PSMAIRESFCY1TK真核表达载体的构建及在前列腺癌细胞中的表达
关键词:前列腺肿瘤,,前列腺特异性膜抗原;,FCY1;,HSVTK;,前列腺肿瘤,0引言,1材料和方法,2结果,3讨论,【
Construction of PSMAIRESFCY1TK eukaryotic expression vector and its expression in prostate cancer cell line
YIN Ying1, LU Fan2, SU MingQuan1, HAO XiaoKe1
1Department of Clinical Laboratories,Xijing Hospital, 2Department of Biochemistry & Molecular Biology, school of Basic Medicine.Fourth Military Medical University, Xian 710033, China
【Abstract】 AIM: To construct the eukaryotic expression vector that specifically drives FCY1/TK double suicide gene by prostatespecific 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 prostatespecific 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 PSMAPIRES vector was obtained. The recombinant plasimd PSMAPIRES was identified by restriction analysis and DNA sequence. Then the gene FCY1 and TK was cloned into corresponding sites of PSMAIRES and the pPSMAIRESFCY1TK 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.RTPCR was used to demonstrate successful transcription.The toxic effects of 5FC, 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 RTPCR.The growth inhabition rate (GIR) in double gene group were higher than that of single gene.(P<005)CONCLUSION: pPSMAIRESFCY1TK double suicide gene system has significant toxic effect on LNCaP cells in vitro, which was superior. ......
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