c-erbB-2特异性核酶基因体外转录载体的构建与快速鉴定

Construction and fast identification of in vitro transcription vector of cerbB2 specific ribozyme genes

目的：构建ｃｅｒｂＢ２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ｐＧ３ＺＲＺ１，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．１６ｋｂ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 cerbB2 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 pG3ZRZ1 The DNA sequencer analysis proved that the synthesized RZ1 genes were correct A 16 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 