Construction of eukaryotic expressing plasmid of human PSCA and establishment of stably transfected B16 cell line

Objective:To construct the eukaryotic expressing plasmid pcDNA3.1-PSCA and establish a stably transfected B16 cell line which expresses human prostate stem cell antigen(PSCA) with high efficacy.Methods: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.Results:The eukaryotic expressing 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.Conclusion:The established B16 cell line can highly express PSCA gene,which has provided a solid experimental foundation for further studies on the function of the anti-prostate cancer vaccine.