内部核糖体进入位点控制hytk基因和绿色荧光蛋白的表达

构建一种带有绿色荧光蛋白基因（ｇｆｐ）和ｈｙｔｋｃＤＮＡ（潮霉素磷酸转移酶和ＨＳＶ－ｔｋ的融合基因）的新型真核表达载体，用于简便、快速地了解自杀基因在恶性肿瘤听转染效率。利用脑炎心肌炎病毒的内部核糖体进入位点，将ｇｆｐ的ＣＤＮＡ与ｈｙｔｋ真核表达载体重组，构建获得含ｇｆｐ和ｈｙｔｋ基因的重组质粒；Ｌｉｐｏｆｅｃｔｉｎ介导下分别转染ＣＯＳ－７细胞及人膀胱癌细胞株ＥＪ，并检测表达情况。结果示该载体在

Expression of hytk cDNA and green fluorescent protein gene regulated by an internal ribosome entry site

To facilitate the suicide gene delivery into neoplasms, a bicistronic eukaryotic vector carring gfp and hygromycin phosphotransferase-thymidine kinase fLlsion (hytk) gene was constructed. The internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV) which could coordinate expression of two genes in a single vector was optioned. By using Liposollleunlediated transfection, eukaryotic expression vector tgCMV/hytk-IRES-gfP was transfected into COS7 cell and human bladder carcinoma cells EJ. The results of PCR and microscopy detection show that the hytk-IRES-GFP gene was successfully transferred into COS7 cell and EJ cells. There were no differences in the growth pattern or the morphology between EJ and EJ/hytk-IRES-GFP cells. In vitro experiments demonstrated dose- and time-dependent cell killing by transduction of the hytk-IRES-gfP gene followed by GCV treatment. The IC50 (the concentration required to elicit 50% growth inhibition) was 2.16 mg/L in teatment with 72 hours. Results suggest that this new kind of eukaryotic vector could serves as a new tool and method for bladder neoplasms therapy gene.