Gene expression of prostate-specific membrance antigen

Abstract

Objective The aim of this research is to clone cDNA of human prostate-specific membrane antigen(PSMA),construct pcDNA3-PSMA eukaryotic expression vector, and then identify the expression of PSMA protein. We hope this experiment will give a good basis on the research of the prostate therapy based on DC vaccine. Methods Total RNA was extracted from prostate cancer tissues. A pair of primers were designed according to M99487 in Genebank and obtain the PSMA cDNA fragment by reverse transcription-polymerase chain reaction. The PCR products were then cloned into empty plasmid pcDNA3.Further sequence analysis was carried out and the recombinant plasmids PcDNA3.0-PSMA were transfected into PC-3 cells. In G418 selective medium(400μg/ml),two positive clones expressing PSMA mRNA, named D4 and B3 respectively, were identified by RT-PCR. Using rabbit anti-human PSMA monoclonal antibody, we identified the location of expressed PSMA protein by indirect immunofluorescence analysis. To verified the molecular weight of expressed PSMA protein, western blotting analysis of total cell extracts was carried out using chemiluminescence western blotting kit. Results Sequence analysis confirmed the homogeneity between cloned PSMA cDNA and M99487 was 99.7%.After transfection. two cell clones expressing PSMA mRNA were identified by RT-PCR.The indirect immunofluorescence assay confirmed that expressed PSMA protein was located on membrane and had good immunoreactivity. Western blotting analysis showed the molecular weight of expressed PSMA was 100kD.as was PSMA protein from LNCaP cells. Conclusion We have obtained the PSMA cDNA from prostate cancer tissues. The PcDNA3.0-PSMA eukaryotic expression vector was constructed and the target PSMA protein which had immunoreactivity expressed on cell membrane. The molecular weight of expressed PSMA was 100kD,the right molecular weight as we expected. Our research layed foundation for treatment of prostate neoplasms by DC vaccines transfect with PSMA. Key words: Prostate; Antigeson