表达翻转重组酶的真核载体的构建

Construction of eukaryotic vector expressing flippase recombinase

目的构建表达翻转重组酶（FLP）重组表达的真核载体。方法以质粒pFRT为模板，用聚合酶链反应（PCR）扩增出两端包含了Xbal和BamHI内切酶识别位点的全长FLP基因片段，将该片段插入用NheI和BamHI双酶切后的载体质粒pBK—CMV上，构建出重组质粒pBK—CMV—FLP。经过转化、挑选菌落，PCR鉴定且测序均正确。结果成功构建重组质粒pBK—CMV—FLP。结论构建好的pBK—CMV—FLP质粒为生物工程上删除筛选标记物提供了良好的条件。

Objective To construct a cukaryotic vector expressing flippase recombinase （FLP）. Methods The full - length sequence of FLP gene was PCR - amplified with Xba I and BamH I endonuclease sites added using plasmid pFRT as the template. The sequence was digested with Xba I and BamH I and then inserted into Nhe I and BamH I - di- gested pBK - CMV vector, to construct the recombinant eukaryotic expression vector pBK - CMV - FLP. After trans- formed into E. coli and selected single colony, the expected FLP - expressed vector was confirmed by PCR, restriction analysis and sequencing. Results The FLP - expressed recombinant plasmid vector pBK - CMV - FLP were constructed successfully. Conclusion The recombinational plasmid pBK - CMV - FLP will provide a useful tool for marker gene de letion in biological engineering.