含hTRAIL及LUC基因的腺病毒双表达载体的构建

发布时间：2019-01-14 11:19

【摘要】：目的: 构建含人肿瘤坏死因子相关调亡诱导配体(human TNF-Related Apoptosis Inducing Ligand,hTRAIL)和萤火虫荧光素酶(firefly luciferase,LUC)的腺病毒载体,为用影像学方法在活体上间接监测治疗基因hTRAIL 的表达奠定基础。方法: 用Bgl II 和XbaI 酶切质粒PDC-LUC 后,回收1700bp 大小LUC 的DNA 片段;用BamH I 和XbaI 酶切含内核糖体进入位点( interal ribosoe entry site, IRES)基因的质粒Pclon5 后,回收其线性化片段。用T4 DNA 连接酶连接上述两片段,LUC 基因就插入到Pclon5 的IRES 基因的下游,命名为Pclon5-LUC。Pclon5-LUC 用Kpn I 和Xba I 酶切鉴定。将Pclon5-LUC 用Bgl II 和XbaI 酶切,回收IRES-LUC基因,与用BamHI/SpeI 酶切后线性化的Pclon9 连接,获得含多克隆位点的Pclon9.5-LUC,先用EcoR I 与Age I 酶切鉴定,再用Sca I 酶切鉴定。 Pclon9.5-LUC 用EcoRI 和Xho I 酶切,回收含多克隆位点IRES-LUC基因片段。PDC312-CMV用EcoRI和Sal I酶切,回收线性化片段,将含多克隆位点的IRES-LUC 基因片段插入到PDC312-CMV 中两个绝缘子中间, 即PDC312-CMV-9.5-LUC,用Pst I 酶切鉴定。将PDC312-CMV-9.5-LUC 用EcoRI 和SalI 酶切,回收线性化片段;PDC-humanTRAIL 用EcoRI 和SalI 酶切,回
[Abstract]:Aim: to construct adenovirus vector containing human tumor necrosis factor-related apoptosis inducing ligand (human TNF-Related Apoptosis Inducing Ligand,hTRAIL) and luciferase (firefly luciferase,LUC) of firefly. To provide a basis for indirect monitoring of therapeutic gene hTRAIL expression in vivo by imaging methods. Methods: after the plasmid PDC-LUC was digested with Bgl II and XbaI, the DNA fragment of the 1700bp size LUC was recovered, and the plasmid Pclon5 containing the internal ribosome entering the (interal ribosoe entry site, IRES) gene was digested with BamH I and XbaI, and the linearized fragment was recovered. The LUC gene was inserted into the downstream of the IRES gene of Pclon5 with T4 DNA ligase, and named Pclon5-LUC.Pclon5-LUC was digested by Kpn I and Xba I. Pclon5-LUC was digested with Bgl II and XbaI, and the IRES-LUC gene was recovered and ligated with the linearized Pclon9 digested by BamHI/SpeI. The Pclon9.5-LUC, containing polyclonal sites was identified by EcoR I and Age I restriction digestion, and then by Sca I restriction digestion. Pclon9.5-LUC was digested with EcoRI and Xho I, and polyclonal site IRES-LUC gene fragment was recovered. PDC312-CMV was digested with EcoRI and Sal I enzyme, and linear fragment was recovered. The IRES-LUC gene fragment containing polyclonal sites was inserted into the two insulators in PDC312-CMV, that is, PDC312-CMV-9.5-LUC, was identified by Pst I digestion. PDC312-CMV-9.5-LUC was digested with EcoRI and SalI, and linearized fragments were recovered. PDC-humanTRAIL was digested with EcoRI and SalI.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

【共引文献】