反向克隆法大鼠anti-ERK2腺病毒载体构建

Construction of recombinant adenoviral vector carrying rat anti-ERK2 gene by reverse orientation clone technology

目的正义基因反向克隆法构建大鼠anti-ERK2腺病毒载体。方法酶切质粒p3XFLAG-CMV7.1-ERK2,得到ERK2cDNA片段,连接到T载体进行测序,经测序正确反向克隆法插入质粒pShuttle中,最后转入AdEasy(tm)XLAdenoviralVectorSystem系统中得到anti-ERK2基因腺病毒载体。结果DraI和NotI双酶切鉴定anti-ERK2基因腺病毒载体,发现插入的基因序列与插入方向均符合预期目标。结论成功构建了大鼠anti-ERK2腺病毒载体,为研究移植排斥中ERK2基因治疗的作用提供了良好工具。

[Objective] To construct recombinant adenoviral vector carrying the anti-ERK2 gene of rat. [Methods] The plasmid of p3XFLAG-CMVT.1-ERK2 was digested with enzymes, obta/ned fragment of ERK2cDNA, then connected it to T-vector. After sequencing, it was reversely inserted into the plasmid of pShuttle, at last transcribed it to AdEasy（tm） XL Adenoviral Vector System. [Results] The plasmid of pshuttle-anti-ERK2 was digested with DraI and NotI enzymes, and found that the sequence and transcription orientation were identical with expectation. [Conclusion] Recombinant adenoviral vector carrying the anti-ERK2 gene been constructed successfully by reverse orientation cloning the target fragment into pshutfle. It provided tools for researching the role of anti-ERK2 gene therapy in graft rejection.