Characterization Of DNA Vaccines Encoding The Domains Of BAP31 For Their Ability To Elicit Tumor-specific Immunity

The global burden of cancer continues to increase largely because of the aging and cancer-causing behaviors. Based on the GLOBOCAN 2008 estimates, about 12.7 million cancer cases and 7.6 million cancer deaths occurred in 2008; of these, 56% of the cases and 64% of the deaths occurred in the economically developing world. Standard therapeutic procedures currently in practice, including surgery, radiation, and chemotherapy have not greatly impacted the spread and recurrence of progressive malignancies. There is an urgent need for new therapeutic strategiesWolff research team first discovered in mice by plasmid DNA directly injected exogenous to the mice, the exogenous DNA encoding the protein can be long-term expression in skeletal muscle, which is the development of DNA vaccines provide a practical basis.The first vaccination studies utilizing plasmid DNA in protection scenarios involving influenza and HIV-1. In this application, found that plasmid DNA by subcutaneous or intramuscular injection can be effective in stimulating the immune system response, which prompted DNA vaccines can stimulate the immune system to achieve the effect of preventing disease, which makes mass vaccination becomes more meaningful , Especially for mass production of vaccines are difficult, such as recombinant protein or whole tumor cell vaccine. Is well known that tumor cell is not easy to remove the immune system is an important reason for this is the low immunogenicity of tumor antigens, and compared with DNA vaccines offers the possibility to overcome the disadvantages, making it an effective cancer immunotherapy strategies.DNA vaccine in animal studies found that although the vaccine can induce specific proteins for the purpose of CTL immune response, but the animals did not result in high titer antibody to consider the reasons for the DNA vaccine into the body, is After cellular uptake expressed relative to body endogenous protein antigen, MHC I class offered through the channels; the purpose of antigen presentation by MHC II class was handed approach is a CD4 + T cells induce the production of the target antigen in both cellular immunity Reaction and produce high titers of specific antibodies necessary conditions. Because DNA vaccines into cells expressed endogenous protein can not enter the MHC II class presentation pathway, it can only be through the introduction of targeted molecular targeting to MHC II presentation pathway class to effectively activate CD4 + T cells.In 1985, monoclonal antibodies discovered through the lysosome located in the outer membrane of the lysosomal associated membrane protein (lysosome associated membrane protein, LAMP), followed by dendritic cells and other antigen presenting cells (antigen presenting cells, APC) Cells, also found that LAMP and MHC II molecules were localized in the MHC class II compartment (MIIC), MIIC is the degradation of antigen-presenting cells and assembly of peptide antigen processing and MHC II molecules of the important places. LAMP molecules through the C terminal short cytoplasmic tail of the recognition sequence Tyr-XX-? (? behalf of any one hydrophilic amino acids) to target specific proteins transported from the cytoplasm to the lysosomes. Therefore, if the tumor antigen genes into the LAMP lysosomal molecules between the lumen and the transmembrane construct DNA vaccine, expected by the role of LAMP molecules targeting the tumor antigen to the MHC-II class presentation pathway, inducing strong Cellular and humoral immune responses. Cancer / testis antigen (CTA) is a class expression in various tumor tissues, in addition to the normal tissues other than testis almost no expression of antigen. CTA in cancer patients is immunogenic and can induce efficient humoral and cellular immunity, for the purpose of the CTA molecules for the treatment of cancer vaccines provides a new strategy for cancer,Such as NY-ESO-1 as a therapeutic melanoma vaccine target molecules.My term found BAP31 molecule highly expressed in testis, positive products were mainly located in the spermatogonia and primary spermatocytes, also found in cervical cancer, ovarian cancer, breast cancer, esophageal cancer, liver cancer, colon cancer, rectal cancer and Highly expressed in lung cancer, BAP31 protein gene mapping and MAGE-A, NY-ESO-1, LAGE-1, SAGE and other gene adjacent to the CTA. BAP31 expression under the restrictive characteristics and gene mapping, suggesting that BAP31 may be a family for the CTA can be used as targets for cancer gene vaccine.The carboxyl terminus of the BAP31 gene as a 366 nucleotide (ΔBAP31) molecules into the LAMP luminal domain and encoding transmembrane / cytoplasmic tail between genes to form chimeric gene construct new cancer vaccine. Set DNA vaccines p-LAMP-ΔBAP31 as the experimental group, p-ΔBAP31, p-LAMP, PBS as a control group, immune 6-8 week old female C57BL/6J mice. Subcutaneous injection of the way to the end of the root, each 50μg / only by an interval of 3-week cycles were immune 3 times, the first 5,8 weeks of tail vein blood serum was separated for ELISA detection of anti-BAP31 antibody production, 2 nd and last time 2 weeks after immunization and anti-ELISPOT test line. ELISPOT results showed that,ΔBAP31/GST fusion protein can stimulate the secretion of spleen cells in experimental group-specific IFN-γ, and the levels were significantly higher (P <0.05). Anti-trial, the experimental group on B16 spleen cells isolated cells (mouse melanoma cell line) were significantly higher killing rate (P <0.05). Indirect ELISA results showed that, p-LAMP-ΔBAP31 and p-ΔBAP31 group to produce specific antibodies againstΔBAP31, and with the increase in the number of immunization, antibody titers increased gradually to peak after the 3rd immunization. In vivo experiments confirmed that, p-LAMP-ΔBAP31 and p-ΔBAP31 Group to tumor growth in mice was significantly inhibited.In short, BAP31 and LAMP chimeric gene vaccine composed of molecules through the LAMP targeting the BAP31 molecular transport to the MIIC, and turned into one, in order to achieve protein expressed by DNA vaccines derived from the class of MHC I antigen processing Way out derived MHC II antigen processing pathway in the transformation of class, so as to effectively activate CD4 + T cells to induce efficient cellular and humoral immune response to cancer therapeutic vaccine has laid a solid foundation.