摘要
目的 :构建一种带有绿色荧光蛋白 (gfp)、潮霉素磷酸转移酶和HSV -tk的融合基因cDNA(hytk)的新型真核表达载体。方法 :利用脑炎心肌炎病毒 (EMCV)的内部核糖体进入位点 (IRES) ,将gfp的cDNA与hytk基因连接 ,构建获得含gfp和hytk基因的重组质粒 ;lipofectin介导下转染人膀胱癌细胞株EJ,并检测其表达情况。结果 :成功地构建了含gfp和hytk基因双顺反子真核表达载体 ,PCR检测证实转基因细胞的gfp与hytk基因的整合 ,荧光显微镜下可观察到发绿色荧光的转基因细胞 ,MTT法测定示丙氧鸟苷 (GCV)对EJ/hytk -gfp细胞有明显的杀伤作用 ,此杀伤效应呈时间和剂量依赖性 ,72hIC5 0值为 2 16mg/L ,而对野生型的EJ细胞无明显毒性 (P <0 0 5 )。结论
AIM: To facilitate the suicide gene delivery into neoplasm, a chimeric gene of HSV-tk and green fluorescent protein (gfp) was constructed. METHODS: Molecular cloning technique was used to construct this kind of eukaryotic vector. 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 liposome-mediated transfection, eukaryotic expression vector tgCMV/hytk-IRES-gfp was transfected into human bladder carcinoma cells EJ. RESULTS: A bicistronic eukaryotic vector carrying gfp and hygromycin phosphotransferase-thymidine kinase fusion (hytk) gene was constructed. The results of PCR and microscopy detection show that the hytk-IRES-gfp gene was successfully transferred into EJ cells. There were no differences in the growth pattern or the morphology between EJ and EJ/hytk-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 treatment with GCV for 72 hours. CONCLUSION: These results suggest that this new kind of eukaryotic vector could serves as a new tool and method for neoplasm gene therapy.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2001年第9期825-829,共5页
Chinese Journal of Pathophysiology
基金
福建省科委科研基金资助 (No .99-Z - 16 7)
