pSUPER-2H1重组双靶向干扰载体的构建及鉴定  

The Construction and Evaluation of Recombinant pSUPER-2H1 Plasmid Vector

作　　者：王辉[1] 张义军[1] 陈伟娟[2] 

机构地区：[1]潍坊医学院药理学教研室,山东潍坊261053 [2]潍坊医学院病理学教研室

出　　处：《潍坊医学院学报》2009年第5期356-358,共3页Acta Academiae Medicinae Weifang

基　　金：山东省自然科学基金资助课题(课题编号:Y2008C75)

摘　　要：目的构建含有双H1启动子的重组pSUPER-2H1质粒载体,为进一步构建同时干扰LRP和P-gp基因的RNAi载体做基础。方法以pSUPER质粒中原有的H1启动子为模板PCR扩增H1启动子,将扩增的H1启动子连接到Pumc-T载体上,重组的质粒载体命名为Pumc-T-H1。将pSUPER质粒与Pumc-T-H1利用xho Ⅰ和sal Ⅰ进行双酶切,将两者的酶切产物连接后转化大肠杆菌DH5α,抽提后的重组质粒载体命名为pSUPER-2H1,并对其进行酶切鉴定和测序。结果经酶切鉴定及测序证实克隆入pSUPER质粒的H1启动子与pSUPER质粒中原有的H1启动子序列一致,从而成功构建了pSUPER-2H1重组质粒。结论 pSUPER-2H1重组质粒的构建为下一步LRP和P-gp基因双干扰RNAi重组质粒的构建奠定了基础。Objective To construct the recombinant pSUPER-2H1 plasmid with double Hlpromoter in order to lay a foundation for further building interference LRP and P-gp gene RNAi vector. Methods HI promoter was amplyfied by polymerase chain reaction （ PCR ） utilizing the H1 promoter containing pSUPER plasmid as template and joining Taq DNA polymerase. The purified products of H1 promoter was ligated with Pumc-T vector and then it was transformed into DH5ct. The white colne was selected to be amplified and identified. The recombinant plasmid was named Pumc-T-Hl. pSUPER plasmid and H1 promoter were further double-enzyme digested by xho Ⅰ and sal Ⅰ . Respectively,the purified linear fragment of the former and the purified 250bp DNA fragment was ligated and transfered into DH5α. The recombinant plasmid was named after pSUPER-H1 ,which was identified by double-enzyme digested and sequenced. Results The H1 promoter was cloned into pSUPER plasmid,which was identified by restriction enzyme analysis and sequencing. The H1 promoter was coincided with the one pos- sessed by pSUPER plasmid formerly. Conclusion The construction of recombinant pSUPER-H1 plasmid vector laid the foundation for the construction of LRP and P-gp double interference RNAi recombinant plasmid.

关 键 词：RNA干扰 基因重组 基因治疗 

分 类 号：R730[医药卫生—肿瘤]