Amplification And Eukaryotic Expression Of Human Prostate Tumour Associated Antigen Gene

Prostate cancer is the common malignancy in men and surpassed lung cancer as the most common cancer in U.S.men.Prostate cancer is also a significant and growing health problem in the Chinese population.In this study,prostate tumour associated antigens:PSCA,PAP,PSMA gene was specifically amplified and cloned into the eukaryotic expression plasmid,which will provide a solid foundation for research of prostate cancer vaccine.Objective:To amplify human PSCA,PAP,PSMA genes and construct the eukaryotic expression plasmids express human prostate tumor associated antigens.Methods:The genes of PSCA,PAP,PSMA were amplified by RT-PCR from human prostate cancer cell line LNCaP,and then cloned into pIRES-neo eukaryotic expression vactor to construct the pIRES-neo-PSCA-His,pIRES-neo-PAP-His, pIRES-neo-PSMA-His plasmids.Under the mediation of lipofectamine,the plasmids were transfected into COS-7 cells,and then detected protein expression by flow cytometry.Then pIRES-neo-PSCA-His plasmid was transfected B16 cells and preliminarily selected by G418.The transcription of PSCA mRNA and the expression of protein in B16 cells were respectively detected by RT-PCR and flow cytometry as well as immunofluorescence assay.Results:The target genes:PSCA,PAP and PSMA were obtained and identical with the sequences of GenBank.The plasmids were constructed and transfected into cells successfully.The eukaryotic expression was approved.The pIRES-neo-PSCA-His plasmid was successfully transfected B16 cells and preliminarily selected by G418.The expression of protein was confirmed.Conclusion:The eukaryotic expression plasmids ware constructed successfully. After preliminarily selected by G418,these constructed plasmids were proved that can be used to establish transgenic cell lines,which will contribute to the functional evaluation of prostate cancer vaccine.