The Preparation Of Chitosan-pcDNA3.1(-)/MAGE-3-HSP70 Nanoparticles And Research Of The Anti-tumor Immune Response

Tumors are always a big threat against people's health and life,and the traditional treatments are far from satisfaction. Cancer vaccines that include tumor cell vaccines,dendritic cell vaccines,peptide based vaccines, protein vaccines,and DNA vaccines,are regarded as the most ideal therapies because these vaccines can elicit antigen-specific antitumor immunity and play an important role in prevention therapy of tumor.DNA vaccines have become an very attractive approach for generating antigen-specific immunotherapy.It is safe, stable, has low immunogenicity, easy of preparation and storage. But they can only induce low or moderate immune response in clinical trials. So, how to construct a more effective DNA vaccine has become a hot research spot for cancer immunotherapy.The identification of tumor specific antigen is the key to induction of specific antitumor immunity. The melanoma antigen,MAGE) was the first reported example of tumor specific antigens. MAGE-3 is a number of MAGE. MAGE-3.It expresses in most malignant tumors but not in normal tissue except for testis and placenta so that can be the ideal antigen in antitumor immunity. The MAGE-3 vaccines can induce specific CTL and show potency in tumor immunotherapy. However, it is still necessary to enhance the potency of MAGE-3 vaccines. Heat Shock Proteins (HSPs), a highly conserved protein in vivo, participate in the folding, transport and assembling of proteins, so named "molecular chaperone".Recently, immunization with HSP complexes isolated from tumor cells has been shown to be able to induce potent antitumor immunity. The immunogenic HSP-peptide complexes can also be reconstituted in vitro by mixing the peptides with HSPs,and the immune response can be induced with MHC either identical or different to the MHC of donor HSPs. Theseinvestigation have made HSPs more attractive for use in immunotherapy.The two main types of vectors that are used in gene therapy are based on viral or non-viral gene delivery systems. The viral gene delivery system shows a high transfection yield but it has many disadvantages, such as oncogenic effects and immunogenicity. However, cationic polymers, like chitosan, have potential for DNA complexation and may be useful as non-viral vectors for gene therapy applications.Chitosan is a natural non-toxic polysaccharide, it is biodegradable and biocompatible, and protects DNA against DNase degradation.So chitosan has great potential in gene delivery.In this study, we Constructed pcDNA3.1(-)MAGE-3-HSP70 and prepared chitosan-pcDNA3.1(-)/MAGE-3-HSP70 nanoparticles by complex coacervation method.Then we evaluated the antitumor immunity and investigated the therapeutic effect of the nanoparticles against established tumor expressing MAGE-3.Objective:To construct and identificate eukaryotic expression vector of MAGE-3-HSP70. Methods:Signed a pair of MAGE-3 primers containing Xho I restriction site, and HSP70 primers containing Kpn I restriction site, pINCY/MAGE-3 and pOTB7/HSP70 were used as template to amplify MAGE-3 and Hsp70 gene by polymerase chain reaction (PCR). Signed MAGE-3 upstream primer (containing Xho I restriction site) and HSP70 downstream primer(containing Kpn I restriction site), used the gene fragments as template to amplify MAGE-3-HSP70 fusion gene fragments. The PCR products and empty plasmid pcDNA 3.1(-) were digested by Xho I and Kpn I,ligated, and transformed competence E. coli DH5α. Positive clones were identified by restriction endonuclease digestion and sequencing.Results: sequencing results showed that the MAGE-3 gene in the vector was in conincidence with the sequence registered in Gene Bank and the HSP70 has three synonymous mutations.Conclusion:pcDNA3.1 (-) /MAGE-3-HSP70 fusion gene eukaryotic expression vector has been constructed successfully. Objective:To prepare chitosan nanoparticles loaded with pcDNA3.1(-)/ MAGE-3-HSP70 by complex coacervation method and to research its characteristics.Methods:Chitosan nanoparticles loaded with pcDNA3.1(-)/ MAGE-3-HSP70 were prepared by complex coacervation method.Then the naparticles were transfected into B16 cells, and the level of MAGE-3-HSP70 mRNA was tested using RT-PCR technology. The in vitro cytotoxicity of the nanoparticles was determined by the MTT assays.Results:The mean diameter of chitosan plasmid DNA nanoparticles was223 nm, its Zeta potential was 16 mV. The encapsulation efficiency of DNA was 92.3%. The transfection efficiency of chitosan plasmid DNA nanoparticles by B16 cells were about equivalent to that of the Lipofectamine 2000 reagent. Chitosan plasmid DNA nanoparticles were much less cytotoxic when compared with Lipofectamine 2000-pDNA complexes.Conclusion:The results showed that chitosan plasmid DNA nanoparticle were nontoxic to cultured cells and plasmid DNA can be efficiently Objective:To evaluate the immunological effects in vivo,and antitumor effects of chitosan-pcDNA3.1(-)/MAGE-3-HSP70 nanoparticles on tumor.Methods:The C57BLI6 mice were immunized with DNA vaccines and their therapeutic effects against B 16-MAGE-3 tumors were investigated. ELISPOT, LDH release assay and ELISA evaluated the immunity responses elicited by vaccines. Results:After the C57BL/6 mice were immunized with DNA vaccines,there were more T cells releasing IFN-y, higher cytotoxicity of CTL to B16-MAGE-3 cells in chitosan-pcDNA3.1(-)/MAGE-3-HSP70 nanoparticle group than those in other groups.However the anti-MAGE-3 antibody titer in serum had no significant difference in all groups. In mice established with B16-MAGE-3 tumors,the tumor volume was significantly lower;the survival time was longer in the chitosan-pcDNA3.1(-)/MAGE-3-HSP70 nanoparticle group than those of other groups. Conclusion: chitosan-pcDNA3.1(-)/MAGE-3-HSP70 nanoparticle can elicit more stronger MAGE-3-specific cellular immunity and have better therapeutic effects against the MAGE-3 expressing tumors than naked DNA vaccine.