Construction of PSMAª²IRESª²FCY1ª²TK eukaryotic expression vector and its expression in prostate cancer cell line

    YIN Ying1, LU Fan2, SU Mingª²Quan1, HAO Xiaoª²Ke1

    1Department of Clinical Laboratories£¬Xijing Hospital, 2Department of Biochemistry & Molecular Biology, school of Basic Medicine.Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To construct the eukaryotic expression vector that specifically drives FCY1/TK double suicide gene by prostateª²specific membrane antigen promoter(PSMAP)and to investigate the toxic effects of single or double gene on transfected prostate cancer cell line LNCaP after prodrug treatment.METHODS: Genomic DNA was extracted from benign prostate tissues and prostateª²specific membrane antigen promoter(PSMAP) fragment was obtained by polymerase chain reaction (PCR).The pCMV promoter on eukaryotic expression vector pIRES was replaced by PSMA promoter and PSMAPª²IRES vector was obtained. The recombinant plasimd PSMAPª²IRES was identified by restriction analysis and DNA sequence. Then the gene FCY1 and TK was cloned into corresponding sites of PSMAª²IRES and the pPSMAª²IRESª²FCY1ª²TK eukaryotic expression vector was obtained.Lipofection- mediated genetransfers were performed with the obtained vector and a control plasmid, pIRES, in human prostate cancer cell line LNCaP cells.RTª²PCR was used to demonstrate successful transcription.The toxic effects of 5ª²FC, GCV, and both of them on transfected LNCaP cells were explored by MTT assay.RESULTS: A 1400 bp DNA fragment was amplified with PCR.Sequence and restriction analysis showed that PSMA promoter by amplified was identical with that of GenBank reported(GenBank accession number AF007544). The expression of suicide genes TK and FCY1 could be detected by RTª²PCR.The growth inhabition rate (GIR) in double gene group were higher than that of single gene.(P<0ª±05)CONCLUSION: pPSMAª²IRESª²FCY1ª²TK double suicide gene system has significant toxic effect on LNCaP cells in vitro, which was superior. ......