摘要
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。
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.
出处
《基础医学与临床》
CSCD
2000年第4期31-36,共6页
Basic and Clinical Medicine
基金
福建省科委科研基金!(99-Z-167)
关键词
HYTK基因
基因治疗膀胱肿瘤
IRES
gfg
green flurescent protein
thymidine kinase
internal ribosome entry site
gene therapy
