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构建一种带有绿色荧光蛋白基因（ ｇｆｐ）和 ｈｙｔｋ ｃＤＮＡ（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中的转染效率。利用脑炎心肌炎病毒 （ｅｎｃｅｐｈａｌｏｍｙｏｃａｒｄｉｔｉｓ ｖｉｒｕｓ， ＥＭＣＶ）的内部核糖体进入位点（ ｉｎｔｅｒｎａｌ ｒｉｂｏｓｏｍｅ ｅｎｔｒｙ ｓｉｔｅ， ＩＲＥＳ），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，并检测其表达情况。结果示该载体在瞬时表达及稳定表达时均可获得ｈｙｔｋ 及 ｇｆｐ的良好表达。说明含 ｇｆｐ和 ｈｙｔｋ基因双顺反子真核表达载体的构建成功，为临床应用自杀基因治疗膀 胱恶性肿瘤提供了新的治疗工具及随访检测手段。
A novel eukaryotic expression vector with green fluorescent protein (gfp) and hytk cDNA (fusion gene of hygromycin phosphotransferase and HSV-tk) was constructed for the simple and rapid understanding of suicide genes in malignant tumors Transfection efficiency. Recombinant plasmids containing gfp and hytk genes were constructed by recombining the gfp cDNA with the hytk eukaryotic expression vector using the internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV) Lipofectin were transfected COS-7 cells and human bladder cancer cell line EJ, and detect the expression. The results showed that the vector can obtain good expression of hytk and gfp both in transient expression and stable expression. The successful construction of bicistronic eukaryotic expression vector containing gfp and hytk genes provided a new therapeutic tool and follow-up test for the clinical application of suicide gene in treatment of bladder cancer.