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目的构建小鼠T—bet基因重组真核表达载体pcDNA3一T—bet,为T—bet基因治疗研究提供有效的生物表达系统。方法采用内切酶切从质粒T—bet/GFP—RV中获得约1.7 kb的小鼠T—betcDNA片段,以单酶切非定向克隆方式与真核表达质粒pcDNA3连接,构建真核表达载体pcDNA3-T-bet。结果经PCR筛选阳性重组子,双酶切鉴定T—bet基因重组方向,并测序鉴定无错配及插入移位等DNA顺序改变。结论本实验成功构建了小鼠T—bet基因真核表达载体pcDNA3-T-bet。
Objective To construct a recombinant T-bet eukaryotic expression vector pcDNA3-T-bet, which provides an effective biological expression system for T-bet gene therapy. Methods T-bet cDNA fragment of about 1.7 kb was obtained from the plasmid T-bet / GFP-RV by restriction endonuclease digestion and ligated with the eukaryotic expression plasmid pcDNA3 in a single digested non-directional cloning manner to construct the eukaryotic expression vector pcDNA3 -T-bet. Results The positive recombinants were screened by PCR. The direction of T-bet gene recombination was identified by double enzyme digestion, and the DNA sequence changes without mismatch and insertion were identified by sequencing. Conclusion This study successfully constructed the mouse T-bet gene eukaryotic expression vector pcDNA3-T-bet.