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目的 构建hTERTPromoter/HSV tk靶向性胶质瘤基因治疗系统。方法 采用TSS法在大肠杆菌JM 10 9中扩增 pGL2 hTERTPromoter和STK质粒。DNA提取纯化试剂盒提取细菌中生长的质粒 ,经电泳 ,紫外分光光度计检验后用HindⅢ ,ClaⅠ核酸内切酶对这两个质粒同时进行双酶酶切。琼脂糖电泳凝胶分离酶切片段 ,切取其中 8.5 ,1.7kbp两个片段长度的凝胶。凝胶DNA提取试剂盒提取其中所含DNA片断 ,T4DNA连接酶连接这两个片段 ,琼脂糖凝胶电泳检测连续结果。结果 通过连接反应获得 10 .2kbp的重组DNA片段。结论 成功构建hTERTPromoter/HSV tk重组表达单位。
Objective To construct hTERTPromoter / HSV tk targeted glioma gene therapy system. Methods The pGL2 hTERTPromoter and STK plasmids were amplified by using the TSS method in Escherichia coli JM109. DNA extraction and purification kit for the growth of bacterial plasmid extraction, electrophoresis, UV spectrophotometer test with Hind Ⅲ, Cla Ⅰ endonuclease digestion of these two plasmids simultaneously. The agarose gel was electrophoresed on gel to separate the fragments and the gel was cut between two fragment lengths of 8.5 and 1.7 kbp. Gel DNA extraction kit to extract the DNA fragments contained therein, T4 DNA ligase to connect the two fragments, agarose gel electrophoresis to detect continuous results. As a result, a l.2 kbp recombinant DNA fragment was obtained by ligation reaction. Conclusion The hTERTPromoter / HSV tk recombinant expression unit was successfully constructed.