| PSCA(Prostate stem cell antigen, PSCA), which contains 123 amino acids, is a surface glycoprotein belonging to Thy-1/Ly-6 family. Studies have shown that PSCA is low-expressing in normal prostate and high-expressing in prostate cancer,bladder cancer and pancreatic cancer; Meanwhile, the antibody of PSCA can inhibit the growth of prostate cancer. With the suitable adjuvant molecule, recombinant protein vaccine based on PSCA may probably has a positive effect on the therapy of prostate cancer. However PSCA can hardly be expressed in soluble condition in E.coli because of its complicated spatial structure, which make it difficult to develop the vaccine for treating prostate cancer. So how to enhance the soluble expression of PSCA and elevate the immunogenicity of PSCA is key point. Construction of a mouse model of human prostate cancer for evaluating the immune effect of vaccine is our main destination, too.In this study, HSP70 was chosen as adjuvant molecule to be coupled with PSCA to construct recombinant protein vaccine for prostate cancer treatment. Different fusion genes were constructed to investigate whether they could increase the soluble expression of PSCA and decrease the antigenicity of HSP70 with the activity of enhancing the immunoresponse.Firstly, the PSCA and HSP70 genes were amplified by PCR. PSCA(T), only containing nucleotides encoding the active structural domain, and PSCA(FL) were separately cloned to pET21a(+) vector with HSP70 to construct recombinant plasmids pET21-PSCA(T)-HSP, pET21-PSCA(FL)-HSP, pET21-HSP-PSCA(T) and pET21-HSP-PSCA(FL). Only PSCA(T)-HSP was comfirmed to be expressed in soluble condition in E.coli by SDS-PAGE electrophoresis analysis. Furthormore, for reducing the antigenicity of HSP70, HSP70 was devided into HSPI and HSPII to be constructed into recombinant plasmids pET21-PSCA(T)-HSPI and pET21-PSCA(T)-HSPII with PSCA(T). Both of them were confirmed to be expressed in soluble condition in E.coli by SDS-PAGE electrophoresis analysis. In this study, PSCA(T)-HSP,PSCA(T)-HSPI and PSCA(T)-HSPII were chosen as candidates for prostate cancer treatment.For evaluating the immune effect of recombinant protein, a traditional mouse model of prostate cancer was constructed. RM-1 cells were transfected stably with pcDNA-PSCA, then they were enoculated to C57BL/6 mice. Results indicated that tumor was observed in all the mice and the mean survival time of mice was 37 days. So the PSCA-expressing cancer model in mice was successfully established.However, the growth,metastasizing of the tumor tissue and the time expression and spatiality expression of gene are unable to be observed through the traditional animal model. Besides animals have to be killed to get experimental data. So prostate cancer animal model with luciferase expression was constructed to evaluate the immune effect of recombinant protein.The RM-1 cells with Luc expression and human PSCA expression were enoculated to C57BL/6 mice and the prostate cancer animal model with luciferase expression was constructed successfully. Results indicated that tumor was observed in all the mice and the mean survival time of mice was 38 days. Tumor cells with luciferase gene grew stabley. Luc expression in mice could be detected by in vivo bioluminescence imaging and the activity of luciferase could reflect the size of tumor. In conclusion, the prostate cancer animal model with luciferase expression is a bettetr choice to evaluate the immune effect of vaccine, which lands the foundation for development of vaccine. |
PDF Full Text Request