Basic Research On A Replication-competent DNA Vaccine PSCK-PSCA3 For Anti-prostate Cancer

Background and Objective:Human prostate stem cell antigen(PSCA) is highly expressed in advanced prostate cancer and metastasis, and is closely related to the genesis and progression of prostate cancer, thus has caught much attention in the diagnosis and treatment of the disease. In this research, we intend to choose PSCA as a potential target for the immunotherapy of prostate cancer and construct a replication-competent DNA vaccine pSCK-PSCA3 based on Semliki Forest Virus(SFV) replicon vector pSCK. To evaluate the function of the vaccine, we expressed a PSCA fusion protein in E.coli BL21 and established a stably transfected B16 cell line which can highly express PSCA. This research has provided a solid experimental foundation for the function evaluation of the vaccine pSCK-PSCA3, and has offered a novel strategy for the immunotherapy of prostate cancer.Methods:(1)The fragment of PSCA was cloned into prokaryotic expression vector pET42a which contains a glutathione s-transterase(GST) tag. Following the double restriction enzyme digestion analysis, the recombinant plasmid pET42a-PSCA was transformed into E.coli BL21(DE3). GST-PSCA fusion protein was expressed under IPTG, and purified by passing over Glutathione Sepharose 4B column. The purified fusion protein was identified by SDS-PAGE and Western Blot.(2)The full length of PSCA cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector pcDNA3.1. The recombinant plasmid pcDNA3.1-PSCA was identified by double restriction enzyme digestion and DNA sequencing. Then the plasmid pcDNA3.1-PSCA was transfected into B16 cells by lipofection. After screening culture by G418, a stably transfected B16 cell line was established. The expression of PSCA gene was identified by Flow Cytometry, Immunofluorescence and Western Blot. (3)The genetic fragment sig-PSCA3-Fc-GPI-GM/B7 was acquired from pVAXl-PSCA3-FcGB plasmid, then the fragment was cloned into the SFV replicon vector pSCK to construct the final DNA vaccine pSCK-PSCA3. The expression of the vaccine in eukaryote cells was identified by Flow Cytometry and Immunol Histochemistry.Results:(1) The prokaryotic expression plasmid pET42a-PSCA was successfully constructed. The GST-PSCA fusion protein was expressed effectively and its relative molecular weight was 43kD analyzed by SDS-PAGE and Western Blot.(2) The eukaryotic expression plasmid pcDNA3.1-PSCA was successfully constructed. A stably transfected B16 cell line was established and the expression rate of PSCA gene was virtually 100 percent.(3) The replication-competent DNA vaccine pSCK-PSCA3 was successfully constructed. The DNA vaccine can effectively express in eukaryotic cells analyzed by Flow Cytometry and Immunol Histochemistry.Conclusion:The PSCA fusion protein is expressed effectively in the BL21 strains. The established B16 cell line can highly express PSCA gene. A new type replication-competent DNA vaccine pSCK-PSCA3 was successfully constructed. The research has provided a solid experimental foundation for further studies on the function of the vaccine.