【摘要】 目的:构建以GFP做标志基因的放射敏感基因Egr-1调控单纯疱疹病毒胸苷激酶基因(herpssimplexvirusthymidinekinase,TK)的腺病毒载体。方法:以PCl-neo-Egr-1-TK(pET)为模板扩增Egr-1,插入到pAdTrack的XbaI和XhoI位点,构建重组质粒pAdTrack/Egr-1;与pET酶切获得TKcDNA相连,构成pAdTrack/Egr-1-TK;用PmeI酶切后与pAdEasy-1混合,应用细菌内同源重组法获得重组表达质粒pAdEgr-1-TK。结果:重组表达质粒pAdEgr-1-TK的酶切鉴定符合预期结果,感染肿瘤细胞可见绿色荧光表达。结论:通过细菌内同源重组成功构建含GFP做标志基因的pAdEgr-1-TK重组表达质粒,为研究Egr-1调控自杀基因及对肿瘤细胞进行灭活打下了基础。更多还原

【Abstract】 Objective:To construct adenoviral vector carrying the TK suicide gene regulated by Egr-1 promoter, containing a green fluorescent protein(GFP) gene incorporated into the adenoviral backbone. Methods:Egr-1 was amplified by polymerase chain reaction from pET.Egr-1 gene was inserted into pAdTrack to construct pAdTrack/Egr-1.pAdTrack/Egr-1 was joined in TKcDNA from pET to construct pAdTrack/Egr-1-TK.Adenoviral vecter of pAdEgr-1-TK was generated through homologous recombination in bacteria. Results:Restriction endonucleases digestion of pAdEgr-1-TK was the same as expected.The green fluorescent was observed in carcinoma cells.Conclusion:The construction of pAdEgr-1-TK is successful,which would benefit the studies on the TK suicide gene regulated by irradiation via Egr-1 promoter.更多还原