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目的:在靶向克隆酶的作用下,利用PCR靶向克隆构建携带人神经生长因子的重组腺病毒穿梭质粒pShuttle-hNGFβ-EGFP。方法:按PCR引物设计原则设计两条引物,分别在上下游引物的5′端加上12个碱基与线性化载体切口两端同源载体序列,引物合成后PCR反应扩增目的基因hNGF-β,提纯后与EcoRⅤ酶切的穿梭质粒pShuttle-IRES-hrEGFP在靶向克隆酶(LPReccoenzyme)的作用下37℃孵育15min,转化感受态大肠杆菌,碱裂解法制备及纯化质粒DNA。结果:EcoRⅤ酶切鉴定证实pShuttle-hNGFβ-EGFP构建成功。结论:在不需要限制性内切酶的情况下用靶向克隆酶连接可快速、准确得到穿梭质粒pShuttle-hNGFβ-EGFP。
OBJECTIVE: To construct a recombinant adenovirus shuttle plasmid pShuttle-hNGFβ-EGFP carrying human nerve growth factor (NGF) by PCR-targeted cloning under the action of targeted cloning enzyme. Methods: Two primers were designed according to the principle of PCR primer design, and 12 bases and linearized vector nicked ends vector sequences were added to the 5 ’end of the upstream and downstream primers, respectively. PCR amplification of the target gene hNGF- β. The purified shuttle plasmid pShuttle-IRES-hrEGFP digested with EcoRⅤ was incubated with LPReccoenzyme for 15 min at 37 ℃. The recombinant plasmid was transformed into competent E. coli and plasmid DNA was prepared and purified by alkaline lysis. Results: EcoRⅤ digestion confirmed pShuttle-hNGFβ-EGFP was successfully constructed. Conclusion: The shuttle plasmid pShuttle-hNGFβ-EGFP can be rapidly and accurately obtained by targeting cloning enzyme without the restriction enzyme.