摘要
目的:构建cerbB2特异性的核酶(ribozyme)基因及其靶基因的体外转录载体并探讨快速简便的筛选方法.方法:在原设计的核酶RZ1基因的3′端加上新的限制性酶切位点EcoRV,合成修饰后的RZ1基因并将之定向克隆于载体pGEM3Zf(-),经用EcoRV正筛选和XbaI负筛选快速鉴定出重组子并测序鉴定.结果:核酶RZ1基因合成后与pGEM3Zf(-)连接,经用正负筛选法很快鉴定出了重组子并命名为pG3ZRZ1,DNA测序证实合成的核酶基因序列正确并已被准确克隆入pGEM3Zf(-)的BamHI和SalI位点.16kb的RZ1的靶基因也被克隆于pGEM3Zf(-).结论:合成带有与表达载体多克隆位点相匹配的粘末端核酶基因可容易地克隆于载体.正负筛选法能简便、快速、准确地鉴定出含有核酶基因的重组子.
Aim:To construct the in vitro transcription vectors of genes of cerbB2 specific ribozyme and its substrate; to develop a simple and fast way in screening the recombinant vectors Methods: According to the computer design, a specific restriction site EcoR V was added to the 3′ end of the RZ1 genes,then the modified RZ1 genes were synthesized and were cloned into the vector pGEM3Zf(-) The recombinants were first selected by EcoR V/Xba I digestion and were analyzed by automatic sequencing Results: The recombinants including the RZ1 genes were successfully selected by the EcoR V/Xba I digestion and designated pG3ZRZ1 The DNA sequencer analysis proved that the synthesized RZ1 genes were correct A 16 kb long target gene of RZ1 was also cloned into the pGEM3Zf(-) Conclusion:The synthesized ribozyme genes with the flanking sites can be easily cloned into the in vitro transcription vector The screening methods appear to be a simple, fast and accurate way in the identification of interested recombinants
出处
《第四军医大学学报》
1998年第1期25-27,共3页
Journal of the Fourth Military Medical University
基金
国家自然科学基金