| Gastric cancer is one of the most commonly seen malignant tumors in digestive system, which poses a great threat to the public health. Conventional therapeutic methods such as surgery and chemotherapy work poorly in combating gastric cancer. In recent years, immunotherapy has become an attractive intervention. However lack of tumor-specific or -associated antigen hinders the development of gastric cancer vaccine. MGv-Ag, identified by our institute, was found to be a marker molecule of gastric cancer and closely related to the progress of gastric cancer. The antigen determinant of MG7-Ag was found to locate in a sugar chain, suggesting that the MG7-Ag was some kind of carbohydrate antigen. By screening the phage display library, we have identified the mimicry peptide of MG7-Ag, competitive antibody binding assay showed that the mimicry peptide could mimic the primary antigen well, which provided the premise for further using it to develop vaccines.Oligopeptides are degraded easily in vivo and require carrier proteins to protect them. Heat shock protein 70 (hsp70) is a molecule chaperone that keeps oligopeptides from degrading, and also it can promote antigen presenting to MHCI >. MHC II molecules . Hsp70 was also found to be a kind of antigen presenting molecule as the MHC molecules, which presents peptide directly to T cells. Hsp70 is a highlyconservative protein among various species, so it won't induce immune response between individuals. All these indicate that Hsp70 is a good carrier for vaccine development, and indeed hsp70-based vaccine has been tested in clinic trials.DNA vaccine is a relatively new kind of vaccine. It can induce specific, potent and persisting immunity against given antigen and can be easily constructed. The immunizing routes of DNA vaccines include: intramuscular injection, intradermal injection, skin gene gun bombard and vein injection. In recent years, hydrodynamics-based vein injection is applied to gene delivery, which employs a physical force to transiently disrupt the cell membrane barrier to allow the entrance of DNA molecules into cells and then be expressed efficiently. This methodproved a new approach to study functions of gene and develop vaccines. Objective To construct a fusion DNA vaccine of gastric cancerMG7-Ag mimotope and heat shock protein 70 and investigate the cellular and humoral immunity induced by the fusion gene vaccine and its protective effect in vivo in mice.Methods (1) The coding sequence of MG7 mimotope (GC23) was included in the reverse primer, and by PCR incorporated with HSP70 gene at the 3' terminus. PCR product was cloned into pUCm-T vector and confirmed by enzyme digestion and sequencing; (2) PCR product was subcloned into prokaryotic expression vector pET-21a(+), and then transduced into E Coli. The protein yielded was identified by SDS-PAGE, Western blot and dot blot. (3) The eukaryotic expression vector pcDNA3.1(+)-HSP70/MG7 was constructed by subcloning the fusion fragment into pcDNA3.1(+) vector, and the recombinant vector was sequenced to confirm the proper encoding. BALB/c mice were immunized with the pcDNA3. l(+)-HSP70/MG7 (namely the fusion genevaccine), and pcDNA3.1-hsp70 and empty pcDNA3.1 vector were used as controls. The sera were collected at the 3rd week after every immunization. Cellular ELISA was performed to evaluate the humoral immunity induced. The splenocytes were separated at the 9th week, and 51Cr release assay was performed to investigate the antigen specific CTL response. At the 9th week, murine EAC cells expressing the MG?-Ag were used as a model to challenge the mice. 25 days after the challenge, tumor-bearing status of mice was observed. And we also investigate the antinuclear antibody in mice to evaluate safety of the DNA vaccine.Results: (1) A fragment of 2.0 kb in size was amplified by the PCR. Sequence analysis revealed that the sequence of GC23 was successfully fused into the 3' terminus of hsp70, and the sequence of HSP70 was as reported. (2) When expressed in the prokaryotic E coli, the f... |
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