| Cancer vaccines,including tumor cell vaccines, dendritic cell vaccines, peptide based vaccines, protein vaccines, and DNA vaccines, could be one of the ideal therapies because those vaccines can induce cellular and humoral immune responses and specificly kill tumor cells by targeting tumor-specific antigens. DNA vaccines have become an attractive approach for generating antigen-specific immunotherapy. The naked plasmid DNA is safe, stable, has low immunogenicity, simple of delivery, and can be repeatly administered. DNA vaccines can be easily prepared in large scale with high purity. 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 reseach spot for cancer immunotherapy.The identification of tumor specific antigen is the key to induction of specific antitumor immunity. MAGE-3 is one of the tumor-specific antigens, it expresses in a high proportion of melanomas and in many human malignant tumors, including hepatocellular carcinoma,head and neck squamous cell carcinoma, testicular germ cell tumor, breast cancer,non-small cell lung cncer, gastric carcinoma, colorectal carcinoma, etc. MAGE-3 doesn' t express in normal tissues except testis and placenta. So MAGE-3 is regarded as an ideal target for specific antitumor immunotherapy.Recently, immunization with HSP complexes isolated from tumor cells has been shown to be able to induce potent antitumor immunity. The immunogenic HSP-peptidecomplexes can also be reconstituted in vitro by mixing the peptides with HSPs. The HSP-based protein vaccines can also be administered by fusing antigens to HSPs. These experiments demonstrate that HSP-peptide complexes derived from tumor cells,but not from normal tissue, can stimulate tumor specific immunity;the specificity of this immune response is caused by tumor-derived peptides that are bound to the HSPs, not by the HSPs themselves, and the immune response can be induced in mice with MHC either identical or different to the MHC of donor HSPs. These investigation have made HSPs more attractive for use in immunotherapy.In this study, we chose MAGE-3 as a model antigen, and investigated whether MAGE-3 cDNA linking to HSP70 can enhance the potency of DNA vaccines.We compared DNA vaccines containing MAGE-3 fused to human HSP70 with DNA vaccines containing MAGE-3 or HSP70 for their immune response generation and their ability of antitumor immunity against established tumor.Section one Construction of pcDNA3. 1/HSP70, pcDNA3. 1/HSP70-MAGE-3 eukaryotic expression plasmidsObjective: To construct pcDNA3. 1/HSP70, pcDNA3. 1/HSP70-MAGE-3 eukaryotic expression plasmids. Methods: The cDNA fragment encoding HSP70 was amplified by RT-PCR. The PCR product was cloned into pGEM-T vector and was sequenced. The HSP70 cDNA was subcloned into pcDNA3. 1+ and pcDNA3. l/MAGE-3 eukaryotic expression vector. The recombinant plasmids were confirmed by restriction enzyme analysis with Afl II and Kpn I. Results: The size of PCR product of HSP70 gene was 1923bp. Sequencing result revealed the sequence of amplified HSP70 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that the recombinant expression vector pcDNA3. 1/HSP70, pcDNA3. 1/HSP70-MAGE-3 were constructed collectively. Conclusion: The eukaryotic expression plasmids pcDNA3. 1/HSP70, pcDNA3. 1/HSP70-MAGE-3 were constucted successfully.Section two Transfection of recombinant eukaryotic expression plasmids by liposome and their expressions in B16 cells Objective: To identify if the recombinant eukaryotic expression plasmids pcDNA3. 1/HSP70, pcDNA3. l/MAGE-3, pcDNA3. 1/HSP70-MAGE-3 can expression HSP70, MAGE-3, HSP70-MAGE-3 in B16 cells respectively. Methods: The recombinant plasmids were transfected into B16 cells by liposome, the expression of HSP70,MAGE-3 or HSP70-MAGE-3 were checked by RT-PCR, Immunocytochemistry and Western blot. Results: The transcription of HSP70, MAGE-3 or HSP70-MAGE-3 in transfected B16 cells were confirmed by reverse transcripta...
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