pWPXL-EGFP-IRES-hTfR慢病毒载体的构建和鉴定

Construction and Identification of pWPXL-EGFP-IRES-hTfR Lentivirus Vector

【目的】将人转铁蛋白受体(hTfR)基因克隆到慢病毒表达载体pWPXL,并进行鉴定,为活体MR分子成像提供实验基础。【方法】利用聚合酶链反应(PCR)扩增hTfR基因,并克隆到pWPXL载体;通过菌落PCR初步筛选、MluⅠ/SpeⅠ双酶切和测序鉴定构建的pWPXL-EGFP-IRES-hTfR重组载体。【结果】hTfR基因片段重组到pWPXL载体;菌落PCR和双酶切鉴定,电泳结果显示均能得到与理论大小相符的片段;测序结果与Genebank序列完全一致。【结论】pWPXL-EGFP-IRES-hTfR慢病毒表达载体构建成功,可以用于下步的活体MR分子影像学研究。

[Objective] To clone the human transferrin receptor （hTfR） into the lentivirus vector pWPXL and identificate the reconstructed plasmid, which will provide experimental foundation for in vivo MR molecular imaging. [Methods] hTfR gene cDNA was amplicated by polymerase chain reaction （PCR） and cloned into the pWPXL vector. The reconstructed pWPXL-EGFP-IRES-hTfR plasmid was identified by colony PCR, Mlu Ⅰ/Spe Ⅰ enzyme digestion and sequencing analysis. [Results] hTfR gene fragment was constructed into the pWPXL vector. The reconstructed plasmid was identified correct by colony PCR and enzyme digestion. DNA sequencing analysis confirmed that hTfR gene sequence was exactly the same with that reported by Genbank. [ Conclusion] pWPXL-EGFP-IRES-hTfR lentivirus vector has been successfully constructed, and could be applied for in vivo MR molecular imaging.