral vaccine, anti-tumor immune responses were elicited in mice.As a vaccine vector, the recombinant adenovirus has many advantages: it can replicate to high titers;it can infect and express of genes in replicating and non-replicating cells; high expression level o f functional proteins; no insertional mutagenesis; free from inactivation by complement. Adenovirus is a safe and efficient vector in vaccine development. It infectsantigen-presenting cells, breaks the immune tolerance, activates dendritic cells and enhances their ability to migrate to local lymphonode and present antigen to T cells. Besides, antigens expressed by adenovirus are presented in MHC I and MHC II pathways. As a result, both CD4+ and CD8+ T cells are activated, inducing potent immune response.The pAdEasy system of adenovirus vector is a more convenient system in construction recombinante adenovirus, which would be used in our experiment.Heterologous prime-boost immunization protocol is a potent strategy in enhancing the immunity induced by vaccines. In this protocol, vectors and adjuvants in priming vaccine are different from the boosting vaccine, avoiding the immune response against the same vector or adjuvant to be too strong. The strategy has been proved to be highly effective in generating protective immune responses, especially in virus vector-based carcinoma vaccine, which would also be adopted in our experiment.Objective To construct a recombinant adenovirus vaccine based on MG7-Ag mimotope of gastric cancer using replication defective recombinant adenoviruses as a carrier, and immunize BALB/c mice with the vaccine and investigate the induced cellular and humoral immunity. Using heterologous prime-boost immunization protocol to immunize BALB/c mice with the vaccine and an oral DNA vaccine based on MG7-Ag, the induced cellular and humoral immunity were investigated.Methods (1) Subcloned the fusion gene of the MG7-Ag mimotope and HBcAg into the pAdTrack-CMV shuttle vector. (2) The resultant plasmid (pAdTrack-CMV - MG7) was linearized by restriction endonuclease Pme I, and subsequently cotransfected into E. coli. BJ5183 cells with pAdEasy-1 plasmid to undergo homologous recombination. (3)Then, the linearized recombinant plasmid (pAd-MG7) was transfected into 293 cells. The recombinant adenovirus was detected by examining the expression of the green fluorescence protein in the 293 cells. (4)Presence of the recombinant adenovirus was confirmed by Western blot and PCR. (5) Immunized BALB/c mice with the recombinant adenovirus and reinforced the immunity every 2 weeks. (6)At 3, 6,9 weeks, serum titer of antibody was detected by ELISA, and at the 9th week cellular immunity was detected by 51Cr release test. (7)Ehrlich ascites carcinoma cells expressing MG7-Ag were used in tumor challenge assay as a model to evaluate the protective effect of the immunization. (8) Heterologous prime-boost immunization protocol was used to immunize BALB/c mice with the recombinant adenovirus vaccine and an oral DNA vaccine based on MG7-Ag, and then investigate the induced cellular and humoral immunity as above.Results (1)Restriction e...
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