体外转录系统质粒pNI-11和pNI-12的构造

Construction of in vitro transcription system plasmids p^(NI-11)and p^(NI-12)

本文介绍将大鼠心房肽(ANP)前体的互补DNA(proANP—cDNA)的PstI片段再克隆到具有双向转录启动子的质粒pGEM—2中,构建了体外转录系统的质粒pNI—11和pNI—12。用限制性內切酶AccI消化重组的质粒,结合电泳比较酶谱,区别出插入段cDNA的方向。用这种质粒可以体外合成高灵度的探针,可供基因调控、原位杂交等多方面研究。判断插入基因在重组质粒中方向的方法有普遍意义。

In order to construct a plasmid that can be transcripted in vitro to prepare ribo-probe, the proANP-cDNA was subcloned into Pst I site of plasmid pGEM-2 .The proANP-cDNA was digested from pANP3A with Pst I , and pGEM-2 was also digested with Pst I .After digestion, the plasmids were furthermore (5 '-p) dephosphated with alkaline phosphatase. The inserted fragment and vector were recombined with T4 lagase. DH-1 E.coli were transformed by the recombined plasmids. The authors selected more than ten colonies and had their plasmids digested with Pst I , and thus, most of them were new plasmids with Pst J insert. To determine the orientation of the insert in recombined plasmids, the positive plasmids were then digested with Acc I , thus some of them had nearly 500 bp pieces, and these were designated as pNI-11, in which the antisense strand of proANP-cDNA was subcloned into SP6 RNA polymerase coding strand. After linearizing of the plasmids, the ribo-probe could be synsthesized. Other plasmids,with a fragment of about 100 bp pieces were designated as pNI-12, in which the antisense strand of proANP -cDNA was combined into T7 RNA polymerase coding strand. The strategy to determine the orientation is simple and useful for transcription system.