Research Of Recombinant Protein Vaccine And Recombinant Adenovirus Vector Vaccine For Prostate Cancer Based On Prostate Stem Cell Antigen

In view of the prophase study of therapeutic DNA vaccine for prostate cancer based on PSCA, in this study, HSP70 was chosen as adjuvant molecule to be coupled with PSCA to construct recombinant protein vaccine and recombinant adenovirus vector vaccine for prostate cancer treatment, expecting to increase the soluble expression of PSCA with the activity of enhancing the immune response. Furthermore, combined vaccines were inoculated to C57BL/6 mice on account of the immune strategy of "prime-boost". The immune responses and therapeutic efficacy of these vaccines based on PSCA were evaluated in mice model, which lays the foundation for development of vaccines for prostate cancer and clinical research.For construction of recombinant protein vaccine, the PSCA and HSP70 genes were amplified by PCR. PSCA, only containing nucleotides encoding the active structural domain, was cloned into prokaryote expression vector pET21a(+) with HSP70 gene to construct recombinant plasmid pET21-PSCA-HSP. PSCA-HSP was confirmed to be expressed in soluble condition in E.coli by SDS-PAGE electrophoresis analysis. For evaluating the immune effect of the recombinant protein vaccine, PSCA,HSP70 and PSCA-HSP were expressed in E.coli system and purified with chromatography methods. After immunization with these recombinant protein, the PSCA-specific cellular and humoral immune responses were detected by using ELISPOT assay, intracellular cytokines staining assay and ELISA assay. The tumor growth and survival of vaccined mice were observed. The result showed that the mice vaccinated with recombinant protein PSCA-HSP could induce a PSCA-specific cellular immune response. Meanwhile, the mice vaccinated with recombinant protein PSCA+HSP,PSCA and PSCA-HSP could induce a PSCA-specific humoral immune response. They all generated high level of anti-PSCA antibody without difference. Furthermore, recombinant protein PSCA+HSP,PSCA and PSCA-HSP could inhibit the growth of PSCA-expressing tumors and prolong the mice's survival. Meanwhile, PSCA-HSP could obtain the optima antitumous effect.After being obtained from pcDNA-PSCA plasmid and pcDNA-PSCA-HSP plasmid by digesting with restriction enzyme, PSCA gene and fusion gene PSCA-HSP were separately cloned into pDC316 shuttle vector to construct recombinant shuttle plasmids pDC316-PSCA and pDC316-PSCA-HSP. Recombinant shuttle plasmids were packed with recombinant adenovirus, then recombinant Ad5-PSCA and Ad5-PSCA-HSP were prepared. For evaluating the immune effect of recombinant adenovirus vector vaccine, ELISPOT assay, intracellular cytokines staining assay and ELISA assay were used to detect the PSCA-specific cellular and humoral immune responses. The tumor growth and survival of vaccined mice were observed. The result showed that the mice vaccinated with Ad5-PSCA and Ad5-PSCA-HSP both could induce a strong PSCA-specific cellular immune response. However, the level of anti-PSCA antibody was low among these groups. Furthermore, they both could inhibit the growth of PSCA-expressing tumors and prolong the mice's survival. Meanwhile, Ad5-PSCA-HSP could obtain the optima antitumous effect.On account of the immune strategy of "prime-boost", in this study, recombinant protein vaccine and recombinant adenovirus vector vaccine were inoculated to C57BL/6 mice respectively. The PSCA-specific cellular and humoral immune responses were detected by using ELISPOT assay, intracellular cytokines staining assay and ELISA assay. The tumor growth and survival of vaccined mice were observed. The result showed that the mice vaccinated with vaccines could induce a PSCA-specific cellular and humoral immune response. Furthermore, all these vaccines could inhibit the growth of PSCA-expressing tumors and prolong the mice's survival. Meanwhile, Ad5-PSCA-HSP+PSCA-HSP could obtain the optima antitumous effect.In this study, the recombinant Ad5-PSCA-HSP and the recombinant protein PSCA-HSP were successfully constructed. Cellular and humoral immune responses induced by different vaccines were evaluated in mice, which lays the foundation for development of vaccines for prostate cancer and clinical research.